Adrenal and extra-adrenal production of 11-deoxycorticosterone
Monica Ann Schneider
A thesis submitted for the degree of Doctor of Philosophy
University of London
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The conversion of progesterone to 11-deoxycorticosterone (DOC), a steroid known to be hypertensive, by adrenal and extra-adrenal 21-hydroxylase enzyme activity was investigated. For assessment of DOC production, a method for determining the excretion rate of tetrahydro-11-deoxycorticosterone (THDOC), a urinary metabolite of DOC, was developed. Various chromatographic techniques were employed, with detection of the steroid by gas chromatography coupled to a mass spectrometer (GC-MS), using selected ion monitoring (SIM), after MO-TMS ether formation. Inspection of the mass spectrum of THDOC showed Ion m/z 476 to be the obvious candidate for SIM, with the molecular ion (M+, m/z 507) providing confirmatory evidence for the steroid. Ion 476 proved to be unsuitable for quantitative analysis, in pregnancy and some other clinical situations, due to the presence of co-eluting steroids. The molecular ion was therefore used for quantification. These data highlighted the need for users of SIM in general to take care in the selection of the monitored ions. During pregnancy, serial urine samples were analyzed in normal women, patients with raised progesterone (for example from ovarian theca lutein cysts) and hypertension (pre-eclamptic toxaemia - PET). Ranges for various urinary steroid metabolites were established. Urine samples from patients undergoing in vitro fertilization with oocyte donation were also investigated, both during and after progesterone and oestrogen administration, which allowed assessment of the exogenous steroids. Correlation of excretion rates of THDOC with pregnanediol, a main urinary metabolite of progesterone, offered some support for extra-adrenal 21- hydroxylase activity. Many urinary metabolites of progesterone, generally hydroxypregnanolones (including THDOC) were found, particularly in pregnancy. The relative importance of some of these metabolites, using the Ion 476 SIM response, was found to be different in PET and placental sulphatase deficiency (PSD). One of the
2 hydroxypregnanolones, in pregnancy urine extracts, was found to co-elute with THDOC on gas chromatography. Various methods of separation of this co-eluting steroid, prior to the GC analysis were explored. The use of Celite gradient elution chromatography eventually facilitated almost complete separation and allowed tentative identification of 3,16-dihydroxypregnan-20-one. A hypothesis, put forward in the literature, that extra-adrenal DOC production is promoted by oestrogens, was not supported by data from pregnant subjects with PSD (who have low oestrogen production), as they were found to have THDOC excretion rates similar to normal pregnant subjects. Doubt was also raised, from mass spectral evidence, as to the accuracy of progesterone to DOC conversion rates (measured by the rate of THDOC excretion) quoted in a number of published papers, that used radioactive isotope ratios to suggest the presence of extra-adrenal DOC production. THDOC excretion rates were quantified in a number of further clinical situations with elevated progesterone and/or DOC production. A patient with a recurring DOC secreting tumour, followed over 53 months, along with subjects with congenital adrenal hyperplasia (due to 116-, 17- and 21-hydroxylase deficiency), were studied. The separate function of the zona glomerulosa (ZG) and fasciculata (ZF) of the adrenal cortex is described in a patient with 116-hydroxylase deficiency. DOC production was stimulated in the ZF after stimulation of the renin-angiotensin system, following suppression of ACTH stimulation of the ZG. A further objective of the project was to explore the use of deuterium labelled steroids in metabolic studies, thus avoiding the use of radioactivity, with its inherent risks, particularly during pregnancy. Deuterium labelled progesterone was obtained and its purity and enrichment assessed by GC-MS; unfortunately there was too little for further studies. A pilot study with 2H-cortisol showed that this could be useful in studying cortisol metabolites in vivo.
3a List of abbreviations
11BOHSD llfl-hydroxysteroid dehydrogenase 170HPr 17-hydroxypregnanolone (3a, 17a-dihydroxy-5fi-pregnan-20-one) ACTH Adrenocorticotrophin hormone And. Androsterone (3 a-hydroxy-5a-androstan-17-one) Aet. Aetiocholanolone (3a-hydroxy-5fl-androstan-17-one) A,S and C Internal standards for quantification - 5a-androstane-3a,17a-diol, stigmasterol and cholesteryl butyrate BO Benzylhydroxylamine BSA Body surface area CAH Congenital adrenal hyperplasia CBG Corticosterone binding globulin CRF Corticotrophin releasing factor dex. Dexamethasone DHA[-S] Dehydroepiandrosterone [-sulphate] (3fl-hydroxy-15-androsten-17-one) DOC 11-deoxycorticosterone (21-hydroxy-4-pregnene-3,20-dione) E Cortisone (17a,21-dihydroxy-4-pregnene-3,11,20-trione) EMS Early morning sample EO Ethoxylamine Eyal Oestradiol valerate F Cortisol (1 lfl,17a,21-trihydroxy-4-pregnene-3,20-dione) FID Flame ionization detector foil. Follicular (phase of the menstrual cycle) GC-MS Gas chromatography-mass spectrometry HBV Hold back volume hCG Human chorionic gonadotrophin HMDS Hexamethyldisilazane HO Hydroxyl amine HPLC High performance liquid chromatography IA Immunoadsorption I.D. Internal diameter IRMS Isotope ratio mass spectrometry IS Internal standard IVF In vitro fertilization KCH Kings College Hospital LDL Low density lipoproteins M+ Molecular ion MIS Mullerian inhibitory substance MO-HC1 Methyloxime hydrochloride MO-TMS Methyloxime trimethylsilyl ether MSD Mass Selective Detector MU Methylene unit
3b NMR Nuclear magnetic resonance oc. Oral contraceptives 0E3 Oestriol (1,3,5(10)-oestratrien-3,16a, 1715-triol) -OH Hydroxylase P Progesterone (4-pregnene-3,20-dione) PCO Polycystic ovary PD Pregnanediol (5fi-pregnane-3a,20a-diol) PET Preeclamptic toxaemia PRA Plasma renin activity Prl, Pr2 and Pr3 Additional hydroxypregnanolones found in SIM runs, using ion 476, in pregnancy urine samples PSD Placental sulphatase deficiency PT Pregnanetriol (5fi-pregnane-3a, 17a,20a-triol) RER Rough endoplasmic reticulum RIA Radioimmunoassay SD Standard deviation SER Smooth endoplasmic reticulum SIM Selected ion monitoring S 11-deoxycortisol (17a,21-dihydroxy-4-pregnene-3,20-dione) SV Simple virilizing SW Salt wasting TIC Total ion chromatogram THDOC Tetrahydrodeoxycorticosterone (unless otherwise stated the 3a,2 l-dihydroxy-5B-pregnan-20-one isomer) THE Tetrahydrocortisone (3a, 17a,2 l-trihydroxy-56-pregnane-l 1,20-dione) THF Tetrahydrocortisol (3a, 1 IB, 17a,21-tetrahydroxy-5fi-pregnan-20-one) THS Tetrahydrodeoxy cortisol (3a, 17a,21-trihydroxy-5B-pregnan-20-one) TLC Thin layer chromatography TMSI Trimethylsilyl imadazole
3c Table of contents
Chapter Index
T itle ...... 1 Abstract ...... 2 Table of contents...... 4 Acknowledgements...... 19
Chapter 1 - Introduction...... 20 1.1 Initial aims of the project 1.2 Maturation and inhibition of the human adrenal cortex 1.2.1 Embryology 1.2.2 Cytology 1.2.3 Vasculature and innervation 1.2.4 Pathways of adrenal steroid biochemistry 1.2.5 Enzymes of adrenal steroid biosynthesis 1.2.6 Control of steroidogenesis 1.2.7 Cell differentiation and adrenal growth 1.2.8 Fetal steroidogenesis and the "feto-placental steroidogenic unit" 1.2.9 Parturition 1.2.10 Neonatal life 1.2.11 Adrenarche 1.2.12 Sexual differentiation and congenital adrenal hyperplasia 1.2.13 Polycystic ovary (PCO) syndrome 1.2.14 Ectopic adrenal tissue and tumours 1.2.15 Adrenopause Clinical significance of deoxycorticosterone 1.3.1 Introduction 1.3.2 The menstrual cycle 1.3.3 Pregnancy 1.3.4 Other clinical situations with altered patterns of plasma DOC concentrations
4 1.4 Extra-adrenal 21 -hydroxylase enzymes 1.5 The renin-angiotensin system 1.6 Profiling steroid hormones using glass capillary gas chromatography 1.6.1 Introduction 1.6.2 Extraction 1.6.3 Hydrolysis 1.6.4 Additional separation of steroids 1.6.5 Derivatization of steroids 1.6.6 Gas chromatographic conditions and detection 1.7 The use of stable isotopes in Endocrinology 1.7.1 Radioactive versus stable isotopes 1.7.2' Analytical techniques available 1.7.3 Availability :s , ■, i > > > > 1.7.4 Toxicity --N N % 1.7.5 Quantitative applications 1.7.5.1 Internal standards and isotope dilution 1.7.5.2 Studies of metabolites of endogenous compounds 1.7.5.3 Pharmacological studies 1.7.5.4 Other quantitative methods 1.7.6 Qualitative applications 1.7.6.1 Isotope cluster technique (or ion doublet/twin ion technique) 1.7.6.2 Mechanistic studies 1.8 Altered plan of investigation
Chapter 2 - Materials and m ethods...... 76 2.1 Urinary steroid profiles 2.1.1 Extraction of steroids 2.1.2 Hydrolysis of steroid conjugates and re-extraction of free steroids 2.1.3 Derivative formation and sample clean up 2.1.4 Conditions for gas chromatography (GC)/mass spectrometry (MS) 2.1.4.1 GC analysis for urinary steroid profiles 2.1.4.2 Mass spectral data acquisition for confirmation and identification of steroids in urinary steroid profiles 2.1.5 Quantification of steroid profiles 2.2 THDOC quantification 2.2.1 Addition of internal standard and extraction of steroids 2.2.2 Hydrolysis of steroid conjugates and re-extraction of free steroids 2.2.3 Sephadex LH-20 chromatography
5 2.2.4 Derivatization formation and sample clean up 2.2.5 Quantification of 3a5fl THDOC by SIM using GC-MS
Chapter 3 - Development of the GC-MS method for quantitative determinations of tetrahydrodeoxycorticosterone in urine ...... 88 3.1 Characteristics of 3B5a THDOC as internal standard 3.2 Choice of selected ions and method of quantification 3.3 Sensitivity 3.4 Introduction of a Sephadex LH-20 chromatography step 3.5 Use of alternative derivatives to separate THS from 3a5B THDOC 3.6 Inconsistency in 476:507 ratios for "3a5B THDOC" 3.7 Method validation
Chapter 4 - Separation of the co-eluting steroids ...... 107 4.1 Immunoadsorption 4.1.1 Introduction 4.1.2 Precipitation of immunoglobulins 4.1.3 Cyanobromide activation of Sephadex G25 4.1.4 Coupling of antibodies to activated polymer 4.1.5 Urine sample processing 4.1.6 Progesterone antisera immunoadsorption 4.1.7 THDOC antisera immunoadsorption 4.2 Change in GC conditions 4.3 Alternative GC derivatives 4.4 Celite columns 4.4.1 Introduction 4.4.2 Experimental work 4.4.2.1 Hydrolysis 4.4.2.2 Extraction 4.4.2.3 Gradient elution chromatography 4.4.2.4 Partition chromatography 4.4.2.5 First thin layer chromatography (TLC) 4.4.2.6 Second TLC (separation of diacetates) Discussion
Chapter 5 - Normal subjects and the menstrual cycle ...... 158 5.1 Introduction 5.2 Experimental 5.3 Subjects and results
6 5.3.1 Adult males 5.3.2 Adult females and the menstrual cycle 5.3.3 Children 5.3.4 Inter-subject variation 5.3.5 Pregnanediol:THDOC ratios 5.3.6 Cortisol metabolites 5.3.7 Influence of body surface area on excretion rate 5.3.8 Use of creatinine corrected excretion rates and early morning urine samples 5.4 Discussion
Chapter 6 - 1113-hydroxylase deficiency congenital adrenal hyperplasia.... 181 6.1 Introduction 6.2 Experimental 6.3 Subjects and results 6.3.1 Two brothers with 116-hydroxylase deficiency (Subjects Y1 and Y2) 6.3.2 Other cases of 116-hydroxylase deficiency 6.4 Discussion
Chapter 7 - Mineralocorticoid secreting tumour...... 198 7.1 Introduction 7.2 Case history and results 7.3 Discussion
Chapter 8 - Normal pregnancy...... 207 8.1 Introduction 8.2 Experimental 8.3 Subjects 8.4 Steroid excretion rates (from urinary steroid profiles) 8.5 THDOC excretion rates 8.6 Post partwn excretion rates 8.7 Pregnanediol:THDOC ratios 8.8 476:507 ratios 8.9 Other hydroxypregnanolones in SIM runs 8.10 Discussion
Chapter 9 - Placental sulphatase deficiency...... 238 9.1 Introduction 9.2 Experimental 9.3 Subjects
7 9.4 Steroid excretion rates (from urinary steroid profiles) 9.5 THDOC excretion rates 9.6 Pregnanediol:THDOC ratios 9.7 476:507 ratios 9.8 Other hydroxypregnanolones in SIM runs 9.9 Discussion
Chapter 10 - Pre-eclamptic toxaemia and hypertension in pregnancy 253 10.1 Introduction 10.2 Experimental 10.3 Subjects 10.4 Steroid excretion rates (from urinary steroid profiles) 10.5 THDOC excretion rates 10.6 Pregnanediol:THDOC ratios 10.7 476:507 ratios 10.8 Other hydroxypregnanolones in SIM runs 10.9 Discussion
Chapter 11 - Other clinical situations with raised progesterone...... 261 11.1 Pregnancies maintained with exogenous hormones after in vitro fertilization using donated oocytes 11.1.1 Introduction 11.1.2 Subjects 11.1.3 Experimental 11.1.4 Urinary steroid profile and plasma hormone concentration results 11.1.5 THDOC excretion rates 11.1.6 Pregnanediol:THDOC ratios 11.1.7 476:507 ratios 11.1.8 Other hydroxypregnanolones in SIM runs 11.1.9 Discussion Ovarian theca-lutein cysts (hyperreactio luteinalis) 11.2.1 Introduction 11.2.2 Case history 11.2.3 Results 11.2.4 Discussion 11.3 Pregnancy complicated by 21-hydroxylase deficiency congenital adrenal hyperplasia 11.4 Pregnancy complicated by suspected late-onset 21-hydroxylase deficiency CAH
8 11.5 Pregnancy complicated by suspected Cushing’s syndrome 11.6 Pregnancy in a post pituitary operation patient 11.7 Ovarian progesterone secreting tumour 11.8 Congenital adrenal hyperplasia due to 21-hydroxylase deficiency 11.8.1 Introduction 11.8.2 Classical C AH results 11.8.3 Late-onset CAH results 11.8.4 21-hydroxylase deficiency CAH with extra raised progesterone metabolites due to a steroid secreting tumour 11.8.5 Discussion 11.9 Congenital adrenal hyperplasia due to 17-hydroxylase deficiency 11.9.1 Introduction 11.9.2 Results 11.9.3 Discussion
Chapter 12 - General Discussion...... 305
Appendix 1 - The use of deuterated cortisol to investigate the action of llfi-hydroxysteroid dehydrogenase - a pilot study ...... 317
Appendix 2 - Additional work involving deuterium labelled steroids.... 331
Appendix 3 - Mass spectral data on deuterium labelled progesterone .... 337
Appendix 4 - Initial DOC radioimmunoassay work ...... 346
R eferences...... 351
Source of m aterials...... 380
9 List of Figures
Chapter 1 - Introduction 1.1 Summary of adrenal steroidogenesis and the main urinary metabolites 1.2 Winter’s feto-placental model 1.3 Structures of steroids involved in DOC production and metabolism 1.4 The control of aldosterone secretion
Chapter 2 - Materials and methods 2.1 Cross section through solid injector device and connection for capillary column to gas chromatograph (FID) 2.2 Urinary steroid profile from a female subject in the luteal phase of the menstrual cycle 2.3 Temperature programme for THDOC SIM runs 2.4 Ion 507 and 476 responses from a SIM run of MO-TMS ether derivatized urine extract from a female in the luteal phase of the menstrual cycle
Chapter 3 - Development of the GC-MS method for quantitative determinations of tetrahydrodeoxycorticosterone in urine 3.1 GC run of the two isomers of THDOC 3.2 Partial mass spectra (m/z = 98 - 520) of the two isomers of THDOC 3.3 Height vs area - Quantification from GC FID traces 3.4 Partial mass spectrum (m/z = 98 - 600) of the MO-TMS ether derivative of tetrahydrodeoxycortisol (THS) 3.5 Partial mass spectrum (m/z = 98 - 600) of the MO-TMS ether derivative of oestriol (OE3) 3.6 Sephadex LH-20 fractions from urine of an adrenalectomized patient after tritiated DOC administration 3.7 Sephadex LH-20 fractions - Pregnancy sample (1.2g column) 3.8 Structures of different carbonyl derivatives of THDOC (all as TMS ethers) 3.9 Initial 476:507 ratios 3.10 Possible partial structures of steroids co-eluting with 3a5J3 THDOC 3.11 476:507 ratios - Longitudinal pregnancy urine samples 3.12(a-d) Standard curves
10 Chapter 4 - Separation of the co-eluting steroids 4.1 Standard curves with and without progesterone immunoadsorption 4.2 Effect of varying quantities of immunoadsorption gel on pregnanediol 4.3 Temperature programmes - Change in GC conditions 4.4 Summary of Winkel et al. (1980a) method 4.5 Typical gradient elution curve and experimental set up 4.6 Comparison of elution patterns using 20g and 30g Celite gradient elution columns 4.7 The effect of column height on THDOC elution from gradient elution columns 4.8 Fraction analysis - Ion 507 (3a5B THDOC) - Gradient elution columns 4.9 Fraction analysis - Ion 476 (3a515 THDOC) - Gradient elution columns 4.10 Fraction analysis - 17-OHPregnanolone using ion 476 - Gradient elution columns 4.11 Fraction analysis - 476:507 ratios - Gradient elution columns 4.12 Fraction analysis Prl, Pr2 and Pr3 (gradient elution) from Pregnancy 2 using ion 476 4.13 Partition chromatography 3H-THDOC and 3H-DOC 4.14 Fraction analysis - Ion 507 (3a5B THDOC) - Partition chromatography 4.15 Fraction analysis - Ion 476 (3a5B THDOC) - Partition chromatography 4.16 Fraction analysis - 17-OHPr (using ion 476) - Partition chromatography 4.17 Fraction analysis - 476:507 ratios - Partition chromatography 4.18 Fraction analysis - Prl, Pr2 and Pr3 (partition chrom.) from Pregnancy 2 70-90 using ion 476 4.19 Partial mass spectrum (m/z = 98 - 520) of the MO-TMS ether derivative of the steroid co-eluting in pregnancy with THDOC 4.20 Sections and results off TLC plate 1 4.21 Sections and results off TLC plate 2 4.22 Partial mass spectrum (m/z = 98 - 500) of the MO-TMS ether derivative of THDOC diacetate 4.23 Partial mass spectrum (m/z = 98 - 347) of MO-TMS ether derivative of pregnanediol diacetate 4.24 Partial mass spectrum (m/z = 98 - 480) of MO-TMS ether derivative of 17-hydroxypregnanolone acetate
Chapter 5 - Normal subjects and the menstrual cycle 5.1 THDOC excretion rates - Males 5.2 THDOC excretion rates - Females in follicular phase or taking oral contraceptives
11 5.3 THDOC excretion rates - Females in luteal phase 5.4 THDOC excretion rates - Changes in a normal menstrual cycle (Subject N18) 5.5 THDOC excretion rates - Children 5.6 THDOC excretion rates - Intrs.-subject variation: Male 5.7 THDOC excretion rates - Intra-subject variation: Female taking oral contraceptive 5.8 PD/THDOC Normal adults (THDOC using Ion 507) 5.9 THE/THF Normal adults: range and median 5.10 THF/aTHF normal adults: range and median 5.11 Creatinine corrected early morning sample compared to 24 hour collection - THDOC (Ion 476) 5.12 Creatinine corrected early morning sample compared to 24 hour collection - THE
Chapter 6 - 110-hydroxylase deficiency congenital adrenal hyperplasia 6.1 Urinary steroid profile from a patient (Yl) with congenital adrenal hyperplasia due to 110-hydroxylase deficiency 6.2 Change in THDOC excretion rates and blood pressure with treatment (Subject Yl) 6.3 SIM run from a patient with 118-hydroxylase deficiency CAH 6.4 Effect of Na+ restriction on urinary THDOC and THS excretion rates 6.5 Effect of Na+ restriction on plasma DOC, S and ACTH 6.6 Effect of Na+ restriction on plasma renin activity (PRA) 6.7 Model for the regulation of adrenocortical steroidogenesis in 118- hydroxylase deficiency CAH
Chapter 7 - Mineralocorticoid secreting tumour 7.1(a+b) Urinary steroid profiles (a) pre-op and (b) 34 months post-op from a patient with a recurring mineralocorticoid secreting tumour 7.2 THDOC excretion rates - Mineralocorticoid secreting tumour
Chapter 8 - Normal pregnancy 8.1 Urinary steroid profile from a normal pregnancy (30 weeks gestation) 8.2 Pregnanediol excretion rate - Normal pregnancies 8.3 Oestriol excretion rate - Normal pregnancies 8.4 17-hydroxypregnanolone excretion rate - Pregnancy (week 18 - term) 8.5 Pregnanetriol excretion rate - Pregnancy (week 18 - term) 8.6 THS excretion rate - Pregnancy (week 18 - term)
12 8.7 THE excretion rate - Pregnancy (week 18 - term) 8.8 Total cortisol metabolites excretion rate - Pregnancy (week 18 - term) 8.9 THE/THF - Pregnancy (week 18 - term) 8.10 THF/5aTHF - Pregnancy (week 18 - term) 8.11 PD/OE3 - Normal Pregnancies 8.12 The effect of the Sephadex LH-20 chromatography step on pregnancy urine extracts 8.13 Example of a SIM run for THDOC quantification using normal pregnancy urine (30 weeks gestation) 8.14 THDOC excretion rate - Ion 507 response - Normal pregnancies 8.15 THDOC + co-eluting steroid - Ion 476 response - Normal pregnancies 8.16 THDOC - comparison of 476 and 507 response 8.17(a-d) THDOC Ion 476 response : Ion 507 response - Normal pregnancies 8.18 THDOC excretion rate - post partum - Ion 476 response 8.19 THDOC excretion rate - post partum - Ion 507 response 8.20 PD:THDOC (ion 507 response) - Normal pregnancies 8.21 476:507 ratios - Pregnancy and post partum - Subject P3 8.22 Partial mass spectrum (m/z = 98 - 520) of the MO-TMS derivative of Prl (additional hydroxypregnanolone seen in pregnancy) 8.23 Partial mass spectrum (m/z = 98 - 520) of the MO-TMS derivative of Pr2 (additional hydroxypregnanolone seen in pregnancy) 8.24 Partial mass spectrum (m/z = 98 - 520) of the MO-TMS derivative of Pr3 (additional hydroxypregnanolone seen in pregnancy) 8.25 Prl - Ion 476 response relative to analyte peak response 8.26 Pr2 - Ion 476 response relative to analyte peak response 8.27 Pr3 - Ion 476 response relative to analyte peak response
Chapter 9 - Placental sulphatase deficiency 9.1 Urinary steroid profile from a pregnancy complicated by placental sulphatase deficiency 9.2 Pregnanediol excretion rate - Placental sulphatase deficiency 9.3 Excretion rates of summed oestriol precursors - Placental sulphatase deficiency 9.4 THDOC excretion rate - Ion 507 response - PSD vs normal pregnancies 9.5 THDOC excretion rate - Ion 476 response - PSD vs normal pregnancies 9.6 THDOC Ion 476 : Ion 507 response - Placental sulphatase deficiency 9.7 PD:THDOC (Ion 507 response) - Placental sulphatase deficiency 9.8 SIM run from a pregnancy complicated by placental sulphatase deficiency
13 9.9 Prl - Ion 476 response relative to analyte peak response - PSD vs normal pregnancies 9.10 Pr2 - Ion 476 response relative to analyte peak response - PSD vs normal pregnancies 9.11 Pr3 - Ion 476 response relative to analyte peak response - PSD vs normal pregnancies
Chapter 10 - Pre-eclamptic toxaemia and hypertension in pregnancy 10.1 Pregnanediol excretion rate - PET vs normal pregnancies 10.2 Oestriol excretion rate - PET vs normal pregnancies 10.3 THDOC excretion rates - Ion 507 response - PET vs normal pregnancies 10.4 THDOC excretion rates - Ion 476 response - PET vs normal pregnancies 10.5 Prl - Ion 476 response relative to analyte peak response - PET vs normal pregnancy 10.6 Pr2 - Ion 476 response relative to analyte peak response - PET vs normal pregnancy 10.7 Pr3 - Ion 476 response relative to analyte peak response - PET vs normal pregnancy
Chapter 11 - Other clinical situations with raised progesterone 11.1 Treatment regime and plasma hormone results from four successful donated oocyte in vitro fertilization pregnancies maintained in the first trimester by exogenous hormones 11.2 Pregnanediol excretion rate in exogenous hormone maintained pregnancies with oocyte donation 11.3 Oestriol excretion rate in exogenous hormone maintained pregnancies with oocyte donation 11.4 THDOC Ion 507 excretion rate in exogenous hormone maintained pregnancies with oocyte donation 11.5 THDOC Ion 476 excretion rate in exogenous hormone maintained pregnancies with oocyte donation 11.6 Prl - Ion 476 response relative to analyte peak response - Ovum donation vs normal pregnancy 11.7 Pr2 - Ion 476 response relative to analyte peak response - Ovum donation vs normal pregnancy 11.8 Pr3 - Ion 476 response relative to analyte peak response - Ovum donation vs normal pregnancy 11.9 Urinary steroid profile from a patient with theca-lutein cysts 11.10 Urinary steroid excretion rate - Theca-lutein cysts
14 11.11 THDOC excretion rate - Theca-lutein cysts 11.12 Urinary steroid profile from a pregnancy complicated by suspected late- onset 21-hydroxylase deficiency CAH 11.13 Urinary steroid profile from a patient with a progesterone secreting ovarian tumour 11.14 Urinary steroid profile from (a) a child with classical 21-hydroxylase deficiency CAH, and (b) a patient with 21-hydroxylase deficiency and a steroid secreting adrenal tumour 11.15 SIM runs from (a) a patient with classical 21-hydroxylase deficiency CAH, and (b) a patient with 21-hydroxylase deficiency and a steroid secreting adrenal tumour 11.16 Main urinary steroid metabolites seen in a patient with CAH due to 21- hydroxylase deficiency with a steroid secreting adrenal tumour 11.17 Urinary steroid profile from a patient with 17-hydroxylase deficiency CAH 11.18 SIM run from a patient with 17-hydroxylase deficiency CAH
Appendix 1 - The use of deuterated cortisol to investigate the action of llfi-hydroxysteroid dehydrogenase - a pilot study A l.l Cortisol and cortisone metabolites A1.2 Partial mass spectrum (m/z = 98 - 650) of the MO-TMS ether derivative of 1 la-2H-cortisol A1.3 Temperature programme for SIM quantification of cortisol A1.4 Standard curve A1.5 SIM run of cortisol and 2H-cortisol standards A1.6 SIM run of 0.1ml serum spiked with lOng of 2H-cortisol A1.7 605:606 ratios from plasma samples basal, and with carbenoxolone treatment in a normal subject A1.8 2HF:F ratios from plasma samples basal, and with carbenoxolone treatment in a normal subject A1.9(a+b) Cortisol (RIA results) A1.10 605:606 ratios from plasma samples in an adult subject with 11B-OHSD deficiency
Appendix 2 - Additional work involving deuterium labelled steroids A2.1 Isotope enrichment in 3a515 THDOC (unlabelled and deuterium labelled) A2.2 Partial mass spectra (m/z = 98 - 520) of the MO-TMS ether derivatives of (a) pregnanediol and (b) 2H3 pregnanediol from the urine extract of an adult male subject loaded with 2H4-pregnanolone
15 A2.3 Partial mass spectra (m/z = 98 - 520) of the MO-TMS ether derivatives of (a) pregnanetriol and (b) 2H5 pregnanetriol from the urine extract of an adult male subject loaded with 2H8-17-hydroxyprogesterone A2A Part of the total ion chromatogram from the urine extract of an adult male subject loaded with 2H8-17-hydroxyprogesterone (MO-TMS ether derivatives)
Appendix 3 - Mass spectral data on deuterium labelled progesterone A3.1 Partial mass spectra (m/z = 98 - 380) of the MO derivative of 11,11,12,12-2H4-progesterone (upper panel) and unlabelled progesterone (lower panel) A3.2 Total ion chromatogram of the MO derivative of ll,12,16-2H3-5a- pregnanedione A3.3 Partial mass spectra (m/z = 98 - 400) of MO derivatives of 11,12,16- 2H3-pregnenedione (upper panel) and 11,12,16-2H3-5a-pregnanedione (lower panel) A3.4 Partial mass spectra (m/z = 98 - 400) of MO derivative of 15,15,16-2H3- progesterone A3.5 Partial mass spectra (m/z = 98 - 400) of MO derivative of 15,15,16-2H3- pregnanedione A3.6 Total ion chromatogram of MO-TMS ether derivative of crystals of 1,11,12,16-2H4-progesterone A3.7 Partial mass spectrum (m/z = 98 - 400) of MO derivative of 1,11,12,16- 2H4-progesterone A3.8 Partial mass spectra (m/z = 98 - 400) of MO derivative of deuterated steroids in the crystals containing 1,11,12,16-2H4-progesterone. Possible identification (a) 2H2-progesterone and (b) mixture of 2H3- and 2H2- pregnanedione A3.9 Partial mass spectrum (m/z = 98 - 400) of MO derivative of 18,18,19,19-2H4-progesterone
Appendix 4 - Initial DOC radioimmunoassay work A4.1 Standard curve for DOC RIA and the effect of antibody concentration A4.2 Tritium content of HPLC fractions - labelled DOC vs background
16 List of Tables
Chapter 1 - Introduction 1.1 Some stable isotopes of interest 1.2 Estimated possible rate constant ratios at 25°C for various stable isotopes
Chapter 2 - Materials and methods 2.1 Methylene units (MU) of some MO-TMS ether derivatives of urinary steroids on OV-1 type capillary columns
Chapter 4 - Separation of the co-eluting steroids 4.1 Analysis of mass specta of alternative GC derivatives of THDOC (all as TMS ether derivatives) 4.2 Analysis of mass specta of alternative GC derivatives (all as TMS ether derivatives) of the steroid peak at the GC retention time of 3a5fl THDOC
in pregnancy and comparison with ions from a (3),16-dihydroxy-20-one C 2 1 steroid 4.3 Variation in Celite columns used for gradient elution chromatography 4.4 Variation in Celite columns used for partition chromatography
Chapter 5 - Normal subjects and the menstrual cycle 5.1 Urinary steroid excretion in adult males (fig/24 hours) 5.2 Urinary steroid excretion in adult females in the follicular phase or taking oral contraceptives (fig/ 24 hours) 5.3 Urinary steroid excretion in adult females in the luteal phase (fig/24 hours) 5.4 Urinary steroid excretion in children (fig/24 hours) 5.5 Intra-subject variation in steroid excretion (fig/24 hours) 5.6 Steroid excretion (fig/24 hours) corrected for body surface area (BSA) 5.7 Steroid excretion (ftmol/24 hours) corrected for creatinine excretion
Chapter 6 - llh-hydroxylase deficiency congenital adrenal hyperplasia 6.1 Plasma results from subject Yl (llB-hydroxylase deficiency CAH)
Chapter 7 - Mineralocorticoid secreting tumour 7.1 Laboratory investigations on admission to KCH 7.2 Additional steroid results
17 Chapter 11 - Other clinical situations with raised progesterone 11.1 Details of subjects undergoing in vitro fertilization with donated oocytes 11.2 Cyclical regimen of hormone replacement therapy 11.3 Urinary steroid profile results from a patient with ovarian theca-lutein cysts 0*g/24h) 11.4 Urinary steroid excretion rates from subjects with 21-hydroxylase deficiency CAH 11.5 Urinary steroid excretion rates from subjects with late-onset 21-hydroxylase deficiency CAH
Appendix 1 - The use of deuterated cortisol to investigate the action of llfl-hydroxysteroid dehydrogenase - a pilot study A 1.1 Spiking experiment using deuterated cortisol A1.2 Ratios of cortisol symanti peak areas using ions 605 and 606
18 Acknowledgements
I would like to thank Dr. John Honour for his valuable help and advice as supervisor of these studies.
I am grateful to the staff, past and present, of the Cobbold laboratories and the Department of Chemical Pathology for their help and support, in particular
1C Peter Holownia. I also wish to thank Prof. Howard Jacobs for his helpful critism and advice throughout the period of these investigations.
Thanks also go to my parents for their continuing help and support, both moral and financial.
Finally I thank Nigel, for his advice and help with the word processing and diagrams of this thesis, and for his love and support throughout my academic career.
Cobbold Laboratories MAS Middlesex Hospital May 1991 Mortimer Street London
A large part of this project was funded by the Medical Research Council to whom I am also grateful.
19 1 - Introduction
1.1 Initial aims of the project
Deoxycorticosterone (DOC) is a mineralocorticosteroid, generally thought to be of adrenocortical origin. It is produced from progesterone by the enzyme 21- hydroxylase (E.C. 1.14.99.10). In normal subjects an acute effect of adrenocorticotrophic hormone (ACTH) is to increase plasma concentrations of DOC by direct adrenal secretion. Studies in pregnant women show that production of DOC rises progressively in parallel with the increase in progesterone. In the third trimester of pregnancy plasma concentrations of DOC do not fall in response to dexamethasone or increase in response to ACTH treatment. An alternative extra-adrenal source of DOC is therefore suggested. Extra-adrenal formation of deoxycorticosterone from progesterone has been described by various groups in the literature. Current methods of measurement of this phenomenon rely upon the use of radioactive steroids, which are inappropriate and unethical to use in pregnancy and childhood. The plan for the proposed research was therefore to assess peripheral steroid metabolism, by administration of steroids labelled with stable isotopes and to quantify their metabolite by mass spectrometry. The funding for this project included the manufacture of progesterone labelled at specific sites with deuterium. The fate of this stable isotope labelled steroid after administration by injection could then be studied quantitatively and qualitatively by measuring changes in the mass spectrometric pattern of metabolites isolated from biological fluids (plasma and urine). The conversion (adrenal or extra-adrenal) of progesterone to DOC represents a change in biological activity from a natriuretic hormone to a salt retaining hormone. Changes in the extent of this transformation were hoped to help in the understanding
20 of salt and water retention seen in some pregnancies. The pathological significance of this conversion has not been studied, but if contributory to hypertension, or to premenstrual disturbances, it would explain some features of these clinical problems and suggest possible appropriate treatments. In subjects with 21-hydroxylase deficiency congenital adrenal hyperplasia (CAH), DOC production can be normal even with deficiency of the adrenal enzyme required for its production. Study of the peripheral 21-hydroxylase activity of the simple virilising and salt losing forms of this condition could help to clarify differences between the two forms. Once the labelled progesterone was synthesised it was planned to follow its fate after an initial priming injection and then constant infusion, in both plasma and urine samples. The following groups (with age matched controls) were of interest: salt and non-salt losing forms of CAH due to 21-hydroxylase deficiency, normal pregnancy, pregnancy in women with a family or personal history of high blood pressure in pregnancy, and women with premenstrual syndrome.
21 1.2 Maturation and inhibition of the human adrenal cortex
The adrenal glands, which together in the adult human weigh less than 15 grams, play essential roles in many stages of development and in body homeostasis, starting early in gestation and continuing throughout life. Steroid production is a function of the outer portion of the adrenal, the adrenal cortex. This gland undergoes much change in structure, mass and function during the life of a human being. The adrenal cortex is involved in the synthesis of mineralocorticoids, glucocorticoid and androgenic steroids. The normal scheme of events for adrenal steroidogenesis is summarised in Figure 1.1.
1.2.1 Embryology Embryologically the adrenal is derived from two components, ectodermal neural crest cells that form the medulla, and mesothelial cells that give rise to the cortex (O’Riordan et al ., 1985). During the fifth week of development mesothelial cells, located at the cranial ends of the mesonephros (i.e., between the root of the mesentery and the development urogenital ridge), proliferate and penetrate the underlying retroperitoneal mesenchyme. They form an acidophilic mass of cells, the adrenal blastema, which is penetrated by phaeochromaffinoblasts at about the seventh week of development. This primitive or fetal adrenal cortex is then surrounded by a second wave of mesothelially derived cells, which eventually become the cortex of the adult gland. The differentiation of the latter neocortex and the inner fetal cortex is seen at six to eight weeks of gestation. The inner fetal zone occupies approximately 85% of the total volume of the gland at this stage. Mesenchymal cells that surround the fetal cortex differentiate into fibroblasts and lay down the collagenous capsule of the gland. Blood and nerve supply also starts to develop during this stage. The adrenal continues to enlarge, mainly due to the expansion of the fetal zone, assuming an extended, flattened shape, which allows growth without any further increase in cortical thickness. The latter is thought to be important with respect to the blood supply - the venous end of the capillary bed cannot be too distant from its arterial supply. At term the adrenal gland can be as much as twenty times larger
22 Figure 1.1 - Summary of adrenal steroidogenesis and the main urinary metabolites 2 o o _J LU 0 1 LU oc o _l — , ■g CO ■O 0 tr ■c c <5 05 o o o o o o o o o g o CD x E o C CD (/) > Q_ cn LU 0 Z LU 2 o o _l ^ LU •6 o . Q. 8 8 i TO
I a eO, co €>*3 Q. 0 oc O LU 0 h- LU oc O 2 LU o . §> § involution of the fetal adrenal, and then slowly rise again during childhood (Dickerman et al ., 1984).
1.2.2 Cytology The three cortical adrenal zones in the adult are structurally distinct. The outermost zona glomerulosa, which occupies 5 - 10% of the cortex is composed of closely packed ill defined clusters of cells. They contain little rough endoplasmic reticulum (RER), but abundant smooth endoplasmic reticulum (SER). There is a relatively high nuclear to cytoplasmic ratio and some lipid droplets are present. The zona fasciculata on the other hand has an abundance of lipid droplets containing cholesterol esters and ascorbic acid. This zone occupies 75 % of the cortex and the lipid droplets give the gland its yellow colour. The fasciculata cells are larger than cells in the zona glomerulosa and are in long cords arranged radially with respect to the medulla. The cords are separated by straight cortical capillaries. In the fasciculata cells there is again an abundance of SER. The innermost cortical zone - the zona reticularis - is composed of a network of short cords with interdigitating capillaries. There is less extensive SER and fewer lipid droplets, but the cells are seen to contain more lysosomes and larger lipofucin granules, which increase in number with age.
24 Cell contacts between cortical cells in all three zones involve desmosomes. Large and numerous gap junctions are found functionally coupling the cells of the two inner zones.
1.2.3 Vasculature and innervation The adrenal gland, although only 0.02% of the total body weight in an adult, may receive 0.14% of the cardiac output. The blood supply for each gland is derived from a circle of different arteries arising from superior, middle and inferior adrenal arteries. Smaller vessels from these main trunks pierce the adrenal capsule and break up into a plexus. Three types of vessel are found, capsular, cortical and medullary arterioles. The cortical vessels descend from the capsular plexus and form a capillary bed which supplies the cortical parenchyma. Straight capillaries between the fasciculata cells join in the zona reticularis and empty into the medullary vascular bed. The medullary venules collect the blood, which then empties to a central vein. Blood from the left adrenal drains into the left renal vein and from the right adrenal into the inferior vena cava. Studies of micrographs of rat adrenal glands fixed under conditions of stress or ACTH administration have shown that every cortical cell is adjacent to a blood vessel (Hinson et al., 1986). Some cortical blood can leave the organ via the alar into an emissary vein, p&rcfcutarl/ veins, Awhen the other small veins are contracted (and hence obstructed) by longitudinal muscle columns* As blood vessels are constricted there is a build up of blood at first in the zona reticularis and inner zona fasciculata and then eventually outwards through the cortex. The increase in blood space and temporary slowing down of the passage of blood enables adrenocorticotrophic hormone (ACTH) to come in contact with the cells with greater ease. This explains changes in the morphology seen in the adrenal cortex from the zona reticularis outwards in response to stress, with for example, lipid droplet depletion (Dobbie and Symington, 1966). The innervation of the gland is derived from the splanchnic nerves that arise from the lateral horn preganglionic sympathetic neurones at spinal cord levels T8 to T il. Some of these fibres innervate the blood vessel walls of the gland, as mentioned above, while others enter the medulla and end in cholinergic synapses. The cells of the adrenal cortex are thought not to have any secretomotor innervation.
25 1.2.4 Pathways of adrenal steroid biochemistry Steroid hormones are derived from cholesterol. De novo biosynthesis of cholesterol however from acetate yields only a small proportion of cholesterol necessary for steroid hormone production, the majority coming from low density lipoproteins (LDL). LDL is taken into cells by endocytosis and acted upon by proteolytic and lipolytic enzymes to release cholesterol esters, which are stored in lipid droplets of the cortex cells. The importance of LDL cholesterol is demonstrated by cases of abetalipoproteinaemia in whom there is impaired secretion of adrenal steroids (Illingworth et al ., 1980). Increased intracellular cyclic AMP levels, due to ACTH stimulation, cause activation of protein kinase activity, that in turn results in phosphorylation of the enzyme cholesterol ester hydrolase. The latter in its active form catalyses the hydrolysis of stored cholesterol esters in the lipid droplets. Binding of cholesterol to cytochrome P-450 side chain cleavage enzyme results in the formation of pregnenolone. This is the rate limiting step in steroidogenesis. Increase rates of flux through subsequent steps in the pathway largely result from an enhanced entry of cholesterol. The division of adrenal steroidogenesis into three separate zones reflects some of the zonal differences in function and regulation. Along with the histological differences of the zones, each cortical layer produces a different spectrum of steroids. Aldosterone (a mineralocorticoid) is produced only by the zona glomerulosa. The zona fasciculata produces mainly cortisol, but other steroids, such as deoxycorticosterone (DOC), deoxycortisol and corticosterone are secreted, as well as some androgens. Adrenal androgens are produced in the adrenal largely in the zona reticularis. There is a good correlation between plasma concentrations of dehydroepiandrosterone-sulphate (DHA-S) and the development of the reticularis during childhood (Reiter et al., 1977). In order to produce active steroid hormones, there must be changes in the structure of cholesterol, this being achieved by several enzyme-catalysed reactions which involve cytochrome P-450 dependent enzymes. These are located specifically either microsomally or mitochondrially and their organisation within cells contributes a level of regulation for steroid biosynthesis.
26 1.2.5 Enzymes of adrenal steroid biosynthesis Hydroxylases The steroid hydroxylases are mono-oxygenase P-450 cytochromes, hence enzymes that reductively activate molecular oxygen for insertion into steroids and other lipids. All these enzymes require oxygen and NADPH for activity. In hydroxylation steps NADPH is the primary electron donor to the cytochrome and flavin-haem electron transfer is used finally resulting in the hydroxylation reaction and water being produced as a by-product. Carbon monoxide, nitrous oxide and cyanide ions may compete with oxygen binding sites. 21-hydroxylase, used both in the mineralocorticoid and glucocorticoid pathways has a relative molecular mass (Mr) of 52,000 and requires NADPH- dependent cytochrome reductase as its electron carrier. 21-hydroxylase is found in the microsomal function. The human gene for this enzyme is located on the short arm of chromosome 6 within the region coding for complement C4. Part of this region has been duplicated and hence two genes ’A’ and ’B’ are found, •functionally tMu'fe etaly fttfl a ) 21-hydroxylase A being a pseudogene that is not^ expressed^. In patients with a deficiency of 21-hydroxylase mutation or deletion of the 21-hydroxylase B gene prevents transcription. 21-hydroxylase is inhibited by pregnenolone and its
CCJowfery 1984- metabolites, and also by androstenedione and to a lesser degree by testosteroneA- 1115-hydroxylase (E.C. 1.14.15.4) occurs in the inner membranes of mitochondria of all zones of the adrenal cortex (Gower, 1984b; Arai et al . , 1972). It has an Mr of 60,000 and the structural gene is located, as a single copy, on the long arm of chromosome 8 (White et al ., 1987b; Arai et al ., 1972). 1115-hydroxylase is inhibited to varying degrees in the adrenal by certain C19 steroids (for example DHA, DHA-S, testosterone and androstenedione), so that synthesis of corticosterone, cortisol and 1115-hydroxyandrostenedione are controlled by tissue levels of adrenal androgens (Gower, 1984a; Baird etal., 1983; Sharma et a l , 1963). Nicotine inhibits 1115-hydroxylase (and 21-hydroxylase) in fetal adrenal cells, and may account for the altered pattern of steroidogenesis seen in fetuses of smokers (Barbieri et al . , 1989). Ketoconazole (an orally active antifungal drug) inhibits 1115-hydroxylase, as well as other cytochrome P-450 dependent enzymes in steroidogenesis (Loose et al., 1983). 18-hydroxylase (E.C. 1.14.15.5) is coded by the same gene. Parallelism of 1115- and 18-hydroxylation has been demonstrated (Rubin, 1988; Sonino et a l, 1980). The
27 enzyme is mainly involved in aldosterone production, but acts in the mitochondria of the zona glomerulosa and fasciculata. The conversion of 18-hydroxycorticosterone to aldosterone is restricted to the zona glomerulosa. 17-hydroxylase (E.C. 1.14.99.9) is active in the microsomal fraction of the inner two adrenal cortical zones (zona reticularis and zona fasciculata). This enzyme is not active in the zona glomerulosa and this explains why biosynthesis of aldosterone is possible in this zone (Gower, 1984b). The gene coding for 17- hydroxylase is located on chromosome 10 (wkte t£a(. /^ 7 0 - Cleavage of carbon - carbon bonds The conversion of cholesterol to pregnenolone is co-ordinated in three catalytic cycles of cytochrome p450scc (20,22 desmolase) that occur in fairly rapid succession. Hydroxylation at C-20, C-22, and side chain cleavage are catalysed by the same protein. In humans this activity is coded for by a single gene on chromosome 15. 20-hydroxylation of cholesterol is inhibited by several steroids including its own product 20-hydroxycholesterol, C2i steroids such as pregnenolone and progesterone, and by testosterone. 17,20-lyase is involved in the cleavage of C2i steroids to C19 steroids, i.e., in androgen production. Recent molecular studies have shown that the activities of this enzyme and 17a-hydroxylase are encoded in a single protein by a gene on chromosome 10, but how their separate activities are controlled still remains unclear (Chung et al ., 1987). This microsomal enzyme requires NADPH and oxygen for full activity with a requirement for cytochrome P-450. 17,20-lyase is competitively inhibited by pregnenolone, progesterone and 17-hydroxypregnenolone all to varying Awongsb etker druas recerHy degrees (Gower, 1984a). ^Ketoconazole hasAbeen shown to inhibit testicular 17,20- lyase (Ayub and Levell, 1987). 3B-hydroxysteroid dehydrogenase - 5,/f 'isoywe,rase. This enzyme is responsible for conversion of 5-ene-3B-hydroxysteroids to their respective 4-ene-3-keto forms, for example pregnenolone to progesterone. This enzyme is located in the smooth endoplasmic reticulum. The reaction is thought to be NAD specific. Relatively small amounts of endogenous oestrogens can inhibit this reaction (Gower, 1984a; Hirato et al ., 1982; Voutilainen and Kahri, 1980) and this is particularly important in pregnancy (sec
28 1.2.6 Control of steroidogenesis Cortisol and androgens The rate of cortisol production in the adrenal cortex is regulated by ACTH. Human ACTH is a single polypeptide of 39 amino acids that is secreted by the pituitary in irregular bursts throughout the day, with the highest values in the morning. Trauma, emotional stress, and certain drugs initiate nerve endings in the posterior hypothalamus to secrete corticotrophic releasing factor (CRF). CRF is transported via the hypophyseal portal vessels to the adenohypophysis causing the release of ACTH. Cortisol concentrations in plasma tend to fall and rise in parallel to ACTH levels. Insulin induced hypoglycaemia, vasopressin, bacterial pyrogens and glucagon all stimulate ACTH secretion. ACTH may itself exert a negative feedback on its own release by the pituitary (Beckford et al ., 1983; Mahmoud et al ., 1984) but the main locus of the feedback is the action of cortisol in suppressing ACTH secretion, presumably by suppressing the release of CRF. If ACTH stimulation is maintained for prolonged periodSj cytoplasmic lipid droplets and ascorbic acid in the fasciculata cells decrease and eventually the two inner adrenocortical zones increase in thickness due to hypertrophy of the tissue. The zona glomerulosa, however, does not appear to be affected. In hypophysectomised animals the lack of ACTH results in the shrinkage of the zona fasciculata and reticularis to less than half the original thickness, along with the reduction of glucocorticoid production. The zona glomerulosa responds to the acute effects of ACTH with the release of aldosterone, but this is not sustained and is not a feature of chronic stimulation of ACTH. In Addison’s disease with adrenocortical failure that causes a deficiency of both cortisol and aldosterone, the lack of aldosterone causes hypotension, excessive pigmentation is also seen. The latter is due to melanocyte stimulating hormone, which is part of the same precursor protein, pro-opiomelanocortin (POMC) as ACTH. The over production of ACTH is due to the absence of the negative feedback of cortisol. Androgens, unlike cortisol, vary in basal serum concentrations throughout life. Their secretion is also under ACTH control, although other hormones are thought to influence their secretion.
29 Aldosterone Although physiological levels of ACTH regulate acute changes in aldosterone concentration in the circulation, the primary regulatory mechanism of aldosterone secretion is via the renin-angiotensin system, which is responsive to the electrolyte balance and plasma volume. Renin (a proteolytic enzyme stored in the kidney) is released with a decrease in renal arterial pressure or depletion of body sodium concentration. Renin releases a decapeptide (angiotensin I) from circulating renin substrate. Angiotensin I has little intrinsic biological activity, but is further hydrolysed to angiotensin n, an octapeptide, by the action of angiotensin converting enzyme. Converting enzyme is present in high concentrations in the lung, although widely distributed in the vasculature and other tissues. The local production of angiotensin II may be physiologically important in certain circumstances. Renin release is stimulated by a reduced renal perfusion pressure, by hyperkalaemia and by reduced sodium concentration in the macula densa. Renin release indirectly stimulates angiotensin synthesis. Angiotensin II stimulates aldosterone production, which then causes sodium reabsorption causing an expansion in plasma volume. The synthesis of aldosterone is regulated at two steps in steroidogenesis, firstly at the rate limiting step of cholesterol to pregnenolone (a step also stimulated by angiotensin II) (Mulrow et a l , 1987). Secondly the conversion of corticosterone to aldosterone, which is stimulated by potassium, high concentrations of angiotensin II and sodium depletion. The adrenal cortex possesses specific dopamine receptors, and dopamine has been shown to modify aldosterone biosynthesis in vitro (Fraser et al ., 1989). Aldosterone response to angiotensin II is selectively inhibited by dopaminergic mechanisms which may affect sodium balance.
1.2.7 Cell differentiation and adrenal growth During the time of embryonic and post-natal growth, most of the cell division in the adrenal cortex takes place in the outer zona glomerulosa and outer zona fasciculata (Dhom, 1973; Wright, 1971; Wright et al ., 1973), or in the definitive zone of the fetal adrenal cortex (Johannisson, 1979). The actual mechanism restricting cell division to the outer region of the cortex is not known, but is probably
30 related to the pattern of blood supply. Once the adrenal cortex has reached its mature size the rate of cell division in the outer cortex decreases to that required to balance the rate of loss of cells due to death. Initially it was assumed that each zone of the adrenal cortex was self maintaining, i.e., that cells in each zone were derived solely from other cells in that zone, as there were functional differences recognized between the zones - a concept known as the "zonal theory". However, the observed distribution of mitosis in the adrenal cortex makes this concept unlikely. The zona glomerulosa exceeds its rate of production of cells for self maintenance, while the rate of cell division in the zona reticularis is insufficient to maintain the zone. In addition to the high rate of cell death in the zona reticularis deposits of age pigment (lipofuscin) are found in this zone. The lipofiiscin in probably the result of lipid peroxidation. A number of observations support the concept that cells in the inner zona glomerulosa are pushed inwards down through the cortex by the pressure of cell division to become fasciculata cells and then further into the gland to become reticularis cells. Adrenal cells arise from stem cells or adrenocytes which can secrete aldosterone, after leaving the glomerulosa the cells produce corticosteroids and on reaching the reticularis produce sex hormones. Half the cells are thought to die on the way, while the rest are finally eliminated in the reticular zone. This concept of centripetal migration of adrenocytes has been named the "escalator" or "cell migration theory" (Jones, 1948; Crowder, 1957) and more recently the term of "the streaming adrenal cortex" has been introduced (Zajicek et al ., 1986). The adrenal cortex is capable of dramatic regenerative growth when required. A functional, zoned adrenal cortex can regenerate from fragments of adrenocortical tissue when transplanted elsewhere in the body. The pattern of growth is similar to that occurring naturally during normal development, with the formation of a morphologically disorganised cell mass followed by a period of growth with mitosis in the outer region of the regenerating tissues, and finally re-establishment of functional zones. ACTH is the major hormonal regulator of adrenocortical growth. Excess pituitary secretion of ACTH as in Cushing’s disease, or the various forms of congenital adrenal hyperplasia (CAH) are associated with a large adrenal gland, and conversely diseases of ACTH deficiency with adrenal atrophy. Chronic ACTH
31 administration in rats stimulates the growth of the zona glomerulosa and partially transforms it to functional fasciculata cell types (Nussdorfer et al., 1982). The removal of one adrenal gland stimulates compensatory growth of the other. The mechanism for regulation of adrenocortical growth is still being elucidated. Some polypeptide hormones (for example fibroblast growth factor) are thought to be direct mitogens of the adrenal gland (Waterman and Simpson, 1985), whereas ACTH is probably an indirect mitogen (as it directly inhibits replication) acting to increase the delivery of growth factors to adrenocortical tissue by, for example effects on adrenal vasculature. The growth stimulation effect of ACTH is usually accounted for by an increase in cell size rather than an increase in cell number or DNA content. Blocking ACTH secretion from the pituitary results in a decrease in adrenocortical growth including DNA synthesis and cell division. This is later followed by adrenal atrophy with the loss of protein, DNA and RNA content.
1.2.8 Fetal steroidogenesis and the "feto-placental steroidogenic unit" A close metabolic interrelationship exists between the fetal adrenal, the fetal liver, the placenta and the maternal circulation (see Figure 1.2) (Winter, 1985). This, in the past, has been interpreted as evidence for a fetoplacental steroidogenic unit. According to this concept the fetal adrenal is intrinsically deficient in 3B- hydroxysteroid dehydrogenase and therefore entirely dependent on the placental progesterone for the synthesis of cortisol. Since the placenta is deficient in the ACTH dependent enzymes 17-hydroxylase and 17,20-desmolase, the major purpose of the fetal adrenal was seen to be the provision of dehydroepiandrosterone sulphate (DHA-S) as essential precursors for oestrogen biosynthesis. Winter has now proposed that the fetal pituitary adrenal axis is linked to the placenta more by circumstance than by necessity. Cortisol production is carefully regulated by the fetus and serum cortisol concentrations depend on four variables. (i) pituitary ACTH secretion (ii) inhibition of adrenal 3B-hydroxysteroid dehydrogenase by placental and maternal steroids, particularly the oestrogens (Bryne et al., 1985; Hirato et al., 1982). Co-culture of adrenal with pituitary and placental tissue has shown this inhibition to be placental in origin (Voutilainen and Kahri, 1980). (iii) rapid placental clearance of cortisol by conversion to cortisone; and
32 Figure 1.2 - Winter’s feto-placental model
Mother Placenta Fetus ACTH
Lipoprotein Cholesterol Pregnenolone l Oestrone DHA-S DHA-S Oestradiol ♦ Oestriol 160H-DHA-S
Progesterone Progesterone
Cortisol (iv) placental transport of maternal cortisol. Although fetal low density lipoprotein (LDL) cholesterol appears to be the principal substrate, placental progesterone may be utilized to some degree to circumvent the relative deficiency of 36-hydroxysteroid dehydrogenase (Winter, 1985). Up to 30% of fetal adrenal steroidogenesis may be derived from de novo cholesterol biosynthesis in the adrenal itself (Carr and Simpson, 1981). Placental progesterone is used as a substrate for glucocorticoid and mineralocorticoid production by the fetal adrenal, as one of the key enzymes for their production, 313-hydroxysteroid dehydrogenase is inhibited in the fetal zone throughout pregnancy by placental oestrogen. In the human fetus ACTH appears in the pituitary by five weeks gestation. Since ACTH does not cross the placenta fetal plasma concentrations depend entirely on the integrity of the fetal hypothalamic pituitary unit (Winter, 1985). As early as 8-12 weeks gestation negative feedback regulation of fetal ACTH secretion can be demonstrated. A fall in cord cortisol and DHA-S levels and maternal oestrogen is seen following administration of glucocorticoids to the mother (Arai et al., 1972). This is the basis of treatment of a fetus affected with congenital adrenal hyperplasia, so as to prevent early virilization. ACTH administered to the fetus usually increases total cortisol production and maternal oestrogen excretion. The occasional lack of an acute response may indicate that the adrenal is already maximally stimulated (Winter, 1985). Experiments of this nature have been done mainly on early gestation fetuses immediately prior to termination of pregnancy. The adrenal glands are small in anencephalic (brainless) fetuses as little or no corticotrophin releasing factor (CRF) is released from the grossly malformed or absent hypothalamus. ACTH values in such fetuses are markedly reduced and as a result adrenal steroidogenesis shown by, for example, serum DHA-S levels or maternal oestriol excretion are negligible. The fetal cortex is capable of steroid production at an early stage of gestation and glucocorticoids produced by the adrenal are involved in a number of important processes including: (i) production of surfactant from type II cells in the alveoli of the lung. A deficiency of surfactant results in respiratory distress syndrome in the newborn (ii) development of hypothalamic function and of the pituitary thyroid axis
34 (iii) sequential changes in placental structure and in the ionic composition of amniotic fluid (iv) induction of thymic involution, and (v) initiation of endocrine changes for parturition.
1.2.9 Parturition The initiation of parturition is not fully understood yet. Most work has been performed in sheep. The sequence of events and the nature of signals that lead to the onset of parturition in the ewe are associated with an increase in fetal cortisol production. This is thought to act on the placenta to reduce progesterone secretion (and to increase oestrogen formation). The reduction in progesterone is again at least partially controlled by the inhibition of 36-hydroxysteroid dehydrogenase. Just before and during labour there is also an increase in production of certain prostaglandins and (Casey ef /W 5) these are thought to be critical in the initiation of labour^ In man this relationship is not as clear, although anencephaly is frequently associated with extended pregnancy and intra-amniotic cortisol administration may initiate labour in women with prolonged pregnancy (Winter, 1985). In the last third of human pregnancy the oestrogen production can be 1000 times that of a non-pregnant woman. The rise in cortisol production provides the essential signal for parturition in several species.
1.2.10 Neonatal life By term, fetal steroidogenesis is proceeding at a rate higher than at any other stage of development, at least five time that of the adult gland (Shackleton, 1984). The gland is capable of responding to signals mediated by pituitary ACTH or the renin-angiotensin system. Comparison of mean cord plasma ACTH concentrations after vaginal birth and Caesarian section, the former being very much higher, demonstrates that the fetal pituitary at term can respond to stress, the rise presumably mediated by increased secretion of CRF (Winter, 1985). During the newborn period placental steroids such as progesterone and oestradiol are rapidly cleared from theneonatal circulation; sulphated conjugates are cleared more slowly. As theplasma concentrations of inhibitory steroids in the newborn infant fall there is a striking improvement in adrenal 3B-hydroxysteroid dehydrogenase activity and a corresponding increase in the ability of the neonate to
35 secrete steroids such as cortisol and aldosterone without excessive production of the 5-ene-315-hydroxysteroids. A study in children up to 2 years of age has shown that 10 times more androstenedione is found in the adrenals than in the testes. After the first six months the adrenals are the main source of testosterone in the infant (Bidlingmaier et al ., 1986).
1.2.11 Adrenarche As the child develops there is a progressive rise in the circulating concentration of the adrenal C19 steroids DHA and DHA-S starting at around seven years of age until the middle of the second decade of life. A 10 to 20 fold rise in plasma levels of these steroids is seen in this period (Cutler and Loriaux, 1980) whilst urinary cortisol remains constant when corrected for body weight (Forest, 1978; Honour et al ., 1991). These changes occur in parallel with progressive development of the zona reticularis (Dhom, 1973). The process of adrenal androgen production during childhood is known as adrenarche and plays a part in normal sexual maturation with the development of pubic and axillary hair. Adrenarche occurs independently of puberty (Cutler and Loriaux, 1980; Sklar et al ., 1980) during childhood. These changes have been calculated from the ratio of metabolites with substrate and product interrelationship. The activities of the enzymes 17,20 desmolase and 17a-hydroxylase increase during childhood, whilst there is a decline in 315-hydroxysteroid dehydrogenase activity and a fall in 1115-hydroxylase activity. The overall effect is a reduced potential for cortisol synthesis. This accounts for the increased ACTH secretion to maintain normal levels, and in turn results in a rise in adrenal androgen secretion. Adrenal 1115-hydroxylase activity is inhibited by androgens (Gower, 1984a). This could lead to greater deoxycorticosterone production. The importance of DHA in initiating the early physical signs of sexuality in children is different in the two sexes with serum DHA in girls being double that seen in boys when compared according to stage of pubic hair growth (Apter et al . , 1979). Recent work in pre-adrenarchal dogs (Perez-Femandez et al ., 1987) demonstrated that the morphological and functional development of the zona reticularis may be subject to dopaminergic control and therefore could represent an important step in the initiation of adrenarche.
36 1.2.12 Sexual differentiation and congenital adrenal hyperplasia Androgens play an important role in sexual differentiation in a fetus. Normal differentiation of the male genitalia depends on two functions of the fetal testes: (i) the leydig cells secrete testosterone which stimulate Wolffian ducts to develop into the male internal sex organs. (ii) the sertoli cells secrete the glycoprotein Mullerian inhibitory substance (MIS) that inhibits the development of the female internal genitalia. The fetal ovary secretes neither testosterone nor MIS, and thus does not influence sexual differentiation. The normal pattern of development in females can, however, be disturbed by exposure to high levels of androgens. This could be a consequence of CAH (see below), an androgen producing tumour in the mother, or maternal ingestion of progestins. Inherited adrenal enzyme deficiencies are recognised to cause the condition known as congenital adrenal hyperplasia (CAH) (White et al ., 1987a). The commonest enzyme deficiency is 21-hydroxylase and this can manifest itself in a number of ways - simple virilizing (SV), salt wasting (SW) and non classical (late onset). The latter will be considered later. Deficiencies are also recognised of the enzymes 118-hydroxylase, 3B-hydroxysteroid dehydrogenase and 17a-hydroxylase. Impairment of 21-hydroxylation results in decreased cortisol synthesis, which induces increased ACTH secretion and thus over production of cortisol precursors and sex steroids, which do not require 21-hydroxylase for their biosynthesis. Prenatal virilization of females results in labioscrotal fusion, with a urogenital sinus in addition to an enlarged clitoris, all to varying degrees. In extreme cases the urethra is penile. However as no Mullerian inhibitory substance is present, the female with CAH is bom with a uterus and fallopian tubes and hence is still potentially fertile. The infants present with pseudohermaphroditism. Males with 21- hydroxylase deficiency do not normally have genital abnormalities at birth, but during early childhood there is rapid somatic growth, progressive penile enlargement, early appearance of facial, axillary and pubic hair and even acne. Without treatment, early epiphyseal closure and eventual short stature results. Some infants fall into a second group of 21-hydroxylase deficiency in being salt losers as opposed to the simple virilizing group described above. Salt losers show degrees of incorrect sexual differentiation, but also develop signs of adrenal
37 insufficiency with low serum sodium and high potassium concentration. This may lead to a life threatening crisis and is the result of reduced aldosterone production. It is the presence of these two distinct conditions that has led to the concept that the adrenal fasciculata and glomerulosa function as two separate glands. All patients show defective 21-hydroxylation of 17-hydroxy steroids leading to elevated serum 17a-hydroxyprogesterone and decreased cortisol levels. The nature of the 21- hydroxylase deficiency in the mineralocorticoid pathway is less clear. Two hypotheses have been proposedA. According to the first "one enzyme theory" one enzyme exists for both pathways and the difference between SW and SV adrenal hyperplasia is due to degrees of enzyme deficiency or different substrate affinities of the abnormal enzyme. This may be related to changes in structure due to gene deletion or point mutation. The second "two enzyme theory" postulates that two enzymes regulate the two pathways and that simple virilizers only lack the 17-hydroxy pathway while salt wasters are deficient in both pathways. Hence in both SW and SV subjects there is a defect of 21-hydroxylase in the 17-hydroxy pathway, while in the SW subjects there is an additional defect of 21-hydroxylase in the zona glomerulosa which is not present in SV subjects (Cutler and Loriaux, 1980; Kuhnle et al . , 1981). This proposal does not conform with the cell migration theory, as it implies that in SV subjects the 21-hydroxylase is expressed in the adrenocyte when in the glomerulosa, but is then not expressed when the adrenocyte reaches the zona fasciculata. There is therefore, if the two enzyme theory is correct, fine control of gene expression possibly by promoters, the details of which are still to be elucidated. oHier There areAless common enzyme deficiencies causing CAH, firstly 11ft- hydroxylase deficiency which results in hypertension, female pseudohermaphroditism and postnatal virilization. Secondly 3ft-hydroxysteroid dehydrogenase deficiency presents as male and female pseudohermaphroditism, salt wasting and in less severe forms disordered puberty, menstrual irregularity, hirsutism, acne and infertility. This is due to excess of weak androgens. With deficiency of 17a-hydroxylase, cortisol and sex steroids are diminished, and there is an increased secretion of precursor steroids such as corticosterone, progesterone and deoxycorticosterone resulting in hypertension, sexual infantilism and male pseudohermaphroditism. Prenatal diagnosis of 21-hydroxylase deficiency is now possible by measurements of amniotic fluid levels of 17a-hydroxyprogesterone or by DNA
38 analysis using chorionic villus biopsy material. Prenatal treatment is controversial and involves suppression of the fetal pituitary adrenal axis by administration of dexamethasone to the mother which can cross the placenta. At 8 to 16 weeks gestation such suppression can prevent ambiguity of the external genitalia in the female fetus. Continued treatment will prevent clitoromegaly. There is a broad spectrum of clinical and biochemical abnormalities in CAH. The forms recognised at birth are the most severe and are referred to as classical. Mild forms (late-onset) are increasingly diagnosed in adults with 21-hydroxylase and 315-hydroxy steroid dehydrogenase deficiencies. Patients with the non-classical forms of CAH do not have ambiguity of the genitalia or salt wasting, but for example, in 21-hydroxylase deficiency during childhood or even later, show signs of androgen excess associated with the biochemical defect. The majority of cases are diagnosed in adult female patients, but this can be attributed to the less obvious signs of androgen excess in males. The late presentation of these biochemical defects raises the question whether there is a separate disorder with delayed presentation or a separate acquired disorder. Classical and non classical CAH are considered to be allelic variants, as HLA linkage seen in classical CAH is consistent in patients with the non-classical disorder. An ACTH stimulation test can be performed to show the presence of the mild enzyme defects (Brodie and Colston Wentz, 1987).
1.2.13 Polycystic ovary (PCOI syndrome In this condition where one of the first clinical presentations noted is often hirsutism, there is excessive secretion of androgens, mainly of ovarian source, although there is evidence of adrenal involvement (Brooks, 1984). ACTH stimulation tests and dexamethasone suppression tests back this up (Dewis and Anderson, 1985), although it should be remembered that ACTH stimulation may not be confined to the adrenals only, e.g. ovarian theca cells can also be stimulated (Dewis and Anderson, 1985). In mild adrenal hyperplasia due to 21-hydroxylase or 118-hydroxylase deficiency maintenance of normal cortisol secretion is at the expense of increased adrenal androgens and progesterone production (Lachelin et al ., 1979). This is •fine case in thought to beAone of the groups of patients with PCO. On sustained ACTH
39 stimulation such cases show increased DHA-S, 17-hydroxyprogesterone, progesterone and 17-hydroxypregnenolone. Increased androgen levels may be modulating adrenal enzyme activity by inhibition of various enzyme steps (Dewis and Anderson, 1985). Gross et al. (1986) measured adrenal iodocholesterol accumulation as an index of adrenal function. All the women he studied showed normal dexamethasone suppression of plasma cortisol and urinary 17-hydroxycorticoid steroid production, but excessive adrenal cortical uptake of the labelled iodocholesterol was seen in women with PCO compared to normal women.
1.2.14 Ectopic adrenal tissue and tumours Ectopic adrenal cortical tissue occurs relatively frequently, with isolated groups of cells being found in adult spleen, below the kidneys, along the aorta, in the pelvis and associated with gonadal structures. However it is not normally significant unless it becomes hyperplastic or malignant. Tumours of the adrenal are relatively common appearing in as many as 5% of some autopsy studies (Bondy, 1985). Infiltration of the adrenal with carcinomas is probably due to its generous blood supply and the local high concentration of corticosteroids which promote implantation of metastases. These may become functionally inactive if the tumour grows to such a size as to block major blood vessels. Adrenal adenomas secrete mainly cortisol, whereas a carcinoma tends to produce a variety of steroids in addition to cortisol (James, 1984). Some tumours and ectopic tissue secrete ACTH (ectopic ACTH syndrome). In such cases feedback mechanisms do not operate since the source of ACTH is unresponsive to the elevated cortisol levels produced (James, 1984) resulting in adrenal hyperplasia.
1.2.15 Adrenopause In the normal human as life continues through the third to the sixth decade serum cortisol levels remain constant and then a small decrease in production rates is seen in old age. On the other hand serum concentrations of adrenal androgens DHA and DHA-S fall markedly in old age, to about 20% of young adult values, in both sexes. This appears to be due to a marked fall in production rates as there is no age related change in ACTH concentrations to explain the decrease (Parker, L. et
40 al ., 1981). These findings could be explained by (i) an age related partial or complete loss of a adrenal enzyme such as 17,20 desmolase or cell populations which produce adrenal androgens, (ii) a loss or decrease in a subpopulation of ACTH receptors specific for adrenal androgen production, or (iii) a loss of a pituitary factor necessary for adrenal androgen secretion. A fairly recent study has confirmed the reduced ACTH response for 5-ene-steroids (DHA, pregnenolone, 17-hydroxypregnenolone), but has found that the response of 4-ene-steroids (androstenedione, progesterone and 17-hydroxy- progesterone) was maintained in older people (Vermeulen et al ., 1982). This fall off in some adrenal steroidogenesis has been named "the adrenopause" and ends the life story of this fascinating gland whose functions, mass and structure vary dramatically during the lifetime of a human being.
41 1.3 Clinical significance of deoxycorticosterone
1.3.1 Introduction Deoxycorticosterone is an important steroid, known to have salt retaining properties, produced in the mineralocorticoid pathway of the human adrenal (which leads to aldosterone production), by 21-hydroxylation of progesterone (see Figure 1.1). An alternative biosynthetic pathway, at least in the rabbit liver, has been demonstrated, with production of DOC from 21-hydroxypregnenolone as a result of 3B-hydroxysteroid isomerase dehydrogenase (Trant et al ., 1990). DOC is normally an ACTH-dependent steroid, a rise in plasma steroid concentration being seen in response to ACTH administration (Tuck et al ., 1981; Biglieri et al . , 1969; Oddie et al ., 1972). The zona fasciculata is the main source of DOC, but when ACTH is suppressed with dexamethasone, a contribution from the zona glomerulosa is uncovered (Tan and Mulrow, 1975) .bcc has been postulated to have a modifying effect on the renin-angiotensin system under certain circumstances, such as the one just mentioned (New, 1985). Diurnal variation in plasma DOC concentration has been recorded in normal subjects, with the highest values at approximately 8 AM (Tan and Mulrow, 1975). DOC in plasma is found primarily (84%) in plasma protein bound form (Zipser et al., 1980). DOC is normally converted into corticosterone in the adrenal by adrenal 116- o? Doc hydroxylase activity, though 18-hydroxylationAhas been demonstrated. Work using ofboc bovine adrenals has also suggested an alternative metabolic pathway by oxidation^at the C-19 position (Kobayashi et al., 1987). In the liver tetrahydrodeoxycorticosterone (THDOC) (conjugated in the form of a glucuronide), a principal urinary metabolite, is formed prior to excretion in urine, by reduction of the A-ring of DOC to form the 3a-hydroxy 58-tetrahydro metabolite (see Figure 1.3). This reduction is similar to aldosterone (the 3a5B metabolite being formed), but different to corticosterone (3a5a metabolites predominate). In rat liver the enzyme corticosteroid side chain isomerase has been shown to utilize DOC, and may have importance in some side chain metabolism of corticosteroids (Marandici and Monder, 1990). Work on only limited numbers of subjects has been done in quantification of excretion rates of THDOC (for example Eberlein and Bongiovanni, 1956; Crane and
42 Figure 1.3 - Structures of steroids involved in DOC production and metabolism
CH3 I c«o CH3 I HCOH Progesterone O
!i- Pregnanediol CH20H HO I H c-o 4 (gut) CH20H C-0
DOC (liver) O
THDOC CH20H HO I H C-0 HO
Corticosterone O
43 Harris, 1966; Harris et al. , 1967; New et al. , 1969; Biglieri et al. , 1969; Sizonenko et al. , 1972; Ehrlich et al. , 1974; Romanoff and Baxter, 1975; Romanoff and Brodie, 1976; Nolton et al. , 1979a; Levine et al. , 1980; Zachman et al. , 1983;). Most work with this urinary metabolite has been directed to production rates calculated from the ratios of 3H and 14C in THDOC after the infusion of the radioactive tracers 3H- progesterone and 14C-DOC (various studies mainly by Casey, Winkel, MacDonald and co-workers, see section 1.4). Some THDOC is excreted into the bile and then is metabolised in the gut, by the resident flora, where it can be 21-dehydroxylated to form pregnanediol (Hoffman et al. , 1943; Schneider and Horstmann, 1951; Eriksson et al. , 1969a; Gower and Honour, 1984). The percentage of DOC that is eliminated as THDOC glucuronide is low, and may be variable between individuals depending on the activity of the intestinal flora. 19-nor-DOC another potent mineralocorticoid is also present in humans and co aid be other species, but it is thought its production A from a parallel pathway, with 19- hydroxyprogesterone undergoing renal 21-hydroxylation to form 19-hydroxy-DOC, followed by / ° / - c k ti/yidboo (Griffmg et al. , 1989). Various clinical situations exist where raised DOC may contribute to hypertension and hypokalaemia. Impaired llfl-hydroxylation due to a genetic deficiency or inhibition, or excess ACTH drive of steroidogenesis are the most obvious. Other situations in normal subjects, however, for example the menstrual cycle and pregnancy, also show evidence of raised DOC and the latter’s contribution to hypertension seen in these circumstances is less understood. Excessive increases of DOC production, from what ever source, could lead to manifestations of mineralocorticoid excess, particularly sodium retention and hypertension.
1.3.2 The menstrual cycle The production rate of DOC in the luteal phase of the menstrual cycle compared to the follicular phase has been shown to be raised by various studies (for example Winkel et al. , 1980b; Parker, C.R. et al. , 1981; Schoneshofer and Wagner, 1977; Antonipillai et al ., 1983b), a 2 - 3 fold rise in plasma DOC concentrations being seen. This rise is not too surprising as the main substrate, progesterone, is also higher in the luteal phase. The higher levels of DOC, due to the sodium retention
44 properties and possible hypertensive nature of this steroid may have implications in the symptoms seen in pre-menstrual syndrome, though further work is required to elucidate this fully. Adult men have plasma DOC concentrations of around 40 - lOOpg/ml * (Antonipillai et al., 1983b; Schoneshofer and Wagner, 1977; Tan and Mulrow, 1975; Sippell et al., 1978; Wilson and Fraser, 1971; Brown and Strott, 1971), which are similar to the levels seen in women in the follicular phase of the menstrual cycle.
* to convert plasma concentrations pg/ml to pmol/1: DOC multiply by 3.03 aldosterone multiply by 2.78
1.3.3 Pregnancy Plasma concentrations of the two potent mineralocorticoids aldosterone and 11- deoxycorticosterone are increased markedly in pregnancy. Despite these elevated levels, normal pregnant women are not hypertensive and do not show other manifestations of mineralocorticoid excess. Aldosterone secretion starts to rise early in pregnancy and increases progressively reaching very high levels during the last two trimesters. The result is that mean plasma concentrations of aldosterone are 2100pg/ml plasma in the third trimester, as opposed to 94pg/ml in the non-gravid (Nolton et al ., 1978)). The elevated aldosterone level in the third trimester has been shown to respond readily to changes in salt balance and postural stimuli (Ehrlich et al., 1976). For this reason, it has been assumed that aldosterone secretion in pregnancy is governed by normal mechanisms, such as the renin-angiotensin system, and that aldosterone only increases to the extent required to maintain normal homeostasis. On the other hand results in various studies have shown this not to be the case with total plasma DOC. The elevated DOC secretion in women in the third trimester (Nolton and Ehrlich, 1980; Dorr et al., 1989) is unresponsive to changes in salt intake (Ehrlich et al ., 1976) and is not influenced greatly by ACTH stimulation or dexamethasone (Nolton et al., 1978). Sequential measurements of plasma DOC during normal gestation reveal that only small increases occur in the first two trimesters and that this can be stimulated with ACTH administration. The most
45 dramatic increase occurs in the third trimester when mean plasma DOC concentrations rise to 6.1ng/ml compared with l.lng/ml in the non pregnant state (Nolton et al ., 1978). DOC is bound to corticosterone-binding globulin (CBG) to a similar degree as cortisol (Slaunwhite and Sandberg, 1959) and progesterone (Rosner, 1990). Thus it is not immediately apparent whether the measured increments in total plasma DOC concentrations are accounted for entirely by the higher levels of CBG that occur in pregnancy, or whether the biologically active free moiety is raised above non pregnant levels. The free DOC index, which more accurately reflects relative differences in plasma concentrations of free DOC, is greatly increased above the normal non-pregnant range when measured in third trimester women (Nolton et al ., 1979a). The markedly elevated rate of urinary free DOC excretion (770ng/day in late pregnancy compared to 90ng/day in the non-pregnant subjects) provides additional evidence that the plasma free DOC concentration is greatly increased, because the amount of unconjugated, free steroid excreted in the urine is related directly to the circulating levels of unbound steroid (Nolton et al ., 1979b; Beisel et al ., 1964). This is also shown by the fact that urinary THDOC excretion increased in the third trimester of normal pregnancy (Nolton et al ., 1979a; Ehrlich et al ., 1974). THDOC excretion rate increases substantially during ACTH stimulation (Biglieri et al ., 1969). As already stated ACTH stimulation does not significantly increase total plasma DOC, so the extra plasma free DOC must be due to the displacement of bound DOC from CBG by the increase in ACTH stimulated production of cortisol (Nolton et al ., 1979a). These observations emphasise the need to use measurements that reflect the free steroid fraction, for example by urinary excretion, particularly in pregnancy where increased quantities of circulating cortisol and progestins compete for binding sites on elevated CBG levels. The source of increased DOC secretion in pregnancy has not been fully localised. The non-suppressibility of DOC by dexamethasone administration or during high salt intake diets suggests that the increased DOC does not arise from either the glucocorticoid or mineralocorticoid pathways in the maternal adrenals. The higher concentrations of DOC and DOC-sulphate found in mixed cord blood, compared with maternal venous blood (16.6 and 55.3ng/ml cp. 5.8 and
46 3.8ng/ml respectively) seem to point to the feto-placental unit as a source of the increased DOC (Nolton et al., 1979b). High levels of DOC and DOC-sulphate are found in amniotic fluid (Nolton et al., 1981; Sippell et al., 1981; Schweitzer et al., 1969). The feto-matemal gradient for total plasma DOC is even steeper because CBG levels are negligible in fetal blood (Nolton et a l, 1979b; Tulchinsky et al., 1972). The extremely high concentrations of DOC-sulphate noted in cord blood are also consistent with increased DOC production within the feto-placental unit, steroid sulphate conjugation leading to the inactivation of DOC (Schweitzer et al., 1969). The above data does not however exclude the possibility of significant extra- adrenal maternal production of DOC. Various papers documenting extra-adrenal 21- hydroxylase enzymes have been presented by a number of groups of workers. These are reviewed in the next section. In view of the very high plasma levels of progesterone that occur in pregnancy, pathological elevation of DOC from the maternal sources may be important. The mineralocorticoid activity of increased DOC is mitigated to some extent by the high levels of progesterone, but pregnant women are still quite sensitive to the sodium-retaining effect of administered DOC. Administration of ACTH to normal third trimester pregnant women results in marked sodium retention, whilst aldosterone excretion declines to below pre-treatment levels. This is similar to the response in non-pregnant subjects, where this is due to increased ACTH dependent mineralocorticoid, including DOC, secretion. In the pregnancy study a two fold increase in THDOC is measured (Ehrlich et al., 1974). The very high levels of mineralocorticoid activity, which seem to be well tolerated in normal pregnancy might begin to express themselves in abnormal circumstances and lead to pathological consequences. For example, in the development of pre-eclampsia, aldosterone is reduced, but non-suppressible DOC, although reported to be no higher (Brown et al ., 1972b) or lower (Weir et al., 1976) than in normotensive subjects, might be excessive, relative to the existing state of sodium balance.
47 1.3.4 Other clinical situations with altered patterns of plasma DOC concentrations Impaired 116-hydroxylation due to a genetic deficiency or inhibition, or excess ACTH drive of steroidogenesis are the most common reasons for excessive DOC production. Alternatively some mineralocorticoid secreting tumours may be the source of excess DOC production. The latter is covered in Chapter 7. ACTH excess can be a result of lack of cortisol negative feedback on the pituitary, or a result of an ACTH secreting tumour. In 1113- and 17-hydroxylase deficiency CAH, cortisol production is greatly reduced, as both these enzymes are necessary in adrenal cortisol biosynthesis. DOC production in these two conditions is raised, and may be the cause of the hypertension seen (refer to Chapters 6 and 11 for further discussion of these conditions). The same effect can be seen with drugs that inhibit 1113- hydroxylase such as metyrapone. DOC production with this drug is greatly raised (Antonipillai et al., 1983a; Brown and Strott, 1971; Crane and Harris, 1966; Sonino et al., 1981; Seth et a l, 1972; Sonino et al., 1980). Metyrapone can be used to aid diagnosis of the cause of Cushing’s syndrome, with pituitary dependent Cushingoid patients responding with an excessive increase in 11-deoxy steroid metabolites. Free DOC levels are however raised in Cushing’s syndrome (Casser et al ., 1980). Various other studies with patients with hypertension and/or hypokalaemia, some with low renin, have been reported (for example Cope and Loizou, 1975; Messerli et al ., 1976; Brown et al ., 1972a; Tan and Mulrow, 1979), but there is great range in DOC levels compared to normals, so each case should be carefully considered. Reduced DOC production is reported in adrenalectomized patients (New et al., 1969). Patients with 21-hydroxylase deficiency CAH would be expected to have greatly reduced plasma DOC concentrations, progesterone and 17-hydroxy- progesterone metabolites being in excess. DOC production rates in such patients have been reported to be normal (Antonipillai et al., 1983a; Winkel et al . , 1983a), and this has provided more evidence for an extra-adrenal source of DOC production (see next section).
48 1.4 Extra-adrenal 21-hvdroxvlase enzymes
In the 1980’s work by various groups, though mainly by a group in Dallas, demonstrated tissue sites of 21-hydroxylation of plasma progesterone. The ovary was investigated, due to the increase in DOC seen in the luteal phase of the menstrual cycle (Antonipillai et al. , 1983b; Winkel et al., 1980b; Schoneshofer and Wagner, 1977). The presence of a 21-hydroxylase enzyme was confirmed by the use of ovarian tissue slices, and by finding high levels of DOC in follicular fluid and ovarian vein samples (Dehennin et al ., 1987b; Nahoul et al. , 1988). The extra-adrenal conversion of plasma progesterone to DOC must be in an organ with a high blood flow as it has been calculated that in some people 75 litres of plasma were cleared of progesterone each day by way of conversion to DOC (Winkel et al. , 1980c). Kidney and liver are ideal candidates for alternative enzyme sites, due to their high blood flow, and as potential sites of mineralocorticoid action. Both human adult kidney (Winkel et al ., 1980c) and fetal kidney tissue (Casey et al. , 1981; Winkel et al ., 1981) demonstrate 21-hydroxylase enzyme activity. Steroid 21-hydroxylase activity is however not detectable in human liver tissue (Winkel et al., 1980c). Animal studies show the Rhesus monkey to be a suitable extra-adrenal DOC production animal model (Winkel and Brooks, 1984). 21-hydroxylase activity is also demonstrable in guinea pig spleen (Winkel et al., 1983b), and rabbit liver (Dieter et al., 1982a, b; Trant et al., 1990). The latter is found to have a high affinity for pregnenolone, but reduced affinity for 17-hydroxyprogesterone, and a bimodal distribution of activity. Further sites of 21-hydroxylase activity in the human have been demonstrated in the adult aorta (Casey and MacDonald, 1982a), intestinal bacteria (Eriksson and Gustafsson, 1971), and in the fetus in the testes (Acevedo et al., 1963), thymus, spleen, skin, urinary bladder, pancreas, ovary and intestine (Casey et al., 1983; Casey and MacDonald, 1983). The sites of extra-adrenal production of DOC are likely to be of importance, as progesterone to DOC conversion has been shown to be within the normal range in both adrenalectomised subjects (Winkel et al., 1980a) and patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (Antonipillai et al., 1983a; Winkel et al., 1983a), who lack the adrenal 21-hydroxylase enzyme. In new-boms
49 with 21-hydroxylase deficiency CAH, the presence of highly elevated 21- pregnenolone excretion, relative to normal new-boms has been shown (Shackleton et al . , 1987). The enzyme responsible for this 21-hydroxylation could be (a) a distinct fetal 21-hydroxylase enzyme, or (b) the enzyme was structurally different from the normal adrenal enzyme and had a high activity towards pregmne)c»iej(but not towards 17-hydroxyprogesterone) as seen in rabbit liver (see above). Plasma levels and the production rate of DOC are significantly greater in women during the luteal phase than in the follicular phase of the ovarian cycle (Antonipillai et al ., 1983b; Winkel et al ., 1980b; Schoneshofer and Wagner, 1977). DOC levels are only partially suppressible by dexamethasone, whilst cortisol is fully suppressed. Parker et. al. (1983b) therefore conclude that luteal phase DOC is derived from both the adrenal and an extra-adrenal source not affected by dexamethasone suppression of ACTH secretion. During late pregnancy, another situation where progesterone is raised, DOC levels are high. Plasma DOC levels are not appreciably altered by ACTH or dexamethasome treatment, and do not correlate with maternal diurnal cortisol, again indicating an extra-adrenal source of DOC (Nolton et al ., 1978; Brown et al ., 1972b; Nolton et al ., 1981). Parker et al. (1980) showed that plasma levels of DOC in women who develop pregnancy-induced hypertension are similar to normal pregnant subjects at all stages of pregnancy and parallel progesterone levels. However changes in DOC levels again do not follow cortisol. Thesedata indicate that a fraction of the circulating DOC arose from extra-adrenal hydroxylation of progesterone, rather than through adrenal secretion. Other work on the origin of DOC suggests that the fetus may be one of the extra-adrenal sites of 21-hydroxylase activity (Casey et al . , 1984). Administration of glucocorticosteroids (dexamethasone or betamethasone) to the mother results in a decrease in plasma DOC in the newborn infant, but does not affect umbilical cord plasma progesterone, suggesting that the fetal adrenal glands play a role in the production of DOC, at least in the fetal compartment (Parker et al . , 1983c). A study of fetal plasma corticosteroid sulphates showed a significant correlation of DOC-sulphate with cortisol-sulphate, and suggests that the adrenal, in the fetus at least, prevails over extra-adrenal production of either the sulphated or unconjugated DOC (Nahoul et al ., 1989). On examination of human messenger RNA from fetal adrenal, liver, lung, brain, heart, spleen, testis, and placenta little or no
50 adrenal 21-hydroxylase (P450c21) mRNA is detectable (Mellon and Miller, 1989). This data suggests that extra-adrenal 21-hydroxylase is a seperate gene product. Oestrogen has been implicated as a stimulator of the progesterone to DOC conversion (MacDonald et al ., 1982). Levels of DOC are found to be lower (Parker et al ., 1984; Diver et al ., 1973) and can be stimulated by administered oestrogen (Casey et a l , 1987), in particular in pregnancies which have anencephalic or dead fetuses (Parker et al . , 1983a; MacDonald et al . , 1982), where oestrogen production is reduced. It is these studies on extra-adrenal DOC production that inspired some of the work presented in this thesis. It was hoped to look at DOC levels in pregnancy and in the menstrual cycle, and to investigate further the contribution of oestrogen to the conversion from progesterone to DOC in another group of hypo-oestrogenic pregnancies - placental sulphatase deficiency.
51 1.5 The renin-angiotensin system The most important regulator of the secretion of aldosterone is the renin- angiotensin system. Renin is synthesized in the juxtaglomerular cells in the nephrons of the kidney, and stored intracellularly in granules. Release of renin is activated by among other factors, a fall in extra-cellular fluid volume. It is discharged from the cells by exocytosis, and then diffuses into the lumen of the arterioles and thus into circulation, where it has a half life of around 20 minutes. Renin is a proteolytic enzyme which splits a leucine-leucine bond, the usual substrate being a circulating a2-globulin of hepatic origin, angiotensinogen. The resulting product angiotensin I, a decapeptide, is largely biologically inactive. It is converted to angiotensin II, an octapeptide, in a number of tissues principally the lung, by an endopeptidase. Angiotensin II is one of the most potent pressor substances known, raising both systolic and diastolic blood pressures, with no alteration in pulse pressure. It has a short half-life in circulation of about 1 minute. Angiotensin II stimulates aldosterone secretion by an effect on the zona glomerulosa cells. It also has action on peripheral arterioles and so can help maintain blood pressure both directly and indirectly. During sodium depletion, angiotensin II has a potent effect on renal circulation as it reduces the rate of glomerular filtration, and hence the renal excretion of sodium. Renin, or at least closely related substancesyare present in other organs than the kidney, but renin disappears from circulation almost completely after nephrectomy, indicating that the kidney must be the main source of circulating renin. The brain appears to possess an intrinsic renin-angiotensin system, which is independent of the renal system, and angiotensin II does not cross the brain-blood barrier (O’Riordan et al ., 1985). Renin is also present in the salivary glands and the genital tract. Four factors control the secretion of renin by the kidney: (i) neural - in the vicinity of the juxtaglomerular apparatus of the kidney is a sympathetic innervation, destruction of which leads to blunting of the renin response to sodium depletion, (ii) the flux of sodium across the macula densa of the distal tubule, (iii) the mean transmural pressure, when low renin being stimulated, and (iv) the concentration of angiotensin II, by product inhibition.
52 Angiotensin II, potassium and ACTH can directly stimulate the release of aldosterone (see Figure 1.4). The effects of potassium can be seen when plasma volume is constant, small increases of potassium concentration within the physiological range causing a rise in aldosterone secretion. The dependence of the secretion rate on extra-cellular volume, is mediated by the renin-angiotensin system, which can override the effects of raised plasma concentrations of potassium and ACTH. The secretion of aldosterone rises during the morning (because of the fall in plasma volume on assuming an upright posture) even though the secretion of ACTH falls during the day. Plasma concentrations of renin, angiotensinogen, angiotensin II and aldosterone show cyclical fluctuations in a normal menstrual cycle, activity peaking at or shortly after ovulation. If conception occurs these raised concentrations continue to rise (Sippell et a l, 1981). In early pregnancy a significant relationship exists between plasma angiotensin II and aldosterone concentrations, but no correlation between these hormones and plasma progesterone is seen (Weir et al., 1976). Chorionic cells have been shown to synthesize renin in vitro, which is immunological and enzymatically indistinguishable from renal renin (Acker et al., 1982). The amniotic fluid of human pregnancy contains high concentrations of inactive and active renin, which presumably originates from the chorion. Women with hypertension and proteinuria in the third trimester have significantly lower plasma concentrations of renin, renin substrate and angiotensin II, compared with normal pregnant women (age parity and gestation matched). Plasma aldosterone and DOC concentrations are also lower in the hypertensive group, whilst cortisol, corticosterone and vasopressin show no significant difference (Weir et al ., 1976). Pre-eclamptic toxaemia (a form of hypertension in late pregnancy, see Chapter 10 for more details) has been reported to be associated with lower levels of plasma renin activity and urinary excretion of aldosterone, when compared to matched normotensive pregnant subjects (Coyle et al., 1962). Beyond its primary role in the preservation of normal fluid volume, the renin- angiotensin system is involved in pathogenesis of some forms of hypertension. In primary hyperaldosteronism there is excessive aldosterone (and in many cases also DOC and corticosterone) secretion (Biglieri et al., 1968), accompanied by low or subnormal levels of plasma renin and angiotensin II. This condition is normally
53 Figure 1.4 - The control of aldosterone secretion
Juxtaglomerular - Renal nerve cells stimulation, Na+ flux in macula densa, ReninT changes in transmural J© pressure Renin -►Angiotensin I Substrate Changest in AngiotensinI II extracellular i© fluid volume ChangesI in Potassium Adrenal -►Aldosterone sodium and water cortex excretion t ACTH
54 caused by some form of mineralocorticoid secreting tumour. Secondary hyperaldosteronism can be caused by renal or liver disease, when hypoproteinaemia occurs. Due to redistribution of extra-cellular fluid, plasma volume is reduced, stimulating the production of renin and consequently angiotensin II. In the 1115-hydroxylase deficiency form of CAH there is a virtual absence of cortisol production. This results in the increased production of ACTH and excessive excretion of DOC and corticosterone from the zona fasciculata. Plasma renin activity is low and there is a reduction in aldosterone secretion from the zona glomerulosa. Pituitary suppression of ACTH leads to a return to normal of plasma renin activity. Metyrapone, an 1115-hydroxylase inhibitor, which can be used in the diagnosis of the cause of a lesion in patients with Cushing’s syndrome, is a similar situation.
55 1.6 Profiling steroid hormones using glass capillary gas chromatography
1.6.1 Introduction The analysis of steroids by chromatographic methods has been well documented in a number of reviews (Shackleton, 1985a, b, 1986; Robards and Towers, 1990), so this will only be summarised briefly here. A number of chromatographic methods have been used for the separation of steroids for quantification and identification including paper chromatography, thin layer chromatography, Celite chromatography, packed column gas chromatography and high performance chromatography, but glass capillary gas chromatography (GC) particularly in combination with mass spectrometry (GC-MS) remains one of the most specific. Before injection into a gas chromatograph steroids are normally extracted from the biological fluid or tissue, conjugates are hydrolysed (though not in all cases, eg. Shackleton et al ., 1983) and free steroids undergo derivatization reactions (as many steroids, in particular Qi steroids, are thermally unstable). Further group separation of steroids may be necessary, before derivatization, by for example chromatography or immunoadsorption.
1.6.2 Extraction While solvent extraction (liquid/liquid partitioning) is still favoured for the extraction of unconjugated plasma steroids, solid phase extraction has become the most common method of choice for recovery of steroid conjugates and free steroids from aqueous solution. Non-polar hydrophobic steroids (eg androgens and oestrogens) are best extracted with solvents such as diethyl ether (and benzene), while more polar solvents such as dichloromethane, chloroform and ethyl acetate are useful for a broader range of steroids including corticosteroids. Various solid phase extractions have been used for steroids, including resins, Lipidex 1000 and Amberlite XAD-2 (for example Shackleton and Honour, 1976; Honour et al ., 1982, 1983a; Eriksson and Gustafsson, 1970; Brooks and Harvey, 1970). More recently solid phase extraction on reverse phase bonded silica cartridges has provided faster recovery of steroids. Sep-pak C18 and Bond Elut C18 are the most widely used. Sep-pak C18 cartridges give equal or better recoveries than those obtained by solvent extraction, or with Amberlite XAD-2 (Shackleton and Whitney,
56 1980). Following priming with ethanol or methanol, and a water rinse, the sample can be applied directly onto the cartridge. Rinsing with an aqueous solution removes salts and other polar substances. The steroid can then be eluted with methanol or ethanol.
1.6.3 Hydrolysis Steroids are often found in biological samples particularly in urine as glucuronides and sulphates. Various glucuronidases are reported in common use, for example bacterial (Escherichia coli ), limpet (Patella vulgata) lyophilized powder, and the digestive juice of the Roman snail (Helix pomatia). The latter two also contain sulphatase activity. The enzyme activities have respective optimal pH, Helix pomatia for example pH 4.5 - 5.0, which can be achieved by the use of an appropriate buffer solution. The majority of extract preparation time is often taken up by enzyme hydrolysis, normally 16 - 72 hours. Although optimal conditions are probably best achieved by prolonged hydrolysis, compromises can be made in some situations in order to produce faster results. An increase of temperature, for example, from the typical 37°C to 55°C can reduce hydrolysis time from 24 to 3 hours, though there is some loss of efficiency of hydrolysis for some conjugates (Shackleton, 1986). A further method for sulphate hydrolysis is solvolysis, achieved by the addition of ethyl acetate saturated with sulphuric acid to the dried sample and incubation at 40°C for at least 1 hour. Acid can be neutralized in ethyl acetate (containing the hydrolysed steroids) using sodium hydroxide.
1.6.4 Additional separation of steroids Biological fluid extracts inevitably contain a large number of closely related steroids, along with other nonspecific unwanted compounds, so under certain analytical circumstances further separation is necessary. Immunoadsorption has been used to isolate the steroid of interest (Gaskell et al . , 1983, 1984). Chromatography provides an effective means of fractionation of groups of steroids; the separation of steroids that co-elute, or whose peaks merge after gas chromatography may be necessary in quantitative methods. Both paper and thin layer chromatography have had limited application, whereas column chromatography has been exploited in various modes, for example anion exchange (Fotsis and Adlercreutz, 1987; Fotsis et
57 al ., 1981), Celite, and bonded reverse phase materials such as Sephadex LH-20 (for example Setchell and Shackleton, 1973; Shackleton and Taylor, 1975; Shackleton et al., 1973; Gustafsson et al . , 1972; Honour, 1986; Axelson and Sahlberg, 1983; Eriksson et al., 1970; Philip et al., 1989).
1.6.5 Derivatization of steroids Many underivatized steroids cannot be analyzed by gas liquid chromatography, the preparation of volatile thermostable derivatives of corticosteroid metabolites, for example, being essential for such an analysis. Numerous combinations of derivatization reactions have been reported, but the formation of trimethylsilyl ethers and methoxime-trimethylsilyl ethers, reported in the original profile analyses by Gardiner and Homing (1966), remains the preeminent technique. The condensation of steroid carbonyl groups with methyloxime hydrochloride (in pyridine) resulting in the formation of methoxime derivatives serves two functions (a) the products are more thermally stable and less polar than the underivatized steroids and (b) enol-ether formation is prevented during the following trimethylsilylation. All steroid oxo-groups with the exception of the sterically hindered 11-oxo-group can be quantitatively converted into oxime derivatives. Aldosterone, its metabolites, and 18-hydroxycorticosterone require careful conditions for successful analysis, as the natural form contains a hemiacetal or hemiketal ring that is best opened during derivatization (Shackleton and Honour, 1977; Honour and Shackleton, 1977). Other derivatization reactions reported include the use of N,N- dimethylhydrazine (VandenHeuvel and Homing, 1974), ethanedithiol (Zmigrod and Lindner, 1966), ethyloxime (Brooks and Harvey, 1970), bistrimethylsilylacetamide (Chambaz and Homing, 1969; Fennessey et al., 1983; Homing et ah, 1969), O- benzyloxime (Devaux et al., 1971; Joannou, 1981; Homing et al., 1971), O- methylhydroxylamine (Dray and Weliky, 1970; Fales and Luukkainen, 1965; Gardiner and Homing, 1966; Homing et al., 1986; Land and Ulick, 1987), hexadecafluoronanoyl (Mickan and Zander, 1979c), and hexamethyldisililazane with trimethylchorosilane (Sjovall and Sjovall, 1968; Baillie et a l, 1976).
58 The use of alternative derivatization reactions can be useful in separation of co-eluting steroids, for example benzyloxime derivatives allow the separation of carbonyl containing steroids from components containing C-20 hydroxyl groups, a situation that occurs for example with some of the marker steroids in the urine of patients with 17-hydroxylase deficiency CAH (Honour et al ., 1978). After derivatization is complete, excess reagent must be removed. This is often achieved by evaporation of the pyridine under nitrogen (to prevent oxidation), and chromatography using a Sephadex derivative Lipidex 5000. The apolar steroid derivatives are eluted rapidly from mini columns of this material prepared with a solvent system based on hexane or cyclohexane, whilst the polar (and sometimes immiscible) derivatization reagents remain on the top of the column.
1.6.6 Gas chromatographic conditions and detection There are a variety of stationary phases that can be used for coating the glass or fused silica capillary columns used in the GC separation of steroids. They fall into two main categories (a) non-selective phases (including SE-30 and OV-1) in which separation is based on volatility effects, and (b) selective phases (including SE-52, QF-1, OV-17, XE-60, and OV-210) in which the separation based on volatility is modified by the presence or absence of characteristic groups in the steroid molecule. The introduction of phenyl groups to the stationary phase in SE-52, for example, permits the resolution of B-cortolone from B-cortol, two steroids which co-elute on OV-1 and SE-30 GC columns. The most common injection techniques are split injection, splitless injection, and on column injection, but solid injection (Shackleton and Honour, 1976) has also been reported. Splitless injection allows relatively large volumes of solvent to be injected onto the instrument, so is particularly useful in trace analysis. The sample is injected into a wide-bore glass insert, and cold trapping on the analytical column is necessary to recondense the sample in a small band at the top of the column. After a short interval the splitter is opened to purge the injector of residual solvent vapour. Split injection is suitable when larger amounts of steroid are present. It can be used either with isothermal or temperature-programmed operation, since cold trapping (injection into the hot injector with the column at a lower temperature) is not necessary.
59 Two types of detectors feature prominently in the GC of steroids. Flame ionization detection (FID) is particulary useful in combination with trimethylsilyl ethers permitting detection down to nanogram levels in relatively impure biological extracts. Electron capture detection is a more sensitive method, but difficult to maintain at good performance. GC-MS is increasingly being used for the determination of steroids in biological samples, due to the high specificity and sensitivity that this method allows, including as a reference method for simpler less specific procedures such as radioimmunoassay (Gaskell et al ., 1983, 1984).
60 1.7 The use of stable isotopes in Endocrinology
The use of the stable isotopes of hydrogen, carbon, nitrogen and oxygen in biological science has undergone in the past twenty years, and continues to undergo, dramatic expansion. This is mainly due to three factors, firstly the growing demand for the development of non-radioactive tracer techniques for human studies, secondly the greater availability of isotopically enriched compounds, and finally the increasingly refined technology for the analysis of stable isotope labelled compounds.
1.7.1 Radioactive versus stable isotopes Increasing awareness of the possibility of biological damage at dose levels of radioactivity previously considered acceptable has resulted in gradual restriction of the use of long lived radionucleotides such as 14C, 3H and 32P. Such biological damage is now considered unethical, thus restricting studies particularly on neonates, children, during pregnancy and generally any adult still within reproductive age ranges. The use of stable isotopes offers alternatives that are acceptable to both ethical committee and patient/subject, and also to the scientist as it permits safer handling. Stable isotopes have "infinite" lifetimes unlike their radioactive equivalents. In the case of nitrogen (15N) and oxygen (170, lsO), there are no radioactive equivalents of long enough half life for detection following metabolic studies of several hours duration. Scintillation counting, used frequently in radioisotope studies, especially when used to distinguish multiple labelling within a single molecule, does not always allow the same precision, which stable isotope quantification can achieve using selected ion monitoring mass spectrometry (see below). Hydrogen isotopes are frequently used in metabolic studies. Kinetic isotope effects due to the replaced hydrogen atoms have been found to be less pronounced for the stable isotope deuterium ^H) than for radioactive tritium fH). A disadvantage however of stable isotopes, is the high cost of isotope labelled substances, and the requirement for expensive high technology measuring equipment, such as a mass spectrometer. An additional point to consider when using stable isotopes as biological tracers is the natural background against which measurement of label must be made. This
61 background level can be as high as 1.1% in the case of 13C (Halliday and Rennie, occurs t o -fte- c f i 1982). Deuterium ^ about 1 part in 6400 in nature (Blake et al., 1975) with deuterium oxide being 150 ppm in tap water (Halliday and Miller, 1977), a little less in fresh water (approx. 140 ppm) (Blake et al . , 1975). A summary of the abundances of some stable isotopes h .shcw#i Thhle 1.1«
1.7.2 Analytical techniques available A variety of physiochemical methods have been applied to the determination of compounds labelled with stable isotopes. Mass spectrometry (MS) in general and gas chromatography - mass spectrometry (GC-MS) in particular are by far the most important analytical techniques for the use in studies, as they provide the investigator with as yet, in combination, the greatest sensitivity, specificity and versatility of detection. Two main approaches of MS have been used, (i) isotope ratio mass spectrometry (IRMS) and (ii) selected ion monitoring (SIM GC-MS). (i) IRMS The isotope ratio mass spectrometer is designed for precise heavy isotope measurement of low molecular weight permanent gases which retain their molecular identity under the conditions of analysis. The compounds containing the labelled atoms of interest therefore requires initial "cleaning up", isolation, purification and then wet chemistry, combustion or enzymatic degradation to provide a gaseous product suitable for isotope analysis. This type of instrument is capable of detecting 1 molecule of 2H2, 13C02 or 15N2 in 105 unlabelled molecules. It is thus ideal where sample size is not limiting, but high precision isotopic measurements are essential. This method allows high precision of measurement (0.01%), but requires large amounts of sample (0.1 - lOmg). This limitation makes it almost impractical in many endocrinological studies where milligrams of some hormones the scientist is interested are often only ever seen in bottles ordered from suppliers, and the quantity of plasma or tissue required from subjects would be unethical or impossible.
(ii) SIM GC-MS In this approach isotope enrichment within a molecule or ion fragment can be examined, the ionization of which is accomplished in the mass spectrometer "source" by bombardment with electrons. After initial "clean up", a
62 Table 1.1 - Some stable isotopes of interest
Element Atomic Mass Relative abundance (atoms %)
1.00783 99.985 2H 2.01410 0.015 12c 12.00000 99.89 13c 13.00335 1.11 14N 14.00307 99.63
15n 15.00011 0.37 16o 15.99491 99.759 17o 16.99914 0.037 180 17.99916 0.024
Based on Baillie (1981)
Table 1.2 - Estimated possible rate constant ratios at 25°C for various stable isotopes
Natural abundant Heavy isotope Rate ratio isotope (1) (2)
!H 2H 18 !H 3H 60 12c 13C 1.25 ,2C 14C 1.5
14N 15n 1.14 16o 180 1.19
Based on Blake et al. (1975)
63 sample mixture can be treated to form volatile derivatives and individual compounds. The resultant complex mixture has components sequentially transferred into the mass spectrometer, following separation on the gas chromatograph. Focusing of selected ions by rapid switching of the electrical field in a quadrapole, or the magnetic field in a magnet sector mass spectrometer, permits quantification of the ion intensities of labelled and unlabelled fragments. Isotopic enrichment can then be calculated from the relative ion current intensities. This direct analysis method, which has poorer precision of measurement (1 - 10%) than IRMS, offers the endocrinologist a variety of advantages. It can be used in small samples (< lng) and can be specific for a particular molecule, even in a mixture of labelled and unlabelled compounds. Nuclear magnetic resonance (NMR) spectrometry is the most important analytical technique other than MS that is used in the study of stable isotope labelled compounds. In this technique magnetic moment is monitored and has some importance in biosynthetic studies owing to the relative ease with which the precise position of heavy atoms can be detected in the intact molecule. The possibility now exists with the modem NMR machines of monitoring the metabolism of 13C compounds in vivo. Other methods for the analysis of deuterium content of aqueous fluids or of organic molecules include freezing point evaluation, infrared spectrometry and emission spectrometry (Halliday and Rennie, 1982). These methods however require isolation and purification of compounds from samples making them impractical in most biological studies.
1.7.3 Availability Developments in GC-MS instrumentation for the determination of stable isotope enrichment, coupled with an increased demand for deuterium labelled NMR solvents of high isotopic purity^ are factors which have greatly stimulated the production of stable isotope labelled compounds on a commercial basis. A wide variety of simple organic compounds of high purity are now available labelled with 2H, 13C, 15N or 180. These commercial products go hand-in-hand with highly enriched deuterium labelled steroids produced in laboratories for specific projects. A number of papers have been published in the last 20 years on "simple"
64 methods for production of such steroids (for example Dyer and Harrow, 1979; Dehennin et al., 1980; Johnson et al ., 1981; Baillie, 1981; Kirk et al . , 1990a, b; Ohnishi et al., 1990; Wudy, 1990; Linberg et al ., 1991).
1.7.4 Toxicity Biological effects of stable isotopes were noticed early on in their experimental era, a year after (1933) the discovery of deuterium. The difference in mass between deuterium and hydrogen causes the vibrational frequencies of carbon, oxygen and nitrogen bonds to deuterium to be lower than the corresponding bonds to hydrogen. As a result the chemical bonds involving 2H will generally be more stable than those of !H, thus significantly affecting the rate of bond cleavage and hence affecting the relative rates of metabolic reactions. This is however only apparent when the bonds involved are in rate-determining steps. It is these kinetic isotopic effects resulting from the substitution of deuterium that are implicated in the above biological effects. The difference between larger atoms, such as oxygen and carbon and their respective stable isotope equivalents are proportionately smaller and hence these isotopes have comparatively lower effects. A summary of these effects on reaction rates is shown in Table 1.2. Mice which had 60% of their body oxygen as 180 survived and reproduced normally (Baillie, 1981), deuterium only being tolerated up to 15%. No ill effects were noted over 4 months in humans when body water was replaced with deuterium oxide to the extent of 0.5%. This and other studies reviewed by Blake et al. (1975) show that in mammals it appears that up to 15% deuterium enrichment of body hydrogen can be tolerated, but severe toxic effects and possibly death may result at the 30% level and higher. Although toxic effects appear to correlate with the overall level of deuterium, harmful effects in subtle ways are possible at lower concentrations. The ultimate cause of death from deuterium is not clear, but in animal studies numerous disturbances were observed including renal function impairment, central nervous system disturbances, cardiac involvement, enzymatic interference, hormonal imbalance and glucose metabolism disturbance (Blake et al., 1975). However no factor appears to be the principal cause of death. Notwithstanding the damaging
65 effects of high isotope levels, deuterium can be considered a non-toxic substance at physiological concentrations used in endocrinological studies.
1.7.5 Quantitative applications 1.7.5.1 Internal standards and isotope dilution Isotope labelled hormones (in particular steroids) are ideal for internal standards in quantitative work as their physicochemical properties approximate their unlabelled counterparts. Resolution of the two species usually occurs only in the final step of investigation (usually SIM GC-MS), when the labelled and unlabelled forms can be distinguished from one another on the basis of their difference in molecular weight, or other relevant ions. Quantification is achieved by reference to a standard curve previously prepared by measuring ion peak ratios from differing amounts of the steroid of interest with a fixed amount of internal standard. The ratio between the responses can be measured accurately to below nanogram levels. A number of considerations must be taken into account in selecting these internal standards: (i) the internal standard should differ in molecular weight from the compound of interest by at least 2 (preferably 3) so that mass/charge (m/z) values monitored for the internal standard are basically free of contribution from unlabelled molecules containing heavy isotopes at the level of their natural abundance (for example 13C natural abundance = 1.1%); (ii) isotopic label(s) should be incorporated at chemically stable positions in the molecule so that there is no loss of isotope in the analytical/extraction procedures; (iii) internal standards should be labelled to a high degree of purity, with less than 1 % of residual unlabelled molecules; and (iv) the site of labelling must be such that the fragment ion(s) chosen for monitoring contain the heavy atoms. A number of studies have been undertaken, mainly on steroids, using stable isotopes as internal standards and also in isotope dilution studies, in which stable isotopes administered to the patient/animal are compared to endogenously produced equivalents.
66 Chapman and Bailey (1974) report a quantitative method for the determination of testosterone in human male peripheral plasma. Deuterium labelled testosterone is added to the plasma samples to act as the internal standard. Plasma samples are then submitted to a simple extraction procedure using diethyl ether, derivatization, and GC-MS. The precision of the method (CV=7.2 %) from replicates (n=6) of a plasma pool is acceptable. Values obtained from 6 male individuals ranged from 0.42 -1.37 /xg/100ml plasma, and are similar to those reported in the literature by alternative techniques. The method proved sensitive enough to measure female plasma testosterone levels which are at least an order of magnitude lower than in males. Esteban and Yergey (1990) quantified cortisol production rate in adults and children using isotope dilution mass spectrometry. Circadian variations were observed and the results suggested lower cortisol production rates in normal children and adults (9.9 and 9.5mg/day respectively) than previously reported in the general literature. Similarly a reference method for the measurement of DHA-sulphate was developed by Shackleton etal. (1990) using high performance liquid chromatography- mass spectrometry in conjunction with a deuterated internal standard. Results obtained using this method were approximately half of those obtained using radioimmunoassay techniques. Steroid hormone determinations in human ovarian follicular fluid are mostly performed by RIA derived for blood analysis, without taking into account sufficiently the considerable quantitat/Ve and qualitative differences of the two body fluid steroid compositions. For this reason Dehennin et al. (1987a) developed isotope dilution mass spectrometric methods for the measurement of various androgens and 19-nor steroids in pre-ovulatory follicles. Various deuterium labelled androgen analogues and (3,4-l3C2)-testosterone were used in quantification. The steroids were confirmed to be present by the comparison of retention time using GC-MS and by the presence of and relative abundance of the same ions in both follicular fluid and the authentic compound. They identified and quantified 19-nortestosterone and 19- norandrostenedione for the first time in follicular fluid, with mean values of 6.81 and 14.2ng/ml respectively. DHA-S was found to be the most abundant androgen quantified in this study. Testosterone levels measured by RIA were higher than using
67 the MS method. This was thought to be due to the cross reaction with 19- nortestosterone confirming the specificity of the MS method. Progesterone has also been studied in quantitative assays using SIM (Broom et al ., 1983; Johnson et al . , 1983). Johnson and co-workers used the isotope dilution principle with (T^lb-^J-progesterone coupled with GC-MS analysis to evaluate daily metabolic clearance and urinary production rates. The minimum plasma concentration of deuterium labelled steroid required to achieve accurate quantification was 3 - 6^mol/l, so an infusion of 20ml/h of at least 30/xmol/l of deuterated steroid was used. Administration of the progesterone did not affect endogenous levels. Broom et al. (1983) in closely related work looked at the menstrual cycle and pregnancy and confirmed the value of using pregnanediol to indirectly monitor progesterone production. Oestrogen studies in normal pregnancy using isotope dilution determinations (Pinkus et al ., 1971) and added internal standard methods (Bjorkhem et al ., 1975) have been undertaken. The latter showed comparable excretion rates to routine photometric analyses of urinary oestriol. Injection of 17il-4-2H-oestradiol by Pinkus et al. (1971) allowed more varied analysis of oestrogens (oestrone, 17B-oestradiol, oestriol and 16-epi-oestriol) in urine by means of tritiated internal standards - oestriol being the main metabolite excreted (1300 and 1700/xg/24h in two women of 14 and 17 weeks gestation respectively were measured), but with 16-epi-oestriol containing the larger proportion of deuterium. Cortisol (4-14C) was successfully used as an internal standard for GC-MS measurement of plasma cortisol, giving comparable results to routine competitive binding methods (Bjorkhem et a l, 1974). The group concluded however that although this method was more precise, due to the binding of steroids other than cortisol in the competitive binding method, overall for routine analysis the latter is probably preferred due to the higher throughput of samples. Gaskell et al. (1983) used an isotope dilution method coupled with GC-MS, for external quality control assessment of assays for cortisol in plasma, using 2H3-cortisol. Routine laboratory assays, as judged by comparison with GC-MS data were generally positively biased in the range 100 - 650nmol/l investigated. An isotope dilution assay of serum cholesterol using 3,4-13C2-cholesterol (Pelletier et al ., 1987) was found to be highly reproducible (CV<0.5%). In this, as
68 in all such methods, sources of inaccuracy arising from hydrolysis, instability and decomposition are compensated for by using the ratio of the internal standard and endogenous steroid. Interfering steroids present in the serum, due to the specificity of the method, are eliminated from the quantification. Various other internal standards and isotope dilution investigations - including some reference methods - have been documented. Breuer & Siekmann (1975) have specifically determined using SIM oestrogens, testosterone, 5a-dihydrotestosterone, cortisol and aldosterone in human plasma. RIA of melatonin has been validated using GC-MS with stable isotope as internal standard in a number of species (Kennayway et al ., 1977). Gaskell (1983) has produced an extensive list of quantitative determinations of at least 16 steroids in human fluids and tissues using isotope dilution with GC-MS .
1.7.5.2 Studies of metabolites of endogenous compounds The biosynthesis and metabolism of endogenous compounds in vivo and the effects of physiological or pharmacological factors on the rates of these processes have been studied. Baba et al. (1980) have used stable isotope labelled testosterone as a metabolite tracer using plasma and urine in humans. Further studies by the same group (Shinohara et al ., 1980) showed that testosterone metabolites in urine could be quantified. Greater than 30% of an administered dose (20mg powdered 19-2H3- testosterone orally) was excreted over 24 hours as glucuronide conjugates of 2H3- androsterone and 2H3-aetiocholanolone. Testosterone underwent extensive "first pass" metabolism in the liver, as shown by the fact that only small amounts appeared in the circulation, even though the testosterone was completely reabsorbed from the gastrointestinal tract. Dehennin et al. (1987a) working with androgen and 19-nor steroid profiles in human pre-ovulatory follicles from stimulated cycles using 13C in an isotope dilution - MS study, identified and quantified 19-nor-androstenedione and 19-nor-testosterone in human follicular fluid. They observed an accumulation of 19-nor-steroids in follicular fluid confirming that these are weakly active intermediates in enzymatic conversion of androgens to oestrogens. In association with this observation, strong positive correlations between 19-nor-testosterone and 17B-oestradiol, and 19-nor-
69 androstanedione and oestrone concentrations were observed, giving evidence for common cellular origin. Production rates and metabolism of sulphates of 315-hydroxy-5a-pregnane derivatives in pregnant women were followed using 2H labelling in the 3a, 11,11- or 3a, 11,11,2015- positions (Baillie et al ., 1980; Anderson et al., 1990). Four main metabolic reactions were observed - oxidoreduction at C-20, 16a-hydroxylation, 21- hydroxylation and sulpho-conjugation at C-20. Injection of 2H labelled steroids in pregnant women was used to show various metabolic reactions. C21 steroid monosulphates in plasma undergo interconversion and further metabolism in vivo. Rapid oxidoreduction at C-20 being the major reaction. About 15% of the pregnenolone/pregnanediol monosulphates became 16a-hydroxylated, and then also underwent oxidoreduction at C-20. Pregnanediol-disulphate was found to be a metabolic end product accounting for a large part of the elimination of deuterated steroids injected into the pregnant women. Studies of glucose turnover important in diabetes have been performed using 6,6-2H2-glucose in studies by Bier et al. (1977) and Robert et al. (1982) based on isotope dilution. Bier found that glucose production was related to childhood brain size. Robert and his group studied elderly patients, using various isotopes including 6-3H, 6,6-2H2- and 13C-glucose, and found the amount of exogenous insulin required to maintain normoglycaemia was about two times that necessary in young adults. This study suggested that reduced glucose tolerance in these elderly healthy subjects was associated with changes in uptake by peripheral tissues and possibly impaired insulin sensitivity.
1.7.5.3 Pharmacological studies The determination of drug levels in biological fluids is now very important, with the development of highly potent therapeutic agents that can be administered in less than milligram doses, resulting in concentrations in plasma in the nanogram per millilitre or less range. The study of tolerance of drugs and potential differential metabolism of optical isomers of drugs in racemic mixtures must also be undertaken to ensure patient safety. Differences in pharmokinetics occurring between acute and chronic administration is of importance.
70 Conjugated equine oestrogens are widely used for treatment of syndromes associated with oestrogen deficiency in menopausal, post menopausal and ovariectomized women. From the pharmacological as well as toxicological point of view, it is important to determine the concentrations of various steroids in the blood of patients on such treatments and to investigate the pharmodynamic behaviour of the oestrogenic substances after oral administration. Drug levels were originally monitored using RIA, however due to cross reaction of these hormones in such assays and the low concentrations in the blood, the results are not always reliable. A method using isotope dilution-mass spectrometry for quantification was developed by Siekman et al. (1983). Their work showed that non-conjugated oestrogens concentrations in serum were low compared to sulphate esters. Maximum free oestrogens were seen at 8 - 11 hours after administration, and were cleared slowly from the peripheral blood, falling to half maximum levels after a further 12-28 hours. The SIM technique allowed measurement in the pg/ml range. In a study by Baba et al. (1980) it was shown that after oral administration of 20mg of 19,19,19-2H3-testosterone, a slight but significant amount of the deuterated testosterone (peak value 2.5ng/ml) appeared in plasma one hour after oral administration representing about 25% of testosterone measured and was rapidly cleared to less than 5% in 6 hours. The results (quantified using SIM GC-MS) showed that administration of this oral exogenous testosterone did not influence the plasma levels of endogenous testosterone.
1.7.5.4 Other quantitative methods Though internal standards and isotope dilution - mass spectrometry are the main stay of quantitative stable isotope work in endocrinology, another assay must be mentioned. Total body water can be measured using isotope dilution. In this case deuterium oxide (^ O ), as opposed to a very selectively labelled more complex molecule such as a steroid, is quantified. The most relevant applications to endocrinology are during pregnancy and post-partum, and in milk intake estimations in breast fed babies (from Blake et al . , 1975).
71 1.7.6 Qualitative applications 1.7.6.1 Isotope cluster technique (or ion doublet/twin ion technique! In this methodology the compound under investigation is enriched at a level of approximately 50% with one or more atoms of a suitable heavy isotope. The mass spectra of the molecule of interest and its metabolites exhibit "twin" ions for the molecular ion and all fragments retain the isotope atoms. By this approach metabolites of compounds containing the stable isotopes may still be detected against a complex background of endogenous metabolites by virtue of the conspicuous feature of their mass spectra. The enrichment is often achieved by a mixture of pure unlabelled analogue in a ratio of approximately 1:1. Alternatively if synthetic pathways allow, the heavy isotope is introduced into the substrate from a reagent that is labelled at a level of only 50 atoms percent excess. Early applications centred on investigations of androgen metabolites in vitro where 7-2H-androst-4-ene-3,17-dioneand 7-2H-testosterone (see review, Baillie, 1981) were used as substrates and products of oxidation or A-ring aromatization, identified by GC-MS. Braselton et al. (1973) worked on intermediates in oestrogen biosynthesis by human placental microsomes, and confirmed by direct isolation and identification the formation of 19-oxo compounds from androstenedione and testosterone during oestrogen synthesis. It should be noted that deuterium is not ideal, though not totally unusable, for this method because: (i) loss of label due to oxidation at the site of binding, (ii) loss of label due to oxidation on an adjacent carbon to the site of labelling, as keto-enol tautomerism may be introduced, (iii) due to (i) an alternative pathway may be favoured compared to the unlabelled form; and (iv) if extensive labelling is used GC retention time may be affected. 13C, 170 and 180 are not so readily affected by these problems.
1.7.6.2 Mechanistic studies A number of studies to elucidate metabolic steps/rate determining steps have been under taken. Pitman et al. (1972) for example studied hydrocortisone labelled with two deuterium atoms at the C-21 position and compared this compound’s
72 stability against unlabelled hydrocortisone by degradation in aqueous solution. Under oxidative conditions the 2H compound was more stable than the form. They suggested that the explanation of this was that the reaction mechanism for degradation involved a rate determining enolization in the C-17 position of the dihydroxyacetone side chain. A study, using deuterium labelling, on the effect of ethanol was reported by Cronholm et al (1972). The rate of excretion of different corticosterone metabolites, and the transfer of the deuterium to these metabolites was measured, after 1-2H2- ethanol was administered. Excretion of corticosterone metabolites increases after injection of ethanol, but with relative amounts of these steroids remaining fairly constant. In studying the results it was concluded that the co-enzyme pool(s) used in different reductions at C-3 was metabolically related to NADH formed in the alcohol dehydrogenase reactions. Since little or no deuterium was found at C-5 or C-20J other co-enzyme pools were probably used for the reduction of these positions. In a study in rats (Ringold et al ., 1961) the androgenic activity of 3a-2H-17a- methyl-5a-androstane-3J3,1713-diol was compared to its *H analogue. The 3-hydroxy form is basically androgenically inactive and must be oxidised to its keto form in vivo to become a potent androgen. If oxidation to the 3-ketone with the loss of the 3a-1!! or 3a-2H was the rate determining step, then the 3a-2H compound was expected to be oxidised at slower rate (due to isotope effects mentioned earlier), therefore exhibiting a lower androgenic activity than its unlabelled counterpart. This was tested in castrated male rats and on the basis of ventral prostrate responses and seminal vesicle responses, the *11 compound was 3.4 and 4.7 times more active respectively than the 2H compound. It was therefore concluded that oxidation to the keto form is a necessary step for androgenic activity in the rat. The interaction of several steroids with the glucocorticoid receptor protein of chick thymus cytosol has been tested in water and deuterium oxide by Ardnyi (1984). Substitution of deuterium for hydrogen did not influence association rate constants, but dissociation rate constants decreased two-fold in 2H20 in the case of steroids wtre containing a 1113-hydroxyl group, butAunaffected when this was absent. These findings suggested that the 1113-hydroxy group, known to be in every optimal glucocorticoid agonist molecule, participates in having a kinetically relevant hydrogen bond and this bond may have a role in glucocorticoid action.
73 Zachmann et al. (1979) looked at the modification of 15N balance by growth hormone, testosterone and thyroxine in patients with growth hormone deficiency and hypothyroidism. From their results they concluded that long term human growth hormone therapy does not increase basal nitrogen balance, but markedly enhanced the response to acute testosterone. By contrast long term testosterone increased basal nitrogen balance, but did not influence the response to acute human growth hormone. Long term thyroxine treatment increased the basal nitrogen balance, but abolished the response to acute testosterone. Gas chromatography of steroids, particularly when in conjunction with MS has been well established for years. The use of loading tests with deuterium compounds has been used to investigate steroid metabolism (Curtius et al ., 1975) especially of the enzyme deficiencies associated with the congenital adrenal hyperplasia 21- hydroxylase and 1 IB-hydroxylase deficiencies.
From this short review it can be clearly seen that stable isotopes can play an important role in the study of endocrinology. They are useful in both quantitative and qualitative applications, due to the specificity and sensitivity of the GC-MS technology used. Internal standards and isotope dilution has allowed quantitative work, and confirmed (or disputed) other assay (such as RIA) results. Qualitatively metabolic steps have been elucidated and new compounds not expected or known have been identified. The incorporation of stable isotopes into compounds allows sites of metabolic attack to be defined due to loss or retention of the heavy isotopes at the specific sites of labelling. Stable isotopes have proved invaluable in studies in ethically sensitive groups, such as infants and pregnant women in whom radioactive isotopes are no longer acceptable. With the growing technology that is appearing in the analytical market, and the increasing fall in the relative cost of this equipment, stable isotope work will increase in the future.
74 1.8 Altered plan of investigation
During the period of time during which the deuterium labelled progesterone was being synthesized (two years had been allowed for this), the urinary steroid quantification methodology was developed, along with some preliminary plasma radioimmunoassay (RIA) work. As time passed it became apparent that due to technical difficulties encountered in the synthetic work, beyond my control, insufficient 2H-progesterone would be available for the proposed project (see Appendix 3 for analytical work). The work involving the quantification of the excretion rates of THDOC, a principal urinary DOC metabolite, and other progesterone metabolites had meanwhile become very interesting. This was therefore pursued as the main stay of the practical work to be submitted. Some pilot and related studies involving deuterium labelled steroids are still however included.
75 2 - Materials and methods
Analytical methods for identification and quantification of steroids in urine
2.1 Urinary steroid profiles
This method is based on that described by Shackleton (1986) and is routinely used in this laboratory for patients’s samples.
2.1.1 Extraction of steroids Steroids (free and conjugated) were adsorbed from aqueous solution by passing them through a C18 Sep-Pak (Waters) previously primed with 100% ethanol (5 ml) and rinsed with distilled water (5ml). 10 or 20ml of urine were usually used. The cartridge was washed with water (50 - 100% of the sample volume), before the adsorbed steroids were eluted with 100% ethanol. This method was used in preference to solvent extraction because of improved recovery of polar steroids and also because the use of toxic solvents could be avoided.
2.1.2 Hydrolysis of steroid conjugates and re-extraction of free steroids The ethanol was removed, by means of reduced pressure, using a rotary evaporator, with a water bath at 50°C. Steroid sulphates and glucuronides were hydrolysed by incubation of the dried urine extract with approximately 25mg "Sulfatase" enzyme (from the snail Helix pomatia , Sigma HI) for 18 - 24 hours at 37°C in acetate buffer (0.5M) at pH 4.6. The steroids were re-extracted using an ethanol primed and water rinsed C18 Sep-Pak, again steroids being eluted with ethanol. This ethanol was then evaporated off under reduced pressure using a rotary evaporator. Extracts were redissolved in a known volume of ethanol (2ml) and, after thorough sonication to ensure maximum recovery, stored in a 2ml glass vial.
76 2.1.3 Derivative formation and sample clean up For derivitization an aliquot of extract, normally equivalent to 5ml urine was transferred to an 8ml glass stoppered derivatization tube. To this, 50/xl of a mixture of three internal standards, prepared at 100^g/ml in ethanol, i.e. 5/tg of each standard, was added. The standards were 5a-androstane-3a,17a-diol (from MRC Steroid Reference Collection at Queen Mary and Westfield College, London), stigmasterol and cholesterol n-butyrate (from Sigma) and were referred to as A, S and C. Ethanol was evaporated off under nitrogen and two steps of derivitization followed: (a) Oxime formation The condensation of carbonyl groups in steroids was achieved by reaction of dry standard compound or extract with 200^1 of pyridine containing 2% (w/v) methoxyamine hydrochloride (MOHC1) for 1 hour at 60°C, in a stoppered glass tube.
(b) Trimethylsilyl ether formation The silylation of hydroxyl groups was achieved by adding 100/ri of trimethylsilyl imidazole (TMSI) to the oxime reaction mixture and incubating the stoppered mixture at 100°C for a minimum of 4 hours, usually overnight, or at room temperature for 2 - 3 days. The pyridine was then evaporated from the derivative and approximately 1ml of "Lipidex solvent" (cyclohexane:pyridine:hexamethyldisilazane (HMDS) 98:1:1 (v/v/v)) was added to the slightly viscous residue. After sonication this mixture was pipetted onto a previously prepared 2ml column containing Lipidex 5000 swollen in "Lipidex solvent", above a glass collecting tube. Once the solvent had fully penetrated the column, a further 1ml of solvent sonicated in the sample tube was pipetted onto the column, followed by 0.3ml of pure solvent. The 2.3ml of derivative in solvent was dried under nitrogen at 60°C and immediately stored in cyclohexane in a glass 2ml vial.
77 2.1.4 Conditions for gas chromatography (GCVmass spectrometry (MSI
2.1.4.1 GC analysis for urinary steroid profiles A Packard 437A gas chromatograph fitted with a flame ionization detector (FID) was used for analytical chromatography. The ends (having been carefully trimmed to give flat end profiles) of an open tubular fused silica capillary column (25m x 0.32mm internal diameter (I.D.), WCOT Sil 5 (= OV-1) coating stationary phase from Chrompak UK Ltd) were coupled to the injector and the detector using Swagelock couplings with graphite ferrules. Dead space was kept to a minimum. The cyclohexane solution of derivatized steroids was injected into a pre-silanised open ended glass tube (approximately 1 x 5mm) and allowed to dry in air. Normally 1 - 2id of the steroids in approximately 250^1 cyclohexane was found to give measurable results. The glass tubes were hand cut from glass capillary tubes. Before use the tubes were heated (60°C) with dimethyldichlorosilane (2% in 1,1,1 trichloroethane) for at least one hour, and then rinsed with acetone three times. The tubes had then been placed in the GC oven for the duration of at least 3 GC runs in order to evaporate off any contaminants that may have remained after the solvent treatment. Up to 15 such samples were placed simultaneously into the rotary magazine of the injector (24 sample "holes” were available, but due to the increasing distance from the flash heater as the tubes collected in the glass insert, normally no more than 15 were loaded at one time). At the start of each operational cycle the rotary magazine advanced 15° to allow the sample vial to fall into the glass insert which was situated within the flash heater maintained at 250°C (Figure 2.1). The detector was also maintained at 250°C. The oven temperature was increased from 60°C to 185°C at 25°C/min and then from 185°C to 260°C at 2.5°C/min, whilst the helium carrier gas was maintained at lml/min. Each cycle lasted approximately 40 min because of the isothermal (260°C) period at the end to ensure column clearance, and then a 10 - 15 minute period was required for the oven to cool and equilibrate to the starting temperature. The flame ionization detector (FID) response was charted with a pen recorder.
78 Figure 2.1 - Cross section through solid injector device and connection for capillary column to gas chromatograph (FID)
L id — f O-Ring Rotary Magazine
Sample vial Carrier gas inlet
Removable glass insert
Flash heater
v:;r-,r -Sample vial from previous GC run l-2mm gap between insert and column to ensure Helium flow
Locknut Swagelock couple with ferrule
Capillary column
79 2.1.4.2 Mass spectral data acquisition for confirmation and identification of steroids in urinary steroid profiles A Hewlett Packard 5890 gas chromatograph connected to a Hewlett Packard 5870 Series Mass Selective Detector (MSD) was used for this analysis. The ends of a capillary column of the same type as described above were coupled to the injector and the detector using nuts enclosing graphite ferrules. The injector end consisted of a split/splitless injector port, whilst the detector end of the column was inserted to be within the source of the MSD by direct insertion. Helium gas flowed continuously through the system at lml/min. Samples were injected (0.5 - 2/xl of approximately 250/xl solution of steroids in cyclohexane) directly through the septum, using a glass 10/xl Hamilton syringe, into a glass insert within the injector. The temperature programme was similar but not identical to the FID detector GC - 2 minutes isothermal at 70°C to allow column loading during which the injector was in splitless mode, 60°C/min rise to 200°C, and then 3°C/min rise to 280°C to give a total run time of 32 minutes. All ions between m/z = 98 and 800 were measured, acquisition time normally being between 12 and 32 minutes of the GC run. A complete scan of ions m/z = 98 to 800 required 0.61 seconds. The ion responses were then totalled for each scan and displayed together to produce a total ion chromatogram (TIC), which was equivalent to the FID pen recorder chart of the same sample. The data could be displayed for each scan as a mass spectrum.
2.1.5 Quantification of steroid profiles Steroids were quantified from the FID pen recorder plots by comparison of their peak heights to those of the known amount of internal standards added. An example of a steroid profile from a normal female in the luteal phase is shown in Figure 2.2. A line was drawn between the tops and bases of the internal standards. Stigmasterol was found to be less stable than cholesterol butyrate (C) therefore the line was normally drawn between androstanediol and C.
80 Results were calculated according to the formula:
X=-x-xExSx— H y A R
where X = Steroid excretion /zg/24 hours Hx = Height of steroid peak Hy = Distance between lines joining internal standards at the same retention time as Hx V = Total 24 hour urine volume A = Volume of aliquot used in extraction (eg. 20ml) E = Correction for the portion of extract used in the derivative (eg. 4 for 1/4 of total extract) S = Amount of internal standards added (normally 5 fig) R = Correction factor for non 100% responses
Shackleton & Honour (1976) found that tetrahydrocortisone (THE), tetrahyrocortisol (THF) and allo-tetrahydrocortisol (aTHF) did not have 100% responses and correction factors were used, R=0.8 for THE and R= 1.2 for THF and aTHF. Relative retention times of steroids were constant and were characterised as methylene units (MU) by linear interpolation between the nearest alkanes when a sample is co-injected with a series of even numbered alkanes. These are shown in Table 2.1.
81 Figure 2.2 - Urinary steroid profile from a female subject in the luteal phase of the menstrual cycle
7
"Total urinary cortisol metabolites'
10
1 = Androsterone 2 = Aetiocholanolone 3 = Dehydroepiandrosterone (DHA) 4 = lip-hydroxy androsterone 5 = lip-hydroxy aetiocholanolone 6 = 16a-hydroxy DHA 7 = Pregnanediol 8 = Pregnanetriol 9 = Androstenetriol 10 = Tetrahydrodeoxycorticosterone (3a5p THDOC) 11 = Tetrahydrocoitisone 12 = Tetrahydrocortisol 13 = 5a-tetrahydrocortisol 14 = a-cortol 15 = p-cartol + P-cortolone
82 Table 2.1 - Methylene units (MU) of some MO-TMS ether derivatives of urinary steroids on OV-1 type capillary columns
STEROID MU STEROID MU
Androsterone 25.11 5-pregnene-3 B, 16a,20a-triol 29.20
Aetiocholanolone 25.28 5a-pregnane-3a, 1 lB,20a-triol 29.31
Dehyhdroepiandrosterone (DHA) 25.75 16a-hydroxypregnanolone 29.38
11-oxo-androsterone 25.95 3B, 16a-dihydroxy-5a-pregnan- 29.42 20-one
11-oxo-aetiocholanolone 25.95 5-pregnene-3B, 17a,20a-triol 29.46
17a-hydroxypregnanolone 27.00 Hexahydro-substance S 29.54
1 lB-hydroxyandrosterone 27.00 Tetrahydrocortisone (THE) 29.65
1 lB-hydroxyaetiocholanolone 27.18 Tetrahyro-compound A (THA) 30.16/ 30.65
16a-hydroxyDHA 27.38/ Tetrahydrocorticosterone 30.00/ 27.42 30.16
Pregnanediol (PD) 27.64 allo-tetrahydrocorticosterone 30.16/ 30.80
Pregnanetriol (PT) 28.00 Tetrahydrocortisol (THF) 30.24
5 -pregnene-3 B, 20a-diol 28.16 allo-tetrahydrocortisol (aTHF) 30.39 ' Androstenetriol 28.46 a-cortolone 30.51
Tetrahydro-substance S (THS) 28.62 B-cortolone 30.73
16,18-dihydroxyDHA 28.65/ B-cortol 30.73 28.81
3a,20a-dihydroxy-5a-pregnan-l 1-one 28.66 Hexahydro-compound A 30.76
Oestriol 28.75 5B-pregnane-3a, 1 IB,20a,21- 31.15 tetrol
3B, 15a-dihydroxy-5a-pregnan-20-one 28.75 or-cortol 31.20
3a,20a-dihydroxy-5a-pregnan-l 1-one 28.75 5a-pregnane-3 a, 1 IB,20a,21- 31.23 tetrol
5fi-pregnane-3a,16a,20a-triol 28.75 6a-hydroxytetrahydro- 31.82 corticosterone
3a,21-dihydroxy-5B-pregnan-20-one 28.75/ Cortisol 32.54/ (THDOC) 29.25 32.63
allo-tetrahydro-substance S 29.05 20a-dihydrocortisol 32.45/ 32.83
11 -oxo-pregnanetriol 29.10
5B-pregnane-3a, 1 lB,20a-triol 29.19 2.2 THDOC quantification
This method was developed from a combination of a SIM GC-MS method described by Honour and Shackleton (1977), and the above urinary steroid profile method. The former method quantified the major urinary aldosterone metabolite, 3a515 tetrahydroaldosterone, using SIM by use of the 3155a isomer as internal standard. The development of the sensitive quantification method for THDOC is (pages 93-iofc) described in the next chapter^, and the final methodology used for sample measurement is described below.
2.2.1 Addition of internal standard and extraction of steroids A fixed quantity (150 or lOOOng) of the internal standard (IS) 3B5a THDOC (Ikapharm, Israel) was added to a known volume of urine (0.5 - 10ml) and the total volume was made up to 20ml with distilled water. Extraction was achieved using C18 Sep-Pak cartridges as described above in Section 2.1.1.
2.2.2 Hydrolysis of steroid conjugates and re-extraction of free steroids As described in Section 2.1.2, with the extracts in ethanol going straight on to Sephadex LH-20 chromatography. THDOC glucuronide or sulphate was not available to test the efficiency of hydrolysis. The conditions were very similar to those used for the analysis of tetrahydroaldosterone in urine (Honour and Shackleton, 1977), where the conditions of hydrolysis were optimized to give the greatest yield of free steroid.
2.2.3 Sephadex LH-20 chromatography Elution and separation of steroids was achieved with cyclohexane: ethanol (4:1 v/v). Sephadex LH-20 (1.2g) was swollen in the eluting solvent and loaded into a glass column 8 x 0.5cm, and allowed to settle under gravity, with excess solvent flow from the base of the column. The sample was dissolved in 0.2ml 100% ethanol and sonicated. Cyclohexane (0.8ml) was added, mixed and re-sonicated. The 1ml extract was applied carefully, to avoid disturbance, to the dry surface of the gel. A further lml of elution solvent was sonicated in the sample tube and applied to the column followed by a further 2ml of elution solvent. These 4ml of solvent were discarded.
84 The flow rate of the column was approximately 8ml/h. Solvent (6ml) was loaded onto the top of the column and the fraction 4 - 10ml containing THDOC was collected directly into an 8ml glass derivatization tube.
2.2.4 Derivatization formation and sample clean up As described in Section 2.1.3, without the addition of A, S and C. The above mentioned 6ml of column eluent from the Sephadex LH-20 column were dried down, under nitrogen at 60°C, followed by 0.5 ml of ethanol used to rinse the sides of the derivatization tube.
\ 2.2.5 Quantification of 3o?5B THDOC by SIM using GC-MS Sample (or standards) in cyclohexane were concentrated under nitrogen and injected onto the gas chromatograph connected to the MSD. The temperature Csee pages*) programme is shown in Figure 2.3. Ions m/z = 507 and 476 were monitored^ The ratio of the main peak areas (see next chapter) - determined by MSD integration - of the analyte and internal standard was calculated. A corrected ratio was then read off a previously prepared standard curve appropriate for the quantity of internal standard added. The total 24 hour excretion was calculated using the following formula:
X = - x R c x S A where X = Steroid excretion /-eg/24 hours V = Total 24 hour urine volume A = Volume of aliquot used in extraction Rc = Ratio of analyte to IS after correction onstandard curve S = Amount of internal standard added (ie. 0.15 or 1 fig)
This was calculated for both ions 507 and 476 responses. An example of a typical SIM run for an adult female in the luteal phase is shown in Figure 2.4, the two main THDOC isomer peaks used for quantification being indicated.
85 Figure 2.3 - Temperature programme for THDOC SIM runs
Oven Temperature (°C) 300 i Data 280- Acquisition
260-
240-
2 2 0 -
2 0 0 - Approx. time for Injection 385a3a58 180- THDOC THDOC 160-
140 0 1 2 3 4 5 6 7 8 9 10 11 12 Time (min)
86 Figure 2.4 - Ion 507 and 476 responses from a SIM run of MO-TMS ether derivatized urine extract from a female in the luteal phase of the menstrual cycle
Ion 507
£l0000: ~o 365a THDOC JQ=» 8000- CE ; G000"
4000
2000
Ion 47G.
17-hydroxypregnanolone 1 .2E5 (170HPr)
1 .0E5
8.0E4
G.0E4
4.0E4
2.0E4
- I — I— I— I— I— I— I— I— I— I— I—
87 3 - Development of the GC-MS method for quantitative determinations of tetrahydrodeoxycorticosterone in urine
3.1 Characteristics of 3155a THDOC as internal standard This non-naturally occurring (in humans) isomer, in MO-TMS ether derivatized form, gave two peaks (syn and anti forms) the former being larger, similar to the analyte (3a515 THDOC), see Figure 3.1. The second peak of the internal standard had a retention time slightly closer to the first compared with the two peaks of the analyte, MU values for MO-TMS ether derivatives being: 3a515 THDOC 28.75 29.25 3155a THDOC 29.65 30.05 The presence of two peaks due to isomers of derivatives has been reported for other steroids (for example 16-hydroxyDHA, 16,18-dihydroxyDHAand20a-dihydrocortisol (Shackleton and Honour, 1976)). The mass spectra of the main peak of MO-TMS derivatives of the analyte and the internal standard are shown in Figure 3.2. Similar ions were present in both isomers and the ions of choice for selected ion monitoring 507 (M+) and 476 (M+- 31), were clearly seen. These ions were chosen because ion 476 was the most prominent high value ion, and 507 was the molecular ion and was large enough to be detected. R efer fco page 116 for explanation t/on patter'#- The absence of 3155a THDOC from urine was confirmed. No peaks for ions 476 or 507 were detected at the relevant GC retention time in control urine sample extracts analyzed without the addition of the internal standard.
3.2 Choice of selected ions and method of quantification When measuring peaks using the mass spectrometer the option of measuring peak height or area was available. In order to decide on which to use, the heights and areas of various quantities of the analyte to a fixed quantity of internal standard
88 Figure 3.1- GC run of the two isomers of THDOC
3a58 THDOC I
---- 3B5a THDOC /
15 20 25 30 Ti me (mi n . )
Figure 3.2 - Partial mass spectra (m/z = 98 - 520) of the two isomers of THDOC
2.0E5 175 478 188 / 1 . 5E5 75 2.0E5 188 78 1 . 5E5 507 . 0E5 404 (M+) 385 a THDOC 35B -o 5. 0E4 0 ■f 100 200 300 400 500 Mass/Charge 89 were measured using FID GC. Examples of the results are shown in Figure 3.3. Peak height response ratios plotted against concentration ratios did not give a linear relationship, when calculated from the direct ratio of the main peaks of the two isomers. A curve was also plotted from the responses when each isomer was first quantified using the A, S and C internal standards (urinary steroid profile quantification). Areas, on the other hand, gave a good linear fit up to the range (maximum measured) 5:1 for analyte:internal standard. Area was measured by running the pen recorder on the FID GC at a fast speed, photocopying the relevant peaks and weighing the cut out areas on an accurate balance. These area ratios were found to be highly reproducible e.g. 5:1 ratio, mean = 5.05:1, CV = 2.8%, n=10. The areas of the first larger peaks of the two isomers were found to give the same linear standard curves as when both the syn and anti peaks were summed for each isomer, so it was decided to use only the first larger peak for quantification. Ion 507 and 476 responses were then analyzed using selected ion monitoring (using the temperature program for steroid profile GC-MS analysis). Both ions gave near linear standard curves for area up to the ratio 5:1, similar to those obtained from the GC traces when using area. Ion 507 gave a slightly better standard curve but was too small to be easily detected in some samples using GC-MS SIM, so it was decided initially to use ion 476 for quantification. Various standard curves were prepared the lowest using lOng internal standard (5 - 50ng analyte), and the highest using 10/xg IS (1 - 50/xg analyte). On examination of the higher value standard curves, there was some loss of linearity above unity. In further standard curves, including the Sephadex LH-20 step (see later), the best reproducibility of the ratio of 3a5fi:3B5a THDOC was found to be around unity, eg. 3a5B:3B5a THDOC ratio CV% (n=5 injections each) 0. 1:1 4.1 0.5:1 8.0 1:1 3.1 2.5:1 10.8 5:1 13.5 Standard curves were therefore set up so that the internal standard (IS) added was close to unity when in ratio to the analyte. A convenient aliquot (normally between 1 and 10ml) of urine was thus used. Quantification using 150ng IS for normals and 90 Figure 3.3 - Height vs area Quantification from GC FID traces ■o _ Q CD to TD 05 Q. co o . a ■03 r ■*^ Q> -0 c — ™ -< ttJ +-< *1 ® k ^ k- ^ 0 £ *0 <0 (0 — O) ? 3 O - + CO 03 co .c CM o m O - T M co CM o t CO ■D Q C QQ O X co in +-» CO o 0 X Q. 0 a) co _ k co 91 samples with lower values, and 1/xg IS for samples with high THDOC concentration was decided upon. Up to this point in the evaluation of the method, the 32 minute long temperature programme, used for urinary steroid profiles had been used. As many hundreds of SIM runs would be required for the work in this project a shorter temperature programme was developed. Samples were injected into the GC connected to the MSD with the oven at 160°C. An isothermal 2 minute period, to allow column loading (the injector was at 250°C), was followed by a fast rise of 40°C/min up to 260°C. The remainder of the run was then isothermal (260°C). A total run time of 12 minutes was used, though in most cases 9 minutes would allow total clearance of loaded steroids from the capillary column (see Figure 2.3). The two main peaks of the two isomers of THDOC eluted from the GC column at approximately 7 and 8 minutes. 3.3 Sensitivity In order to assess sensitivity, a double dilution analysis of standards was performed. Starting with 200:100ng 3a5B THDOC:3B5a THDOC being injected, sequential dilutions (1 in 2) and further injections were made until reaching 0.4:0.2ng. Consistent ratios of ion responses from 3a5B:3B5a THDOC were found with 507 and 476 ion responses (2.51 ± 0.31 and 1.96 ± 0.23 respectively, n = 9, CV = 12%). Using the 0.25mm internal diameter column initially installed in the MSD the minimum sensitivity, defined by 3:1 signal to noise ratio, using the ion 476 response, was approximately 300pg injected. When the column was exchanged for a 0.35mm internal diameter column this sensitivity increased to approximately lOOpg injected. Normally more than 500pg was injected onto the GC column. Initially the reproducibility of injection was found to be relatively poor, CV for 476 peak area ratios = 8.7%, n = 10. This CV was reduced to <5% by improved injection technique, i.e. pulling up some cyclohexane, then some air followed by sample and then further air. Using this technique the sample volume can be rechecked further up the scale of the Hamilton syringe. 92 3.4 Introduction of a Sephadex LH-20 chromatography step In order to evaluate the method for clinical samples a number of urines were processed in a similar way to that described for steroid profiles, with 3B5c* THDOC added as the internal standard at the start of urine processing, but analyzed using GC- MS SIM for ions 476 and 507 responses. It was found in some samples, in particular from patients with 1 lB-hydroxylase deficiency and Cushing’s disease (markedly so when they were receiving metyrapone) that there were large amounts of tetrahydro- 11-deoxycortisol (THS) present. One major ion of THS is 474, and due to the natural occurrence of stable heavy isotopes of carbon, nitrogen, oxygen, and hydrogen the ion 476 was present in significant amounts (see Figure 3.4). In pregnancy samples, oestriol which has a retention time almost identical to 3a5B THDOC, has a strong ion 504 response present in its mass spectrum (Figure 3.5). Ion 507 was present (at around 5% of the 504 value, again due to he natural abundance of stable heavy isotopes), and similarly interfered in quantification if the ion 507 response were used, as oestriol in pregnancy was present in great excess of THDOC. The peaks originating from THS, oestriol and 3a5B THDOC were not totally separated by the gas chromatography performed. To resolve THS and OE3 from THDOC, urine extracts were subjected to Sephadex LH-20 chromatography, based on the method described by Setchell and Shackleton (1973), Shackleton et al. (1973), and Honour et al. (1978). Elution with cyclohexane:ethanol (4:1 v/v) enabled separation according to polarity of the steroids. The method described used 6g of the Sephadex LH-20. When using a 6g column THDOC eluted in the 20 - 40ml of solvent fraction. This was clearly shown in the urine of an adrenalectomized patient, on DOC acetate treatment, who had had tritium labelled DOC administered prior to urine collection. The urinary steroid profile of this patient showed 3a5B THDOC to be the main, and almost only, excreted steroid metabolite present, and hence associated with a high proportion of the radioactivity present. Fractions (2ml) from a 6g Sephadex LH-20 column were collected and aliquots of these were counted using a scintillation counter, see Figure 3.6. As large numbers of samples were to be analyzed the method was scaled down and 1.2g of Sephadex LH-20 was used satisfactorily. Using the scaled 1.2g columns and a urine sample from a patient with Cushing’s syndrome, collecting 0.5ml 93 derivative of tetrahydrodeoxycortisol (THS) 64 476 2.0E5- aj u 474 c rd T 3 c 13 147 255 _Q 384 CE 168 294 435 / jLikii Jii„i)ii,. ^ 4 .i,iL 100 200 300 400 500 Mass/Ch arge Figure 3.5 - Partial mass spectrum (m/z = 98 - 600) of the MO-TMS ether derivative of oestriol (OE3) 7.0E5 04 507 6.0E5 5.0E5 “ 4.0E5 T 3 147 386 I 3.0E5 _Q CE 29 2.0E5 203 231 1 .0E5 200 400 500 Figure 3.6 - Sephadex LH-20 fractions from urine of an adrenalectomized patient after tritiated DOC administration CO counts/50 min (x1000) CM o CD E O) o O D c CM CO CO CM n i CD ^ co in o 00 CM CO in CM j v r CO CO CO 1'1w nc CM • • O 10 95 CO CM 5 fraction (ml) fractions, the majority of THDOC was found to be eluted in the 4.5 - 9.5ml of solvent fractions. A small quantity of THS was eluted in the fraction 8.5 - 10ml, but it was found on analysis of the 0.5ml fractions that some 3B5a THDOC was also found in these fraction and it was therefore retained. The first 4ml of solvent eluted from a 1.2g LH-20 column was therefore discarded and the 4 to 10ml of solvent fraction retained. This removed most (>95%) of the THS and all of the oestriol. A similar experiment was performed using a 36 week gestation pregnancy urine extract. Fractions were collected as follows: 0 - 4ml 1ml fractions 4 - 10ml 0.5ml fractions 10 - 15ml 1ml fractions Each fraction was derivatized and run on the MSD using the same temperature programme as the SIM runs (refer to Figure 2.3), but in scan mode to identify the steroids present. The results for each fraction are shown longitudinally in Figure 3.7, relative to GC retention time. 3.5 Use of alternative derivatives to separate THS from 3a5fl THDOC An alternative approach for separating off THS was attempted using alternative GC derivatives. The structures of the D-ring using MO and three other chemical alternatives, all as TMS ether derivatives are shown in Figure 3.8. Ethoxyamine (EO) instead of MO gave similar retention times for the THDOC isomer peaks to the MO-TMS ether derivative, but separation of THS to 3a5B THDOC was reduced. Similar retention times for the main THDOC isomer peaks were seen, when hydroxylamine (HO) was used, with the second minor peaks showing poorer separation than using MO-TMS ether derivative. Separation from THS was not improved. Using benzylhydroxylamine (BO), syn and anti forms of the isomers were no longer resolved as separate peaks (i.e. two instead of four peaks) and THS was slightly better resolved from 3a513 THDOC. The overall run time using this derivatization, however, was 150% that of the MO derivative and was considered impractical for the large number of samples that would be run. 96 00 > » o c a> C)^ |-(0^ u CO c ti *N c _o CO o c uu O a ■S'S? X x O C ym CD. - Is- uu CD CO •SI X oil 8 X ^ 2 _ j c \ i 0 Q § X g « o ni o) in “O _0 ] * H.E CD 0 3 Q . Q a. in o Q - l CD r JSCO Qo CD ^ a> !C CO w -*— « > * ic o o 2 T3 Q r - c CL !C CD ■ 0 i i time (min) GC retention Approximate CO C c d Q 00 D) 00 0 0 CO Q_ 0 X < O X O in T> CD. 0 CD in (/) CD 1 x: co coO« 1 TJCDi^ c O c C c> » £: (0 o 03 "-+—> CO "O o LU c aj O < I I I I I I I in in co "ST a> oo n co in co C\J The elution volumes, from a 1.2g Sephadex LH-20 column, of individual steroids are shown (vertical bars against ordinate). Fractions of 1ml or 0.5ml were derivatized and separated by capillary GC. The position of the vertical bars on the abscissa are aligned with the GC retention time for each steroid detected. Steroids not named 97 have a major ion indicated. The fraction 4 - 10ml (as indicated by the large arrow) was collected for THDOC quantification. Figure 3.8 - Structures of different carbonyl derivatives of THDOC (all as TMS ethers) H2C-0-Si-(CH3)3 I C-N-0-CH3 methyloxime (MO) (CH3)3-Si-0 H H2C-0-Si-(CH3)3 I C-N-0-CH2-CH3 ethyloxime (EO) H2C-0-Si-(CH3)3 I C-N-0-Si-(CH3)3 hydroxylamine (HO) H2C-0-Si-(CH3)3 O N -0-C H 2- benzylhydroxylamine (BO) 98 3.6 Inconsistency in 476:507 ratios for "3a56 THDOC" Using standards, it was found that the ratio of the area under the main SIM peak for ions 476 and 507 was consistent. The exact values varied, but 3135a THDOC always gave a slightly higher value than 3a56 THDOC. Typical ratios were in the range 2.8 - 4.3 (3a56 THDOC) and 3.1 - 4.7 (365a THDOC). These values were sensitive to the slight differences in tuning of the MSD. As the source becomes "dirtier" (oxidation of metal surfaces), the higher ions in the spectrum reduced in intensity relative to lower ions in the mass spectrum. This change was sufficient enough to be noticed in this sensitive analysis. In order to avoid errors due to this phenomenon, standard curves were regularly produced along with sample analysis. After a number of clinical samples had been analyzed, it was discovered that the consistent 476:507 ratio for 3a56 THDOC seen in the standards, was not seen in all the clinical samples, whereas 365a THDOC was not affected, see Figure 3.9. On inspection of full spectra of steroids present in these samples after the LH- 20 chromatography step, the following ions were found at the retention time of 3a56 THDOC, that were increased relative to ion 507, as seen in the analyte: In non-pregnant samples 117, 131, (188), 386 and 476 In pregnant samples 100, 188, 386 and 476 These ions were consistent with the possible D-ring structures shown in Figure 3.10. This strongly suggested that steroids were co-eluting from the GC column with 3a56 THDOC. Samples from patients with 116-hydroxylase deficiency CAH and Cushing’s disease had 476:507 ratios equivalent to the standard. The variation in 476 to 507 ratio exceeded those seen when the MSD source needed cleaning as mentioned above. This was shown to be the case by a set of 476:507 ratio results obtained later from a series of pregnancy urines (Figure 3.11). Up until week 21 and then again after week 29 the 3a56 THDOC and 365a THDOC 476:507 ratios were consistently at approximately 10 and 4 respectively. The samples between weeks 21 and 28 were injected on a different day to the others already mentioned, and the 476:507 ratios were greatly raised for the 3a56 THDOC peak. A rise was however also seen for the internal standard. Both isomers fell back into the expected range on re-injection after the source had been cleaned. It can therefore be seen that the 476:507 ratio for the internal standard was a good guide to whether 99 Figure 3.9 - Initial 476:507 ratios cq co in . o cvi in in in oo o o in 100 standards 11p-0H def. Cushings male female pregnancy Figure 3.10 - Possible partial structures of steroids co-eluting with 3oSI3 THDOC MO-TMS derivative THDOC CH20H H2C-0-Si-(CH3)3 I OO » 175 Pregnancy CH3 I C-N-0-CH3 CH3 CH3 I C-0 C-N-0-CH3 OH 0-Si-(CH3)3 Non-pregnant CH3 CH3 i HC-OH HC-0-Si-(CH3)3 GC analysis of the MO-TMS derivatives of steroids at the GC retention time of THDOC showed different prominent ions in pregnant and non-pregnant females, which were thought to be related to the above D-ring and side chain structures. The steroids were thought to be hydroxypregnanolones. 101 o co co CO CO CO CO 0 CM 4ff o CO Q. o CO E CO 03 CO .2 co + -H- X <] CM +-* X <&> K 03 0 -HH CO + + X :<] c w c h- ‘i- o o ^ CM ' 3 _, X <3 CM CO + -+H X <] CM CO co o + -Hff o o O) ^ 503 -Hh 6 CM C CO I O) - t - T-CO - (1) * r - <1> <$> CD 1— k- £ £ • Q . CO _ + 0 03 -H- o C < £ > 3 ■" <> CM U> ^ o o o O c C O L ^ o O 13 13 I X 1 1 H f <&> D) i 102 |! there was any co-eluting steroid present at the retention time of 3a5B THDOC as the 476:507 ratio was expected to be lower than for the internal standard, and also if the source needed cleaning. The use of ion 188 for quantification was also investigated, as it was a strong ion for the analyte and internal standard (see Figure 3.2). This ion, like ion 476, was however also affected by the presence of co-eluting steroids. Quantification was therefore achieved by analysis of ion 476 and 507 responses, the latter being assumed to be nearer the true result for pure 3a5B THDOC. 3.7 Method Validation Reproducibility of extraction Adult male sample extracted 5 times (using 150ng standard curve and ion 476 for quantification) mean + SD 36.6 ± 2.8/*g/24h range 33.6 - 40.3/xg/24h CV 7.6% Duplicate extractions of various clinical samples n=8 (using 1/xg standard curve) excretion rate (jug/24h) CV 717 2.2 760 3.9 5762 4.4 3370 7.2 2390 8.8 2782 10.3 138 11.0 88 11.3 Excretion rates (ion 507 response) in pregnancy (n > 100 samples) up to 600/xg/24h gave CV <8% for two extracts injected twice into the MSD (ie 4 injections). 103 Retention time reproducibility on MSD n=75 injections of different standards and samples 3a515 THDOC 3B5a THDOC range(minutes) 7.703 - 8.091 8.877 - 9.124 mean ± SD 7.748 ± 0.066 8.936 ± 0.063 CV 0.86% 0.71% The retention times varied slightly over time as different columns were used. Reproducibility of single sample (repeated injection) Injections onto GC-MS: Standards n=5 (5 injections) Range of analyte to fixed IS (150ng) = 15 - 750ng CV = 1.2 -5.6% Samples n=5 (5 injections) Range of analyte to fixed IS (150ng) = 10 - 700ng CV = 4.4 - 9.2% Samples n=4 (2 injections) Range of analyte to fixed IS (l/*g) = 0.5-5.3/zg CV = 2.2 - 8.8% Recovery A sample from an adult female (follicular phase) was spiked with analyte and recoveries calculated (n=5 injections) amount of 3a515 THDOC 3a513 THDOC measured Recovery added (ng) (ng) mean ± SD (%> 0 100.2 ± 6.9 - 50 148.5 ± 13.9 95 100 256.9 ± 22.7 147 250 360.3 ± 20.7 104 regression slope = 1.01 ± 0.27 regression intercept = -0.05 ± 37.3 r = 0.996 104 Reproducibility of standard curves A total of 26 standard curves were made, 11 for Ipg and 15 for 150ng IS. The mean and standard deviation of all values are shown in Figures 3.12(a-d), with the best fit through the first 4 standards as per experimental work. 105 Figure 3.12(a) - Standard curves Figure 3.12(b) - Standard curves 150ng 366a THDOC Ion 507 150ng 3B5a THDOC Ion 476 *o e O © <0 m ol 3 o £ © ctf 80 3 COCD CM o o 3 ^ ^ I ? o ? I CO TJ TJ LL CO OJ -•-» T>O J T CO CO £ CJ 5 3 3 w © > © » CO C CO . k O <0 s 3O CO Q 5 ° ^ . k o t > o a> C © OIO 1 O C o o o o CO IO - » X Q T“ K CO N / c Ol o C as CO TJ E © © o 3 ctf o E © <0 8» 3 o to to CO CM o o 106 3a5B:3B6a THDOC ratio 3a6B:3B5a THDOC ratio 4 - Separation of the co-eluting steroids In urine collected during pregnancy and the menstrual cycle the specificity of the THDOC measured by SIM GC-MS was hampered by the presence of steroids with the same GC retention properties. Several approaches were tried in order to isolate or remove the compound(s) co-eluting with 3a5B THDOC: (i) Immunoadsorption (ii) Change in GC chromatography conditions (iii) Alternative GC derivatives (iv) Celite chromatography, coupled with thin layer chromatography based on the method described by Winkel et a/.^from one of the papers that inspired the project. 4.1 Immunoadsorption 4.1.1 Introduction Immunoadsorption is a method of extracting, with a high degree of specificity, a biological compound from a complex mixture such as plasma or urine. From an antiserum, raised in an animal against the steroid of interest, antibodies are precipitated and then coupled to a gel matrix backbone, such as agarose or cross- linked dextran (Sephadex). The compound of interest can then be extracted from the complex mixture by the antibodies, and then can be released if necessary for further analysis. Alternatively the biological molecules remaining in the mixture can proceed to further steps of analysis once an interfering compound has been removed. It was hoped that the co-eluting steroid was sufficiently different to allow separation from 3a5B THDOC. 107 Experimental 4.1.2 Precipitation of immunoglobulins This method was based on standard methods using salt fractionation of proteins, and offered some degree of purification of antibodies in the antisera. A known amount of serum containing the antibody of interest was cooled on ice (30 min). Ammonium sulphate (3.8M (NH4)2S04) was then added dropwise to the serum, vortexing the tubes between drops. The amount added was calculated from the formula: 3.8xS=1.6x(K+S) where V = volume of serum used S = volume of 3.8M (NH4)2S04 required The mixture, after 30 minutes on ice, was centrifuged for 20 minutes (at 2000y) After removal of the supernatant the pellet was resuspended in approximately twice its volume of 1.6M (NH^SC^ and vortexed gently. After centrifugation (20 min, 2Q00y) the supernatant was discarded. The precipitated proteins, mostly immunoglobulins, were redissolved in 0.5M NaHC03 buffer (1ml). To desalt the immunoglobulins, the latter were passed through a PD-10 Pharmacia 9.1ml gel column previously equilibrated with 10ml of buffer. The immunoglobulin fraction was pipetted gently on to the top of the column, and allowed to run through. 15 - 20 fractions of between 0.5 and 1ml were collected by adding buffer gradually to the top of the column. Acidified Coomasie Blue (ACB) reagent and distilled water (100/ri respectively) were pipetted into an appropriate number of clean glass tubes. After vortexing, 10/d from each fraction was added to a tube containing ACB. The presence of protein caused a colour change from yellowy- brown to blue. All fractions indicated by unequivocally blue tubes were pooled. Subsequent fractions were rejected as they contained salt. The column was washed with a mixture (10ml) of NaHCC > 3 buffer, phosphate buffer, bovine serum albumin and sodium azide, and stored at 4°C for use again. 108 4.1.3 Cvanobromide activation of Sephadex G25 This method is based on that described by Cuatrecasas (1970). All work was carried out in a well ventilated fume cupboard. Cyanobromide (CNBr, 5g, Sigma) was weighed in a stoppered glass conical flask and 200ml distilled water was added to give a 2.5% w/v concentration, and then was gently stirred until dissolved using a magnetic stirrer (30min). A CNBr solution (200ml) was constantly stirred as 5g of Sephadex G25 fine polymer was added. The pH was checked continually as the mixture was brought to pH 10.5 by rapidly adding 1M NaOH, to avoid toxic fumes being produced. After 2 minutes further stirring, the activated polymer was filtered using a Buchner funnel and washed immediately with 500ml ice cold distilled water. Further washes with 50% acetone/water, 75% acetone/water and 100% acetone followed. All washes were disposed of according to the prescribed University procedures for hazardous solvent waste. The polymer was dried at room temperature, weighed and stored at -20°C. 4.1.4 Coupling of antibodies to activated polymer This method was based on that described by Wide (1970), as modified by Seth and Brown (1978). In the ratio of 1:4:1 the following were constantly mixed at room temperature for 2 days: antisera (ml, equivalent to original antibody serum) sodium borate buffer pH 0.1M (ml) activated polymer (g) This mixture was centrifuged (20min, 2000j>) and carefully decanted. This supernatant was kept as it may contain some uncoupled antibodies. The immunoadsorbent was then washed in various solutions (i - vi below). After mixing for 20 minutes (unless otherwise stated) and centrifugation (20min, 2000p) the supernatant was carefully decanted and discarded: (i) 10ml 0.5M NaHC03 (ii) 10ml 0.5M NaHC03 (iii) 10ml 0.1M acetate buffer pH 4 - 1 hour mix (iv) 10ml 0.1M acetate buffer pH 4 - ultrasonic bath 30 seconds 18-20 hour mix (v) 10ml assay buffer (see below) 109 (vi) 10ml assay buffer The immunoadsorbent was then made up in 100ml assay buffer and stored at 4°C. Assay buffer: 500ml 0.1M phosphate buffer pH 7.5 500ml 0.9% (w/v) NaCl 10ml 5% (w/v) NaN3 5M Tween 20 2g Bovine Serum Albumin 4.1.5 Urine sample processing This method, originally used for serum, was adapted from Gaskell et al. (1984). The extract of partially purified steroids from a urine sample (normally after the Sephadex LH-20 chromatography step of the THDOC method) was dried down under nitrogen and redissolved in 1ml citrate buffer (pH 4.0, containing disodium hydrogen orthophosphate - 75mmol/1 - and citric acid - 62.5mmol/1). This was placed in a silanised 20ml glass conical flask. Glassware was treated to minimise adsorption by rinsing with dimethyldichlorosilane (2% in 1,1,1 trichloroethane) followed by methanol, and then dried at 60°C. The specimen tube was rinsed with a further 0.5ml of citrate buffer and added to the conical flask along with 1ml (or the volume otherwise stated) of coupled antiserum (previously washed 5 times with 100% ethanol followed by distilled water and citrate buffer). This mixture was shaken at room temperature for 1 hour. After transfer to a glass tube, with the addition of further 1ml citrate buffer rinsing of the conical flask, centrifugation was performed (lOmin, 2000y)» The supernatant was decanted and retained with 2 x 2ml water washes. The aqueous solution was then dried down either under a continuous stream of air or by solvent extraction using 35 - 40ml dichloromethane, followed by evaporation using the rotary evaporator, after the aqueous fraction had been discarded. Once dried the steroids were reconstituted in 100% ethanol, transferred to a derivatization tube prior to MO-TMS ether derivative production as normal. The coupled antiserumwas washed with ethanol, methanol, water, citric buffer, water and citric buffer once more before possible further use. 110 Results 4.1.6 Progesterone anti serum immunoadsorption As one of the possible structures of the co-eluting steroid present in pregnancy was thought to have a D-ring/side chain structure similar to progesterone, immunoadsorption using coupled progesterone antisera was attempted. The antiserum, kindly donated by Guildhay Antisera, was raised in sheep against a progesterone 11a- hemisuccinate-albumin conjugate. (i) Effect on THDOC standard curve Various quantities of 3a56 THDOC (0.1 - 5/xg) were processed up to the Sephadex LH-20 step. The 6ml collected from the Sephadex LH-20 column were split into two equal fractions and one fraction underwent immunoadsorption and then both fractions proceeded to the derivatization step after the addition of 365a THDOC (l^cg). The results are shown in Figure 4.1. There was no significant difference between the two standard curves, indicating that progesterone immunoadsorption did not adsorb THDOC. (ii) Effect of varying quantities of immunoadsorption gel Initial attempts at immunoadsorption showed a slight improvement in 476:507 ratio in one pregnancy sample, but not in another. Varying amounts (0.2 - 5ml) of gel were then used in the second sample that had given poor improvement in ion ratios. No change in 476:507 ratio was seen between the different amounts of gel, indicating that immunoadsorption using progesterone antisera had had little or no effect at removing the co-eluting steroid. When the samples (which had had standards A, S and C added) were analyzed using GC steroid profiles a decrease of pregnanediol was seen with increasing gel quantity, with a plateau adsorption at gel quantities above 2ml (see Figure 4.2). (iii) Released products from immunoadsorption of pregnancy urine Steroids were liberated from the immunoadsorption gel using methanol. After a MO-TMS reaction the derivatives were found to contain mainly pregnanediol. A small amount of a steroid possibly 20-dihydroprogesterone (M+ = 419) was detected, 111 Figure 4.1 - Standard curves with and without progesterone immunoadsorption measured ratio 1.5- O without IA ~+— with IA 385a THDOC added 0.5- just prior to derivatization IA* ItMWNwv'Oadsorpti’oK) 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 expected 3a56:385a THDOC Figure 4.2 - Effect of varying quantities of immunoadsorption gel on pregnanediol Pregnanediol (mg/1000ml) 14 n------ 12 0 1 0 - 8 - 6 - 0 H i i i------1------r 0 1 2 3 4 5 Quanity IA gel used (ml) 112 ions 117 (loss of secondary hydroxyl group as TMS ether derivative from the side chain), 388 (M+-31) and 298 (M+-[31+90]) being detected. No steroid could be identified at the retention time for 3a5B THDOC. (iv) Menstrual cycle urine samples Four urine samples (collected by one subject on days 7, 14, 21 and 27 of one menstrual cycle) were analyzed with and without immunoadsorption. No difference was found between the 476:507 ratios in the two sets of results. This indicated that progesterone antisera immunoadsorbent did not bind the co-eluting steroid found in the menstrual cycle urine samples. Similarly no change was seen in the 476 and 507 response ratio in the THDOC peak of an adult male urine extract. 4.1.7 THDOC antiseiwiiimmunoadsorption As immunoadsorption using progesterone antisera had not been successful an alternative approach was then pursued, adsorbing the THDOC and examining steroids remaining. THDOC antiserunwas kindly donated by Prof. P. Vecsei (University of Heidelberg). The anti sen^i was similar to the one reported by Gless et al. (1976) raised in rabbits against THDOC-20-oxime coupled to Bovine Serum Albumin. Even though the coupling of the antibody to the activated polymer was performed twice, no change in the standard curves was achieved in either case after immunoadsorption. This methodology was therefore abandoned. 113 4.2 Change in GC conditions With the GC temperature programme on the GC-MS used for a urinary steroid profile, 3a5B THDOC had a retention time of approximately 20 minutes. Using an extended run time (see Figure 4.3) the retention time was increased to 146 minutes. There was no improvement in the separation of 3a5B THDOC from the co eluting steroids, even with an almost threefold increase in theoretical plates (160,000 to 430,000), calculated using the formula below: n=16x (— f Wb where n = theoretical plates Tr = retention time (seconds) of steroid of interest Wb = width (in seconds) of base of steroid of interest The presence of the co-eluting steroid was confirmed by inspecting the spectrum of the peak of interest. 114 Figure 4.3 - Temperature programmes Change in GC conditions Oven Temperature (°C) 300 n 250- 3°C/min 200 60°C /min 0.75°C/min 150 1 0 0 - 5°C/min 50 Extended run Injection Normal run n------r 0 30 60 90 120 150 180 210 240 270 Time (min) 115 4.3 Alternative GC derivatives The next approach was to see if using different derivatives would enable separation of the co-eluting steroids. Several derivatives (n = 4) of the carbonyl groups, in conjunction with TMSI were analyzed. The mass spectrum of the MO-TMS ether derivative of 3a5B THDOC is shown in Figure 3.2 (see previous chapter). The molecular ion (M+) was m/z = 507. Loss of the methyl oxime group (m/z = 31) at C-20 resulted in a strong 476 ion response. The further loss of a trimethylsilyl group (m/z = 90), either at C-3 or C-21, resulted in an ion 386 response. Similarly loss of the second trimethylsilyl group resulted in an ion 296 response. Two further strong ions were seen at m/z 175 and 188 (Ions A and B). These were ions resulting from fragmentation of the D-ring (see structure below Table 4.1). Substitution of ethyloxime (EO), hydroxylamine (HO), or benzyl- hydroxylamine (BO) for methyloxime (MO) (always in conjunction with TMSI) resulted in the production of different characteristic fragmentation ions for Ions A and B. These are shown in Table 4.1, along with the molecular ion (M+) of each alternative derivative. On loss of the methyl oxime, or equivalent fragment, the remaining chemical structure was almost identical in the four derivatives, hence all had ion 476, 386 and 296 responses though these varied in relative intensities. For each alternative derivative Table 4.1 also shows the ratios of ion 476:M+, ion 386:M+, Ion A:M+ and Ion B:M+. The steroid peak at the GC retention time of 3a515 THDOC from an urine extract from an 1115-hydroxylase deficiency CAH patient had been shown previously to have no co-eluting steroids present, by virtue of the 476:507 ratio being equivalent to standard THDOC (refer to Section 3.6 and Figure 3.9). Analysis of the four alternative derivatives of this urine extract resulted in mass spectra which were equivalent to those seen for the standard. This demonstrated that THDOC could be identified by all four derivatives in urine extracts, and further confirmed that no interfering co-eluting steroid was present in this clinical sample. When steroids in an extract from a pregnancy urine sample were analyzed with these derivatives there was evidence of incomplete separation of the co-eluting steroid from 3a515 THDOC. Differences in the relative intensities of certain ions were 116 apparent. The respective ratios of ions 476, 386, Ion A and Ion B to the M+ (of pure THDOC), in the mass spectrum of the GC steroid peak at the retention time of 3a5B THDOC, for each of the alternative derivatives are shown in Table 4.2. The ion 476:M+ and ion 386:M+ ratios in all four alternative derivatives were greatly raised compared to those seen in the THDOC standard. The relative intensities of the two ions (A and B) resulting from the fragmentation of the D-ring were also altered. The intensity of Ion B relative Ion A was greatly increased (see Ion A:Ion B ratios). The significant presence of ion 476 (resulting from the loss of the methyl oxime or equivalent group) and ion 386 (further loss of a trimethylsilyl group) in all four derivatives suggested that the co-eluting steroid, like THDOC, contained two hydroxyl groups and one carbonyl group. The location of one hydroxyl group outside the D-ring was assumed to be at C-3. The ions that would be expected from the D- ring fragmentation of such a C2i steroid with a 16-hydroxyl and 20-ketone structure are also shown in Table 4.2. Ion D (see structure below Table 4.2) from such a derivatized steroid had the same m/z value as Ion B in THDOC, and was raised relative to M+ (THDOC) in the MO and BO derivatives. Ion D was raised relative to Ion A of THDOC in all four derivatives (see above). The presence of the prominent ion m/z = 188 (but lack of ion 175 found in THDOC) in the MO-TMS derivative limited the position of a hydroxyl group attached to the D-ring to carbon 16, or 17. Significant levels of Ion C (see Table 4.2) in the three of the alternative derivatives, (lower levels in the MO-TMS ether derivative), supported the structure of the side chain, a carbonyl group at C-20 only, and excluded C-17 as the position of the D-ring hydroxyl group. A (3),16-dihydroxy-20-one C 2 1 steroid (16- hydroxypregnanolone) was therefore the most likely candidate for this co-eluting steroid. A further prominent ion of m/z = [Ion C + 14] was seen in the mass spectra of the four alternative derivatives of the pregnancy urine extract. This could have possibly indicated that the D-ring hydroxyl group was at position C-15, and that this ion represented "Ion D" for that structure. The prominence of ion m/z 188 in the MO-TMS derivative, however, did not fit this hypothesis, and suggested rather that the hydroxyl group was at C-16. Furthermore the mass spectrum of the MO-TMS ether derivative of 15-hydroxypregnanolone reported various papers (Gustafsson and Sjovall, 1968; Janne and Vihko, 1970b; Boumot and Ramirez, 1989) did not match 117 the co-eluting steroid in pregnancy. As expected ions 507, 476 and 386 were present in the mass spectrum of 15-hydroxypregnanolone, however the prominence of ions 170, 201 and 241 rather than 188, further confirmed that in pregnancy the hydroxyl group of the co-eluting steroid was not at C-15. An alternative explanation was that there was a further co-eluting steroid in pregnancy at the GC retention time of the main peak of 3a5fi THDOC. When the presence of co-eluting steroids was first detected by raised 476:507 ratios two possible D-ring structures for the steroids co-eluting with the analyte in pregnancy were put forward (see Figure 3.10). The second of these has been shown likely to be present (see above). The first, a C2i steroid with a carbonyl group at C-20 and no hydroxyl group attached to the D-ring would produce the relevant m/z = [Ion C + 14] ion and an ion of the same m/z as Ion C in all four alternative derivatives. The position of any groups outside of the D-ring cannot be deduced from this data. This evidence would therefore indicate that there may be two steroids co-eluting with the main peak of 3a5B THDOC in pregnancy. The levels of 3a5J5 THDOC and its co-eluting steroid were too low in urine extracts from the menstrual cycle for clear mass spectra to be obtainable with the full range of all the alternative derivatives, preventing further confirmation of the structure of that co-eluting steroid. 118 Table 4.1 - Analysis of mass spectra of alternative GC derivatives of THDOC (all as TMS ether derivatives) Ion Ion 476 386 Ion A Ion B Ion A Derivative M+ ★ A B :M+ :M+ :M+ :M+ :Ion B MO 507 175 188 31 2.70 0.66 2.37 3.58 0.66 EO 521 189 202 45 1.95 0.49 6.56 7.87 0.83 HO 566 233 246 89 0.36 0.11 6.77 2.30 2.94 BO 584 251 264 108 2.12 0.40 9.30 5.63 1.65 MO = Methyloxime EO = Ethyloxime HO = Hydroxylamine BO = Benzylhydroxylamine M+ = Molecular ion ★ Methyl oxime or equivalent fragment (see side chain of D-ring structure below) Ions A and B refer to the D-ring structure below CH2-OSi-(CHj)j > Methyl oxime or equivalent fragment — ► Ion A Ion B X = group relevant to respective derivatives (see Figure 3.6) 119 Table 4.2 - Analysis of mass spectra of alternative GC derivatives (all as TMS ether derivatives) of the steroid peak at the GC retention time of 3<*5B THDOC in pregnancy and comparison with ions from a (3), 16- dihydroxy-20-one Q i steroid 476 386 Ion A Ion B Ion A Other Derivative Ion C Ion D :M+ :M+ :M+ :M+ :Ion B ions it MO 90.16 30.10 2.56 27.45 0.09 100® 86 188 EO 11.95 6.62 0.20 1.76 0.11 100 114 100 202 HO 2.35 2.03 0.60 1.11 0.54 145 159 144 246 BO 76.29 12.94 9.56 53.64 0.18 163 177 163 264 MO = Methyloxime EO = Ethyloxime HO = Hydroxylamine BO = Benzylhydroxylamine # Other ions raised relative to M+ (THDOC) @ The relative ion m/z=100 response varied between analyzed samples (MO-TMS); the ion m/z=86 response was relatively small Ions A and B refer to the D-ring structure in Table 4.1 Ions C and D refer to the D-ring structure shown below CH3 v I \ C=N-0-X ^ Ion C 0-Si-(CH3)3 * Ion D X = group relevant to respective derivatives (see Figure 3.6) 120 4.4 Celite columns 4.4.1 Introduction The work in this thesis was undertaken as a result of interest generated by the data in various papers by Winkel, Casey, MacDonald and co-workers, and a number of other groups, who showed evidence for extra-adrenal 21-hydroxylase activity (for references see Introduction). I have shown that what might have been assumed to be exclusively 3a5B THDOC in mass spectral analysis of steroids in urine, may actually in pregnancy be largely one or more co-eluting steroids, probably hydroxypregnanolones. The conversion rate of progesterone to 3a5B THDOC shown by Winkel et al. could be invalidated by the presence of co-eluting steroids, so their method was repeated. The method quoted by Winkel et al. (1980a) was followed as closely as possible up to the final compound, checking the products after each chromatography step by mass spectrometry, rather than using the original method of specific activity and recrystallization. The first two chromatography steps used Celite columns, a form of chromatography used for many years for profiling unconjugated steroids and their metabolites. A summary of the method is shown in Figure 4.4. The details published were rather imprecise in places, so extra experimental work had to be undertaken. Two types of clinical urine sample were used in the experimental work: (i) 1115-hydroxylase deficiency (11150H def.) - so that pure THDOC could be followed through all the steps; (ii) Pregnancy - so that both THDOC and its co-eluting steroid could be monitored. 4.4.2 Experimental work 4.4.2.1 Hydrolysis This was performed overnight or longer at 37 - 45°C, rather than the 3 days at room temperature, and using the enzyme Sulfatase (Sigma, HI) as normally used in the laboratory, the enzyme powder containing both sulfatase and B-glucuronidase activity, rather than pure B-glucuronidase quoted in the Winkel method. 121 Figure 4.4 Summary of Winkelet al. (1980a) method Urine Sample i Hydrolysis i Extraction using ethyl acetate i Chromatography by gradient elution on ethylene glycol- Celite i Chromatography by partition chromatography on Celite using isooctane:t-butanol:methanol:water (25:10:8:7) i Thin Layer chromatography (TLC)-silica gelG using methylene chloride:diethylether (7:3 v/v) i Pyridine/acetic anhydride 1:1 v/v 2hr 37°Cto form acetate i TLC using isooctane:ethyl acetate (1:1 v/v) i Crystallization 4.4.2.2 Extraction Ethyl acetate in approximately the ratio 2 - 3:1 to the aqueous sample was used for extraction and the solvent was evaporated off in stages using a rotary evaporator. Where standards were used, the standard was loaded (in isooctane) onto the Celite column without the hydrolysis or extraction step. 4.4.2.3 Gradient elution chromatography (i) Initial method This was based on that of Siiteri (1963), who developed the method for the separation of oestrogen metabolites from pregnancy urine. Celite was used as the supporting material for the columns. Acid washed Celite (Sigma), was washed further with distilled water, methanol, and diethyl ether before use. The solvents were removed by suction using a Buchner funnel. The diethyl ether remaining in the Celite was evaporated off and the dry Celite stored in an oven (60°C) prior to use to minimise water uptake. Following Siiteri’s method closely, lOg of Celite was mixed thoroughly with 5ml distilled water and gradually loaded into a glass column (approximately 1.6 x 60cm) by first settling the damp powder by tapping the lower end of the column on a rubber pad and then tightly packing with a closely fitting tamper. The final height of each segment was 1 -1 .5 times the column diameter. The next section of the column consisted of 30g Celite mixed with 15ml ethylene glycol packed in the same manner. Ethylene glycol was used as the stationary phase as it has a high affinity for the support material - Celite. The aqueous stationary phase section served as a "trap" to minimise ethylene glycol elution from the column. The sample was mixed with in 2ml of the stationary phase (ethylene glycol), Celite (4g), and an equal or lesser amount of mobile phase (isooctane). This was again loaded onto the column using a foil covered tamper. Mobile phase was then carefully introduced to the top of the column until it emerged from the base, the volume required being the hold back volume (HBV). Two columns of this type were made. The variation in heights of Celite in the columns (543 and 491mm), and the HBV (73 and 64ml), were comparable to those quoted in the Siiteri paper (mean 540mm and 57ml respectively). Flow rates of only 3.4 and 3.8ml/h were however obtained, whereas the published method suggested 123 flow rates of 60 - lOOml/h for gradient elution chromatography. Even with the application of pressure this sort of flow rate could not be obtained, so an alternative had to be adopted. (ii) Adapted method Celite was thoroughly mixed with ethylene glycol (2:1 w/v) and acetone (approximately 100ml to 30g Celite). The acetone was then evaporated off using a rotary evaporator. This achieved a uniform coating of the Celite with the stationary phase. The ethylene glycol coated Celite was then swollen in a beaker with excess isooctane for at least 30 minutes, with frequent mixing. A Pharmacia P-3 pump was used to achieve a gradient mixture of isooctane and ethyl acetate. The experimental set up is shown in Figure 4.5. Pump tubing of internal diameter (I.D.) 3.1mm, connected to capillary tubing (I.D. 1mm), allowed variable flow rates between 60 and > 300ml/h to the top of the column. A starting volume of 900ml pure isooctane was used in all the columns, except the first 20g column that used 600ml (2/3 by direct scale). The volume of mobile phase on the top of the Celite in the column was kept to a minimum, but without letting the column dry out. The proportions of ethyl acetate and isooctane were continually changing, so this methodology ensured that a constant gradient was achieved. Fractions (10ml) were collected in glass tubes using a fraction collector. The typical shape of a gradient elution curve produced from this experimental set up was obtained by spiking the ethyl acetate with radioactivity fH-progesterone) and collecting fractions just prior to the point where the solvent mixture was loaded onto the column. Every fifth fraction was counted in a scintillation counter for 30 minutes, after the solvent had been dried down and 10ml scintillation fluid had been added. The resultant gradient of solvent mixture was near linear, as also shown in Figure 4.5. The first column employed used 20g of Celite. The total slurry of Celite was loaded into the glass column and allowed to settle with gravity. Excess isooctane was run off from the base of the column. Tritiated progesterone and DOC in isooctane (approximately 1ml) were loaded onto the top of the column. The first 60 fractions (10ml each) from the gradient elution were collected. 124 Figure 4.5 - Typical gradient elution curve and experimental set up LU .C < +-» +-• O 126 Table 4.3 - Variation in Celite columns used for gradient elutution chromatography Celite Approximate Height used flow rate Analysis (mm) (g) (ml/h) 20 452* >100 3H-Progesterone/3H-DOC 30 564 100 3H-Progesterone/3H-DOC 30 570 85 3H-Progesterone/3H-THDOC 30 496 95 3H-Progesterone/3H-THDOC 30 511 90 llfiOHdef urine (~215fig THDOC) 30 501 95 Pregnancy urine (1) («95 /-ig THDOC) 30 528 95 Pregnancy urine (2) (*260 fig THDOC) 30 553 85 Pregnancy urine (3) (* 65 fig THDOC) & 3H-THDOC * Non-compacted. Table 4.4 - Variation in Celite columns used for partition chromatography Celite Approximate Height used flow rate Analysis (mm) (g) (ml/h) 20 380 70 3H-DOC 20 380 95 3H-THDOC 20 390 80 3H-THDOC 20 400 95 11 BOH def. THDOC fractions 20 405 90 Pregnancy (2) THDOC (fractions 40-55) 20 385 95 Pregnancy (2) coeluting steroid (70-90) 127 Figure 4.6 - Comparison of elution patterns using 20g and 30g Celite gradient elution columns counts (x1000/20min) 120 20g 100 - 30g 3H-Progesterone 80 - 60 - 40- 3H-DOC 20 - 0 10 20 30 40 60 60 70 80 90 100 Fraction Figure 4.7 - The effect of column height on THDOC elution from gradient elution columns Counts (x1000\20min) 14 - 570mm height 3H-Progesterone - 1■- 496mm height 1 0 - 3H-THDOC 0 10 20 30 40 60 60 70 80 90 100 Fraction 128 urine was available only low levels of radioactivity could be used. The 3H- progesterone, which acted as an extra means of checking reproducibility, was run in total 4 times. The peak of the 3H-progesterone in the 30g columns was seen in fraction 13-15, and in fraction 9 in the 20g column. (iii) Clinical urine samples (a) llft-hydroxylase deficiency CAH The steroids in urine from an 11 BOH deficiency patient (285ml pooled urine calculated to contain * 95 pig THDOC) underwent hydrolysis and extraction as described above. The sample in isooctane was loaded onto a 30g Celite column and 75 x 10ml fractions were collected. An aliquot (0.5ml) was taken from selected fractions and 50ng 3B5a THDOC was added to each. These samples then underwent the Sephadex LH-20 chromatography and derivatization steps described in Materials and methods (Chapter 2). The response ratios of the ions 507 and 476, at the GC retention time of 3a5B THDOC to the internal standard, as measured by SIM, are shown in Figures 4.8 and 4.9 respectively. The maximum of the broad peak was found at fraction 58, which corresponded fairly well with the retention volume determined with the radioactive THDOC described above. Steroid recovery up to this stage (ie, after hydrolysis, extraction and gradient elution) was 46%. 17-hydroxypregnanolone (170HPr) was present in the urine of this patient and also gave an ion 476 response on the SIM runs. This was monitored as the ratio of it s ion 476 response to that of the 3B5a THDOC internal standard added. The 170HPr GC-MS peak has an earlier retention time in the SIM runs, being fully separated from 3a5B THDOC (and any co-eluting steroid), see Figure 2.4. The peak for 170HPr eluted from the gradient elution Celite column was at almost the same retention volume as THDOC starting at only 2 fractions earlier, see Figure 4.10. (b) Pregnancy (third trimester) urine Urine (470ml) was pooled from stored frozen urine aliquots from one pregnant woman (Subject P2), all samples being after gestational week 29. This was called Pregnancy 1. When 2 x 5ml of the pooled urine were analyzed for THDOC, approximately 95pig was calculated to have been present in the 460ml sample that underwent hydrolysis and extraction. This was then loaded onto a prepared Celite 129 Figure 4.8 - Fraction analysis Ion 507 (3a5l3 THDOC) Gradient elution columns Ratio to 50ng IS dotted area 2.5 - 116 OH def. represents 3a58 THDOC peak 0 .5 - Ratio to 100ng IS Pregnancy 1 0.8 - 0.6 - 0.4 - 0.2 - 1.2 Pregnancy 2 0.8 - 0.6 - 0.4 - 0.2 - 1.8 Pregnancy 3 0.9 - 0.6 - 0.3 - 20 30 40 50 60 70 80 90 100 Fraction 130 Figure 4.9 - Fraction analysis Ion 476 (3a5l3 THDOC) Gradient elution columns Ratio to 50ng IS dotted area 2.5 - 11(3 OH def. represents 3a5B THDOC peak 0.5 - Ratio to 100ng IS Pregnancy 1 0.8 0.6 0.4 0.2 6 Pregnancy 2 4.5 12 10 Pregnancy 3 8 6 4 2 0 0 10 20 30 40 50 60 70 80 90 100 Fraction 131 Figure 4.10 - Fraction analysis 17-OH Pregnanolone using ion 476 Gradient elution columns Ratio to 50ug IS 3 .5 3 118 OH def. 2.5 2 1.5 1 0.5 0 Ratio to 100ng IS 40 Pregnancy 1 30 20 10 0 12 - Pregnancy 2 Pregnancy 3 0 10 20 30 40 50 60 70 80 90 100 Fraction 132 Figure 4.11 - Fraction analysis 476:507 ratios Gradient elution columns iatio 10 116 OH def. 8 6 4 3a56 THDOC 2 3B5aTHDOC 0 J______L 10 Pregnancy 1 8 6 4 2 0 J______I______I______I______I______L 80 Pregnancy 2 60 40 20 0 80 Pregnancy 3 60 40 20 j—^ i_____ i i_____ 0 30 40 50 60 70 80 90 100 Fraction gradient elution column. Fractions (86 x 10ml) were collected. Selected aliquots (0.5ml) had lOOng 3B5a THDOC added to them, before undergoing Sephadex LH-20 chromatography and derivatization. A peak of THDOC, as measured by ion 507 and 476 responses, was found at around fraction 74 (see Figures 4.8 and 4.9), which was later than that seen with the tritiated compound and in the 11BOH deficiency patient’s urine sample. However the 170HPr peak (see Figure 4.10) co-eluted with THDOC from the Celite column in the same fashion, starting two fractions earlier, (as seen in the 11 BOH deficiency patient sample), suggesting that there was a shift in elution volume in this column. The ion 476 response ratio to the IS showed a further rise starting at fraction 80. The 476:507 ratio for 3c*5fi THDOC, which at first was at the correct expected level, then rose above that expected for the analyte value (®4), at a similar retention volume, ie, fraction 80 (Figure 4.11). This suggested that a co eluting steroid was possibly partially separating from the analyte of interest. Unfortunately fraction collection had been stopped at fraction 86 and further analysis was not possible with this pooled sample. A further pregnancy sample (Pregnancy 2) was therefore analyzed, calculated to contain 260 fig 3a5B THDOC. This consisted of 1900ml of pooled third trimester normal pregnancy urine sample aliquots from 9 different women. One hundred fractions of 10ml were collected. Analysis of the fractions from this sample also gave a peak of THDOC using ions 507 and 476 responses (Figures 4.8 and 4.9) concurrent with a peak of 170HPr measured using ion 476 (Figure 4.10). This was followed by a rise in both ion 476 and 476:507 ratio, see Figures 4.9 and 4.11. The peak of pure THDOC started earlier than the tritiated compound and that seen in the 11BOH deficiency patient’s sample, peaking at fraction 50 (the 170HPr peak peaking earlier at around fraction 42). This earlier elution is probably due to the fact that 800ml of isooctane was used, instead of 900ml in the gradient elution. Recovery of THDOC, quantified using ion 507, was less than 50%. After MO-TMS derivatization, aliquots of fractions 42, 48, 58 and 72 were run using scan mode on the MSD. Pregnanediols were the most prevalent type of steroid present in the first three of the above fractions, but were still present in at least the same order of magnitude to the co-eluting steroid in fraction 72. The mass spectrum of the co-eluting compound found in fraction 72 corresponded to the 134 differences seen in other pregnancy samples, ie, raised ions 100, 188 and 476 compared to ion 507. The 476:507 ratio of this steroid was 102:1. To confirm that the first peak was pure 3a5B THDOC, and that THDOC was only a minor proportion of, or absent from, the fractions containing the co-eluting steroid, it was decided to chromatograph together a mixture of an extract from pregnancy urine (Pregnancy 3, calculated to contain approximately 65fig THDOC) and 3H-THDOC. This total 24 hour sample (1800ml) came from a patient with mild pre-eclamptic toxaemia (PET). The 3H-THDOC peaked in the same fraction as the ion 507 and 476 responses of the THDOC from the urine, as measured by SIM, the peak being seen at around fraction 52. 170HPr eluted with the analyte peak starting one fraction before the analyte. The co-eluting steroid was again separated from the analyte, see Figures 4.9 and 4.11. Csee pa*}* In later chapters (8^10 and 11) referring to pregnancy, three further hydroxypregnanolone type steroids are discussed. These steroids - Prl, Pr2 and Pr3 - were also measured after gradient elution chromatography in the 3 pregnancy samples and the results from Pregnancy 2 are shown in Figure 4.12. This data indicates that these three steroids eluted from a gradient elution column after THDOC, but concurrently with the latter’s co-eluting steroid, and are included here for completeness. 4.4.2.4 Partition chromatography (i) Method This chromatography was again based on that described by Siiteri (1963). The solvent systems for this next step were prepared by thoroughly mixing isooctane (500ml), t-butanol (200ml, Sigma), methanol (160ml) and distilled water (140ml) in a separating funnel. The two phases were allowed to remain in contact (at least 1 hour) until the upper mobile phase (isooctane and most of the t-butanol) became clear in appearance. Celite (20g) was mixed thoroughly with the stationary phase (10ml) in a glass beaker, and then swollen in excess mobile phase with frequent mixing for at least 30 minutes. The Siiteri method again used the approach of tamping down small quantities of Celite for loading the column (same size as above), but I opted to load small sections (height 1.5 - 3 x column diameter) as a slurry and allow the Celite 135 Figure 4.12 - Fraction analysis Pr1, Pr2 and Pr3 (gradient elution) from Pregnancy 2 using ion 476 Ratio to 100ng IS 20 Pr1 5 0 5 0 35 -r 30 - Pr2 25 - 20 - 10 Pr3 0 10 20 30 40 50 60 70 80 90 100 Fraction 136 to settle by gravity with the excess solvent flowing from the base of the column. Samples or standards were loaded in mobile phase (3 x 1ml) to the top of the column and fractions (5ml) collected using the fraction collector. The column head space was topped up frequently to maintain head pressure to the column, lower flow rates being obtained if the volume of mobile phase above the Celite reduced. The method was first tested using 3H-DOC, which gave a peak at fraction 26. Two columns using 3H-THDOC were then run to determine the retention volume of the analyte, and its reproducibility. The peaks of elution of the 3H-THDOC were determined to be in fractions 26 and 29. The earlier peak fraction was in the column with the higher flow rate and slightly smaller column height. The results from all three columns are shown in Figure 4.13. A summary of the columns used for this type of chromatography are shown in Table 4.4. (ii) Clinical samples (a) llfi-hydroxylase deficiency CAH Fractions 47-75 inclusive from the gradient elution chromatography column were pooled and submitted to partition chromatography. Aliquots (0.5ml) of fractions 18 to 50 were each mixed with lOOng 3B5a THDOC and proceeded to the MO-TMS derivatization steps and SIM analysis. The peak of THDOC elution, as determined by 507 and 476 response, occurred in fraction 31, ie, similar to the tritiated standard (see Figures 4.14 and 4.15). 170HPr elution was also monitored (Figure 4.16), the peak being seen 3 fractions before THDOC. Neither steroid showed a sharp peak, rather a definite rise to a peak followed by slow tailing off. This was also seen to a lesser degree in the 3H-THDOC columns (see Figure 4.13). Using ion 507, recovery was calculated at approximately 55% for this chromatography. GC-MS scan mode runs of fractions 30 and 48 from this column showed pregnanediol, 170HPr and THDOC to be the major steroids present, the former being present in excess of the others. (b) Pregnancy As the co-eluting steroid was partially separating from the analyte it was decided to pool two sets of fractions from the gradient elution chromatography 137 Figure 4.13 - Partition chromatography 3H-THDOC and 3H-DOC 3H-THDOC Counts (x1000/20min) 3H-DOC 16 50 14 — 3H-THDOC O 3H-DOC - 40 12 10 - 30 8 6 - 20 4 2 ©O 0 10 20 30 40 50 60 7080 90 100 Fraction (5ml) 138 Figure 4.14 - Fraction analysis Ion 507 (3a5B THDOC) Partition chromatography Ratio to 100ng IS 4.5 - 116 OH def. dotted area represents 3a5G THDOC peak 2 Pregnancy 2 40-55 0.5 - 0.75 Pregnancy 2 70-90 0.5 - 0.25 - 0 10 20 30 40 50 60 70 80 90 100 Fraction 139 Figure 4.15 - Fraction analysis Ion 476 (3a5l3 THDOC) Partition chromatography Ratio to 100ng IS 3.5 1113 OH def. dotted area represents 2.5 3a5B THDOC peak 0.5 2 Pregnancy 2 4 0 -5 5 0.5 3.5 Pregnancy 2 70-90 2.5 0.5 0 10 20 30 40 50 60 70 80 90 100 Fraction 140 Figure 4.16 - Fraction analysis 17-OHPr (using ion 476) Partition chromatography Ratio to 100ng IS (ion 476) 3.5 113 OH def. 2.5 - 0.5 - 50 Pregnancy 2 40-55 40 - 30 - 20 - 5 Pregnancy 2 70-90 0 5 0 0 10 20 30 40 50 60 70 80 90 100 Fraction 141 Figure 4.17 - Fraction analysis 476:507 ratios Partition chromatography Ratio 0 1113 OH def. 8 *> 3a5(3 THDOC - 3B5a THDOC 6 4 2 0 10 Pregnancy 2 4 0 -5 5 measured on different day Pregnancy 2 70-90 0 10 20 30 40 50 60 70 80 90 100 Fraction 142 column of Pregnancy 2, and to investigate these separately. Fractions 40-55 (pure THDOC) and 70-90 (co-eluting steroid) were selected in order to have the best separation of the two steroids. SIM analysis (ion 507 and 476 responses) of the Pregnancy 2 F40-55 fractions (lml aliquot plus lOOng IS) after partition chromatography and MO-TMS derivatization gave a similar pattern to the tritiated standard and the 116-hydroxylase deficient patient’s sample, with the peak of the THDOC at fraction 32 (Figures 4.14 and 4.15). 170HPr elution peaked in fraction 30, similar to the 116-hydroxylase deficient patient’s sample (Figure 4.16). Tailing off of the analyte was again noted. 476:507 ratios remained constant in all the fractions analyzed, with a very small rise after fraction 76 (see Figure 4.17). The fractions obtained after partition chromatography from Pregnancy 2 F70- 90 gave a very different pattern. The 170HPr peaked in fraction 22, a few fractions earlier than the other samples run on the same system. A small rise in ion 507 and 476 responses were seen two fractions later for pure THDOC as expected, indicating that this was an elution volume shift. The small rise in the GC-MS ion 476 response at the retention time of 3a56 THDOC was followed by a larger rise in ion 476 response in fraction 34 (Figure 4.15). This, like the THDOC in the other columns, did not give a sharp peak, but rather tailed on over the next 64 fractions collected. These results were mirrored in the 476:507 ratio results (Figure 4.17), if anything showing a general upward trend. Peaks of the hydroxypregnanolones Prl, Pr2 and Pr3 were also detected in the fractions from this column and are shown in Figure 4.18. The three steroids were not as clearly separated from THDOC in this chromatography system as they were in the gradient elution chromatography system. Some fractions from the two partition chromatography columns loaded with steroids from extracts of pregnancy urine, were run in scan mode. Pregnancy 2 F40- 55 had very high 170HPr and pregnanediol levels in all the fractions (32, 48 and 68) looked at, 170HPr reducing in fraction 68 compared to pregnanediol. Pure THDOC was detectable in comparatively very small amounts in fraction 32. Pregnanediol was present in larger amounts than the co-eluting steroid in fractions 38, 60 and 90 from the Pregnancy 2 F70-90 sample partition chromatography column. Pure 3a56 143 Figure 4.18 - Fraction analysis Pr1, Pr2 and Pr3 (partition chrom.) from Pregnancy 2 7 0 -90 using ion 476 Ratio to 100ng IS 0 8 Pr 1 6 4 2 0 30 - 25 - Pr 2 20 - 3.5 Pr 3 2.5 - 0.5 - 20 30 40 50 60 70 80 90 100 Fraction 144 THDOC was seen in low quantity in fraction 26 of this column, still being dominated by 170HPr and more so by pregnanediols. The scan analysis of Pregnancy 2 F70-90 fractions allowed the production of a good mass spectrum of what is believed to be the co-eluting compound and this is shown in Figure 4.19. 4.4.2.5 First thin layer chromatography (TLC1 (i) Method Fractions from the partition chromatography were pooled as follows: 22 - 50ml llflO H def. 22 - 50ml Pregnancy 2 40-55 34 - 90ml Pregnancy 2 70-90 One twentieth of the pooled fractions was derivatized and run in scan mode on the GC-MS to determine the nature and amount of steroid loaded onto the TLC plate. The remainder was loaded, with authentic 3a5fi THDOC as the fourth spot, 4cm apart, 2cm from the base, onto a precoated Silica G thin layer chromatography plate (20 x 20cm, 0.25mm thickness, from Merck, Darmstadt, Germany). One hundred millilitres of dichloromethane: diethyl ether (7:3 v/v) was vigorously shaken in the chromatography tank, and then left to equiliberate for 10 minutes. The plate was then run (approximately 70 minutes) in the solvent, until the solvent front was approximately 1cm from the top of the plate. After complete evaporation of the solvent off the plate at room temperature the plate was inspected under UV light. Sections of the plate were scraped off and thoroughly mixed with 100% ethanol and allowed to stand for 60 hours. An aliquot of between 1/10 and 1/4 of the total was used for identification of the steroids present and approximate quantification of THDOC in each section. (ii) Results Inspection of the steroids present in the pooled samples that were loaded onto the plate revealed the following: (a) 1113 OH def. - Approximately 6/xg of THDOC was loaded onto the plate, along with 170HPr and large quantities of pregnanediol (>350/*g, which was * 60 x THDOC present in the extract). 145 Figure 4.19 - Partial mass spectrum (m/z = 98 - 520) of the MO-TMS ether derivative of the steroid co-eluting in pregnancy with THDOC 7.0E5 47G G.0E5 5.0E5 188 4.0E5 100 i 3.0E5 3BG 2.0E5 492 200 500 146 (b) Pregnancy 2 40-55 - Approximately 22/xg of THDOC was loaded onto the plate, along with >5 times the amount of 170HPr, and relatively enormous amounts of pregnanediol (> 100 x THDOC). (c) Pregnancy 2 70-90 - Approximately 180/xg of the co-eluting steroid was loaded onto the plate, along lesser amounts of 170HPr («1/3), a similar quantity of pregnanetriol and about twice the quantity of pregnanediol. Some other steroids of lesser interest were also present. After running the TLC plate and inspection under UV light only a very faint spot was seen in the lane with the THDOC standard. In a second plate 10 times the quantity of standard (500/xg) was run under identical conditions to confirm the Rf of the analyte, where Rf = Distance of steroid of interest from the starting point Distance of solvent front from the starting point The standard was located between Rf values 0.48 and 0.56. Two UV absorbing spots were seen for the 1 IB OH deficiency sample at Rf = 0.54 - 0.56 and 0.36 - 0.40. The two pregnancy pooled fraction samples contained pink and brown pigments, which obscured any UV spots present. Some tailing of the pigment was noted suggesting that the steroids were doing the same, so it was decided to inspect small sections (Rf 0.06 wide) either side of the section thought to contain the standard for the presence of the analyte. The sections off the plate are shown in Figure 4.20. Internal standard (lOOng) was added to aliquots of each section and SIM runs (ions 476 and 507) were analyzed and THDOC quantified. The quantity of THDOC present in each section is also shown in Figure 4.20. Most of the detectable standard (78%) was found in Section B, showing that some tailing had occurred. However this detectable standard only represents approximately 35% of that loaded onto the plate. Some of the sections were also analyzed using scan runs: (a) 1113 OH def. - Both sections E and F contained pregnanediol in quantities in excess of the analyte ( » 25 and 8 times respectively). The fact that there was more THDOC in section F than section E suggests there had been some overloading of the plate. 147 Figure 4.20 - Sections and results off TLC plate 1 Numbers represent pg THDOC as measured using ion 507 0.95 H M <0.05 <0.05 <0.05 <0.05 0.64 012 0.\1 0.58 B 127.6 0.3 0.3 OH 0.45 0.5 3.3 35.6 5.3 K 0.39 1.5 0 . 1 0.3 <0.05 0.1 o O O O Preg 2 Preg 2 Rf value Std 11BOH def 40-55 70-90 148 (b) Pregnancy 2 40-55 - Sections J and L contained pregnanediol and 170HPr in quantities greatly in excess of the THDOC, in ratios fairly comparable to before running on the TLC plate. A small amount of the co-eluting steroid was also present in section L. Section H revealed no THDOC, but still contained a large amount of pregnanediol. 170HPr was lower compared to pregnanediol than in the other two sections. (c) Pregnancy 2 70-90 - A small amount of THDOC was detected in section O. The 476:507 ratio (<4) suggested that this was almost free of the co eluting steroid. Pregnanediol and 170HPr were present in large amounts, comparable to section J. Section Q contained the "co-eluting steroid" as the dominant steroid, with its 476:507 ratio >40. Pregnanediol was present at approximately half the quantity of the co-eluting steroid. Pregnanetriol and 170HPr were also detected along with some other steroids of lesser interest. 4.5.2.6 Second TLC (separation of diacetatesl (i) Method The residues of the following sections of TLC plate 1 were pooled for loading onto the next TLC plate: Standard B 11J3 OH def. E + F Pregnancy 2 40-55 J + K Pregnancy 2 70-90 O + P Pregnancy 2 70-90 Q (for co-eluting steroid) The ethanol was evaporated completely off under nitrogen. The steroids were then acetylated by reaction with acetic anhydride:pyridine (1:1 v/v, 300/d), for two hours at 37°C. The pyridine was present as a catalyst. Differential acetylation of steroids occurs due to steric hindrance of hydroxyl groups, 1 IB, 15B, 12 and tertiary hydroxyl groups, for example, failing to acetylate (Bush, 1961). All excess reagent was then evaporated off under nitrogen and 75/cl of dichloromethane was added, in which the steroids were loaded onto a Silica G TLC plate. The plate was run (approximately 90 min) in a chromatography tank containing 100mlisooctane: ethyl acetate (1:1 v/v). 149 After complete evaporation of the solvent, the plate was inspected under UV light. Sections of the silica were scraped off the plate and thoroughly mixed with 5ml dichloromethane and left to stand overnight. Half of the supernatant was dried down and the steroids reconstituted in 1ml of ethyl acetate. To this, 1ml of saturated NaHC03 was added and thoroughly shaken. The two layers were allowed to separate and the lower aqueous layer was discarded. The ethyl acetate was then evaporated off and the steroids underwent derivatization after the addition of lOOng 3B5c* THDOC. (ii) Results Inspection of the TLC plate under UV light revealed several visible spots. These are shown in Figure 4.21 along with the sections scraped off the plate. The spot of standard THDOC diacetate was very faint, so a second plate under identical conditions was run with 5 times the quantity of THDOC diacetate (500/xg THDOC before acetylation) to confirm the position of the analyte. The mass spectra of the acetate derivatives of THDOC, PD and 170HPr, (produced from the free steroids by the method described above); were determined by scan runs are shown in Figures 4.22 - 4.24. Approximately equivalent amounts of non-acetylated and acetylated 170HPr were found in the MO-TMS ether derivative, whereas the PD and THDOC showed complete acetylation. The ion 416 was chosen for SIM analysis for THDOC diacetate, along with ion 476 for the detection of the added internal standard. THDOC diacetate was found to have a retention time after that of the internal standard in SIM runs, and was detected clearly in the standard (section 2B). No ion 476 from 3a5B THDOC was detected, showing that the attempt to remove the acetate groups with sodium hydrogen carbonate had not worked. Section 2C revealed a very small amount (less than 2% of that found in section 2B) of the THDOC diacetate. The sections (2E, 21 and 2M) from the 1 IB OH deficiency patient’s and pregnancy extracts also contained low level detectable 416 peaks, in varying degrees of magnitude, at the correct retention times. Scan runs of some of the sections revealed the following: Section 2B (Standard) - THDOC diacetate detected Section 2E (section with Rf of standard in 11BOH def.) - THDOC diacetate was just detectable, along with some PD diacetate and a lesser amount of 170HPr acetate. 150 Figure 4.21 - Sections and results off TLC plate 2 0.97 2A 2D 2H 2L 2P 0.85 2B2E 2M 2Q 0.73 2 JO 2N: 2R2C 0.59 2G 2K 20 2S O o 0.06 Preg 2 Preg 2 Rf value Std 11B0H def co-eluting 40-55 70-90 steroid 151 derivative of THDOC diacetate 2.5E5 145 158 2.0E5 241 298 358 460 ce 5.0E4 300 400 500 Mass/Ch arge Figure 4.23 - Partial mass spectrum (m/z = 98 - 347) of the MO-TMS ether derivative of pregnanediol diacetate 5.0E5 284 4.0E5 3.0E5 216 230 107 344 2.0E5 147 174 288 £ 1.0E5 150 200 250 300 Mass/Charge Figure 4.24 - Partial mass spectrum (m/z = 98 - 480) of the MO-TMS ether derivative of 17-hydroxypregnanolone acetate 1 .0E6 46 8.0E5 188 6.0E5 334 T 3 255105 4.0E5 _Q 2.0E5 386 100 200 300 400 Mass/Ch arge 152 Section 21 (section with Rf of standard in Pregnancy 2 F40-55) - THDOC diacetate was too low to detect in this mode. However two prominent peaks whose ions correspond to those of 170HPr acetate and PD diacetate were found. Section 2J (section with Rf just below that of standard in Pregnancy 2 F40-55) - THDOC diacetate (or the co-eluting steroid in diacetate form, as they would probably not be distinguishable as diacetates) was detected along with very large amounts of 170HPr acetate and a small quantity of non-acetylated 170HPr. PD diacetate was not present in any great amount. Section 2M (section with Rf of standard in Pregnancy 2 F70-90) - THDOC diacetate was too low to be detected. PD diacetate was the most prominent peak along with 170HPr acetate (approximately 1/4 that of PD diacetate). 4.5 Discussion The separation of the co-eluting steroid in pregnancy proved to be formidable. Immunoadsorption, a large change in GC conditions, and the use of alternative GC derivatives did not allow separation of the steroid from THDOC. Immunoadsorption might have achieved selective uptake of related steroid. The method was shown to work for pregnanediol. The adsorption of PD is consistent with the cross reaction of 20-dihydroprogesterone with the progesterone antiserum. Although the progesterone antiserum tried was expected to bind to the side chain of the co-eluting steroid in pregnancy, the binding may have been reduced by the of -rite fact presence of a hydroxyl group (presumed to be at the C-16 position) or by virtue^that the A and B rings were saturated. It is not clear why immunoadsorption using THDOC antisera was not successful, but only one antiserum was tested, which had not previously been subjected to the conditions needed to make an immunoadsorbent. Even with the increase in already high values of theoretical plates, separation on GC of THDOC and its co-eluting steroids was not achieved. This showed the importance of the further specificity that mass spectrometry with multiple ion detection offers this situation to show the presence of the co-eluting steroid. Alternative GC derivatives did not separate the two co-eluting steroids found with MO-TMS, but offered further clues into the identification of the co-eluting steroids (see Section 4.3 and below). 153 Use of the Celite gradient elution chromatography system, which separates steroids by partition and polarity, finally allowed a fair degree of separation of the co-eluting steroids from THDOC, although, since the number of theoretical plates for this type of column is only in the region of 200, the peaks were diffuse. The gradient elution columns were difficult to manufacture in a consistent manner, as there was a fine balance between over-tamping the stationary phase coated Celite, resulting in columns with too slow flow rates, and not being able to fit the 30g of Celite into the size of glass column recommended. The reproducibility of column heights (496 - 570mm, n = 7, CV = 6%) and flow rates (85 - lOOml/h, CV = 6%) was similar to that quoted by Siiteri for HBV (3%, n = 10). Variation in column height made a difference in the elution volume, adding to possible errors in pooling of the fractions for the next step of chromatography and to the already high losses of the method. Variation in the fraction with the peak elution of the analyte (CV = 6%, n = 5, if Pregnancy 1 is ignored as an outlier) was also similar to those quoted by Siiteri (CV = 4 - 5%, n = 10). Winkel et al. state that after this first chromatographic step THDOC was contaminated by 3H-pregnanediol (ie, a direct metabolite of the 3H-progesterone administered to their subjects). This fits with the large amounts of PD seen using GC-MS. Partition chromatography offered poorer separation of the co-eluting steroid from THDOC, but as most of the co-eluting steroid was removed with appropriate fraction rejection after the first column chromatography, this was not so important. Pregnanediol however, whether at high (pregnancy) or low (1113 OH deficiency) concentrations, was not "separated from THDOC during this second column chromatographic procedure" as quoted by Winkel et al. (1980a, footnote below Table II on page 806 of the paper). Similar numbers of theoretical plates were found for this chromatography step as the gradient elution chromatography. As pregnanediol was found in all sections inspected of the first TLC plate loaded with sample, this method of chromatography was unsuitable for separation of this steroid metabolite. 170HPr also co-migrated with THDOC, with considerable tailing, so again little was gained in removal of this steroid. Pregnanediol diacetate, in the second TLC step, also co-migrated with THDOC diacetate, but overall showed better separation on this plate than on the first TLC plate. Little PD diacetate Was .seen 154 the section thought to contain THDOC diacetate. 170HPr acetate also co-migrated with THDOC diacetate, but was found in addition at Rf values less than the analyte. The number of theoretical plates, as calculated for the standards, on the two TLC plates was higher than the Celite chromatography steps at 670 and 550 respectively. These values could be lower for the mixtures of compounds seen in the extracts, in particular in pregnancy, as excess material may affect the chromatography and change the rate of migration. Recoveries were low if the total method was considered, less than 5% after the 6 steps (hydrolysis, extraction and 4 chromatography). This suggests this methodology is not suitable for direct quantification methods, but rather, as Siiteri and Winkel employed, methods that measure the ratios of two different isotopes fH and 14C). PD and 170HPr were present in all the fractions retained for THDOC, if anything being retained in preference to THDOC. This suggests that purification of THDOC was not as complete as thought by Winkel et al.. The method did however allow almost total separation of the co-eluting steroids from THDOC in pregnancy. From the evidence available these steroids were thought to be a 3,16-dihydroxypregnane-20-one, and a C2l steroid with the D-ring structure of progesterone. The use of alternative GC derivatives information would fit these structures (see Section 4.3). The mass spectrum found for the MO-TMS ether derivative of the co-eluting steroids (Figure 4.19) was similar to (i) that shown for 313,16a-dihydroxy-5 a-pregnane-20-one shown in a paper by Boumot and Ramirez (1989), with the addition of ion 100 in the mass spectrum presented here, and (ii) and a standard of 3B,16a-dihydroxypregnane-20-one (not known if it was 5a or 5B) analyzed on the GC-MS. The latter eluted from the GC column at the approximate retention time of the second peak for 3a5fl THDOC and the additional hydroxypregnanolone Pr3 (MU = 29.25) in both SIM and scan runs on the mass spectrometer, and is similar to that quoted by the former reference. Using SE-30 coated capillary columns Janne and Vihko (1970a) showed the relative retention times of 3,16-dihydroxy-5-pregnane-20-one isomers as to be as follows: 3a5B < 3B5B < 3a5a < 3115a 155 My work was performed on OV-1 type coating, but similar relative retention time values are obtained from capillary columns usingAnon-polar coatings with methyl silicone gums, such as SE-30 (Shackleton, 1985a). With further information on the retention times of 3a5B THDOC (Janne and Vihko, 1970a) the peak containing 3a5B THDOC and its co-eluting steroid was found to fall between the middle two of the 4 isomers quoted above, nearest to the 3a5a isomer, strongly suggesting the 3a5a isomer as the candidate for the 16-hydroxylated co-eluting steroid in pregnancy. In addition Anderson et al. (1990) in deuterium labelled progesterone metabolism studies showed that 50% of the metabolites of the compound were in the 3a5a configuration. The isolation of the co-eluting steroids in pregnancy has shown that quantification using ion 507 response in a SIM run was close to being accurate for pure 3a5B THDOC, as the ratio of 476:507 ion response in the isolated co-eluting steroid was approximately 100:1. The co-eluting steroids’ relative contribution would therefore only be around 1 % if present in equal quantities to 3a5B THDOC in an extract. If only the ion 476 response had been used for quantification very inaccurate results for THDOC excretion rates would have been obtained, again emphasising the importance in the selection of ions for SIM quantification. In conclusion,two steroids were thought to co-elute with THDOC (ie. had the same relative GC retention time) in pregnancy. One was thought to be (3), 16- dihydroxypregnan-20-one, whilst the other was tentatively identified as a C2i steroid (possibly a hydroxypregnanolone) with a D-ring and side chain structure similar to progesterone. 156 Clinical Results The following seven chapters investigate urinary steroid excretion, in particular THDOC excretion, in various clinical groups: Chapter 5 - Normal subjects and the menstrual cycle Chapter 6 - 1 IB-hydroxylase deficiency CAH Chapter 7 - Mineralocorticoid secreting tumour Chapter 8 - Normal pregnancy Chapter 9 - Placental sulphatase deficiency Chapter 10 - Pre-eclamptic toxaemia (PET) and hypertension in pregnancy Chapter 11 - Raised Progesterone in (a) pregnant and (b) non pregnant subjects, including 17- and 21-hydroxylase deficiency CAH These clinical situations were chosen for one or more of the following reasons: (i) DOC production was known to be raised (ii) The precursor, progesterone, was known to be raised (iii) To investigate if DOC production in pregnancy, was reduced in the absence of normal oestrogen production (iv) The clinical group was known to have hypertension (v) Unusual metabolites were present allowing validation of the method 157 5 - Normal subjects and the menstrual cycle 5.1 Introduction Urine samples from normal non-pregnant subjects were analyzed to obtain reference values for steroid excretion rates with which to compare various physiological and clinical situations. Several groups of subjects were considered - adult males, adult females in the luteal phase of the menstrual cycle, females in the follicular phase and those taking oral contraceptives, and children. Excretion rates in the laboratory were normally expressed in p.%!24h. The influence of body surface area (BSA) on the results was considered, along with expressing excretion rates with respect to creatinine excretion. The latter was also used in assessing the accuracy of an early morning sample (EMS) compared to a 24 hour urine collection, in an attempt to use an EMS for ease of collection for subjects involved in the studies. 5.2 Experimental Urinary steroid profiles and THDOC excretion (using 150ng IS) were analyzed by the methods described in Chapter 2. Creatinine was measured by standard methods based on the kinetic Jaffe reaction using a discrete auto analyzer (Department of Chemical Pathology). 5.3 Subjects and Results 5.3.1 Adult males The steroid excretion rates of ten healthy adult males (aged 19 - 45 years) as quantified from urinary steroid profiles is shown in Table 5.1. THDOC excretion rates quantified using both ions 476 and 507 (range = 15.3 - 47.7 and 9.1 - 48.1 /xg/24h respectively) are shown in Figure 5.1. All subjects, except Subject N8, showed 476:507 ratios (3.6 - 6.9) greater than the pure standard (ratio * 3). 158 5.3.2 Adult females and the menstrual cycle Urinary steroid metabolite excretion rates for five women in the follicular phase and five women taking oral contraceptives are shown in Table 5.2. No difference was seen between the excretion rates of the two groups, except for a-THF excretion was lower in the oral contraceptive group (see section 5.3.6 below), so they were considered one group for this analysis. THDOC excretion rates for ions 476 and 507 (range = 5.8-35.5 and 1.8 - 17.5 /xg/24h respectively) are shown in Figure 5.2. 476:507 ratios were raised above that expected for the pure standard in the follicular phase (range = 6.7 - 8.6, median = 7.9), as were the 476:507 ratios for women taking oral contraceptives, though significantly lower, p<0.01 (range = 4.4 - 5.2, median = 4.6). Table 5.3 shows the results of urinary steroid excretion in six women in the luteal phase of the menstrual cycle, with three samples from one subject (N18). Four of these women had regular 28 day cycles, and the other two had 34 and 23 day cycles. The day in the cycle for each of the samples is indicated. THDOC excretion rates for ions 476 and 507 (range = 57.5 - 260.0 and 16.1 - 83.3 /xg/24h respectively) are shown in Figure 5.3. 476:507 ratios were considerably raised (range = 6.7 - 16.5, median = 10.6) above the pure standard, and higher than most of the follicular phase results (p<0.25). Subject N18 collected 5 samples in total, four from one 28 day menstrual cycle (N18A-C, plus one on day 27 of the cycle not shown in Tables 5.2 or 5.3). THDOC excretion in this menstrual cycle is shown in Figure 5.4 for both ions 476 and 507. THDOC excretion rose rapidly between days 7 and 14, and then decreased at a slower rate over the next 13 days, with THDOC quantified using ion 507 following approximately the same pattern as ion 476 quantified THDOC, but at a lower level. 476:507 ratios varied considerably over the 28 day period - 7.5, 11.7, 16.5 and 11.7 for days 7, 14, 21 and 27 respectively. 5.3.3 Children Many of the patients with CAH who were investigated, see Chapters 6 and 10, were children. In order to establish approximate ranges of THDOC excretion appropriate for subjects of smaller body size than adults, a number of urine samples from children sent into the laboratory for urinary steroid profiles were analyzed. 159 Table 5.1 - Urinary steroid excretion in adult males (jjlg/24 hours) « O CQ e o 110H 110H 160H And’ e a- total Subject Age And Aet DHA PD PT THE THF And Aet DHA triol cort cort. 096 § o 00 00 o N1 24 1 1170 <50 740 200 180 100 350 490 600 610 670 710 009 O © 099 o ON N2 30 1240 1670 650 310 360 n 310 3330 1390 1100 1010 7490 N3 ON 1670 1600 260 780 280 240 150 200 290 2920 1130 910 360 1230 6550 09 © o N4 34 1070 780 1110 450 400 160 330 1790 820 610 640 750 4610 cs 099 N5 ON 1350 2030 210 610 180 260 520 250 2390 840 380 1370 5640 006 v~> 8 N6 o c 'pH 1300 1150 < 50 680 <50 130 220 250 430 1810 1040 550 800 00 N7 45 O o 870 240 670 290 230 100 300 290 1900 940 630 480 840 4790 009 o z 00 o 38 1040 760 630 cn 220 110 290 1860 950 770 410 810 4800 VO 00 © N9 1060 930 210 610 150 230 90 290 500 2550 1420 950 860 1030 N10 CO 1370 620 VO o 590 120 310 80 130 500 1600 920 860 220 410 4010 00 o 066 ON o median CO 1200 940 210 630 160 230 270 390 640 650 870 4950 O 45 1620 2030 1110 780 310 400 600 3330 1420 910 1100 1370 7490 w-4cn © 069 mean CO 1230 1140 310 640 00 © 260 260 380 2200 1010 620 910 5430 b I 1 I n c o § 160 Table 5.2 - Urinary steroid excretion in adult females in the follicular phase or taking oral conraceptives (/xg/24 hours) 8 3 110H 110H 160H And’ . Be & total Subject Age And Aet DHA PD PT THE THF ts And Aet DHA triol Be cort 06 (N vo 099 N il* 910 190 140 80 280 140 350 1220 740 250 470 370 3050 | o 096 CO 00 CN o N12* 00 840 780 50 390 200 410 110 1810 390 460 510 4130 09 06 CO r- ■'3-o VO o CO * CO 30 410 650 50 o 130 70 320 1180 700 1000 700 j t * 8 - 28 260 270 <50 140 <50 <50 <50 100 1110 560 300 310 350 2630 09 * 09 n c 580 830 230 200 230 100 350 1040 440 170 460 640 2750 N16 34 650 450 310 300 <50 260 06 110 150 980 720 520 390 610 3220 o o o o N17 n c 460 470 <50 350 140 130 <50 170 110 2080 640 1330 1090 6220 N18A 30 630 360 <50 280 170 © 06 80 190 1070 570 550 210 290 2690 00 © N19 29 1250 1420 100 150 140 430 260 110 1410 800 400 390 410 3410 00 00 © \ C © N20 n c 1210 1390 680 390 200 310 210 310 3170 1610 530 1100 7300 06 VO © n c © median 650 720 80 840 CN © 280 170 1260 880 400 470 560 3320 © © ss s ^ V 26- 260- 270- <50- <50- 80- <50- ■ 100- 980- 440- 160- 210- 410- 2630- range 34 1250 1420 680 200 430 190 350 3170 1610 640 1330 1100 7300 mean 30 700 750 180 250 CN © 200 100 150 210 1510 820 390 590 610 3910 161 Table 5.3 - Urinary steroid excretion in adult females in the luteal phase (/xg/24 hours) «Q * CQ O Subject 110H 110H 160H And’ a - total Age And Aet DHA PD PT THE THF o n And Aet DHA triol cort cort o 006 n c N18B 14 580 300 50 330 8 260 590 160 210 440 480 160 220 2190 | 066 n c o CN 0 0 N18C 21 30 640 350 50 320 170 550 1350 310 200 470 490 190 250 o « 0£I£ | o o N21 24 32 1270 970 970 570 CN 2110 470 150 1410 530 280 400 510 N22 22 33 1040 1130 400 VO 0 0 o 200 480 2060 480 200 3240 1180 510 630 1070 6630 N23 25 26 820 1080 o 120 190 o 340 390 360 1200 550 260 320 390 2720 CN o CN N18D 16 30 760 560 70 450 410 480 1280 380 410 1130 430 370 300 290 06 s N24 30(34) 39 500 1150 650 1230 1160 3000 440 2740 1540 530 610 670 6090 N25 15(23) 5 490 970 140 780 390 160 069 380 70 1310 810 550 370 610 3450 m O o median 680 970 130 510 n 370 1320 390 210 1260 680 490 350 450 2930 n i on © o i 26- 490- 300- 50- 120- 100- n i 340- 160- 70- 900- 550- 260- 160- 220- 2190- range 44 1270 1150 970 1230 1160 3000 480 410 3240 1540 550 630 1070 6630 mean 33 760 810 310 560 340 330 1430 380 220 1620 740 430 370 500 3640 <4H B •3 l l CN 00 73 4 o o pn o ° I fi ° <0 S -» g S | | ? S H *2 ** f ? f Q 8 cqmH J £ * d O B S* 8 I I I I II II II II II < 8 is £ 2 2 •2 X 2 X 2 162 Table 5.4 - Urinary steroid excretion in children (/xg/24 hours) op CN VO vo VO 00 o oo o CN in in O o Ov o 00 cn o o VO o CN 00 o in o o Ov © n c o CN OV in o n c 00 VO o o Ov o CN t"- o 00 o f O f O in o in o o n c 00 O v O n c in m r- O CN CN f O oo o 00 tj o CN o Ov o o o CN - f O f O f O f O v O oo CN CN in n c © oo CN o o CN Tt o 00 NCN CN CN n c oo o in O VO oo m o o o CN CN n c v O 00 Tt"oo 00 CN CN o CN CN O ^ VO O ON VO o o ' ' t 68 p o CN o CN ov cn r- o n c VO x> 3 *8 ■a H in n c O O VH O V 8 g § $ © h n -» i 163 Figure 5.1 - THDOC excretion rates Males THDOC (pg/24h) 50 1------ N1 N2 N3 N4 N5 N6 N7 N8 N9 N10 164 Figure 5.2 - THDOC excretion rates Females in follicular phase or taking oral contraceptives (*) THDOC (iig/24h) 300 WMk Ion 476 250- Ion 507 200 - 150 - 100 - 50 - HfH mm mpi\ mv- mr~ J N11* N12* N13* N14* N15* N16 N17 N18A N19 N20 Figure 5.3 -THDOC excretion rates Females in luteal phase THDOC (ug/24h) 300 i------ N18B N18C N21 N22 N23 N18D N24 N25 Figure 5.4 - THDOC excretion rates Changes in a normal menstrual cycle (Subject N18) THDOC (pg/24h) 100 on 476 8 0 6 0 - 4 0 - 20 - D7 D14 D21 D 27 166 Figure 5.5 - THDOC excretion rates Children THDOC (ug/24h) 50 H Ion 476 40 - EH3 Ion 507 30- 20- UlTil HI i l WiM. N26 N27 N28 N29 N30 N31 N32 N33 N34 N35 N36 Males Females 167 These samples were selected as the adrenal function of these children was appropriate for age or body surface area, "normal profile" being sent as the result to the requesting clinician. Urinary steroid excretion rates are shown in Table 5.4. THDOC excretion is shown in Figure 5.5 for ions 476 and 507 (range = 5.2 - 22.3 and 1.9 - 20.0 /xg/24h respectively). 476:507 ratios were slightly raised in all 11 children (3.3 - 6.6) above the pure standard (« 3). No direct correlation of age with THDOC excretion was seen, and all values were equivalent to the adult female follicular phase range. 5.3.4 Intra-subject variation Two of the normal adult subjects provided many further 24 hour urine collections, N2 the male with the highest values for urine cortisol metabolites, and N il a female taking oral contraceptives (n = 10 and n = 8 respectively). Each sample was analyzed for THDOC excretion and had urinary steroid profile analysis performed on it. The results are shown in Figures 5.6 and 5.7, and Table 5.5. Excretion rates of the various steroid metabolites measured in a urinary steroid profile showed coefficients of variance (CV) between 13 and 55% (mean = 25%). THDOC excretion rates had a CV of 18% and 21 % (Subject N2) and 13% and 24% (Subject N il) for ions 476 and 507 respectively. Ion 507 produced ranges of 25.3 - 48.3 and 3.3 - 6.8 jng/24h, and ion 476 ranges of 39.4 - 67.0 and 12.8 - 18.2 /-cg/24h for Subjects N2 and N11 respectively. 476:507 ratios were raised at 4.1 - 7.7 (Subject N2) and 4.5 - 6.8 (Subject N il) as compared to the pure standard (« 3 ). 5.3.5 Pregnanediol:THDOC ratios Pregnanediol (PD) excretion rates of adult males were in the same range (< 250/xg/24h) as females in the follicular phase and those taking oral contraceptives (see Tables 5.1 and 5.2). Women in the luteal phase of the menstrual cycle had statistically higher (p<0.05) PD excretion rates of between 340 and 3000jng/24h. THDOC in the luteal phase was also statistically higher (p<0.05) than in the follicular phase, the women taking oral contraceptives and the males. PD:THDOC ratios in the luteal phase were again statistically significantly higher (p<0.05) than the other three groups, although there was overlap between the luteal phase and the other female results (Figure 5.8). 168 Figure 5.6 - THDOC excretion rates Intra-subject variation Male THDOC (ug/24h) 70 n------ 123456789 10 Figure 5.7 - THDOC excretion rates Intra-subject variation Female taking oral contraceptive THDOC (yg/24h) 70 HI Ion 476 60 - 111 Ion 507 50- 40- 30 - 2 0 - 10 - 1 2 3 4 5 6 7 8 169 Table 5.5 - Intra-subject variation in steroid excretion (jig /24 hours) Adult male n= 10 samples Female n= 8 samples (on o.c.) Variable Range Median cv% Range Median CV% And 1110-2040 1590 17 450-800 710 16 Aet 1680-2580 1850 15 850-1650 1140 23 DHA 260-880 600 33 260-730 460 35 llOHAnd 640-1120 850 21 90-260 180 33 11 OH Aet 380-1000 630 31 60-220 140 41 160HDHA 150-400 280 28 140-430 290 30 PD , 100-250 180 25 80-170 110 32 PT 280-540 410 30 70-390 180 55 And’triol 390-560 470 13 240-380 300 15 THE 2080-3470 2950 18 830-1780 1130 31 THF 960-1960 1660 21 360-910 580 36 aTHF 680-1650 1040 28 90-200 120 27 or-cort 410-700 530 21 280-550 530 25 6c & 6c 190-540 330 32 170-370 250 22 total cort. 4790-7770 6390 18 2070-3460 2530 21 Urine 1010-3660 2040 41 930-1590 1210 12 volume (ml) For abbreviations see Table 5.3 170 Figure 5.8 - PD/THDOC Normal adults (THDOC using Ion 507) PD/THDOC 90 8 0 - 70 - 60 50 4 0 H 30 20 - 10 - 0 male female female female oc. foil. luteal n-10 n-5 n-5 n-8 171 5.3.6 Cortisol metabolites Total cortisol metabolites (ie. the total of THE, THF, aTHF, a-cortol, B- cortol and B-cortolone) excretion rates were not statistically different between the males, luteal phase females and follicular phase and oral contraceptive taking females (Tables 5.1-3). THE to THF ratios were also not significantly different (Figure 5.9) with a mean of 2.0. THF(5B) to 5a-THF ratios, with the exception of the subjects taking oral contraceptives, were not statistically different (Figure 5.10), with a mean of 1.6. The five subjects taking oral contraceptives (and the further eight samples from subject N il) showed a higher THF: aTHF ratio (p<0.05), the 5a metabolite being lower in comparison to normal in this clinical state. Interestingly, the androsterone (5a) to aetiocholanolone (5B) ratio was also lower in the oral contraceptive taking group (range = 0.63 - 1.08, median = 0.73) than in the other adult groups - follicular phase (0.87 - 1.75, median = 0.98), luteal phase (0.44 - 1.93, median = 1.12) and males (0.67 - 2.21, median = 1.18). 5.3.7 Influence of body surface area on excretion rate Body surface area was calculated from nomograms of height and weight in all of the males and 10 of the female (a mixture of luteal phase, follicular phase and those taking oral contraceptives) subjects (Table 5.6). Comparison of steroid excretion rates corrected for BSA excretion rates between the two sexes revealed differences in only (i) higher PD excretion range in the females, due to the menstrual progesterone surge, and (ii) the androgen metabolites androsterone, aetiocholanolone and androstenetriol in the males were in the upper half of the range seen in the female subjects (androsterone p< 0.2, aetiocholanolone p< 0.6, androstenetriol p<0.05). When uncorrected excretion rates were compared^the same steroids differed between the sexes, at more statistically significant levels (androsterone p < 0.005, aetiocholanolone p<0.15, androstenetriol p<0.1), plus the ranges of the cortisol metabolites in the males were shifted to the upper end of the female range (p<0.1). Correction for BSA resulted in similar ranges (p<0.5). It was interesting to note however that there was no clear correlation of body surface area to cortisol excretion in the subjects studied, one of the smallest females, for example, having a greater cortisol excretion than the female with the largest BSA. 172 Figure 5.9 - THE/THF Normal adults range and median THE/THF 3.5- 2.5- 1.5- 0.5- n-10 n-5 n-5 n-8 n-10 n-8 male female female female male female menstrual oc. foil. luteal oc. cycle Inter-subject variation Figure 5.10 - THF/aTHF Normal adults range and median THF/aTHF 8 7 6 5 4 3 2 1 n-10 n-5 n-5n-8 n-10 n-8 n-4 0 male female female female male female menstrual oc. foil. luteal oc. cycle Inter-subject variation 173 Table 5.6 - Steroid excretion (ftg/24 hours) corrected for body surface area (BSA) Adult males (n= 10) Adult females (n= 10) Steroid Corrected for BSA *tg/24h Corrected for /tg/24h /ig/m2/24h BSA jig/m2/24h And 540-860 1040-1620 180-830 260-1270 Aet 360-1190 620-2030 180-940 270-1390 DHA <50-600 <50-1100 <50-630 <50-970 llOHAnd 240-420 450-780 80-400 130-680 11 OH Aet <50-170 <50-310 <50-140 <50-200 160HDHA 70-220 130-400 <50-330 <50-510 PD 50-120 80-220 <50-1380 <50-2110 PT 60-310 110-520 <50-280 <50-480 And’triol 150-330 290-600 50-240 100-350 THE 920-1810 1600-3330 680-2140 980-3240 THF 320-760 600-1420 310-1090 440-1610 aTHF 220-490 380-910 90-360 160-640 a-cort 130-600 220-1100 210-600 310-1330 Be & Be 240-800 410-1370 240-750 350-1110 total cort. 2300-4070 4010-7490 1780-4930 2630-7300 BSA (m2) 1.74-1.98 1.44-2.22 For abbreviations see Table 5.3 174 5.3.8 Use of creatinine corrected excretion rates and early morning urine samples When creatinine excretion was measured in the 24 hour urine collections of the same 20 subjects as in section 5.3.7, the males had significantly higher excretion rates (p<0.05) than the females (Table 5.7) with all values within the normal laboratory range, even though there was overlap of BSA between the two groups (refer to Table 5.6). Without correction for creatinine excretion,but conversion to ^mol/24h, higher androgen metabolites and cortisol metabolite excretion rate ranges in the males and higher PD excretion rates wOejra- sitoilav'-fo a&ove. in the femalesA. With creatinine correction of total excreted urinary cortisol metabolites, males and females were very similar (p=0.9), with the females having slightly higher upper ranges, presumably due to the over compensation by the higher male creatinine excretion rates. Androgen excretion rates, however, with creatinine correction resulted in equivalent ranges in the sexes (androsterone p < 0.5 and aetiocholanolone p = l, compared to P < 0.005 and p < 0.2 respectively uncorrected). It was then attempted to see if accurate results for steroid excretion could be obtained from early morning samples. The eight 24 hour urine samples from Subject N il and the four samples from a single menstrual cycle (Subject N18) had been collected such that the EMS was separate from the remainder of the 24 hour collection. A 20ml aliquot of the EMS was reserved and the remainder pooled with the "24 hour" collection. The EMS and an aliquot of the pooled 24 hour collection were analyzed separately and creatinine in each was quantified. Inter-subject variation for creatinine excretion was lower for the 24 hour collections (13 and 22%) than for the early morning samples (27 and 27% respectively for subjects N il and N18). Comparison of THDOC and THE excretion rates corrected for creatinine excretion are shown in Figures 5.11 and 5.12. The EMS results were inconsistent with the total 24 hour collection. Inter-subject variation for creatinine corrected THDOC (quantified with ion 476) was similar to non-corrected THDOC results for the 24 hour collections (CV = 13%), but higher for the early morning samples (CV = 17%). 5.4 Discussion Excretion rates of the most prominent urinary steroid metabolites, and of THDOC were determined for normal subjects. The results obtained from the urinary 175 Table 5.7 - Steroid excretion (^mol/24 hours) corrected for creatinine excretion Adult males (n=10) Adult females (n= 10) Steroid />imol/24h/nimol jimol/24h/mmol /unol/24h /imol/24h Creatinine Creatinine And 3.59-5.59 0.24-0.43 0.90-4.37 0.11-0.49 Aet 2.13-7.00 0.13-0.57 0.93-4.79 0.12-0.53 DHA 0.17-3.85 0.01-0.29 0.17-3.37 0.01-0.37 llOHAnd 1.47-2.55 0.11-0.20 0.42-2.22 0.04-0.21 11 OH Aet 0.16-1.01 0.01-0.08 0.16-0.65 0.02-0.07 160HDHA 0.41-1.25 0.03-0.09 0.16-1.59 0.02-0.18 PD 0.26-0.72 0.02-0.05 0.16-6.90 0.02-0.77 PT 0.33-1.56 0.02-0.13 0.15-1.44 0.02-0.16 And’triol 0.95-1.97 0.06-0.15 0.33-1.15 0.04-0.12 THE 4.40-9.15 0.27-0.70 2.69-8.90 0.27-0.96 THF 1.64-3.88 0.13-0.29 1.20-4.40 0.12-0.48 aTHF 1.04-2.60 0.08-0.19 0.44-1.75 0.04-0.21 of-cort 0.60-3.03 0.04-0.23 0.85-3.63 0.11-0.44 Be & fie 1.12-3.74 0.07-0.30 0.96-3.03 0.09-0.36 total cort. 12.24-20.54 0.67-1.56 7.54-19.99 0.76-2.20 Creatinine 11.5-16.3 8.0-11.8 (mmol/24h) For abbreviations see Table 5.3 176 Figure 5.11 - Creatinine corrected early morning sample compared to 24 hour collection - THDOC (Ion 476) limol THDOC/mmol creatinine 25 20 15 10 5 0 Subject N11 Subject N18 Figure 5.12 - Creatinine corrected early morning sample compared to 24 hour collection - THE umol THE/mmol creatinine 500 24 hour t = l E.M.S 400 300 - 200 - 826^16481^676701 100 0 2 3 4 5 6 D7 D14 D21 D27 Subject N11 Subject N18 177 steroid profiles were lower than some other reported ranges (Weykamp et al., 1989; van de Calseyde et al., 1972; Bevan et al., 1986), being in the lower half of the results quoted, except for PD in females which had a wider range in my results, and cortisol metabolites which varied between groups relative to my results. These other reports used varying hydrolysis and extraction methods and GC conditions. As all results in this thesis were achieved with the identical method throughout, comparison of the different clinical results to these measured in normals allowed accurate analysis of clinical differences. The results showed that the excretion rates of various urinary steroid metabolites varied between the groups of normal subjects studied, though all excretion rates were in the same order of magnitude, except THDOC and PD. These steroids were raised in the luteal phase of the menstrual cycle, although there was not a direct correlation of rise between the two, as demonstrated by the PD:THDOC ratios being higher in this group than in the other normal adults. Pregnanediol excretion rates in fertile women parallel the progesterone levels in plasma (Metcalf and Livesey, 1988). Pregnanediol has been shown to parallel plasma progesterone levels, and no age related changes in pregnanediol are seen in premenopausal women (Metcalf and Livesey, 1988). Urinary THDOC excretion rates in normal humans have not been widely measured. THDOC excretion rates of 7.1 - 40/zg/24h, n = 27 subjects (Schambelan and Biglieri, 1972; Harris et al., 1967; Romanoff and Brodie, 1976), which are equivalent to my male range, and 15 - 153 ugl 24h, n = 45 (Romanoff and Baxter, 1975; Crane and Harris, 1966; New et al., 1969) have been reported. The latter group of results was measured by specific activity based quantifications which I have shown may be inaccurate (see Chapter 4 and General discussion). Conversion rates be> of progesterone^DOC and to THDOC have been determined by Casey and co- workers, and other groups, in their investigations into extra-adrenal 21-hydroxylase (see Introduction), but absolute levels were not determined by these groups. DOC production has been shown to rise significantly between the follicular and luteal phase of the menstrual cycle (Parker, C.R., Jr. et al., 1981; Schoneshofer and Wagner, 1977; Antonipillai et al., 1983b), so it is likely that THDOC would follow the same pattern of excretion. 178 One interesting anomaly found in the steroid metabolite excretion pattern was the decrease in 5a metabolism seen in the females taking oral contraceptives. The ratios of THF:aTHF and androsteroneraetiocholanolone are similar to those reported in the literature (for example Stewart et al ., 1990a; Imperato-McGinley et al . , 1990; Wang et al . , 1976; De Slypere et al . , 1983). Inhibition of 5a-reductase activity has been shown to raise the 13:a isomer ratios (Imperato-McGinley et al., 1990), suggesting that the women taking oral contraceptives are affected by a degree of enzyme inhibition, probably due to the action of progestogens. Children, at least those studied here, had THDOC excretion rates similar to those of adult female follicular phase women. From these limited data there was no obvious correlation with age. This contrasts with the findings of Kelnar and Brook, (1983) who showed a steady rise in levels of THDOC in a longitudinal study of normal boys (n = 127) between the ages of 7 and 17. Excretion rates reported in this thesis, in particular of androgens and cortisol metabolites were in general lower than in the adults. Honour et al. (1991) showed that urinary excretion rates of cortisol metabolites when corrected for body size remain constant with age. The results obtained in this study were within the ranges quoted by the latter paper. Androgen metabolite excretion rates rise sharply in childhood to approach adult levels at the end of puberty, as the adrenal zona reticularis and gonads mature. Steroid excretion rates as measured in samples from one subject varied considerably (CV = 13 - 55%, n = 8 or 10 samples), so a single sample result should be considered to be an approximation for the subject in question rather than an absolute result. Cortisol excretion rates were higher in adult males than females, but showed close correlation when corrected for BSA. However, as a good correlation between BSA and excretion rates was not seen in the female subjects analyzed, and the majority of the clinical subjects studied were female, it was decided not to correct for BSA in this study. Although correction of steroid excretion rates for creatinine excretion produced improved agreement between male and female results, the purpose of creatinine correction had been to use an EMS instead of a 24 hour collection. The early morning urine samples were shown to give insufficiently accurate results to be considered a reflection of the steroid excretion pattern of a particular subject. It was 179 therefore decided that all urine samples would have to be from a 24 hour collection as the creatinine correction produced no improvement, as demonstrated by poorer inter-subject variation seen in the early morning samples. The use of creatinine as a measure of completeness of 24 hour collections has fk is correcfaoiA been addressed by many groups over the years, most finding ^too imprecise ^ &ag I fcakW as a standard to compare excretion rates of other substances to (Vestergaard and Leverett, 1958; Scott and Hurley, 1968; Chatterway et al., 1969; Paterson, 1967; Webster and Garrow, 1985; Knuiman et al., 1986; Bingham et al., 1988; James et al., 1988). Creatinine excretion rates can be influenced by various factors including diet compositioi^in particular meat consumption (Chatterway et al . , 1969; Bingham et al., 1988; Delanghe et a l, 1989), the subjects muscle mass and lean body mass (James et al., 1988; Webster and Garrow, 1985), renal activity (Chatterway et a l, 1969; James et al., 1988), exercise rate - up to 50% increase in creatinine excretion rate during exercise (Calles-Escandon et a l, 1984), and the subjects mental state or stress level (Chatterway et a l , 1969). The latter paper also shows some correlation between urine volume and creatinine excretion. The inter-subject variation in urinary creatinine excretion rate has been reported by various groups with CVs of between 2 and 20% quoted for 24 hour collections (Vestergaard and Leverett, 1958; Bleiler and Schedl, 1962; Scott and Hurley, 1968; Knuiman et a l , 1986; Waterlow, 1986; Bingham et al., 1988; James et al., 1988). The EMS creatinine excretion proved to be more imprecise. Zorab et al. (1969) showed that excretion rates per hour, when there was no restriction on activity, fluid intake or diet, could vary 2 - 3 fold in samples collected throughout the day, confirming this problem. The diurnal variation of cortisol and DOC (Tan and Mulrow, 1975) would also contribute to the variation seen between the EMS and 24 hour collection, as strict control of collection times of the EMS yjas not possible in this study. Progesterone is raised in the luteal phase of the menstrual cycle. The corpus luteum has been shown, by ovarian vein sampling, to be the main source of this raised progesterone (Khan-Dawood et al., 1989). This raised progesterone enters the general blood circulation and is hence available for 21-hydroxylation at extra-adrenal sites where such an enzyme is present. The luteal phase therefore offers the opportunity for extra-adrenal DOC production. 180 6 - llft-hydroxylase deficiency congenital adrenal hyperplasia 6.1 Introduction Deficiency of adrenal 1 18-hydroxylase results in one of the hypertensive forms of CAH. This enzymatic deficiency results in decreased cortisol synthesis, which in turn induces increased ACTH secretion, with the resultant overproduction of androgens and precursors of cortisol and aldosterone, in particular 11-deoxycortisol and DOC. The most prominent feature of the condition is virilization. The external genitalia of the female fetus are masculinized by the excessive fetal adrenal androgens and female pseudohermaphroditism results. Internal female genitalia are normal. Postnatally, in both males and females the excessive androgen production results in rapid somatic growth, advanced epiphyseal maturation, progressive penile or clitoral enlargement, early appearance of facial, axillary and pubic hair, and acne (Hochberg et al ., 1985; New and Levine, 1984). Prepubertal gynaecomastia is sometimes seen in genetic males (Zachman and Prader, 1975; Zachman et al ., 1983). Without replacement treatment, early epiphyseal closure and short stature result. Salt loss in the neonatal period, and in older patients, is reported in some cases, often when on glucocorticoid replacement treatment (Zachman et al . , 1983; Holcombe et al . , 1980; Hochberg et al ., 1984, 1986; Zadik et al ., 1984). The onset of hypertension, characteristic in most, though not all patients, is often delayed until late childhood or adult life. The raised DOC level seen in these patients is the most likely candidate for this hypertension (Vallotton and Favre, 1985), although these levels correlate poorly with the severity of hypertension (Zachman et al ., 1983). Unless suitable steroid precursors and their metabolites are determined in plasma or urine (plasma 17-hydroxyprogesterone can be raised above normal particularly in the neonatal period in both 21- and 118-hydroxylase deficiency), the incorrect diagnosis of 21-hydroxylase CAH may be made. The exact enzymatic 181 defect may only be realized when the patient becomes hypertensive in later life (Glenthoj et al . , 1980). This is true of a number of the patients, whose urines were analyzed in this laboratory and are described below. The characteristic urinary steroid profile of these patients has high androgen excretion, and a very prominent double peak consisting of THS and THDOC, the latter not fully separating from the trailing end of the THS peak (Shackleton et al ., 1980b). This pattern of steroid excretion is not evident in the first few days of life, whilst the adrenal gland matures. 11-deoxycortisol was found to be undetectable in plasma until day 5 of life in a report by Hughes et al. (1986), and THS is not a clear urinary marker for this defect until after this time. Subjects with partial or mild llB-hydroxylase deficiency have also been described, in particular amongst hirsute women and patients with hypertension (Newmark et al ., 1977; De Simone et al., 1985; Tan et a l, 1978), in whom diagnosis was sometimes only possible with an ACTH stimulation test. A further form of CAH attributed to combined 21- and 1113-hydroxylase deficiency was described in 3 families by Hurwitz et al. (1985). Two explanations for the latter were offered: (i) that raised androgens from a partial 21-hydroxylase deficiency were inhibiting 1 16-hydroxylation, and (ii) a genetically inherited abnormal 1113- hydroxylase existed in these families with a lower affinity for its normal substrate, 17-hydroxyprogesterone being preferred, resulting in 11-deoxycortisol and 21- deoxycortisol being raised. The opportunity arose to study two brothers with complete 116-hydroxylase deficiency at length, and urine samples from 8 further 1113-hydroxylase deficient subjects. 6.2 Experimental Urinary steroid profiles and THDOC quantification were achieved using the methods described in Chapter 2. Renin and aldosterone were measured by the Supra Regional Assay Service using radioimmunoassay (RIA). ACTH was measured (by Mr. P. Holownia) using a RIA kit marketed by Eurodiagnostics, kindly donated by Organon Teknika. Plasma 11-deoxycorticosterone and 11-deoxycortisol were kindly measured by Dr. R. Fraser in the MRC Blood Pressure Unit, Glasgow, using RIA. Plasma cortisol was measured by RIA (in-house method). 182 6.3 Subjects and results 6.3.1 Two brothers with 1 lfl-hvdroxylase deficiency (Subjects Y1 and Y21 Subject Y1 - A 12 year old boy of Turkish origin had been treated since early childhood for CAH, presumed to be 21-hydroxylase deficiency. A complete defect of 1115-hydroxylase was confirmed by GC urinary steroid profile analysis, which showed an absence of cortisol metabolites, but high excretion of 11-deoxycortisol, DOC and androgen metabolites (Figure 6.1). His progress during treatment was monitored using urinary steroid profiles. When inadequately treated^the THDOC excretion rates (upper panel Figure 6.2, closed circles) were > 3000j*g/24h. This was in great excess of the upper limit for normal adult males (50jxg/24h). An example of a SIM run for THDOC excretion is shown in Figure 6.3. Blood pressure was difficult to control despite five drugs being prescribed on admission to the Middlesex Hospital (lower panel Figure 6.2). Dexamethasone, a more potent ACTH suppressant than hydrocortisone, was utilized and he was then reasonably well controlled on only this and two antihypertensives. In the first four urine samples THDOC excretion was over 100 times normal, the highest excretion rate being 5700/xg/24h. On raising the dexamethasone dosage from 0.75 to lmg/day, THDOC was suppressed but was still higher than normal, the lowest excretion rate shown being 140^cg/24h. Spironolactone facilitated only a marginal fall in blood pressure, but aggravated the tendency to hyperreninaemia when mineralocorticoid (DOC) production was adequately suppressed. During the same period of time a number of blood samples were analyzed (see Table 6.1). ACTH levels were initially well above the normal range. On changing from hydrocortisone to dexamethasone, ACTH dropped into the normal range and then on the higher dosage was fully suppressed. No cortisol or aldosterone was ever detected, in the latter’s case despite renin levels being initially suppressed to within the normal range. Renin rose with increasing time on treatment. Plasma DOC values were extremely high on hospital admission and fell in parallel with ACTH on administration of dexamethasone. Paradoxically though ACTH was fully suppressed, the DOC level initially also suppressed, subsequently rose over the next month and a half. Pregnanediol and 17-hydroxypregnanolone were seen in all these urine samples; PD excretion rates were seen at higher levels, range 420 - 630/xg/24h 183 Figure 6.1 - Urinary steroid profile from a patient (Y l) with congenital adrenal hyperplasia due to lip-hydroxylase deficiency 1 = Androsterone 2 = Aetiocholanolone 3 = 17-hydroxypregnanolone 4 = Pregnanediol 5 = Tetrahy drodeoxycortisol (THS) 6 = Tetrahy drodeoxycorticosterone (3a5|3 THDOC) 7 = 5a-THS 8 = Hexahydro-S 184 Figure 6.2 - Change in THDOC excretion rates and blood pressure with treatment (Subject Yl) Blood pressure (mm Hg) THDOC (mg/24hr) 300 250 - - 5 2 00 - - 4 150 - - 3 100 - - 2 50 - - 1 0 Hydrocortisone Dex. 30mg 0.75mg Spironolactone 100 50mg 200 Amiloride 5mg Frumil 1 tab. Metoprolol 200mg Prazosin 9 mg 4.5 Enalapril 15mq daily dose max 15/6 22/8 25/8 27/8 30/8 10/9 16/9 28/9 4/10 11/10 b|oo Change in blood pressure (vertical bars) and THDOC excretion (closed circles) is shown in response to treatment. The time axis is not linear, with the lower panel showing doses of each treatment and duration. The subject was admitted to the Middlesex Hospital on 25/8. 185 The upper panel shows a typical SIM Ion 507 response, with the two THDOC isomers being clearly identified. The lower panel shows the corresponding Ion 476 response, again clearly showing the two main peaks of the two THDOC isomers, but with the addition of further steroids. The Sephadex LH-20 chromatography had eliminated the majority of the large THS peak, that would otherwise have merged with 3a5J3 THDOC. Figure 6.3 - SIM run from a patient with 116-hydroxylase deficiency CAH Ion 507 6.0E4- 5.0E4- 2 .0E4 H 1 0000 Ion 47G 170HPr 1 .4E5 3a5B THDOC 1 .0E5 remammg THS T3 8.0E4 365a THDOC . 0E4 2.0E4 186 Table 6.1 - Plasma results from Subject Y1 (llB-hydroxylase deficiency CAH) Normal range 25/8 31/8 7/9 14/9 28/9 5/10 11/10 0800 < 80 64 <13 <13 ACTH (pg/ml) 213 <13 2400 < 30 <43 <13 <13 0800 150-650 13 13 <27 <43 < 32 <45 Cortisol (nmol/1) 2400 < 170 23 <27 <43 < 32 <45 Plasma Renin R 1.1-2.7 1.0 0.9 1.0 2.5 6.4 Activity A 2.4-4.5 1.6 1.4 1.6 4.1 10.4 11.1 (pmol/h/ml) Aldosterone 100 - 500 <20 < 20 <20 < 20 < 20 < 20 < 2 0 (pmol/1) DOC (nmol/1) 0.12 - 0.48 54 4.2 11 8.4 11 R = Recumbent A = Ambulant Samples were taken on the dates indicated, cp. with Figure 6.2 187 (greater than those seen in normal adult males), when THDOC excretion rates were raised (range 3165 - 5760^g/24h). Subject Y2 - This is the 5 year old younger brother of Subject Yl, who had had precocious pubic hair since infancy. He had been empirically treated by his mother with the occasional use of Subject Y l’s hydrocortisone tablets. He was finally taken to the referral hospital where his brother was first seen in Britain, and then transferred to the Middlesex Hospital. A further 3 siblings (including a set of twins) had died in infancy, with hypertension implicated in the death of the older single child. Six other siblings appear asymptomatic or unaffected. A urinary steroid profile confirmed the diagnosis of 1 lB-hydroxylase deficiency and optimization of treatment was also attempted in this patient. Initially when taking hydrocortisone, THDOC excretion was high at 83^g/24h. Change of replacement treatment to dexamethasone reduced this to 37^g/24h. This was not complete suppression, but was almost into the normal range for children. PD was detected in both of these samples. The initial treatment regime for both brothers included dexamethasone with a salt restricted diet, which was variably adhered to. As a result of the subsequent hyperreninaemia the effect of sodium intake on these two subjects was also investigated. Both boys were allowed normal diets and were maintained on 0.5 and 0.3mg dexamethasone respectively for body size. They were then provided with a diet with reduced sodium (50mmol/24h) for 7 days. Following this they were treated with fludrocortisone (mineralocorticoid replacement). Urinary THS and THDOC excretion rates over this period are shown in Figure 6.4. Blood samples were also taken over this period. The results are shown for plasma 11-deoxycortisol, DOC, ACTH and renin activity in Figures 6.6 and 6.7. The data showed that Subject Y2 was not properly suppressed during the study period, with compliance problems noted in medication, even when in hospital. Plasma ACTH, DOC, S, and urinary THDOC and THS were all very high. Plasma renin activity was within the normal range when on a normal diet, but rose with Na+ restriction. His older brother on the other hand was well suppressed on dexamethasone. In the first 3 days of Na+ restriction a rise in plasma DOC and S was seen, with a parallel fall in serum Na+ (results not shown), but this then reduced again. Plasma renin levels were raised well above normal ranges and rose 188 Figure 6.4 - Effect of Na+ restriction on urinary THDOC and THS excretion rates (a) Subject Y1 THDOC or THS (ug/24h) 300- THDOC THS Fludrocortisone 250 - 200 - 150 - 100 - 50 - Na+ 50mmol/24h 0 2 4 6 8 10 Day (b) Subject Y2 THDOC (ug/24h) THS (mg/24h) 300 - - O - THDOC THS 250 - - 2.5 200 - 150 - Fludrocortisone 1 0 0 - 50 - 0.5 Na+ 50mmol/24h 0 2 4 6 8 10 Day 189 Figure 6.5 - Effect of Na+ restriction on plasma DOC, S and ACTH (a) Subject Y1 DOC or S (ng/dl) ACTH (pg/ml) 300 250 - - O - DOC ■■ S - 250 - 0 - ACTH 200 - Nat 50mmol/24h - 200 150 - - 150 Fludrocortisone 1 0 0 - - 100 50 - - 50 0 2 4 6 8 10 Day (b) Subject Y2 DOC or S (ug/dl) ACTH (pg/ml) 6 300 Na+ 50mmol/24h DOC - S - 2505 - 0 - ACTH 4 - 200 3 Fludrocortisone - 150 2 - 100 1 - 50 0 0 2 4 6 8 10 Day 190 Figure 6.6 - Effect of Na+ restriction on plasma renin activity (PRA) (a) Subject Y1 PRA (pmol/hr/ml) 40- A m bulent R esting 30- Fludrocortisone 2 0 - 1 0 - Na+ 50mmol/24h 0 2 4 6 8 10 Day (b) Subject Y2 PRA (pmol/hr/ml) 10 A m bulent R esting Fludrocortisone Na+ 50mmol/24h 0 2 4 6 8 10 Day 191 particularly on salt restriction except on day 3, parallel with the rise in plasma DOC and S. Administration of fludrocortisone reduced plasma renin activity in both subjects. 6.3.2 Other cases of 1 lfl-hvdroxvlase deficiency Eight further subjects, with 116-hydroxylase deficiency were investigated, supplying one to three urine samples each. (i) Subject Y3 - Adult male This patient was diagnosed at birth to have 21-hydroxylase deficiency. This diagnosis was later changed to 116-hydroxylase deficiency with the development of hypertension, and confirmed by this urinary steroid profile. THS and the urinary androgens (androsterone and aetiocholanolone) were greatly raised at > 11000, 4800 and 8200/ig/24h respectively. The THDOC excretion rate was also greatly raised at 717^g/24h. A further sample from this patient on treatment showed complete suppression of THS and excessive androgens. This patient proved to be fertile, the male offspring having a normal urinary steroid profile. (ii) Subject Y4 - Adult male Two samples were analyzed, using urinary steroid profiles, from this subject with mild hypertension. The first urine sample off treatment, showed the characteristic raised THS (29100/*g/24h), but normal androgens (androsterone 3330/zg/24h and aetiocholanolone 1930/xg/24h). The second on dexamethasone treatment confirmed reasonable suppression, with the THS excretion rate reduced, though still above normal at 700/xg/24h (2.5% of unsuppressed value), and androsterone and aetiocholanolone, 2690 and 2030/*g/24h, in the normal adult male range. THDOC excretion rates were 462 and 97/xg/24h respectively. When on treatment,THDOC was not fully suppressed (21 % of unsuppressed value), at still twice the upper end of the normal range for adult males. (iii) Subject Y5 - Male age 12 years, brother of Subject Y4 This boy presented with malignant hypertension, partial blindness and evidence of myocardial damage. Off treatment this patient, had greatly raised androgens (11780 192 and 26000/xg/24h) and THS (17640/xg/24h) excretion rates. Suppression with dexamethasone resulted in partial suppression of androgens (880 and 3750^ig/24h) and THS (1250/xg/24h - 7% of untreated). The THDOC excretion rate was suppressed on treatment to 15% of the untreated excretion rate, at 22^g/24h (with the normal adult range), compared to 143/ig/24h when untreated. Both brothers had refused treatment for 4 years prior to collection of the above urine samples. They are discussed further in a report by Hague and Honour (1983). (iv) Subject Y6 - Female age 15 years This patient was diagnosed at birth to have 21-hydroxylase deficiency, but then became hypertensive. Two 24 hour urine samples were supplied, before and after dexamethasone treatment. Untreated, this patient gave the characteristic urinary steroid profile of raised androgens (2510 and 2370^g/24h) and THS (5120^cg/24h) excretion rates, diagnosing llfl-hydroxylase deficiency. On dexamethasone treatment the androgens were suppressed to levels (240 and 360/xg/24h respectively) below normal for age. THS excretion was also reduced but not fully suppressed ( * 18% of untreated excretion rate), quantified at 940jxg/24h. The excretion rates of THDOC, using SIM quantification, were 265 and 208/xg/24h respectively off and on treatment, a reduction to only 78% of the untreated excretion rate seen when on treatment. (v) Subject Y7 - Male age 13 years THS (13490/*g/24h) and androgens (3400 and 3490/xg/24h) were greatly raised in the single sample obtained from this child. The THDOC excretion rate was calculated at 499ftg/24h. (vi) Subject Y8 - Male age 4 years Androgen excretion rates (870 and 1470^g/24h) were very high for a child of this age (normally <50/*g/24h). THS was also raised at 660/zg/24h. A second who* the subject sample,Aon dexamethasone, resulted in reasonable suppression of the metabolites of interest - THS 220/xg/24h, and androgens 60 and 160^g/24h. A final urine sample 193 was supplied when on prednisolone. All the normally detected steroid metabolites found between the two internal standards A and S were completely suppressed. Prednisolone related metabolites (confirmed by mass spectrometry) were seen in the urinary steroid profile between the internal standards S and C. THDOC excretion rates in these three urine samples were calculated at 12, 10 and undetectable ^g/24h respectively. (vii) Subject Y9 - Female age 17 months Three urine samples were obtained from this infant with ambiguous genitalia. The first was an incomplete 24 hour collection, calculated using an estimated 190ml 24 hour collection (based on the volume of the subsequent complete collection), and the second a complete 24 hour collection. Both of these had no detectable steroids using urinary steroid profiles. THDOC excretion rates measured by SIM, this being a more sensitive method, produced results of * 5 and 7.9/*g/24h. An ACTH stimulation test was therefore performed and the characteristic urinary steroid metabolite pattern for llfi-hydroxylase deficiency was seen with THS at 940/xg/24h and small amounts of androgen (25 and 50^g/24h), which are not normally produced at this age. THDOC excretion also rose, to 35.6/xg/24h (greater than follicular phase excretion rates seen in adults). (viii) Subject Y10 - Newborn Three random urine collections were obtained from this newborn female with ambiguous genitalia on days 1, 3 and 5 of life. The profiles were similar to normal newborns, but with reduced cortisol metabolites. No THS was detected in any of the samples. THDOC was not quantified. This confirmed the results of Hughes et al. (1986), that urinary THS was not detectable in the first few days of life in these patients. All patients described above had very low or totally absent urinary cortisol metabolites. The specificity of the THDOC measurements by SIM runs, as judged by the ratios of the response of the ions 476 and 507 (476:507 ratios) was high in these patients, with the 476:507 ratios equal to THDOC standard. 194 6.4 Discussion THDOC excretion rates have been measured in 1 lB-hydroxylase deficiency by a number of groups using RIA, paper chromatography, double isotope dilution derivative techniques and gas chromatography (Levine et al ., 1980; Eberlein and Bongiovanni, 1956; Sizonenko et al . , 1972; Zachman etal., 1983). Results obtained ranged between 200 and 12000/zg/24h. These are equivalent to those determined in this work (up to 5700/*g/24h). In Subject Y1 DOC and THDOC levels were still above normal even when ACTH was fully suppressed by the higher dose of dexamethasone. This high DOC production complicated treatment to normalize blood pressure. The possibility that the renin-angiotensin system was the stimulus for the raised DOC was considered. In CAH due to 1 lB-hydroxylase deficiency ACTH levels are high due to the lack of negative feedback of cortisol production. As a result DOC, 11-deoxycortisol, and androgen production are greatly stimulated. The increase in DOC causes sodium retention and hence plasma volume expansion. Under these circumstances the renin- angiotensin system is suppressed by the excessive DOC (see Figure 6.7(a)). With appropriate replacement of glucocorticoid ACTH is suppressed, but DOC production was noted to continue at a rate in excess of normal. Initially the Spironolactone was aggravating the tendency to hyperreninaemia, but even later when off Spironolactone treatment the DOC suppression resulted in reduced negative feedback, due to plasma volume expansion being absent or minimal, since aldosterone was never produced in this patient, causing the renin-angiotensin system to be stimulated. Angiotensin II stimulated the glomerulosa cells in the adrenal cortex in an attempt to produce aldosterone. Due to the llB-hydroxylase deficiency, DOC is the principal secretory product of the zona glomerulosa (see Figure 6.7(b)). This then appears to become the source of the excess DOC, seen as there is minimal zona fasciculata activity since ACTH is suppressed. A similar discrepancy in DOC production when on glucocorticoid replacement therapy was seen in Subjects Y4, Y5, and Y6. THS excretion rates were suppressed to a higher degree than THDOC excretion rates. This extra THDOC would be the urinary metabolite of DOC produced by the zona glomerulosa during stimulation by the renin angiotensin system. 195 Figure 6.7 - Model for the regulation of adrenocortical steroidogenesis in 1113-hydroxylase deficiency CAH (a) Untreated Renin "TO © I ACTH Angiotensin II l Glomerulosa Fasciculata HI DOC DOC S (Androgens) Na+ retention I Plasma volume expansion (b) Treated o Angiotensin II ACTH * Glomerulosa Fasciculata I 1 I DOC DOC S (Androgens) Na+ retention Plasma volume expansion 196 The hypothesis described above was put forward by New (1985) and the data presented here supports the idea that the fasciculata and glomerulosa zones of the adrenal cortex behave as two separate endocrine glands under separate regulation (Sizonenko et al ., 1972). The latter paper pointed out that 116-hydroxylase deficiency can be expressed to a greater degree in the fasciculata. In the zona glomerulosa 116-hydroxylase may not be rate limiting and sodium depletion may lead to an appropriate increase in aldosterone secretion if the enzyme deficiency is incomplete. Subjects Y1 and Y2 reported here had complete deficiencies of both adrenal zones, but reports in the literature show varying degrees of deficiency, often incomplete, in the zona glomerulosa (Kowarski et al . , 1968; Sizonenko et al . , 1972; Zachman et al . , 1983; New, 1985; Rodiguez Portales et al ., 1988). 116-hydroxylase deficiency congenital adrenal hyperplasia offers an example of adrenal DOC production, but with additional evidence that, in this clinical condition, this steroid production is made up of two sources, the ACTH driven fasciculata, and the renin angiotensin system controlled glomerulosa zones of the adrenal cortex. 197 7 - Mineralocorticoid secreting tumour 7.1 Introduction Clinical syndromes produced by adrenal cortical hyperfunction are frequently associated with electrolyte and water imbalance - sodium retention, increased potassium excretion, blood volume expansion and frequently hypertension. These are often caused by mineralocorticoid excess. Causes of mineralocorticoid excess include: (i) Aldosterone producing adenoma (Conn’s syndrome) (ii) Bilateral nodular hyperplasia (iii) B arth s syndrome (iv) CAH due to 17-hydroxylase (and llB-hydroxylase) deficiency (v) Pregnancy (vi) Massive glucocorticoid excess producing mineralocorticoid effect eg in ectopic ACTH syndrome (vii) Rare renin secreting tumours of the kidney (viii) Mineralocorticoid secreting tumour (eg. corticosterone or DOC) DOC secreting tumours, in particular those involving the adrenal gland, are relatively rare in the literature (for example Foye and Fechtimeir, 1955; Lipsett and Wilson, 1962; West et al ., 1964; Biglieri et al ., 1968; Solomon et al ., 1968; Powell-Jackson et al . , 1974; Kondo et a l, 1976; Kelly et al ., 1979, 1982; Karpf et al ., 1986; Irony et al ., 1987; Saadi et al ., 1990; Imperato-McGinley et al ., 1981). Surgical resection is the normal treatment of choice, but op’DDD (1,1- dichlorodiphenyldichloroethane, also known as mitotane) has been associated with cures, long-term remissions, regression of metastases, and increases in survival (Luton et a l, 1990). 198 A further case of a mineralocorticoid secreting tumour was investigated. The original diagnosis was reported by Drury et al. (1987). This is outlined in the case history. Results after the initial diagnosis are reported, including THDOC excretion rates. 7.2 Case history and results A 43 year old woman was referred (1986) to King’s College Hospital (KCH), London for a hepatic mass, hypertension and hypokalaemia. Fourteen months previously she had developed headaches and was found to be severely hypertensive (blood pressure 210/130mmHg). Records of previous blood pressure readings were not available, however as she had been taking oral contraceptives when in her 20’s, she was presumed to be normotensive at that time. There was no family history of hypertension. Her blood pressure was eventually controlled, after trying both oxprenolol and nifedipine on which she developed side effects, with enalapril (20mg daily) and bendrofluazide (lOmg daily). Six months before referral she had collapsed suddenly at home and was taken to her local hospital. On arrival she was unconscious and shortly after sustained cardiac arrest caused by ventricular fibrillation. Investigations at this time showed profound hypokalaemia (1.1 mmol/1), with normal serum magnesium and calcium concentrations. A CT scan of the abdomen described two hepatic masses, one in the right lobe, and the other in the left lobe, with calcification in the former. The patient was transferred to KCH for further investigation. On investigation at KCH the patient reported an increase in the rate of hair growth on her arms and legs, and an abrupt cessation of menstruation 3 months prior to admission to the local hospital. There were some retinal hypertensive changes. She was not Cushingoid and had had no recent weight gain. Laboratory investigations from KCH are shown in Table 7.1. The hypokalaemia and clinical features - the acute onset, severity and resistance to single line therapy - were all consistent with the severe hypertension being secondary in nature. The acute episode of collapse could be explained by the severe hypokalaemia, as this is known to cause weakness and confusion, and in 199 Table 7.1 - Laboratory investigations on admission to KCH Investigation Result Normal range Serum sodium 145 mmol/1 Serum potassium * 4.2 mmol/1 Serum bicarbonate 27 mmol/1 Serum glucose 5.4 mmol/1 4.2-6.4 Serum creatinine 53 fimol/l post menopausal < 6 Serum progesterone 25.3 nmol/1 luteal peak > 16 Serum DHAS 9.3 fimol/l 2-9 Serum testosterone 2.3 nmol/1 <3 9am 470 nmol/1 Plasma cortisol 150-700 midnight 340 nmol/1 recumbent <0.2 pmol/h/ml 1.2-2.4 Plasma renin activity ambulant <0.2 pmol/h/ml 3.0-3.4 recumbent 147 pmol/1 100-500 Plasma aldosterone ambulant 157 pmol/1 600-1200 Urine free cortisol 160 nmol/day <300 * Note the patient was on supplements at this time On stopping supplements: Serum potassium 24hr 3.2 mmol/1 60hr 2.7 mmol/1 with urine sodium 22 mmol/1 potassium 50 mmol/1 Based on Drury et al. (1987) 200 a extreme cases fits and loss of consciousness. As^chest X-ray and ECG at the time were normal, the hypertension was of recent onset. Hypokalaemia has several possible causes. Immediately eliminated in this case were severe vomiting, under eating, diarrhoea causing potassium loss and purgative abuse, as none were admitted to by the patient, or indicated by evidence of increased urinary losses (see note Table 7.1). The most likely cause therefore was mineralocorticoid excess, which fitted with the observed hypertension. The causes (i) - (vii) of mineralocorticoid excess leading to possible hypertension cited in the introduction above were however eliminated as the cause, due to contradictory laboratory findings or not fitting into the patient’s medical history. The plasma renin and angiotensin did not increase normally when the patient was ambulent, implying that firstly the renin-angiotensin-aldosterone axis was suppressed by an increased circulating blood volume, and secondly that aldosterone was not the cause of the hypertension. Another mineralocorticoid must therefore be responsible, deoxycorticosterone and corticosterone being the most likely candidates. At this point additional steroids (serum and urinary, the latter measured by the method described in Chapter 2) were measured, see Table 7.2 and Figure 7.1(a). From this data it was concluded that the hypertension was caused by secretion, from a tumour, of 11-deoxycorticosterone. Some other steroid metabolites, in particular 11-deoxycortisol, were also raised. Further investigation by CT scanning and ultrasound showed that the mass on the right side arose from an enlarged adrenal gland displacing the liver and that the mass in the left lobe of the liver was a metastasis. The right adrenal gland was surgically removed along with two segments of the left side of the liver. The patient recovered well and for the next 9 months was asymptomatic, normotensive and normokalaemic. She also had a normal urine steroid profile, and her remaining liver segments on ultrasound appeared normal. A year after the operation, follow up urinary steroid profiles showed an increase in mineralocorticoid excretion; all urinary steroid profiles from this point being done by the author. The patient was put on chemotherapy with op’DDD 18 months post-op. 201 Table 7.2 - Additional steroid results Steroid Result Normal range (1) Serum (measured by Dr. Fraser, MRC Blood Pressure Unit, Glasgow) All values ng/dl Deoxycorticosterone 895 < 18 Corticosterone 980 <800 11-deoxycortisol 2820 <400 (2) Urinary Steroid Profile (measured in Dr. Honour’s laboratory) All values /xg/day Androsterone 5980 260-1270 Aetiocholanolone 480 270-1390 11 B-hydroxyandrosterone 1650 130-680 1115-hydroxyaetiocholanolone 1520 <50- 200 16a-hydroxy DHA 2550 <50-510 Pregnanediol 12120 <50-2110 Pregnanetriol 1580 <50-480 Tetrahydrodeoxycortisol (THS) 14680 <50 Tetrahydrodeoxycorticosterone (THDOC) 5930 <50 Tetrahydrocorticosterone (THE) 12530 980-3240 Tetrahydrocortisol (THF) 2930 440-1610 allo-tetrahydrocortisol (5aTHF) 1580 310-1330 B-cortol + B-cortolone 2740 350-1110 202 Figure 7.1 - Urinary steroid profiles (a) pre-op and (b) 34 months post-op from a patient with a recurring mineralocorticoid secreting tumour (a) Pre-op (1/1350 of thetotal 24 hour urine collection) (b) 34 months post-op (1/300 of the total 24 hour urine collection) 1 = Aetiocholanolone 2 = Pregnanediol 3 = Pregnanetriol 4 = Tetrahydrodeoxycortisol (THS) 5 = Tetrahydrodeoxycorticosterone (3a5p THDOC) 6 = Tetrahydrocortisone (THE) 7 = Tetrahydrocortisol (THF) 8 = 5a-THF 203 The patient was followed up for 53 months post-op with a number of urinary steroid profiles (Figure 7.1(b) shows one such profile). Tetrahydrodeoxycortisol (THS) fell from over 14mg/24h, to less than 200/xg/24h four months post-op. THS excretion rates then rose steadily over the next three years to a maximum of 3mg/24h. Improved treatment after this time then kept excretion rates to levels below 2mg/24h. All of the available urines were also analyzed for THDOC excretion rates by GC-MS SIM (using the method described in Chapter 2). These results are shown in Figure 7.2. The THDOC excretion rate was just above the normal range at 4 months post-op. When the samples at 10 and 11 months post-op were analyzed, THDOC excretion rates were raised to 2 and 7 times the upper limit of the normal follicular range respectively. After this time THDOC excretion rates in all samples analyzed were at least 30 times the upper limit of the follicular phase range. There was a steady rise in THDOC excretion rates, until 39 months post-op when optimization of treatment had been improved. Later there was a slight drop, then a plateauing in excretion rates was seen. All samples from this patient produced results with no disruption of the 476:507 ratios. THS:THDOC excretion rates changed in ratio with time; pre-op the ratio was approximately 4.5, for the first year post-op THS:THDOC ratios of between 5 and 7 were seen, but as time progressed the ratio fell to between 1 and 2.5. A scan at 3 years post-op showed metastases in the remaining liver which the surgeons have indicated would be difficult to remove due to their extensive nature. 204 Figure 7.2 - THDOC excretion rates Mineralocorticoid secreting tumour X O H Q O '— CM E o> co CM in o o co in in CM co in o C\J in O IO JZ +■* *-> •* Q. o CO 0 Q. E o c CO 1 O Q. d> <8 205 7.3 Discussion THDOC measurement in this woman was undertaken initially as a means of verifying the quantification method. The results show however that the method could be useful in such cases for monitoring DOC excretion. Resection of the tumour in the short term improved the clinical picture of this patient. Within a year of surgery a steady return of the excess mineralocorticoid secretion was seen, with an upward trend that would have returned the patient to pre- op THDOC excretion rates within 60 months. Treatment optimization has slowed down this trend, and hopefully improved the prognosis of this patient. Continued urinary metabolite profiles will be useful for monitoring the progress of tumour secretion rates. The fact that there was no rise from the expected 476:507 ratio in the samples from this patient, indicates that only THDOC was being measured at the relevant retention time of the SIM runs. The origin of the DOC in this patient can be considered to be virtually exclusively tumour in origin as immediately post-op the THDOC excretion rate was only just above the normal range for adult follicular phase excretion rate. This DOC is therefore an example of extra-adrenal 11- deoxycorticosterone production. 206 8 - Normal pregnancy 8.1 Introduction Pregnancy adds an additional endocrine organ into the body of a woman - the feto-placental unit. Many steroids, and factors controlling their production, are introduced into the already complex interactions occurring in homeostasis of the individual. The plasma concentrations of a number of steroid hormones are strikingly increased during the course of human pregnancy. Two of the major steroids produced in pregnancy are progesterone and oestriol. Progesterone is secreted by the corpus luteum (early pregnancy) and in the placenta, whilst the oestrogens are produced in the placenta from DHA-sulphate and 16-hydroxy-DHA-sulphate of fetal origin (refer to Figure 1.2). The origin and metabolism of these hormones is fairly well established, but precise information on the origin of DOC, a potent mineralocorticoid is not. The significance of the latter in hypertension in pregnancy is also not understood. Various studies have suggested that the some of the DOC seen in pregnancy is the result of extra-adrenal 21-hydroxylase of progesterone (see Introduction). It was hoped to establish normal ranges in pregnancy urine for various of the most prominent steroid metabolites and to investigate further the production of DOC, using THDOC excretion rates. 8.2 Experimental Urinary steroid profiles were performed on all samples according to the method described in Chapter 2. Between 1 and 5 ml of urine were analyzed in combination with 2, 5 or Kfyig of the internal standards (A,S and C), in order to obtain the best profile possible for quantification. The results shown are the mean calculated excretion rates from 2 (and in some cases 3) FID traces of each sample. 207 THDOC quantification was performed as described in Chapter 2, using 1/xg 3fi5a THDOC (IS) for most of the samples. Only small urine aliquots were available from Subjects P4 and P6, so 150ng IS were used with small urine volumes (1 - 2ml). Two extracts were made from each sample and each of these injected twice onto the MSD, the mean of these four results were plotted. Gestational week was considered to be weeks after the last menstrual bleed. 8.3 Subjects Eight healthy women in various stages of pregnancy were recruited to collect serial 24 hour urine samples at weekly intervals. Six volunteers were hospital staff, whereas Subjects P7 and P8 were patients under the care of the Department of Reproductive Endocrinology (Middlesex Hospital) recruited in order to obtain early pregnancy results. All were in their first or second pregnancy and in the age range 26 - 38 years (median = 31). None of these subjects suffered from clinically significant hypertension in the period during which samples were supplied. No medication was taken by any of the subjects. 8.4 Steroid excretion rates (from urinary steroid profiles! A typical urinary steroid profile in pregnancy (week 30, Subject P2) from the FID pen plotter is shown in Figure 8.1. Pregnanediol (PD) and oestriol (OE3), the two largest metabolite peaks in the analysis of steroids excreted in the second two trimesters, and the excretion rates of these steroids for the eight normal subjects are shown in Figures 8.2 and 8.3 respectively. Other metabolite excretion rates were quantified from gestation week 18. Two of the subjects provided urine samples only in early pregnancy. The profiles from two of the patients (OV3 and OV4) undergoing oocyte donation IVF (see Chapter 11), were added. These two women were perimenopausal, but, after week 18 when their treatment had ceased, had steroid excretion rates similar to normal for pregnancy as measured for PD and OE3 (see Chapter 11) so were considered normal in pregnancy. The results are shown for 17-hydroxypregnanolone (170HPr), pregnanetriol (PT), tetrahydrodteoxy cortisol (THS), tetrahydrocortisone (THE), total cortisol metabolites, THE: 5fl-tetrahydrocortisol (THE:THF) ratios and THFiaTHF ratios in Figures 8.4 - 8.10. 208 Figure 8.1 - Urinary steroid profile from a normal pregnancy (30 weeks gestation) 11 1 = Androsterone 2 = Aetiocholanolone 3 = 17-hydroxypregnanolone 6 = Pregnanediol 7 = Pregnanetriol 9 = Hydroxypregnanolone (^retention time of Prl) ' 0 = Tetrahydrodeoxycortisol (THS) + hydroxypregnanolone (=Pr2) 1 = Oestriol (OE3), 3a5fJ THDOC + co-eluting hydroxypregnanolone 2 = Hydroxypregnanolone (=Pr3) 4 = Tetrahydrocortisone (THE) 5 = Tetrahydrocortisol (THF) 6 = 5a-THF 7 = a-cortol 8 = (3-cortol + P-cortolone 4,5,8, 3 = PD like progesterone metabolites 209 Figure 8.2 - Pregnanediol excretion rate Normal Pregnancies PD (mg/24h) • P1 30 H + P2 * P3 * 25 □ ...... Q □ P4 + X P5 S 0 20 - - *■ * 0 * ... 0 + x P6 K+ 15 - V P7 * ft P8 □ ^ ^ X x + £ 10 - + <> -K> * x " 5 Jk * * x V V x 0 n 10 15 20 25 30 35 40 weeks gestation Figure 8.3 - Oestriol excretion rate Normal Pregnancies OE3 (mg/24h) 35 ■ P1 30 + P2 * P3 25 □ P4 + X P5 20 ...ito .. 0 P6 + □* n+ 15 V P7 ft P8 10H g ..... * 5 0 10 15 20 25 30 35 40 weeks gestation 210 Figure 8.4 - 17-hydroxypregnanolone excretion rate Pregnancy (week 18 - term) 170HPr (mg/24h) ■ P1 5- + P2 * P3 +x...... □ 4- □ P4 + X P5 X ' o 3- 0 P6 o- °E T o OV3 0 * X0 A .0.0 ^ D.+ * £... 2 - OV4 AQ ® ^0 □ + * * +* - 0 10 15 20 25 30 35 40 weeks gestation Figure 8.5 - Pregnanetriol excretion rate Pregnancy (week 18 - term) PT (mg/24h) • P1 5 - + P2 * P3 + 4 - □ P4 □ X P5 + + 3- 0 P6 O OV3 4- 2 - A OV4 oo 1 - A-A^-a 0 B A* *• t J $ * * ...... & pfi^AA£ fl$*$Xx**.^X * i------1------1------1------1------r 10 15 20 25 30 35 40 weeks gestation 211 Figure 8.6 - THS excretion rate Pregnancy (week 18 - term) THS (mg/24h) ■ P1 + P2 * P3 □ P4 □ X P5 + 0 P6 O ... x i + o OV3 x X A OV4 * ------O ? l |-X ~W o A &^X X+X±#. o a M *_ ^ 9 * * -A 5 O 'o 0 n------1------r 10 15 20 25 30 35 40 weeks gestation 212 Figure 8.7 - THE excretion rate Pregnancy (week 18 - term) THE (mg/24h) - P1 5 - + P2 + * P3 □ P4 +±... X P5 4- + + 0 P6 + X + Ip 3- "T" X o OV3 + ¥ Ox Dx A OV4 # ± .X ■ r ytr . fi *o £ + f 0 * * - * * ^ BA — a t-----o ...... * * * 10 15 20 25 30 35 40 weeks gestation Figure 8.8 - Total cortisol metabolites excretion rate Pregnancy (week 18 - term) Cortisol metabolites (mg/24h) 14 . P1 + 12 - _ 4 _ + P2 * P3 1 0 - +-f-...... -4=- □ P4 □ 8 - X P5 -t ±4-# D □O 0 P6 ++ x -. 6 - O OV3 41. +... ■ x ©O®o°o o x A OV4 ¥ 4 - o % A* « * ? * - ..... ** * * 10 15 20 25 30 35 40 weeks gestation 213 Figure 8.9 - THE/THF Pregnancy (week 18 - term) THE/THF 10 * - P1 8 — + P2 * P3 X xo □ P4 X X P5 "o * 0 P6 -7^ > o OV3 + * 0 m l ...... ± ...... I.... A OV4 ^ x x+ +* + 9 . o * + 2 - •o a AO □ □ □ 10 15 20 25 3 0 3 5 4 0 weeks gestation Figure 8.10 - THF/aTHF Pregnancy (week 18 - term) THF/aTHF ■ P1 + 5 — + P2 + * P3 □ P4 + X P5 + 0 P6 + ....+...... o OV3 + +? o+ A OV4 2 - o * * 0 - □ . 4* i' +i t x +++ - ...... X 10 15 20 25 30 35 40 weeks gestation 214 (i) Pregnanediol (Figure 8.2) Pregnanediol excretion rose from approximately 3mg/24h at 5-10 weeks gestation, equivalent to the upper end of the luteal phase excretion rate range in non pregnant women, up to between 12 and 30mg/24h at near term. The rise in steroid excretion rates started at 15 - 20 weeks gestation continuing until near term and was a near linear trend, with some fluctuations, for each subject. A slight fall in levels was seen in the last 3 - 4 weeks in some of the subjects. (ii) Oestriol (Figure 8.3) Excretion rates of OE3 started to rise at around 12 weeks gestation from less than 0.5mg/24h up to between 14 and 24mg/24h near term. Normal non-pregnant excretion rates were < 50/xg/24h. The results from the eight subjects were very closely matched, up until approximately gestation week 30, rising in a near linear trend to 7.5mg/24h. After this point between weeks 30 - 35 the start of a sharper rise was seen, with a wider divergence of excretion rates. (iii) 17-hydroxypregnanolone (Figure 8.4) 170HPr rose gradually from between 0.5 - 1.4mg/24h at 18 weeks gestation up to maximum of between 2.6 and 5.5mg/24h in the second half of the final trimester. All but one (n = 5) of the subjects who had supplied urine samples in the last 5 weeks before parturition showed a fall in excretion rates of approximately lmg from the maximum in this period. This quantification is affected by 11 13-hydroxyandrosterone which has the same GC retention time as 170HPr, and so may be contributing to the excretion rates quoted. In the pregnancy urine steroid profiles analyzed on the MSD the contribution of 11 B-hydroxyandrosterone was less than 10%. In non-pregnant normal subjects 170HPr is almost undetectable or less than 20% of the measured 1113- hydroxyandrosterone excretion rate. (iv) Pregnanetriol (Figure 8.5) Pregnanetriol excretion rates were slightly higher than in non-pregnant subjects (< 0.5mg/24h), and remained fairly constant between gestation weeks 18 - 30, in the approximate range 0.3 to 1.5mg/24h, except Subject OV3 who showed a slow rise 215 up to 2.15mg/24h. After week 32 five of the six subjects who had supplied samples in this period showed varying degrees of rises in excretion rates - up to a maximum of 5mg/24h, and then a fall to between 3 and 4mg/24h in two of the subjects, and to around 2mg/24h in the other three subjects. Subject P2 showed only a small rise to lmg/24h excretion rate near term. (v) THS (Figure 8.6) THS excretion rates were below lmg/24h in gestation week 18. These then rose to varying degrees up to between 1.5 and 4.1mg/24h in the last few weeks before parturition. All values measured were above normal non-pregnant values (normally < 0.1mg/24h). These results may not be totally accurate in all samples, as other compounds at the same retention time as THS were noted in some samples run on the MSD in scan mode. A reasonable picture, however, of the changes occurring in the course of pregnancy was seen. (vi) THE (Figure 8.7) Tetrahydrocortisone excretion rates at gestation week 18 (0.9 - 2mg/24h) were in the lower two thirds of the normal range for non-pregnant females. Half of the subjects’ excretion rates rose only very slightly or fluctuated around a certain value throughout the 2nd and 3rd trimester - PI and OV3 at around 2mg/24h, and P6 and OV4 around 1.5mg/24h. The other four subjects showed varying degrees of rise in excretion rate to between 2.0 and 4.6mg/24h in the last 6 weeks of pregnancy. (vii) Total urinary cortisol metabolites (Figure 8.8) All subjects showed some degree of excretion rate rise over the study period, though the same four subjects as above with fairly constant THE excretion showed the smallest rise in total cortisol metabolite excretion. Excretion rates rose from 1.4 - 3.1mg/24h at week 18 or 19, up to 4.9 - 12.6mg/24h in the last two weeks prior to parturition. Most values, except those for Subjects P2 and P4 in the third trimester, were within the normal range for non-pregnant adult females. 216 (viii) THE:THF ratios (Figure 8.9) THErTHF ratios were fairly constant during weeks 18 to 26 in the 1.3 to 3.3 range, which was wider than, but similar to the range for non-pregnant subjects. After 26 weeks two subjects had fairly constant THE:THF ratios - P4 and PI (1.5 and 2.5 respectively. The other subjects showed varying degrees of rise in THErTHF ratios reaching maximums of 4.3 - 9.5 during the last 6 weeks of pregnancy. (ix) THFraTHF ratios (Figure 8.10) THF:aTHF ratios in the subjects excluding P2 were in the range 0.7 - 2.9 in weeks 18 to 20, roughly equivalent to normal non-pregnant subjects. The pregnant subjects shown a decline in THFraTHF ratio with gestational age, with a range of 0.2 to 1.0 in the last 5 weeks of pregnancy. Subject P2 at week 19 had a THFraTHF ratio of 5.5. This fluctuated up and down until week 29, but with a declining trend, ending with a fairly constant ratio between 1.0 and 1.5 for the 10 weeks prior to parturition. PD:OE3 ratios were also calculated in the eight normal pregnancies. Ratios greater than 10 (maximum measured = 50) were seen in the first trimester (not shown), but as the placenta established itself and produced oestrogens in larger amounts the ratio rapidly fell to below 3 by week 18 (Figure 8.11). After week 20 each individual maintained a fairly constant ratio in the range 1.0 - 2.3 until the last sample supplied before parturition, with a small fall seen in the last 5-6 weeks of pregnancy. It was noted that certain subjects had consistently high or low steroid excretion rates in the metabolites quantified here. Subjects P2 and P4, for example, both had one of the highest values throughout the pregnancy period measured for PD, OE3, 170HPr, PT, THS, THE and total cortisol metabolites, P2 showing a slight fall in all but the total cortisol metabolite excretion rate in the last 2 - 3 weeks of pregnancy. Subjects PI and P3 were fairly consistent in having one of the three lowest excretion rates for OE3, PT and THS, with the addition of 170HPr for Subject P3, and PD for Subject PI to this list. 217 Figure 8.11 - PD/OE3 Normal Pregnancies PD/OE3 * 14 ■ P1 + P2 12 6 +...... *<> + □ 4 0 +xT , + 2 ■ 0 1------1------1------r 5 10 15 20 25 30 35 40 weeks gestation 218 The androgen metabolites androsterone and aetiocholanolone were at low levels compared to the other metabolites of interest and the internal standards A,S and C. This made accurate quantification difficult, unless new extracts and derivatives had been made, but the value of the results compared to the time necessary for this was not high enough, therefore approximate ranges were calculated. The calculated excretion rates (n = 115 samples) of androsterone (range = 190 - 1490j*g/24h) and aetiocholanolone (range = 220 - 1310/xg/24h) were equivalent to the normal adult female non-pregnant range (see Tables 5.2 and 5.3). The excretion rate in each subject did not show great changes, for example rise or fall in values, or a significant change in androsterone to aetiocholanolone ratio, through the course of the 2nd and 3rd trimesters. 8.5 THDOC excretion rates All samples underwent Sephadex LH-20 chromatography before derivatization and SIM analysis. This was very important in order to remove THS and OE3 from the steroid mixture, the OE3 being a possible interference in the quantification of the analyte with ion 507, and THS with ion 476, due to the natural abundance of heavy isotopes (refer to Chapter 3 for mass spectra). The GC traces from a pregnancy urine steroid extract with and without the LH-20 chromatography step are shown in Figure 8.12. The main differences seen were the loss of the large OE3 peak at the retention time of the major peak of 3a5B THDOC, and the loss of some of the cortisol metabolites and THS. A typical result for a SIM run for a pregnancy sample is shown in Figure 8.13. This is in fact the SIM run from the same urine sample as that used for the GC urinary steroid profile shown in Figure 8.1. The analyte and the internal standard (3ji5oc THDOC) can be clearly seen in the 507 response, whilst additional peaks are seen in the 476 response, but the analyte and its co-eluting steroid peak was almost fully resolved from the peak eluting just prior to it. In a few samples there was a small amount of merging between the analyte and the peak just prior to it. In these cases the quantification was achieved by considering the start of the peak of interest a line drawn vertically down from the base of the valley between the peaks. The peak of interest in the 476 response could always be identified by comparison to the 507 response. 219 Without Sephadex LH-20 chromatography PD OE3 THE DHA 6.0E6- _Q cr And Aet 3a5B THDOC 20 22 24 26 28 With Sephadex LH-20 chromatography PD No A,S or C was added to this urine extract 8.0E6 7.0EG DHA 6.0EG 5.0EG 170H Pr 4.0EG 3.0EG 2.0EG And Aet 12 14 1G 18 20 22 24 2G T i me (mi n .) And = Androsterone 170HPr = 17-hydroxypregnanolone Aet = Aetiocholanolone PD = Pregnanediol DHA = Dehydroepiandrosterone OE3 = Oestriol pregnancy urine (30 weeks gestation) Ion 507 2.0E4- QJ (J C rd "O c D _D CL 10000“ T r~ TT T -1 7 8 9 10 i i 12 T i me (m i n. ) Ion 47G Prl Pr2 3a5B THDOC + co-eluting steroid 0) G C Pr3 rd “O c □ _Q CE 7 8 9 10 1 1 12 T i me (m i n . ) see section 8.9 for expla nation of P rl, Pr2 and Pr3 170HPr = 17-hydroxypregnanolone 221 The means of the results from the four injections into the mass spectrometer (from two extracts of urine) for all the normal pregnancy samples were plotted for ions 507 and 476 after correction for total urine volume, see Figures 8.14 and 8.15 respectively. THDOC excretion, using the ion 507 response,remained in the luteal phase of the menstrual cycle range (16 - 83/xg/24h) for the first trimester. In the 2nd and 3rd trimesters there was a rise in values to > 100^g/24h in all cases. The results show a divergence into two groups after week 25. Two subjects (PI and P2) had higher values (200-550/*g/24h) than those of the other four (P3 - P6)„ Ap33*! fr°m the latter sutyecis P3 (who peaked to 280/xg/24h in the last 4 weeks of pregnancy), ^did not exceed values above 200^g/24h throughout pregnancy. Analysis of the 476 response shows that Subject P2 again diverged from the main group of results peaking at 3500/zg/24h of 3a5fl THDOC and its co-eluting steroid. Subject P3 also showed a rise above the major group of results in the last four weeks of pregnancy. Overall, excluding the latter two subjects there was a gradual rise in excretion rate from luteal phase values (58 - 260/xg/24h) in the first trimester, up to between 300 and 1000/xg/24h. When the THDOC, calculated from the 476 response and the 507 response in an individual, was compared, a similar shape to the plots was seen, with the 476 response being higher than 507 (Figure 8.16). The ratio of 476 response:507 response calculated THDOC was plotted for each individual (Figures 8.17a-d). Fairly constant ratios, with obvious outliers (see Subject P6), were seen in all subjects. The mean ratios varied from subject to subject (range 3 -5 .5 ). 8.6 Post partum excretion rates Three of the pregnant women (PI, P2 and P3) also supplied between three and seven 24 hour urine samples post partum starting on day 3 or 4 after the birth, and continuing to day 15 (n=2) or day 32. One sample on day 21 post partum from Subject P5 was also supplied. A total of 15 samples were analyzed. Pregnanediol fell to within the normal follicular range (< 250/zg/24h) by day 6-10 post partum , whereas OE3 fell more rapidly to <50/*g/24h by day 3 - 4 in Subjects PI and P3, and day 9 in Subject P2. PD:OE3 ratios rose post partum until OE3 was no longer detected. 170HPr, PT and THS returned to normal lower values within 7 days of parturition. THE and the total urinary cortisol metabolites remained 222 Figure 8.14 - THDOC excretion rate Ion 5 07 response Normal pregnancies THDOC (ug/24h) 600 500- —I— P2 - P3 400- -B- P4 P5 300- - 0 - P6 -V- P7 200 - P8 100 - Luteal phase range 0 5 10 15 20 30 35 4025 weeks gestation Figure 8.15 - THDOC + co-eluting steroid Ion 4 76 resp onse Normal pregnancies •THDOC’ (yg/24h) 4000 —I— P2 3000- P3 -B- P4 - X - P5 2000 - -0- P6 P7 P8 1 0 0 0 - Luteal phase range 0 5 10 15 20 25 35 4030 weeks gestation 223 Figure 8.16 - THDOC comparison of 476 and 507 response THDOC (mg/24h) 3.5 3 2.5 2 1.5 1 0.5 0 15 20 25 30 35 40 weeks gestation Figure 8.17 THDOC ion 476 response : ion 507 response Normal Pregnancies 476 THDOC:507 THOOC 476 THDOC:507 THDOC 10 10 -X - P6 (b) -ifc- P3 0 5 10 15 20 25 30 35 40 0 5 10 « 20 .30 35 4025 waalta gestation waalta gaatatlon 476 THDOC:507 THDOC 476 THDOC:507 THDOC 10 10 - 0 - P6 M P6 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 weeka gaatatlon waalta gaatatlon 224 the same, or fell down to normal non pregnant values, with a drop below the individuals average excretion rate in the first 5 days post partum (Subjects PI and P3). THE:THF ratios had fallen in all four women to below the mean of non pregnant values (« 2 ) to a mean of 1.5 (range = 0.8 -1.7) by the first sample supplied post partum in each subject. THF:aTHF ratios on the other hand rose to values between 13.8 and 18.3 for Subject P2 and between 2.3 and 8.8 for the other three women. Subject P3 showed first a rise (maximum 8.8 on day 9) and then a fall in THF:aTHF ratios over the 32 day period studied, with a final ratio of 2.7 on day 32. These results were higher than normal non-pregnant subjects (mean = 1.6), but consistent with those of the women taking oral contraceptives. The THDOC excretion rate, measured using the 476 ion response, fell rapidly from a mean value >50(tyig/24h (> 1500/xg/24h for Subject P2) in the last week of pregnancy to less than 100/zg/24h by day 3 - 5 post partum , and within the follicular phase range by day 6 -10 post partum (Figure 8.18). The ion 507 response showed a similar decline in magnitude from > 100/*g/24h (> 250/zg/24h for Subject P2) in the last week of pregnancy to < 30^g/24h by day 3 -5 post partum , and within the follicular phase range by day 3-10 post partum (Figure 8.19). The ratio of the ion 476 response calculated:ion 507 response calculated THDOC fell with respect to number of days post partum from pregnancy levels (> 3) between 1 and 2 by day 15. 8.7 Pregnanediol: THDOC ratios PD:THDOC (ion 507 response) ratios (Figure 8.20) fluctuated during pregnancy, but showed either a constant or rising trend with gestation. Most values were within the luteal phase range (<85) or a factor of 2 greater than this. The exceptions to this, with higher values (up to 300) were Subject P6, who had the lowest THDOC excretion rate, and Subject P4 who had the highest PD excretion rates of all the normal pregnancy subjects. Post partum PD:THDOC (ion 507) ratios fell rapidly to within or just above the follicular phase range by day 15. 225 Figure 8.18 - THDOC excretion rate Post partum Ion 4 7 6 resp onse THDOC (ug/24h) 120 1 0 0 - —I— P2 P3 80 - 60 - 40 - Follicular phase range 20 - 0 5 10 15 20 25 30 35 Days post-partum Figure 8.19 - THDOC excretion rate Post partum Ion 6 0 7 resp onse THDOC (pg/24h) 30 25 - —I— P2 P3 20- 15 - Follicular phase range 10 - 0 5 10 15 20 25 30 35 Days post-partum 226 Figure 8.20 PD:THDOC (ion 507 response) Normal Pregnancies PD:THDOC (ion 507) PD:THDOC (ion 507) (b) 3 0 0 - 3 0 0 - - o - pa P 3 250 - 250 2 0 0 - 2 0 0 - 150 150 100 100 Luteal phase 5 0 - 5 0 - range 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 weeks gestation weeks gestation PD jTHDOC (ion 507) PD:THDOC (ion 507) P6 (d) pa 300 - 250 2 5 0 ----- 200 160 100 50 60 - 0 5 10 15 20 25 30 35 40 0 510 15 20 25 30 35 40 weeks gestation weeks gestation 227 8.8 476:507 ratios 476:507 ratios for the peak at the retention time of 3a5B THDOC were raised in all the normal pregnancies. The ratio remained fairly constant for an individual throughout pregnancy with a small rise or fall being seen in the 2nd half of the third trimester in some cases. Ratios ranged from 7 - 18, with a few outliers at 20 - 28. The mean for each subject fell within the range 9 - 15. 476:507 ratios from one subject, showing all four results for each sample, are shown in Figure 8.21, including post partum. During the latter period there was a decline in 476:507 ratios to approximately that of the pure standard by day 15 post partum. The 476:507 ratios of the internal standard used for quantification (3B5a THDOC) remained constant throughout. 8.9 Other hydroxypregnanolones in SIM runs On inspection of the ion 476 response in SIM runs of pregnancy samples, (a typical result shown in Figure 8.13), it was noted that there were consistently three additional prominent peaks present apart from those of 170HPr, 3<*5B THDOC and its co-eluting hydroxypregnanolone, and the added internal standard 3135a THDOC. From scan mode analysis it was determined that they were also hydroxypregnanolones and were named Prl, Pr2 and Pr3. Mass spectra of these additional hydroxypregnanolones are shown in Figures 8.22 - 8.24. These steroid metabolites became of interest when it was noted that their relative ratio to 3a5i3 THDOC and its co-eluting steroid (when quantified using ion 476) appeared different in the pregnancies complicated by placental sulphatase deficiency (PSD) and pre-eclamptic toxaemia (PET). The results of the latter two (See p*3*s -2AS an d Z£+) will be discussed in chapters 9 and 1 (^respectively. The ratio of the 476 response of each of these hydroxypregnanolones, in normal pregnancy, to that of the analyte and its co-eluting steroid are shown in Figures 8.25 to 8.27. In considering changes in levels of the three additional hydroxypregnanolones, it must be remembered that the excretion rates of 3a513 THDOC and its co-eluting steroid are rising throughout the same period (see Figure 8.15). Prl and Pr2 rose at a slightly faster rate than 3a5B THDOC and the co-eluting steroid. Pr3 rose at the same rate as 3a5B THDOC and its co-eluting steroid, as shown by the constant ratio throughout pregnancy. 228 Figure 8.21 - 476:507 ratios Pregnancy and post partum Subject P3 476:507 ratio 24 + 3a5B thdoc 0 305a thdoc (IS) 2 0 - 8 - 10 15 20 25 30 35 40 45 50 weeks gestation post partum 229 ngUlC O .^ " ruiuoi uiadj um ^ tii/— y u - ui mv^ invz-im j uuivauvc ui Prl (additional hydroxypregnanolone seen in pregnancy) 1 .2EGi 478 1 . 0EB (D U 8.0E5 C rd 1 17 188 ■a G.0E5 478 c / / D 4 . 0E5 _Q a: 2.0E5 U jLjL iiIJL ■**!.. I. iXf 100 200 300 400 500 Mass/Charge Figure 8.23 - Partial mass spectrum (m/z = 98 - 520) of the MO-TMS derivative of Pr2 (additional hydroxypregnanolone seen in pregnancy) 478 1 . 5E5 462 188 ~n 386 5.0E4 357 cr 298 ftdrjLlnrlin"^ V" 100 200 300 400 500 Mass/Charge Figure 8.24 - Partial mass spectrum (m/z = 98 - 520) of the MO-TMS derivative of Pr3 (additional hydroxypregnanolone seen in pregnancy) 1 . 2E5 476 100 1 .0E5: 386 (U / a 8.0E4: ~a£ 8 . 0E4i c D 4.0E4: 159 241 331 463 / 298 £ 2 . 0E4 d / 0 irt f'rf Will j .1* iIj it. 1 ■M 200 300 400 500 Mass/Charge 230 Figure 8.25 - Pr1 Ion 476 response relative to analyte peak response ratio to analyte peak t------1------r 10 15 20 25 3 0 3 5 weeks gestation Figure 8.26 - Pr2 Ion 476 response relative to analyte peak response ratio to analyte peak T 15 20 25 weeks gestation 231 Figure 8.27 - Pr3 Ion 476 response relative to analyte peak response ratio to analyte peak 6 5 —I— P2 P3 4 - B - p 4 P5 3 - 0 - P6 P7 2 P8 1 0 0 5 10 15 20 25 30 35 40 weeks gestation 232 8.10 Discussion In the first few weeks of pregnancy following implantation of the embryo progesterone secretion from the corpus luteum helps to maintain pregnancy. Previous work (Csapo et a l., 1972) in which the corpus luteum was surgically removed in early pregnancy showed that miscarriage occurred if surgery was performed before 7 weeks. (Before this time the corpus luteum thus serves as the major source of progesterone and is indispensable in the maintenance of pregnancy). As pregnancy advances, the relative contribution of the corpus luteum to circulating progesterone concentrations falls and the output from the placenta increases. In the present study a constant excretion rate of PD was seen, for the first 15 weeks of gestation, in the upper range for the luteal phase (ie, mainly corpus luteum production and then the start of placental production). This was followed by a steady rise, as the placenta completely took over the additional steroid production seen in pregnancy. PD excretion rates reported here are lower than those quoted by Klopper et al. (1969), but similar to those reported by others (Kaplan and Hreshchyshyn, 1972; Shearman, 1959). Pregnanediol excretion rates were shown to correlate with plasma progesterone production rates (Broom et al., 1983) in a study using deuterium labelled progesterone, so PD excretion rates represent a satisfactory method for evaluating progesterone output in pregnancy. Urinary 24 hour collections in fact allow a more integrated view of what the level of production is than blood samples, as there is episodic progesterone release throughout the day in luteal phase of the menstrual cycle (Veldhuis et al . , 1988), that presumably continues into pregnancy, and diurnal variation in late pregnancy (Allolio et al., 1990). Levels of oestriol excretion rates measured in normal pregnancies were similar to those reported by various groups (Klopper et a l., 1969; Maner et a l., 1963) and of a pregnant women measured weekly using GC from weeks 26 to 40 (Adessi et a l., 1975), but on the lower end of the ranges, measured by GC, reported by others (Kaplan and Hreshchyshyn, 1972; Heikkila, 1971; Brown, 1956; Lee and Wood, 1970). The range of oestriol levels reported here is much narrower than in most other reports. This is attributed to the higher resolution of gas chromatography and hence specificity of measurement used in the present studies. Goebel and Kuss (1974) showed the presence of diurnal variation of serum unconjugated oestriol in late 233 pregnancy, again showing the use of 24 hour urine collection to assess true 24 production. Little work on other urinary steroid metabolites in pregnancy has been reported. A study of urinary excretion of corticosteroid G>i sulphates during pregnancy, measured by a radioactive double isotope assay (Klein et a l. , 1971) showed similar excretion rates, of for example THE, to those in this study. That study had similar THE:THF ratios for third trimester pregnancy and post partum , THF:aTHF values were not reported. 5a-reductase activity in vitro in placentae was found to be similar in early and late pregnancy (Milewich et a l. , 1978). Pregnancy influenced the ratio of excretion of these cortisol metabolites, as shown by the change in ratios seen post partum. The fact that the THFraTHF ratio rose post partum to levels similar to those seen in women taking oral contraceptives indicates that the 5a- reductase system was being affected by the lingering progesterone and oestrogen metabolites, or the influence of hormones during lactation (all the women supplying post partum urine samples breast fed their offspring). Urinary excretion rates post partum returned to normal follicular phase ranges of pregnanediol at around six days and this matches data reported by Shearman (1959). Oestriol was reported by Brown (1956) not to reach follicular phase ranges until two weeks post partum , somewhat longer than found in the women investigated here. Data on blood oestrogen and progesterone levels suggest that they decline much more rapidly than their metabolites in urine. Within 24 hours of the delivery of the placenta serum progesterone fell to <10% of the level just prior to parturition, and serum oestradiol to <2% of the pregnant level (Willcox et al ., 1985; Kettel et al ., 1991), or <50% in the first 1-2 hours (Nachtigall et a l. , 1966). Investigations in this time of stress has focused more, in the last few years, on the use of salivary oestriol and progesterone concentrations, rather than blood samples, in order to evaluate endocrine changes prior to parturition. Salivary steroid concentrations show good correlation with the circulating concentrations of the free hormones (Lewis et al . , 1987; Meulenberg and Hofman, 1989). Salivary oestriol fell to below the assay sensitivity (<3% of pregnant levels) within 24 hours of birth (Lewis et al ., 1987). This is in marked contrast to the urinary clearance and reflects the more rapid clearance of the circulating free oestriol, and the much slower clearance and enterohepatic recycling of oestriol conjugates, which are excreted eventually in the 234 urine. Similarly there is slower clearance of conjugated pregnanediol metabolites into the urine, compared to circulating free hormone concentrations which falls into the follicular phase range by the 3rd day post partum (Lewis et al ., 1987). Salivary free hormone concentrations have also been used to evaluate progesterone to oestriol ratios in late pregnancy and around the time of birth. This ratio was found by McGarrigle and Lachelin (1984) to remain constant at approximately 1.4 in the first half of the last trimester, and then in the last four weeks of pregnancy fall from 1.4 to 0.7 on the day prior to the birth, although Lewis et al. (1987) found no change in the progesterone to oestriol ratio in the last 2 weeks of pregnancy, which does not support claims that parturition is preceded by a significant fall in the concentrations of progesterone (Turnbull et al ., 1974). The urinary PD:OE3 ratios measured in this work were initially in agreement with plasma for progesterone: OE3; a small fall was seen in the last five weeks of pregnancy in urine, though not as great as that seen in some of the saliva results. The change in ratio reflects the continuing rise in oestriol, and plateauing of progesterone seen in the last weeks before birth. The difference in clearance rate of progesterone metabolites and oestriol explains the rise in PD:OE3 ratios seen post partum. Cortisol production has also been evaluated from the salivary concentrations in the human (Scott et al . , 1990; Allolio et al . , 1990). Diurnal variation of cortisol production was preserved in pregnancy. Saliva cortisol levels in late pregnancy were found to be significantly higher than in non-pregnant women, unlike the urinary THE and total urinary cortisol metabolites in my study, except Subjects P2 and P4, the latter two subjects’ urinary cortisol metabolites decreasing in a manner similar to the salivary cortisol (Scott et al ., 1990). Urinary THDOC excretion rates have not been reported throughout pregnancy before. Third trimester levels were reported by Nolton et al. (1979b) and Ehrlich et al. (1974). Excretion rates of "pure THDOC", as measured by the ion 507 response, rose steadily from the luteal phase range up to as great as 7 times the upper limit of the latter range. When the co-eluting steroid was considered in conjunction with THDOC (the ion 476 response), a similar steady rise was seen up to a maximum of 4 times the upper limit of the luteal phase range. Although 3135a tetrahydro metabolites of steroids are rarely excreted in human urine (for example an adrenal adenoma was reported by Axelrod et al. (1969) to 235 produce 3B5a tetrahydrocortisol), the natural occurrence of the 3B5a THDOC used as the internal standard in this work had to be excluded, particularly as Pasqualini et al. (1975) reported that in late pregnancy urine the excretion of 3B5a THDOC was 2 - 3 times that of 3a5B THDOC in urine. The current studies however do not substantiate this, with almost undetectable peaks at the retention time of the internal standard, when no 3B5a THDOC had been added to a pregnancy sample. THDOC has also been reported in the liver of 2nd trimester fetuses, with approximately 10% in the form of the 3B5a isomer, after the adminstration of radioactively labelled DOC into the intact feto-placental circulation (Pasqualini et al., 1970). The fact that Subject PI did not diverge from the main group in her ion 476 response, but did in her ion 507 response provides further evidence that the co-eluting steroid is a separate steroid from an alternative metabolic pathway. The ratio of the THDOC calculated from the ion 476 response to that of the ion 507 response can be considered to approximate the ratio of the co-eluting steroid to THDOC. As fairly constant ratios, that varied between subjects, were seen, this suggests that the two steroids were the result of two biochemical pathways that metabolise progesterone (or a precursor) at a constant rate relative to one another in an individual. Post partum THDOC excretion returned to the follicular phase range at approximately the same rate as PD. PD:THDOC (ion 507 response) remained fairly constant, or rose only slightly, during pregnancy and were similar to the ratios seen in the luteal phase, confirming that there was a close correlation between progesterone and THDOC production. The difference in PD:THDOC ratios between individuals suggests metabolic pathways were not used to the same degree, or that THDOC was metabolized by gut flora to varying degrees in each individual. The presence of the co-eluting steroid was first detected by the difference in 476:507 ratio for the peak at the retention time of the analyte. This ratio was seen to be raised in pregnancy to the highest levels seen in normal subjects. Post partum the co-eluting steroid disappears rapidly from urine, and presumably even more quickly from the circulation, with 476:507 ratios returning to that expected for the pure standard of THDOC. The low levels of PD in the month post partum in conjunction with these low 476:507 ratios indicate that the re-establishment of the menstrual cycle has not occurred (and hence the presence of the co-eluting steroid 236 present under those circumstances), and that the co-eluting steroid in pregnancy was produced as a result of pregnancy, and presumably the presence of the fetus and/or the placenta. Metabolism of C2i pregnane steroids is complex in pregnancy, with at least 6 prominent hydroxypregnanolones being detected by ion 476 SIM runs after extraction and Sephadex LH-20 chromatography. The three additional hydroxypregnanolones detected other than the analyte, its co-eluting hydroxypregnanolone and 17-hydroxypregnanolone, all rose during pregnancy. Pr3 rose at the same rate as THDOC and its co-eluting steroid, suggesting it may be a metabolite from a closely related pathway. The presence of ion 117 in the mass spectra of Prl and Pr2 suggested the progesterone-like D-ring side chain structure of a C-20 reduced product, in these two hydroxypregnanolones. The stereochemistry of the A-ring is uncertain as is the location of the carbonyl group, although this cannot be at position C -ll. 237 9 - Placental sulphatase deficiency 9.1 Introduction MacDonald and co-workers suggest that the rise in DOC concentrations in the plasma of pregnant women in the third trimester was partly due to extra-adrenal 21- hydroxylation of plasma progesterone, and that oestrogen stimulates the steroid 21- hydroxylase activity in extra-adrenal tissues (MacDonald et al., 1982; Parker et al., 1983a, 1984; Casey et al., 1987). The "transfer constant of conversion" of progesterone to DOC was shown to increase in response to the action of oestrogen. Evidence to support this came from the following observations: (a) the administration of diethylstilbesterol to women pregnant with a dead fetus was associated with an increase in the transfer constant for the conversion of progesterone to DOC (MacDonald et al . , 1982); (b) the levels of DOC in the plasma of women with pregnancies that were characterized by hypo-oestrogenism, eg. those in which there were anencephalic fetuses, were decreased compared to those in women with normal pregnancies of comparable gestational age (Parker et al., 1983a); (c) the concentrations of DOC in umbilical cord plasma of anencephalic fetuses were decreased compared with those of normal fetuses, whereas plasma levels of progesterone were similar (Parker et al., 1983a); (d) the administration of diethylstilbesterol to a women pregnant with an anencephalic fetus led to an increase in plasma levels of DOC (Parker et al., 1983a); and (e) in two women with pregnancies characterized by hypo-oestrogenism, ie. fetal death, the transfer constant of conversion of progesterone to DOC was similar or decreased compared with that which was determined in the same women several months post partum (Winkel et al . , 1980a). 238 There are various causes of low urinary oestriol excretion, apart from incomplete urinary collections, including: (i) fetal death (ii) severe intrauterine growth retardation (in) fetal adrenal hypoplasia (iv) anencephaly (v) maternal glucocorticoid ingestion (vi) antibiotics (vii) maternal renal failure (viii) fetal hepatitis (ix) placental insufficiency W placental sulphatase deficiency (PSD). The opportunity arose to study PSD as a further model of the effect of hypo- oestrogenism on DOC production. The low levels of oestrogen excretion in PSD are similar to pregnancies with intra-uterine fetal death and pregnancies with an anencephalic fetus (Oakey et al., 1974; Diver et al., 1973; Parker et al., 1983a, 1984; MacDonald and Siiteri, 1965), where oestriol C19 steroid precursors are low. The work by MacDonald above, suggested that low levels of oestrogens, but normal progesterone levels, in the third trimester would result in reduced levels of DOC production. If this group, with hypo-oestrogenism, also showed similar reduced DOC production, as measured by reduced THDOC excretion, further evidence to support the MacDonald hypothesis of oestrogen stimulation of extra-adrenal DOC production would be obtained. Placental sulphatase deficiency (PSD) is characterised by markedly low maternal oestrogen excretion during pregnancy, in the presence of normal fetal growth and development, and commonly with difficulties in parturition (extended gestation, failure of cervical softening and dilation, failure to undergo spontaneous labour). It is a fairly rare condition affecting between 1 in 2000 and 10000 births (Rose, 1982; Bradshaw and Carr, 1986). PSD prevents hydrolysis and aromatization of fetal 3B-hydroxy-5-ene sulphates, these fetal products therefore being excreted almost unchanged in maternal 239 urine, as steroid monosulphates (Shackleton et al., 1983). A characteristic urinary steroid profile with gas chromatography reveals raised excretion rates of the oestrogen precursors (Taylor and Shackleton, 1979; Shackleton et al., 1980b). In normal pregnancy, pregnenolone is converted to DHA-S and 16a-hydroxy-DHA-S in the fetus (refer to the Introduction for Winter’s model of the feto-placental unit). The latter two steroids are the two main precursors of oestrogens, the conversion occurring in the placenta, requiring the enzymes steroid sulphatase, 315-hydroxysteroid dehydrogenase and aromatase. The deficiency of the active sulphatase enzyme results in very low oestrogen production. Haning et al. (1990) showed that steroid sulphatase activity was also present in the human ovarian corpus luteum, stroma and follicle, but at levels significantly lower than in the placenta. Steroid sulphatase deficiency is a genetically determined inborn error of metabolism. Originally identified as an enzyme disorder of the placenta, it was found that other tissues, of an affected subject, also lack steroid sulphatase enzyme activity. It is now known that the condition is X-linked (Shapiro et al., 1989; Tiepolo et al., 1980; Bradshaw and Carr, 1986; Crawfurd, 1982), and the offspring (almost exclusively male) of affected pregnancies have generalized sulphatase deficiency, and that the enzyme defect persists throughout life (Lykkesfeldt et al., 1985). Steroid sulphatase is firmly bound to endoplasmic reticulum (Rose, 1982; Salido et al., 1990). The nature of steroid sulphatase deficiency has been attributed to various biochemical alterations, for example, a defect in the membrane-enzyme structure (McNaught and France, 1980), a disorder of lipid metabolism, shown by reduced phospholipid content (McKee et al., 1981), and decreased amount of steroid sulphatase protein (and not the presence of an inhibitor) (van der Loos et al., 1983), possibly due to deletion of part of the short arm of the X chromosome (Shapiro et al., 1989). The latter explanation may also explain the genital anomaly cryptorchidism (Traupe and Happle, 1983), seen in some patients. Investigations of skin cells from affected subjects have provided valuable information on the association between steroid sulphatase deficiency and the dermatological condition X-linked ichthyosis, which most of the offspring suffer from (Rabe et al., 1984; Lykkesfeldt et al., 1984; Honour et al., 1985; Hameister et al., 1979; Shapiro et al., 1977; Muller et al., 1980; Harkness, 1982). Studies during pregnancy and post partum have yielded further information of the roles of oestrogens 240 in the normal processes of parturition, abnormal accumulation of cholesterol sulphate in the cervix possibly being responsible for cervical dystocia (Harkness et al . , 1983), and lactogenesis, where the low antenatal levels of oestrogens had little effect (Lykkesfeldt and Bock, 1985). There has also been some work that has shown that other placental enzymes involved in steroid biosynthesis, eg, 3B-hydroxysteroid dehydrogenase-isomerase and the aromatase complex, are deficient to some degree in some affected subjects, at least in vitro (Marton and Oakey, 1980; Oakey et al . , 1974). 9.2 Experimental Urinary steroid profiles were performed on all samples according to the method described in Chapter 2. Only small volumes of urine were available, so 1ml of urine was extracted. The results shown are the mean of two FID traces for each sample. The quantification of THDOC excretion rate was performed as described in Chapter 2, using 150ng 3B5a THDOC (IS) for all samples with 1ml urine in duplicate. Each extract was injected twice onto the MSD, the mean of the four results plotted. 9.3 Subjects Serial urine samples, collected at weekly intervals from one subject PSD1, and one urine sample, from each of 12 other women with placental sulphatase deficiency (PSD2 - PSD 13), in the third trimester (weeks 31 - 38), were studied. The number of weeks gestation of three of the subjects was not available and these three results are shown at week 41. Subject PSD1 was aged 31 years. 9.4 Steroid excretion rates (from urinary steroid profiles! The pattern seen in third trimester urinary steroid profiles, in the subjects with PSD (Figure 9.1) was different from the normal subjects described in Chapter 8. Pregnanediol again dominated the urinary steroid profile, but oestriol was all but absent. Several unusual metabolites were also seen, in particular 16a-hydroxy DHA, androstenetriol and 16a-hydroxypregnenolone. The excretion rate of the latter 241 Figure 9.1 - Urinary steroid profile from a pregnancy complicated by placental sulphatase deficiency A 14a 14b 21 23 9 16 18 1 = Andros terone 13 = Androstenetriol 2 = Aetiocholanolone 14a+bi= 16,18-dihydroxy DHA 3 = Dehydroepiandrosterone (DHA) 15 = Hydroxypregnanolone (? = Pr2) 4 = 17-hydroxypregnanolone + 16 = PD like (+ oestriol) 11 p-hydroxyandrosterone 17 = Progesterone metabolite 5 = Hydroxypregnanolone 18 = PD like 6+7 = 16a-hydnoxy DHA 19 = 16a-hydroxypregnenolone 8 = Pregnanediol (PD) ^ 20 = 3p,16a-dihydroxy-5a-pregnane-20-one (? 9 = Hydroxy androsterone like 21 = Tetrahydrocortisone 10 = Pregnanetriol 22 = Progesterone metabolite 11 = 11-oxo-androstenediol 23 = Tetrahydrocortisol + unknown compound 12 = PD like 24 = PD like + pregnenolone 25 = Cortolone 242 steroid, a metabolite of pregnenolone, was summed with those of the two other mentioned steroids to give an estimate of oestrogen precursors excretion rates. Pregnanediol excretion rates (Figure 9.2) in Subject PSD1 were within the normal range for pregnancy, starting at gestational week 13 at the top end of the normal pregnancy range, and ending up in the last few weeks of pregnancy at the lower end of the range. Subjects PSD2 - PSD 13 had PD excretion rates at the lower end of the normal range or just below it. PD was particularly low in Subjects PSD2, PSD4 and PSD13. The estimated summed excretion rates of oestrogen precursors (see above) rose steadily upward with gestational age (Figure 9.3), the androstenetriol excretion rate ending up at between 2 and 3 times the excretion rate of 16a-hydroxy-DHA and 16a-hydroxy-pregnenolone. Compared to oestriol excretion rates in normal pregnancy the summed oestrogen precursor excretion rates in Subject PSD1 were close to the upper limit of the normal pregnancy oestriol excretion rate range throughout the 2nd and 3rd trimesters. The other subjects had summed oestrogen precursor excretion rates equivalent to the normal oestriol excretion rate range or just below. The ratio of PD to the estimated summed oestrogen precursors showed a similar pattern to that seen for PD:OE3 in normal pregnancy. 170HPr excretion rates in subject PSD1 rose steadily throughout pregnancy at the same rate as in normal pregnancies, at the upper limit of normal. The other subjects had 170HPr excretion rates within the normal range, except PSD4 which was lower. Pregnanetriol excretion rates in the third trimester were within the normal range, except for Subjects PSD2 and PSD4, which were low. Subject PSD1 had average pregnanetriol excretion rates of around 1.8mg/24h (range 1.1 - 2.7mg/24h) throughout the 25 weeks studied, starting above the normal range, and then falling into the normal range in the third trimester. The other subjects, except PSD2 and PSD4 who had low PT excretion rates, were in the normal pregnant range. THS was not detected clearly enough to be quantified. Tetrahydrocortisone was in the normal pregnant range for subject PSD1 (remaining fairly constant throughout the study period), and four of the other subjects with PSD. The other eight subjects had THE excretion rates below the normal range. THF and aTHF were not clearly distinguishable in the urinary steroid profile, due 243 Figure 9.2 - Pregnanediol excretion rate Placental sulphatase deficiency PD (mg/24h) 35 — - PSD1 30- O other PSD subjects 25 - 20 - 15 - 5 - 20 25 30 35 40 | weeks gestation unknown gestation Figure 9.3 - Excretion rates of summed oestriol precursors* Placental sulphatase deficiency mg/24h 35 sum of 16a-OH-DHA, androstenetriol — PSD1 and 16a-hydroxypregnenolone 30 - O other PSD subjects 25 - 2 0 - 10 - 20 25 30 35 40| weeks gestation unknown gestation 244 to the presence of co-eluting steroids and low excretion rates, so data on total cortisol metabolites, THE:THF ratios and THF:aTHF ratios were not available. Androgens (androsterone and aetiocholanolone) excretion rates were, like in the normal pregnancies, similar to non-pregnant female subjects. 9.5 THDOC excretion rates THDOC excretion rates for Subject PSD1 were, using both ion 507 and 476 responses (Figures 9.4 and 9.5 respectively), within the normal range following the lower excretion rate group of normal pregnancy THDOC excretion rates (refer to Figure 8.14 and 8.15). A parallel fall in excretion rates to that seen for PD was seen, measured using ion 507 and 476 responses, after the steady rise in excretion rates seen until gestation week 32, but with the values still within the normal range. The other subjects with PSD had excretion rates within the normal range or slightly below it, Subjects PSD4 and PSD13 had the lowest values. The ratios of THDOC calculated in the PSD subjects, from the 476 ion response to those of the 507 ion response, were similar to those seen in normal pregnancies. Subjects PSD9 and PSD 13 had higher values than the others at 8.3 and 9.8 respectively. All the other subjects had ratios in the range 2 - 5, see Figure 9.6. 9.6 Pregnanediol:THDOC ratios PD:THDOC (ion 507 response) ratios were similar to those seen in normal pregnancy, ranging from 52 - 218 (Figure 9.7). 9.7 476:507 ratios Subject PSD1, like normal pregnant subjects had fairly constant 476:507 ratios in the second trimester (mean » 11). There was then a slight rise in the third trimester up to a mean of approximately 13. The other PSD subjects had 476:507 ratios in the range 9-23 (median = 13.5). 9.8 Other hydroxypregnanolones in SIM runs It was while analyzing the results of SIM runs from these patients that it became apparent that the additional hydroxypregnanolones seen in the ion 476 response, were not present in the same ratios to the peak at the retention time of 3a515 245 Figure 9.4 - THDOC excretion rate Ion 507 response PSD vs normal pregnancies THDOC (ug/24h) 600 Normals 600 - - a - p s d i O other PSD subjects 400 - 300- 200 - 100 - io 20 25 30 35 40 | weeks gestation unknown gestation Figure 9.5 - THDOC excretion rate Ion 4 7 6 resp on se PSD vs normal pregnancies THDOC (ug/24h) 4000 3500- Normals - B - PSD1 3000- O other PSD subjects 2500- 2 0 0 0 - 1500- 1000 - 500- 20 25 30 35 401 weeks gestation unknown gestation 246 Figure 9.6 - THDOC Ion 4 7 6 resp on se : Ion 6 07 resp on se Placental sulphatase deficiency 476 THDOC:507 THDOC 10 — PSD1 8- O other PSD subjects 7 - 5- 2 . . . 20 26 30 36 40| weeks gestation unknown gestation Figure 9.7 - PD:THDOC (Ion 507 response) Placental sulfatase deficiency PDiTHDOC (Ion 507) 350 — PSD1 300 - O other PSD subjects 250 - 200 - 150 - 100 - luteal phase 50- range 20 25 30 35 401 weeks gestation unknown gestation 247 THDOC as in normal pregnancy. A typical SIM result for a patient with PSD is shown in Figure 9.8. The ratio of Prl to the analyte peak response was found to be identical with normal pregnancies (Figure 9.9). Pr2 and Pr3, on the other hand, were found to be higher in ratio to the analyte peak (Figures 9.10 and 9.11), than in normal pregnant subjects, except in a few samples were they were in the upper half of the normal range (Subjects PSD6 and PSD9 having lower values in both cases). 9.9 Discussion The low urinary excretion rates of oestriol in PSD, agreed with those published in the literature, in which oestriol excretion rates rarely exceeded 3mg/24h at any point of gestation (France, 1979; Braunstein et al . , 1976; McKee et al . , 1981; Sherwood and Rocks, 1982; Tabei and Heinrichs, 1976; Honour etal . , 1985; France et al., 1973; Fliegner et al., 1972; Chadwick and Mumain, 1976). Oestriol precursors (16a-hydroxy-DHA and androstenetriol) and 16a-hydroxypregnenolone were found to be in similar ranges to those published by other groups (Lykkesfeldt et al., 1984; Taylor and Shackleton, 1979; Honour et al., 1985; Sherwood and Rocks, 1982; Taylor, 1982). Pregnanediol excretion rates in urine were unaffected, and within the normal range, this being in agreement with published reports (France, 1979; France et al., 1973; Oakey et al., 1974). Subject PSD1 showed a slight drop in excretion rates in the last few weeks of pregnancy, though still within the normal range. A similar drop though more dramatic was seen in a pregnant PSD patient followed serially in the third trimester by Oakey et al. (1974). Subject PSD4 was consistently low in excretion rates of all the urinary steroid volume. metabolites considered, and had a relatively low 24 hour unne collection^suggesting that this was possibly an incomplete urine collection. The change in ratio of the peak response using ion 476 of the additional hydroxypregnanolones Pr2 and Pr3 relative to the analyte peak, suggests that these two steroid metabolites are excreted at higher rates as a consequence of PSD. 3B,16a-dihydroxy-5a-pregnane-20-one, which I believe may be Pr3, is documented to be raised in this condition (Shackleton et al., 1980b). Pr2 has the same retention time as a hydroxypregnanolone raised in PSD (see Figure 9.1, peak 15). The mass spectrum of Pr2 (refer to Figure 8.23), does not fully match with the mass spectrum 248 ** v'*“ 1V,J VV“ 1Jt/AAVUVVU |/AUW11UU JU1^/1 IUU1JV deficiency Ion 507 . 2E4_ 10000" 4000- 2000 - Ion 47G 2.2E5 Pr2 2.0E5 1 . 8E5 Pr3 1 .BE5 1 .4E5 Prl 1 .2E5 c 1.0E5 13 170HPr £ 8.0E4 G.0E4 4.0E4 2.0E4 0 G 8 10 12 249 Figure 9.9 - Pr1 Ion 476 response relative to analyte peak response PSD vs normal pregnancies ratio to analyte peak 12 N orm als 1 0 - PSD1 -0 - Other PSD subjects 0 5 10 15 20 25 30 35 40 weeks gestation Figure 9.10 _ Pr2 Ion 476 response relative to analyte peak response PSD vs normal pregnancies ratio to analyte peak N orm als 10 - PSD1 -0 - Other PSD subjects 0 5 10 15 20 25 30 35 40 weeks gestation 250 Figure 9.11 - Pr3 Ion 476 response relative to analyte peak response PSD vs normal pregnancies ratio to analyte peak 12 ■ Normals O □ PSD1 O - e - Other PSD subjects o ■ : ■ 1 . . . ■ . ; I : I • : H------1------1------1------1— 5 10 15 2 0 25 30 35 4 0 weeks gestation 251 seen at this retention time in PSD. The ion 117 response was reduced, while ion 156 response was raised, relative to the strong ion 476 response, suggesting possibly a hydroxypregnanolone with a 16- or 17-hydroxyl group, along with a C-20 ketone structure. This suggests that two steroids may be co-eluting in subjects with PSD at this retention time. The Prl ion 476 response relative to the analyte peak ratio was similar to normal pregnancy, indicating that this steroid metabolite is not raised above normal in PSD, and Hie excretion rate was not affected by this condition. Subjects PSD6 and PSD9 who had the lower Pr2 and Pr3 responses relative to the other subjects, were found to have the highest two ion 476 analyte peak responses of all the PSD subjects, thus forcing the Pr2 and Pr3 relative responses down. Overall the change in responses, compared to normal pregnancies would appear to be a result of the biochemical block in these patients increasing the excretion of some additional hydroxypregnanolones, probably excreted as sulphate conjugates in urine. THDOC excretion in placental sulphatase deficiency has not been previously published. THDOC and its co-eluting steroid excretion rates were within the normal pregnant range or just below it, as determined from the subjects in Chapter 8. A similar fall in THDOC excretion rates were seen in Subject PSD1 as was seen in PD excretion rates in the last few weeks of pregnancy, this parallel drop being confirmed by relatively consistent PD:THDOC ratios (Figure 9.7). This would suggest that the DOC production rate was closely linked to the circulating progesterone levels. These findings suggest that the low levels of DOC, previously reported in pregnancies with dead and anencephalic fetuses, were possibly not the result of the extra-adrenal produced contribution to the circulating DOC pool being reduced due to the lack of oestrogen stimulation. Instead it was another undetermined factor, possibly of brain or placental origin, that was responsible. The observations in hypo- oestrogenic pregnancies may also relate to lower cortisol binding globulin than in normal pregnancy, the protein being a transport mechanism for progesterone in addition to cortisol. Oestrogen may contribute to stimulation of extra-adrenal DOC production, but is unlikely from the evidence presented here that it is the main factor. 252 10 - Pre-eclamptic toxaemia and hypertension in pregnancy 10.1 Introduction Pre-eclamptic toxaemia (PET) is a condition in pregnancy involving hypertension. DOC is a fairly potent mineralocorticoid (Harris et al ., 1967), and its production rate is raised in pregnancy (see Chapter 1). This group of subjects therefore offered an opportunity to investigate if DOC was involved in this form of pregnancy hypertension. Clinical features of PET include raised blood pressure, oedema (in particular facial), proteinuria, elevated serum uric acid, a fall in blood platelet count, increased haemoglobin concentrations and increased plasma corticotrophin releasing hormone (Rubin, 1988; Sarrel et al . , 1990; Jeske et al . , 1990). Increased levels of angiotensin I, angiotensin II, active and inactive renin are reported in pregnancy (Freund and Arvan, 1990; Symonds, 1976; Broughton Pipkin, 1988). The vascular smooth muscle of blood vessels within the maternal cardiovascular system normally exhibits resistance to angiotensin II and its pressor effects. Women with PET, on the other hand, appear to have increased responsiveness to pressor hormones including angiotensin II and vasopressin (Freund and Arvan, 1990). They also have reduced renin, angiotensin II and aldosterone levels (Weir et al., 1976). Oestriol levels in amniotic fluid were found to rise exponentially throughout normal pregnancy (Wame et al., 1978), but were significantly lower in pregnancies complicated by pre eclampsia, especially those with heavy proteinuria (Allem et al., 1969). PET occurs in approximately 5 % of pregnancies (Jeske et al . , 1990), affecting mainly primagravids, and usually develops after the 20th week of gestation, though may occur earlier in pregnancies where a hydatidiform mole or extensive molar degeneration of the placenta is present. Additional risk factors include a history of chronic hypertension, twin or multiple fetus gestation, diabetes mellitus and co existing renal disease (Freund and Arvan, 1990). 253 These pregnancies are sometimes accompanied by reduced oestrogen production (Axelsson et al . , 1978; Cleary et al . , 1969; Coyle et al . , 1962; Heikkila and Luukkainen, 1971) Loading tests using DHA-sulphate in normal uncomplicated pregnancies result in a rapid and pronounced rise in plasma oestriol. Patients with PET show a lower increase and later appearance of the peak value of oestriol (Axelsson et al . , 1978). Similarly the conversion in vitro of androgen precursors to oestrone and oestradiol is reduced in placentae from PET pregnancies compared to normal pregnancies (Rahman et al ., 1975). 10.2 Experimental Urinary steroid profiles and THDOC quantification were performed as described in Chapter 2. The results shown are the mean of duplicate results. 10.3 Subjects 24 hour urine collections from twelve women (Subjects PET1 - PET12) with hypertension in the third trimester of pregnancy were analyzed. Subject PET1 supplied two samples, A and B at weeks 37 and 38 respectively. The women were aged between 25 and 41 years. Subjects PET1, PET2, PET4 and PET5 were thought to have mild PET, and subject PET3 severe PET. The source of the hypertension . * was unknown in the other 7 subjects. 1/ ^ r 10.4 Steroid excretion rates ffrom urinary steroid profiles! The urinary steroid profiles showed a pattern similar to normal pregnancy. On examination of the steroid excretion rates, PD and OE3 were low for subjects PET1 - PET5, and in the lower half of or below the normal range for the other hypertensive subjects (see Figures 10.1 and 10.2). THE and total cortisol metabolites in all the subjects were within the normal ranges for normal pregnancy, as was 170HPr. Pregnanetriol was low for subjects PET1 - PET5, the other subjects having excretion rates within or below the normal range. 10.5 THDOC excretion rates Subjects PET1 - PET3 had low THDOC excretion rates quantified using ions 507 and 476 responses (see Figures 10.3 and 10.4). The other subjects had THDOC 254 Figure 10.1 - Pregnanediol excretion rate PET vs normal pregnancies PD (mg/24h) 3 5 ■ N orm als 3 0 o PET □ O th ers 2 5 - 2 0 - . □ 15 ■ □ : 10 H □ □ o ° o 0 d □ 5 0 T 10 15 20 25 3 0 35 40 weeks gestation Figure 10.2 - Oestriol excretion rate PET vs normal pregnancies OE3 (mg/24h) ■ N orm als 3 0 - o PET □ O th ers 25 - 20- 1 5 - □ 10 - °Qd ■i . Q& □ O 5 ■ ■ ■ a ■ □ ■ : ’ 0 -I------■ ■ 1------1— 10 15 20 25 30 35 40 weeks gestation 255 Figure 10.3 - THDOC excretion rates Ion 507 response PET vs normal pregnancies THDOC (yg/24h) 600 - ■ N orm als 500 - o PET □ O th ers 400 - 300 - □ 200 - . . . o " ■ ■: ■■■ 100 - 8 ° . - I D o§ o 0 - 0 5 10 15 20 25 30 35 40 weeks gestation Figure 10.4 - THDOC excretion rates Ion 4 7 6 resp on se PET vs normal pregnancies THDOC (ug/24h) 4000 3500 ■ N orm als o PET 3000 □ O th ers 2500 2 000 1500 1000 ■ 9 m 9 • a ■ . ■ ■ : v ' ® ■ 500 ■ : . : . . \ ■ 1 = ■©■ = ■ ' ■ - Dq 0 i------1------r 10 15 20 25 30 35 40 weeks gestation 256 excretion rates within the normal range for pregnancy except Subject PET9 who had low THDOC excretion rates using both ion 507 and 476 responses, and Subject PET6 who was low using ion 507 response. The ratio of ion 476 response : ion 507 response THDOC ranged from 2.8 to 10.6, all values within the range calculated in normal pregnancy except for in Subject PET6 which was higher. 10.6 Pregnanediol:THDOC ratios The PD:THDOC ratios of the 5 subjects with known PET did not differ significantly from the other subjects with hypertension in pregnancy, with similar values to normal pregnancy. 10.7 476:507 ratios 476:507 ratios were similar to normal pregnancy, the 5 known PET subjects having a range of 11.7 - 13.6 (median = 13.0), and the other subjects a range of 8.5 - 24.7 (median = 14.4). 10.8 Other hvdroxvpregnanolones in SIM runs The additional hydroxypregnanolones seen in normal SIM runs were also quantified in these subjects. Pr2 excretion rates for all twelve subjects were in the upper half of the normal range for pregnancy (Figure 10.6). Prl was in the upper half of the normal range or raised for the 5 known PET subjects, with subjects PET1A and PET3 having particularly high values (Figure 10.5). The other 7 subjects, except Subject PET9 had values within the normal range. All the subjects had Pr3 excretion rates in the upper half of the normal pregnancy range or were slightly raised, PET1A and PET3 again having the highest values (Figure 10.7). 10.9 Discussion The subjects investigated here had, in general, reduced PD and oestriol excretion rates, compared to normal pregnancies of the same gestations. This was also reflected in the THDOC excretion rates. This would suggest that DOC production was not raised above normal in these subjects, if anything lower, and was not contributing to the hypertension seen in pregnancy to any extent above normal. 257 Figure 10.5 - Pr1 Ion 476 response relative to analyte peak response PET vs normal pregnancy ratio to analyte peak 12 ■ N orm als 10 o PET o o □ O th ers 8 6 o 1 4 B > a ■ ■ ■ o i■ g ■ ■ ■ □ □ 5 ■ 2 . ■ 0 0 5 10 15 20 25 3 0 35 40 weeks gestation Figure 10.6 - Pr2 Ion 476 response relative to analyte peak response PET vs normal pregnancy ratio to analyte peak 12 ■ N orm als 10 o PET □ O th e rs 8 6 . O . 4 2 0 I I I I I I I I 0 5 10 15 20 25 3 0 35 4 0 weeks gestation 258 Figure 10.7 - Pr3 Ion 476 response relative to analyte peak response PET vs normal pregnancy ratio to analyte peak 12 ■ N o rm als 10 o PET □ O th e rs 8 6 O 4 o n □ 0 □ 1 0 1 1 ■ ■1 aaBllaaa|aalBj« : l • : 1 1 T 10 15 2 0 25 30 3 5 4 0 weeks gestation 259 Weir et al. (1976) reported lower (approximately half) plasma DOC levels in women with hypertension and proteinuria in pregnancy compared to normal pregnancies. Similarly the same paper reported lower levels of renin, angiotensin II and aldosterone in women with PET. These women, at least during the pregnancy, have increased responsiveness to angiotensin n , so often fail to keep their blood pressure normal due to the incorrect levels of responsiveness and hormone levels. The results for the relative ion 476 response for the additional hydroxypregnanolones seen in the SIM runs differed from both normals and subjects with PSD (see Chapter 9). Prl was raised in three of the PET samples. The high values seen in Subjects PET1A and PET9 could possibly be explained by the fact that these two samples had the two lowest ion 476 responses for the analyte peak, so that the actual excretion rate of Prl was normal and the low THDOC excretion rate had skewed the result. Subject PET3, who had severe PET, also had a high ratio, even though ion 476 response THDOC excretion rate in this sample (303/zg/24) was near to the median value for the 5 known PET subjects (320/xg/24h). Pr2 excretion in PET subjects was similar to normals, unlike in PSD where it was raised. Pr3, on the other hand, was slightly raised, unlike in PSD where it was greatly raised. Subjects PET1A and PET3 again had the highest values. From this limited data it could be postulated that pregnant subjects with hypertension have similar excretion rates relative to normal pregnancy of the additional hydroxypregnanolones quantified, except perhaps in severe PET, where Prl (and possibly Pr3) are raised. Further work would be required to determine if one of these or a precursor hormones are active in relation to water and electrolyte homeostasis. 260 11 - Other clinical situations with raised progesterone (A) RAISED PROGESTERONE IN PREGNANT SUBJECTS 11.1 Pregnancies maintained with exogenous hormones after in vitro fertilization using donated oocvtes 11.1.1 Introduction The development of steroid replacement regimes in agonadal women provides an insight into the endocrine requirements for implantation and early pregnancy. Previously it has only been possible to address early pregnancy by intervention experiments such as the classic surgery performed by Csapo et al. (1972) who removed the corpus luteum in early spontaneous pregnancy. The first pregnancy in an agonadal women on steroid replacement therapy was achieved, using a fresh 2-cell embryo, in 1984 by Lutjen et al.. Other workers have introduced alternative techniques of ovum transfer (Asch et al ., 1987; Yovich et al ., 1987) and differing HRT regimes (Serhal and Craft, 1987; Cameron et al., 1988, 1989; Devroey et al., 1989, Rosenberg et al., 1989). Steroid excretion rates in four such successful pregnancies were analyzed and the doses of hormone administered assessed. Peripheral serum oestradiol and progesterone concentrations were also monitored during the treatment period. Two further unsuccessful pregnancies were also analyzed, one of which involved triplets. 11.1.2 Subjects Six patients with ovarian failure treated by in vitro fertilization with donated oocytes were followed during pregnancy. Details are shown in Table 11.1. Pregnancies OV1 - OV4 resulted in singleton live births. 261 Table 11.1 - Details of subjects undergoing in vitro fertilization with donated oocytes Subject Age Gravidity Diagnosis Embryos OV1 24 1 Turner’s syndrome 2 x PN OV2 25 1 Gonadal dysgenesis 2 x 4-cell OV3 40 2 Perimenopausal 3 x PN OV4 39 1 Perimenopausal 3 x PN OV5 40 1 Perimenopausal 3 x PN OV6 38 1 Perimenopausal 5 x PN PN = Pronuclear embryo Table 11.2 - Cyclical regimen of hormone replacement therapy Day of cycle Evai (mg/day) P (mg/day) 1-5 2 - 6-9 4 - 10-13 6 - 14 4 - 15-16 4 25 17-26 4 50 27-28 2 - 262 11.1.3 Experimental (il Clinical Patients awaiting oocyte donation received hormone replacement with oral oestradiol valerate (E^: Progynova; Schering) and intramuscular progesterone (P: Gestone; Paines and Byrne) according to the protocol shown in Table 11.2. Treatment was formulated to mimic the ovarian steroid pattern of a normal menstrual cycle using a regime based on that of Lutjen et al. (1984). Donors were recruited from patients undergoing ovarian stimulation for IVF. Oocytes were fertilized in vitro and embryos cryopreserved at the pronuclear stage as described by Davies et al. (1990). All pregnancies arose from frozen-thawed embryos transfered on the 3rd or 4th day of P administration. In subject OV1 the "follicular phase" was extended by 3 days of E ^ administration and transfer performed on the 4th day of P administration. Subject OV6 had embryo transfer performed at another centre to the other 5 subjects, who were under the care of Dr. M.C. Davies at the Hallam Medical Centre and the Middlesex Hospital in London. Pregnancy was diagnosed by elevated serum hCG concentration 14 days after embryo transfer and confirmed by pelvic ultrasound scan. Exogenous steroid support was increased on diagnosis of pregnancy to E ^ 8mg/day and P lOOmg/day. Dosage regimes for the four successful pregnancies are shown in Figure 11.1. Monitoring of pregnancies was performed by (a) measurement of circulating steroid concentrations in serial blood samples taken weekly during the period of steroid administration, and (b) measurement of steroid excretion rates in 24 hour urine collections weekly throughout pregnancy. (ii) Analytical Plasma progesterone and oestradiol levels were measured from peripheral blood serum using established methods in the Department of Chemical Pathology (radioimmuno assay technique, kits from Immunodiagnostic Systems Ltd., UK and Baxter Healthcare, UK respectively, by Mr P. Holownia). Urinary steroid profiles were performed as described in Chapter 2. The mean of data from two FID pen plots was plotted. THDOC was quantified using the 263 method described in Chapter 2, in duplicate for each urine sample. The mean of results from 4 injections into the MSD, two from each extract, was plotted. 11.1.4 Urinary steroid profile and plasma hormone concentration results (al Successful Pregnancies Serum progesterone and oestradiol levels at intervals up to week 20 gestation for the four successful ovum donation pregnancies are shown in Figure 11.1. The excretion rates, throughout pregnancy, of the two main urinary steroid metabolites pregnanediol and OE3, are shown in Figures 11.2 and 11.3 respectively. Rising serum concentrations of oestradiol were seen whilst the dosage of exogenous hormone support was constant, and continued to rise when E ^ was withdrawn. A rise in urinary oestriol was seen from about 15 weeks. Urinary oestriol levels were indistinguishable between the ovum donation and normally conceived pregnancies. During the first 14 weeks of pregnancy with exogenous progesterone administration, pregnanediol excretion in the ovum donation pregnancy group was higher than in the normal pregnancy group if all samples before gestation week 15 were considered ( p< 0.001). Urinary pregnanediol excretion rates were similar after week 16 of gestation in the two groups of pregnancies. On withdrawal of treatment there was no effect on circulating serum oestradiol but the progesterone concentrations fell in Subjects OV1, OV3 and OV4, although pregnancy continued. Subject OV1 also supplied a sample 7 months after the birth of her son. OE3 was not detected and PD excretion was 200/xg/24h (within the normal range of the follicular phase of the menstrual cycle). This woman was unique in having excretion rates of DHA, during pregnancy, in excess of normal. There was a slow general decline, from week 5 (3.4mg/24h) to week 34 (1.7mg/24h). This was followed by a sharp fall to the 0.34mg/24 seen in gestation week 38. 7 months post partum the DHA excretion rate was 2.2mg/24h (above the normal non-pregnant range). 264 iue 11 ramn rgm n lsahroe eut fo fu successful four from results hormone plasma and regime Treatment - 11.1 Figure Dose E«i (m g ) Serum E 2(pmol/l) • Dose Eyai (m g ) Serum E 2(pmol/l) • 24000 20000 12000 16000 16000 12000 8000 000 8 4000 4000 10 10 _ J _ I trimester by exogenous hormonesexogenous by trimester donated oocytedonated Weeks of pregnancy Weeks of pregnancy i -- 1 in vitroin 20 00 £ 0 30 0 40 300 200 200 100 0 S 100 150 150 100 100 | fertilization pregnancies maintained in the first first the in maintained fertilization pregnancies 8000 « • 16000 12000 12000 16000 4000 0 0 0 8 0 0 0 4 14 10 I --- Weeks of pregnancy Weeks of pregnancy ----- 1 8 0 2 18 20 20 c 0 0 2 300 100 100 300 150 200 0 S 100 150 100 265 Figure 11.2 - Pregnanediol excretion rate in exogenous hormone maintained pregnancies with oocyte donation PD (mg/24h) OV1 -3K- OV2 OV3 OV4 X OV5 OV6 10 15 20 25 30 35 40 weeks gestation Figure 11.3 - Oestriol excretion rate in exogenous hormone maintained pregnancies with oocyte donation OE3 (mg/24h) OV1 OV2 -B- OV3 OV4 X OV5 OV6 "i------r 10 15 20 25 30 35 40 weeks gestation 266 £b) Unsuccessful pregnancies Only two urine samples were provided by subject OV5, in gestation weeks 5 and 8. In the first urine sample the OE3 excretion rate was in the normal range for gestation, and the PD excretion range was in the expected range compared to the four pregnancies described above. Three weeks later, however, PD had fallen to less than 0.5mg/24h, less than in normal pregnancy. Low plasma hCG concentrations confirmed that the pregnancy was no longer viable. Subject OV6 maintained her triplet pregnancy until week 22, when she had a spontaneous miscarriage. Urinary excretion rates of PD and OE3 from gestation weeks 9 to 21 for this patient are shown in Figures 11.2 and 11.3 respectively. OE3 excretion rates were slightly raised until week 16 above those seen in the normal and the successful singleton ovum donation pregnancies, whilst PD excretion rates were at the lower end of the range of PD excretion rates seen in the other ovum donation pregnancies, though still above that seen in normal pregnancies up until week 13. A gradual decline in PD excretion rates was seen in this patient after week 16, up until her miscarriage. 11.1.5 THDOC excretion rates THDOC excretion rates, using ions 507 and 476, for all the ovum donation pregnancies are shown in Figures 11.4 and 11.5. Subject OV5 showed a marked drop in THDOC excretion between gestation weeks 5 and 8, paralleling the PD excretion rates. Subjects OV1 - OV3 had, from the ion 507 response, THDOC excretion rates similar to normal pregnancies, whilst Subject OV4 had slightly raised THDOC excretion rates, and Subject OV6 was greatly raised above the normal range up until week 16. The ion 476 response THDOC excretion rate for the 4 successful pregnancies was in the upper half or just above the normal pregnancy range in the first trimester, after which they were similar to normal pregnancy, again being in the upper half of the normal range in the 3rd trimester. The post partum sample from Subject OV1 had an excretion rate of 9/zg/24h (within the normal range for the follicular phase of the menstrual cycle). The calculated ion 476 response : ion 507 response THDOC ratios were similar to normal pregnancies, with ratios in the range 2.9 to 8.8, results for each 267 Figure 11.4 - THDOC Ion 507 excretion rate in exogenous hormone maintained pregnancies with oocyte donation THDOC (ug/24h) 300 OV1 -5K- OV2 - B - OV3 200 OV4 X OV5 —I— OV6 100 5 10 15 20 25 300 35 40 weeks gestation Figure 11.5 - THDOC Ion 476 excretion rate in exogenous hormone maintained pregnancies with oocyte donation THDOC (ug/24h) 2000 OV1 OV2 1500- - a - OV3 OV4 X OV5 1000- I— OV6 500- 0 5 10 15 20 25 30 35 40 weeks gestation 268 subject remaining fairly constant. No difference was seen between the times the subjects were on and off replacement hormone therapy. 11.1.6 Pregnanediol:THDOC ratios The PD:THDOC (ion 507 response) in the successful pregnancies were similar to those seen in normal pregnancies with all ratios in the range 49.5 - 258.5, each subject remaining fairly constant. Subject OV6 had the lowest PD:THDOC ratios in the range 40.7 - 59.5. No difference was seen in the ratios between the times when the subjects were on and off replacement hormone therapy. 11.1.7 476:507 ratios 476:507 ratios were raised above that expected for THDOC, with mean values in the range 10 - 14.5. The post partum sample from pregnancy OV1 had a 476:507 ratio identical to that of the THDOC internal standard. 11.1.8 Other hydroxypregnanolones in SIM runs The relative excretion rates of the three additional hydroxypregnanolones seen in the SIM runs were similar to ratios in normal pregnancy (Figures 11.16- 11.18). Prl and Pr2 in Subjects OV1 and OV4 between weeks 5 and 12 were lower than the results seen in the normal pregnancies. This could however be due to the limited number of data points in the first trimester in the normal pregnancies, and the fact that the ovum donation pregnancies had THDOC excretion in the upper half of the normal pregnancy range, thus making the relative Prl and Pr2 excretion rates lower. The three pregnancies that had samples extending beyond the cessation of hormone replacement showed a plateau of relative Prl excretion immediately the progesterone was stopped, just as in three of the normal pregnancies, which also show a plateau of relative Prl excretion at around the same time in gestation. 11.1.9 Discussion Pregnancy without ovaries provided a model to study the endocrine development of the feto-placental unit in the absence of the corpus luteum. The only previous work was performed by Csapo et al . (1972) who surgically removed the corpus luteum in early pregnancy; miscarriage occurred if surgery was performed 269 Figure 11.6 - Pr1 Ion 476 response relative to analyte peak response Ovum donation vs normal pregnancy ratio to analyte peak 6 Norm als 5 OV1 OV2 OV2, OV3 & 0V6 4 -a- OV3 OV4 3 OV6 2 1 f Progesterone stopped 0 0 5 10 15 20 25 30 35 40 weeks gestation Figure 11.7 - Pr2 Ion 476 response relative to analyte peak response Ovum donation vs normal pregnancy ratio to analyte peak 6 Norm als 5 OV1 OV2 4 -S- OV3 OV4 3 OV6 2 1 0 0 5 10 15 20 25 30 35 40 weeks gestation 270 Figure 11.8 - Pr3 Ion 476 response relative to analyte peak response Ovum donation vs normal pregnancy ratio to analyte peak 6 N orm als 5 OV1 OV2 4 -B- OV3 OV4 3 OV6 2 1 0 0 5 10 15 20 25 30 35 40 weeks gestation 271 before 7 weeks. Before this time the corpus luteum thus serves as the major source of progesterone and is indispensable in the maintenance of pregnancy. As pregnancy advances, the relative contribution of the corpus luteum to circulating progesterone concentrations falls and the output from the placenta increases, a rise being seen at 6 - 8 weeks (Yoshimi et al., 1969; Tulchinsky and Hobel, 1973; Radwanska et al., 1978). In order to maintain pregnancy in women without functional ovaries, and hence no corpus luteum, exogenous progesterone and oestradiol need to be administered. In this study four such successful pregnancies are described. Serum oestradiol concentrations are slightly lower than those reported for normal pregnancies (Tulchinsky et al. 1972; Tulchinsky and Hobel, 1973), but similar to those of exogenous hormone maintained embryo transfer pregnancies monitored by Ben-Nun et al (1989). The use of the non-invasive urinary steroid profile method for monitoring steroid excretion rates has not been previously described in this clinical situation. The levels of oestriol measured in normal pregnancies were similar to those reported by Klopper et al. (1969) and of a normal pregnant women measured weekly using GC from weeks 26 to 40 (Adessi et al ., 1975), but on the lower end of the ranges, measured by GC, reported by Kaplan & Hreshchyshyn (1972) and Heikkila (1971). The range of oestriol levels reported here is much narrower than in other reports. cap* M ry This is attributed to the higher resolution of^gas chromatography and hence accuracy of measurement used in the present studies. The rising serum concentrations of oestradiol, whilst the dosage of exogenous hormone support was constant (from 10 -12 weeks), indicated that the feto-placental unit was contributing to circulating hormone levels. This was corroborated by the rise in urinary oestriol seen at about 15 weeks. Pregnancies without functional ovaries achieved with the help of IVF had identical oestriol levels to normal pregnancies showing that the dosage of oestradiol used for hormone replacement was probably ideal. The high levels of pregnanediol excretion seen in the exogenously maintained pregnancies indicated that the daily dose of progesterone administered was supraphysiological. The majority of blood samples were taken just prior to the injection of the intramuscular progesterone. Serum concentrations measured in this 272 study were thus lowest of the day. The serum progesterone levels were however similar to those reported by Yoshimi et al. (1969), but higher than, or at the upper end of the ranges, reported by others using RIA (Tulchinsky et al ., 1972; Tulchinsky and Hobel, 1973; Radwanska et al ., 1978). A fall in serum progesterone concentration similar to that reported here was also seen by Ben-Nun et al. (1989) on treatment withdrawal. The use of a daily depot preparation minimised discomfort to the patient and gross fluctuations in serum concentration. Reassessment of the dose must take into consideration that the steroid administered reduces with time. Serum concentrations of circulating progesterone between consecutive injections would have to be measured more frequently to avoid prolonged periods of progesterone below that required for the maintenance of early pregnancy. There are theoretical risks of fetal malformations following progesterone therapy during pregnancy (Rock et al ., 1985; Check et al . , 1986). The incidence in those studies was not found to be higher than in other pregnancies and is extremely unlikely given the doses of natural progesterone used in this study. As successful pregnancies for women requesting treatment for infertility are so precious, it is unlikely that clinicians would reduce the hormone support so avoiding any possibility of miscarriage due to insufficient progesterone. The results should however warrant the confidence of clinicians that the dosage is sufficient, but the possibly excessive progesterone needs to be explored further. The PD: THDOC (ion 507 response) in the successful pregnancies were similar to those seen in normal pregnancy, suggesting that the raised circulating progesterone was converted to DOC at a similar rate as in normal pregnancies. Subject OV6 however showed a slightly lower ratio, as a result of greatly raised THDOC but the lowest PD excretion rate of the hormone replacement pregnancies (though still above normal in the first 15 weeks of gestation). This greatly raised THDOC may reflect the variability in the extra-adrenal conversion of progesterone to DOC, or relatively less metabolism of THDOC in the gut and hence greater recovery of THDOC in the urine. Twin or multiple pregnancies have been shown to have raised urinary and serum oestriol (Westergaard et al ., 1985; Heikkila and Luukkainen, 1971). This is the result of more than one fetus supplying the precursors to this oestrogen. Subject OV6 (triplet pregnancy) did appear to have slightly raised urinary OE3 excretion until 273 week 16, after which time it fell to the lower limit of results seen in normal pregnancy. The PD excretion rate in subject OV6 was initially similar to the other hormone replacement pregnancies, but as the progesterone was withdrawn the feto placental unit did not adequately take over the production of oestrogens and progesterone required for the maintenance of pregnancy, possibly contributing to the miscarriage. Extension of replacement therapy may possibly have saved this pregnancy, but multiple pregnancies are always more at risk than singleton pregnancies for miscarriage. Subject OV1 had raised 476:507 ratios, indicating the presence of the co eluting steroid seen in pregnancy, as is seen in all the pregnancy urine samples analyzed. 7 months post partum the 476:507 ratio indicated that only pure THDOC was being quantified in the analyte peak. This woman had Turner’s syndrome, so had non-functional ovaries. The lack of co-eluting steroid in this patient suggests that the co-eluting steroid seen in the menstrual cycle was of functional ovarian origin. 274 11.2 Ovarian theca-lutein cvsts (hvperreactio luteinalis) 11.2.1 Introduction This is a condition, in pregnancy, where there are multiple ovarian luteinized cysts, usually enlarging both ovaries and is normally a result of very high secretion rates of chorionic gonadotrophin (hCG). Raised androgens cause the virilization seen in 25% of cases. Progesterone and 17-hydroxyprogesterone levels are also greatly raised. 11.2.2 Case history A history of hirsutism, in this 22 year old woman, prior to pregnancy was reported. Oral contraceptives had been taken until 1-2 months prior to conception. The patient had been admitted to hospital during her pregnancy because of generalized oedema. At 28 weeks gestation serum testosterone was found to be > 30nmol/L. Marked hirsutism, vulval swelling and bilateral large ovarian cysts were also reported on ultrasound at this time. A diagnosis of hyperreactio luteinalis was made. Pregnancy continued to 37 weeks when an elective caesarian section was performed. The offspring was a healthy male. Four 24 hour urines were supplied for steroid profile analysis with the patient on the following treatments: weeks gestation therapy 30 2nd day dexamethasone suppression test lmg qid. 30 4/7 No therapy 30 5/7 No therapy 31 4/7 Prednisolone 7.5mg/day for 4 days Further urine samples were analyzed from both mother and child at one week post partum. 11.2.3 Results The steroid profile results are shown in Table 11.3. The steroid profile for 30 4/7 weeks gestation (no therapy) is shown in Figure 11.9. Androgens (androsterone and aetiocholanolone) were well above the normal range (between 3 and 9 times normal) in all 5 samples. The progesterone metabolites 17-hydroxy- 275 Table 11.3 - Urinary steroid profile results from a patient with ovarian theca-lutein cysts (/*g/24h) o o X PL, weeks gestation And Aet l-H PD PT OE3 THS THE THE/THF —Hi t 8 VO 00 CO o 9870 7430 13620 19000 15000 2850 1720 H - 30 4/7 11020 6890 21450 25000 15820 0966 3670 2780 1.4 30 5/7 9060 4670 15000 5590 3090 2330 9*0 098 31 4/7 3820 2470 0006 7320 7990 2680 2240 0.5 | 2 e C/5 8>la * o a l 00 8 3 8 normal range 30-32 ON 8 1 1 12000-25000 850-2200 7500-10000 1250-4300 1.8-6.8 weeks gestation 0006 P 2^ 2 2 c 00 h O oo B 00 a o 1 276 Figure 11.9 - Urinary steroid profile from a patient with theca-lutein cysts 3 6 1 = Androsterone 2 = Aetiocholanolone 3 = 17-hydroxypregnanolone 4 = PD like 5 = Pregnanediol (PD) 6 = Pregnanetriol 7 = Oestriol 8 = Tetrahydrocortisone (THE) 9 = Tetrahydrocortisol (THF) 10= 5a-THF 277 pregnanolone (170HPr) and pregnanetriol (PT) (see Figure 11.10) were raised above the normal range for gestational age. Pregnanediol (PD) excretion was in the normal range for the first three samples, but slightly below normal when the patient was taking prednisolone. Post partum all three progesterone metabolites were excreted at rates well above normal. Oestriol was within or just below the normal range for all samples except when the subject was taking prednisolone, when it was below the normal range for gestation. Tetrahydrodeoxycortisol (THS) was above the normal range both during pregnancy (when taking prednisolone in the normal range) and post partum. Cortisol metabolite excretion, as measured by tetrahydrocortisone (THE), was partially suppressed when the subject was taking prednisolone, but otherwise within the normal range. The THE/THF ratio was low in all the pregnancy samples but high in the post-partum sample, as was found in normals immediately post-partum (refer to Chapter 8). The values of urinary steroid excretion measured in the second sample when off treatment (weeks gestation 30 5/7 weeks) were a mean value of 74% of other off treatment sample, suggesting that the second sample was perhaps not a complete 24 hour collection. THDOC values, as measured by ion 507, follow the same pattern as PD excretion, with all samples except when the subject was taking prednisolone being above the normal range. Using ion 476 all the pregnancy samples fall within the normal range, but the value post partum was high. The results for THDOC are shown in Figure 11.11. The steroid profile of the male offspring was that of a normal newborn and will not be discussed in this thesis. 11.2.4 Discussion One cause of virilization in pregnancy is hyperreactio luteinalis (or theca-lutein cysts). This is a condition where there are multiple luteinized cystic tumours, usually affecting both ovaries. The condition occurs when there is an abnormally high rate of secretion of chorionic gonadotrophin (hCG), such as in pregnancies associated with large placentas, for example multiple pregnancy, trophoblastic disease and erythroblastosis (van der Spuy and Jacobs, 1984). The source of the raised hCG was not determined in the patient described here. Furthermore in hyperstimulation syndrome, such as in infertile patients treated with gonadotrophins or clomiphene 278 Figure 11.10 - Urinary steroid excretion rate Theca-lutein cysts mg/24h 30 i d e x prednisolone 1mg qid n o t h e r a p y 7 .5 m g /d 25- 2 0 - 1 0 - * 30 31 p o s t Weeks Gestation p a rtu m -B- 170HP' PD PT OE3 Figure 11.11 - THDOC excretion rate Theca-lutein cysts THDOC (mg/24h) d e x prednisolone 1mg qid no th e r a p y 7 .5 m g /d 0.8 - 0 . 6 - 0.4 - 0 . 2 - 30 31 p o s t Weeks Gestation p a rtu m Ion 476 I...I Ion 507 279 citrate of the ovary, has a similar histological appearance to theca-lutein cysts (Hensleigh and Woodruff, 1978). Prolonged and elevated exposure to hCG may stimulate the sensitive gonadal stroma, a known source of androgens, to produce this pathological lesion (Berger et al., 1984). Ovarian follicles are known to possess the capacity for side chain cleavage of C2i steroids and also have 17-hydroxylase and 16a-hydroxylase activities. They however lack 1 IB- and 21-hydroxylase activities (Sano et al ., 1983). Steroid levels have been reported to be very similar in fluid from pre-ovulatory follicles and in theca-lutein cysts (Vanleuchene et al . , 1983). The progestins present, in particular progesterone and 17-hydroxyprogesterone, but also 16a-hydroxyprogesterone and 20- dihydroprogesterone are potential precursors of the raised progesterone metabolites seen in the urinary steroid profiles of these patients (Bevan et al . , 1986). Likewise the principal androgens dehydroepiandrosterone-sulphate and androsterone-sulphate account for the high concentrations of urinary androgen metabolites, androsterone, aetiocholanolone, and in some cases 16a-hydroxydehydroepiandrosterone. There are few published reports of steroidogenesis in patients with theca-lutein cysts. Stitch et al. (1966) reported of a case of a molar pregnancy associated with increased urinary PT excretion. In that case report it was noted that, on removal of the mole, the ovaries were seen to contain massive theca-lutein cysts, which enlarged further, along with a rise in urinary PT output. The latter only returned to normal as the cysts regressed, making the cystic ovaries the most likely source of the PT precursor. Testosterone was also been found to be raised by the largest amount in women with trophoblastic disease in whom the ovaries were greatly enlarged (Samaan etal., 1972). Work by Abraham (1974) showed that the steroids progesterone, 17- hydroxyprogesterone and oestradiol which were found to rise to a mid cycle peak were not diminished by dexamethasone suppression, indicating non-adrenal, probably ovarian origin. The former two steroids are those present in excess in the cases of theca-lutein cysts reported in the literature and in the case reported here. Theca lutein cysts, similarly ovarian tissue, possess the enzymes required for progestin, androgen and oestrogen synthesis. Bevan et al. (1986) therefore suggested that the excess steroid secretion resulted from the enormous proliferation of the theca-lutein cells under the influence of hCG. They also suggested that the additional steroids in 280 the profiles of the cystic subjects they studied were a result of the saturation of the normal metabolic excretion pathways, activating other normally minimally used pathways. In the patient described here, the production of PD when off treatment, or on low dose dexamethasone, was in the upper end of the normal range, and when on prednisolone treatment only just fell below the normal range. PT and 170HPr, the main urinary 17-hydroxyprogesterone metabolites, were both raised above the normal ranges for gestation, in particular when off treatment. This could suggest that the pathway leading to PD production was saturated as suggested by Bevan et al. (1986). With this amount of progesterone present in the body DOC levels as measured by THDOC excretion, 0.53 and 0.85mg/24h when off treatment and 0.63mg/24h on dexamethasone, were raised above normal as measured for the same gestation by ion 507 (0.1 - 0.35mg/24h). The level fell to within the normal range when the subject was taking prednisolone. A fall in PD excretion was also seen in this sample, indicating circulating progesterone levels were not as high. Interestingly in all four samples during pregnancy THDOC as measured by the ion 476 response in SIM fell within the normal range, indicating that the production of the co-eluting steroid was possibly independent of THDOC production. Post partum the progesterone metabolites 170HPr, PD and PT were all still raised as was the excretion of THDOC as measured by both ions 507 and 476 responses. This would indicate that the co-eluting compound is likely to be of progesterone origin, though again independent of THDOC production as the ratio of ion 476 to ion 507 measured THDOC had risen (0.95 - 1.75 during pregnancy, 3.54 post partum). The fact that the metabolites were all still raised post partum was due to the fact that the cysts had not regressed fully at one week post partum and were still secreting steroids. Unfortunately a sample was not available after this point so that full regression could not be demonstrated. Cortisol excretion as measured by tetrahydrocortisone (THE) was found to be within the normal range, and slightly decreased when on prednisolone. The THE/THF ratio however was reduced in all four pregnancy samples, both on and off treatment. The conversion of cortisol (F) to cortisone (E) is mediated by the enzyme 1 lJ3-hydroxysteroid dehydrogenase (11BOHSD) (see also Appendix 1). In normal subjects, the kidney using this enzyme, may protect the mineralocorticoid receptors 281 from cortisol by converting F to E before it reaches the distal nephrons. The enzyme has also been detected amongst other tissues in the placenta. Using homogenates of placenta at full term the conversion in vitro of F to E was detected (Ldpez Bernal et al., 1980). The fetus can convert F to E and the above metabolism may serve as a mechanism to help protect the fetus against high levels of maternal cortisol, ensuring the fetal adrenal has control of circulating cortisol levels in the fetus. Ldpez Bernard found that in vitro that 11BOHSD in the placental homogenates was inhibited by cortisone (product inhibition), dexamethasone and prednisolone (being llB-hydroxy competitors of cortisol) and high levels of progesterone. Oestriol and 178-oestradiol had little or no effect. In this patient high levels of progesterone are certainly present. This could possibly be the reason for high F to E ratios as shown by the THF:THE ratio, due to inhibition of 11BOHSD. The male offspring was found to be normal. In pregnancy when androgen levels are high fetal mascularization often occurs. This does not usually happen in pregnancies complicated by hyperreactio luteinalis even though androgens are high and virilization of the mother can occur (Hensleigh and Woodruff, 1978). It is thought that the conversion of androgens to oestrogens in the enlarged placenta is responsible for the protection of the fetus against the maternal high androgen concentrations. 282 11.3 Pregnancy complicated bv 21-hvdroxvlase deficiency congenital adrenal hyperplasia Pregnancy in a a patient with 21-hydroxylase deficiency CAH offers the opportunity to investigate two simultaneous sources of progesterone. A urine sample was obtained from one such patient (aged 31 years) at 37 weeks gestation, who was being treated with dexamethasone. The efficiency of the treatment had been questioned by the referring hospital, because plasma 17-hydroxyprogesterone concentrations were not fully suppressed. In early pregnancy the excess steroid excretion due to the CAH had been controlled. The urinary steroid profile at 37 weeks gestation showed that the androgens, androsterone and aetiocholanolone, were almost completely suppressed at 220 and 660/xg/24h respectively. Cortisol metabolite excretion rates were also low. Pregnanediol and oestriol excretion rates were appropriate for gestation. However 17-hydroxypregnanolone and pregnanetriol excretion rates were both raised significantly above those seen in normal pregnancy, at 11800 and 13060/xg/24h respectively. 11-oxo-pregnanetriol was also detected but was not particularly raised. THDOC excretion in this sample was calculated using ion 507 and 476 ion responses at 259 and 1315^g/24h respectively. This was at the upper limit of normal in both cases. Two urine samples two weeks post partum on dexamethasone were also supplied. All steroids were almost fully suppressed in both samples. THDOC was difficult to measure, see Section 11.8 of this chapter. Approximate values of 60 and 39/ng/24h using ion 476 were calculated. If these values are accurate this is above normal for post partum. The almost completely suppressed steroids seen post partum ruled out that the raised 17-hydroxyprogesterone metabolites (170HPr and PT) were of a tumour source in this patient. The normal adrenal ACTH drive was well suppressed, as evidenced by the low androgens. The adrenal function in this patient in pregnancy could possibly have been maintained by some other factor, for example an ACTH like hormone produced by the placenta. 283 11.4 Pregnancy complicated bv suspected late-onset 21-hvdroxvlase deficiency CAH A sample from a 26 year old pregnant women was analyzed. She had a normal menarche at age 13 with fairly regular periods, but subsequently at age 18 had developed amenorrhoea. This was diagnosed as polycystic ovary syndrome, due to the mild androgen excess. She was treated originally with dexamethasone, but stopped the medication on wishing to start a family. Regular menstruation was reestablished after two irregular cycles. She was normotensive with modest evidence of androgen excess, and had normal genitalia. Her identical twin sister, living in Chile, had been diagnosed as having late-onset CAH. The urinary steroid profile (Figure 11.12) of the urine sample supplied in gestation week 12 showed appropriate pregnanediol and oestriol excretion rates. The striking feature of this urinary steroid profile was the greatly raised DHA excretion rate, at 5180^g/24h. The DHA excretion rate in women is normally less than that seen for androsterone and aetiocholanolone, which in this patient were raised at 2390 and 2050^g/24h respectively. 170HPr and PT excretion rates were also raised above the normal range. The THDOC excretion rate was slightly raised using the ion 476 response at 472/>ig/24h, and at the upper limit for normal pregnancies using the ion 507 response at 77/xg/24h. The initial pattern of raised androgens, 170HPr and PT seems to point to a diagnosis of late-onset CAH. The androgen excretion rate, however, in particular that of DHA, was disproportionately high for a metabolic block in steroid synthesis, making the diagnosis of conventional late onset 21-hydoxylase CAH unlikely. An undetermined alternative source of androgen was more likely. 284 Figure 11.12 - Urinary steroid profile from a pregnancy complicated by suspected late-onset 21-hydroxylase deficiency CAH 3 1 = Androsterone 2 = Aetiocholanolone 3 = Dehydroepiandrosterone (DHA) 4 =s 17-hydroxypregnanolone 5 = Pregnanediol 6 = Pregnanetriol 7 = Oestriol 8 = Tetrahydroc or tisone 9 = T etrahydrocortisol 10 = 5a-tetrahydrocortisol 11 = a-cortol 12 = P-cortol + P-cortolone 285 The following two cases were not investigated due to raised progesterone, but are included in this section as they are two interesting cases involving pregnancy. 11.5 Pregnancy complicated bv suspected Cushing’s syndrome A urine sample was analyzed from a women in her 27th week of gestation, who presented with Cushingoid features. Cortisol metabolites were calculated to be at the upper end of the normal range for gestation in normal pregnancy. Pregnanediol and oestriol were just below normal for gestation at 7730 and 4520^g/24h respectively. PT and THS were above the normal range at 3800 and 2010/*g/24h respectively, whilst 170HPr and the androgen metabolites were normal. The THDOC excretion rate in this sample using the ion 507 response was 623 jtg/24h. This was almost double the highest value seen in normal pregnancy at the same gestation. Ion 476 response calculated THDOC was at the upper limit of normal for the same gestation at 1350/*g/24h. The 476:507 ratio was raised above that expected for THDOC at 9.2. For comparison, THDOC excretion rates were also measured in 3 adult non pregnant subjects with Cushing’s syndrome, on and off metyrapone. Subject A (d) had a basal THDOC excretion rate of 760^g/24h, which rose to 2390^g/24h on metyrapone. Subject B (9) supplied two basal and two urine samples when on treatment, which gave excretion rates of 138, 88, 3370 and 2783fig/24h respectively. In subject C (9) the basal THDOC excretion rate rose from 56^g/24h to excretion rates of 846 and 5960^g/24h on the following days on metyrapone. All of these samples had 476:507 ratios equivalent to THDOC. In all three non-pregnant subjects the basal THDOC excretion rates were raised above the normal non-pregnant range. The raised DOC and S production in the pregnant women described here could therefore be a result of mild Cushing’s syndrome superimposed on top of the raised DOC seen in pregnancy. 286 11.6 Pregnancy in a post pituitary operation patient A urine sample from this pregnant woman was sent to our laboratory to assess her adrenal status as she had undergone a pituitary operation prior to pregnancy. We were not informed of the number of weeks gestation, but using the pregnanediol and oestriol excretion rates, she was estimated to be in gestation week 27. The cortisol production rate was appropriate for this gestation. All other steroid metabolites quantified were within the normal pregnant range. The THDOC excretion rate in this sample (973/xg/24h) was almost four times as high as the upper limit of normal for the ion 507 response at gestation week 27, and double the maximum seen at any point in normal pregnancy. The ion 476 response THDOC excretion rate was also raised at 3039/xg/24h, approximately double that seen at gestational week 27 in normal pregnancy. The adrenal status would appear to be near normal, based on the cortisol excretion rate, despite some disruption of the pituitary. The source of her greatly raised THDOC excretion rate remains unclear, but if the adrenal were normal an extra-adrenal source of DOC would be indicated in this patient. 287 (B) RAISED PROGESTERONE IN NON-PREGNANT SUBJECTS 11.7 Ovarian progesterone secreting tumour A 24 hour urine sample was analyzed from a woman, age 38 years, with an ovarian granulosa cell tumour. The urinary steroid profile is shown in Figure 11.13. Androgen and cortisol metabolites were within the normal range, but 17-hydroxy- pregnanolone, pregnanediol and pregnanetriol were all raised above normal at >400(Vg/24h. The THDOC excretion rate was quantified, using the method described in Chapter 2. Ion 507 and 476 responses gave excretion rates of 393 and 540^g/24h respectively. These results were higher than the upper end of the normal range for the luteal phase of the menstrual cycle (83 and 260/xg/24h respectively), and fell within the upper end of the normal range for ion 507 response, and within the normal range for ion 476 response, in the third trimester of pregnancy. The 476:507 ratio was slightly raised (6.1), but not to the extent seen in pregnancy, or the luteal phase of the menstrual cycle. The source of the majority of the DOC in this patient was thought to be the ovary, as it is known that follicular fluid contains DOC (Dehennin et al ., 1987b; Bijault and Dehennin, 1991; Vanluchene et al., 1990, Mr.P.Holownia personal communication) and secreted into the blood circulation (Nahoul et al., 1988). DOC production in this patient was therefore, to a large extent extra-adrenal in origin. 288 Figure 11.13- Urinary steroid profile from a patient with a progesterone secreting ovarian tumour 4 5 6 V\ 1 = Androsterone 2 = Aetiocholanolone 3 = Dehydroepiandrosterone (DHA) 4 = 17-hydroxypregnanolone 5 = Pregnanediol 6 = Pregnanetriol 7 =Tetrahydrocortisone 8 = Tetrahydrocortisol 9 = 5a-tetrahydrocortisol 10 = a-cortol 11= P-cortol + P-cortolone 289 11.8 Congenital adrenal hyperplasia due to 21-hvdroxvlase deficiency 11.8.1 Introduction Deficiency of the adrenal 21-hydroxylase enzyme causes the most common type of congenital adrenal hyperplasia (New, 1985). Decreased cortisol synthesis induces increased ACTH secretion, and thus overproduction of cortisol precursors and sex steroids which do not require 21-hydroxylase for their biosynthesis. The source of these steroids has been shown to be adrenal in origin by adrenal venous catheterization (Wajchenberg et al ., 1979). Plasma 17-hydroxyprogesterone levels are raised (Franks, 1974; New and Levine, 1984; Lippe et al., 1974; Hughes and Winter, 1978; Atherden et a l, 1972), even in the neonate (Cacciari et al., 1982; Murphy et al., 1983; Wallace et a l, 1986, 1987), though care in the methodology used and interpretation of the results in neonates must be taken. Circadian variation in plasma 17-hydroxyprogesterone in patients with CAH has been shown, with high morning levels (Atherden et al., 1972; Meyer et al., 1976). Heterozygote carriers do not usually show any of the typical symptoms of CAH, and their plasma 17- hydroxyprogesterone concentrations are normal, but with ACTH stimulation values above those seen in normal controls (Krensky et al., 1977; Gutai et al., 1977). Three urinary steroid metabolites of 17-hydroxyprogesterone - 17- hydroxypregnanolone (170HPr), pregnanetriol (PT) and 11-oxo-pregnanetriol (11- oxo-PT) - are characteristically seen in 21-hydroxylase deficient patients (Shackleton et al., 1980b), in some cases to the apparent exclusion of almost all other steroids other than androgens. The presence of raised urinary 170HPr and PT is also seen in neonates, over the age of 3 - 4 days, though the pattern is somewhat different due to the presence of steroids of fetal adrenal origin. This is well described elsewhere (Honour, 1986; Gustafsson etal., 1972; Steen etal., 1980; Joannou, 1981), and was not pursued in this work. Measurement of plasma 17-hydroxyprogesterone, or urinary pregnanetriol (in patients over 1 month of age (laboratory experience)) provides a good index of the biochemical defect. Some groups prefer to use growth rates for monitoring control with replacement glucocorticoids (Appan et al., 1989). The high androgen production in untreated cases results in clitoromegaly, early appearance of facial, axillary and pubic hair, initial fast growth but eventual short stature due to early epiphyseal closure before expected normal height has been 290 reached, and can result in female infertility. Salt loss due to reduced or complete lack of aldosterone production is seen in some patients (known as salt losers), but not all cases (these patients being known as simple virilizers). CAH was reviewed further in Section 1.2.12. A variant of the classical CAH also exists, known as late-onset 21-hydroxylase deficiency CAH. In this condition the typical symptoms are not detected at birth, but rather by premature adrenarche or rapid growth in children, or amenorrhoea and excessive androgen production manifested as virilization in young women (Kohn et al ., 1982; Shackleton et al. , 1986; Brodie and Colston Wentz, 1987). Infertility is fairly common in female patients with CAH due to 21- hydroxylase deficiency. This is usually the result of the high androgen levels seen in many such subjects, due to insufficient glucocorticoid replacement, or poor compliance with treatment to suppress adrenal activity (Mulaikal et al ., 1987; Jones Klingensmith et al ., 1977). It has also been suggested that the androgens affect the psychosexual behaviour of same patients (Federman, 1987). There are however some reports of amenorrhoea related to progestin excess in CAH (Rosenfield et al . , 1980; Eden, 1989). Rosenfield et al. (1980) showed that there were episodic bursts of progestin secretion in many patients whose plasma androgen levels were well controlled. Treatment with low dose dexamethasone was found to be a suitable treatment in most cases (Rosenfield et al ., 1980; Richards et al ., 1978). Among the CAH patients studied, a woman with 21-hydroxylase deficiency is described here, in whom progesterone levels could not be controlled sufficiently to overcome her infertility. The source of this progesterone was finally tracked down to being adrenal tumour in origin. A number of tumours, mostly progestin and androgen secreting, have been described in 21-hydroxylase deficient patients in the adrenal (Pang et al. , 1976, 1981; Van Seters et al. , 1981; Korth-Schutz et al. , 1977; Hamwi et al. , 1957; Daeschner, 1965; Bauman and Bauman, 1982) and testes (Radfar et al. , 1977; Kirkland et al. , 1977). A pattern of steroid secretion resembling CAH to dueA21-hydroxylaseAhas also been described in two patients with lipoid cell tumours of the ovary (Rosenfield et al. , 1987; Imperato-McGinley et al. , 1981). The progress of treatment in the patient described here was monitored by both plasma steroid measurements and urinary steroid profiles. The latter analytical method was also used by Grondal et al. (1990) in the diagnosis and follow up of adrenocortical carcinomas. 291 The 21-hydroxylase enzyme defect offers an interesting set of circumstances for the investigation of extra-adrenal DOC production. The deficiency of adrenal 21- hydroxylase results in little or no adrenal DOC production, even on ACTH stimulation (Kater et al ., 1985). At the same time there are large amounts of circulating progesterone. It has been reported that the conversion of progesterone to DOC in 21-hydroxylase deficient CAH patients was similar to normals, so implying the presence of an extra-adrenal 21-hydroxylase enzyme (Antonipillai et al . , 1983a; Winkel et al ., 1983a). If this is correct, THDOC production in these patients would be expected to be in the normal range. To investigate this further, urine from a number of 21-hydroxylase deficient patients, both classical form and late-onset were analyzed for THDOC excretion rates. 11.8.2 Classical CAH results Eight subjects with classical CAH due to 21-hydroxylase deficiency were investigated, four of the subjects providing two urine samples. All subjects showed the typical urinary steroid profile of greatly raised 17-hydroxypregnanolone, pregnanetriol and 11-oxo-pregnanetriol (see Table 11.4). One such profile is shown in Figure 11.14(a). Cortisol metabolites were detectable in the profiles. THDOC excretion rates were also quantified in these urine samples, the results from ion 507 and 476 responses are shown Table 11.4. When correlated to the corresponding PD excretion rate of the subject, and compared to either luteal and follicular phase results in normal adults, most of the results fell at the lower end of, or below, the normal range of THDOC excretion rates for both the ion 507 and 476 responses. Only two (CAH6 and CAH8) of the 7 samples with luteal range PD excretion rates had THDOC excretion rates well within the corresponding normal THDOC excretion rate. Some of the SIM results were very difficult to interpret confidently, due to the presence of additional compounds, presumably further progesterone metabolites, giving either 507 or 476 responses. An example of one of the better traces (Subject CAH4) is shown in Figure 11.15(a). 11.8.3 Late-onset CAH results Seven subjects with late-onset CAH due to 21-hydroxylase deficiency were investigated. All were female, and were detected due to early pubic hair growth, 292 Table 11.4 - Urinary steroid excretion rates from subjects with 21-hydroxylase deficiency CAH '5b CN ' S o u 3 H K Q *c 'Sb <+H£ • ?"N 3 3 Tt 3 c* I CO 00 2 CO CO 6 D 1 cx Subject Sex r- o Age E a< Ion 507 Ion 476 PD u PT 11-oxo-PT *o 8 f ON CN CAH1 9.2 ^H 1400 800 8 = £ = 1.6 7.7 1200 1500 750 VO 8 f *o i o -H i 51.0 1250 >5000 3580 = £ £ 18.6 70.7 1000 9320 1680 7040 f O i-H 8 in CAH3 2.0 1 970 >2000 8 = = = 2.0 710 2500 1110 8 f O 00 »n o m A CAH4 i 17.6 100 1800 o CAH5 on 25.0 39.7 1230 >8000 >10000 >6000 f O r-H o CAH6* 30.6 I 1320 >10000 >10000 5090 f O VO oo Os o CAH7* cn 15.3 38.6 830 14900 >20000 = £ £ 3.9 26.9 12000 >18000 >6000 | f O CAH8 Adult 20.5 151.9 2320 22720 >37000 18140 » i-H » 3 * a VI to III II 293 3 3 ■a -4—> -4-» 2 fl C 0 0 to c o D 3 THDOC(/xg/24h) Results from urinary steroid profiles (/zg/24h) Subject Age Sex i—Hc- O & >-i 0 o X H Ion 507 Ion 476 PD PT 1 i o VO LOl 5.3 9.8 300 2060 620 <100 o+ 8 8 o L02 oo 5.5 950 500 <100 f O m r- H-) O 11.3 oo 390 970 <100 o 3 o j l i 1 54.3 370 340 340 <100 f O in O o 23.0 i-H m o 450 390 <100 901 o Adult 11.1 1780 1270 780 <100 f O o L07 Adult 33.8 35.0 870 480 <100 O IO o IO o o Child 2-23 5-22 <50-200 <50-160 V V V Normal © Follicular 2-18 6-35 <50-190 <50-500 range Luteal 16-83 58-260 590-3000 160-480 — *—H l—H 3 g ‘*3 $3 *o m3 c § •a € S € •> 3 -•-> g B {? 2 V > O 0> 1/3 Js CO G V G S e o ed ,_H 43O 2 g W> G s 1 Vi O g G 294 Figure ii.i4 - urinary sieroia prome rrom (a; a cnua wirn classical zi-nyaroxyiase deficiency CAH, and (b) a patient with 21-hydroxylase deficiency and a steroid secreting adrenal tumour 6.0E61 5.0E6 4.0EG CD a c rd 3.0E6 T 3 c _Q CE 2.0EG 1 . 0EG 20 25 30 35 l = 170HPr 2= P D 3= P T 5.0EG 4=ll-oxo-PD 5=11-oxo-PT 6="X" (see text) 4.0EG a) a c 3.0EG cd ~o c □ _Q CL 2.0EG 1 . 0EG 15 20 30 35 295 figure 11.13 - MM runs irom (a; a paueni witn classical z±-iiyuruxyiase ucnuicncy CAH, and (b) a patient with 21-hydroxylase deficiency and a steroid secreting adrenal tumour Io n 5 0 7 - 1.0E5 5.0E4- 3B5<* THDOC T5 - 0E:4 (U CJ Ion 47B T 3 DC 170HPr _Q CE . 0E5 5.0E4 5.0E4 G 8 97 10 Ion 507 8.0E5H I-8.0E5 -G.0E5 4.0E5- :4.0E5 :2.0E5 03 O Ion 47G | 8.0E5: 170HPr -8.0E5 -Q CE G.0E5- G.0E5 4.0E5 2.0E5- 2.0E5 296 excessive height for age (in the children) or amenorrhoea. Urinary steroid profiles produced profiles similar to normal subjects, but with greatly raised 17- hydroxypregnanolone, raised pregnanetriol, and in some cases raised androgens (androsterone and aetiocholanolone). 11 -oxo-pregnanetriol, though usually detectable using the mass spectrometer, was not raised to the degree seen in classical CAH. Results are shown in Table 11.5. THDOC excretion rates in these subjects were within, or close to the normal range for their corresponding PD excretion rate, Subjects L04 and L07 being slightly higher than the normal range, and Subject L06 slightly lower. SIM results were again difficult to interpret in some samples, results appearing similar to the classical CAH subjects, but with lower 170HPr ion 476 responses. 11.8.4 21-hydroxylase deficiency CAH with extra raised progesterone metabolites due to a steroid secreting tumour Subject CAHP - This subject was bom with ambiguous genitalia and was admitted into hospital at three weeks of age in a salt losing crisis, when the diagnosis of 21-hydroxylase was made. One sibling had died as a neonate, but there was no family history of CAH. She underwent vaginoplasty and clitoromegaly at age 2 years. Treatment was initially with deoxycorticosterone acetate, and later with cortisone. She was amenorrhoeic until the age of 26, when she was investigated for primary infertility, and had menarche induced with clomiphene. Induction of ovulation was attempted with the same drug. Raised plasma progesterone was detected and interpreted as evidence of ovulation. Further attempts at induction of ovulation with clomiphene were monitored with repeated hormone measurements and abdominal ultrasound. The latter showed poor follicular growth with little endometrial proliferation. At this time her daily treatment consisted of dexamethasone (0.5mg nocte) with fludrocortisone (0. lmg). Plasma progesterone was raised throughout the cycle although 17-hydroxyprogesterone and androstenedione were adequately suppressed, at <20 and <3nmol/l respectively. An endometrial biopsy in the follicular phase showed minimal proliferative activity with microglandular hyperplasia consistent with progestogenic stimulation. The high plasma progesterone concentrations were found to be episodic, making 297 pharmacological suppression appear successful. It was shown by chromatographic methods that the raised serum progesterone levels were not due to cross reaction of the characteristic adrenal steroids seen in 21-hydroxylase deficiency. Ovulation induction was attempted over three cycles with follicle stimulating hormone (FSH), during pituitary suppression with Buseralin. Ultrasound monitoring showed poor follicular development, in a typical polycystic ovary. There was little endometrial thickening, and ovulation after hCG administration was not persued because of cyst formation. Serum progesterone concentrations were suppressed and did not rise with exogenous gonadotrophins. To confirm adrenal suppression in this patient, and to alleviate some doubt of patient compliance, a 24 hour infusion of 300mg cortisol was given. This failed to suppress plasma progesterone concentrations. A computerized tomography scan of the adrenal glands then revealed a 4cm mass lying superior to the left kidney. The patient underwent unilateral adrenalectomy, but follicular phase serum concentrations remained high. The right adrenal was then removed, after which time the patient has ovulated spontaneously with normal follicular phase progesterone concentrations. Urine samples from this subject were investigated in the laboratory over a period of 4 years. In total forty-four 24 hour urine collections were analyzed, all but 3 by the author. Urinary steroid profiles showed 6 main steroid metabolites, the three typical metabolites 170HPr, PT and 11-oxo-PT, and three atypical metabolites attributed to high progesterone production (see Figure 11.16). Steroid "X" was identified as a 3,X,20-trihydroxy-pregnan-ll-one, with X probably at C-15 or C-16. An example of a urinary steroid profile from this patient is shown in Figure 11.14(b). During treatment for adrenal suppression and ovulation induction there were some occasions when the metabolite markers for CAH were totally suppressed, but the atypical progesterone metabolites were rarely fully suppressed. The degree of suppression varied greatly on different treatments, PD excretion rates quantified at levels between 200/xg/24h and 5000^tg/24h, and correlated fairly well with the plasma progesterone concentrations if measured in a blood sample from the same day. After the first adrenalectomy, all six urinary metabolites of interest were still present, PT > 4000/xg/24h and PD 480/zg/24h. However after the second adrenal had been removed all six metabolites were at levels < 100^g/24h. 298 Figure 11.16 - Main urinary steroid metabolites seen in a patient with CAH due to 21-hydroxylase deficiency with a steroid secreting adrenal tumour Classical CAH Metabolites C H 3 I progesterone c-o ■-OH 17-hydroxy progesterone HO 21-deoxycortlsol 17-hydroxypregnanolone C H 3 C H 3 I I HC-OH HC-OH -OH ■-OH HO HO 11-oxo-pregnanetriol pregnanetriol Atypical Steroid Metabolites C H 3 I HC-OH progesterone HO 11-hydroxy progesterone pregnanediol C H 3 C H 3 I I HC-OH HC-OH OH HO HO 11-oxo-pregnanediol 299 THDOC excretion rates were measured in this patient, in urine samples with both high and low levels of pregnanediol and pregnanetriol. In all samples analyzed the same problems were encountered as in the 21-hydroxylase deficient patients described above (Sections 11.8.2 and 11.8.3), in an even more marked manner, making quantification impossible (see Figure 11.15(b)). THDOC was not detected in scan mode using the mass spectrometer. 11.8.5 Discussion The use of urinary steroid profiles in the laboratory has proved useful in the detection 21-hydroxylase deficiency in numerous cases, in particular in newborns. A number of late-onset CAH cases have also been diagnosed in women presenting with virilization or amenorrhoea. Urinary steroid profiles also proved invaluable in the adaption of treatments in Subject CAHP, who was eventually diagnosed as having an adrenal tumour. This group of patients are interesting in the area of extra-adrenal DOC production. The method developed in this work was not totally suitable for the quantification of THDOC excretion in these subjects, due to the presence of additional progesterone metabolites that interfered with the assay. Results obtained indicated that in classical CAH due to 21-hydroxylase deficiency THDOC excretion rates were on average at the lower limit of the normal range, while in late-onset CAH the THDOC excretion rates were equivalent to normal subjects. In the experience of the or laboratory, cortisol metabolites are detected in classical CAH patients, usually atnear normal levels, suggesting that 21-hydroxylase activity exists in these patients, either adrenal or extra-adrenal in origin. This enzyme activity would also account for the DOC production seen. In late-onset CAH the enzyme deficiency is less complete. THDOC excretion similarly appears unaffected by the degree of enzyme deficiency manifested in these patients. In new-boms with 21-hydroxylase deficiency CAH, a paradox of highly elevated 21-pregnenolone production levels, relative to normal new-boms has been shown (Shackleton et al . , 1987). This is probably the result of the action of a fetal or an extra-adrenal 21-hydroxylase enzyme. 300 11.9 Congenital adrenal hyperplasia due to 17-hvdroxvlase deficiency 11.9.1 Introduction The enzyme 17<*-hydroxylase is essential, not only for adrenal biosynthesis of cortisol, but also for the formation of androgens and oestrogens by both gonads and adrenals. Male pseudohermaphroditism, with incomplete differentiation of the androgen dependent external genitalia, results from the inability to synthesize the gonadal hormones. Female patients have sexual infantilism. Patients with a defect of this enzyme are normally hypertensive and hypokalaemic, due to ACTH stimulation of the intact mineralocorticoid pathway resulting in excessive secretion of DOC. This steroid excess causes plasma volume expansion, suppressed renin secretion and a consequent failure of aldosterone synthesis. Dexamethasone suppression of ACTH results in diminished DOC secretion, resulting in renin- angiotensin stimulation of the glomerulosa and a rise in aldosterone secretion (New, 1985; New and Levine, 1984), a pattern similar to that seen in 116-hydroxylase deficiency (see Chapter 6). A case of a young woman with 17-hydroxylase deficiency CAH has been reported without hypertension, but with amenorrhoea and undeveloped secondary sex characteristics at the age of 19 years (Wei et al . , 1987). In all cases there is overproduction of 17-deoxysteroids. This is reflected in the characteristic urinary steroid profile seen in these patients (Honour et al ., 1978; Bumstein et al ., 1983; Fennessey et al ., 1983; Shackleton et al ., 1980b). The 21- deoxy, 11-oxygenated steroids seen are metabolites of corticosterone formed by bacterial metabolism following biliary excretion (Gower and Honour, 1984). THDOC excretion as high as 1200/xg/24h has been recorded, after paper chromatography of urine steroids, in a patient with 17-hydroxylase deficiency (Lim etal., 1969). 11.9.2 Results Urine from a 51 year old male patient, off dexamethasone treatment for three weeks prior to collection was analyzed. This is the same patient described by Toumiaire et al. (1976) and Honour et al. (1978). The urinary steroid profile (Figure 11.17) is similar to that shown by Honour et al. (1978), even though the sample had 301 Figure 11.17 - Urinary steroid profile from a patient with 17-hydroxylase deficiency CAH 1 2 11 13 10 7 8 ii 12 JUL JL 1 =Pregnanediol (PD) 2 =5-pregnene-3p,20a-diol 3+4=3a,20a-dihydroxy-5p-pregnane-11-one (11 -oxo-PD) + 3a,20a-dihydroxy-5a-pregnane-l 1-one + 3a5p THDOC 5 =5p-pregnane-3a,1 ip,20a-triol 6 =5a-pregnane-3a, 11 P,20a-triol + 5-pregnene-3oc, 16a,20a-triol 7 =3a,21-dihydroxy-5p-pregnane-11,20-dione (THA) 8 =Tetrahydrocorticosterone 9 =5a-telrahydrocorticosterone 10 = 1,3,20-trihydroxypregnane-l 1-one 11 =Hexahydro-A 12 =5P-pregnane-3alip,20a,21-tetrol 13 =5a-pregnane-3alip,20cx,21-tetrol 302 Figure 11.18 - SIM run from a patient with 17-hydroxylase deficiency CAH Ion 507 365a THDOC G000 (U 5000 u c -a 4000 c D £ 3000 2000 1000 G 7 8 9 10 Ion 47G 0E4- 10000“ G 7 8 9 10 303 been stored frozen for over 10 years and Amberlite XAD-2 extraction and Sephadex LH-20 chromatography was used in the earlier methodology. THDOC was eluted from the GC column with 11-oxo-pregnanediol (3a,20a- dihydroxy 5a-pregnan-l 1-one). Using SIM they are however distinguished as the latter has a molecular ion of 478, and two strong ions of 388 (M+-90) and 334 (M+- 144) (Honour et al ., 1978), as compared to the ions 507 and 476 used for THDOC quantification. The SIM result is shown in Figure 11.18. THDOC excretion rates calculated from ion 507 and 476 responses were 105 and 134/xg/24h respectively, slightly lower than the estimated 160^g/24h calculated by Honour et al. (1978). Ion 476 response peaks were also seen from 17-hydroxypregnanolone and the hydroxypregnanolone Pr3 described in pregnancy (see Chapters 5 and 8). The presence of other C-16 hydroxylated steroid metabolites in the urinary steroid profile suggested that the a similar compound as in pregnancy (Pr3) was excreted. The presence of the 170HPr response indicates that a small amount of 17-hydroxylase activity is present in this patient. 11.9.3 Discussion Subjects with 17-hydroxylase deficiency supply another example of raised adrenal production of DOC. The urine excretion of THDOC is not as high as would be expected for a condition with excess DOC production, though still well above the normal male range. This may be due to 21-dehydroxylation of DOC by bacterial gut metabolism, with the excretion of pregnanediol. The latter is certainly raised, above normal male excretion rates, at 1680^g/24h. This metabolism is discussed in more detail in the next chapter. 304 12 - General Discussion This project aimed to investigate adrenal and extra-adrenal production of DOC. This represents the conversion of a natriuretic hormone, progesterone, by 21- hydroxylase activity, to the salt retaining hormone under investigation. A highly sensitive and practical selected ion monitoring GC-MS method for the quantification of the urinary metabolite of DOC, tetrahydrodeoxycorticosterone, was developed. THS and oestriol (in pregnancy) were known to be interfering compounds, because of their similar GC retention times as MO-TMS ether derivatives, and the presence of naturally occurring heavy isotopes of the atoms in their derivatized forms. These two steroids were easily removed from urine extracts using Sephadex LH-20 chromatography. This methodology added about 3 hours to the extraction procedure, but with the simultaneous use of many (up to 12) mini columns at one time, did not add too greatly to the complete analysis time. Initially only one ion (m/z = 476) was to be used for selected ion monitoring (SIM), to give maximum sensitivity. The addition of other ions (m/z = 507 and m/z = 188) to facilitate the identification of the peaks of interest, highlighted the presence of further co-eluting steroids, with strong 476 ion responses, in some clinical conditions. This emphasized the importance of the selection of appropriate ions for use in SIM quantification, to obtain correct results. The quantification of THDOC in all samples was judged from the responses of both ions 507 and 476. There were large discrepancies in the calculated amount of "THDOC" excreted in some urine samples when comparing ion 507 and 476 results (up to 9 times as high using ion 476 response compared to ion 507 response in normal pregnancy). Co-eluting steroids were indicated and when finally isolated, at least in pregnancy, mass spectra where the 476:507 ion response ratio was > 100:1, as opposed to the * 4 seen in pure THDOC, were seen. The contribution of the co-eluting steroids to the result, when quantified using the ion 507 response, 305 was therefore assumed to be negligible, except perhaps in the samples with very high 476:507 ratios for the peak containing THDOC. The ion 507 response was therefore considered to be near accurate in all samples, even those with raised 476:507 ratios. On discovery of the co-eluting steroids, attempts were made to remove or separate them to allow accurate quantification. This proved to be surprisingly difficult. Most effort was put into isolating the steroid in pregnancy samples as the levels of excretion were highest in these samples, and it was these samples from which the potentially most interesting results would be obtained. Immunoadsorption was not successful as the co-eluting steroid did not cross react with the progesterone antisera used. The use of alternative GC derivatives to the MO-TMS ether used routinely in the laboratory, and alteration of GC conditions, and the consequent increase in theoretical plates both proved unsuccessful. The former did however offer further evidence as to the identity of the co-eluting steroids in pregnancy. The use of more sophisticated mass spectrometry methods, such as metastable peak monitoring (Gaskell et al . , 1980), MS-MS (Gaskell, 1988) or thermospray LC-MS (Esteban and Yergey, 1990) would probably be the next analytical step that could be undertaken to help confirm the structure of the co-eluting steroids. The co-eluting steroid in pregnancy was finally isolated using the chromatography methodology described by Winkel and co-workers in some of the papers that inspired this project. Overall the methodology would however not be suitable for incorporation into the quantitative method developed in this project as the overall recovery of steroids was poor (and probably inconsistent) and would have increased sample preparation time greatly. The work presented here did not determine whether the co-eluting steroids were conjugated, and if so, as sulphates or glucuronides. THDOC is found in urine in the form of a glucuronide conjugate. Separation of sulphate and glucuronide conjugated steroids, by means of for example Sephadex LH-20 chromatography and the solvent system chloroform: methanol (1:1 v/v saturated with salt) as described by Honour (1986) may have resulted in the separation of some or all of the co-eluting steroids if they were in the form of sulphate conjugates. Time did not allow this possibility to be explored. The use of mass spectrometry allowed close inspection of the steroids present at each stage of chromatography described by Winkel and co-workers. Their paper 306 suggested that the final crystals of steroid obtained after 4 chromatography steps was pure THDOC as consistent 3H-THDOC:14C-THDOC ratios were established. They stated that the 3H labelled PD, the main urine metabolite after 3H-progesterone administration, was still present after the first gradient elution chromatography step, but that this PD was removed during the second column chromatography step (Celite parturition chromatography). My work showed that PD (and 17-hydroxy- pregnanolone) were in fact still present after each of the four chromatography steps. They appeared to be retained preferentially to THDOC, shown by the fact that there was more PD and 170HPr present relative to THDOC after extraction and the four chromatography steps than in the original urine from the 1 lB-hydroxylase deficient patient. What could be the source of the PD seen, if all or most of the PD from direct progesterone metabolism had been removed? In the experiments performed by Winkel and co-workers, 14C-DOC was also administered simultaneously with the tritiated progesterone. PD excretion after DOC administration in rabbits and rats has been reported (Hoffman et al., 1943; Schneider and Horstmann 1951). This could be the result of 21-dehydroxylation by intestinal organisms. Steroids are not only filtered from the general circulation in the kidney from where they are either reabsorbed or are excreted in the urine, but also enter the hepatic circulation. From the liver they can be excreted in bile and enter into the gut. Here they are further metabolised by the varied bacteria present. Some of the steroids are excreted along with the faeces, and the rest returned to the liver after intestinal reabsorption. Urine is the main route for elimination of steroids from the body, but the intestine and enterohepatic circulation represent a large pool of steroids. In rats it has been shown that administered THDOC can be converted to pregnane-3,20-diol, and that there is an absence of 21-hydroxylated steroids in the faeces. Germ free rats on the other hand, showed no such conversion (Eriksson et al ., 1969a, b). In a study where radioactively labelled (14C) corticosterone was administered to an adult patient with 17-hydroxylase deficiency the majority of radioactive metabolites in the urine were 21-hydroxylated on the first day of urine collection. By the third day at least 75 % of the excreted activity was associated with 21-deoxysteroids. Bacterial metabolism in the intestinal tract would have been responsible for the dehydroxylation (Shackleton et al ., 1979a). The bacterium 307 Eubacterium lentum in humans has been documented to convert DOC to progesterone, THDOC to pregnanolone, and dihydro-DOC to pregnanedione (Gower and Honour, 1984). These products are then reabsorbed into the enterohepatic circulation, and can be excreted from the body, via the kidney route, in urine. This would appear to be a likely source of some of the PD remaining after the four chromatography steps following the Winkel method. Therefore, although they demonstrated a consistent 3H:14C ratio in what they regarded as purified THDOC, contamination of that material with 14C-pregnanediol was not excluded. It is ironic that in the Winkel paper they explored the possibility of using an additional HPLC step in their methodology, which they showed separated PD from THDOC. This chromatography step was however not routinely included in the quantitative methodology, as they had obtained reasonably consistent 3H:14C ratios after the initial gradient elution chromatography step. It is interesting to note that most of the 118-hydroxylase deficient patients, investigated in the work reported here, also had PD present at quantities higher than normal in their urine. Some of this steroid was likely to be from the same source (gut metabolism), due to the high quantities of DOC as substrate in these patients. Winkel and co-workers did not consider in their work the possibility that DOC could be converted to PD by enterohepatic metabolism. If 14C-PD was produced in their studies, and not fully removed by the four chromatography steps (which I have shown to be possible and likely) quantification of THDOC production using the ratio of 3H:14C (in what was assumed to be pure THDOC) would be inaccurate. THDOC was purified by the same group (Winkel et al ., 1983a), using the same methodology, in two women with 21-hydroxylase deficiency CAH. Similar conversion rates, to those seen in normal women, of progesterone to DOC were computed. This offered them further evidence of a site of extra-adrenal 21-hydroxylase activity. In the same study however they also considered 17-hydroxyprogesterone (170HP) conversion to 11-deoxycortisol (S), by means of 14C-170HP and 3H-S administration, using similar chromatographic methodology. They found lower 170HP to S conversion rates than progesterone to DOC conversion rates. They suggested the reason for this may be that the of the extra-adrenal enzyme was lower for 170HP than for progesterone, or that plasma 170HP did not enter the extra-adrenal cellular site of 21-hydroxylation as readily as did plasma progesterone. Neither of these possibilities can be excluded, 308 but the inaccuracy of THDOC production, as a result of the presence of PD (adrenal or enterohepatically produced) in the "pure THDOC" quantified, must be considered, and offers more evidence that the gut metabolism of DOC occurred. This does not of course exclude the presence of extra-adrenal sites of 21-hydroxylation, as evidenced by the progesterone to DOC conversion in adrenalectomized subjects shown in the original paper (Winkel et al . , 1980a), but the work based on the methodology used must be interpreted carefully. The method, even though now shown to be probably inaccurate, did however allow the separation in pregnancy urine extracts of the co-eluting steroids, which probably did not interfere with their calculation. One of the co-eluting steroids in pregnancy was thought to be a 3,16- dihydroxypregnane-20-one (refer to Chapter 4). This type of steroid has been reported to be present in pregnancy in the plasma as glucuronide or monosulphate conjugates (Axelson and Sahlberg, 1983; Anderson et al ., 1990; Baillie et al ., 1976), in urine (Eriksson and Gustafsson, 1970), in amniotic fluid (Dawood, 1977), in bile (Laatikainen and Kaijalainen, 1972), and in faeces (Eriksson et al ., 1970; Eriksson and Gustafsson, 1971). Anderson et al. (1990) in steroid metabolism and kinetic studies using stable isotopes showed that the formation of 16a-hydroxylated steroids from pregnane-3 a , 20a-diol monosulphate was very limited, but that 16-hydroxylated steroids appeared to be formed mainly by hydroxylation of pregnanolone monosulphate, followed by oxidoreduction at C-20. 16-hydroxylation is known to be an important metabolic step in the fetus (see Introduction - Winter’s feto-placental unit model). It is important in the production of oestrogens in pregnancy, 16- hydroxy-dehydroepiandrosterone-sulphate being one of the precursors of oestriol. The feto-placental unit is possibly the site of 16-hydroxylation seen in the co-eluting steroid in pregnancy. This statement is endorsed by the finding that in the menstrual cycle the steroid co-eluting with THDOC was tentatively identified (see Chapter 3) as a hydroxypregnanolone with no hydroxyl group directly attached to the D-ring (in particular at C-16). The fall in the raised excretion rates of the pregnancy co-eluting steroids (as evidenced by the fall from raised 476:507 ratios during pregnancy) to those expected for pure THDOC, during first days after elimination of the feto placental unit at parturition, would further confirm this suggestion. An instant fall was not seen in the steroids produced as a result of pregnancy due to the pool of steroids presumably residing in the intestine and enterohepatic circulation. The co 309 eluting steroids and oestriol for example taking 10 - 15 and 3 - 9 days respectively in this study to be reduced to non-pregnant levels in the mixture of steroids excreted in urine. Lewis et al. (1987) suggested that the slow decline of, for example progesterone, may be due to leaching out of the hormone from body fat stores. Various sites of hydroxylation of the steroid nucleus have been reported apart from the commonly seen C-3, C -ll, C-17, C-20 and C-21 positions. Hydroxyl groups have been reported at C-l, C-2, C-4, C-6, C-7, C-15, C-16, C-18 and C-19 (many references, some not included in the reference list, those for position C-16 include Anderson et al ., 1990; Baillie et al ., 1976; Axelson and Sahlberg, 1983; Eriksson et al ., 1970; Eriksson and Gustafsson, 1971; Laatikainen and Kaijalainen, 1972; Eriksson and Gustafsson, 1970; Dixon, 1969; Numazawa et al., 1985; Fotsis, 1987; Honour et al., 1978; Boumot and Ramirez, 1989). 16a-hydroxylation was also demonstrated, by the presence of 16a- hydroxyprogesterone, in long term cell cultures of steroid secreting cells from adrenals of a patient with 1 IB-hydroxylase deficiency (Miller and Morel, 1989). The properties of 16a-hydroxyprogesterone and its metabolites concerning hypertension are not fully understood, but 16-hydroxyprogesterone was shown by Jacobs (1969) to have a natriuretic effect in adults. This compound was not detected in the urine of the 1 lB-hydroxylase deficiency patients studied for this thesis, and there was no interference from the 3,16-dihydroxylated steroid found co-eluting with THDOC in pregnancy. In 17-hydroxylase deficiency CAH further urinary steroid metabolites have been shown to co-elute with THDOC. 5B-pregnane-3a,16a,20a-triol, 5-pregnene- 3a,16a,20a-triol and 5a(and 58)-pregnane-3a,20a-diol-l 1-one were reported by Bumstein et al., 1983 and Honour et al., 1978. The daily synthesis of progesterone, in particular in late pregnancy can be very high at approximately 250 - 300mg/day (Anderson et al., 1990), and there is some evidence of cyclical production (Paaby et al., 1989, 1990). The normal metabolism of progesterone in pregnancy involves reduction of C-4 to C-5 double bond and the 3- and 20-ketone groups. These reactions result in the formation of isomeric pregnanolones and pregnanediols. Metabolites formed by hydroxylation at various points in the steroid nucleus are also known, see above, C-16 and C-21 sites being the most common. The metabolites are excreted as complex mixtures in urine 310 (Philip et al . , 1989), in faeces (Eriksson et al., 1970; Eriksson and Gustafsson, 1971), via bile (Laatikainen and Kaijalainen, 1972), and into amniotic fluid (Schweitzer et al., 1969; Sippell et al., 1981; Dawood, 1977). The mixtures of progesterone, oestrogens and other steroid metabolites excreted in urine, bile and faeces are extremely complex. This complexity is increased by the possibility of oxidation of C-21 acids (Senciall and Roberts, 1989), and by bacterial metabolism in the gut (Gower and Honour, 1984), including hydrolysis of conjugates, epimerization at C-3, 16-dehydroxylation leading to 17a- pregnane derivatives (Eriksson et al., 1968, 1970), and 21-dehydroxylation (Eriksson et al., 1969a), as well as deconjugation. The excretion pattern of progesterone metabolites and oestriol in faeces and urine changes during antibiotic treatment. Normally the bulk (85 - 90%) of faecal oestriol is unconjugated (Adlercreutz and Jarvenpaa, 1982). With antibiotic administration large amounts of conjugated oestriol appear (Martin et al., 1975). A decrease in excretion rates of oestriol and pregnanediol glucuronides, caused by the interruption of the enterohepatic circulation of steroids as a result of inhibition of intestinal steroid metabolism was seen, though this may be transitory (Martin et al., 1974). During pregnancy, a variety of pregnanolones, pregnanediols, and hydroxylated derivatives thereof appear in high concentrations in plasma (numerous references including Anderson et al., 1990; Axelson and Sahlberg, 1983; Brooks and Harvey, 1970; Baillie et a l, 1976; Sjovall et al., 1968; Sjovall and Sjovall, 1968; Laatikainen and Peltonen, 1975; Mickan and Zander, 1979a, b). This was also reflected in the steroids found excreted in pregnancy urine. The presence of at least six hydroxypregnanolones (170HPr, 3a5B THDOC, the latter’s 16-hydroxylated co eluting steroid, Prl, Pr2, and Pr3) excreted throughout pregnancy was shown by their presence in the SIM runs of pregnancy urine extracts quantified in this work. Urinary steroid profiles also showed the presence of pregnanediols (at least five, refer to Figure 8.1) and pregnanetriol. Additional C21 metabolites, many 16-hydroxylated, were seen in subjects with placental sulphatase deficiency (PSD). Placentae from these patients are not only deficient in the sulphatase activity for steroid 3-sulphates (required for the production of oestrogens from DHA-sulphate and 16-hydroxy DHA-sulphate), but also for steroid 311 21-sulphates (Guerami et al ., 1988; Egyed and Oakey, 1985; Mathis et al ., 1983), and it has been suggested that these activities in fact derive from a common enzyme. In normal non-pregnant subjects plasma DOC-sulphate is not hydrolysed to DOC except in the intestine, by way of the action of bacterial enzymes (Casey and MacDonald, 1982b). Desulphurylation can be followed by 21-dehydroxylation to give progesterone. In normal pregnancy on the other hand, steroid 21-sulphatase activity is present in the placenta. The placenta in a normal individual may therefore play a unique role in the biogenesis of DOC, from fetal DOC-sulphate. The deficiency of placental sulphatase activity results in greatly reduced oestriol production. This offered the opportunity to investigate a further situation of hypo-oestrogenism, other than that involving a dead or anencephalic fetus, which had provided evidence to suggest that oestrogen promoted extra-adrenal DOC production (MacDonald et al ., 1982; and others). The data obtained in my work (see Chapter 9) did not support this hypothesis, as normal (or only slightly reduced) THDOC excretion was seen despite the greatly reduced oestrogen levels. The initial data reported here on pregnant subjects with hypertension, in particular those with PET, demonstrated slightly raised Pr3 excretion rates. Pr3 is thought to be a 16-hydroxylated C2i steroid metabolite. 16-hydroxyprogesterone has been shown to have a natriuretic effect in adults (Jacobs, 1969). It is possible that production rates of certain 16-hydroxylated steroids may therefore be contributing to symptoms seen in some clinical situations with hypertension in pregnancy. Various workers have put forward evidence for the concept that the adrenal zona glomerulosa and zona fasciculata function as separate glands. Analysis of blood and urine results in Subject Y1 with 1 lB-hydroxylase deficiency CAH demonstrated that DOC production was switched from the zona fasciculata to the zona glomerulosa, when glucocorticoid replacement therapy suppressed the excessive ACTH drive of the adrenal, as a result of stimulation of the renin-angiotensin system (see Chapter 6). 21-hydroxylase deficiency CAH patients show a variable degree of salt wasting. All the patients show a high degree of defective 21-hydroxylation of 17- hydroxy steroids, leading to elevation of serum 17-hydroxyprogesterone and diminished production of cortisol. The nature of the 21-hydroxylase defect in the 17- deoxy pathway remains less clear, although there is evidence, from ACTH stimulation tests, that aldosterone secretion may be more deficient in those with the salt wasting 312 form (Esteban and Yergey, 1990; Kuhnle et al., 1981; Bartter et al., 1968). Plasma renin activity is raised in this condition, particularly so in patients with the salt losing form. ACTH stimulation (with a low sodium diet and dexamethasone suppression) resulted in a rise of plasma renin in the simple virilizers, but not in the already high salt losers (Kuhnle et al., 1981). The reduced or absent aldosterone levels result in stimulation of the renin-angiotensin system in a drive to increase mineralocorticoid production. These CAH patients therefore benefit from treatment with mineralocorticoids as well as glucocorticoid replacement for optimal control of their disease (Schneider et al., 1975). This is also true of llB-hydroxylase deficient subjects. Oversecretion of aldosterone, secondary to increased plasma renin activity may act as a compensatory response to the natriuretic hormones secreted by the adrenals in at least the simple virilizers. Mineralocorticoid receptor binding (antagonist) substances have been reported in the non salt-losing form of 21-hydroxylase deficiency. Land and Ulick (1987) found however that the majority of such material was in fact DOC, an agonist, but of lesser potency than aldosterone. The DOC may be a result of extra-adrenal hydroxylase activity. In a large review study (Kater et al ., 1989), covering many clinical conditions of primary and secondary adrenocortical disorders (including ACTH excess and deficiency, increased and reduced plasma renin activity and subclinical adrenal abnormalities, including AIDS), it was found that treatment which modified the action of the renin-angiotensin system stimulated or suppressed only aldosterone and 18- hydroxycorticosterone levels. This is also shown in pregnancy where aldosterone rises during pregnancy (Nolton et al., 1978), and is influenced by salt intake and postural stimuli (Ehrlich et al., 1976), both of which affect the renin-angiotensin system (renin rising during pregnancy), whilst this is not the case for DOC. The mineralocorticoid effect of DOC however, when in excess, does suppress the renin-angiotensin system and can, in the long run, result in salt depletion due to the reduction of the production of the more potent mineralocorticoid aldosterone. 313 In the original thesis project plan deuterium labelled progesterone was to be administered to various clinical subjects of interest and its metabolic fate investigated by the presence of deuterium in steroids extracted from plasma and urine. DOC and THDOC production were of particular interest. Mass spectral data of some deuterated progesterones, synthesized in small quantities, are shown in Appendix 3. The establishment of the quantitative methods for determining urine steroid excretion rates which was extensively covered in the work presented here, has advanced our knowledge in some of the problems that we would encounter when the intended stable isotope project is further pursued. The relatively low levels of THDOC excretion rates in normal subjects, including potentially those with premenstrual syndrome, and patients with 21- hydroxylase deficiency would require very large quantities of urine to be extracted in order to detect the deuterium labelled THDOC, as only a small percentage of b e - a s 3 x ? C circulating plasma prcysW ooe e * le n v ih t ache^al u;cvilcj^(< 1 %, Winkel et al ., 1980a). The presence of co-eluting steroids in the urine extracts would also contribute to the necessity of large urine volume for extraction „ To obtain the maximum accuracy, the molecular ion response would be needed to be used, and this quadruples the quantity of steroid required to be analyzed to obtain a comparable result to if the ion [476 + number of incorporated deuterium atoms] response were used. The GC-MS SIM methodology developed was not totally suitable for use with urine extracts from 21-hydroxylase deficiency CAH subjects, due to the presence of additional interfering progesterone metabolites. Further purification of urine extracts from these patients would be required. The two simplest possibilities, may be (i) to achieve a working THDOC antisera immunoadsorption method, or (ii) incorporation of an additional high performance liquid chromatography (HPLC) step. An HPLC method using an alumina adsorption column for HPLC was reported by Senciall and co-workers (1989, 1990) which separated 21-hydroxysteroid (including THDOC) and 21-deoxysteroid metabolites of DOC and progesterone in rabbit urine and tissues. Progesterone and ten commonly encountered metabolites, in bovine tissues and milk, were successfully separated using a diol HPLC column by Purdy et al. (1980). A method similar to this is being used for the separation of DOC from progesterone in plasma for use in deuterated studies and plasma DOC quantification (see 314 preliminary work in Appendix 4). Adaption of these methodologies may prove successful. In the studies by Winkel and co-workers normally 190/xCi of 3H-progesterone and 1/xCi of 14C-DOC over 6 hours were infused into their subjects. The final recoveries of THDOC from urine, ranged between 100 and 52000 d.p.m. for the tritiated material, which represented a conversion rate of around 0.5 - 2.2% of the administered dose. In the IVF programme which I studied (see Chapter 11), the injection of progesterone at 25 - lOOmg/day was associated with a rise of THDOC excretion above the luteal phase levels of less than 100/xg/day. If deuterated progesterone were to be used the incorporation of 2H into THDOC should be detectable. The levels of progesterone used in the IVF subjects would therefore be an adequate dose for pilot studies of 21-hydroxylation. In normal pregnancies (where a corpus luteum is a source of progesterone in the first weeks of pregnancy) higher levels may be necessary, and the pool of progesterone would need to be labelled before study could be undertaken. Alternatively stable isotope labelled DOC could be infused and the effect of non-labelled progesterone loading could be investigated to estimate progesterone to DOC production. If time had allowed I would have liked to have done work to identify the co eluting steroid seen in the normal non-pregnant subjects. It may also have been possible to isolate this using the gradient elution Celite chromatography. Other progesterone metabolites could also have been investigated further. The deuterated progesterone metabolite study might also have thrown further light on this. In this study I used an isomer of the steroid of interest as the internal standard for quantification. Although this gave a very satisfactory result, further development incorporating a stable isotope labelled internal standard would have been of interest. This would have allowed investigation into various phenomenon such as the carrier effect seen with such internal standards. An attempt with a very old 2H-labelled compound was made, but the isotope enrichment had deteriorated with time (see Appendix 2). Overall, the project has produced some interesting new results in the area of progesterone metabolism and DOC production in various clinical situations, and highlighted some areas of research where care must be taken in the interpretation of 315 results. Even though the original plan of investigation could not be achieved, the work submitted here will prove very useful to any scientist pursuing a similar project. In the subjects described in this thesis, both adrenal and extra-adrenal production of DOC, as measured by THDOC excretion, has been suggested. 11J5- and 17B-hydroxylase deficiency congenital adrenal hyperplasia, and Cushing’s syndrome offer examples of pure adrenal production of DOC, whereas the mineralocorticoid secreting and ovarian progesterone secreting tumours offer examples of extra-adrenal production of DOC. Pregnancy, and possibly the menstrual cycle, are a mixture of the two sources of DOC, with extra-adrenal DOC production being shown in at least the pregnant patient with 21-hydroxylase deficiency CAH, as she had normal range THDOC excretion (confirmed by mass spectrometry), but was on dexamethasone suppression of her adrenals. The importance of the contribution of extra-adrenal DOC production in pregnant subjects with normal adrenals is still however unclear. Subjects with PSD who are hypo-oestrogenic, which according to the hypothesis of oestrogens promoting extra-adrenal DOC production put up by MacDonald and co-workers, should have low DOC production, had THDOC excretion rates within, or just below, the normal pregnancy range. 316 APPENDIX 1 THE USE OF DEUTERATED CORTISOL TO INVESTIGATE THE ACTION OF 1 lfl-HYDROXYSTEROID DEHYDROGENASE - A PILOT STUDY. Al.l Introduction Hypertension with suppressed renin and hypokalaemic alkalosis can be caused by overproduction of aldosterone, or one of the other known mineralocorticoids such as DOC or 18-OH-DOC. In some cases however no mineralocorticoid excess can be demonstrated. Impaired conversion of cortisol (F) to cortisone (E) has been associated with a similar syndrome including low renin, low aldosterone hypertension with hypokalaemia in children known as "apparent mineralocorticoid excess". The interconversion of cortisol and cortisone is carried out by a microsomal enzyme 1113-hydroxysteroid dehydrogenase (llfi-OHSD) and another independent enzyme 11-oxo-reductase (Abramovitz et al ., 1982; Lakshmi & Monder, 1985). Cortisol binds to renal mineralocorticoid (type 1) receptors, to which aldosterone normally binds. Cortisone does not bind to these receptors, so in normal subjects the kidney is thought to protect these receptors from cortisol, by conversion of the steroid to cortisone before it reaches the distal nephrons by the action of the relatively abundant renal 11B-OHSD. Further work in rats has confirmed the presence of the two separate enzymes, and shown that they are (at least in rats) kinetically distinct forms of llfl-OHSD in the liver. It was also suggested that 11B-OHSD protects various tissues from the deleterious effects of cortisol (Edwards et al ,, 1988; Lakshmi & Monder, 1988; Monder & Lakshmi, 1989, 1990; Phillips et al., 1989). Apparent mineralocorticoid excess (AME) has been reported in the literature in around 15 children, some siblings (Shackleton et al., 1980a, 1985; Fiselier et al., 1982; Honour et al., 1983b; Harinck et al., 1984; Monder et al., 1986), and one adult (Stewart et al., 1988). A characteristic urinary steroid metabolic profile 317 Figure A 1.1 - Cortisol and cortisone metabolities CH2-OH CH2-OH c=o OO HO. OH 11-OHSD OH 11-OXO-STEROID REDUCTASE O O CORTISOL (F) CORTISONE (E) THF aTHF THE aTHE ✓ N /N oc-cortol p-cortol a-cortolone p-cortolone llp-OH-Aet. llp-OH-And. 11-oxo-Aet. 11-oxo-And. And. = Androsterone Aet. = Aetiocholanolone OHSD = Hydroxysteroid dehydrogenase 318 of this syndrome is well described, with elevated urinary 1 IB-hydroxy metabolites of cortisol (THF and cortols) and low 11-oxo metabolites (THE and cortolones). Figure A 1.1 shows some of the possible metabolites. A second form of the syndrome (designated Type II) has been described, with normal E to F ratios, but decreased cortisol metabolic clearance rates (Ulick et al ., 1989, 1990). 11B-OHSD occurs in many tissues in man including the liver, kidney, gastrointestinal tract, prostate, muscle, lung, thyroid (various sources see Stewart et al ., 1988), and the placenta (Ldpez Bernal et al . , 1980). The latter is interesting as it is possible that in some cases of hypertension in pregnancy a high cortisol to cortisone ratio may be found. Potent inhibitors of 11B-OHSD have been described - liquorice and derivatives of it such as carbenoxolone sodium and glycyrrhetinic acid - which enhance mineralocorticoid action of glucocorticoids in several tissues (Epstein et al . , 1978; Monder et al . , 1989; Stewart et al . , 1987, 1990b). The drug carbenoxolone (used initially for peptic ulceration treatment) not only inhibits 11B-OHSD, but also the 11- oxo-reductase activity (Stewart et al ., 1990b), so the cortisol to cortisone ratio is perturbed to a lesser extent than seen in AME patients. The opportunity arose to participate in a pilot study (with Prof. Edwards and Dr. B. Walker of the Western General Hospital, Edinburgh), looking at the use of lla -2H-cortisol in the measurement of the activity of 11B-OHSD in man, including a few samples from the first published surviving adult case of 11B-OHSD deficiency (Stewart et al.f 1988). A1.2 Experimental work (1) Selection of ion for analysis London Cortisol and lla -2H-cortisol (supplied by Prof Kirk, QM\V^) were derivatized and cleaned up using Lipidex 5000, as described in Materials and Methods. A single doublet peak of cortisol (syn and anti forms of MO-TMS ether derivative) was found 'Uae is in each sample, ^niass spectral ^ shown in Figure A 1.2. Ion 605 was expected to have a strong ion response in cortisol, but due to rounding up of the exact mass by the MSD computer, 606 was assigned. When more accurate values were requested, 605.55 was found to be the true value. Similarly 606.55 was rounded up to 607. 319 Figure A 1.2 - Partial mass spectrum (m/z = 98 - 650) of MO-TMS ether derivative of lla -2H-cortisol 31 3.0E4- G07 24G / o2. 0E41 c rtf 3G2 T3 207 3C / CE_Q 5 1G 10000" 42G 457 36 , , 100 200 300 400 500 G00 Mass/Charge 320 Ions 605.55 and 606.55 were selected for SIM quantification. The ions are referred to as 605 and 606 from now on. (2) Establishment of SIM method The temperature program used was based on that for THDOC SIM runs, but with an extended run time, see Figure A 1.3. The two samples prepared for mass spectral analysis were run using this SIM program and the ratio of ions 605:606 was found to be: cortisol 1.904 ie. ion 606 =52% of ion 605 2H-cortisol 0.165 ie. ion 605 = 6% of ion 606 (94% enrichment) (3) Standard curve Two standard curves were prepared with 2HF ranging from 0 - 200ng and fixed F of 200ng - see Figure A1.4. An example of a SIM run using standards is shown in Figure A1.5. The sum of the areas of the syn and anti peaks was used in the calculation of results. (4) Extraction of cortisol from plasma The following method of extraction was adopted for all samples: Plasma made up to 2ml with distilled water I 20ml dichloromethane added,thoroughly mixed I Two layers allowed to separate, top layer and emulsion discarded * Dichloromethane dried off on rotary evaporator I MO-TMS I GC-MS 321 Figure A1.3 - Temperature programme for SIM quantification of cortisol Oven Temperature (°C) 300-i Data 280- Acquisition 260- 240- 2 2 0 - 40 C/m in 2 0 0 - Approx. Injection time for 180- cortisol 160- 140 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Time (min) Figure A1.4 - Standard curve 2.1 X 1st curve 4- 2nd Curve 1.7 - 1.3- 0.9- 0.7 0 0.1 0.2 0.3 0.4 0.6 0.6 0.7 0.8 0.9 1 2HF:F Ratio 322 ruguic /\i.j - oim iuu ui uuiusui aiiu n-curusui suuiuaius Ion 605.55 1.5E4; 1 . 5E4 l 0000: 10000 -5000 Ion 606.55 1 . 5E4 i 0000: ;5000 Figure A1.6 - SIM run of 0.1ml serum spiked with lOng of 2H-cortisol Ion 605.55 1200: 1000: 800: 600: 400: 200: CDU Ion606.55 c 1200: r 1 200 £ 1000: 1000 800: 800 600 400 200 9.5 0.5 11.0 Ti me (mi n . ) 323 Table A 1.1 - Spiking experiment using deuterated cortisol Calculated Quantity 2H-F measured equivalent ratio quantity of F added to 1ml 605:606 ratio ^ - F : F present in 1ml serum * serum (ng) # lOOng 1.152 0.55:1 182 50ng 1.500 0.26:1 192 10ng 1.860 0.05:1 200 Ong 1.987 0:1 - * Mean of values from two extractions for each quantity of 2H-F added # Mean = 191ng CV = 4.7% Table A1.2 - Ratios of cortisol synranti peak areas using ions 605 and 606 Ion 605.55 Ion 606.55 mean ± SD CV mean + SD CV Standards 0.601 ±0.049 8.2% 0.594+0.044 7.4% (n=16) Control Subject - No Drugs 0.564 +0.042 7.5% 0.583±0.045 7.7% (n=18) Control Subject - On Carbenoxolone 0.528+0.049 9.3% 0.545 ±0.052 9.6% (n=18) 324 Spiking experiments with human plasma were performed. Using 0 .1ml of serum, the ion response results were very poor due to insufficient sample, with signal to noise ratio being unacceptable (see Figure A 1.6). The use of 1ml of serum produced better results, as shown in Table Al.l. (5) Analysis of plasma from a normal subject, and the effect of carbenoxolone. after 2H-cortisol administration A normal healthy male was administered (by injection) with 0.7mg lla - 2H- cortisol and regular blood samples (n=9) were taken for two hours. The procedure was repeated after 7 days of carbenoxolone therapy. Plasma (1ml) was processed as described above and injected twice into the mass spectrometer. This was repeated a few days later to give duplicate results for all samples. The 605:606 ratio (mean of two injections) was calculated (see Figure A 1.7) and using the standard curve the 2HF:F ratio calculated (see Figure A 1.8). The ratio of the syn and anti peaks were also calculated for standards and samples to check the specificity of the results (see Table A 1.2). The total cortisol (in-house method, as measured by Mr. P. Holownia) was measured in each sample by RIA, results are shown in Figure A 1.9a. Calculation of the 2HF (nmol/1) using the RIA results still did not smooth out the decay curve sufficiently to allow the calculation of the half-life. In the hope that it would be a more appropriate measure of enrichment of a plasma sample "atoms percent enrichment” (ie. [sample 606/605 - basal 605/606] x 100) was calculated. A linear standard curve (not shown) was obtained by this calculation. The apparent smoothing of the decay curve of 2HF after i.v. injection was however considered artifactual because of plotting on a smaller Y axis. (6) Analysis of plasma from an adult subject with 11B-OHSD deficiency on dexamethasone. after 2H-cortisol administration After injection with 0.7mg lla -2H-cortisol, blood samples were taken at regular intervals (n=14) from a patient with 11J5-OHSD deficiency whose endogenous cortisol had been suppressed with dexamethasone. Cortisol was measured by RIA (in-house method at Western General Hospital), see Figure A 1.9b. 605:606 ratios were calculated after extraction, derivatization, and SIM runs on the MSD (see Figure A 1.10). As the 2HF was metabolized (not exclusively by llfi-OHSD) the 325 Figure A1.7 - 605:606 ratios from plasma samples basal, and with carbenoxolone treatment in a normal subject 605:606 ratio 2.2 Basal Carbenoxolone 1. 8 - 1. 6 - 1.4- 0 10 20 30 40 50 60 70 80 90 100 110 120 Time (min) Figure A1.8 - 2HF-.F ratios from plasma samples basal, and with carbenoxolone treatment in a normal subject 2HF:F ratio 0.7 Basal 0 . 6 - Carbenoxolone 0.5- 0.4- 0.3- 0.2 - s-e- 0 10 20 30 40 50 60 70 80 90 100 110 120 Time (min) 326 Figure A1.9 - Cortisol (RIA results) (a) Normal subject Cortisol (nmol/L) 400 300 - 2 0 0 - 100 - Basal Carbenoxolone 0 20 4060 80 100 120 Time (min) (b) 11B-OHSD deficient patient Cortisol (nmol/L) 600 500 - 400 - 300 - 200 - 100 - 0 20 6040 80 100 120 140 160 180 200 Time (min) 327 Figure A1.10 - 605:606 ratios from plasma samples in an adult subject with 11B-OHSD deficiency 605:506 ratio 0.6 0.5- 0.4 - 0.3- Levels too low to measure 0.2 - 0 10 20 30 40 50 60 70 80 90 100 110 120 Time (min) 328 signal to noise ratio decreased to a point, after the sample at 60 min post injection, where accurate values could not be obtained. Insufficient sample was available to repeat the experiment with a known amount of non-labelled cortisol added to each sample in order to allow quantification. A1.3 Results and Discussion In the normal subject the labelled cortisol was easily detectable during its distribution phase, showing that sufficient substrate for these analyses was injected. The labelled cortisol declined rapidly over the first 10 min in the basal test, with the enrichment approximating a plateau level by 7 minutes when the first post-injection sample was taken, indicating that more frequent sampling, possibly every minute for the first 10 - 15 minutes would have been more informative. The level of enrichment declined at a slower rate in the same subject when on carbenoxolone, with the level of enrichment (ie 2HF:F) being higher at all time points, despite lower cortisol (by RIA) levels than basal. The half life of cortisol in both tests was considerably shorter than the equivalent experiments using lla -3HF (personal communication from Dr Walker). In the patient with 11B-OHSD deficiency the decline of 2HF was comparable over the same time scale with that of the normal subject on carbenoxolone. It must be remembered that when measuring the decline of the labelled substrate that other metabolic pathways other than just 11B-OHSD activity may be involved. In studies where isotopically labelled H20 is measured a truer reflection of the action of 116- OHSD could be assessed. This was not possible with the experiments undertaken in this pilot study. The measurement of 2H20 would also require a large volume of sample (>20ml), (greater than that needed with a tritiated compound and scintillation counting), making it routinely impractical in particular if frequent sampling was wanted. The influence of isotopic effects of deuterium in the metabolism of cortisol are also uncertain. The problem of the fluctuating endogenous cortisol must also be considered. RIA measurements of cortisol (see Figure A 1.9) showed a number of fluctuations, and this endogenous cortisol would effect the calculated amount of 2HF present in the sample. Even with correction after RIA measurements in the samples the decay curve was not appreciably smoothed. This could possibly be overcome by (1) abolishing 329 endogenous cortisol with dexamethasone suppression, and then spiking the sample with a constant quantity of unlabelled cortisol before GC-MS, or (2) performing the study during constant rate infusion of cortisol. The latter would however not allow the study of physiological variation in 11B-OHSD activity which may be controlled by ACTH or glucocorticoid exposure. It was interesting to note that in the 11B-OHSD deficient patient that, after the distribution phase, there was a period of plateau of 605:606 ratio. This ratio then rose indicating that either endogenous cortisol was being produced (though unlikely due to the dexamethasone suppression) or that the reverse enzymatic reaction was occurring where unlabelled cortisone was converted to unlabelled cortisol by 11-oxo- reductase. Patients with 11B-OHSD deficiency still have full 11-oxo-reductase activity. The use of multiple 2H labelled cortisol would reduce the reliance of a ratio with endogenous cortisol and allow both dehydrogenase and reductase activity to be followed. Three (or possibly four) deuterium labelled sites would probably be ideal, with a least one deuterium atom in a site that would be cleaved, allowing cortisol turnover measurements without altering the metabolic kinetics by molecule size too much. Since this pilot study was undertaken a synthesis of deuterium labelled cortisol (9,11,12,12-2H4) has been reported by Linberg et ah (1991) for the use in the study of the rate of 11B-hydroxydehydrogenation in man. This labelling was chosen for use in determination of the residual deuterium content, rather than attempting to measure deuterated water or the fall in lla -2H-cortisol. 330 APPENDIX 2 ADDITIONAL WORK INVOLVING DEUTERIUM LABELLED STEROIDS A2.1 Deuterium labelled THDOC In quantitative GC-MS an isotope labelled internal standard is an alternative to using an isomer. The internal standard is chosen so that it does not separate from the unlabelled analyte, rather the relative isotope distribution, after the addition of a known quantity of labelled steroid by means of a prepared standard curve, can be used for for reference in quantification. The presence of 3 deuterium atoms is usually the best compromise between maximizing the difference in masses of the ions quantified for the internal standard and the unlabelled analyte, and the increase in mass causing separation of the two compounds by GC. An example of such separation is described in Section A2.2, though the 2H5 steroid was not used as an internal standard in that case. THDOC labelled with three deuterium atoms at C-17a, C-21, and C-21 had been synthesised in 1979 and used in experimental work for initial experiments to determine the use of deuterium labelling in mineralocorticoid analysis (Shackleton et al . , 1979b). This work reported that the 2H3-THDOC was stable during normal methodological procedures for urinary analysis. The molecular ion (M+) of the methyloxime trimethylsilyl ether derivative of 2H3-THDOC was 510 (3 greater than unlabelled THDOC), and the ion 479 (M+-31) with the loss of part of the oxime group was also easily detected. This labelled steroid had been stored in ethanol and after 10 years I reassessed the isotope enrichment of the compound. The isotope enrichment is shown along with that of the 331 Figure A2.1 - Isotope enrichment in 3a5B THDOC (unlabelled and deuterium labelled) % of total response 1 0 0 - 90 - original d3 steroid 80- old d3 steroid unlabelled steroid 70- 60- 50 - 40 - 30 - 20 - 10 - ^ 476 478 480 482 476 478 480 482 476 478 480 482 ion (m/z) % of total response 100 90 - original d3 steroid 80 _ old d3 steroid Liiil unlabelled steroid 70 60 d0 50 40 30 20 10 0 l l l l 1111 i ii ii i 607 609 611 613 607 609 611 613 607 609 511 ion (m/z) freshly made compound and unlabelled THDOC in Figure A2.1. The freshly made deuterium labelled compound and the unlabelled THDOC standard showed similar patterns of isotope enrichment for both the 476/479 and 507/510 clusters of ions, with the deuterium labelled compound consistently 3 mass units higher. The stored labelled compound on the other hand showed a different pattern of enrichment. The two freshly prepared standards (labelled and unlabelled) had the base peaks of the ion clusters at d3 and d0 respectively, and then show a steady fall in response with rise in m/z, these ions being present due to the naturally occurring heavy isotopes of carbon, hydrogen, silicon, oxygen and nitrogen. In the stored labelled standard the base ion response of the ion clusters was at do + 1 (m/z = 477 or 508). The d0 + 2 ion was as expected the next largest ion response each ion cluster, but was larger than expected, at 81 % of the base ion response of the ion cluster, rather than the 42% (mean value) of the base peak response as seen in the freshly made standards. The d0 + 2 ion response was therefore made up of a mixture of d2 and the heavy isotope found naturally. This would suggest that deuterium enrichment had been lost over the 10 year period, possibly by exchange of deuterium with hydrogen atoms in the storage ethanol or any water in the solvent. Varying degrees of exchange had occurred resulting in the presence of d0, dlf d2 and only a small amount of d3 THDOC in the solution. This degraded labelled steroid was therefore not suitable for use as an internal standard for quantification analysis. 333 A2.2 Identification of deuterium labelled metabolites after labelled steroid administration Steroid metabolism can be investigated by loading a subject with a particular deuterium labelled steroid and analyzing biological fluids using GC-MS. Examples of this type of analysis were reported by Curtius et al. (1975). They looked at the deuterium content of steroid metabolites in urine, after separate loading with deuterated progesterone, pregnenolone and cholesterol. Urine was available from two normal male subjects who had been loaded intravenously with either [17,21,21,2 l]-2H 4 -pregnanoloneor [2,2,4,6,6,21,21,21]-3H8- 17-hydroxyprogesterone. Urinary steroid profiles (using the method described in Chapter 2) were analyzed in scan mode using GC-MS. After 2H4-pregnanolone administration only pregnanediol (PD) and allo- pregnanediol were seen to be deuterated. One deuterium atom was lost, as was shown by only an increase of 3, rather than 4, of the 117 base ion response seen in unlabelled PD. The mass spectra of unlabelled and the d3-pregnanediol are shown in Figure A2.2. After 2H8-17-hydroxyprogesterone loading pregnanetriol was the prominent deuterated metabolite seen. This metabolite and unlabelled pregnanetriol are shown in Figure A2.3. The large number of deuterium atoms present in the molecule resulted in sufficient change in the polarity the compound to cause some separation from unlabelled pregnanetriol as shown in Figure A2.4. 334 urine extract of an adult male subject loaded with 2H4-pregnanolone 3.0E51 2.0E5H 255 269 368 475 347 449 _Q 100 200 300 400 500 Mass/Ch arge 20 1 . 0E5 T 3 5.0E4 : 155 217 369 / / 2P 349 453 504 100 200 300 400 Mass/Ch arge Figure A2.3 - Partial mass spectra (m/z = 98 - 520) of the MO-TMS ether derivatives of (a) pregnanetriol and (b) 2H5 pregnanetriol from the urine extract of an adult male subject loaded with 2H8-17- hydroxyprogesterone 4 . 0E5 4 255 435 3.0E5 2.0E5 147 159 345 1 . 0E5 283 _Q 501 100 200 300 400 500 Mass/Ch arge 4.0E5 440 260 3. 0E5 147 2.0E5 350 \ 161 220 _Q . 0E5 CE 502 200 300 400 500 Mass/Ch arge 335 Figure A2.4 - Part of the total ion chromatogram from the urine extract of an adult male subject loaded with 2Hg-17-hydroxyprogesterone (MO-TMS ether derivatives) 4.0E6- pregnanetriol pregnanediol 3.0EG- androstenetriol 2.0EG" allo-pregnanediol 336 APPENDIX 3 MASS SPECTRAL DATA ON DEUTERIUM LABELLED PROGESTERONE A3.1 Introduction The initial aim of this project was analyze the metabolic fate of progesterone, using GC-MS analysis of the excreted products by means of a deuterium labelled analogue. During the attempt to produce sufficient stable isotope labelled material I analyzed the products and some compounds in their production, in order to check purity and isotopic enrichment. This section details the results from these analyses. All compounds were analyzed as methyl oxime derivatives (no hydroxyl groups being present in progesterone), the derivatives being prepared as described in Chapter 2, using both MO-HC1 and TMSI, to allow silylation of hydroxyl groups in any reduced products. Unlabelled progesterone as a MO derivative had a strong molecular ion (M+) of 372, and another strong ion at m/z 341 (M+-31), from the loss of part of an oxime group. A doublet peak with incomplete separation is seen for progesterone, both peaks giving similar mass spectra. Isotope enrichment of the deuterium labelled progesterone was calculated by either simultaneous SIM of the ions between 370 and 379 inclusive, and comparison of the ion response areas, or measurement of the relative heights of the ion responses from mass spectra. Problems were encountered in the production of sufficient amounts of any of the deuterium labelled compounds described below, due to large losses encountered in some of the many steps involved in labelling this steroid at specified carbon atoms. A3.2 H1.11.12.121 -Pro gesterone This labelled compound was synthesized as described by Kirk et al. (1990a). The partial mass spectra of this compound and unlabelled progesterone (as MO 337 derivatives) are shown in Figure A3.1. The deuterium content of this compound was found to be d4 61%, d3 25%, d2 7%, dl 1%. A by-product of this synthesis was 11,12,16-2H3-5a-pregnanedione. When a derivatized sample of the crystals was analyzed by GC-MS four peaks (two doublet peaks), were found. The total ion chromatogram is shown in Figure A3.2. Both peaks in the first doublet had similar mass spectra (Figure A3.3(a)) including ions m/z = 343 and 374. The mass specbwfrom the second doublet (Figure A3.3(b)) contained ions m/z = 345 and 376. This would suggest that the former doublet had an unsaturated bond (hence 2 mass units less), but other wise had a similar structure, so was probably ll,ll,1 6 -2H3-pregnenedione (ie. progesterone). A3.3 fl5 .15.16-2H,1-Progesterone This deuterated progesterone had poorer isotope enrichment than the above compound, with d3 44%, d2 46% and dj 13%. The partial mass spectrum of this compound is shown in Figure A3.4. The deuterated pregnanedione by product was also analyzed, the mass spectrum of one of the peaks of the doublet is shown in Figure A3.5. A3.4 f l. 11.12.16-^1-Progesterone The synthesis of this deuterated compound was less successful. Analysis of the derivatives of the compounds in the steroid crystals in scan mode revealed at least 3 different deuterated compounds (Figure A3.6). A large peak of the expected deuterated compound was seen, the mass spectrum of this compound is shown in Figure A3.7. The isotope enrichment of this compound was d4 48%, d3 39%, d2 11%, dj 1%. On the trailing edge of the progesterone doublet peak a doublet peak was detected, the spectrum of which would match a 2H3-progesterone (Figure A3.8(a)). The presence of a strong m/z = 101 ion response suggests it was not the C-16 deuterium atom was not one of the two deuterium atoms not incorporated. The other doublet peak had a retention time prior to the deuterated progesterone, and it’s mass spectrum (Figure A3.8(b)) had some similarities to deuterated pregnanedione. Ion responses of m/z = 374 and 343 are similar in intensity to ion responses m/z = 373 and 343, suggesting this peak is a mixture of 2H3 and 2H2-pregnanedione. 338 Figure A3.1 - Partial mass spectra (m/z = 98 - 380) of the MO derivative of ll,ll,12,12-2H4-progesterone (upper panel) and unlabelled progesterone (lower panel) 100: 345 80- 378 100- 341 80- 72 0) M+) o 80- c 273 (0 T5 40- 153 286 c 125 / 3 / / 172 XI 20 - 220 CE / 0 I .- i. i 150 200 250 300 350 Mass/Charge CH C=NrO-CH Progesterone 339 iu 32 oa in hoaormo h O eiaieo ll,12,16-2H3-5a- of derivative MO the of chromatogram ion Total - A3.2 Figue Rb u n d a n c gG j . 0 E 6 : 2.0E6: 2.0E6: 3.0E6: 3.0E6: 4.0E6: 5.0E6: .0EG:8 7.0E6: 7.0E6: 9. 0E6 1.0EG: 1 . 0E7i 0E7i . 0^ pregnanedione pregnenedione 11,12,16-2H3- ~ r — 15 TI me pregnanedione 11,12,16-2H3- (min.) 20 25 i— — 30 340 Figure A3.3 - Partial mass spectra (m/z = 98 - 400) of MO derivatives of ll,12,16-2H3-pregnenedione (upper panel) and ll,12,16-2H3-5a- pregnanedione (lower panel) 0E5 259 aj 343 o 0E5: c rd 101 374 *o d 0E5: 3 S 2 CL 0 LUu 100 200 300 400 Mas s/Ch arge 4. 0E5 101 345 Qj 0E5 O c nd 2 . 0E5 26 2 376 3 0E51 \ _Q \ / CL 0 100 200 300 400 Mass /Ch arqe 341 A iU M4*i A AAUkJkJ kJ|/W V l U y iil/ M-t ----- \J ~T\J\J J V/l 1»AV/ U V i A. ▼ UVA » V V I A*/ ^ A«/^ A V 2H3-progesterone \ 343 8.0E5 374 S 6.0E5 74 C ■o 4.0E5: c a: 2.0E5H 0 yk n4 i,iLX.«fc JlJ i t i iJ .. i 200 400 Mass/Ch arge Figure A3.5 - Partial mass spectra (m/z = 98 - 400) of MO derivative of 15,15,16- 2H3-pregnanedione 101 6.0E5- 291 346 342 Figure A3.6 - Total ion chromatogram of MO-TMS ether derivative of crystals of 1,11,12, ^-^-progesterone 2.0E7- -Q 20 22 24 26 Figure A3.7 - Partial mass spectrum (m/z = 98 - 400) of MO derivative of 1,11,12, ^-^-progesterone 345 76 155 276 229 272 200 300 400 Mass/Ch arge 343 Possible identification (a) 2H2-progesterone and (b) mixture of 2H3- and 2H2-pregnanedione 74 1G7 200 300 400 Mass/Ch arge B.0E5 259 359 oj G.0E5 374 4.0E5 15G Xd m -, 296 -a 2.0E5 ll ^ 200 300 400 Mass/Ch arge Figure A3.9 - Partial mass spectrum (m/z = 98 - 400) of MO derivative of 18,18,19,19-2H4-progesterone 4.0E41 345 37G 3.0E4 125 277 Jd2. 0E4 137 \ 17G 207 272 / 1 0000 /\ ill 111 ii 1 ii il 0 iiiiiinitiilillll iiikiiiiLik ■l.i »■ M.Mu -4huulv —r 100 150 200 250 300 350 Mass/Charge 344 A3.5 f!9.19.19-2ILl -Pro gesterone The synthesis of this compound and [18,18,18-2H3]-progesterone are described in the paper by Kirk et al. (1990b). Only the [19,19,19-2H3]-progesterone was analyzed. Isotope enrichment was good at d3 87%, d2 4%, dt 2%. Strong ion responses were seen for m/z = 375 and 344, confirming the presence of three deuterium atoms in the compound. A3.6 [18.18.19.19-^1-Progesterone Two batches of this deuterated compound were synthesised. They gave comparable isotope enrichment with d4 62%, d3 25%, d2 6%, dj 2% and d4 60%, d3 24%, d2 8%, dj 3% respectively. The mass spectrum of this compound is shown in Figure A3.9. In the last two deuterated compounds contamination of the compound with 17- iso-progesterone and 1,2-dehydroprogesterone was checked, to satisfy ethical committee criteria. None was detected in scan or SIM mode indicating <0.001% contamination. 345 APPENDIX 4 INITIAL DOC RADIOIMMUNOASSAY WORK A4.1 Introduction A partly developed quantitative method for plasma DOC was explored. Due to the DOC antibody’s relatively high cross-reactivity with progesterone («12%, Mr P. Holownia, personal communication), and the desirability of having an as pure material for radioimmunoassay (RIA) as possible, plasma was first extracted and then separated using high performance liquid chromatography (HPLC). The RIA was based on second antibody separation, and scintillation counting of the tritium for quantification. A4.2 RIA Reagents The following reagents were prepared for each assay: (1) Assay buffer - NaCl 8.75g Na2HP04 1.1757g NaH2P04.2H20 0.25g NaN3 l.Og Bovine Serum Albumin l.Og Made up to 11 with distilled water (could be stored at 4°C up to 1 month). (2) Standards - 11-deoxycorticosterone 1.28mM/l (stock solution in ethanol). 50/xl of stock solution was dried down (under nitrogen) and reconstituted in 5ml assay buffer (= 12.8/tM/l). 50/xl of this solution was made up to 5ml with assay buffer to give a final concentration of 128nM/l of DOC. A series of doubling dilutions was performed giving "working standards" of 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25nM/l. 346 (3) Antibody - The initial antiserum was diluted to 1:100 with assay buffer and stored at -20°C until further use. When required a further 1:33.3 dilution with assay buffer was made (150/zl in 5ml assay buffer). (4) Label - Neat label from Amersham (specific activity 250/zCi/250/xl) was diluted 1:20 with ethanol, giving a "stock label" of activity 50/xCi/ml. This was stored at -20°C until further use. The stock label was further diluted 1:100 in ethanol, and 300/xl of this solution was dried down and resuspended in 3ml of assay buffer to give the "working label". (5) Separation buffer -8% Polyethylene glycol (PEG) in assay buffer (6) Separation reagent - 24/zl Normal rabbit serum 300/xl donkey anti-rabbit serum made up to 15ml with separation buffer A4.3 RIA method Glass tubes used throughout, with all samples and standards done in duplicate. (i) The following was added to labelled tubes: 50/xl assay buffer for 0 standard 100/zl assay buffer for total counts (TC) and non-specific binding (NSB) 50/zl standards or samples (ii) 50/zl antibody was added to all tubes (except TC and NSB), and all tubes were vortexed. (iii) 50/zl of working label was added to all tubes, and all tubes were vortexed. Tubes were incubated at 37°C in a water bath for 2 hours. (v) 250/zl of separation reagent was added to all tubes except TC (250/zl of assay buffer was used instead). All tubes were vortexed, and then incubated at room temperature for 30 minutes. (vi) All tubes were centrifuged (2000xG) for 30 minutes at 4°C. (vii) 300/xl of supernatant was transferred from each tube to scintillation vials. 5ml of scintillant was added, and the vials were thoroughly vortexed, before counting for 20 minutes in a 13-counter. 347 A4.4 RIA Standard curves and the effect of alteration of antibody concentration Standard curves were prepared from the counts/20min, of % bound vs DOC concentration where: % bound = (total counts - "standard” counts x 100 total counts A typical result is shown in Figure A4.1, along with the standard curve obtained when the antibody dilution was altered from the usual 1:33.3 dilution of the stock antibody to 1:42.2. The more dilute antibody (1:33.3) resulted in the better standard curve - a slightly higher % bound for the low value standards and a larger working range ( « 1-32 nmol/1) than the more concentrated antibody standard curve. Typical non-specific binding (NSB) results were between 2.1 and 5% bound. A4.5 Initial HPLC results To establish the retention time of DOC, tritium labelled DOC was run on the HPLC. A 1:2500 dilution of neat label was made. 60 fx\ of this solution was dried down and resuspended in 0.6ml 25% isopropyl alcohol (IPA) in hexane. 400/zl of this was transferred to an HPLC injection vial. The HPLC was set up to automatically inject 200/fi onto the 25cm Lichrosorb diol column (Altech), with a guard column. The column was maintained at 40°C. Solvent was set to run in a gradient of 5 to 40% isopropanol to hexane over 50 minutes, at lml/min. Initially less than 100% recovery was measured for the injected labelled DOC (recovery = 83%). When the labelled material was run initially through the HPLC and the fractions containing DOC (as determined from counting 1 minute fractions) were collected, and these were used for recovery experiments, the recovery was improved. In a further set of experiments 50 x 1 minute fractions of the eluent off the column were collected in scintillation vials. Scintillant (5ml) was added to the vials and they were counted for 10 minutes. A second run with 200/xl of 25 % IPA in hexane injected onto the HPLC column was also analyzed, 5 minute fractions of eluent collected in this case. The results of these two runs are shown in Figure A4.2. Both sets of results showed a parallel rise in baseline, but a clear peak of DOC was seen at 16 - 18.5 minutes. 348 Figure A4.1 - Standard curve for DOC RIA and the effect of antibody concentration % Bound 70 O 1:33.3 Ab dilution 60 8 o o □ 1:42.2 Ab dilution o o 60 B B 8 □ □ 40 Q □ □ 30 20 8 10 B O 0 0.1 1 10 100 DOC (nmol/l) Figure A4.2 - Tritium content of HPLC fractions - Labelled DOC vs background Counts/10min (x1000) (Thousands) 16 14 — DOC © blank run 12 10 8 6 4 2 0 0 6 10 15 20 25 30 35 40 45 50 Time (min) 349 Some of the labelled DOC was also counted without passing through the HPLC system, and this allowed confirmation of recovery of all radioactivity. The cumulative count of the total run was 109% of that calculated for the equivalent quantity of labelled DOC counted directly. 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