THE EFFECT OF OXIDATION PHENOTYPE
ON THE DISPOSITION OF
CERTAIN ENDOGENOUS STEROIDS
HIKMAT HAMDI NADIR
A Thesis Submitted
For The Degree Of
DOCTOR OF PHILOSOPHY
In The
UNIVERSITY OF LONDON
Department of Pharmacology,
St. Mary's Hospital Medical School,
London, W2 IPG. March 1985 To My Wife
and
In Memory ot My Father » -3
ABSTRACT
1. This thesis addresses the question as to whether or not the
allelomorphic gene which governs the oxidative metabolism of a
number of drugs including debrisoquine, influences the oxidative
metabolism of certain steroids in both man and rat. ft
2. Human subjects, both male and female, of known oxidation phenotype
were investigated for urinary excretion of seven individual 17-
oxosteroids and three progesterone metabolites by gas-liquid
chromatography. Persons of the recessive poor metaboliser phenotype
excreted only half the pregnanetriolone of extensive metabolisers.
3. Urinary oestrogen excretion did not differ between phenotypes,
including for E^, E^, Eg and their 2- and 16-oxygenatea metabolites.
4. Serum cholesterol concentration in extensive and poor metaboliser
volunteers did not differ. In patients with excessively high or
low serum cholesterol concentrations, there was no association with
oxidation phenotype.
5. It was concluded that, in man, no simple determinant of endogenous I steroid disposition had been found that could be used as a non-
invasive phenotyping test.
6. Similar investigations were also carried out in rats of known
oxidation phenotype. No differences between female DA
(phenotypically poor metaboliser) and Lewis (phenotypically
extensive metaboliser) rats were found for urinary 17-oxosteroid
and progesterone metabolite excretion. » * -4
7. DA rats given [i^C]-oestrone (20 mg i.p.) recovered four-fold more 14 C in the first day urine than Lewis rats. However, Lewis rats
excreted 50% more water, Na+ and K+ in the first day than DA rats. l Whether or not this finding has a metabolic origin is not known.
Biliary cannulated Lewis rats excreted 2-3-fold more 2-hydroxy-
oestrone than DA rats in the 0-1 h bile after a dose of 16 pg 14 [ C]-oestrone.
8. Additionally, serum cholesterol of DA rats was 50% greater than
Lewis rats and a number of other extensive metabolising rat strains.
Neither K nor V for cholesterol 7ci-hydroxyl ase showed an m max interphenotype difference in rats. However, 3-methylcholanthrene
treatment increased the K and V approximately twice as much in m max J DA than Lewis rats.
9. It is concluded that no non-invasive phenotyping test was
uncovered during the course of this work. However, a number of
clues emerged which may lead to such a test in the future.
* »
CONTENTS
Abstract
List of Tables
i List of Figures
Acknowledgements
CHAPTER ONE
INTRODUCTION
1. Introduction
1.1 Drug absorption
1.2 Drug distribution
1.3 Drug excretion
1.4 Drug metabolism
1.4.1 Involvement of different forms
of cytochrome P-450
1.4.2 Steroid metabolism
1.5 Pharmacogenetics
1.6 Regulation of drug oxidation by the
debrisoquine hydroxylation locus
I 1.7 Animal model for drug oxidation
1.8 Aims of the present study ...
CHAPTER TWO
EFFECT OF POLYMORPHIC OXIDATION ON URINARY
STEROID PROFILES IN MAN AND RAT ...
2.1 Introduction ... f -6-
Contents, continued Page
2.1.1 Urinary 17-oxosteroids 42
2.1.1.1 Androsterone 45
2.1.1.2 Aetiocholanolone 45
2.1.1.3 Dehydroepiandrosterone 46
2.1.1.4 The 11-oxo- and 118-hydroxy-
17-oxosteroids 46
2.1.2 Urinary progesterone metabolites 47
2.1.3 The involvement of cytochrome P-450 in
steroid hydroxylations 49
2.1.4 The genetic abnormalities of steroid
hydroxylation 54
2.1.5 Aims of the investigation 55
4 2.2 Materials and Methods 58
2.2.1 Materials 58
2.2.2 Animals 59
2.2.3 Human subjects 59
2.2.4 Methods 60
2.2.4.1 Determination of oxidation
phenotype ... 60 t 2.2.4.2 Determination of individual 17-oxosteroids, pregnanediol,
pregnanetriol and pregnanetriolone
in urine 62
2.2.4.3 The colorimetric quantitative determination of creatinine in
urine 70
2.2.4.4 Determination of total urinary
17-oxosteroids 71
¥ Contents, continued Page
2.3 Results 74
2.3.1 Population study 74
2.3.2 Steroid profiles in man 74
2.3.2.1 The effect of sex 74 2.3.2.2 The effect of phenotype 87
2.3.3 Steroid profiles in rats 87
2.3.4 Total 17-oxosteroids in a panel of
subjects arid two different phenotyped
strains of rats 88
2.3.5 Analysis of the 5$/5a and DEA/A + Ae
ratios 88
2.3.6 Comparison of urinary 17-oxosteroid
concentration determined by colorimetry
and by summation of individual 17-oxosteroids
determined by gas chromatography 94
2.4 Discussion 98
CHAPTER THREE
EFFECT OF POLYMORPHIC OXIDATION ON URINARY
OESTROGEN EXCRETION IN MAN AND RAT 103
3.1 Introduction 104
3.2 Materials and Methods 113
3.2.1 Materials 113
3.2.2 Animals 113
3.2.3 Human subjects 114
3.2.4 Dosing of animals and collection of
excreta 114 -8-
Contents, continued Page
3.2.5 Preparation of faeces for g.c.
analysis 114 14 3.2.6 Preparation of faeces for C
analysis 114 » 3.2.7 Counting of [14C]-oestrone 116
3.2.8 Analytical procedures 116
3.2.8.1 Determination of oestrogens and
metabolites in urine employing
gas-liquid chromatography ... 116
3.2.8.2 Determination of biliary, faecal
and urinary metabolites of labelled
oestrone in rats by HPLC 120
3.2.8.3 Collection of bile from rats.. 128
3.2.8.4 Determination of sodium and
potassium excretion in rat urine 128
3.3 Results ...... 131
3.3.1 Human urinary oestrogen profiles -
effect of phenotype ... 131
3.3.2 Faecal and urinary oestrogen metabolite
profiles after administration of labelled
oestrone to DA and Lewis rats 134
3.3.3 Oestrogen profiles in bile of DA and
Lewis rats after an intramesenteric venous
dose of labelled oestrone 139
3.3.4 Sodium and potassium excretion and urine
volume in Lewis and DA rats after oestrone
administration 142 - 9 -
Contents, continued Page
3.3.5 Administration of 2-hydroxyoestrone and
16oi-hydroxyoestrone to Lewis and DA rats 146
3.4 Discussion ... 147 ► CHAPTER FOUR
EFFECT OF POLYMORPHIC OXIDATION ON SERUM
CHOLESTEROL IN MAN AND RAT AND CHOLESTEROL
7a-HYDROXYLATION IN THE RAT ... 156
4.1 Introduction 157
4.1.1 Cholesterol biosynthesis 157
4.1.2 Metabolism of cholesterol in vivo 159
4.1.3 Metabolism of cholesterol in vitro 161
4.1.4 The involvement of cytochrome P-450 in
cholesterol 7a-hydroxylation 164
4.1.5 Deficiency of the LDL receptor leads to
hypercholesterolaemia and premature
atherosclerosis 165
4.1.6 Aims of the study 167
4.2 Materials and Methods 168
4.2.1 Materials 168
4.2.2 Animals ... 168
4.2.3 Human subjects 169
4.2.3.1 Healthy subjects 169
4.2.3.2 Volunteers with "high" or "low"
serum cholesterol 169 - 10-
Contents, continued Page
4.2.4 Methods ...... 170
4.2.4.1 Determination of human/rat cholesterol by colorimetry ... 170
4.2.4.2 Determination of the activity of
cholesterol 7a-hydroxylase in the liver t of DA and Lewis rats ... 171
4.2.4.2.1 Preparation of liver cell fractions
and incubations ... 171
4.2.4.2.2 Determination of protein ... 172
4.2.4.2.3 Isolation of radioactive products
by thin layer chromatography ... 172
4.2.4.2.4 Radiochromatogram scanning ... 173
4.2.4.2.5 Quantitation of radioactive peaks 173
4.3 Results ...... 176
4.3.1 Serum cholesterol levels of different
strains of rats ...... 176
4.3.2 Effect of 3-methylcholanthrene induction on
the serum cholesterol in the rat ... 180
4.3.3 Relationship between oxidation phenotype
and serum cholesterol in human ... 180
4.3.4 Determination of cholesterol 7a-hydroxylase I activity in rat liver fraction ... 183
4.3.5 Derivation of Km and Vmax of the cholesterol
7a-hydroxylase system ...... 187
4.3.6 The effect of 3-methylcholanthrene induction
on the activity of cholesterol 7ot-hydroxylase 189
4.4 Discussion ...... 196 Contents, continued Page
CHAPTER FIVE
GENERAL DISCUSSION AND CONCLUDING REMARKS ... 200
5.1 General Discussion ...... 201
BIBLIOGRAPHY 208
APPENDIX 252 % - 12-
LIST OF TABLES
Table Page
1.1 Polymorphic drug oxidation ...... 34
1.2 Regulation/inf1uence of the debrisoquine i hydroxylation locus on various metabolic pathways ...... 36
2.1 Urinary excretion (ranges, pg/24h) of individual
17-oxosteroids in male and female children and
adults ...... 44
2.2 Spectral dissociation constants (Ks) for
compounds interacting with human foetal adrenal
microsomes ...... 53
2.3 Classification of the adrenal hyperplasias ... 56
2.4 Different methods used for determination of
steroids ...... 63
2.5 Gas chromatographic characteristics of derivatized
17-oxosteroids, pregnanediol, pregnanetriol and
prenanetriolone on 3% OV-17 ...... 66
2.6 Mean (+ S.D.) of 17-oxosteroids, pregnanediol,
pregnanetriol and pregnanetriolone/creatinine i ratios (pM/mM) in human urine. The effect of sex ...... 85
2.7 Mean (+S.D.) of 17-oxosteroids, pregnanediol,
pregnanetriol and pregnanetriolone/creatinine
ratios (pM/mM) in human urine. The effect
of phenotype ...... 86 Table Page
2.8 Mean (+S.D.) values of individual 17-oxosteroids/
creatinine in rats 89
2.9 Urinary excretion of total 17-oxosteroids
(mg/24h) in human 90
2.10 Urinary excretion of total 17-oxosteroids
(mg/48h) in rats 91
2.11 Mean (+ S.D.) of Ae/A and DEA/A + Ae ratios in
human. The effect of sex 92
2.12 Mean (+ S.D.) of Ae/A and DEA/Ae + A ratios in
human. The effect of phenotype 93
2.13 Comparison of colorimetric and gas chromatographic
determinations of 17-oxosteroid concentration in
human urine 95
3.1 Dose, route of administration and vehicles
used for oestrogens given to rats 115
3.2 Gas chromatographic characteristics of some
silylated steroids, including oestrogens,
on 1% 0V-1 119
3.3 HPLC characteristics of oestrogens on
Lichrosorb diol column eluted with 7%
ethanol in hexane 129
3.4 Volunteers used for urinary oestrogen
determination ... 133
3.5 % Dose recovered by DA and Lewis rats as 14 C in urine and faeces after administration
of 20 mg (+ 2 pCi) oestrone p.o. 135
3.6 % Dose recovered by DA and Lewis rats as
14C in urine and faeces after administration
of 20 mg (+ 2 pCi) oestrone i.p. 137 Table Page
3.7 % Dose recovered by DA and Lewis rats as 14 C in urine and faeces after administration
of 54 pg (10 pCi) oestrone i.p. ... 1
3.8 Mean (+ S.D.) of urine volume, urinary sodium
and potassium excretion before and after
administration of high dose oestrone i.p. in
Lewis and DA rats 145
4.1 Suppliers of rats used and some physical
characteristics 169
4.2 Rp values of cholesterol and its relevant
derivatives in different solvent systems 174
4.3 Serum cholesterol levels of rats of
various strains 177
4.4 Serum cholesterol levels of F^ generation
hybrids 178
4.5 Physical properties together with metabolic
ratios and serum cholesterol levels of
individual generation hybrids 181
4.6 Effect of 3-methylcholanthrene induction
on serum cholesterol 182
4.7 Effect of oxidation phenotype on serum
cholesterol in man 184
4.8 Relationship between metabolic ratio and
serum cholesterol in subjects with
abnormally "high" or "low" cholesterol 185
4.9 Effect of 3-methylcholanthrene induction
on cholesterol 7a-hydroxylation in Lewis
and DA rats i - 1 5 -
Table Page
5.1 Summary of the measurements made
in EM and PM ...... 203
5.2 Summary of the measurements made
in DA and Lewis rats ...... 204
i
>
* » - 16-
LIST OF FIGURES
Figure Page
1.1 Schematic electron transport chain during
microsomal drug oxidation ...... 27
• 2.1 Structures of the common urinary
17-oxosteroids ...... 43
2.2 Structures of progesterone and its common
urinary metabolites ...... 48
2.3 Early steps in the synthesis of corticosteroids
and testosterone from cholesterol ... 51
2.4 Principal pathways for adrenal corticosteroid
biosynthesis with cytochrome P-450-dependent
» reaction ...... 52
2.5 Typical chromatogram of debrisoquine and
4-hydroxy-debrisoquine ...... 61
2.6 Typical chromatogram of 17-oxosteroids,
pregnanediol, pregnanetriol and
pregnanetriolone ...... 65
2.7 Calibration curve for androsterone ... 67
2.8 Calibration curve for aetiocholanolone ... 67
1 2.9 Calibration curve for dehydroepiandrosterone ... 68
2.10 Calibration curve for pregnanediol ... 68
2.11 Calibration curve for steroids ... 69
2.12 Calibration curve for creatinine ... 72
2.13 Frequency distribution histogram of metabolic
ratio (debrisoquine/4-hydroxy-debrisoquine) ... 75
* - 17-
Figure Page
2.14 Frequency distribution histogram of urinary
creatinine 76
2.15 Frequency distribution histogram of A/Cr 77
2.16 Frequency distribution histogram of Ae/Cr 78 2.17 Frequency distribution histogram of DEA/Cr 79
2.18 Frequency distribution histogram of PD/Cr 80
2.19 Frequency distribution histogram of
11-oxo/Cr 81
2.20 Frequency distribution histogram of
il-$0H/Cr 82
2.21 Frequency distribution histogram of PT/Cr 83 2.22 Frequency distribution histogram of PTone/Cr ... 84
2.23 Correlation between colorimetric and gas
chromatographic determinations of 17-oxosteroid
concentration in human urine ... 96
2.24 Correlation between the rank of urinary
17-oxosteroid concentration determined by
colorimetry and gas chromatography 96
3.1 Chemical structure of oestrogens employed and
the internal standard 105
3.2 Some pathways in the metabolism of oestrogens ... 107
3.3 Typical chromatogram of principal oestrogens
and oestrone metabolites as their TMS derivatives 118
3.4 Calibration curves for oestrone 121
3.5 Calibration curves for 2-methoxyoestrone 122
3.6 Calibration curves for 2-hydroxyoestrone 123
3.7 Calibration curves for 16a-hydroxyoestrone 124
3.8 Calibration curves for 16-oxo-oestradiol ... 125 - 18-
Fi gure Page
3.9 Calibration curves for oestriol 126
3.10 Calibration curve of oestradiol and its
glucuronide and sulphate conjugates 127
3.11 Typical high performance liquid chromatogram
of oestrogens and oestrone metabolites 130
3.12 Urinary oestrogen gas chromatograms from
three human subjects 132
3.13 Bile flow in DA and Lewis rats 140 14 3.14 The rate of elimination of C into bile
of DA and Lewis rats as semi logarithmic
piots ...... 141
3.15 Frequency distribution histogram of the
0-1 h bile from a Lewis rat 143
3.16 Frequency distribution histogram of the
0-1 h bile from a DA rat 144
4.1 Key steps involved in cholesterol
metabolism in the li.ver 158 14 4.2 Typical radiochromatogram of [ C]-
cholesterol incubated with rat liver
S.. fraction ... 175 i y 4.3 Frequency distribution histogram of
serum cholesterol for various Lewis X
DA hybrids 179
4.4 Correlation between cholesterol concentration
rank and metabolic ratio rank in subject with
abnormally "high" or "low" cholesterol 186
4.5 A typical time course of cholesterol
7a-hydroxylase activity 188 - 19-
Fi gure Page
4.6 Lineweaver-Burk plots showing the activity
of cholesterol 7ot-hydroxyl ase in DA rat ... 190
4.7 Lineweaver-Burk plots showing the activity
of cholesterol 7a-hydroxylase in Lewis rat ... 191
4.8 Lineweaver-Burk plots showing the activity
of cholesterol acyl transferase in DA rat ... 192
4.9 Lineweaver-Burk plots showing the activity
of cholesterol acyl transferase in Lewis rat ... 193
4.10 Lineweaver-Burk plots showing the effect of
3-methylcholanthrene induction on 7a-hydroxylase
activity in Lewis and DA rats ...... 195 -20-
ACKNOWLEDGEMENTS
I am very grateful to Professor R.L. Smith for giving me the opportunity to work and use the facilities in his department and for his guidance during my research time.
I would like to express my sincere gratitude to my supervisor
Dr. O.R. Idle for his invaluable advice, unlimited help and encouragement throughout the course of my work.
I am indebted to Mr. L.A. Wakile and Dr. N.S. Oates for their skillful preparation of the figures.
I wish to thank Mr. L.A. Wakile, Dr. O.C. Ritchie and Dr. S.G. A1-
Dabbagh for their assistance in the surgical procedures and dosing of the rats.
Special thanks are also due to Drs. J.C. Ritchie and N.S. Oates for their useful advice and discussion at different times.
My thanks are to Mr. M. Crothers and Mr. J. O'Gorman and to my colleagues who have helped me on particular points and who agreed to participate in normal volunteer studies.
I should also like to express my thanks to Dr. R. Unwin in the
Medical Unit for his collaboration in the measurement of electrolyte concentration, ana to Dr. P. Hirom in the Biochemistry Department for his assistance in bile experiments in rats.
I would like to thank Dr. T. Meade of C.R.C., Northwick Park
Hospital for providing me with urine samples from patients with "high" or "low" serum cholesterol.
Additionally, I would like to give special thanks to Miss Gerry
Bartlett for the excellent typing of this thesis. - 21-
I am indebted to the University of Mosul for financial support.
Finally, I am grateful to my mother for her patience and encouragement, my wife Nada for bearing with me, and to my daughters for their understanding and appreciation during this period of study. CHAPTER ONE
INTRODUCTION -23-
1. Introducti on.
The pharmacological effect of a drug is partly dependent upon its concentration at its site of action, which in turn is determined by several factors such as absorption, distribution and elimination. The rate of elimination of many drugs is mainly controlled by the rate of metabolism, and therefore any change in the activity of the drug metabolizing enzymes may result in an alteration of drug action.
1.1 Drug absorption.
Most drugs given orally are absorbed from the stomach or the small intestine. A number of factors can alter the absorption of a drug
(Grover, 1979), the most important of which are drug ionisation
(dependent upon pH and pKa) and lipid solubility. The drug formulation, for example particle size, can also influence the bioavailability of a drug. Concurrently administered drugs may also alter the absorption of drug. Sometimes drug toxicity is related to the vehicle used or contaminations introduced during synthesis of a drug. Ingredient-, vehicle interactions are especially important in cases of solutions used for giving slow intravenous infusions of a drug. Contamination of a drug can also be produced from disintegration of active ingredients into toxic substances through inappropriate storage. Gastric emptying time, intestinal motility, intestinal pathology or changes in gut enzymes are among other significant factors influencing drug absorption.
1.2 Drug distribution.
After absorption of a drug administered orally, it passes through the liver and is distributed throughout the body. Many drugs are highly protein bound and protein binding determines its distribution. The -24-
unbound fraction is responsible for the pharmacological effect of a drug. Variation in distribution leads to abnormal drug responses and can emerge for a number of reasons like the presence of unusual plasma proteins or interaction between two or more drugs at a common protein binding site. Rapid change in body fluid volumes, such as in dehydration, can furthermore give rise to changes in drug distribution.
1.3 Drug excretion.
For the majority of drugs which are metabolized to more polar metabolites prior to their renal elimination, impaired renal function contributes to metabolite-related pharmacological and/or toxic effects
(Orme, 1977). The fact that adverse drug reactions are more common in patients with renal diseases underlines the importance of drug renal handling. The factors that contribute to producing adverse drug reactions in these patients include decreased drug/metabolite renal clearance, altered receptor sensitivity, plasma protein binding, volume of distribution, impaired renal metabolism of certain drugs or electrolyte imbalances. It is now well recognised that drugs excreted unchanged by the kidneys may present special problems in elderly patients with compromised renal function.
1.4 Drug metabolism.
Drug metabolism is a mechanism by which lipid soluble drugs are rendered polar, which in turn aids their renal elimination (Williams &
Millburn, 1975). The metabolism of a drug can lead to the production of metabolites which a) are inactive, b) are toxic or c) produce secondary pharmacological effects of the drug (Williams & Mi 11 burn, 1975). Some drugs require metabolical activation for their primary pharmacological effects (Brodie & Reid, 1967). - 2 5 -
For the majority of drugs, metabolism occurs in the liver (Brodie &
Reid, 1967; Williams, 1967). Shannon had identified the role of drug metabolism in drug response as early as 1945/1946. However, it is becoming increasingly apparent that other tissues such as white blood cells, placenta, skin, lung and especially the gut may play an important role in the metabolism of drugs.
A number of factors affect drug metabolism including age and sex, hepatic blood flow, consumption of a heavy meal or alcohol consumption, smoking habits and concurrent drug administration (Prescott, 1979). The effect of concurrent drugs is either by induction, inhibition or competition for drug metabolizing enzymes.
Drug metabolic reactions are divided into two phases, Phase I and
Phase II. Most drugs are metabolized by these two phases, although there are some drugs which are metabolized by only one of these two phases (Williams, 1967; Williams & Millburn, 1975). During Phase I metabolism, insertion of a polar group such as a hydroxyl group takes place. Other mechanisms can be the removal of an alkyl radical from oxygen, nitrogen and sulphur alkyl ethers to generate a polar group.
All these reactions are classified as oxidation reactions. The insertion of a hydroxyl group at an aromatic, aliphatic or alicyclic carbon centre is the major Phase I oxidation reaction.
The preponderance of these oxidative reactions is carried out by enzymes located in the hepatic microsomes, a fraction obtained from the endoplasmic reticulum of the hepatic parenchymal cells (Fouts, 1971).
These microsomes contain a haemoprotein known as cytochrome P-450 which acts as the terminal oxidase for a variety of oxidative reactions which drugs undergo. The term P-450 points to the ability of the reduced form of the haemoprotein to react with carbon monoxide, producing a complex -26-
with an absorption peak at 450 nm (Garfinkel, 1958; Klingenberg, 1958;
Omura & Sato, 1962). Microsomal oxidation has a specific requirement for NADPH and molecular oxygen. The system fits into the class of mixed function oxidases which catalyse the consumption of one molecule of oxygen per molecule of substrate, namely the NADPH oxidising flavoprotein known as NADPH-cytochrome P-450 reductase and a cobinding haemoprotein called cytochrome P-450. A scheme for the electron transport chain in microsomal oxidation is given in Fig. 1.1. Other
Phase I metabolic reactions include reduction or hydrolysis of a compound.
Phase II metabolism includes conjugation or synthetic reactions.
Glucuronic acid conjugation is the most prevalent reaction and it can occur with compounds containing reactive groups like hydroxyl, carboxyl, amino and sulphydryl radicals. Other conjugation reactions exist, such as acetylation, sulphation, glycine conjugation and mercapturic acid synthesis. The reactions require ATP for energy, activated nucleotide and transferring enzymes (Williams & Millburn, 1975).
1.4.1 Involvement of different forms of cytochrome P-450.
The ability of carbon monoxide and other chemicals to selectively inhibit some microsomal hydroxylation reactions more than others (Conney,
1967; Conney £t £l_., 1968; 1969) suggested that more than one CO-binding haemoprotein may participate in microsomal hydroxylation reactions. Two cytochrome P-450 fractions with different spectral an catalytic properties were originally separated and purified from rats pretreated with either phenobarbitone or 3-methyl chol anthrene (Ryan et £l_., 1975).
Comai Si Gay!or (1973) separated three cytochrome P-450 fractions with different cyanide binding affinities from rat liver microsomes. Mailman F i g . 1.1 Schematic electron transport chain during microsomal drug oxidation.
[Taken from Holtzman et al., 1968]
FP Flavoprotein
ox = Oxidised
red = Reduced
x - Non-haem iron pigment necessary in some tissues, but probably not in hepatic microsomes
E NADPH - cytochrome P-450 reductase
K>i I et al_. (1975) also demonstrated the presence of multiple forms of
cytochrome P-450 in liver microsomal suspension from untreated rats and mice using a sucrose density gradient. More recently, four forms of rabbit liver microsomal cytochrome P-450 with different physical and catalytic properties have been isolated and purified by Haugen et al.
(1975). Guengerich et al_. (1982) have more recently purified eight different forms of cytochrome P-450 from rat liver to electrophoretic homogeneity. Thus, multiple forms of cytochrome P-450 can be demonstrated not only in different animals, but also in the same animal
Conney £t a]_ (1968) suggested multiple forms of cytochrome P-450 in microsomes prepared from untreated rats when they observed that the ratios of CO to 0^ needed for 50% inhibition of testosterone hydroxylation at the 66, 7a and 16a positions were significantly different.
Cytochrome P-450 and P-448 may be the predominant species in liver microsomes from rats treated with phenobarbitone and 3-methyl- cholanthrene, respectively. The relative catalytic activity of the cytochrome P-450 and P-448 fractions for aniline hydroxylation and 7a- hydroxylation of testosterone follow a similar pattern. This is of interest since aniline hydroxylation and the 7a-hydroxylation of testosterone appear to be under similar regulatory control.
Jefcoate et ^1_. (1970) found that there are two cytochrome P-450 fractions in adrenocortical mitochondria, each possessing different substrate specificities. One fraction showed high 116-hydroxylase activity for li-deoxycorticosterone, but low activity for side-chain cleavage of cholesterol, while the other fraction exhibited high activity for side-chain cleavage, but low 116-hydroxylase acivity.
Isaka et al. (1971) also reported that a cytochrome P-450 fraction -29-
prepared from bovine adrenocortical mitochondria supported side-chain cleavage of cholesterol, but not 118-hydroxyl ation. These observations suggest that more than one cytochrome P-450 exists in adrenocortical mi tochondri a.
1.4.2 Steroid metabolism.
Steroid hormones are hydroxylated in the liver by a microsomal mono oxygenase system. This step is followed by excretion of steroids in bile and urine and effects the inactivation of the hormones and production of more soluble compounds. Sometimes, hydroxylation of steroid hormones may also end with biologically active metabolites with specific physiological functions. Because steroid hormones are natural substrates for the microsomal cytochrome P-450-dependent enzyme systems, they exhibit certain advantages in biochemical studies on liver microsomal cytochrome P-450. Steroids frequently interact with cytochrome P-450 with a higher affinity than xenobiotic substrates and therefore should be preferred in substrate specificity studies. In addition, hydroxylation of steroids occurs at multiple positions around the steroid nucleus, and owing to the fact that different forms of cytochrome P-450 seem to be involved in hydroxylation at different positions (Haugen et £l_., 1975; Gustafsson & Ingelman-Sundberg, 1976), the steroid molecule may be used to study the specificity of many different forms of cytochrome P-450.
The pathway in the adrenal cortex leading from cholesterol to glucocorticoids (cortisol and corticosterone) involves several different forms of cytochrome P-450. Cytochrome P-450 has been reported to be an efficient catalyst of the 7ot-hydroxylation of testosterone induced by phenobarbitone and 3-methylcholanthrene (Ryan et aj_., 1979). 173-Oestradiol was most efficiently hydroxylated in the 2-position by
cytochrome P-450 induced by isosafrol compared to the other
haemoproteins assayed (Ryan et al_., 1982). Bowen (1980) has indicated
that this latter cytochrome P-450 also participates in the metabolism of
lanosterol to cholesterol.
Many endogenous steroid interconversions involve P-450-dependent
hydroxylations. The rate of mammalian bile acid synthesis is believed
to be controlled by the hepatic microsomal enzyme, cholesterol 7a-
hydroxylase (Danielsson et al_., 1967; Shefer et aj_., 1970). 7a-
Hydroxylase activity in humans with various disorders of lipid
metabolism was studied by Nicolau et a/h (1974). The 7a-hydroxylation
of cholesterol appears to be linked to the mixed-function oxidase system
described by Conney (1967) and Gi 1 lette et al_. (1972).
1.5 Pharmacogenetics.
The inter-individual differences in drug metabolism are of
considerable importance in man. This was observed by Shannon (1946) and
further developed by Motulsky (1957), who established the conceptual
basis of a subject which Vogel (1959) termed "pharmacogenetics".
Motulsky observed that drug toxicity may be produced by genetic traits
or enzyme deficiencies and that the detection of such hereditary
biochemical characteristics that expedite drug toxicity may contribute
to the advance of human genetics.
The subject of pharmacogenetics deals with heritable variations in
drug responses and diathesis to diseases. In patients with
pharmacogenetic lesions, there are genetically determined qualitative or
quantitative variations in drug handling ability or receptor sites at
which drugs act. These alterations are represented as therapeutic failure, drug toxicity, atypical drug response or susceptibility to - 3 1 -
disease.
These genetically determined differences in the rate of drug metabolism can be investigated in different ways. A genetically controlled difference in drug metabolism, resulting in increased metabolic clearance of a drug, has been described in one of 1029 male subjects studied by Neitlich (1966). This subject had decreased sensitivity to succinylcholine, a drug whose action is terminated after hydrolysis by plasma pseudocholinesterase. The presence of the enzyme with this increased activity is determined by an autosomal dominant variant allele. This is an example of where population studies can help in defining a genetic polymorphism. However, other methods have been used to demonstrate genetic control of drug metabolism, such as comparison of mono- and di-zygotic twins as used by Vesell (Vesell &
Page, 1968a,b,c). Twin methodology has been particularly useful in dissecting environmental from genetic influences (see Vesell et a!.,
1971), although in the case of antipyrine pharmacokinetics family studies (Blain et aj_., 1982) and twin studies (Vesell & Page, 1968a) have given contradictory results. Obviously therefore, there is still some debate amongst pharmacogeneticists as to the most appropriate genetic methodology to answer particular questions. There has also been considerable debate as to the interpretation of laboratory data as showing polymorphism or not. Since this lies at the centre of all pharmacogenetic studies, a few general comments might not be inappropriate here and will relate to the discussion concerning metoprolol metabolism given later.
Genetic polymorphism is defined as "the occurrence together in the same locality of two or more discontinuous forms of a species in such proportions that even the rarest of them cannot be maintained merely by - 3 2 -
recurrent mutation" (Ford, 1940). The question arises as how best to discover this discontinuity. Several classical examples exist in the annals of genetics, one of which is itself a pharmacogenetic phenomenon.
Firstly, the pioneering work of Harris in the 1960's led to an understanding of the ubiquity of polymorphism. One of the main examples which emerged from his laboratory, that of erythrocyte acid phosphatase, is most revealing on the subject of discernment of genetic polymorphism.
There are five main genotypes for r.b.c. acid phosphatase distinguishable as phenotypes by electrophoresis (A, BA, B, CA, CB).
Assignment of phenotype by electrophoresis of r.b.c. lysates is unequivocal, yet the distribution of enzyme activity in the population is unimodal and would suggest to the uneducated eye the lack of polymorphism (see Hopkinson et a]_., 1963).
Secondly and more appropriately, the distribution of plasma pseudocholinesterase activity is unimodal, individuals who exprienced apnoeia after premedication with succinylcholine being indistinguishable from the population at large (Kalow & Staron, 1957). However, the introduction of the technique of observing the inhibition of this activity using dibucaine (Kalow & Genest, 1957) clearly segregates the population into two phenotypes for pseudocholinesterase activity (Kalow
& Staron, 1957).
In accord with the above therefore, pharmacogenetic traits tend to be elusive almost certainly due to the incorrect measuring devices having been employed in the past and/or because of an intellectual resistance on the part of some researchers, as will be shown later in the case of metoprolol.
Inter-individual diversity in drug metabolic ability for administered drugs has been well illustrated. Kutt et ^1_. (1964a, b) reported an -33-
example by studying a pedigree in which some family members were unable
to effect 4-hydroxylation of phenytoin and Vasko et (1980) published
another similar family. Shahidi (1968) described a family in which two
sisters were unable to effect the £-de-ethylation of phenacetin. These
sisters formed large quantities of alternative metabolites, 2-hydroxy-
phenacetin and 2-hydroxy-phenetidine, the latter being responsible
for marked methaemoglobinaemia. Solomon (1968) reported an uncommon
example of dicoumarol sensitivity, thought to be due to a deficiency of
mixed function oxidase activity. Kalow et aT_. (1977) reported a
polymorphism in the metabolism of amylobarbitone, originally thought to
be due to N-oxidation but then shown to be due to N-glucosidation.
Recently, genetically determined control of alicyclic 4-hydroxylation
of debrisoquine has been described and the effects of the responsible
alleles have been extensively studied (Mahgoub et 1977; Idle &
Smith, 1979; Evans et _al_., 1980). This polymorphism has opened up new
areas of research into drug metabolism and drug response. Table 1.1
shows several exampls of genetic polymorphism of human drug oxidation.
Available data suggest that genetic polymorphism of human drug oxidation
is not an occasional phenomenon.
Debrisoquine metabolism is possibly the best studied example in this
respect. Its metabolism is under genetic control, the predominant 4- hydroxylation pathway being regulated by a single autosomal gene which is allelomorphic, the recessive poor metabolizer trait (PM) being characterised by an almost total inability to effect 4-hydroxylation in
some 9% of the British white population (Mahgoub e^t al_., 1977; Evans et
al., 1980). Autosomal dominants comprise the rest of the population, with varying degrees (10-99%) of hydroxylation ability. Such subjects constitute the so-called extensive metabolizer (EM) phenotype. Table 1.1 Polymorphic drug oxidation.
SUBSTRATE OXIDATION REACTION METABOLITE FORMED REFERENCE
Debrisoquine alicyclic 4-hydroxylation 4-hydroxy-debrisoquine Mahgoub et^ a/L, 1977
Sparteine dehydrogenation 2- and 5-dehydro-sparteine Eichelbaum et _al_., 1979
(S)-Mephenytoin aromatic hydroxyl ation 4-hydroxy-(S)-mephenytoi n Kupfer et a_h, 1981
Phenformin aromatic hydroxylation 4-hydroxy-phenformin Oates jit a K , 1982
Bufuralol aliphatic hydroxylation 1-hydroxy-bufuralol Dayer et_ a\_., 1982
S-Carboxy-methylcystei ne sulphoxi dation sulphoxide Waring et al., 1982
[Taken from Idle & Smith, 1984] -35-
1.6 Regulation of drug oxidation by the debrisoquine hydroxylation
locus.
This polymorphism influences the metabolic disposition of a number of other drugs besides debrisoquine, and this has been extensively reviewed
(Idle & Smith, 1979, 1984; Smith & Idle, 1981) together with its possible toxicological implications (Ritchie et ^1_., 1981). The debrisoquine hydroxylation locus has been shown to regulate or influence the metabolic oxidation of many other drugs (Table 1.2).
Relative to EM subjects, PM volunteers have been shown to be defective oxidizers of phenacetin (Sloan eit al_., 1978; Kong et a!.,
1982), guanoxan (Sloan et aj_., 1978), phenytoin (Sloan et £]_., 1981), metiamide (Idle et^l_., 1979a), 4-methoxy-amphetamine (Kitchen et a!.,
1979) and nortriptyline (Berti 1 sson et aj_., 1980). Polymorphisms of sparteine oxidation (Evans et al_., 1983) and phenformin hydroxyl ati on
(Oates et , 1982) certainly seem to be related to the debrisoquine oxidation polymorphism, if only genetically linked rather than coincidental. The PM phenotype would therefore seem to be considerably disadvantaged versus the EM phenotype, inasmuch that the impaired oxidation status can lead to drug accumulation and a higher prevalence to drug related toxicity. Therefore, protecting poor metabolizers from unwanted side effects of many drugs or even from administration of special drugs is necessary (Lennard et al_., 1984; Idle & Smith, 1984).
Postural hypotension caused by debrisoquine (Idle et al_., 1978), methaemoglobinaemia induced by phenacetin (Kong et ^1_., 1982) and the lactic acidosis associated with phenformin (Idle et al_., 1981) can all be demonstrated in PM subjects at drug doses which have little or minimal effect in individuals of the EM phenotype for whom oxidation is uncompromi sed.
Of interest are patients presenting with untoward drug side-effects Table 1.2 Regulation/influence of the debrisoquine hydroxylation locus on various metabolic pathways
METABOLIC PATHWAYS SUBSTRATES REFERENCES Affected Unaffected
Aromatic hydroxylation debrisoquine Mbanefo et a K , 1980 phenformi n Oates et a]_., 1982 guanoxan Sloan et_ a K , 1978 nortriptyline Mel 1strom et al., 1981 acetani Tide Wakile et al., 1979 methaqualone Oram et^ a K , 1982
Alicyclic and aliphatic perhexiline Cooper et^ a K , 1984 hydroxylation sparteine Eichelbaum et al., 1979 metoprolol Lennard et al., 1982 bufuralol Dayer et_ al., 1982 amobarbital Inaba et_ TT., 1980 cortisol Park et a K , 1982
Oxidative O-dealkylation phenacetin Sloan et al_., 1978 4-methoxyamphetami ne Kitchen ert al_., 1979 encai nide Woos ley ert , 1981 anti pyrine Danhof et al., 1981 theophylline Monks, 1978 pethidine Notarianni, 1979
Angular methyl oxidation tolbutamide Idle et^ al_., 1979b antipyrine Danhof et al., 1981 methaqualone Oram et al., 1982 meti amide Idle et al., 1979a cimetidine Mitchell et a K , 1982
[Taken from Idle & Smith, 1984] u>i CNI -37-
or simply inappropriate plasma levels of certain drugs, who are then
phenotyped. For example, it was demonstrated that 10/20 patients
studied with perhexiline-associated neuropathy were PM (Shah et a!.,
1982).
Alvan e_t al_. (1982) studied two patients and two volunteers with
unusually high plasma concentrations of the e-blocking drugs alprenolol,
metoprolol and timolol and all were found to be of the PM phenotype.
The conclusions drawn by Jack & Wilkins (1984) are contradictory, or at
best, inconsistent with polymorphism in general, especially in the case
of metoprolol, although Lennard et aj_. (1983a) showed in a population
study that a-hydroxylation of metoprolol was polymorphic. An additional
study by Lennard et al_. (1983b) in a panel of phenotyped subjects showed that other routes of metoprolol metabolism should be linked to 4- hydroxylation of debrisoquine, which was confirmed by studies showing a
significant correlation between the carboxylic acid metabolite of metoprolol and the debrisoquine metabolic ratio (Lennard et aT_., 1983c).
Lennard et_ al_. (1984) stated that the point of importance is that poor metabolizers are concentrated in the tails of skewed AUC distributions
and, at least in the case of compounds showing the debrisoquine
polymorphism, it becomes possible to recognize subjects with a high probability of having elevated AUC values. Jack & Wilkins (1984) believe that the clinical importance of polymorphism in drug metabolism remains unclear. At the same time, Lennard et £l_. (1984) agreed with the above statement but contended that genetic polymorphism of the debrisoquirie-type will prove to be important in clinical use. The metoprolol debate, which is only in part described above, is the most recent example of the problem of perception discussed earlier in the case of acid phosphatase and pseudocholinesterase. To state that an -38-
enzyme or enzyme activity is not polymorphic can be both dangerous and
bigoted, until proven otherwise. However, to argue that the clinical
significance of polymorphism is unclear seems both reasonable and simply
a comment of the status quo, and should be interpreted as a directive
for further research. Thus the problem of the existence of genetic
polymorphism and its clinical applicability are two separate issues and
it is helpful not to confuse these. Positive proof of the former and
its breadth of occurrence will hopefully come via the techniques of
molecular biology. Discernment of the latter will depend upon the
perception or resistance of clinical pharmacologists and the drug
i ndustry.
1.7 Animal model for drug oxidation.
The genetic polymorphism of drug metabolic oxidation has more
recently been shown in the rat (Al-Dabbagh et aj_., 1981). The need for
an animal model, having some of the metabolic and genetic
characteristics of the human polymorphism, was apparent. Such a model
meeting these criteria would permit screening of new substrates and
allow pharmacological and toxicological testing of known subtrates of the
human polymorphism, which could not readily be fulfilled directly in
man. Accordingly, females of the DA strain of rat have been observed to
have a relative impairment of debrisoquine hydroxylation and phenacetin
^-deethylation (Al-Dabbagh et aj_., 1981), the same as for the human poor
metabolizer phenotype. Breeding studies using hybrids between DA and
Lewis (extensive debrisoquine hydroxylator) strains, together with
progeny, prove that the failing metabolism discovered in the maternal DA
strain is, as in man, a Mendelian recessive trait (Al-Dabbagh et a!.,
1981; Al-Dabbagh, 1982). -39-
Toxicity of a variety of chemicals is obviously associated with the formation of chemically reactive and electrophilic oxidative metabolites. Thus, individual variability in the pathways leading to such intermediates might be of considerable importance. With polycyclic aromatic hydrocarbons, an arene oxide (epoxide) is frequently implicated
(see Sims & Grover, 1974). Thus, benzo[a]pyrene is metabolized to its
7,8-dihydrodiol-9,10-oxide which is thought to be an ultimate carcinogen of the procarcinogenic benzo[a]pyrene (Sims et al_., 1974). Aflatoxin
B-p also a potent naturally-occurring mycogenic carcinogen, is thought to be activated via epoxidation to the 2,3-oxide (Swenson et al_., 1974).
Safrol (Borchert et _al_., 1973), vinyl chloride (Gorrod, 1979), estragole
(Dri nkwater et aj_., 1976) and acetyl ami nofluorene (Weisburger et al.,
1964) are further examples of chemicals requiring oxidation to exert their full toxic potential.
Spectral binding characteristics of hepatic-derived cytochrome P-450 with debrisoquine and other polymorphicaly metabolized substrates are strongly suggestive of the defect in the DA rat occurring at the cytochrome P-450 drug binding step in the catalytic cycle (Kupfer et_ al., 1982). This molecular defect manifested as impaired oxidation in the DA rats, might have importance for the metabolism of those endogenous substrates, such as various lipids, which are normallly hydroxylated by hepatic and adrenocortical cytochrome P-450. However, interaction of substrate with oxidized cytochrome P-450 has shown to give rise to two distinct types of spectral changes, namely Type I and
Type II spectra. Certain steroids, including oestrogens and androgens, which produce a Type I difference spectrum with rat hepatic microsomes
(EM), but not female DA rat microsomes (PM) (Idle, 1985).
The Lewis/DA model for the human EM/PM phenotypes presents an -40-
opportunity to study aspects of the polymorphism which are not readily
examinable in man. Studies with carcinogens and other toxins is one
obvious area, but another might be to provide additional support to the
notion that a non-invasive phenotyping test could be found.
Accordingly, crude experiments might at first be carried out in rats and
then, in cases where a positive finding obtained, more careful studies
could be executed in human volunteers. At all times, however, the
investigator would have to bear in mind that the model might only apply
up to a point and not absolutely.
1.8 Aims of the present study.
The question asked in this thesis is a simple one and that is - does the polymorphic gene which determines debrisoquine oxidation phenotypes
influence the disposition of an endogenous steroid to an extent that measurement of say a urinary steroid could be used as a phenotyping test. Additionally, such information might provide a clue as to why this and other genes of similar effect exist in allelomorphic forms and what effect, if any, they have exerted on human disease patterns.
In order to answer this question, a number of approaches have been tried, including examination of urinary corticosteroid metabolites, such as 17-oxosteroids, urinary oestrogens and aspects of cholesterol metabolism. Both human subjects of known phenotype and the Lewis/DA rat model have been employed in an attempt to answer, at least in part, the question posed above. The findings are given in the following Chapters
2-4. -41-
CHAPTER TWO
EFFECT OF POLYMORPHIC OXIDATION ON URINARY
STEROID PROFILES IN MAN AND RAT -42
2.1 INTRODUCTION
2.1.1 Urinary 17-oxosteroids.
The laboratory measurement of the 17-oxosteroids is now widespread
due to their diagnostic value in relation to adrenocortical function.
The term 17-oxosteroids, originally called the 17-ketosteroids, refers
to those steroids having a ketone group at C-17 and includes urinary
testosterone metabolites, mainly androsterone and aetiocholanolone, but
primarily metabolic products of the secretions of the adrenal cortex,
such as dehydroepiandrosterone and 11-oxo- and 113-hydroxy-androsterone
and -aetiocholanolone (Fig. 2.1). Low values of urinary 17-oxosteroids
point to adrenal insufficiency, whilst high values are almost always
indicative of adrenocortical hyperactivity or tumour.
In healthy subjects with "normal" adrenocortical function, many factors are known to influence the urinary excretion of 17-oxosteroids,
and these include age, sex (Feher, 1966a; Martin & Hamman, 1966), diurnal variation (Okamoto et al., 1971) and heat stress (Sharma et al.,
1972). Table 2.1 gives ranges of the main individual 17-oxosteroids in the urine of children and adults of both sexes. Noteworthy are the following observations regarding these data, that all these steroids are
lower in childhood, that adult males excrete greater quantities than
adult females, particularly androsterone and aetiochlanolone which are partly produced by the testis, that there is a wide interindividual variation in excretion of, and presumably therefore production of, all the 17-oxosteroids. Of particular note in this respect is the greater than 200-fold observed variation (n = 14) in androsterone excretion in young females (Table 2.1). In order to examine the metabolic origins of individual 17-oxosteroids, each of the major ones is dealt with below. Fig. 2.1 Structures of the common urinary 17-oxosteroids.
O O
i H I II O
O O
V O
I Androsterone V 1 1 (3-Hydroxyaet iocholanolone II Aetiocholanolone VI 11-Oxoandrosterone III Dehydroepiandrosterone VII 11-Oxoaetiocholanolone IV 11$~Hydroxyandrosterone Table 2.1 Urinary excretion (ranges, pg/24 h) of individual 17-oxosteroids in male and female children and adults.
17-oxosteroids
Group Age (yrs) No A Ae DEA 11-OxoAe 116-OHA 113-OHAe
Male 3-12 9 70-820 40-820 <10-330 30-620 90-920 30-880
Male 19-43 10 1500-6000 1400-5100 800-6400 300-1400 1000-2100 300-1100
Female 3-11 14 10-2280 10-1560 <10-340 30-840 100-1740 10-730
Female 18-43 25 900-4800 700-4300 100-2600 200-1400 200-2900 200-1200
A = androsterone; Ae = aetiocholanolone; DEA = dehydroepiandrosterone; 11-OxoAe = 11-oxoaetiocholanolone;
11&-0HA = llB-hydroxyandrosterone; 113-OHAe = 113-hydroxyaetiocholanolone.
Data taken from Feher (1966a).
i -N I -45-
2.1.1.1 Androsterone (5g-androstan-3cro1-17-one).
O
Androsterone is one of the most abundant urinary 17-oxosteroids in men, women and children (Table 2.1), primarily as its glucuronic acid conjugate (Keutmann & Mason, 1967). It is a terminal urinary metabolic product of testosterone, 17a-hydroxyprogesterone and 11-deoxycortisol
(Bongiovanni, 1978). All of these reactions occur in the adrenal cortex and additionally the conversion of testosterone to androsterone occurs in the testis. In the case of testosterone, the 3-oxo group is reduced to a 3a-hydroxy group, the 4,5-double bond is reduced with the 5-proton added in the a configuration giving a trans A, B ring junction and the
17-hydroxy group is oxidized to a 17-oxo group. The first step involves formation of androstenedione (4-androstene-3,17-dione) by 17- hydroxysteroid dehydrogenase (E C 1.1.1.51).
2.1.1.2 Aetiocholanolone (aetiocholan-3a-ol-17-one).
O Aetiocholanolone is also one of the abundant urinary 17-oxosteroids
(Table 2.1). It arises from the same metabolic sources as androsterone but has a 53-proton with a cis A,B ring junction. Like the latter, it is also excreted mainly as its glucuronide (Keutmann & Mason, 1967).
2.1.1.3 Dehydroepiandrosterone (5-androsten-3B-ol-17-one). O
Dehydroepiandrosterone is not only a common urinary 17-oxosteroid
(Table 2.1), but also almost exclusively the only 33-hydroxy steroid found in human urine. Unlike the other 17-oxosteroids which possess a
3a-hydroxy group, this steroid is not precipitable with digitonin and this affords a method for its separation (Sunderman & Boerner, 1949).
The configuration of the 3-hydroxyl group also seems to confer another important difference on dehydroepiandrosterone; that being that this is the only 17-oxosteroid which is predominantly excreted as its sulphate conjugate (Baulieu, 1963). Metabolically, it arises from 17a- hydroxypregnenolone, which itself derives from pregnenolone, the major precursor of progesterone. The reaction involves removal of the 173- methyl carbonyl side-chain and oxidation of the 17a-hydroxy group to the
17-oxo by a desmolase enzyme which breaks this 17,20 carbon-carbon bond.
2.1.1.4 The 11-oxo- and llB-hydroxy-17-oxosteroids.
These are 113-hydroxyandrosterone, 113-hydroxyaetiocholanolone, 11- oxoandrosterone and 11-oxoaetiocholanolone (see Fig. 2.1). They occur -47-
in lesser amounts in human urine than the other 17-oxosteroids (see
Table 2.1) and are also conjugated primarily with glucuronic acid. They
are most likely formed from cortisol in the adrenal cortex, but in low
amounts (Cope, 1972).
2.1.2 Urinary progesterone metabolites.
A number of progesterone metabolites can be detected in human urine which retain the 17B side-chain intact. The most important of these is pregnanediol (Fig. 2.2), although pregnanetriol and pregnanetriolone are also found. Like the 17-oxosteroids, the excretion of these steroids is also highly variable between subjects, for pregnanetriol being 110-970 and 110-450 pg/24 h in adult men and women respectively (Kinoshita et al., 1966). Values as high as 21,700 pg/24 h have been observed in congenital adrenal hyperplasia and adrenal tumours (Martin et al.,
1961). These steroids are apparently excreted as their glucuronic acid conjugates.
The major steroid in this group is pregnanediol which is formed in the adrenal cortex by the reduction of the 20-oxo group and the unsaturated ketone (4-en-3-one) to the two corresponding secondary alcohols at C-3 and C-20. Pregnanetriol, however, is formed by analogous reductions of 17a-hydroxyprogesterone (see Sunderman & Boerner,
1949). Like the 17-oxosteroids, the excretion of progesterone metabolites is also influenced by many factors other than disease. For example, in women, pregnanediol appears in urine immediately after ovulation with a mean excretion of 2-5 mg/24 h, but within days rises to
5-10 mg/24 h. For 1-2 days premenstrually, pregnanediol is often undetectable in urine (see Sunderman & Boerner, 1949).
Pregnanetriolone (Fig. 2.2) was first synthesized by Sarett (1948) -48- Fig 2.2 Structures of progesterone and its common urinary metabolites.
CH3 c=oi
P
PD PT PT one
P Progesterone (4-pregnene-3,20-dione) PD Pregnanediol (5|3-pregnane-3a,20a-diol) PT Pregnanetriol (5$-pregnane-3a,17a,20a- triol) PTone Pregnanetriolone (5$-pregnane-3a,17a,20a-triol 11-one) -49-
and subsequently isolated from the urine of two female pseudohermaphro
dites with congenital adrenal hyperplasia (CAH) by Zondek &
Finkelstein (1952) and Finkelstein et £]_. (1953) and then isolated from
the urine of a male CAH sufferer (Fukushima & Gallagher, 1957). This
steroid metabolite is also abundant in the urine of Cushing's patients
(Finkelstein, 1959), postnatal virilization syndrome (Jayle et al.,
1958; Brooks et £]_., 1960) and patients with polycystic ovaries (Cox &
Shearman, 1961). Pregnanetriolone is excreted by healthy adults (18-59
pg/24 h urine), with slight elevation in Cushing's syndrome (35-290
pg/24 h) and marked elevation in CAH (250-7000 pg/24 h) (Lahoud et al.,
1976). Like pregnanetriol, it arises metabolically in the adrenal
cortex from 17a-hydroxyprogesterone, with the addition of an 11-oxo
grouping.
2.1.3 The involvement of cytochrome P-450 in steroid hydroxylations.
Perusal of the metabolic interconversions of steroids reveals that
the vast majority of reactions are oxidations and reductions, including
hydroxylation. The biosynthesis of corticosteroids, sex hormones and
bile acids in particular, together with their further metabolism to
urinary excretion products, involves many such oxidation and reduction
reactions. Insomuch as cytochrome P-450 is concerned with bile acid formation and oestrogen metabolism will be dealt with in Chapters 4 and
3 respectively. Corticosteroid, testosterone and progesterone metabolism is the subject of this Chapter and therefore a brief outline
of the possible role of cytochrome P-450 in the various interconversions
of these compounds will be given below.
The precursor to the adrenocortical hormones is pregnenolone (4- pregnen-33-ol-20-one). Progesterone, testosterone, cortisol, corticosterone and aldosterone all derive from it. It is synthesized - 5 0 -
from cholesterol in adrenocortical mitochondria by 20,22-dihydroxylation followed by cleavage of the side-chain at C-20 by 20,22-desmolase, leaving an oxo-group at this position. All three steps, 20- hydroxyl ation, 22-hydroxylation and the desmolase-catalysed cleavage utilise NADPH and O2 and require cytochrome P-450 (see Bongiovanni, 1978). Pregnenolone then undergoes two reactions, either 17a- hydroxylation to 17a-hydroxypregnenolone, which is the metabolic precursor of dehydroepiandrosterone (see 2.1.1.3) or oxidation to c A progesterone by 3B-hydroxysteroid dehydrogenase and a , A -isomerase
(see Fig. 2.3). The 17a-hydroxylation is cytochrome P-450 dependent
(Colby & Rumbaugh, 1980) and is involved in the further conversions of progesterone which ultimately lead to testosterone, oestrogens and corticosteroids. The cytochrome P-450 related biotransformations are shown in Fig. 2.4.
The cytochromes P-450 responsible for steroid hydroxylations are located in the adrenocortical mitochondria (Colby & Rumbaugh, 1980).
They are not, as was thought, specific for steroid substrates only, but will metabolize a number of drugs and foreign chemicals. However, the affinities of these cytochromes for steroid substrates is considerably higher than for drugs, as judged by spectral dissociation constants
(Table 2.2). Not surprisingly, the hepatic P-450 system will also metabolize steroids in vitro and considerable interest has centred around the hydroxylation of androgens, such as testosterone by hepatic
P-450. Following soon after the landmark discoveries independently by
Conney and Remmer that treatment of rats with phenobarbitone and methylcholanthrene induce hepatic microsomal drug oxidation (Conney et al., 1956, 1957, 1960, 1961; Remmer, 1959a,b), the hepatic androgen hydroxylases were examined in this respect (Conney & Klutch, 1963). -51 Fig 2.3 Early steps in the synthesis of corticosteroids and testosterone from cholesterol.
20a,22-Dihydroxycholesterol
17a~Hydroxypregnenolone Progesterone
Corticosteroids and testosterone - 5 2 -
Fig. 2.4 Principal pathways for adrenal corticosteroid
biosynthesis with cytochrome P-450-dependent reaction.
(Taken from Colby & Rumbaugh, 1980).
Cholesterol
Pregnenolone ------► 17a-hydroxyprenenolone i l Progesterone ------► 17a -hydroxyprogesterone 1 c lc 11-deoxycorticosterone 11-deoxycortisol 1 ° ID Corticosterone Cortisol 1 E 18-hydroxycorticosterone I Aldosterone
A = Cholesterol sidechain cleavage
B = 17a-Hydroxylase
C = 21-Hydroxylase
D = llp-Hydroxylase
E = 18-Hydroxylase. - 5 3 -
Table 2.2 Spectral dissocation constants (Ks) for compounds
interacting with human foetal adrenal microsomes.
(Data taken from Colby & Rumbaugh, 1980).
Substrate Spectral Change Ks
Aniline Type II 3.5 X 10“2
Nicotinamide Type II 2.5 X 10"2
Progesterone Type I 1.8 X IQ"7 1 i—* 17a-0H Progesterone Type I 6.3 X o
Pregnenolone Type I 1.1 X 10-7
Androstenedi one Type I 5.0 X 10-6
★ Type I spectral change = peak-trough
Type II spectral change = trough-peak -54-
These authors concluded that chronic treatment of rats with
phenobarbitone or chlorcyclizine stimulated several-fold the activity of
hepatic microsomal enzyme systems which hydroxylated testosterone and 4-
androstene-3,17-dione. The hydroxylation pathways observed were 23-,
63-, 7a- and 16a-hydroxylation. More recently, it has been shown that
the 63- and 7a-hydroxylations can be mediated approximately equally by
P-450 (phenobarbitone-inducible) and P-448 (methylcholanthrene-
inducible) cytochromes, while the 16a-hydroxylation of testosterone
seems to be P-450 dependent (Lu _et 1972). None of these sites of
hydroxylation is commonly found as an activity of adrenocortical P-450
which are essentially 113-, 17a-, 18- and 21-hydroxylation (see
Fig. 2.4).
However, in instances where overlap between adrenocortical
mitochondrial P-450 and hepatic microsomal P-450 activity, with respect
to a particular steroid hydroxylation, may occur, no data can be found
for the correlation of these activities in individual animals or human
biopsies. Accordingly, no assessment can be made at present of the
similarity or otherwise in P-450's between the liver and adrenal gland.
It may be, therefore, that no relationship exists between two
hydroxylation enzymes performing the same metabolic function in the two
tissues. Nevertheless, it is interesting to note that the hepatic
activities towards drug oxidation are stimulated by ACTH (see Peterson &
Holtzman, 1980) iri a similar way to which adrenocortical P-450 is
stimulated by ACTH to hydroxylate steroids.
2.1.4 The genetic abnormalities of steroid hydroxylation.
Congenital adrenal hyperplasia, which has been mentioned previously
(Sections 2.1.1, 2.1.2), is a group of similar diseases with different - 5 5 -
metabolic and genetic aetiologies which have been classified as Types
I-V (see McKusick, 1983). All five clinically-distinct conditions are genetically-distinct and also recessive. Apart from Type II (36- hydroxysteroid dehydrogenase deficiency), all types involve genetic deficiencies of corticosteroid and sex hormone-producing enzymes which are known to be P-450 dependent. In some communities, these diseases are often relatively common, presumably due to inbreeding, such as 21- hydroxylase deficiency (Type III) in Zurich Canton, Switzerland (1 in
5041 live births, i.e. 1 in 35 carriers, see McKusick, 1983). The Type
III disease is the best studied genetically and biochemically and is closely linked to the HLA system, particularly the DR locus (Klouda et al., 1980). Multiallelism probably occurs, since it has been estimated that 6-12% of hirsute women have 21-hydroxylase deficiency because of homozygosity for a "mild" allele for the 21-hydroxylase gene of frequency 0.015 - 0.057 (Chrousos et ^1_., 1982).
Finally, the P-450-mediated 18-hydroxylation which leads to aldosterone is also associated with two genetic deficiencies, namely, aldosterone deficiency I (18-hydroxylase deficiency) and aldosterone deficiency II (18-hydroxysteroid dehydrogenase deficiency) (see
McKusick, 1983).
2.1.5. Aims of the investigation.
The aim of the study described in this Chapter was to investigate whether or not the genetic polymorphism of debrisoquine hydroxylation described in man (Mahgoub et al_., 1977) and rats (Al-Dabbagh et al.,
1981) also affects disposition of some endogenous steroids, particularly the formation of 17-oxosteroids and some progesterone metabolites (Fig.
2.1, 2.2). Table 2.3 Classification of the adrenal hyperplasias. (Data taken from McKusick, 1983).
Type Nomenclature Inheritance Enzyme deficient Substrate (s) Product (s)
I Lipoid hyperplasia, Recessive 20,22 desmolase Cholesterol - Pregnenolone congenital, of adrenal (ultimately) cortex with male pseudo hermaphroditism
II 3-Beta-hydroxysteroi d Recessive 33-hydroxysteroid Pregnenolone - Progesterone dehydrogenase deficiency dehydrogenase
III 21-Hydroxylase deficiency; Recessive 21-hydroxylase Progesterone - 11-deoxycortico congenital adrenal sterone hyperplasia-1; CAH 1 17a-hydroxy- progesterone - 11-deoxycortisol
IV 11-Beta-hydroxylase Recessive 113-hydroxylase 11-deoxy- deficiency; hypertensive corticosterone - corticosterone form of adrenal hyperplasia
V 17-Alpha-hydroxylase Recessive 17a-hydroxylase Pregnenolone - 17a-hydroxy- deficiency pregnenolone
Progesterone - 17a-hydroxy- progesterone
i U i csI -57-
The need for a simple non-invasive oxidation phenotyping test is clear and the absence of one of the urinary steroid metabolites in recessive poor metabolizers would obviously provide the basis for such a test. Towards this aim, a gas chromtographic assay for individual 17- oxosteroids and certain progesterone metabolites was developed, in order to uncover whether or not the genetically determined defect of cytochrome P-450 mediated hydroxylation of drugs such as debrisoquine carries over to the hydroxylation of certain steroids. -58-
2.2. MATERIALS AND METHODS.
2.2.1 Materi als.
The steroid nomenclature used throughout this Thesis is that recommended in the "IUPAC-IUB 1967 Revised Tentative Rules for Steroid
Nomenclature" with amendments as described in "Instructios to Authors",
J. Endocrinol 1982; 92: 1-7.
Androsterone (Mr. 290.4) (5a-androstan-3a-ol-17-one), aetio- cholanolone (Mr. 290.4) (aetiocholan-3a-ol-17-one), dehydroepiandro sterone (Mr. 288.4) (5-androsten-38-ol-17-one), pregnanediol (Mr. 320.5)
(58-pregnane-3a,20a-diol), 11-oxoaetiocholanolone (Mr. 304.4)
(aetiocholan-3a-ol-ll,17-dione), 11-oxoandrosterone (Mr. 304.4) (5a- androstan-3a-ol-ll,17-dione), 118-hydroxyandrosterone (Mr. 306.5) (5a- androstan-3a,118-diol-17-one), 118-hydroxyaetiocholanolone (Mr. 306.4)
(aetiocholan-3a,ll8-diol-17-one), pregnanetriol (Mr. 336.5) (58- pregnane-3a,17a,20a-triol), pregnanetriolone (Mr. 350.5) (58-pregnane-
3a,17a,20a-triol-ll-one), cholesterol (Mr. 386.6) (5-cholesten-38-ol),
8-glucuronidase, sulphatase, ^-trimethylsilylimidazole (TMSI), benzyl trimethyl ammonium methoxide (40% in methanol) (anhydrous, Mr.
181.3) and 1,3-dinitrobenzene (Mr. 168.1), were obtained from Sigma
Chemical Company, Poole, U.K. Sodium hydroxide solution (stock No.
555-2), creatinine standard solution (stock No. 925-3) and creatinine standard solution (stock No. 925-15) were supplied as a kit from Sigma
Chemical Company, Poole, U.K. 3,4-Dihydro-2-(lHMsoquinoline carboxamidine hemisulphate (Debrisoquine; m.p. 274-276°C) and 4-hydroxy-
3,4-dihydro-2-(lH^)-isoquinoline carboxamidine sulphate (4-hydroxy- debrisoquine; m.p. 254-255°C) were the gift of Roche Products Limited,
Welwyn Garden City, U.K. 7-Methoxy-2-guanidinomethyl-1,4-benzodioxan (7-methoxy-guanoxan; m.p. undetermined) was a gift of Pfizer Limited,
U.K. Hexafluoroacetylacetone was purchased from Koch Light Laboratories
Limited, Colnbrook, U.K. Crystapen (Benzylpenicillin sodium), 600 mg
(1,000,000 units) and polybactrin (polymyxin B sulphate BP 75,000 IU,
Neomycin sulphate BP 20,000 IU, Bacitracin BP 1,000 IU) in one vial as
sterile dry powder, were obtained from St. Mary's Hospital Pharmacy.
Methanol, chloroform, anhydrous diethyl ether, sodium hydroxide, sodium
acetate, acetic acid, hydrochloric acid, ethanol, anhydrous sodium
sulphate (all Analar grade) were obtained commercially.
2.2.2. Animals.
Inbred female rats (150-200 g body weight) of the two strains (DA and
Lewis) used were maintained on Labshure 41B diet with free access to
water. These animals were supplied from different sources, i.e. St.
Mary's Hospital Medical School, Bantin & Kingman, and Olac. The colour
of the eyes was red for Lewis and black for DA rats, hair colour was white and brown for Lewis and DA respectively.
2.2.3 Human subjects.
Healthy adult male and female volunteers were given orally 10 mg debrisoquine tablets and the 0-8 h urine collected and volume measured.
About 10 ml of each sample was stored at -20°C.
Number of subjects: 76 students, 9 PM and 67 EM, 34 females and 42 males (about 18-20 years old). 14 Pharmacology Department staff and students: 1 PM, 13 EM, 2 of which were females and 12 males (20-34 years old). -6 0 -
2.2.4 Methods.
2.2.4.1 Determination of oxidation phenotype.
This was carried out depending on the method of Idle et al_. (1979c).
To urine (0.1 ml) in a screw-capped septum vial was added 200 pi of internal standard/buffer solution (5 pg/ml 7-methoxy-guanoxan in 1 M sodium bicarbonate solution) followed by 50 pi hexafluoroacetylacetone and 1 ml toluene. The mixture was heated in an aluminium block at 100°C for 1 h, the vial removed and left to cool whereupon 3 M sodium hydroxide (5 ml) to hydrolyse the excess derivatizing agent and 2 ml toluene were added. The reaction vial was vortexed, centrifuged at 2000 rpm for 5 min, and a portion of the toluene layer (1 pi) was injected into a Pye Uni cam GCD gas chromatograph (oven temperature 190°C, injection port temperature 250°C) fitted with an 0V-1 column (3% on
Chromosorb WHP; 1.83 m length, 3 mm internal diameter) with oxygen-free nitrogen carrier gas flow rate of 60 ml min"^. Bis-(trifluoromethyl) pyrimidine derivatives of debrisoquine and 4-hydroxy-debrisoquine were detected using an electron-capture detector (temperature 205°C). A typical gas chromatographic trace is shown in Fig. 2.5.
The measurement of metabolic ratio (% dose excreted as debrisoquine/% dose excreted as 4-hydroxy-debrisoquine) was made and used as a determinant of phenotype. In man, subjects with ratio > 12.6 are phenotypically poor metabolizers (PM), while those with lower values are phenotypically extensive metabolizers (EM) (Evans et al_., 1980). On the other hand in rats, those with ratios > 1 are phenotypically poor metabolizers (PM), whereas those with lower values are phenotypically extensive metabolizers (EM) (Al-Dabbagh et aj_., 1981). -61- Fig. 2.5 Typicol chromatogram of debrisoquine
and 4-hydroxy-debrisoquine -62-
2.2.4.2 Determination of individual 17-oxosteroids, pregnanediol,
pregnanetriol and pregnanetriolone in urine.
The first method for the fractionation of 17-oxosteroids was published by Dingemanse and her colleagues (1946, 1952) and later improved by Kellie & Wade (1957); the individual 17-oxosteroids were separated from each other by chromatography on alumina. Several methods have been used for the determination of steroids in urine (Table 2.4).
These methods were superseded by procedures involving gas chromatography (Wotiz, 1963; Kirschner & Lipsett, 1963; Curtius &
Muller, 1967). Here, the method of Sanghvi et al_. (1974) was adopted with some modification. To urine (2 ml) in a quick-fit tube was added acetate buffer solution (1 ml) containing 6-glucuronidase (500 units), sulphatase (500 units), penicillin G (1000 units), bacitracin (25 units), neomycin (500 units) and polymyxin B (1875 units). The mixture was buffered with 0.2 M acetate buffer (pH 5; 2 ml) and methanolic cholesterol solution (50 pi) as internal standard (25 pg) and 2-3 drops of chloroform as preservative. The sample was shaken and incubated overnight (ca. 16 h) at 42°C. The tube was removed and left to cool whereupon it was extracted with anhydrous diethyl ether (2 X 10 ml).
The bulked ether extracts were washed successively with 2 ml of 0.1 M sodium hydroxide and water. The organic solvent was evaporated to dryness and the residue transferred to a 3 ml screw-capped vial so as to derivatize it with 20 pi of 50% ][-trimethyl si lyl imidazole (TMSI) in pyridine at 60°C for 45 min. 1 pi portions of this solution were used for gas chromatographic analysis. The chromatograph used was a Pye
Unicam 204 equipped with a hydrogen flame ionization detector.
Chromatographic conditions were glass column: 3% OV-17 on Chromosorb WHP from Jones Chromatography Limited, Llanbradach, Wales; length of Table 2.4 Different methods used for determination of steroids.
Method Compound Reference
Thin layer 17-oxosteroids Hamman & Martin chromatography (1964)
Paper 17-oxosteroids Feher (1966b) chromotography
GC'MS 17-oxosteroi ds Curtius et al., (1975)
Col umn Pregnanediol and Bongiovanni & chromatography pregnanetri ol Clayton (1954)
Paper Pregnanediol and Martin et al., chromatography pregnanetri ol (196T) -64-
column: 120 cm and 4 mm internal diameter, carrier gas: nitrogen (flow rate: 35 ml min"*; pressure 18 p.s.i.g.), flow rate of hydrogen and air were 30 ml min“* (pressure 20 p.s.i.g.), and 300 ml min”'*’ (pressure 7 p.s.i.g.) respectively, column temperature: 245°C, detector temperature:
300°C, injector temperature: 300°C. Retention times of emerging peaks were calculated relative to the internal standard reference peak. A typical gas chromatographic trace of 17-oxosteroids is shown in Fig. 2.6 and relative retention times of these compounds are given in Table 2.5.
For androsterone, aetiocholanolone, dehydroepiandrosterone and pregnanediol individual calibration curves were constructed by plotting the height of the substance divided by internal standard peak height against concentration (0.1 - 5 pg ml"*) (Figs. 2.7 - 2.10). In the case of other steroids to be analysed, insufficient authentic material was available to permit construction of full calibration curves.
Accordingly, estimation of their concentration in urine was made by extrapolation from calibration curves for androsterone, aetiocholanolone, dehydroepiandrosterone and pregnanediol by correction for retention time and relative molecular mass (Mr, molecular weight).
Fig. 2.11 shows how in the case of these previously mentioned four steroids, when observed peak height ratio (substance:internal standard) is corrected for small inter-compound differences in relative molecular mass and retention time, one calibration line adequately describes the concentration - response relationship for each steroid. Specifically, this was performed as follows for say “steroid x“:
Peak height X retention time Corrected ratio., (ordinate) = x x X ~ril_ ~ Peak height. X retention time. i.s. i.s. where i.s. = internal stanaard (cholesterol) Fig. 2.6 Typical chromatogram of 17-oxosteroids pregnanediol, pregnanetriol and pregnanetriolone.
+
2 0 18 16 14 12 10 8 6 4 2 0 min 1 Androsterone 2 Aetiocholanolone 3 Dehydroepiandrosterone 4 Pregnanediol 5 11-Oxoandrosterone and 11-oxoaetiocholanolone 4/7 11^"Hydroxyandrosterone and 11$-hydroxyaetiocholanolone 8 Pregnanetriol 9 Pregnanetriolone 10 Internal standard + Unidentified peaks -66-
Table 2.5 Gas chromatographic characteristics of derivatized
17-oxosteroids, pregnanediol, pregnanetriol and
pregnanetriolone on 3% OV-17.
Peak No. Compound Retenti on Relati ve time (min) retention time
1 Androsterone 4.3 0.24
2 Aeti ocholariolone 4.6 0.25
3 Dehydroepi androsterone 5.8 0.32
4 Pregnanediol 6.1 0.34
{ 11-Oxoandrosterone 7 0.38 5 { 11-Oxoaetiocholanolone 7 0.38
7 llg-Hydroxyandrosterone 9 0.49
8 lifc-Hydroxyaeti ocholanolone 9.4 0.52
9 Pregnanetri ol 10.4 0.57
13 Pregnanetriolone 16.6 0.91
14 Cholesterol 18.2 1 (internal standard) Fig■ PERK HEIGHT RRTIO Ljr PERK HEIGHT RATIO . . Clbain uv fr aetiocholanolone for curve Calibration 2.8 2.7 oirto cre o ondrosterone for curve Colibrotion E1C0A00E (/tg/ml) AET10CH0LAN0L0NE ANDROSTERONE ANDROSTERONE l/tg/ml) - 6 8 -
Fig. 2.9 Calibration curve for dehydroepiandrosterone
Fig. 2.10 Calibration curve for pregnanediol Corrected Ratio X Mr Fig. 2.11 airto cre o steroids for curve Calibration ocnrto (ml ml (nmol Concentration -69- -7 0 -
This was plotted against concentration for each steroid (abscissa).
Concentrations of all other steroids were determined from this calibration line in the same manner.
2.2.4.3 The colorimetric quantitative determination of creatinine
in urine.
Most methods used for creatinine determination in biological fluids are based on the Jaffe (1886) reaction with the modification of
Heinegard & Tiderstrom (1973), according to which creatinine forms a characteristic yellow-orange colour when treated with alkaline pi crate.
The colour produced, however, is not specific for creatinine and is subject to interference by a number of substances including proteins in the test specimen (Di Giorgio, 1974; Cook, 1975).
Assays were performed by using a creatinine diagnostic kit (Sigma
555) which involves the addition of 3 ml alkaline pi crate solution
(Reagent B) to three tubes labelled blank, standard arid sample containing 0.3 ml distilled water, 0.3 ml creatinine standard (stock No.
925-3) and 0.3 ml urine, respectively. The tubes are mixed and allowed to stand for 10 min at room temperature. The initial absorbance (A) of standard and sample was measured using the blank as reference at 500 nm.
To all mixtures 0.1 ml acid reagent (stock No. 555-2) was added and mixed immediately and thoroughly, allowed to stand 5 min at room temperature and the final absorbance at the same wavelength was measured. Upon addition of acid, the colour contributed by creatinine is destroyed, while that produced by the non-specific substances remains. The difference in colour intensity measured at 500 nm before and after acidification is proportional to creatinine.
Standards in the range 0-15 mg/100 ml were assayed and the standard -7 1 -
curve found to be linear between the range 0-10 mg/100 ml (see Fig.
2.12). The creatinine values in man and rats were calculated directly from absorbance readings according to the following equation:
Initial (A) test - Final (A) test Creatinine (mg/100 ml) = ______X 3
Initial (A) standard - Final (A) standard where:
Initial (A) of test = Absorbance of test prior to addition of acid
reagent
Final (A) of test = Absorbance of test after addition of acid
reagent
Initial (A) of standard = Absorbance of standard prior to addition of
acid reagent
Final (A) of stanaara = Absorbance of standard after addition of acid
reagent
3 = Concentration of creatinine in standard (3 ml/100 ml)
2.2.4.4 Determination of total urinary 17-oxosteroids.
This was determined according to the method described by King &
Wootton (1964) depending on the Zimmerman reaction (1935). To urine
(4 ml) in a glass-stoppered tube, concentrated hydrochloric acid (0.5 ml) was added and the open tube heated in a boiling water bath for 20 min, the tube removed and allowed to cool, then chloroform (8 ml) was added and the mixture vortexed thoroughly. The aqueous layer was removed as completely as possible, after which 1 M sodium hydroxide (3 ml) was added, followed by thorough shaking, and the upper layer discarded. -7 2 -
Fig. 2.12 Calibration curve for creatinine -73-
This washing was repeated twice with 3 ml of distilled water. After
complete removal of water, the chloroform was dried by the addition of
anhydrous sodium sulphate and 5 ml transferred to a clean dry tube and
evaporated to dryness. The residue so obtained contained the steroids
from 2.5 ml of urine.
Standard solutions were prepared as follows: 0.2 ml of steroid
standard (10 mg/100 ml) was evaporated to dryness so that the tube
contained 20 pg steroid.
Freshly prepared Zimmerman reagent [0.25 ml; a mixture of equal
volume of 1,3-dinitrobenzene solution (1 gm in 100 ml ethanol) together with 40% benzyl trimethylammoniurn methoxide] was added to test, standard
and a clean dry tube for the blank. Furthermore, the tubes were allowed to stand in the dark at 25°C for 1 h, whereupon 3 ml of ethanol was
added to each tube and mixed thoroughly. The extinctions of test and standard at 440, 520 and 600 nm were measured. The following equations were used for calculation of the total 17-oxosteroids (mg/24 h) in the sample:
E = E520 - 1/2 (E440 + E600) corr E for test Then the amount of steroid in the test = corr______X 0.02 mg
E for standard corr
E for test 100 Hence urinary steroid = _____ corr______X 0.02 X ______
E for standard 2.5 corr
And if V ml is the 24 h urine volume:
^corr f°r test V 17-oxosteroids excretion = ______X _____
(mg/24 h) ^corr for standard 125 -74-
2.3 RESULTS.
2.3.1 Population study.
Gas chromatography of urine samples after derivatization with HFAA
revealed two peaks corresponding to debrisoquine and 4-hydroxy-
debrisoquine (Fig. 2.5). From the study of 90 volunteer subjects and
from the histogram explained in Fig. 2.13, it is evident that the
distribution of the metabolic ratio in the population shows a
discontinuous variation, with metabolic ratio ranging from 0.09-153.
A first small group with metabolic ratio from 12-153, consisting of ten
subjects was found. The largest number of individuals (80) tested had
metabolic ratio ranging from 0.09-12. Accordingly, this group contained
ten recessive PM and 80 dominant EM subjects.
2.3.2 Steroid profiles in man.
Urine samples from 90 volunteer subjects which were analysed for
their content of debrisoquine and 4-hydroxy-debrisoquine were also
analysed for their content of individual 17-oxosteroids, pregnanediol,
pregnanetriol and pregnanetriolone (see Fig. 2.1 and Fig. 2.2). The
ratio between each steroid (pM) in the sample to its content of
creatinine (mM) was calculated. Fig. 2.14 - 2.22 show the distribution of the creatinine values and the steroid/creatinine individual ratio for each subject. Tables 2.6 and 2.7 give the mean (+ S.D.) of each
compound excreted to creatinine (pM/mM) for males, females and PM and
EM phenotype.
2.3.2.1 The effect of sex.
It can be seen from Table 2.6 that androsterone was the predominant urinary 17-oxosteroid excreted, the highest amount being in males (2.5 + FREQUENCY FREQUENCY i. .3 rqec dsrbto hsorm of histogram distribution Frequency 2.13 Fig. debrisoquine) ace br rpeet M subjects PM represent bars Hatched eaoi rto (debrisoquine/4-hydroxy- ratio metabolic METABOLIC RATIO METABOLIC
-75- . g i F FREQUENCY FREQUENCY 10- 5 10 5 0 2.14 rqec dsrbto hsorm of histogram distribution Frequency FEMALES MALES ace br rpeet M subjects PM represent bars Hatched rnr creatinine urinary rnr cetnn (mM) creatinine Urinary 2ZZ2 ZZZ2 15 FREQUENCY FREQUENCY Fig. 2.15 ace br rpeet M subjects PM represent bars Hatched androsterone/creatinine rqec dsrbto hsorm of histogram distribution Frequency fl/Cr -77- FREQUENCY FREQUENCY Fig. 10- 0 J 2.16 77, FEMALES MALES 1 W/, aetiocholanolone/creatinine ace br rpeet M subjects PM represent bars Hatched rqec dsrbto hsorm of histogram distribution Frequency 2 777T< Ae/Cr 4 6 5 4 3 7 7 7 7 7//J2 — 78- 8 -7 FREQUENCY FREQUENCY Fig. 2.17 rqec dsrbto hsorm of histogram distribution Frequency dehydroepiandrosterone/creatinine ace br rpeet M subjects PM represent bars Hatched DEfl/Cr FREQUENCY FREQUENCY Fig. 2.18 ace br rpeet M subjects PM represent bars Hatched rqec dsrbto hsorm of pregnanediol/creatinine histogram distribution Frequency FREQUENCY FREQUENCY Fig. 0 2.19 rqec dsrbto hsorm of histogram distribution Frequency J- x-nrseoe n -aetiocholanolone/ and JJ -oxo-androsterone ace br rpeet M subjects PM represent bars Hatched creatinine 1 1 loxo/Cr 2 H 3
- 81 - Fig. 2.20 Frequency distribution histogram of 1 1 B~hydroxy-ondrosterone and -aetio- cholanolone/creatinine
Hatched bars represent PM subjects FREQUENCY FREQUENCY . g i F 0 • 2.21 777i MALES rqec dsrbto hsorm of histogram distribution Frequency ■///< pregnanetriol/creatinine ace br rpeet M subjects PM represent bars Hatched //& 1 77Tt PT/Cr ----- 2 1 i ... 3 FREQUENCY FREQUENCY i. 2.22 Fig. ace br rpeet M subjects PM represent bars Hatched pregnanetriolone/creatinine rqec dsrbto hsorm of histogram distribution Frequency PTone/Cr - 8 5 -
Table 2.6 Mean (+S.D.) of individual 17-oxosteroids, pregnanediol,
pregnanetriol and pregnanetriolone/creatinine ratios
(pM/mM) in human urine. The effect of sex.
Steroid Male Female t 2P
A 2.4 + 1.0 2.1 + 1.1 1.3 n. s.
Ae 1.8 + 0.9 2.0 + 1.0 0.99 n.s.
DEA 0.75 + 0.78 0.92 + 0.71 1.1 n.s.
PD 0.24 + 0.14 0.56 + 0.64 3.57 < 0.001
11-Oxo 0.52 + 0.4 0.59 + 0.38 0.81 n.s.
118-OH 0.62 + 0.39 0.78 + 0.53 1.62 n.s.
PT 0.75 + 0.43 0.73 + 0.44 0.21 n.s.
PTone 0.75 + 0.36 0.84 + 0.42 1.12 n.s.
The following abbreviations are used:
A: Androsterone
Ae: Aetiocholanolone
DEA: Dehydroepi androsterone
PD: Pregnanedi ol
11-Oxo: 11-Oxo-androsterone and -aetiocholanolone
118-OH: 118-Hydroxy-androsterone and -aetiocholanolone
PT: Pregnanetriol
PTone: Pregnanetriolone -86-
Table 2.7 Mean (+ S.D.) of individual 17-oxosteroids, pregnanediol,
pregnanetriol and pregnanetriolone/creatinine ratios
(pM/mM) in human urine. The effect of phenotype.
Steroid EM PM t 2P
A 2.3 + 1.1 2.1 + 0.6 0.56 n.s.
Ae i.9 + 0.96 2.0 + 1.2 0.3 n.s.
DEA 0.83 + 0.78 0.72 + 0.52 0.43 n.s.
PD 0.39 + 0.46 0.22 + 0.18 1.2 n.s.
11-Oxo 0.56 + 0.41 0.42 + 0.19 1.06 n.s.
118-OH 0.71 + 0.46 0.5 + 0.32 1.4 n.s.
PT 0.75 + 0.45 0.68 + 0.27 0.48 n.s.
PTone 0.79 + 0.39 0.44 + 0.28 2.75 < 0.01
The following abbreviations are used:
A: Androsierone
Ae: Aetiocholanolone
DEA: Dehydroepiandrosterone
PD: Pregnanediol
11-Oxo: 11-Oxo-androsterone and -aetiocholanolone
11&-0H: 118-Hydroxy-androsterone and -aetiocholanolone
PT: Pregnanetriol
PTone: Pregnanetriolone -87-
1.2 pM/mM creatinine), rather than females (2.1 + 1.1), although the
difference was not significant. Aetiocholanolone, which was excreted
also in copious amounts, second only to androsterone was found higher in
females (2.0 + 1.0) than males (1.9 + 1.0). Dehydroepiandrosterone
which ranked in third position of importance after the two steroids
mentioned above again was excreted more in women (0.92 + 0.71) than men
(0.75+0.77) according to Table 2.6. Pregnanediol, pregnanetriol and
pregnanetriolone which comprise the metabolites of progesterone, were
excreted in higher amounts in females than in males, with the exception
of pregnanetriol, where it was obvious that there was no sex difference.
Difficulty in chromatographic separation between 11-oxoandrosterone and
11-oxoaetiocholanolone (see Fig. 2.6) and between 116-hydroxy-
androsterone and llB-hydroxyaetiocholanolone meant that each of these
pairs of isomers had to be treated as one compound, for the purposes of
quantitation. Likewise, their quantities were slightly higher in
females than in males.
2.3.2.2 The effect of phenotype.
As Table 2.7 shows, the previously mentioned urinary steroids are
excreted in similar amounts in both phenotypically EM and PM subjects.
When compared by phenotype, the same rank order of excretion was
observed as seen when the volunteers were compared by sex.
Pregnanetriolone was excreted significantly higher in EM (0.79 + 0.39)
than PM subjects (0.44+0.28; t = 2.75; 2P < 0.01).
2.3.3 Steroid profile in rats.
In this study the urine of five female DA and four female Lewis rats was collected for 48 h and a small portion of each was analysed for its
content of the steroids mentioned earlier. It was noticed that neither -88-
androsterone and aetiocholanolone nor progesterone metabolites could be
detected. The ratio of other steroids to creatinine (pM/mM) is shown in
Table 2.8.
Lewis excreted lesser quantity of dehydroepiandrosterone (0.60+0.50
pM/mM creatinine) than DA (0.97 + 0.66), yet Lewis rats excreted
higher amounts of il-oxoandrosterone and 11-oxoaetiocholanolone (0.60 +
0.18), ll&-hydroxyandrosterone and 118-hydroxyaetiocholanolone (0.46 +
0.51) than DA (0.45 +_ 0.16) and (0.29 +0.27) respectively. None of
these differences reached statistical significance however.
2.3.4 Total 17-oxosteroids in a panel of phenotyped subjects and two
different phenotyped strains of rats.
The total 17-oxosteroids for 15 volunteers; two PM and 13 EM was
measured. Table 2.9 shows the values of the total 17-oxosteroids in
mg/24 h in the group. Two females showed lesser amounts of total 17-
oxosteroids excretion (6.7 + 4.6) than males (13.5 + 4.2), the other one
exhibits higher value (18.7) than the mean of the values of the males
(13.5 + 4.2). On the other hand, one PM showed low total 17-oxosteroids excretion (8.0), while the other was similar to EM subjects in this
respect (17.7).
Additionally, Table 2.10 shows the total 17-oxosteroids values in mg/48 h excreted by five female DA and five female Lewis rats with the result of no significant difference in excretion between both strains.
2.3.5 Analysis of the 5fl/5ot and DEA/A+Ae ratios.
The variation of Ae/A together with DEA/A+Ae according to sex and phenotype were described in Tables 2.11 and 2.12 respectively. The average of Ae/A ratio was more than unity in females (1.1 + 0.5), and less than unity in males (0.77 + 0.28), hence the difference was Table 2.8 Mean (+ S.D.) values of individual i7-oxosteroids/
creatinine ratio in rats.
Strai n DEA/Cr 11-Oxo/Cr 116-OH/Cr
DA 0.97 + 0.66 0.45 + 0.16 0.29 + 0.27
Lewi s 0.60 + 0.51 0.60 + 0.18 0.46 + 0.51
The following abbreviations are used:
DEA/Cr = Dehydroepiandrosterone/creatinine ratio (pM/mM)
11-Oxo/Cr = 11-Oxo-androsterone and -aetiocholanolone/creatinine
ratio (pM/mM) ll&OH/Cr = 116-Hydroxy-androsterone and -aetiocholanolone/creatini
ratio (pM/mM) - 9 0 -
Table 2.9 Urinary excretion of total 17-oxosteroids (mg/24 h)
in human.
No. Sex Phenotype Total 17-oxosteroids (mg/24 h)
1 M EM 20.5
2 M EM 7.8
3 M PM 8.0
4 M PM 17.7
5 M EM 14.1
6 M EM 16.8
7 M EM 15.8
8 F EM 9.9
9 M EM 9.2
10 M EM 10.6
11 M EM 10.7
12 M EM 15.8
13 M EM 18.7
14 M EM 15.5
15 F EM 3.4
F = female; M ‘= male
Mean (+ S.D.) of F = 10.1 + 6.3
Mean (+ S.D.) of M = 13.5 + 4.2
Mean (+ S.D.) of EM = 13.0 + 4.8
Mean (+ S.D . ) of PM 12.9 + 4.9 -9 1 -
Table 2.10 Urinary excretion of total 17-oxosteroids (mg/48 h)
in rats
No. Strain mg/48 h
1 DA 0.33
2 DA 0.26
3 DA 0.32
4 DA 0.34
5 DA 0.31
1 Lewi s 0.30
2 Lewi s 0.24
3 Lewi s 0.27
4 Lewi s 0.35
5 Lewi s 0.24
Mean (+ S.D.) of DA = 0.31 + 0.02
Mean (+ S.D.) of Lewis = 0.28 + 0.04 -9 2 -
Table 2.11 Mean (+ S.D.) of Ae/A arid DEA/A+Ae ratios in human.
The effect of sex.
Male Female t 2P
Ae/A 0.77 + 0.28 1.1 + 0.5 4.0 < 0.001
DEA/A+Ae 0.19 + 0.16 0.24 + 0.14 1.5 n. s.
The following abbreviations are used:
Ae/A = Aetiocholanolone/Androsterone ratio
DEA/A+Ae = Dehydroepiandrosterone/Androsterone + Aetiocholanolone
rati o Table 2.12 Mean (+ S.D.) of Ae/A and DEA/A+Ae rati os in human.
The effect of phenotype.
EM PM t 2P
Ae/A 0.88 + 0.4 0.99 + 0.53 0.8 n. s.
DEA/A+Ae 0.21 + 0.16 0.19 + 0.15 0.38 n.s.
The following abbreviations are used:
Ae/A Aetiocholanolone/Androsterone ratio
DEA/A+Ae = Dehydroepiandrosterone/Androsterone + Aetiocholanolone
ratio
EM Extensive metabolizer
PM Poor metabolizer -94-
significant (2P < 0.001). However, the DEA/A+Ae was higher in females
(0.24 + 0.14) rather males (0.19 +_ 0.16) but the difference was not significant. Table 2.12 shows the values related to phenotype, Ae/A slightly higher in PM (0.99 + 0.53) rather EM (0.88 + 0.4), where the
DEA/A+Ae was nearly the same, 0.19 + 0.15 and 0.21 + 0.16 for PM and EM respecti vely.
2.3.6 Comparison of urinary 17-oxosteroid concentration determined
by colorimetry and by summation of individual 17-oxosteroids
determined by gas chromatography.
Some of the 0-24 h urines collected from phenotyped volunteers were used for both total 17-oxosteroid determination by the colorimetric reaction of King & Wooton (1964) (see Section 2.2.4.4) and for gas chromatographic analysis (Section 2.2.4.2) of individual 17-oxosteroids and progesterone metabolites. The opportunity therefore arises to compare these two means of determining 17-oxosteroid concentration by summation of the individual 17-oxosteroid molar concentrations for androsterone, aetiocholanolone, dehydroepiandrosterone, 11- oxoandrosterone, 11-oxoaetiocholanolone, 116-hydroxyandrosterone and
116-hydroxyaetiocholanolone in 0-24 h urines and comparing these aggregate values with the molar concentration of 17-oxosteroids determined directly by colorimetry.
Table 2.13 shows the molar concentration (pM) of total 17-oxosteroids determined by these two methods for 12 of the 15 subjects from Table
2.9. Firstly, the mean (+ S.D.) values by the two methods are extremely similar, 34.5 + 15.0 pM by colorimetry and 35.4 + 20.2 pM by gas chromatography. Additionally, the values from the two methods correlated well, both by least squares regression analysis (Fig. 2.23; r = 0.90, -B5-
Table 2.13 Comparison of colorimetric and gas chromatographic
determinations of 17-oxosteroid concentration in human
uri ne.
Total 17-oxosteroid concentration (pM)
Subject+ Colorimetry G.C. method*
1 34.3 (6) 32.5 (7)
2 30.1 (9) 30.1 (8)
4 31.3 (7) 45.7 (5)
5 50.5 (3) 51.4 (4)
7 56.6 (1) 52.7 (2)
8 30.2 (8) 21.0 (9)
9 16.3 (11) 13.6 (11)
10 55.3 (2) 72.5 (1)
11 36.1 (5) 51.5 (3)
12 22.7 (10) 14.5 (10)
14 43.1 (4) 36.5 (6)
15 7.9 (12) 3.3 (12)
Mean (+ S. D.) 34.5 + 15.0 35.4 + 20.2
6.C. method = Aggregate gas chromatographic determinations of
individual 17-oxosteroids; androsxerone, aetiocholanolone, dehydro-
epiandrosterone, 11-oxoandrosterone, 11-oxoaetiocholanolone, 113-
hydroxyandrosterone and llfS-hydroxyaetiocholanolone.
+Subjects are taken from subjects 1-15 in Table 2.9. Numbers in
parentheses are ranks. Rank No. 1 is the highest concentration. -96-
Fig. 2.23 Correlation between colorimetric and gas chromatographic determinations of 17-oxosteroid
BY COLORIMETRY
BY COLORIMETRY
Fig. 2.24 Correlation between the rank of urinary 17- oxosteroid concentration determined by colorimetr and gas chromatography -9 7 -
P < 0.001) or by Spearman rank correlation analysis (Fig. 2.24; r$ =
0.94, P < 0.001). This is a remarkable finding when one considers that
one method employs the reaction of the 17-oxo group with 1,3-
dinitrobenzene in base after acid hydrolysis of conjugates and colorimetry and the other gas chromatographic determination of the trimethyl si 1y1 ethers of seven individual 17-oxosteroids after enzymic
hydrolysis of conjugates. This correlation must surely validate the gas
chromatographic methods employed here. -9 8 -
2.4. DISCUSSION.
The aim of the work described in this Chapter was to investigate
whether or not the genetic polymorphism of debrisoquine 4-hydroxylation
(Mahgoub et aj_., 1977; Evans et ^1_., 1980) could be observed with
certain endogenous compounds including individual 17-oxosteroids or
progesterone metabolites.
In the study, whereby 90 subjects were examined for 4-hydroxylation
of debrisoquine, it was seen that 80 individuals were EM with metabolic
ratios ranging from 0.09-12, and therefore excreted 4-hydroxy-
debrisoquine in substantial amounts. However, 10 subjects excreted
mainly debrisoquine and a very little amount of 4-hydroxy-debrisoquine
and were thus considered poor metabolizers (PM).
In the study of steroid profiles in man, it was shown that the two
compounds androsterone and aetiocholanolone were excreted in larger
quantities than other 17-oxosteroids, with no significant difference
between EM and PM. Similarly the ratio between aetiocholanol one/
androsterone showed no significant difference phenotypically.
It is interesting that pregnanediol/creatinine ratio showed
significantly higher values in females than males where this is in
agreement with the Klopper et_ aj_. (1955) findings that women in both
proliferative phase and luteal phase excreted higher amounts of
pregnanediol than men in 24 h, although the post-menopausal women excrete lower amounts than men. Scommegna et al. (1967) also found that
pregnanediol excretion in women at luteal phase was much higher (2.68 mg/24 h) than Romanoff et al_. (1966) report for men (0.6 mg/24 h).
Kulpmann & Breuer (1977) moreover found that women excrete higher
amounts of pregnanediol than men. However, the sex cycle is important > -99-
in women and in future studies it might be advisable to collect such
data from female volunteers.
The results presented in Table 2.6 related to dehydroepiandrosterone
which showed its excretion in women higher than men support the findings
of Kulpmann & Breuer (1977), in the same time it is at variance with
results reported by Feher (1966). 11-Oxo-androsterone and
-aetiocholanolone together with llB-hydroxyandrosterone and
-aetiocholanolone are also higher in females than males which is in
agreement with Kulpmann & Breuer's findings (1977).
It has been shown further (Table 2.11) that the Ae/A ratio is also
influenced among other things, by sex, where there is a significantly
higher value in females than males, which is in agreement with Baulieu &
Mauvais-Jarvis (1964) who showed that the amount of 5a metabolites
produced from testosterone and androstenedione was in a greater
proportion in adult males than in females, and the value of Ae/A was
considerably higher in women than men. Feher (1966a) observed a higher
ratio of Ae/A in women than men and stated that the normal value of the
Ae/A ratio depends on different factors:
1) The relative amount of precursors (dehydroepiandrosterone,
androstenedione, testosterone) synthesized by the glands,
2) The activity of enzymes responsible for the two pathways,
3) Sex,
4) The level of other steroid hormones.
The present investigations have shown that the DEA/A+Ae ratio
likewise changes with sex but insignificantly and Dobriner (1953) showed
that, although this ratio was extremely variable, it remained fairly
constant in individual persons. Jayle & Malassis (1958) and Schneider &
Lewbart (1959) demonstrated that administration of DEA in large amounts -100-
increased the DEA content of the urine and raised the urinary output of
A and Ae to a much lesser extent. MacDonald et^ aj_. (1962) claimed that
the DEA to A+Ae conversion was practically constant and hardly depended
on the rate at which DEA was secreted. Wilson & Schenker (1964) have
demonstrated and MacDonald e^t aj_. (1962) concluded that 17-hydroxy- corticosteroids also influence the catabolism of C-jgC^-steroids in a
sense favourable for the production of the 5&-metabolite. It is possible that oestrogen also plays a role in this respect (Herrmann ^t al_., 1960).
Steroid profiles in rat, as was shown in the Results, differ from that of human in the absence of the main 17-oxosteroid peaks, androsterone and aetiocholanolone and progesterone metabolites, pregnanediol, pregnanetriol and pregnanetriolone. According to Table
2.7, dehydroepiandrosterone excretion was higher in EM individuals than
PM, although the difference was not significant, here in rats where DA and Lewis are the animal model of polymorphic drug oxidation (Al-Dabbagh et al_., 1981), it was observed that DEA was excreted higher in DA than
Lewi s.
The results presented in Table 2.9 are in agreement with others
(Kulpmann & Breuer, 1977) whereby the female subjects excreted lesser amounts of total 17-oxosteroids than males.
As it was shown in the Results, pregnanetriol excretion was not significantly different between sexes. Table 2.7 showed the urinary concentration of all compounds relative to creatinine in both EM and PM phenotypes, at the same time Table 2.12 represents the values of Ae/A and DEA/A+Ae ratios in the same phenotypes. It can be noticed that all the values were higher in EM than PM except aetiocholanolone and the
Ae/A ratio. It was shown from Table 2.7 also that pregnanetriolone was -101-
significantly higher (2P < 0.01) in EM (0.79 + 0.39) than PM subjects
(0.44+0.28). Pregnanetriolone is a minor metabolite of progesterone
after 17ot-hydroxyl ati on to 17a-hydroxyprogesterone and 113-hydroxylation
to ll&,17a-dihydroxyprogesterone (21-desoxycortisol) and is finally
converted to pregnanetriolone (all these happen in adrenal cortex).
However, the usefulness of pregnanetriolone measurement as a phenotyping
test is uncertain. These observations nevertheless provide a clue that
the gene responsible for debrisoquine hydroxylation contributes to the
oxidation pathways of certain corticosteroids and thus requires more
detailed study. It would be of great interest to phenotype congenital
adrenal hyperplasia cases, where these pathways can be looked at
individually if there exists an absolute association between
debrisoquine polymorphism and steroid hydroxylation pathways, but
unfortunately such studies fall outside the scope of this thesis.
The major microsomal enzyme system has been termed "mixed function
oxidase" (Mason, 1957) or "monooxygenase" (Hayaishi, 1969) one of whose main metabolic functions appear to be participating in the oxidation of
a number of compounds such as steroids, fatty acids, drugs and
carcinogens (Cooper et ^1_., 1965; Kuntzman, 1969). Adrenal steroid
hydroxylases, like many hepatic drug metabolizing enzymes, satisfy
the criteria for mixed function oxidases. The function of cytochrome
P-450 as the terminal oxidase in mixed function oxidase reactions was first established in adrenal microsomes. The adrenal cortex and other
steroid producing tissues contain an abundance of cytochrome P-450 in
both mitochondria (Harding et aj_., 1964) and microsomes. The
independence of the oxidation pathways of the previously mentioned
steroid metabolites from the gene which regulates the alicyclic hydroxylation of debrisoquine suggests that different forms of cytochrome P-450 must be involved in their metabolism. These results are similar to the observed genetic independence of oxidation of debrisoquine and other exogenous drugs. CHAPTER THREE
EFFECT OF POLYMORPHIC OXIDATION ON URINARY OESTROGEN
EXCRETION IN MAN AND RAT - 104 -
3.1 INTRODUCTION
Oestrogens.
In the human female, oestrogens are produced primarily by the ovary,
although the adrenal cortex is also a source for some steroids which have oestrogenic activity. Doisy (1942) estimated that, during a normal menstrual cycle, the ovary secretes the equivalent of 10 mg of oestrone
(E^). During pregnancy, oestrogens are secreted in rapidly increasing amounts by the placenta. Oestriol (E^) makes up a large fraction of the oestrogen produced by the placenta, but oestrone and oestradiol (E^) are also present. In late pregnancy, 12-50 mg of oestrogenic material may be secreted per day (Pincus & Pearlman, 1943).
In men, it has been postulated that the testes as well as the adrenal cortex secrete oestrogens. This is supported by the observation that in hypogonadal men, oestrogen excretion is diminished, but not entirely absent.
The structure of oestrogens is built on the cyclopentanophenanthrene nucleus (Fig. 3.1). The parent saturated hydrocarbon is oestrane. The natural oestrogens are soluble in ether, alcohol, acetone and many oils, but not in petroleum ether. They are practically insoluble in water, but owing to their phenolic nature they are quite soluble in aqueous alkali, permitting separation from other sex hormones which are nonphenolic. The ketonic oestrogen, oestrone, may be separated from the nonketonic oestrogens oestradiol and oestriol by means of the Girard reagents (l-(carboxymethyl) pyridiriium chloride hydrazide or
(carboxymethyl) trimethylammoniurn chloride hydrazide), while oestriol and oestradiol can be partitioned by their relative solubility between
0.3 M Na2C02 and benzene. The natural oestrogens give the colour reactions obtained with simple phenols with Mill on1s reagent, diazotized aromatic amines and Folin's phenol reagent. - 105 -
Fig. 3.1 Chemical structure of oestrogens employed
and the internal standard
O OH
2-Hydroxyoestrone
OH
16a~Hydroxyoestrone Oes triol
16-0xo-oestradiol Epicoprostanol (internal standard) -106-
The oestrogens induce or influence many physiological responses such
as proliferation of the vaginal epithelium and endometrium. They also
produce growth of the myometrium and duct system of the breast and
nipple. Suppression of certain anterior pituitary hormones,
particularly the follicle-stimulating hormone and prolactin, follows the
administration of large doses of oestrogens. Oestrogens also favour a
retention of salt and water under certain conditions. In the male,
oestrogens, in sufficiently large dosage, inhibit testicular function
and produce the physiological effects of castration, probably through
their action on the pituitary.
The secretion of oestrogens by the ovary is stimulated by the
gonadotrophic hormones. The ovarian oestrogens enter the systemic
circulation and are present chiefly in the free form, although some may be bound to protein. The oestrogens reach the liver where they are rapidly inactivated, but are then apparently reactivated and appear in the bile, subsequently undergoing an enterohepatic circulation during which the bulk of the hormone is probably gradually metabolised in the
liver, small amounts being released into the systemic circulation for many days (Cantarow et £]_., 1943). 4-Androstenedione is believed to be a major precursor of testosterone and of oestrogen biosynthesis in both- tne gonads and the adrenal cortex (Eik-Nes & Hall, 1965). The metabolism of the oestrogenic hormone is oxidative in nature in contrast to the reductive pathway for the neutral steroid hormones (Fig. 3.2).
It has been postulated that oestrone is the precursor of oestriol on the evidence that the required ketol intermediates are normal metabolites of the oestrogenic hormone (Marrian at aj_., 1957a; Layne & Marrian, 1958;
Brown et aj_., 1958). However, it is true that enzymatic hydroxylation of chemically unreactive methylene groups is a common feature of steroid biochemistry (Fried et al., 1955). -107-
Fig. 3.2 Some pathways in the metabolism of oestrogens
OH
O
2-methoxyoestrone -108-
The role of liver in oxidative metabolism leading to hydroxylation in the 2-position (Fishman et ail_., 1960; Kraychy & Gallagher, 1957) and subsequent methylation (Kraychy & Gallagher, 1957; Frandsen, 1959;
King, 1961) of oestrone (Fishman et aj_., 1960; Kraychy & Gallagher,
1957), 17B-oestradiol (Frandsen, 1959), oestriol (King, 1961; Fishman &
Gallagher, 1958) as well as to the formation of 6-oxygenated oestrogens
(Mueller & Rumney, 1957) has been established. Additionally, it has been shown that the liver of several species can effect the conversion of 17&-oestradiol to oestriol (Engel et al_., 1958) and the inter conversion of oestrone and 17B-oestradiol (Ryan & Engel, 1953).
Catecholoestrogens (2-hydroxy and 2-methoxy derivatives) have been detected in brain, pituitary gland, liver and ovary (Paul & Axelrod,
1977) and have been shown to exhibit both oestrogenic and anti oestrogenic properties (Fishman, 1976). The anti oestrogenic properties have been described for the hypothalamus, where 2-hydroxy- oestradiol inhibits the oestrogen-elicited increase in hypothalamic cAMP
(Paul & Skolnick, 1977). In addition^ by inhibiting tyrosine hydroxylase (Lloyd & Weisz, 1978) and catecholamine 0-methyltransferase
(Ball et_ aj_., 1972), enzymes involved in the biosynthesis and metabolism of catecholamines, the catecholoestrogens may further indirectly alter neuronal activity.
There have been reports of elevated 2-hydroxyoestrogen levels in urine in thyroid disease (Fishman et a]_., 1965) and in anorexia nervosa
(Fishman et al_., 1965), a psychosomatic disorder accompanied by altered neuroendocrine homeostasis. The formation of 2-hydroxyoestrone and 2- hydroxyoestradiol, the principal catecholoestrogens, is catalyzed by oestrogen-2-hydroxylase, a microsomal cytochrome P-450-dependent mono oxygenase (Gelbke et , 1977; Paul 1977; Sasame et a!., -109-
1977). In man, it appears that 17a-oestradiol and oestrone can be
converted one to the other and that both can be excreted as oestriol.
There is some evidence that progesterone prevents the excessive
destruction of oestrogens and facilitates a conversion of oestrone to
oestriol.
Oestrogen metabolites are excreted by the kidneys, chiefly in a
conjugated form as water-soluble substances, mainly glucuronides. As
Fishman et aj_. (1979) reported, glucuronides generally represent 90% or
more of the urinary oestrogens. The ability of the liver to conjugate
steroids as glucosiduronates (Dutton & Storey, 1954) and sulphates
(Schneider & Lewbart, 1956) has long been recognized. The formation of
phosphate conjugates of oestrogen metabolites by the liver is also
recorded (Brooks et aj_., 1963).
Mary investigations concerning the metabolism of oestrogens in mammals have shown that hydroxylation at C-2 of the phenolic steroids is
of major importance from the quantitative point of view (Fig. 3.2). The
2-hydroxyoestrogens have a catechol structure and are further metabolized by methylation of the phenolic hydroxy groups; this reaction is catalyzed by a catechol-0-methyltransferase (Knuppen et a!.,
1961; Breuer et^ al_., 1961; Breuer et al_., 1962; Knuppen & Breuer,
1966; Fishman et aj_., 1967). So far, most studies on the formation of
2-hydroxyoestrogens have been restricted to metabolic experiments with radioactive oestrogens in vivo (Fishman, 1963; Zumoff et a]_., 1968a,b;
Watanabe, 1970; Collins et ^1_., 1967; Keith & Williams, 1970) and in vitro (Ball et aT_., 1974; Knuppen & Ball, 1974; Fishman & Dixon, 1967;
Kuss, 1971).
It has been demonstrated that not only 2-hydroxyoestrone (Gelbke et al., 1973) but also 2-hydroxyoestradiol (Gelbke et a!., 1975) and -110-
2-hydroxyoestriol (Gel bke et ^1_., 1975; Gelbke & Knuppen, 1974) are
excreted in the urine of pregnant women.
It is estimated that only 5-10% of the endogenous oestrogen can be
recovered as biologically active hormone from the urine.
During the early weeks of pregnancy, oestrogens continue to be formed
by the corpus luteum of pregnancy, but are gradually replaced by
increasing amounts of oestrogen produced by the placenta. In the urine,
oestriol predominates throughout gestation. This may be accounted for
by the conversion of 17a-oestradiol and oestrone to oestriol (Fig. 3.1),
as well as the endogenous production of oestriol itself. During
pregnancy the rate of destruction of oestriol is apparently low. Almost
all the oestrogen is present in conjugated form until several weeks
before the onset of labour, at which time there is a fall in total
urinary oestrogens, but an increase in free hormone. 16a-Hydroxy-
oestrone is quantitatively a very important oestrogen in pregnancy, its concentration in pregnancy urine being exceeded only by that of oestriol
(Adlercreutz & Martin, 1976). In the non-pregnant woman, 16a-hydroxy-
oestrone is the most abundant oestrogen in bile and it is also one of the more significant oestrogens excreted in urine (Adlercreutz et al.,
1974).
Oestrogens are usually present in such small amounts in the blood and urine that it is not possible to isolate the specific oestrogens present for quantitative chemical determination. In late pregnancy, where the amount of oestrogeris excreted is high, applications of several methods have been made in several laboratories.
Quantitative determinations showed that within the group of these catecholoestrogens the amount of 2-hydroxyoestrone (100-2500 pg/24hr) by far exceed those of 2-hydroxyoescradiol (20-200 pg/24hr) and 2-hydroxy- oestriol (30-250pg/24hr). -111-
Studies of oestrogen metabolism in breast cancer have engaged the
attention of many investigators over the years because of the obvious
clinical correlations between oestrogenic function and the incidence and
natural history of breast cancer. Similarly, MacMahon £t aj_. (1982)
observed that cigarette smoking affects the urinary oestrogen levels and
they have shown that smokers have substantially and significantly lower
levels of all three major oestrogens in the luteal phase of the
menstrual cycle. There have been reports that tobacco smoking reduces
levels of testosterone in men (Surgeon General of the United States), as
well as levels of human placental lactogen (Boyce et aj_., 1975) and
rates of oestriol excretion (Targett et ^1_., 1977) in pregnant women.
More general effects on the ovary have also been postulated (Mattison &
Thorgeirsson, 1978; Weathersbee, 1980).
The products of 16a-hydroxylation, oestriol (Clark et £l_., 1977) and
16a-hydroxyoestrone (Fishman & Martucci, 1980) are reported to be
potent uterotropic agents under physiological conditions, whereas the
alternative 2-hydroxylated compounds, 2-hydroxyoestrone and 2-methoxy-
oestrone, are devoid of such activity (Martucci & Fishman, 1977) but do
exhibit CNS actions (Martucci & Fishman, 1979; Schinfeld et aj_., 1980).
Oestriol is a metabolite of oestrone which is converted by 16a-
hydroxylase to 16a-hydroxyoestrone and finally to oestriol (Genet et
al., 1962). It is well known that all the natural or synthetic
oestrogens which have been tested in a suitable animal model have shown more or less pronounced carcinogenic activity (Lacassagne, 1932;
Lacassagne, 1936; Lacassagne, 1938; Robson & Bonser, 1938; Rudali,
1952; Rudali et al_., 1971). Lemon (1970), however, has stated that
oestriol should be considered a possible inhibitor for mammary
carcinogenesis. -112-
The aim of the study described in this Chapter was to investigate whether or not the genetic polymorphism of debrisoquine hydroxylation in man and rat affects the disposition of some endogenous oestrogens, in particular the formation of 16a-hydroxy-,2-methoxy- and 2-hydroxy- oestrone metabolites. To this end, a gas chromatographic assay for the main oestrogens was developed, in order to show whether or not the genetically determined defect of cytochrome P-450 mediated debrisoquine hydroxylation carries over to the hydroxylation of oestrone at C-2 and
C-16 and thereby providing a possible basis for a non-invasive phenotyping test. In addition, should such an effect be found, it would have implications for a genetic and metabolic component in several diseases of endocrine origin in women. 3.2 MATERIALS AND METHODS
3.2.1 Materi als
Oestrone (E^; 1,3,5 (10)-oestratrien-3-ol-17-one; Mr 270.4), 173- oestradiol (Eg; 1,3,5 (10)-oestratriene-3,173-diol; Mr 272.4), 2- methoxyoestrone ^-CHgOE^ 2-0-methyl-1,3,5 (10)-oestratriene-2,3-diol-
17-one; Mr 300.4), 2-hydroxyoestrone (2-OHE^ 1,3,5 (10)-oestratriene-2
3-diol-17-one; Mr 286.4), 16a-hydroxyoestrone (lCa-OHE^; 1,3,5 (10)- oestratriene-3,16a-diol-17-one; Mr 286.4), 16-oxo-oestradiol (16-oxo E^
1.3.5 (10)-oestratriene-3,17S-diol-16-one; Mr 286.4), oestriol (E^;
1.3.5 (10)-oestratriene-3,16a,173-triol; Mr 288.4), androstenedione (4- androstene-3,17-dione; Mr 286.4), 17a-epitestosterone (4-androsten-17a- ol-3-one; Mr 288.4), testosterone (4-androsten-173-ol-3-one; Mr 288.4), progesterone (4-pregnene-3,20-dione; Mr 314.5), epicoprostanol (53- cholestan-33-ol; Mr 388.7) and bis (trimethylsilyl)-trifluoroacetamide 14 (BSTFA) were obtained from Sigma Chemical Company, Poole, U.K. [4- C]
Oestrone (specific activity 55 mCi/mmol) was supplied by Amersham
International Limited, Bucks. Pyridine was obtained from Aldrich
Chemical Co. Ltd., U.K. Concentrated hydrochloric acid, sodium bicarbonate, 100 vol H^Og, octan-2-ol, ethanol, sodium hydroxide, ethyl acetate and hexane (all Analar grade) were obtained commercially. 17a-
Oestradiol (1,3,5 (10)-oestratriene-3,17a-diol; Mr 272.4), 16- epioestriol (1,3,5, (10)-oestratrierie-3,163,173-triol; Mr 288.4), oestradiol sulphate and oestradiol glucuronide were a gift from the MRC
Steroid Reference Collection, Chemistry Department, Westfield College,
London.
3.2.2 Animals
Inbred female rats (150-200 g body weight) of two strains, DA and
Lewis (Section 2.2.2), were used. -114-
3.2.3 Human subjects
Healthy adult female and male volunteers were asked to collect their
24 h urine and the volume measured. About 200 ml of each sample was
stored at -20°C until analysed.
Seven subjects were studied, six female and one male for comparison.
Of these, two of the females were PM, one of which was also post
menopausal .
3.2.4 Dosing of animals and collection of excreta.
Table 3.1 shows the dose, vehicles and route of administration of
each drug which had been given to the rat. For p.o. administration, the
drug was given directly into the stomach using a modified spinal needle.
After dosing the animals, they were housed in cages which were designed
to separate faeces from urine. Animals were allowed food and water ad
libitum. Urine collections were made after washing the cages thoroughly with water to give a final volume of 50-100 ml and was kept in plastic
screw-capped containers which were stored at -20°C until analysed.
3.2.5 Preparation of faeces for g.c. analysis.
Faeces were homogenized in distilled water in the homogenizer to a final volume of 80 ml.
Following centrifugation for 10 min at 3000 rpm, the supernatant was removed and kept at -20°C until analysed.
14 3.2.6 Preparation of faeces for C analysis
Faeces were homogenized in distilled water in the homogenizer to a volume of 80 ml. The homogenate was bleached by the following method:
Homogenate (5 ml), 3 M NaOH (1 ml), octan-2-ol (0.5 ml) and 100 vol
ml) were mixed and left to stand in a fume cupboard in 100 ml conical flasks covered with aluminium foil. After two days, Table 3.1 Dose, route of administration and vehicles used for
oestrogens given to rats.
Oestrogen Dose Route of Sol vent (mg kg"1) administration
Oestrone 100 p.o. Corn oi1
Oestrone 100 i .p. Corn oil
[4-^C]-0estrone 100 (2 pCi/rat) i .p. Corn oil
[4-^C]-0estrone 100 (2 pCi/rat) p.o. Corn oil
[4-^C]-0estrone 0.27 (10 pCi/rat) i.p. Corn oi1
2-Hydroxyoestrone 25 i .p. Corn oil
16a-Hydroxyoestrone 25 i.p. Corn oil 14 [4- C]-0estrone 0.08 (3 pCi/rat) i .m. v 10% aq. ethanol
l .m. v intramesenteric venous concentrated HC1 (1 ml) was added and the mixture boiled for 30 sec,
cooled and then neutralized to pH 7-8 with solid sodium bicarbonate.
This neutral solution was adjusted to 50 ml with water and then kept in
a plastic container stored at -20°C until counted for 14C.
3.2.7 Counting of [*4C]-oestrone
In cases where [^4C]-oestrone was administered to rats, aliquots of
urine or faecal homogenates were counted for ^4C content as follows:
Urine (1 ml) or faecal homogenate (2 ml) were counted in duplicate in
plastic inserts within glass vials containing Cocktail T Scintran
(triton/toluene-based) obtained from BDH Chemicals Ltd. The vials were
then counted in a liquid scintillation spectrometer using the
external standard method (Packard Tricarb Model 4640).
3.2.8 Analytical procedures
3.2.8.1 Determination of oestrogens and metabolites in urine
employing gas-liquid chromatography
This was carried out essentially according to the method described by
Adessi et al_. (1974) with some modification. To rat urine or faecal homogenate (20 ml or 30 ml respectively), diluted to 50 ml with water or to 50 ml human urine was added epicoprostanol solution (internal standard; 160 pi of a 200 pg/ml solution), 2.5 ml concentrated HC1 and the mixture heated in a screw-topped 50 ml Teflon container at 100°C in an aluminium heating block for 1 h. The samples were allowed to cool to room temperature, whereupon sodium bicarbonate powder was added until pH 5. The samples were extracted with 50 ml ethyl acetate : hexane : ethanol mixture (7:2:1 v/v) by shaking thoroughly for 1 h. The -117-
organic upper layer was separated and evaported to dryness in a rotatory
evaporator. The residue was transferred to a small vial where it was
derivatized with 200 pi of BSTFA in pyridine (1:1) at 60°C for 20-30 min.
Samples (1 pi) were injected into a Pye Uni cam 204 gas chromatograph
equipped with a hydrogen flame ionization detector. Chromatographic
conditions were glass column: 1% 0V-1 on Chromosorb WHP (80-100 mesh)
(Jones Chromatography Limited, Llanbradach, Wales) length of column: 210
cm and 2 mm internal diameter, carrier gas: nitrogen (flow rate: 20 ml
min-^; pressure 30 psig), flow rate of hydrogen and air were 65 ml min“^
(pressure 25 psig), and 180 ml min"^ (pressure 6 psig) respectively.
Column temperature: isothermal 192°C for 20 min then 2°C/min to 220°C,
detector temperature: 300°C, injector: 300°C. Retention times of
emerging peaks were calculated relative to the internal reference peak
(epicoprostanol). A gas chromatographic trace of some authentic
oestrogens extracted from water is shown in Fig. 3.3 and the relative
retention times of these and certain other steroids are given in Table
3.2. Signal integration was performed using Pye Uriicam CDP4 computing
integrator.
Accordingly, the peak area ratio (area of peak interest divided by
area of internal standard peak, epicoprostanol) was determined
chromatographically for a set of aqueous standards hydrolysed and
extracted in the same manner as urine samples. For all oestrogens used, with the exception of oestradiol, two sets of calibration curves were
constructed, one for low concentrations (0.05 - 1 pg ml“^) and one for high concentrations (0.5 - 10 pg ml'^). To achieve this, epicoprostanol
solution (internal standard) was added so that its final concentration in 50 ml water was 0.64 and 3.2 pg ml“^ respectively for the low and high -118-
Fig. 3.3 Typical gas chromatogram of principal
oestrogens and oestrone metabolites as
their TMS derivatives
Min
1 Oestrone 2 Oestradiol 3 2-Methoxyoestrone 4 2-Hydroxyoestrone 5 16a~Hydroxyoestrone 6 16-0xo-oestradiol 7 Oestriol 8 Epicoprostanol (int. std. ) -119-
Table 3.2 Gas chromatographic characteristics of some silylated
steroids, including oestrogens, on 1% OV-1.
Compound Retention +Relative time (min) retention time
Androsterone 6.47 0.19
Aetiocholanolone 6.90 0.21
Dehydroepi androsterone 8.25 0.25
11-Oxo androsterone 8.50 0.25
Androstenedione 8.50 0.25
11-Oxo aetiocholanolone 8.90 0.27
Oestrone (E^) 9.30 0.27
17a-Epi testosterone 10.1 0.30
17a-0estradi ol 11.3 0.34
116-Hydroxyandrosterone 11.5 0.34 ll$-Hydroxyaeti ocholanolone 12.1 0.36
Testosterone 12.4 0.37
17&-0estradiol (E„) 12.6 0.37
2-Methoxyoestrone 15.5 0.46
Progesterone 16.2 0.48
2-Hydroxyoestrone 17.0 0.50
16a-Hydroxyoestrone 19.2 0.57
Pregnanediol 19.2 0.57
16-0xo-oestradiol 21.3 0.63
Oestriol 27.0 0.80
Pregnanetri ol 27.7 0.82
16-Epioestriol 28.3 0.84
Epicoprostanol (int. std.) 33.7 1.00
"^Retention time of peak divided by retention time of internal standard
(epicoprostanol). Oestrogens are italicized. -120-
concentration standard curves. These calibration curves for oestrone,
2-methoxyoestrone, 2-hydroxyoestrone, 16a-hydroxyoestrone, 16-oxo-
oestradiol and oestriol are given in Figs. 3.4 - 3.9. In the case of
oestradiol, where the authentic glucuronide and sulphate conjugates were available (see above), a different type of calibration was made, in order to test the hydrolysis conditions employed. In this case, a set of calibration standards were made up from 0.5 - 10 pg ml“^ oestradiol and duplicate sets containing the molar equivalent of 0.5 - 10 pg ml~* oestradiol as either its B-glucuronide or sulphate conjugate. Fig. 3.10 shows the curves for oes-cradiol and its two conjugates. The purity of the conjugates was not guaranteed by the MRC Steroids Reference
Collection, yet it is interesting that the oestradiol curve falls in between the two curves for its conjugates. Accordingly, it was concluded that complete hydrolysis of conjugates occurs under the conditions employed.
3.2.8.2 Determination of biliary, faecal and urinary metabolites of
labelled oestrone in rats by HFLC
The urine (10 ml), faeces (10 ml) or bile (ca. 0.4 ml) samples were processed in the same manner as for preparation of samples for gas chromatographic analysis (acid hydrolysis and organic solvent extraction). After drying, the extract was dissolved in 200 pi of acetone and injected into a Pye-Unicam high-performance liquid chromatograph (HPLC) fitted with a Lichrosorb diol column (25 X 0.4 cm internal diameter, 10 pm particle size; E. Merck, Darmstadt, West 3 Germany). The use of this column for separating metabolites of H- ethynyloestradiol has been reported by Williams & Goldzieher (1979).
After injection, the metabolites were eluted with 7% ethanol in hexane i. 3.4 Fig.
PERK RRER RATIO PERK RRER RATIO airto cre fr oestrone for curves Calibration pe (- y/l, oe (-0 yg/ml) (0-10 Lower yg/ml), (0-1 Upper 35 airto cre fr 2-methoxyestrone for curves Calibration 3.5 . g i F
PERK AREA RATIO PEAK AREA RATIO pe (- y/l, oe (-0 yg/ml) (0-10 Lower yg/ml), (0-1 Upper i. . Clbain uvs o 2-hydroxyoestrone for curves Calibration 3.6 Fig.
PERK RRER RRTIO PERK RRER RATIO pe (- y/l, oe (-0 yg/ml) (0-10 Lower yg/ml), (0-1 Upper
PERK RRER RATIO PERK RRER RATIO 16g-hydroxyoestrone for curves Calibration 3.7 Fig. 0 . 2
o + O) 00 O - r - f- H- H
TJ O ■n> O Is ) 0 16a-HYDR0XY0ESTR0NE 1 4 0. 8 1 .8 0 .6 0 .4 0 OI t(Q S3 I -125- Fig. 3.8 Calibration curves for 16-oxo-oestradiol Upper (0-1 yg/ml), Lower (0-10 yg/ml) 16-0X0-0ESTRRD10L (/eg/ml) 16-0X0-0ESTRflDI0L ifig/ml) 39 airto cre fr oestriol for curves Calibration 3.9 . g i F PEAK AREA RATIO PEAK AREA RATIO pe (- y/l, oe (-0 yg/ml) (0-10 Lower yg/ml), (0-1 Upper -127- Fig. 3.10 Calibration curve of oestradiol and its glucuronide and sulphate conjugates *■ * 0ESTRADI0L s o GLUCURONIDE CONJUGATE * SULPHATE CONJUGATE -128- (flow rate 5 ml min”^). Eluate fractions (2.5 ml) were dissolved in 4 ml of scintillator and assayed for radioactivity by scintillation counting. Eluates were also monitored at 280 nm with a UV absorbance detector (Pye Unicam LC-UV detector). Peaks of radioactivity were ascribed by comparing their retention times with those of authentic unlabelled steroids (Table 3.3). A typical HPLC chromatogram for authentic oestrogens is shown in Fig. 3.11. 3.2.8.3 Collection of bile rrom rats Female DA and Lewis rats (190 - 230 g) were used. They were maintained on Labshure 41B diet with freeaccess to water. The animals were anaesthetized with ether, and their common bile ducts cannulated. In the experiment, 16 pg (3 pCi) of [^C]-oestrone was administered by i.v. injection into a mesenteric vein. Bile was collected at 1 h intervals for 8 h and stored frozen until assayed. 3.2.8.4 Determination of sodiumand potassiumexcretion in rat urine Female DA and Lewis rats (170 - 180 g) were used. They were maintained on Labshure 41B diet with free access to water. In the first three days, normal 24 h urine were collected and then 20 mg oestrone in corn oil was given i.p. to DA and Lewis rats. The volume of undiluted urine was measured. Aliquots (5 ml) of each sample was analysed for Na+ and K+ by the Medical Unit Laboratory. -129- Table 3.3 HPLC characteristics of oestrogens on Lichrosorb diol column eluted with 7% ethanol in hexane. Peak Number Compound Retention volume (ml) 1 2-Methoxyoestrone 6.0 2 Oestrone 8.0 3 Oestradiol 14.5 4 2-Hydroxyoestrone 18.5 5 16a-Hydroxyoesirone and 23.5 16-oxo-oestradiol 6 Oestriol 46.5 -130- Fig. 3.11 Typical high-performance liquid chromatogram of oestrogens and oestrone metabolites 1 2 3 4 5 6 ml -131- 3.3 RESULTS. 3.3.1 Human urinary oestrogen profiles - effect of phenotype. Ten samples were analysed by gas chromatography by the method described in Section 3.2.8.1 which gives a chromatogram of approx 35 min duration allowing for a better separation of components than that described in Section 2.2.4.2 which had a chromatogram of approx 20 min duration. This was deemed necessary since oestrogens occur in a much lower concentration in urine than corticosteroid and progesterone metabolites (Chapter 2) and therefore larger volumes of urine must be extracted and concentrated for analysis. In this case therefore, large quantities of the aforementioned corticosteroid and progesterone metabolites may carry over in the assay and obscure the relatively sparcely excreted oestrogens. Accordingly, the chromatograms are “stretched out" in an attempt to observe the oestrogen peaks. Accordingly, the results described in this section are merely semi- quantitative and represent an attempt to uncover major differenes between phenotypes in urinary oestrogen excretion. Fig. 3.12 shows gas chromatographic traces for three of the samples analysed. With the exception of the one pregnancy sample (Subject 4, 20/52 pregnant, Table 3.4), the chromatograms were all remarkably similar. In the pregnancy sample (Fig. 3.12 top), a number of peaks are enhanced, almost certainly due to progesterone and its metabolites such as pregnanediol, an observation consistent with the known changes in urinary steroid excretion during pregnancy. However, and of more direct relevance here, the EM and PM traces were also similar and thus at present militate against the notion of urinary oestrogens being used as a rapid phenotyping test. F i g . 3.12 Urinary oestrogen gas chromatograms from -132- Table 3.4 Volunteers used for urinary oestrogen determination. Subject Sex Phenotype Phase"1" 1 F EM F L 2 F EM F L 3 F EM F 4 F EM F 20/52 pregnant 5 F PM F 6 F PM Post-menopausal 7 M EM - +Phase of menstrual cycle; F = follicular, L = luteal -134- In quantitative terms, pregnancy in Subject 4 (20 weeks) caused a rise in Eg from undetectable to 4.32 mg/24 h (normal range for 20 weeks pregnancy = 2-7 mg; Documenta Geigy). The four EM non-pregnant women, in both follicular and luteal phases excreted 6-154 pg/24 h Eg (mean + S.D. 57 + 56 pg/24 h). E^ was not detectable in any sample other than in pregnancy (900 pg/24 h). E^ and Eg were not detectable in the EM male sample, but a little Eg (56 pg/24 h) was observed. For the two PM females (5 and 6 in Table 3.4), neither E^ nor Eg was observed, but the Eg excretion was 15 pg/24 h for subject 5 (follicular phase) and 41 pg/24 h for subject 6 (post-menopausal). As far as the hydroxylated metabolites of E^ and Eg were concerned, 2-hydroxy-E^ and 2-methoxy-E^ were observed in similar amounts in all samples. Non-pregnant EM women excreted 36-112 pg/24 h 2-methoxy-E^ and 72-308 pg/24 h 2-hydroxy-E^. These figures are the same order of magnitude as tnose given by Ball et _al_. (1975). 16a-Hydroxy-E^ and 16- oxo-Eg were found impossible to determine in the urine due to the presence of what is presumably pregnanediol (largest peak in chromatogram Fig. 3.12 top) which has a similar relative retention time (see Table 3.2). 3.3.2 Faecal and urinary oestrogen metabolite profiles after administration of labelled oestrone to DA and Lewis rats. The failure of human studies to show any clear association between the genetic polymorphism of debrisoquine 4-hydroxylation and oestrogen excretion is clear. In order to observe the variation, if any, in the metabolism, fate or urinary and faecal excretion of some oestrogens in rat after administration of oestrone, it was decided to carry out a study on DA and Lewis rats with 14C-labelled oestrone at various doses and routes of administration. Table 3.5 shows the % dose recovered in Table 3.5 % Dose recovered by DA and Lewis rats as in urine and faeces after administration of 20 mg (+ 2 pCi) oestrone p. o. Time period (days) Animal 0i-l ]L-2 2-■3 3i-4 0-4 Lewi s 1 4.2 (30.0) 7.2 (20.0) 2.9 (8.8) 1.2 (3.3) 15.5 (62.1) 2 6.7 (24.6) 11.4 (21.3) 5.8 (11.6) 1.6 (4.4) 25.5 (61.9) DA 1 3.4 (3.6) 7.0 (30.9) 3.3 (22.2) 0.9 (2.0) 14.6 (58.7) 2 4.4 (26.0) 5.7 (34.5) 1.3 (7.2) 1.1 (1.8) 12.5 (69.5) Figures in parentheses represent faecal data -136- urine and faeces in the two strains after administration of a dose of labelled oestrone (20 mg p.o.). The total recovery of in urine after four days was very low in both strains, about 15.5 - 25.5% and 12.5 - 14.6% for Lewis and DA rats respectively. At the same time, the recovery in faeces was about 60% in both strains. In order to investigate whether or not this low urinary recovery was due to incomplete gastrointestinal absorption, it was decided to give the same dose i.p. to give a better chance of absorption. After this dose the recovery in DA urine was significantly higher (10.2 + 3.4%) than Lewis urine (3.0 + 1.4%; 2p < 0.01) (Table 3.6). Additionally, DA rats recovered a higher dose (5.3 + 1.6%) than Lewis (1.4 + 0.5%; 2p < 0.01) in the first day urine. Moreover, no significant difference in recovery throughout the second, third or fourth days was observed (Table 3.6). The total dose recovered in faeces was 37.5 + 10.4% and 31.5 + 9.8% for Lewis and DA respectively. Table 3.7 shows the % dose recovered by two each of DA and Lewis rats after a low dose of 54 pg (10 pCi) oestrone i.p. The recovery in urine and faeces follows the same pattern as in Tables 3.5 and 3.6, where a higher recovery of the dose was seen in faeces than urine. No significant interstrain difference, neither in the total recovery of both urine and faeces nor in the first day urine following 14 administration of [ C]-oestrone. At this lower dose in Lewis rats, the recovery in urine (5.1, 10.7%, 0-4 days) was higher than that seen at the higher dose of 20 mg (3.0 + 1.4%; Table 3.6). Similarly, faecal recoveries (57.1, 70.0%) were higher than for the 20 mg dose (37.5 + 10.4%). For DA rats, the urine recovery was similar (7.9, 11.6%) to that observed at the higher dose (10.2 + 3.4%), but the faecal recovery was greater (48.3, 55.7%) at the lower than the higher (31.6 + 9.7%) -137- Table 3.6 % Dose recovered by DA and Lewis rats as in urine and faeces after administration of 20 mg (+ 2 pCi) oestrone i. p. Time period (days) Animal 0-1 1-2 2-3 3-4 0-4 Lewis 1 1.1 (41.4) 0.3 (4.5) 0.4 (1.3) - (-) 1.8 (47.2) 2 1.0 (33.9) 0.6 (6.1) 0.4 (1.1) - (-) 2.0 (41.1) 3 2.0 (8.8) 2.2 (11.2) 0.4 (2.2) 0.1 (0.7) 4.7 (22.9) 4 1.7 (31.9) 1.4 (4.4) 0.2 (1.6) 0.2 (0.9) 3.5 (38.8) Mean + 1.4 (29 + 1.1 (6.6+ 0.4 (1.6+ 0.2 (0.8+ 3.0 (37.5+ SD +0.5 14.1) +0.9 3.2) +0.1 0.5) +0.1 0.1) +1.4 10.4) DA i 4.8 (25.4) 1.3 (13.7) 0.8 (3.0) - (-) 6.9 (42.1) 2 4.7 (23.6) 1.7 (9.8) 1.4 (3.0) - (-) 7.8 (36.4) 3 4.1 (13.9) 3.2 (9.8) 3.0 (1.7) 1.8 (2.4) 12.1 (27.8) 4 7.7 (8.0) 3.0 (6.7) 1.5 (3.4) 1.7 (1.8) 13.9 (19.9) Mean + 5.3 (17.7 2.3 (10.0+ 1.7 (2.8+ 1.8 (2.1+ 10.2 (31.6+ SD +1.6 +8.2) +0.9 2.9) +0.9 0.7) +0.1 0.4) +3.4 9.7) 2P< (n.s.) n.s. (n.s.) n.s. (n.s. ) 2P< (n.s.) 0.01 0.01 Figures in parentheses represent faecal data - means sample not collected n.s. means not significant -138- Table 3.7 % Dose recovered by DA and Lewis rats as in urine and faeces after administration of 54 pg (10 pCi) oestrone i.p. Time period (days) Animal 0-1 1-2 2-3 3-4 0-4 Lewis 1 4.0 (48.1) 0.9 (8.2) 0.2 (0.8) - (-) 5.1 (57.1) 2 8.8 (59.5) 1.4 (8.4) 0.3 (1.6) 0.2 (0.5) 10.7 (70.0) DA 1 6.9 (41.4) 0.9 (6.3) 0.1 (0.6) - (-) 7.9 (48.3) 2 10.8 (52.3) 0.6 (2.7) 0.1 (0.5) 0.1 (0.2) 11.6 (55.7) Figures in parentheses represent faecal data - means sample not collected -139- dose. Accordingly, up to 80% of the administered dose could be accounted for as urinary and faecal metabolites after administration of 54 pg oestrone to rats. Either metabolism and excretion of oestrone is saturated in rats at a dose of 20 mg. i.p. or a significant proportion of the dose is sequestered by tissues, presumably within the endocrine system, and is thus not available for elimination. In spite of the significant urinary excretion difference described above, the majority of the dose is excreted in the faeces after oral administration. This is therefore likely to arise by biliary elimination of oestrone metabolites. Accordingly, a bile experiment was carried out (see following section 3.3.3) to examine the % recovery of oestrone after injection into a mesenteric vein. 3.3.3 Oestrogen profiles in bile of DA and Lewis rats after an intra- mesenteric venous dose of labelled oestrone. Two DA and two Lewis rats were used. Bile was collected hourly for between 6 and 8 h in the case of three rats and for only 2 1/4 h in the case of one animal which died during the investigation. A dose of 16 pg per animal was administered i.v. into a mesenteric vein after cannulation of the bile duct. The hourly elimination of in bile was determined by scintillation spectrometry. Fig. 3.13 shows that the bile flow does not differ between strains, being 647 and 524 mg h”^ for the two Lewis rats and 562 and 525 for the two DA rats. Fig. 3.14 shows the 14 rate of elimination of C into rat bile in each strain as semi- logarithmic plots. Lewis rat 2, which died after 2 1/4 h has not been included. The slopes of the terminal linear portions of these curves allowed direct calculation of the halt-life for biliary elimination of 14 C, which was 1.7 and 2.8 h for the DA rats and 4.6 h for the Lewis rat. -140- Fig. 3.13 Bile flow in DA and Lewis rats BILE (g) TIME (h) ------* Dfll e------© Dfl2 *------* LEWISI LEWIS2 -141- Fig. 3.14 The rate of elimination of U C into bile of DA and Lewis rats as semilogarithmic plots LOG DPM/h 7t TIME (h) *------* DAI ®------© DA2 LEWIS -142- The nature of the in acid-hydrolysed bile was examined by HPLC and scintillation spectrometry. Figs. 3.15 and 3.16 show the radiochromatograms so obtained for the 0-1 h bile from a Lewis and DA rat respectively. All chromatograms comprised two major peaks, the first corresponding to unchanged oestrone plus 2-methoxyoestrone plus oestradiol and the second to 2-hydroxyoestrone plus 16a-hydroxyoestrone plus 16-oxo-oestradiol. Since [14C]-oestrone was administered, it is likely that the metabolites will be simple hydroxylated derivatives of oestrone, in the 2- and 16-positions, especially in the 0-1 h bile samples. Comparison of the radiochromatograms (Figs. 3.15 and 3.16) with the elution characteristics of authentic standards monitored by UV (Table 3.3) strongly suggests that the majority of the second peak is 2- hydroxyoestrone. Calculation of the proportion of ^ C as this peak reveals as inter-strain differences. Lewis rat 0-1 h bile contained 31 14 and 36% C as "2-hydroxyoestrone" and the corresponding DA samples only 13 and 16%, a difference of 2- to 3-fold. 3.3.4 Sodium and potassium excretion and urine volume in Lewis and DA rats after oestrone administration. 14 It was observed from the experiments where rats were given C- labelled oestrone that after 20 mg oestrone administration to both Lewis and DA rats, both strains excreted a larger volume of urine in the first day post-dose. Furthermore, it was observed as shown in Table 3.8 that Lewis rats excreted significantly larger volume (15.3 + 7.0 ml) of urine 24 h after administration of oestrone than DA (11.8 + 4.7 ml; 2 p < 0.002). However, no interstrain significant difference in the daily urine volume, neither untreated nor in the 2nd and 3rd days following the injection (Table 3.8). Accordingly, it was decided to measure -143- Fig. 3.15 Frequency distribution histogram of the 0-1 h bile from a Lewis rat LEWIS 0 -lh BILE DPM X 10-3 -144- Fig. 3.16 Frequency distribution histogram of the 0-1 h bile from a DA rat DR 0 -lh BILE DPM x 10"3 -145- Table 3.8 Mean (+ S.D.) of urine volume, urinary sodium and potassium excreti on before and after administration of high dose oestrone i,p. in Lewis and DA rats. Urine Vol (ml) Day 0 1 2 3 Lewi s 3.9 + 3.5 15.3 + 7.0 3.4 + 1.5 2.6 + 2.4 DA 2.7 + 2.5 11.8 + 4.7 3.4 + 3.0 4.2 + 4.0 n.s. 2p < 0.002 n.s. n.s. Na+ excretion 0 1 2 3 Lewi s 509 + 160 1213 + 255 378 + 143 217 + 64 DA 429 + 171 880 + 76 400 + 192 280 + 127 n.s. 2p < 0.04 n.s. n.s. K+ excretion 0 1 2 3 Lewi s 873 + 293 2047 + 267 883 + 183 486 +150 DA 720 + 285 1490 + 98 788 + 392 474 + 201 n.s. 2p < 0.01 n.s. n.s. n.s. means not significant (2P > 0.05) -146- urinary electrolyte excretion in Lewis and DA rats. Six DA and six Lewis rats were used. Three days of normal 24 h urine were collected, then 20 mg oestrone in corn oil (i.p.) was administered, and the 24 h urine for three days was collected. The urine volume was measured and the urine analysed for Na+ and K+ concentration by the Medical Unit Laboratory. The results show a high sodium and potassium excretion following 24 h of oestrone dose (Table 3.8), Lewis rats excreted significantly higher sodium (1213 + 255 pmol/24 h) and potassium (2047 + 267 pmol/24 h) than DA rats (880 + 76; 2 p < 0.04 and 1490 + 98; 2 p < 0.01), for sodium and potassium respectively. Again, there was no significant interstrain difference in the days following the first day. 3.3.5 Administration of 2-hydroxyoestrone and 16a-hydroxyoestrone to Lewis and DA rats. It was observed as a result of administration of 20 mg oestrone to the rats, that che urine volume 24 h after administration of the dose was increased. It was decided to give the metabolites, 2-hydroxy oestrone (25 mg kg"'L) and 16a-hydroxyoestrone (25 mg kg’^) to each of three DA and three Lewis rats. The 0-24 h urines was collected and the undiluted urine volume measured. The results showed no significant increase in urine volumes. 2-Hydroxyoestrone produced a 6-15 ml urine volume in Lewis rats and a 12-13 ml diuresis in DA rats in 0-24 h. 16a- Hydroxyoestrone produced 4-18 ml and 13-24 ml urine volumes in Lewis and DA rats respectively. Whilst these volumes are greater than control untreated volumes for these rat strains (see Table 3.8), no interstrain difference in response to these oestrone metabolites was observed. It is therefore concluded that the greater diuresis observed in Lewis rats after oestrone administration (20 mg i.p., section 3.3.4) is due to an effect of oestrone itself or a metabolite other than 2-hydroxy- or 16a- hydroxy-oestrone. 3.4 DISCUSSION. Various investigations described in this Chapter have demonstrated that there is no simple relationship between oestrogen metabolite excretion and the genetic polymorphism of debrisoquine 4-hydroxylation. Accordingly, it will not apparently be possible to phenotype subjects based upon urinary oestrogen profiles. Evidence for the foregoing statement comes from various results obtained. Firstly, investigations were made on the urine of subjects of known phenotype. Unlike the measurement of corticosteroid and progesterone metabolites which are relatively abundant in human urine (Chapter 2), where a population of phenotyped subjects was studied, only a small phenotyped panel of seven subjects was investigated for oestrogen excretion. This encompassed one male and six females of which one was post-menopausal and PM, another pre-menopausal and PM, one was studied both pregnant and non-pregnant, and the remaining were non-pregnant and EM, studied in both luteal and follicular phases of the menstrual cycle In this way, various endocrinological states could be studied together with the effect of phenotype. For a phenotyping test based upon oestrogen excretion to be useful, major differences between EM and PM persons, preferably the complete absence of a metabolite, would be required. The gas chromatographic assay employed measured E^, E^ and E together with several 2- and 16-oxygenated metabolites in urine. Oestrogens are usually measured as their conjugates in plasma by radio immunoassay (RIA) but, because urine analysis was required, for which RIA does not exist, unconjugated (total) levels were required and metabolites were to be determined for which there are no commercial antibodies, a gas chromatographic assay was used. This was not -148- completely satisfactory, due to the low levels of oestrogen excreted in human urine, but it was felt that if a major inter-phenotype difference occurred, this assay would pick it up. No such difference was seen. Secondly, ^C-labelled oestrogens, which could not be administered to human subjects, were given to rats and an HPLC separation of urinary metabolites combined with scintillation spectrometric analysis was employed. As the results showed, only biliary levels represented a significant excretory fraction of the administered doses. A number of as yet unexplained and interesting differences between strains (phenotypes?) were observed. After 20 mg [*4C]-oestrorie (i.p.), the 14 recovery of C in urine in day 1 was about four times greater (5.3 + 1.6%) in DA than in Lewis (1.4 + 0.5%) which was highly significant (2p < 0.01, see Table 3.5). Additionally, the 0-4 day excretion was also highly different between strains. Rates of excretion in DA and Lewis 14 bile for [ C]-oestrone did not differ, but the fraction of polar metabolites in the first hour bile was significantly greater in Lewis than in DA (see Figs. 3.15, 3.16). Finally, major differences in urinary Na+, K+ and water excretion after oestrone administration was observed (section 3.3.4). Whether or not these findings have anything to do with polymorphic oxidation is not known. It is possible that they represent other interstrain differences. The following represents a fuller discussion of the results obtained. It has been shown in non-pregnant women samples that the oestrogen excreted in follicular phase was lower than luteal phase which is in agreement with the results observed by others (Brown, 1955; Knorr et a!., 1970; Kaplan & Hreshchyshyn, 1971). -149- The detection of 16a-hydroxyoestrone in urine lends support to the previous suggestion of Marrian et £]_. (1957b) that this compound may be formed from oestrone by 16a-hydroxylation and may be the metabolic intermediate in the hydration of oestrone to oestriol. It was reported that 16a-hydroxyoestrone may arise in the placenta by biosynthetic routes other than from oestrone and oestradiol (Ryan, 1959). The results described here are ax variance with the results observed by Gelbke et aj_. (1975) where no detectable amount of 2-hydroxyoestrone was observed in pregnant cases. It was observed, however, that a detectable amount of this metabolite could be seen throughout the menstrual cycle, which demonstrated that this hormone is an important product in the metabolism of oestrogens. Assuming that 2-hydroxy oestrone only arises from phenolic steroids, the predominant excretion of 2-substituted oestrogens during the menstrual cycle and not during pregnancy is an additional proof of the fact that during pregnancy the phenolic pathway is of minor importance for the biosynthesis of oestrogens (Ball et a1_., 1975). Generally, it has been agreed that the urinary oestrogen levels do not represent the actual production of these hormones, since most oestrogens are metabolized in the liver and may simply not appear in the urine. It would appear that determination of the circulating oestrogen concentration might represent a more accurate measurement of the ovarian function and also collection of blood samples is a more convenient method, particularly in studies involving the whole menstrual cycle. Although measurement of urinary oestrogens has not produced very profitable information of the aetiology of breast cancer, it should be considered to be of at least a value to determine whether the metabolism -150- of oestrogen may be related to the pathogenesis of breast cancer in humans. Unfortunately, the results from the urine oestrogen profile studies are so contradictory that no conclusions can be made. Since oestrogens are secreted in minute amounts, sensitive and accurate methods for identification and quantification of these compounds are essential for evaluation of a possible correlation of hormone levels with the state of the disease in breast cancer patients. The measurement of urinary oestrogens particularly in pre-menopausal women should be done by multiple urine collections to represent the whole cycle since variation of urinary oestrogen excretion occurs during the cycle. The observation that there is a reduction in oestriol excretion in women with breast cancer led Lemon et al_. (1966) to suggest that oestriol might play a role in the aetiology of breast cancer. These authors calculated the ratio of urinary oestriol to the sum of oestradiol and oestrone, and designated the ratio as the "oestriol quotient" (oestriol/oestrone + 173-oestradiol). It was reported that this quotient was greatly reduced in women with breast cancer as compared to that in non-cancerous women. This led to the suggestion that women with a low ratio of oestriol to other oestrogen fractions are at high risk for breast cancer. The first evidence of abnormal oestrogen metabolism in men with breast cancer was reported by Zumoff et al. (1966) and Heilman et a]_. (1967; 1971) who described a sharp decrease in the formation of 2-hydroxyoestrone and 2-methoxyoestrone, accompanied by a marked increase in oestriol formation. In contrast, in women with breast cancer, the urinary excretion of 2-hydroxyoestrone was only slightly lower, but oestriol excretion was again significantly -151- elevated. This finding seems to suggest that in breast cancer patients, the metabolism of 173-oestradiol favours the pathway of 16a- hydroxylation. The polar oestrogen metabolites are of special interest for several reasons: (1) They are produced in large amounts during pregnancy, but they decline rapidly after parturition and often become non-measurable in non-pregnant women. The exact physiological significance of the presence of these polar metabolites during pregnancy, however, is not known. (2) It is suggested that these oestrogens protect the foetus from the biological effects of potent oestrogens such as oestradiol and oestrone. Although the mechanism by which polar oestrogen might protect the foetus is not understood, their lack of uterotrophic activity and their capacity to bind oestrogen receptors strongly suggest that they are anti-oestrogenic. The concept of polar oestrogens mediating the effects of non-polar "carcinogenic" oestrogens is interesting, and it is important to consider other possibilities, particularly since the validity of the oestriol hypothesis has been severely questioned. Catecholoestrogens are important for the following reasons: (1) 2-Hydroxyoestrone causes an increase in the secretion of luteinizing hormone (Naftolin et a!., 1975); (2) It binds to the specific oestrogen receptor, but without significant uterotrophic activity (Martucci & Fishman, 1976); urinary excretion of 2-hydroxyoestrone during the whole menstrual cycle equals or even exceeds that of oestriol (Ball et al_., 1975); the ratio of 2-hydroxyoestrone to oestradiol is greatly decreased in obese women (Fishman et a]_., 1975). As far as the rat experiments are concerned, the high % of dose recovery in the 0-2 h of bile sample is supported by the evidence that synthetic oetrogens are extensively excreted in the bile of both humans and animals, almost exclusively as glucuronides or sulphate conjugates -1:52- (Fotherby, 1974a,b; Smith, 1974). These conjugates on reaching the gut, are hydrolysed by enzymes present in intestinal micro-organisms to liberate the unchanged drug which then is reabsorbed. This is the basis of the enterohepatic recirculation (Smith, 1974). The first report of the biliary excretion of a steroid hormone was that of Cantarow et al (1943) who observed that oestrogens are excreted in dog bile and moreover that these hormones undergo enterohepatic circulation. Because the relative high molecular weight of oestrogens and their glucuronide conjugates they are excreted in rat bile; since its threshold is much lower than that of human bile for excretion of relatively high molecular weight compounds. It was found in the result as the bile samples of the first hour analysed by HPLC a difference between Lewis and DA strain in 2- hydroxylaticr. of oestrone administered, whereas marked species variations occur in the extent of biliary excretion of some steroids (see Smith, 1974). The observation of 50% of the dose in rats in 4-6 h is an agreement with that mentioned (see Smith, 1974). It has been shown that in the rat, the 16a-hydroxylating pathway of oestradiol is relatively minor compared to that of 2-hydroxylation (Hobkirk, 1979). This supports the result observed with oestrone where 2-hydroxyoestrone was highly excreted among other metabolites. It was reported that C-16 and C-2 hydroxylations of oestrogens partially competitive in nature (Fishman et al_., 1970). Previous investigations have shown the predominance of 2-0- over 3-£-methylation of catechol- oestrogens in vitro and in vivo (Gelbke et _al_., 1977). 2-Hydroxylation is also the predominent pathway of ethynyloestradiol metabolism in the rat (Maggs et aj_., 1982; 1983a) and result in irreversible binding of steroid to hepatic protein in vivo (Maggs et ^1_., 1983b). -153- Catechol oestrogens are deactivated in vivo by catechol-0-methylation (Ball et_ aj_., 1972; Stramentinoli _et a]_., 1981) and by formation of thio-ether adducts with glutathione (Elce & Harris, 1971). Any 2- hydroxyethynyloestradiol escaping into the blood would be rapidly methylated by erythrocyte catechol-0-methyltransferase, for which catechol oestrogens are known to be substrates (Bates et al_., 1977; Merriam et_ aj_, 1980). The possibility that 16a-hydroxyoestrone is readily formed from 16-oxo-oestradiol either directly, or via the triols, is unlikely in view of the studies made by Levitz et al_. (1958). Oestrogen-2-hydroxylase activity has been reported previously in the human placenta (Fishman & Dixon, 1967), human neoplastic breast tissue (Hoffman et ^1_., 1979), rat brain (Sesame et al_., 1977; Ball et al., 19/8; Fishman & Norton, 1975), and human foetal brain and pituitary gland (Fishman et_ a1_., 1976). Oestrogen receptors have been shown to be present in numerous tissues, including brain (Eisenfeld & Axelrod, 1965), pituitary gland (Eisenfeld & Axelrod, 1965), liver (Eisenfeld et al., 1976), heart (Stumpf et ^1_., 1977), lung and placenta (Pasqualini et^jak, 1976) and kidney (DeVries et 1972). Since these tissues are able to convert oestrogen to the catechol derivative in vitro, it is possible that 2-hydroxyoestrogens are involved in modulating the effect of oestrogen in these various target organs. Moreover, microsomal enzymes can bind catecholoestrogens and convert them into reactive, possibly toxic, intermediates (Nelson et aj_., 1976). Unlike some other microsomal P-450-dependent enzymes, oestrogen-2-hydroxylase activity in brain and liver could not be induced by 3-methylcholanthrene, phenobarbitone or testosterone. The female liver has significantly less activity than the male, a common dimorphism in hepatic steroid -154- metabolizing enzymes (Forchielli & Dorffman, 1956; Kuntzman et al., 1964; Einarsson et aj_., 1973). Effects of oestrogens on renal salt, water excretion and volume homeostasis, both in pregnancy and during the menstrual cycle, were described during the 1930s and the 1940s. Determination of electrolyte concentrations in rat urines was adjusted when it was observed that some rats specially Lewis strain, excreted a large 24 h urine volume after high dose of oestrone i.p. The effects of oestrogens on sodium excretion in rats is somewhat controversial. Some investigators have observed a decrease in sodium excretion in rats treated with oestradiol (DeVries et a]_.9 1972), others have noted that the hormone has little (Deming & Luetscher, 1950) or no (Dorfman, 1949; Simpson & Tait, 1952) antinatriuretic action. More studies have shown that oestrogen causes a decrease in food consumption, which may be primarily responsible for the decrease in sodium excretion (Thornborough & Passo, 1975). It has been shown in the Results that increased sodium and potassium excretion occurred in the first day after giving oestrone in both strains of rat, followed by a decrease in sodium and potassium excretion over the ensuing days. This is in agreement with the report of Katz & Kappas (1967) in which they mentioned that extremely high doses of oestradiol (20 mg/day) were initially natriuretic in human, and then salt retention occurred over the ensuing nine to ten days of treatment. The mechanism by which oestrogens reduce the rate of sodium and potassium excretion has been the subject of debate. It has been suggested that oestrogens may affect the filtered load of sodium by altering glomerular filtration rate or may directly increase the sodium reabsorptive activity of the renal tubules. Otherwise, an indirect action via the -155- adrenal mineralocorticoids or some other salt-retaining hormones has been speculated. In other words, the absence of any significant effect on urinary potassium excretion in dogs (Thorn & Engel, 1938) or in man (Preedy & Aitkin, 1956) suggested that the action may not be via the adrenals. Moreover, Thorn & Engel (1938) found that adrenalectomy did not affect the salt-retaining effect of oestrogen in their dogs. The action may therefore be directly in the kidney. Since it has been shown (Dean ejt aj_., 1945) that administered oestradiol does not affect the renal clearances of mannitol or of para-amino-hippuric acid, a direct action on the renal tubules may be involved. The results are at variance with the study of Johnson ^t ^1_. (1972; 1976) in the dog, who most strongly suggest a direct renal antinatriuretic effect of oestrogen. Hinsull & Crocker (1970) stated that sodium and water retention occurs during oestrus, but mineralocorticoid secretion also increases in the rat with oestrus. Nocenti & Cizek (1964) observed that neither the excretion of sodium nor potassium is different in stiIbesterol-treated rats when compared to controls. In summary, therefore, it appears that oestrogens possess antinatriuretic properties both in dog and human, but may have no such effect in the rat. It can be concluded from the results observed in both man and rat that there is no difference between EM and PM phenotypes on one hand and DA and Lewis rats on the other in oestrogen disposition and thus the non-invasive phenotyping test remains elusive. -156- CHAPTER FOUR EFFECT OF POLYMORPHIC OXIDATION ON SERUM CHOLESTEROL IN MAN AND RAT AND CHOLESTEROL 7a-HYDR0XYLATI0N IN THE RAT -157- 4.1 INTRODUCTION Cholesterol, a vital component of living tissues, performs important functions as a structural constituent of most biological membranes and as the direct precursor of a number of essential vitamins, steroid hormones and bile acids (Turley & Dietschy, 1982). The content of sterols in various tissues varies from about 0.5 g/kg (muscle) to 15 g/kg (brain) but averages approximately 1.4 g/kg tissue for the body as a whole (Turley & Dietschy, 1982). 4.1.1 Cholesterol biosynthesis. Generally, cholesterol can be obtained from the environment through the absorption of dietary cholesterol or synthesized from acetyl CoA within the body. An early and important guide concerning the synthesis of cholesterol came from the work of Konrad Bloch in the 1940's. In fact, all twenty seven carbon atoms of cholesterol are derived from acetyl CoA. A pathway for the synthesis of cholesterol from acetate can be outlined (Stryer, 1981) as follows: Acetate------► Mevalonate------► Isopentenyl pyrophosphate------► Squalene------►Cholesterol The major site of cholesterol synthesis in mammals is the liver; appreciable amounts are also formed by the intestine and 800 mg of cholesterol per day can be synthesized by an adult on a low-cholesterol diet (Stryer, 1981). In man, the rate of cholesterol synthesis is 9 mg/day/kg body weight, in the rat is 118 mg/day/kg body weight and the rate of absorption of cholesterol in man is 2-4 mg/day/kg body weight. For the rat, this figure is 220 mg/day/kg body weight (Turley & Dietschy, 1982). Fig. 4.1 shows the biosynthesis together with the utilization of cholesterol in rat liver (Scallen & Sanghvi, 1983). Fig. 4.1 Key steps involved in cholesterol metabolism in the liver. Cholesteryl esters ▲ ACAT* Plasma HMG.CoA (EC 2.3.1.26) lipoproteins reductase Acetyl.CoA -►HMG.CoA------► Mevalonate -►Cholesterol <■ --- Dietary and plasma cholesterol * ACAT = Acyl-CoA-cholesterol acyl transferase -159- 4.1.2 Metabolism of cholesterol in vivo. Dietary cholesterol reduces the activity and the amount of 3-hydroxy- 3-methylglutaryl CoA (HMG-CoA) reductase, which thereby inhibits the de novo synthesis of cholesterol. The LDL (low density lipoprotein) receptor is itself subject to feedback regulation. Pregnenolone is formed from cholesterol by the removal of a C-6 unit from the side chain of cholesterol, which is hydroxylated at C-20 and then at C-22, followed by the cleavage of the bond between C-20 and C-22. The latter reaction is catalysed by desmolase. All these reactions utilize NADPH and 0^. Cholesterol 20a,22-Dihydroxycholesterol Pregnenolone ACTH, a polypeptide synthesized by the anterior pituitary gland, stimulates the conversion of cholesterol into pregnenolone, which is the precursor of all steroid hormones. Cholesterol is also the precursor of vitamin D, which plays an essential role in the control of calcium and phosphorus metabolism. Cholesterol is excreted from the body either as the unaltered molecule or after biochemical modification to other sterol products, such as bile -160- acids and steroid hormones. Bile salts are also the major breakdown products of cholesterol. Cholesterol is converted into trihydroxy coprostanoate and then into cholyl CoA, the activated intermediate in the synthesis of most bile salts. In a few species, and in particular in man, subtle imbalances develop that can lead to elevation in circulating levels of plasma cholesterol or to excessive secretion of cholesterol into bile. In the first instance, this metabolic abnormality may lead to cholesteryl ester accumulation in cells within the walls of arteries and produce clinically-apparent atherosclerotic disease. In the second instance, the bile may become supersaturated with sterol, leading to the precipitation of cholesterol and, ultimately, to clinically-apparent cholelithi asis. Cholesterol and other lipids are transported in body fluids by a series of lipoproteins. Most of the cholesterol in LDL is esterified with linoleate, a polyunsaturated fatty acid. The role of LDL is to transport cholesterol to peripheral tissues and regulate de novo cholesterol synthesis at these sites. HDL (high density lipoprotein), which is synthesized by the liver, is rich in phospholipids and cholesterol. One role of HDL is to transport cholesterol from peripheral tissues to the liver. A number of different enzyme inducers have different effects on lipids, for example, phenobarbitone raises both HDL and LDL cholesterol (Durrington, 1979), while alcohol raises HDL and lowers LDL cholesterol (Castelli et al_., 1977) as does clofibrate (Lehtonen & Viikari, 1979), which has been shown to induce a very specific form of cytochrome P-450 (Parker & Orton, 1980). In 1972, insecticide workers exposed to polychlorinated hydrocarbons were noted to have raised HDL cholesterol -161- levels, although LDL levels were not thought to be changed (Carlson & Kolmodin-Hedman, 1972). 4.1.3 Metabolism of cholesterol in vitro. Studies in vitro from several laboratories (Mendelsohn et £l_., 1965; Danielsson & Einarsson, 1964; Mitton & Boyd, 1965) have shown the conversion of cholesterol into 7a-hydroxycholesterol by subcellular fractions containing microsomes. The identification of endogenous 7a- hydroxycholesterol present in liver microsomal preparations and its increased concentration in the microsomal fractions from the livers of cholestyramine-treated rats provided additional evidence that 7a- hydroxycholesterol is an intermediate in cholesterol catabolism (Mitropoulos et al_., 1972). Cholesterol 7a-hydroxylase [EC 1.14.13.17] belongs to a group of monooxygenases linked to the microsomal cytochrome P-450 oxygenases (Scholan & Boyd, 1968; Wada et aj_., 1968) requiring NADPH and molecular oxygen for activity. This is considered the rate limiting enzyme in the conversion of cholesterol to bile acids (Myant & Mitropoulos, 1977). For optimal catalytic efficiency, hydroxylase requires the presence of thiol protective agents, EDTA and nicotinamide (Scholan & Boyd, 1968; Mitropoulos jet al_., 1972; Van Cantfort & Gielen, 1975). The presence of these reagents prevents the 1ipoperoxidative action on cholesterol and hence minimizes the non-enzymatic formation of 7-oxocholesterol, 7a- and 76-hydroxycholesterol, and cholestane-3B,5a,6B-triol. Since 7- oxocholesterol, 73-hydroxycholesterol and cholestane-3S,5a,6B-triol are not converted into normal bile acids in vivo or in vitro, they presumably have no physiological significance. Changes in physiological conditions and exposure to various -162- biochemicals can exert either an inductive or inhibitory effect on the amount and the catalytic efficiency of the enzyme. Spence & Gaylor (1977) noted that cholesterol 7a-hydroxylase activity may be activated by a non-catalytic cytosolic protein and this is confirmed by Kowk et al. (1981). The enzyme induced by feeding the animals the bile-salt- sequestering agent, cholestyramine, is depressed by feeding sodium cholate and not affected by giving cholesterol or sodium phenobarbitone in addition to cholestyramine (Botham & Boyd, 1979). The activity of the enzyme is decreased by fasting (Myant & Mitropoulos, 1977), ethanol feeding (Lakshmanan & Veech, 1977) and bile acid administration. Furthermore, rat pups exposed to a high cholesterol, high fat diet during lactation had increased levels of 7a-hydroxylase (Naseem et al., 1980). Steroid hormones are known to induce certain hepatic enzymes by binding with high affinity to cytoplasmic receptor proteins (Rousseau et al., 1972), which can then enter the nucleus and interact with the genome in a way which affects the transcriptional process (Lippman & Thompson, 1973). It was shown that pregnenolone-16a-carbonitrile increased the activity by 50%, 16-dehydropregnenolone by 80% and phenobarbitone by 200%. Clofibrate did not affect the intensity of cholesterol hydroxylation (Manankova & Salgavik, 1976). It has been noted that this enzyme is maximally activated, 2-3 fold, by a 15-20 min preincubation (Kwok et_ ^J_., 1981; Goodwin & Margolis, 1978). This activation is probably associated with desensitization of 2+ ATP + Mg inactivation (Rajan et al_., 1978). Longer preincubation time would lead to enhanced conversion of cholesterol to cholesteryl esters in addition to the undesirable effect of increased formation of -1 6 3 - cholestane-3$,5a,6$-triol. Otherwise, preincubation had little effect on the formation of 7&-hydroxycholesterol and 7-oxocholesterol (Kwok et aj_., 1981). A number of studies have shown that ageing affects bile acid metabolism (Story & Kritchevsky, 1974; Li et ak, 1979). Story & Kritchevsky (1974) found that cholesterol 7a-hydroxylase activity decreased with age in rats. The presence of a circadian rhythm running in parallel to cholesterol 7a-hydroxylase and HMG-CoA reductase is well established (Rodwell et al., 1976; Danielsson, 1972; Myant & Mitropoulos, 1977) and homeostasis of these two enzymes is crucial to the balance of cholesterol and bile acid metabolism. This enzyme appeared to saturate when the cholesterol concentration reached 70 pM (Nicolau et aj_., 1974). It appears probable that upon increasing the amount of cholesterol in the acetone solution added to the microsomes, precipitation starts to occur. The conclusion that substrate saturation is probably not achievable has also been drawn by Balasubramaniam et al_. (1973). The experiments of Ahlsten et al_. (1981) showed that the cholesterol 7a- hydroxylase system from rat liver and from human liver have very similar properties, it is hoped that the enzyme assay described in man (Nicolau et_£l_., 1974) will be useful in estimating relative rates of bile acid synthesis in patients with disorders of sterol metabolism and in assessing the effect of treatment. They were able to demonstrate differences in cholesterol 7a-hydroxylase activity among subjects with different disorders of sterol metabolism. For example, enzyme activity was about 50% lower in a group of six patients with cholesterol gallstones in comparison with a control group of five patients with peptic ulcer. This suggests that cholesterol cholelithiasis is -164- associated with a relative reduction of bile acid synthesis and, as previously reported, with a relative increase of hepatic cholesterol synthesis (Nicolau et a]_., 1974). 4.1.4 The involvement of cytochrome P-450 in cholesterol 7a- hydroxylati on. The inhibition of 7a-hydroxycholesterol formation in incubations with CO in tne gas phase (Shefer et £l_., 1968; Scholan & Boyd, 1968; Wada et al_., 1969; Gielen, 1969; Mitropoulos et ^1_., 1972) implicates cytochrome P-450 as the terminal oxidase for cholesterol 7a-hydroxylase. Several studies in the past have indicated that there is no positive correlation with the fluctuations and/or modulations of cytochrome P-450 content and cholesterol 7a-hydroxylase activity (Wada et a1_., 1969; Boyd jit ^1_., 1969; Brown & Boyd, 1974; Bal asubramani am & Mi tropoulos, 1975; Atkin et_ £l_., 1972), but it is generally concluded that cytochrome P-450 is involved in the 7a-hydroxylation of cholesterol (Wadaet aj_., 1968, 1969; Boyd et £]_., 1973). The cholesterol 7a-hydroxylase system has several properties which are different from those of other cytochrome P-450 dependent hydroxylases in liver microsomes (Danielsson & Sjovall, 1975; Einarsson & Johansson, 1968). The 7a-hydroxylase activity is not stimulated by treatment with the common monooxygenase inducers, but is stimulated several-fold by biliary drainage or by feeding cholestyramine, a bile acid binding anion exchanger. It exhibits a circadian rhythm and it is influenced by certain hormones (Danielsson & Sjovall, 1975; Gielen et al., 1975). It is increased by glucocorticoids (Mitropoulos & Balasubramaniam, 1976), thyroid hormones (Myant & Mitropoulos, 1977) and 173-oestradiol (Ferreri & Naito, 1977). Another property, also -165- indieating the involvement of a specific cytochrome P-450 in the 7a- hydroxylation of cholesterol, is the short half-life of a hydroxylase activity. The half-life has been calculated to be 2-3 h in rat liver microsomes whereas that of drug-induced cytochrome P-450 is much longer, about 40 h (Einarsson & Johansson, 1968; Levin et al_., 1970; Ernster & Orrenius, 1965). It appears that cholesterol 7a-hydroxylase is highly regulated and plays an integral role in a metabolic sequence of physiological importance (Shefer et al_., 1968; Danielsson et £]_., 1967; Einarsson, 1968). In this respect, cholesterol 7a-hydroxylation is different from most other known microsomal cytochrome P-450-dependent reactions. Mellon et j^_. (1978) and Wada et aj_. (1969) found that 3-methyl- cholanthrene (3-MC) administration causes a reduction in cholesterol 7a- hydroxylase activity. Ir. contrast, a study by Brown & Boyd (1974) demonstrated no alteration in 7a-hydroxylase activity after prior treatment with 3-MC. These results may be interpreted to mean that the newly formed cytochrome P-448 cannot effectively act as the terminal oxidase for the cholesterol 7a-hydroxylase enzyme system. 4.1.5 Deficiency of the LDL receptor leads to hypercholesterolaemia and premature atherosclerosis. The significance of the LDL receptor is distinguished by studies of familial hypercholesterolaemia. Goldstein et aj_. (1973) gave the name "familial combined hyper!ipidaemia" to the most common genetic form of hyper!ipidaemia recognised in a study of survivors of myocardial infarction, in whom was observed that both cholesterol and triglycerides are elevated in the blood. In the other type, known as hyperlipo- proteinaemia II, in which the blood shows an increase in beta- lipoproteins on a normal diet. In this type, serum cholesterol is increased where phospholipids together with triglycerides stay within normal limits. Most of the biochemical variants can be considered dominant traits, although their predominance makes appearance of homozygotes or genetic compounds relatively frequent. The latter individuals will usually be more severely affected than heterozygotes and their presence in families may suggest autosomal recessive inheritance. Hyperlipoproteinaemia III is a rare phenotype, the affected individuals show increased plasma cholesterol and the presence of abnormal lipoprotein called beta-VLDL. VLDL in general is markedly increased while LDL is decreased. Development of the phenotype depends on age, those under 30 years old are rarely affected. Hyperlipo proteinaemia IV in which the patient shows increased plasma VLDL, plasma triglycerides are increased, but plasma cholesterol and phospholipids are commonly in normal limits. Precocious atherosclerosis, abnormal glucose tolerance, and atheroeruptive xanthoma may arise. The disorder is heterogeneous and the phenotype influenced by environmental factors like carbohydrate and ethanol consumption. Other conditions causing this type are uraemia, hypopituitarism, contraceptive steroids and glycogen storage disease (see McKusick, 1983). Schreibman et a]_. (1969) described hyperprebetalipoproteinaemia behaving as an autosomal dominant with reduced penetrance. Precocious atherosclerosis was not observed in the affected persons. Goldman et_ a1_. (1972) expressed the association of rheumatic manifestations. The final type which is hyperlipoprotein aemia V is characterisd by increased amounts of chylomicrons and VLDL and decreased LDL and HDL in the plasma after a fast. The conditions which cause this phenotype are numerous, including insulin-dependent diabetes mellitus, contraceptive steroids, alcohol abuse and glycogen -167- storage disease (see McKusick, 1983). From study of a 3-generation family, Francois et a[. (1977) concluded that the mode of inheritance was indeterminate. 4.1.6 Aims of the study. The object of the work described in this Chapter is to investigate certain aspects of cholesterol disposition in strains of rat including the drug-oxidation-deficient DA strain, and in a panel of phenotyped subjects. In pursuit of this, a study of 7a-hydroxylation of cholesterol was undertaken in 19,000 g supernatant of rat liver from two phenotypically distinct rat strains; female DA and Lewis. In addition, total serum cholesterol concentrations have been determined for both rats and phenotyped human subjects in an attempt to uncover a relationship between cholesterol disposition and the debrisoquine 4-hydroxylation polymorphism. Should such a relationship exist, it might provide a basis by which a non-invasive phenotyping test could be developed. -1 68 - 4.2 MATERIALS AND METHODS. 4.2.1 Materi als. Kits for the colorimetric determination of human and rat cholesterol in serum were obtained from Boehringer Mannheim and comprised a standard (solution 1) containing 200 mg/100 ml cholesterol in glacial acetic acid and a "colour reagent" (solution 2) which is composed of acetic acid (7 mM) and acetic anhydride (6.5 mM). This was purchased from the Boehringer Corporation (London) Ltd. [4-*4C]-Cholesterol (specific activity 58.4 mCi/mmol) was supplied by Amersham International Limited, Bucks. Cholesterol, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, nicotinamide adenine dinucleotide phosphate monosodium salt, (NADP+), phosphomolybdic acid in propan-2-ol and kits for determination of protein were obtained from Sigma Chemical Company, Poole. Cholesterol palmitate and cholesterol stearate were obtained from the Biochemistry Department. 7a-Hydroxycholesterol (5-cholesten- 3S,7a-diol; m.p. 181-181.5°C) and 76-hydroxycholesterol (5-cholesten- 33,70-diol; m.p. 176-179°C) were from Steraloids Ltd. Ethylenediamine- tetraacetic acid (EDTA), toluene, sodium chloride, cone, sulphuric acid, potassium dihydrogen phosphate, methanol, acetone, sucrose, potassium hydroxide and diethyl ether (all Analar grade) were obtained from BDH Chemicals Ltd. 4.2.2 Animals. Rats as shown in Table 4.1 and weighing 150-200 g were anaesthetized with ether and the blood (3 ml) was withdrawn from each by cardiac puncture, centrifuged at 2000 rpm for about 10 min, the serum was separated and stored at 4-5°C until analysed. -1 69- Table 4.1 Suppliers of rats used and some physical characteristics. Strai n Sex No Supplier Colour of: Eyes Hair DA F 9 Bantin & Kingman Black Brown M 5 Lewi s F 5 SMHMS Red White M 10 Fischer F 5 SMHMS Red White M 5 PVG F 5 Bantin & Kingman Black Brown M 5 F = female; M = male SMHMS = St. Mary's Hospital Medical School colony. 4.2.3 Human subjects. 4.2.3.1 Healthy subjects. Sixteen healthy volunteers from students and staff gave a 10 ml venous blood sample and the serum was separated and stored at 4-5°C until analysed. 4.2.3.2 Volunteers with "high" or "low" serum cholesterol. Eight volunteers from the Northwick Park Ischaemic Heart Disease Trial were investigated with the permission of Dr. T. Meade, Director, MRC Primary Health Care and Epidemiology Unit, Northwick Park. Each subject had a well defined plasma lipoprotein profile and was phenotyped with debrisoquine (see 2.2.4.1). -170- 4.2.4 Methods. 4.2.4.1 Determination of human/rat cholesterol by colorimetry. This is carried out by using the kits which include the addition of the colour reagent (solution 2, 2.5 ml) to three test tubes, the first containing distilled water (0.1 ml), the second containing the standard (solution 1, 0.1 ml) and the last contained the serum (0.1 ml) which correponds to blank, standard and sample, respectively. The tubes were mixed and incubated in water bath at 20-25°C for 5 min. To all mixtures, cone, sulphuric acid (0.5 ml) was then added carefully by pouring down the inside wall of the tubes, and immediately mixed thoroughly with constant cooling, after which they were left to stand in the water bath at 20-25°C for another 10 min. The solutions were poured into a dry 1 cm glass cuvette, in order to measure the absorbances of the sample and standard against the reagent blank in the spectro photometer (Pye Uni cam SP 30) at 578 nm. The principle of this reaction is the formation of a coloured complex by the addition of acetic anhydride and sulphuric acid to cholesterol. Not only serum as the sample material, but heparinized or EDTA- containing plasma can be used also. For determination of the concentration of cholesterol in the sample, the following equation was applied after the measurement of the absorbance (A) of the sample and the standard against the blank: A sample Concentration = 200 X ______[mg/dl] A standard A sample Concentration = 5.17 X ______[mmol/1] A standard -171- 4.2.4.2 Determination of the activity of cholesterol 7a-hydroxylase in the liver of DA and Lewis rats. 4.2.4.2.1 Preparation of liver cell fractions and incubations. For performing this procedure, the methods described by Mitropoulos & Balasubramaniam (1972) and Kupfer et al_. (1982) were adopted with some modification. Many techniques described to estimate the activity of cholesterol 7a-hydroxylase like mass-fragmentography, double-isotope derivative dilution procedures and single-isotope-incorporation techniques were beyond the experimental capability of this project. The rats were anaesthetized with ether and the livers were perfused with 40 ml of ice cold sucrose (0.25 M) through the portal vein to remove contaminating haemoglobin. The livers were chilled with ice for a few min, dried on filter papers and weighed, minced with a stainless steel mincer, homogenized with four volumes of ice cold 1 M potassium phosphate buffer (pH 7.4) containing nicotinamide (30 mM) and EDTA (1 mM) with six up-and-down strokes in a Dounce glass homogenizer. The homogenates were centrifuged at 9000 g for 20 min in order to remove mitochondria, cell debris and nuclei. The supernatant was centrifuged again at 19,000 g for 20 min at 4°C. The volume of the supernatant was then adjusted to 60 ml with buffer. The standard incubation mixture contained 3 ml of the fraction, 1 ml of buffer (pH 7.4) which contained nicotinamide (30 mM), EDTA (1 mM), NADP+ (1 mM), glucose-6- phosphate (10 mM) and 1 unit of glucose-6-phosphate dehydrogenase. Prior to addition of labelled substrate, a 10 min preincubation to utilize endogenous cholesterol was performed in every case, and then the substrate cholesterol, labelled or unlabelled, was added to the incubation mixture as a solution in acetone. The incubations were -172- carried out with air as the gas phase at 37°C with constant shaking for 2, 5 or 10 min at final cholesterol concentrations of 24-1000 pM and 0.072 pCi of [4-^C] cholesterol. The reactions were terminated by the addition of 1 ml of sulphuric acid (0.25 M). The mixtures were shaken vigorously in separating funnels and extracted with diethyl ether (3 X 40 ml). The organic layer was washed with 0.2 Vol of 0.9% sodium chloride and the phases allowed to separate. The aqueous layer contained no radioactivity, therefore it was discarded. The upper etherial phase was evaporated in vacuo to dryness. This residue was used for t.l.c. analysis. 4.2.4.2.2 Determination of protein. In order to carry out the estimation of protein in the fraction, the method described by Lowry et al_. (1951) was followed, in which three test tubes labelled blank, standard and sample containing 0.1 ml distilled water, 0.1 ml standard protein solution (8%) and 0.1 ml of the homogenate solution respectively to which 5 ml of Biuret reagent was added. The tubes were mixed thoroughly and left to stand for 15 min. The optical density at 540 nm was measured after zeroing the spectro photometer with the blank. The following equation was used for the estimation of protein (g/100 ml): Optical density of sample Protein = ______X 8 Optical density of standard 4.2.4.2.3 Isolation of radioactive products by thin-layer chromatography. The residue obtained after the evaporation of the etherial layer was dissolved in 100 pi methanol and applied on Merck 5554 DC-Alufolien -173- Kieselgel TLC plates (B.D.H. Chemicals Ltd., Poole, Dorset) and eluted with diethyl ether at room temperature. At the same time, methanolic solutions of authentic standards, including cholesterol, 7a- hydroxycholesterol and 7B-hydroxycholesterol were chromatographed and visualised with 3.5% phosphomolybdic acid in propan-2-ol. Typical Rp values observed were: 7a-hydroxycholesterol (0.12), 7B-hydroxy- cholesterol (0.16), cholesterol (0.46) and cholesterol ester (0.82). The Rp values of cholesterol and its relevant derivatives are given in Table 4.2 for various solvents. 4.2.4.2.4 Radiochromatogram scanning. Thin-layer chromatograms were scanned to locate bands on a Packard Model 7201 radiochromatogram scanner. Fig. 4.2 shows a typical 14 radiochromatogram of C-labelled cholesterol incubated with rat liver S _ fraction, i y 4.2.4.2.5 Quantitation of radioactive peaks. Each radioactive peak was estimated by cutting up thin-layer plates, run from the extracted residue of each incubation, into bands (1 cm 14 wide) and counting the C content in plastic inserts within glass vials containing a triton-toluene scintillation cocktail (5 ml) of the following composition: Triton X-100 (Koch-Light Laboratories Limited), 33%; 2,5-diphenyloxazole (PPO; Fisons Scientific Apparatus Limited), 550 mg/100 ml; l,4-di-2-(5-phenyloxazoyl)-benzene (POPOP; Fisons Scientific Apparatus Limited), 10 mg/100 ml and toluene (May & Baker Limited), 66.6%. The vials were then counted in a liquid scintillation spectrometer operating in the external standard mode (Packard Model 3385), after sufficient time had passed for cooling of the samples in the spectrometer. Efficiency of counting was commonly 80-90%. Since -174- Table 4.2 Rp Values of cholesterol and its relevant derivatives in different solvent systems. Sol vent Cholesterol 78-hydroxy- 7a-hydroxy- Cholesterol (proporti on cholesterol1 cholesterol ester* by Vol) Ether (room 0.46 0.16 0.12 0.82 temp) Ether (1°C) 0.63 - 0.48 - Acetone 0.66 - 0.58 - Methanol 0.72 - 0.72 - Hexane 0.0 - 0.0 - Acetone/Hexane 0.68 - 0.55 - (3:1) Acetone/Hexane 0.62 - 0.38 - (3:2) Acetone/Hexane 0.55 - 0.31 - (3:4) Acetone/Hexane 0.70 - 0.48 - (1:2) Acetone/Hexane/ 0.50 - 0.11 - Ether (1:4:2) *Cholesterol ester : cholesterol palmitate or cholesterol stearate -175- Fig. 4.2 Typical radiochromatogram of j j ^ c ] - cholesterol incubated with rat liver S-l p fraction. 0 SF min -176- the specific radioactivity of the substrate was known (58.4 mCi/mmol), the radioactivity data could be expressed in terms of pmol of 7a- hydroxycholesterol formed per mg protein per minute. 4.3 RESULTS. 4.3.1 Serum cholesterol levels of different strains of rats. Serum cholesterol concentrations were evaluated in four different strains of rats, female DA rats which are phenotypically poor metabolizers and the other three strains, Lewis, Fischer and PVG, characterised as phenotypically extensive metabolizers (Al-Dabbagh et al., 1981). From the results shown in Table 4.3, it is obvious that female DA rats have elevated serum cholesterol concentrations above the extensive metabolizing strains studied. Male DA rats, however, which are phenotypically extensive metabolizers (Al-Dabbagh et al_., 1981) had serum cholesterol levels considerably lower than female DA rats. Statistical comparison of female DA and Lewis rats showed a highly significant difference (t = 9.8, P < 1 PPM) between the observed cholesterol concentrations. Female DA rat serum cholesterol was some 50% elevated above Lewis, PVG and male DA and about double that of Fischer rats. The serum cholesterol level was also measured in twenty Lewis X DA F^ hybrids, fifteen female and five male. Table 4.4 shows the serum cholesterol of each animal. From the histogram shown in Fig. 4.3, it is obvious that the DA female characteristic of high serum level of cholesterol was lost in these hybrids. In the F^ offspring of brother- sister matings among the F^ generation, twenty two animals were obtained, of which one died prior to metabolic investigation. Table 4.3 Serum cholesterol levels of rats of various strains. Serum cholesterol (pg ml”*) of strain Rat No. DA (F) DA (M) Lewis (F) Lewis (M) Fischer (F) Fischer (M) PVG (F) PVG (M) Phenotype PM EM EM EM EM EM EM EM 1 1150 747 766 593 624 495 784 764 2 1160 720 734 678 475 653 744 693 3 1110 818 755 568 604 495 714 804 4 1060 827 723 678 644 525 724 844 5 950 732 745 619 713 525 744 804 6 1000 619 7 1160 619 8 1120 551 9 1170 602 10 542 Mean + S.D. 1100+78 769+45 746+21 607+44 610+84 542+62 738+27 778+57 M = male; F = female. L- ’ LL Table 4.4 Serum cholesterol levels of generation hybrids. No. Serum cholesterol (pg ml~^) F M 1 925 520 2 766 555 3 687 529 4 846 511 5 722 564 6 907 7 749 8 670 9 731 10 819 11 696 12 837 13 749 14 749 15 775 Mean + S.D. 775 + 77 536 + 23 M = Male, F = Female FREQUENCY FREQUENCY FREQUENCY hybrids DA X Lewis various for cholesterol serum of histogram distribution Frequency 4.3 Fig. - 2 4 0.5 t 0.6 0.7 □ . 0.9 0.8 TOTAL CHOLESTEROL (mg/ml) , F = 2 HYBRID F2 = Fj x F, 1 □ 1.1 1.2 -6L L- -1 80- Add i ti on a 1 ly, eight animals were very small which was thought not to fulfil the requirement of the study. Table 4.5 shows the serum cholesterol levels, together with the metabolic ratios, of these animals. Some of these animals were white, some brown, some black and white in colour and some brown with a white belly. The metabolic ratio and the serum cholesterol did not correlate. The results presented in this investigation are at variance with the classical pattern of Mendelian inheritance of a recessive character. The above data illustrate that the serum cholesterol level which is high in female DA rats and low in female Lewis rats is not inherited in a simple Mendelian pattern. 4.3.2 Effect of 3-methylcholanthrene induction on the serum cholesterol in the rat. Female rats of DA and Lewis strains, weighing 175-200 g, were used. 3-Methyl chol anthrene (80 mg kg-'1’ in corn oil) was given to rats in a single i.p. dose 48 h before taking the serum for cholesterol determination. Corn oil was also given i.p. to control rats. It was shown from Table 4.6 that serum cholesterol level of control Lewis rats was high (1004 + 81) which might be related to the effect of corn oil only. In the same time, the means + S.D. of induced DA rats (1263 + 78) and Lewis rats (1236 + 121) are higher than the control (1147 + 64, 1004 + 81 for DA and Lewis respectively), although the difference was not statistically significant; moreover no inter-strain differences were found. 4.3.3 Relationship between oxidation phenotype and serum cholesterol in human. Sixteen human volunteers, seven PM and nine EM gave blood for serum -181 Table 4.5 Physical properties together with metabolic ratios and serum cholesterol levels of individual generation hybrids. No. Metabolic ratio Cholesterol level Coat colour (pg ml"1) 1 0.65 811 brown and white 2 0.16 1038 white 3 0.15 915 white 4 0.59 934 brown and white 5 1.00 1274 black and white 6 0.83 962 brown and white 7 1.93 849 brown and white 8 0.14 862 wh i te 9 0.2 912 brown with white belly 10 0.07 879 brown with white belly 11 0.22 962 brown with white belly 12 0.13 1163 brown with white belly 13 0.12 762 brown with white belly -182- Table 4.6 Effect of 3-methylcholanthrene induction on serum cholesterol. No. Rat Strain Serum cholesterol (pg m l ) 1 DA control 1192 2 DA control 1102 Mean + S.D. 1147 + 64 1 DA induced 1339 2 DA induced 1273 3 DA induced 1241 4 DA induced 1142 5 DA induced 1322 Mean + S.D. -1263 + 78 1 Lewis control 947 2 Lewis control 1061 Mean + S.D. 1004 + 81 1 Lewis induced 1192 2 Lewis induced 1265 3 Lewis induced 1371 4 Lewis induced 1053 5 Lewis induced 1298 Mean + S.D 1236 + 121 -183- cholesterol determination. Mean (+ S.D.) serum cholesterol for EM and PM subjects, age-adjusted to 20 years of age (see Table 4.7) was 197 + 49 and 161 + 23 mg dl”1. No statistical significance between phenotypes (t = 1.8, 2P > 0.1) in this respect was found. Eight volunteers from the Northwick Park Ischaemic Heart Disease Trial were studied, two of which had abnormally low serum cholesterol concentrations for their age (97 and 135 mg dl"1) and six who had abnormally high serum cholesterols (range 263-348 mg dl”1). All subjects were phenotyped with debrisoquine (see 2.2.4.1) and were found to be EM. Table 4.8 gives the cholesterol concentrations and the metabolic ratios for each of these volunteers and Fig. 4.4 shows the rank correlation between each of these variables. No significant correlation was observed (r = 0.397) between the cholesterol and s metabolic ratio ranks. Accordingly and in the absence of further data, it would appear that the allelomorphic locus controlling debrisoquine oxidation does not cootribute to the variation in serum cholesterol in man. However and because of the rat data, more extensive studies are required on this subject. 4.3.4 Determination of cholesterol 7a-hydroxylase activity in rat liver S-j^ fraction. It has been observed that optimal reaction rates are obtained when the pH of the system was 7.4 - 7.6 (Nicolau et aj_., 1974). The rate of formation of the 7a-hydroxycholesterol was linear with respect to incubation time during the first (30) minutes of incubation (Nicolau et al., 1974) and thus a reaction time of 10 min was chosen to assure optimal assay conditions. Radiochromatogram scans of reaction extracts showed three peaks corresponding to 7a-hydroxycholesterol (Rp q .12 in ether), cholesterol - 184- Table 4.7 Effect of oxidation phenotype on serum cholesterol in man. Serum cholesterol (mg dl"^) Subject Phenotype Age (y) Observed Age-adjusted* AS PM 20 150 150 AM PM 31 188 175 YS PM 20 154 154 KS PM 20 119 119 JD PM 19 188 189 CB PM 20 169 169 AG PM 20 173 173 Mean + S. D. 163 + 24 161 + 23 TE EM 20 108 108 PC EM 36 203 184 JR EM 29 175 164 MC EM 25 244 238 HH EM 35 273 255 SM EM 31 274 263 NO EM 30 216 204 HN EM 33 206 190 LW EM 33 184 168 Mean + S.D. 209 + 52 197 + 49 * Adjusted to 20 years, assumi ng serum cholesterol rises on average by 1.2 mg% per annum after 20 years of age (Documenta Geigy). -185- Table 4.8 Relationship between metabolic ratio and serum cholesterol in subjects with abnormally "high" or “low" cholesterol. i Subject Age (y) Metabolic Ratio Serum Cholesterol (mg dl x) MF 55 2.2 (2) 348 (1) RR 62 0.7 (3) 341 (2) HJ 57 0.4 (6.5) 337 (3) PR 45 0.5 (5) 333 (4) BD 52 4.2 (1) 329 (5) PS 54 0.6 (4) 263 (6) MM 46 0.2 (8) 135 (7) NA 39 0.4 (6.5) 97 (8) Numbers in parentheses are ranks. Rank No. 1 is the highest concentrati on. -186- Fig. 4.4 Correlation between cholesterol concentration rank and metabolic ratio rank in subjectswith abnormally “high" or "low" cholesterol 7- x 6-- x 'X L 2 5-- x cncr Qdo 4-- x I-LU CO LU _Jo 3*- X O2 2- X 1 -- x 0 H------h 0 1 METABOLIC RATIO (RANK) -187- (Rp 0.46) and cholesterol esters (Rp 0.82). The nature of this latter peak was confirmed by isolation of the band from t.l.c. plates by scraping off the silica, eluting the material with methanol and hydrolysing the ^C-labelled material with methanolic KOH. T.l.c. of the ether-extracted hydrolysate gave a single band on t.l.c. with Rp 0.44 corresponding to cholesterol. No hydroxylated products were observed, suggesting that esterification of cholesterol to fatty acid esters occurs in competition to 7a-hydroxylation rather than subsequent to it in rat liver S^ fractions. Additionally, it has been established that providing EDTA is present in the incubation buffer, 78-hydroxycholesterol, 7-oxocholesterol and cholestane-3B,5a,68-triol are repressed relative to 7a-hydroxylation (Mitropoulos & Balasubramaniam, 1972). Thus, enzyme kinetics could be constructed from the observed data both for 7a-hydroxylation and esterification of cholesterol. A typical velocity plot for 7a-hydroxylation is shown in Fig. 4.5. At low concentrations (24 pM), the velocities remained linear over the 10 min period. At higher concentrations (1000 pM), the reaction rates had saturated by 2 min. Accordingly the initial slopes (0-2 min) were taken as the velocities (V) at various cholesterol concentrations (S). Lineweaver-Burk plots of 1/V versus 1/S were constructed from these data to permit estimation of Km and Vmax for cholesterol 7a-hydroxylation in DA and Lewis rat liver fractions. 4.3.5 Derivation of Km and Vmax of the cholesterol 7a-hydroxylase system. The assays were carried out with different amounts of unlabelled cholesterol and [4-^C] cholesterol. The amount of product was -188- Fig. 4.5 A typical time course of cholesterol 7g-hydroxylose activity 7 1000 500 *M 200 *M 72 #M 24 *M -189- determined from the percentage conversion of [4-^C] cholesterol to 7a- hydroxy-[4-^C] cholesterol, presuming all the cholesterol was equilibrated with the ^C-labelled cholesterol. Figs. 4.6, 4.7 show Lineweaver-Burk representations of the Michaelis-Menten kinetics of cholesterol 7a-hydroxyl ation in seven female DA (PM phenotype) and seven female Lewis (EM phenotype) rats respectively. No inter-strain differences in either Km or Vmax emerged as statistically significant. The mean (+ S.D.) Km for Lewis and DA rat liver fraction was 186 + 105 and 145+63 respectively and mean Vmax for Lewis and DA rats was 60 + 34 and 102 + 164 respectively. Similarly, rates of cholesterol esterification showed no apparent inter-strain differences. As Figs. 4.8, 4.9 show, Lineweaver-Burk plots could be constructed for this reaction. Mean Km for Lewis and DA was 484 + 485 and 692 + 573 and mean Vmax for Lewis and DA was 51 + 59 and 95 + 74 respectively. 4.3.6 The effect of 3-methylcholanthrene induction on the activity of cholesterol Jg-hydroxylase. Four rats weighing 175-200 g, two DA and two Lewis, were used. 3-Methylcholanthrene (3-MC; 80 mg kg“^) in corn oil was administered i.p. to one rat from each strain, at the same time the other rat was given corn oil as control. After 48 h the livers were taken and the activity of cholesterol 7a-hydroxylase determined. Table 4.9 showed the Km and Vmax of both control and induced rats in both strains. In the same time Fig. 4.10 showed the Lineweaver-Burk plots of the activity of the enzyme. Whilst the Vmax rose in each strain (20-30%), the Km also was increased (2- to 3-fold), indicating that the first-order rate of reaction (Vmax/Km) actually fell in each strain after 3-MC treatment. The significance of these finaings is not known. -1 9.D - Fig. 4.6 Lineweaver-Burk plots showing the activity of cholesterol 7g-hydroxylose in DA rat 1/V (pmol 7a-0H Cholestero1/mg proteln/mln) DR -191- Fig. 4.7 Lineweaver-Burk plots showing the activity of cholesterol 7g-hydroxylase in Lewis rat 1/V (pmol 7tf-0H Cholesterol/mg proteln/min) LEWIS -192- Fig. 4.8 Lineweaver-Burk plots showing the activity of cholesterol acyltransferase in DA rat 1/V (Cholesterol ester/mg protetn/mln) DR 600 t 0 10 20 30 -193- Fig. 4.9 Lineweaver-Burk plots showing the activity of cholesterol acyltransferase in Lewis rot 1/V (Cholesterol ester/mg protein/mln) LEWIS -194- Table 4.9 Effect of 3-methylcholanthrene induction on cholesterol 7a-hydroxylation in Lewis and DA rats. Strain Vmax Km DA control 25 555 DA induced 34 1111 Lewis control 17 143 Lewis induced 21 500 - 1 9 5 - Fiq. 4.10 Lineweaver-Burk plots showing the effect of 3-methylcholanthrene induction on 7g-hydroxylose activity in Lewis and DA rots 1/V Cpmol 7a-0H Cholestero1/mg proteln/mln) + 3-MC 4.4 DISCUSSION. Hypercholesterolaemia in the rat was observed over 30 years ago by Kohn (1950) whose data are consistent with two co-dominant serum cholesterol phenotypes, characterized by Sprague-Dawly (1200 _+ 60 pg ml-^) and Osborne-Mendel (1320 _+ 40) on the one hand and Tumblebrook Hooded (685 + 33) and Holtzman (653 + 28) on the other, which represent a high cholesterol and low cholesterol serum level, respectively. The results presented in Table 4.3 are in agreement with these findings and possibly offer a basis for these genetic differences in rat serum cholesterol. It has been shown that a significant phenotypic difference exists between female DA rats and Lewis, Fischer and PVG rats with respect to certain drug oxidations (Al-Dabbagh et aj_., 1981). It was thought likely that this polymorphism affects the metabolic disposition of cholesterol, causing hypercholesterolaemia in females of the metabolically-deficient DA strain. It has been shown that the DA female characteristic of high serum cholesterol level was lost in the and F^ generations (Fig. 4.3) where there is no correlation between the metabolic ratios of the hybrids and their cholesterol level, which is at variance with the classical pattern of Mendelian inheritance of a recessive character. Consequently it can be concluded that hypercholesterolaemia in female DA rats together with hypocholesterolaemia in Lewis rats are not inherited in a simple Mendelian pattern. The present observations from Table 4.7 indicate that there is no correlation between oxidation phenotype and serum cholesterol in humans. Although EM volunteers showed higher values of serum cholesterol (197 + 49) than PM volunteers (161 + 23) which did not reach statistical significance, they were on average older than -1 97- the PM subjects and the crude age adjustment made (see Table 4.7) may not have fully eliminated age as a variable. It has been shown from Table 4.6 that the treatment of Lewis and DA rats with 3-MC increase the cholesterol level but not significantly, at the same time no inter-strain difference was observed. Interestingly, it was noticed that control Lewis rats had a high serum cholesterol level (1004 + 81) concluding that corn oil dose might raise the level in these animals, when according to Table 4.3 all the untreated Lewis rats examined showed lower cholesterol levels (746 + 21). It seems fairly well established that the key enzyme in the catabolism of cholesterol to bile salts in the liver is cholesterol 7a- hydroxylase. This enzyme is rate limiting, since changes in the activity of it are associated with parallel changes in the overall rate of bile acid biosynthesis (Mitropoulos et al_., 1973). Hepatic cholesterol, including that reaching the liver from the plasma and that biosynthesized in situ, is converted into bile acids or is secreted into the plasma, as lipoproteins, in the free or in the esterified form. The esterification of hepatic cholesterol (Goodman et_ a]_., 1964) is catalysed by the enzyme acyl-CoA-cholesterol acyl transferase (EC 2.3.1.26). The bile acid synthesis rate in human is estimated as about 0.6 g per day (Vlahcevic et ctl_., 1971; Lindstedt, 1957; Danielsson et a!., 1963), with a range of 265-875 mg reported for ten patients (Vlahcevic et al«, 1971). The role of hepatic cholesterol 7a-hydroxylase in regulating serum cholesterol has been investigated in pigeon strains (Wagner et , 1973; Wagner & Clarkson, 1974). Mittinen has indicated that a similar defect may exist in several types of human hyper cholesterol aemia (see Hulchur & Margolis, 1982). Preincubation has a marked effect on the catalytic activity of cholesterol 7a-hydroxylase (Kwok et aj_., 1981). Long preincubation would lead to enhanced conversion of cholesterol to cholesteryl esters, in the result it has been observed that cholesterol esters were formed in competition to 7a- hydroxycholesterol, since preincubation was performed to utilize the endogenous cholesterol, with the result that cholesterol esters probably were formed as an artefact. Nevertheless, EDTA and nicotinamide were used in order to minimise the formation of non-physiological metabolites like 7&-hydroxycholesterol, 7-oxocholesterol and cholestane 3&,5a,6B- triol which is in agreement with others (Scholan & Boyd, 1968; Mitropoulos et ^1_., 1972; Van Cantfort & Gielen, 1975). Cholesterol 7a-hydroxylase activity has been usually assayed by measuring the extent of conversion of labelled cholesterol into 7a- hydroxycholesterol (Shefer et al_., 1968; Gielen et al_., 1968; Mitton et al_., 1971; Johansson, 1971; Mayer et al_., 1972). In most cases, these assays have been performed with a tracer amount of labelled cholesterol added in acetone, Tween 20 or Tween 80. The results have been expressed as percentage conversion of label or as nmol of 7a- hydroxycholesterol formed, assuming equilibration with endogenous cholesterol. It has been shown in the Results that the mean (+ S.D.) Km for Lewis and DA rat liver S-jg fraction was 186 + 105 and 145 + 63 pM respectively, providing no statistical inter-strain difference. Similarly, the mean (+ S.D.) Vmax for Lewis and DA rats was 60 + 34 and 102 + 164 nmol/min/mg protein respectively, again, no significant difference between both strains was shown. Additionally, no significant inter-strain difference was seen in either Km or Vmax for cholesterol esterification. The Km value for cholesterol 7a-hydroxylase observed - m - was not different from that found by others (150 pM) using liver microsomes (Van Cantf ort et £l_., 1975). It has been shown that the treatment with 3-MC did not alter the activity of cholesterol 7a-hydroxylase (Fig. 4.10) which is in agreement with Brown & Boyd (1974). At the same time it is at variance with the results reported by Wada et al_. (1969) and Mellon et aj_. (1978) who demonstrated a reduction in the 7a-hydroxylase activity after treatment with 3-MC. The observed hypercholesterolaemia in female DA rats thus remains an unexplained curiosity. It cannot be explained by differences in cholesterol 7a-hydroxylation between strains, which is normally thought to be the rate-limiting step in the metabolic disposition of cholesterol. Nor has the observation any parallel in man, between EM and PM phenotypes. Further studies are required to investigate the excretion of cholesterol into bile to see if this is the source of the observed strain differences. Nevertheless, an oxidation phenotyping test for man based upon serum cholesterol concentration is not apparent. CHAPTER FIVE GENERAL DISCUSSION AND CONCLUDING REMARKS -201- 5.1 General Discussion. A number of aspects of endogenous steroid disposition have been investigated in relation to the debrisoquine 4-hydroxylation polymorphism. This has been done in several ways by: (i) investigation of panels of human volunteers of known phenotype and (ii) investigation of phenotypically distinct strains of rat. The aim of these experiments was several fold. Firstly, little if anything is known, but much assumed, about the evolution of the drug metabolizing enzymes and the origin of both inter- and intra-species variation in their occurrence and activities. If an endogenous substrate could be found for a polymorphic drug metabolizing enzyme, then one might gain some insights into why these enzymes exist and why they have been conserved within the human population. Genetic polymorphism fuels the processes of evolution and provides a means by which the species can both adapt to a varying environment and indeed evolve into a more complex and fitter higher species. Genetic polymorphism is often seen as being maintained within a given population in a defined environment through the principle of heterozygous advantage. A classical example of this is that of HbA/HbS polymorphism, where AA homozygotes are at high risk of malaria in malarious regions, SS homozygotes have a high mortality due to sickling and AS heterozygotes (carriers) are at a consequent advantage, with little malaria and sickling and thus propagate the gene within the population (see Bodmer & Cavalli-Sforza, 1976). Whilst other mechanisms might lead to a balanced polymorphism, heterozygous advantage is a wel1-documented and highly plausible means of maintaining an allelomorphic gene in the population. The corollory of this is that a search for the "natural" substrate for the debrisoquine 4-hydroxylase might give a clue as to why 35% of the gene pool is a variant allele and may thus reflect differential diathesis to disease between genotypes. Secondly, inter-phenotype differences in endogenous steroid disposition might provide the basis of a non-invasive phenotyping test, as has been stated throughout this thesis. A simple test is indicated due to the widespread use of debrisoquine phenotyping in epidemiologic studies. Approximately 12,000 phenotyping tests using debrisoquine administration and urine collection have been carried out by colleagues in this Department over the last seven years. Finally, insights into natural disease mechanisms might be obtained by an understanding of the effects of genetically-variable cytochromes P-450, particularly diseases with a known oxidative component such as certain common cancers and inborn errors of hydroxyl ation. Reduction- oxidation (redox) reactions predominate within the cell and it is possible that a cytochrome P-450 which was genetically variable might affect the fitness of that cell in a changing internal environment rather than a changing external environment, as was referred to in the earlier example of heterozygous advantage, polymorphism and malaria. Insight of this nature are beginning now to emerge with the recognition that a genetically variable cytochrome P-450, in this case the debrisoquine 4-hydroxylase, may participate in the aetiology of bronchogenic carcinoma in cigarette smokers (Ayesh et £l_., 1984). Such studies have manifold implications and may herald a new dawn in pharmacogenetics. A discussion of these points will be returned to. A summary of the investigations carried out in this thesis are given in Table 5.1 for the human studies and 5.2 for the rat studies. Of the TABLE 5.1 Summary of the Measurements made in EM and PM. Measurement made Interphenotype difference Androsterone/creatinine ratio None Aeti ocholanolone/creatinine rati o None Dehydroepi androsterone/creati ni ne rati o None 11-Oxoandrosterone + 11-oxoaetiocholanolone/creatinine ratio None 113-Hydroxy-androsterone and -aetiocholanolone/creatinine ratio None Pregnanediol/creatinine ratio None Pregnanetriol/creatinine ratio None Pregnanetri olone/creati nine rati o EM > PM (2-fold) Urinary creatinine concentration None Total urinary 17-oxosteroids None Aeti ocholanolone/Androsterone rati o None Dehydroepiandrosterone/Androsterone + Aetiocholanolone ratio None Urinary E, None Urinary Ei None Urinary E$ None Urinary 2-hydroxy-E, None Urinary 2-methoxy-Ej None Urinary 16a-hydroxy-E-. None Urinary 16-oxo-oestradiol None Serum cholesterol None TABLE 5.2 Summary of the Measurements made in DA and Lewis rats. Measurement made Interstrain difference Dehydroepi androsterone/creati nine ratio None 11-Oxoandrosterone + 11-oxoaetiocholanolone/creatinine ratio None 113-Hydroxyandrosterone + 116-hydroxyaetiocholanolone/creatinine ratio None Urinary creatinine concentration None Total urinary 17-oxostegoids None Urinary excretion of l C]oestrone (20 mg p.o.) None (20 mg i.p.) DA > Lewis (4-fold) (54 pg i.p.) None 24 h urine volume after oestrone (20 mg i.p.) Lewis > DA (1.5-fold) 24 h urine N^ after oestrone (20 mg i.p.) Lewis > DA (1.5-fold) 24 h urine K after oestrone (20 mg i.p.) Lewis > DA (1.5-fold) Bile flow None Biliary elimination of C after [ C]oestrone.(16 pg i.m.v.) None 2- Hydroxy [ x]oestrone in 0-1 h bile after l C]oestrone (16 pg i.m.v.) Lewis > DA (2.3-fold) 24h urine volume after 2-hydroxyoestrone or 16-hydroxyoestrone None Serum cholesterol DA > Lewis (1.5-fold) 3- Methylcholanthrene induction of serum cholesterol None Cholesterol 7a-hydroxylase - apparent affinity constant (K„) None Cholesterol 7a-hydroxylase - maximum velocity (V ) None 3-Methylcholanthrene induction of cholesterol 7a-Sydroxylase K.. DA > Lewis (2-fold) 3-Methylcholanthrene induction of cholesterol 7a-hydroxylase vJJ DA > Lewis (1.5-fold) -205- 20 parameters listed in Table 5.1, only pregnanetriolone/creatinine ratios shows an EM/PM interphenotype difference. The significance, in terms of steroid biochemistry, of this observation is not known. Pregnanetriolone (11-oxo-pregnanetriol, see Fig. 2.2) is a principal urinary metabolite of pregnenolone which itself arises from cholesterol (see Fig. 2.3). The metabolic addition of oxygen at carbons 11-, 17-, 20- and 22- is required to produce pregnanetriolone from cholesterol and the urinary metabolite is at the end of a complex series of metabolic events which have such intermediates as 17a-hydroxyprogesterone and 17a- hydroxypregnenolone (see Bongiovanni, 1978). The significance of the observation that PM subjects have an average only half the urinary excretion of pregnanetriolone as EM subjects is of interest because it is in the 21-hydroxylase defect, the commonest form of the adrenogenital syndrome (adrenal hyperplasia III), that large amounts of urinary pregnanetriolone are found (see Bongiovanni, 1978). The 21-hydroxylase deficiency is essentially a pertubation in the biogenesis of cortisol and thus persons with a diminished excretion of pregnanetriolone (a 21- deoxy steroid) might be interpreted as being efficient 21-hydroxylators. This, at least, is one possible interpretation of the findings and if correct would mean that PM subjects might have elevated cortisol levels. As far as the rat studies are concerned (Table 5.2), several differences between Lewis (EM) and DA (PM) strains were observed and these may reflect the inadequacy of the animal model for the human situation. For example, the differences in serum cholesterol observed in rats were not seen in human studies, although more extensive studies may be required. The origin of these rat serum cholesterol differences is still unclear, since no difference in the 7a-hydroxylase activity could be found between strains. However, it is still possible that it -206- is the disposition of cholesterol, say in bile, rather than a metabolic difference, which is reflected in the serum data. Nevertheless it would now be of great interest to include debrisoquine phenotyping in epidemiologic studies of lipoproteinaemias. Some of the inter-strain differences observed here, such as those after oestrogen administration, may be a little artificial. Doses of 20 mg oestrone per rat are very large indeed and any observation might reflect toxic rather than physiological changes. Nevertheless, it would seem possible to phenotype rats by dosing them with oestrone and simply measuring their urine output or urinary electrolytes! The studies undertaken have produced a number of findings related to the debrisoquine 4-hydroxylation polymorphism, none of which in itself has yielded a new and non-invasive phenotyping test. However, a number of preliminary insights have been found which, after secondary investigations, might lead to such a test, such as via some measurement of 21-hydroxylase activity. During the course of the work carried out for this thesis, many advances have been made in the fields concerning monoclonal antibodies and molecular genetics. It may be possible in future, by isolating biological material from a peripheral blood sample, to phenotype persons using monoclonal antibodies to cytochromes P-450, which have such high affinities for the P-450, that very small amounts of P-450 can be detected, from say lymphocytes. A second approach might be to genotype directly someone using the new techniques of molecular biology by probing for a particular allelomorphic form of the gene in a DNA sample also from lymphocytes using a labelled c-DNA probe. Finally, it might be envisaged that, by understanding the linkage between pharmacogenetic loci and surrounding genes, measurement of some biochemically unrelated event might allow estimation of drug metabolic -207- phenotype. The HLA system is notable in this regard, insomuch as certain HLA alleles seem to be linked to human disease and can be used as predictors of it. Notwithstanding all of the above, it is also quite possible that substrates will remain the best means of discovering enzyme activity and phenotype. Obviously if an endogenous substrate can be found for genetically variable P-450's, phenotyping is likely to become easier to perform in practice and thus of more widespread application. More and more epidemiologists are coming together with laboratory-based scientists to carry out investigations of subjects in epidemiologic studies, rather than just statistical surveys. Pharmacogenetic phenomena could be of immense value in the epidemiology of both natural and iatrogenic disease, but easier ways of phenotyping are needed in practice. 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APPENDIX APPENDIX I Subject details and urinary steroids for volunteers described in Chapter 2 Subjects Sex MR 1 2 3 4 5 6 7 8 9 10 11 Steel A C M F 1.8 10.6 3.8 1.7 0.6 0.3 0.6 1.0 1.5 0.9 0.4 0.1 Taylor M C M 0.5 10.9 3.3 3.9 0.1 0.2 0.8 1.1 1.7 1.2 1.2 0.02 Vyvyan H M 0.4 11.0 2.9 3.4 0.3 0.3 0.7 0.9 1.5 1.0 1.2 0.04 Small N M W M 1.1 9.1 3.3 1.7 0.5 0.3 0.5 0.8 1.1 1.0 0.5 0.1 Stoner J M F 1.1 2.2 4.2 4.0 2.8 0.8 2.0 2.7 0.7 2.0 0.7 0.3 Sweeney P M M 6.5 7.0 2.0 1.3 0.8 0.2 0.4 0.6 0.5 0.7 0.6 0.2 Simmons P A M M 2.9 10.8 4.1 1.5 3.3 0.3 0.4 0.8 0.7 1.6 0.4 0.6 Spencer M M 0.7 10.8 2.0 2.5 0.9 0.5 1.3 0.8 1.6 0.9 1.3 0.2 Simpton C A F 1.3 6.3 3.5 3.4 0.7 0.6 0.7 0.7 0.9 1.5 1.0 0.1 Erskine K F M 0.9 9.0 2.6 2.6 0.2 0.3 0.7 0.6 0.8 1.0 1.0 0.04 Farrar S E F 0.2 10.6 4.1 2.6 0.9 2.3 1.0 1.2 2.3 1.4 0.6 0.1 Githegi D R M M 0.5 10.9 3.4 2.0 0.7 0.5 0.7 1.1 0.9 1.0 0.6 0.1 Hamzah I M 0.7 10.1 1.6 1.3 0.1 0.1 0.3 0.7 0.5 0.8 0.8 0.04 Hasan S M 0.3 6.4 2.4 1.7 0.2 0.2 0.7 0.4 1.3 0.8 0.7 0.04 Gough A K S M >22 10.8 3.0 1.8 0.4 0.2 0.4 0.3 0.9 0.4 0.6 0.08 Iqbal Z M 0.4 8.2 3.0 1.8 1.9 0.3 0.4 0.4 0.4 0.9 0.6 0.4 Johnson J M F 5.3 4.9 2.4 1.9 0.7 0.6 0.7 0.8 0.5 0.7 0.8 0.2 Jeans V C F 0.8 5.4 1.0 1.8 0.3 0.1 0.3 0.3 0.4 0.2 1.9 0.1 Willmott R J F 0.8 3.8 1.6 2.3 0.6 0.4 0.7 0.9 0.4 0.9 1.4 0.2 Winfrey P M M 1.3 9.2 3.1 2.5 0.4 0.3 0.3 0.5 0.9 0.7 0.8 0.08 Wiscombe K A R M 153 7.2 2.0 0.9 1.1 0.2 0.4 0.4 0.8 0.5 0.5 0.4 Wishart K S F 0.7 7.0 0.6 0.6 0.7 0.1 0.7 0.8 0.3 1.2 1.1 0.6 Worlden A F D F 1.1 4.6 1.5 1.7 0.3 0.3 0.2 0.7 0.5 0.7 1.1 0.1 Weadon P K F 1.1 6.8 2.4 3.0 0.4 0.4 0.3 0.5 0.6 0.9 1.3 0.1 -253- m 9 «P Subjects Sex MR 1 2 3 4 5 6 7 8 9 10 11 Warner R J M 0.3 10.2 2.8 1.5 0.3 0.2 0.6 1.5 1.7 0.5 0.5 0.1 Wheeler L J M 1.0 10.2 3.7 2.5 0.4 0.3 0.5 1.1 0.6 1.3 0.7 0.1 Wiggin T R M 0.7 4.2 0.7 0.1 0.2 0.1 0.8 0.3 0.3 0.4 0.2 0.3 Whitworth A G M 1.0 7.5 1.8 1.8 0.8 0.2 0.3 0.5 0.7 0.6 1.0 0.2 Williams H M F 0.3 11.0 2.4 2.2 0.9 0.4 0.6 0.9 0.7 1.5 0.9 0.2 Hughes A S M 1.3 9.3 2.6 1.7 0.4 0.2 0.8 0.9 0.7 1.2 0.7 0.1 Hoyle C F 0.5 5.5 2.0 1.3 1.9 1.4 0.4 0.7 0.7 0.5 0.6 0.6 Hopkinson G P M 3.0 10.3 2.1 2.7 0.2 0.2 0.5 0.5 0.8 0.5 1.3 0.1 Lin Choi B F 1.7 9.4 1.3 1.1 0.4 0.3 0.3 0.4 0.9 0.5 0.8 0.2 Hughes J K F 5.3 3.2 1.9 2.2 1.1 0.3 0.8 0.6 0.9 0.5 1.2 0.3 Ling E A F 0.2 10.5 5.5 4.2 2.0 1.4 1.5 1.9 0.5 1.3 0.8 0.2 Miles 0 F M 1.0 10.3 2.8 2.0 1.6 0.2 0.2 0.5 0.4 0.7 0.7 0.3 Nieman R B M 0.4 10.0 2.5 1.8 1.2 0.2 1.4 1.3 0.4 1.2 0.7 0.3 Dwers D L F 4.0 2.7 1.0 1.3 0.6 0.1 - - - 1.5 1.3 0.3 Lydon A P M F 0.4 3.8 1.9 1.6 0.9 0.6 0.9 0.9 1.7 0.9 0.9 0.3 Nyman D J F 0.8 3.8 2.6 2.3 0.6 0.3 0.4 0.8 0.4 0.6 0.9 0.1 Mee S M F 0.3 2.8 0.6 1.3 0.3 0.2 - - - 0.8 2.3 0.2 Loosemore M P M 2.6 11.7 2.6 1.6 0.5 0.3 0.3 0.3 0.1 0.7 0.6 0.1 Newport M 0 F 0.4 6.9 1.2 2.1 1.4 0.3 0.5 0.5 0.5 1.1 1.7 0.4 Markham A F M 31.7 10.9 2.4 3.4 1.3 0.2 0.6 1.2 0.5 0.7 1.4 0.2 Lumb A B M 1.0 11.5 3.1 1.9 0.3 0.4 0.7 0.6 1.4 0.6 0.6 0.05 Mangat T K F 0.3 10.2 1.1 1.9 0.6 0.3 0.5 0.4 1.1 0.6 1.7 0.2 Evans M J F 0.2 3.9 2.3 1.7 1.3 0.6 0.5 0.7 0.9 0.7 0.8 0.3 - 4 5 2 - T * » 9 Subjects Sex MR 1 2 3 4 5 6 7 8 9 10 11 Beardmore Gray R F 0.6 8.2 2.1 1.1 0.5 0.2 0.2 0.3 0.7 0.9 0.5 0.2 Brinkley D C M 0.8 5.9 3.4 1.7 1.0 0.3 0.5 1.1 1.7 0.3 0.5 0.2 Bench C J M 53.1 8.9 1.7 1.4 0.2 0.1 0.3 0.5 0.4 0.2 0.9 0.1 Collington J A F 0.4 9.9 2.8 2.7 2.3 1.6 0.6 0.6 0.8 0.8 1.0 0.4 Boxter R D M 0.2 7.2 3.5 2.3 0.3 0.6 0.4 0.4 1.0 0.4 0.7 0.1 Goupels M M M 1.3 10.5 1.7 0.9 0.1 0.1 0.3 0.4 0.8 0.7 0.6 0.04 Bayliss E A H F 0.4 2.3 2.6 3.5 1.0 0.4 0.8 0.8 0.7 0.5 1.4 0.2 Cavenagh J D M 4.5 11.0 2.9 2.1 0.5 0.3 0.5 0.7 0.4 0.7 0.7 0.1 Alexander F M F 0.2 7.8 1.9 1.7 0.9 0.2 0.4 0.8 0.5 0.5 0.9 0.3 Davies J A F 36.2 6.0 2.5 1.4 0.4 0.1 0.3 0.8 0.5 0.8 0.6 0.1 Curr D J M 0.5 9.8 2.8 1.5 0.3 0.2 0.6 0.6 0.7 0.7 0.6 0.1 Quiney M J F 0.5 11.6 2.2 1.5 1.0 0.3 0.3 1.2 0.6 0.9 0.7 0.3 Donnelly S F 1.1 7.8 1.7 0.7 0.7 0.2 0.6 1.9 0.6 0.9 0.4 0.3 Sharma N F 0.2 5.4 1.6 1.4 0.8 0.2 0.5 0.5 0.5 0.9 0.9 0.3 Saldanha M B Y F 33.9 5.0 2.0 4.5 1.5 0.7 0.6 0.3 1.1 0.9 2.2 0.2 Sheridan P D M 0.8 5.4 2.2 1.3 1.8 0.1 0.3 0.3 0.6 0.8 0.6 0.5 Parry J I F 0.3 10.3 0.9 2.0 0.3 0.2 0.3 0.2 0.4 0.5 2.2 0.1 Revel! C P F 0.5 4.1 2.6 3.7 0.7 0.8 0.6 0.8 0.9 0.8 1.5 0.1 Reed T I M 0.9 10.4 2.5 3.3 4.3 0.5 0.5 0.8 0.9 1.2 1.3 0.8 Rundell T R M 1.0 8.4 2.8 2.4 0.5 0.3 0.4 0.7 0.9 0.9 0.9 0.1 Rehman A I M 0.09 11.6 5.9 4.3 1.2 0.7 0.7 1.1 1.5 1.5 0.7 0.1 Edwards A F 0.7 4.1 2.9 2.4 3.0 3.0 0.9 0.7 1.1 0.5 0.9 0.6 Eales T D M 0.1 10.7 3.3 1.4 2.0 0.2 0.4 1.4 0.7 0.8 0.4 0.4 -ssz- J • t Subjects Sex MR 1 2 3 4 5 6 7 8 9 10 11 David L A T M 0.2 9.3 1.0 0.9 0.3 0.1 0.2 0.3 0.5 0.1 0.9 0.2 Dunstan M E M 0.2 5.0 2.5 2.0 0.4 0.2 0.7 1.0 0.8 1.5 0.8 0.1 Shennan A S M 33.2 8.9 1.6 0.7 0.2 0.1 0.3 0.2 0.7 0.2 0.5 0.1 Dilkes M G M 43.0 9.3 1.5 1.9 0.3 0.1 0.3 0.4 0.6 0.4 1.3 0.1 Bradbury M M 2.8 10.7 1.7 0.7 0.3 0.1 0.3 0.3 0.8 0.6 0.4 0.1 Ritchie J C M 0.6 11.3 1.1 0.9 0.5 0.1 0.2 0.1 0.3 0.3 0.8 0.2 Idle J R M 0.3 12.4 0.9 0.7 0.5 0.1 0.1 0.2 0.2 0.5 0.8 0.3 O'Gorman J M PM 10.7 1.2 1.4 1.3 0.3 0.2 0.2 0.3 0.05 1.1 0.5 Oates N S M 1.3 12.9 1.4 1.4 0.7 0.4 0.3 0.1 0.9 0.4 1.0 0.3 Crothers M J M 1.0 12.7 1.8 1.6 0.04 0.2 0.3 0.4 0.3 0.5 0.9 0.01 Marsh M V F 0.3 11.1 0.8 0.6 0.1 0.2 0.1 0.2 0.2 0.3 0.8 0.1 Shah R R M 1.2 7.6 0.7 0.7 0.3 0.1 0.1 0.07 0.1 0.07 1.0 0.2 Sinclair K A M 0.7 12.7 2.5 1.7 1.0 0.3 0.5 0.6 0.6 0.8 0.7 0.2 Al-Dabbagh S G M 0.4 11.5 1.8 1.3 0.4 0.2 0.3 0.2 0.3 0.3 0.7 0.1 Sutton D M 0.6 10.3 0.7 0.3 0.3 0.1 0.1 0.1 0.1 0.2 0.4 0.3 Zangorous A M 0.4 12.5 1.2 0.7 0.7 0.2 0.2 0.1 0.3 0.5 0.6 0.4 Sangster S A F 0.6 10.3 0.1 0.1 0.01 0.1 0.05 0.05 0.02 0.05 0.7 0.1 Monks T J M EM 5.7 4.1 4.4 1.4 0.4 2.7 1.6 1.4 0.8 1.1 0.2 T H M PM 10.8 2.9 2.3 0.5 0.2 0.8 0.7 1.0 0.3 0.8 0.1 Wakile L M 0.2 8.7 2.6 1.3 1.1 0.1 0.7 0.8 0.6 0.5 0.5 0.3 -9SZ- Appendix I: The following abbreviations are used: F = Female M = Male MR = Metabolic Ratio 1 = Creatinine in mM 2 = Androsterone/Creatinine ratio in pM/mM 3 = Aetiocholanolone/Creatinine ratio in pM/mM 4 = Dehydroepiandrosterone/Creatinine ratio in pM/mM 5 = Pregnanediol/Creatinine ratio in pM/mM 6 = 11-Oxo-androsterone and -aetiocholanolone/creatinine ratio in pM/mM 7 = llB-Hydroxy-androsterone and -aetiocholanolone/creatinine ratio in pM/mM 8 = Pregnanetriol/creatinine ratio in pM/mM 9 = Pregnanetriolone/creatinine ratio in pM/mM 10 = Aeti ocholanolone/androsterone ratio 11 = Dehydroepiandrosterone/androsterone + aetiocholanolone ratio