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Mass Spec Testing for Steroid Hormone Profiles: Making an Impact on Patient Care

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Mass Spec Testing for Steroid Hormone Profiles: Making an Impact on Patient Care R.J. Singh, Ph.D. Mayo Clinic Objectives •Congenital Adrenal Hyperplasia (CAH) •Sex Steroids •Cushing’s CAH New Born Screening 1 CAH CholesterolBiosynthesis of Steroids Pregnenolone 17- OH Pregnenolone DHEA ase ’ -----------------------------------------------------------------------------3SDH 17,20 desmolase 17 OH Progesterone 17-OH Progesterone Androstenedione 21 OH'ase 17b SDH ---------------------------------------------- Aromatase 21-Deoxycorti- 11-deoxycortisol Testosterone costerone 11 OH'ase Aromatase ---------------------------------------------- Estrone Corticosterone Cortisol Estradiol 17b SDH 18 OH'ase ---------------------- Cortisone --- Aldosterone STEROID PROFILE BY LC MS/MS TIC: from 051200-36 9.5e5 8 9.0e5 1. Cortisone 8.5e5 2. Cortisol, Cortisol d-4 8.0e5 3. 21-Deoxycortisol 4. Corticosterone 7.5e5 5. 11-Deoxycortisol 7.0e5 6. Androstendione 6.5e5 7. DOC 8. 17-Hydroxyprogesterone 6.0e5 17-Hydroxypregnenolone 5.5e5 9. Progesterone 5.0e5 10. Pregnenolone Intensity, cps 4.5e5 4.0e5 3.5e5 3.0e5 6 2.5e5 5 7 10 2.0e5 1.5e5 34 2 1.0e5 1 5.0e4 9 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 Time, min 6 2 Basics of MS Method Basics of MS Method Lack of Standardization 3 RIA vs. LC-MS/MS 14000 12000 10000 8000 6000 4000 Mayo LC/MS/MS ng/dL LC/MS/MS Mayo 2000 0 0 2000 4000 6000 8000 10000 12000 14000 Ext/RIA ng/dL Correlation Between Two Sites 4 Bland Altman Plot (N=76) 1000 + 2 SD = 801.4 500 + 1 SD = 405.8 Mean difference= 10.1 0 (ng/dL) - 1 SD = 385.6 -500 - 2 SD = 781.4 -1000 0 200 400 600 Difference in 17-OHPG; MS/MS -DifferenceMS/MS17-OHPG; in 2.122*Pantex RIA Average 17-OHPG Values 2.122*RIA and LC CholesterolBiosynthesis of Steroids Pregnenolone 17- OH Pregnenolone DHEA ase ’ -----------------------------------------------------------------------------3SDH 17,20 desmolase 17 OH Progesterone 17-OH Progesterone Androstenedione 21 OH'ase 17b SDH ---------------------------------------------- Aromatase 21-Deoxycorti- 11-deoxycortisol Testosterone costerone 11 OH'ase Aromatase ---------------------------------------------- Estrone Corticosterone Cortisol Estradiol 17b SDH 18 OH'ase ---------------------- Cortisone --- Aldosterone Monthly Test Volumes of Testosterone Total Testo + Free Testo Total Testo Total Testo + Bioavail. Testo 15 5 Androgens in Health and Disease • General well-being • Energy • Mood • Libido and sexual satisfaction • Bone physiology • Muscle mass • Hot flashes Labrie F, et al. Endocr Rev. 2003;24:152-582. Dimitrakakis C, et al. Menopause. 2004;11:531-535. 16 Methodologies for Endocrine Analytes: •RIA •GC-FID •CIA •LC-UV/EC •ELISA •GC-MS •FIA •LC-MS •ICMA •LC-MS/MS Low Male Testosterone (<60 ng/dL): ACS-Enhanced vs. LC-MS/MS 50 45 y = 0.8306x - 2.5493 40 R2 = 0.8482 35 n = 46 30 25 20 15 Testosterone by LC-MS/MS (ng/dL) LC-MS/MS by Testosterone 10 5 0 0 102030405060 Testosterone by Enhanced ACS:180 (ng/dL) 6 Correlation between Immunoassays and Gold standard GC-MS method Serum Testosterone nmol/L GC-MS GC-MS method is labor intensive % difference % difference and less sensitive compared to LC-MS/MS method Serum Testosterone nmol/L GC-MS z Quality of The T Lab Test Instrument No. Mean S.D. C.V. Median Low High Labs Value Value Y-01 Abbott Architect 10 717.0 31.9 4.5 726 658 756 Bayer Acs:180 14 718.3 37.1 5.2 717 653 779 Bayer Advia Centaur 354 769.2 77.2 10.0 767 562 996 Beckman Access/2 146 653.3 49.4 7.6 654 212 858 Beckman Unicel DxI 87 620.0 31.1 5.0 622 540 701 Diagnostic Sys Solid 5 784 724 874 DPC Coat-A-Count 31 720.9 62.9 8.7 733 590 855 DPC Immulite 2000 131 986.9 79.5 8.1 986 776 1243 DPC Immulite 2500 12 976.4 79.2 8.1 946 906 1150 DPC Immulite/1000 57 924.6 100.7 10.9 909 636 1189 Roche E170 76 924.8 33.5 3.6 927 843 1007 Roche Elecsys 1010/2010 76 895.4 40.0 4.5 898 776 976 Tosoh AIA-Pack 15 1024.3 210.1 20.5 920 843 1428 Vitros ECi 80 890.3 58.9 6.6 889 765 1030 All Instruments 1144 799.6 145.0 18.1 796 212 1450 Mass Spectrometry 5 586 334 649 20 7 22 An Endocrine Society Position Statement: Precision and Accuracy Matters Laboratory proficiency testing should be based on the ability to measure accurately and precisely samples containing known concentrations of testosterone, not only on agreement with others using the same method J Clin Endocrinol Metab 92:405-413, 2007 23 8 Instrument Parts to TLX4 – MS/MS 4000 TLX4 (Liquid Chromatography-LC) MS/MS (Tandem Mass Spectrometer) Injector 8 valve VIM LX Inj. ports TX Inj. ports LC System 1 LC System 2 LC System 3 Computer LC System 4 25 9 Dynamic range of competitive immunoassays (conc. limited) www.aapsj.org/view.asp?art=aapsj0902029 28 Sex Hormone-Binding Globulin • SHBG is the carrier protein for estrogen and testosterone • SHBG-bound fraction is unavailable for biological activity • Production regulated by estrogen- testosterone balance • Estrogen stimulates SHBG production • Testosterone decreases SHBG synthesis Selby C. Ann Clin Biochem. 1990;27:532-541. 29 Steroids with Mol. Wt. of 288.4 288.42 5, 7-ANDROSTADIEN-3β, 17β-DIOL 288.42 5α-ANDROSTAN-3, 16-DIONE 288.42 5α-ANDROSTAN-3, 17-DIONE 288.42 5β-ANDROSTAN-3, 17-DIONE 288.42 1, (5α)-ANDROSTEN-17β-OL-3-ONE 288.42 2, (5α)-ANDROSTEN-11α-OL-17-ONE 288.42 4-ANDROSTEN-3α-OL-17-ONE 288.42 4-ANDROSTEN-3β-OL-17-ONE 288.42 4-ANDROSTEN-17α-OL-3-ONE 288.42 4-ANDROSTEN-17β-OL-3-ONE 288.42 5-ANDROSTEN-3β-OL-16-ONE 288.42 5-ANDROSTEN-3α-OL-17-ONE 288.42 5-ANDROSTEN-3β-OL-17-ONE 288.42 5-ANDROSTEN-17α-OL-3-ONE 288.42 5-ANDROSTEN-17β-OL-3-ONE 288.42 9(11), (5α)-ANDROSTEN-3β-OL-17-ONE 288.42 9(11), (5β)-ANDROSTEN-3α-OL-17-ONE 30 10 Compound Amount Added % Cross-reactivity 5-dihydrotestosterone 100 ng/mL 5.4 Androstenedione 100 ng/mL 0.94 Methyltestosterone 100 ng/mL 0.68 Estradiol-17 100 ng/mL 0.02 Androsterone 1 g/mL < 0.1 Cortisol 1 g/mL < 0.1 Corticosterone 1 g/mL < 0.1 Cyproterone 100 ng/mL < 0.1 Danazol 1 g/mL < 0.1 DHEA-sulfate 1 g/mL < 0.1 11-deoxycortisol 1 g/mL < 0.1 Dexamethasone 1 g/mL < 0.1 Estrone 100 ng/mL < 0.1 Oxymetholone 100 ng/mL < 0.1 Progesterone 1 g/mL < 0.1 31 Reference Ranges for Testosterone by LC-MS/MS from Two Different Endocrine Laboratories N Total, nmol/L Tanner Stages Reference Range (ng/dL) (ng/dL) Females Females TS1 147 <0.588 (17) TS1 <7-20 TS2 66 0.138-1.35 (4-39) TS2 <7-47 TS3 85 0.346-2.07 (10-60) TS3 17-75 TS4 77 0.277-2.18 (8-63) TS4 20-75 TS5 123 0.346-2.07 (10-60) TS5 12-60 Males Males TS1 143 <0.657 (19) TS1 <7-20 TS2 66 0.069-5.16 (2-149) TS2 8-66 TS3 65 0.242-26.37 (7-762) TS3 26-800 TS4 98 5.71-29.54 (165-854) TS4 85-1200 TS5 42 6.71-27.09 (194-783) TS5 300-950 Inter-individual Variability Harman, S. M. et al. J Clin Endocrinol Metab 2001;86:724-731 Copyright ©2001 The Endocrine Society 11 Testosterone – Representative Sample Analyte Internal Standard Testosterone – Comparison Curve – 1216 Samples 2500 y = 0.9951x - 26.305 2000 R2 = 0.9879 1500 T (ng/ml) 1000 500 0 0 500 1000 1500 2000 2500 T (ng/ml) Poor Inter-Laboratory Agreement of Testosterone Measurements AACC-2008 12 CholesterolBiosynthesis of Steroids Pregnenolone 17- OH Pregnenolone DHEA ase ’ -----------------------------------------------------------------------------3SDH 17,20 desmolase 17 OH Progesterone 17-OH Progesterone Androstenedione 21 OH'ase 17b SDH ---------------------------------------------- Aromatase 21-Deoxycorti- 11-deoxycortisol Testosterone costerone 11 OH'ase Aromatase ---------------------------------------------- Estrone Corticosterone Cortisol Estradiol 17b SDH 18 OH'ase ---------------------- Cortisone --- Aldosterone Estrogens Undergo Extensive CYP Enzymatic Modifications Zhu BT. Lee AJ. NADPH-dependent metabolism of 17beta-estradiol and estrone to polar and nonpolar metabolites by human tissues and cytochrome P450 isoforms. Steroids. 70(4):225-44, 200 Limitations of Steroid Determination by Direct Immunoassay J. Taieb, C. Benattar, A. S. Birr, and A. Lindenbaum Clin. Chem. 2002; 48: 583 - 585 13 E2 by 7 Immuno-assays and GC-MS/MS Lee, J. S. et al. J Clin Endocrinol Metab 2006;91:3791-3797 40 Copyright ©2006 The Endocrine Society Testosterone and Estrogen Circulation in the Body Bioavailable Testosterone Bioavailable Estrogen ~0.5-2% free ~1.8% free ~30% bound ~60% bound to albumin to albumin ~35% bound ~65% bound to SHBG to SHBG Simon JA. Fert Steril. 2002;77:S77-S82. Demers LM. In: Redmond, G, ed. Androgenic Disorders. Raven Press, New York, NY; 1995:21-34. Impact of Drugs Epilepsia, 32(Suppl. 6):S60-S67, I99 I 14 Anti-aromatase Compounds Exemestane AromasinR Atamestane Letrozole Anastrozole FemeraR ArimidixR Chromatographic Separation of Estrogens 1.80E+05 1.60E+05 Estradiol d5 Estradiol d4-E1 d5-E2 1.40E+05 Estrone d4 Estrone 1.20E+05 1.00E+05 8.00E+04 Peak Intensity (cps) Intensity Peak 6.00E+04 E2 E1 4.00E+04 2.00E+04 0.00E+00 01234 Time (minutes) 44 Anastrozole vs.
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  • Adrenocortical Steroid Metabolism and Adrenal Cortical Function in Liver Disease

    Adrenocortical Steroid Metabolism and Adrenal Cortical Function in Liver Disease

    ADRENOCORTICAL STEROID METABOLISM AND ADRENAL CORTICAL FUNCTION IN LIVER DISEASE Ralph E. Peterson J Clin Invest. 1960;39(2):320-331. https://doi.org/10.1172/JCI104043. Research Article Find the latest version: https://jci.me/104043/pdf ADRENOCORTICAL STEROID METABOLISM AND ADRENAL CORTICAL FUNCTION IN LIVER DISEASE BY RALPH E. PETERSON * (From The National Institute of Arthritis and MVetabolic Diseases, Bethesda, Md.) (Submitted for publication July 27, 1959; accepted October 1, 1959) Zondek (1) as early as 1934 demonstrated that to man disappear rapidly from the blood (15, 16). enzymes of the liver destroyed the biological ac- Only minimal quantities are lost via the expired tivity of the estrogens. Since then both in tivo CO2 (17) or the biliary or gastrointestinal tract and in vitro studies have provided much evidence (15, 16). Also, practically all of the administered to show that the liver is the organ primarily re- steroid is metabolized prior to its excretion in the sponsible for the catabolism of the steroid hor- urine (15, 16), and thus it is apparent that urinary mones: estrogens, androgens, progesterone, and excretion plays a relatively minor part in elimi- the corticosteroids (2). However, not until the nating the biologically active steroid. Thus, meta- development of improved methods for measure- bolic transformation by the liver must play the pre- ment of certain of the adrenocortical steroids and dominant role in terminating the action of the their metabolites, and the availability of labeled steroids. radioactive cortisol, corticosterone, and aldosterone Because of the major role of the liver in the has it been possible accurately to evaluate the in- catabolism of the corticosteroids, it might be ex- fluence of liver disease on the rate of degradation pected that this organ could indirectly influence and synthesis of the steroids in man.
  • ANDROGEN ELITE (Dried Urine)

    ANDROGEN ELITE (Dried Urine)

    ANDROGEN ELITE (dried urine) The aging male experiences a decrease in testosterone at a rate of 10% per decade from the age of 30. This reduction of testosterone and other androgens experienced as a consequence of the aging process has been named andropause or androgen deficiency of the aging male (ADAM). Symptoms associated with ADAM can be associated with impaired 5-alpha reductase or aromatase activity, enzymes responsible for conversion of Testosterone to Dihydrotestosterone (DHT) or estrogens. Symptoms of ADAM consist of somatic, sexual and psychological changes including reduced muscle mass, reduced BMD, increased cardiovascular disease, lowered libido, depression, increased Alzheimer’s disease and a general decrease in well-being. This test provides a focused overview of the male, some female hormones, and its metabolites: Estradiol, Estrone, Estriol, 2OHE1, 16αOHE1; Pregnanediol, Allopregnanolone, Testosterone, Epi- Testosterone, 5α-dihydrotestosterone, Androstenedione, DHEA, 5α,3α- Androstanediol, Cortisol, Cortisone, Tetrahydrocortisol, Tetrahydrocortisone. Hormones and Aging Hormones are powerful molecules that are essential for life. Imbalances of hormones affects other organ systems like the adrenals, thyroid and nervous system. What many people fail to recognise however, is that partial deficiencies also have a wide reaching negative effect on the human body. This is understandable however, when a hormone’s mechanism of action is understood. Aging is one process which is associated with hormone decline. In the past it was thought that this reduction was a normal consequence of the aging process. However, more recently an alternative theory has been proposed; that hormones do not decrease because we age, but rather we age because our hormones decrease i.e.