Search

A thin-layer partition method for the separation of urinary metabolites of cortisol is described. From amongst the solvent systems tested the most suitable appeared to be ethylene glycol—dichloromethane. Using this system the authors have obtained very good separation of tetrahydrocortisol, allo-tetrahydrocortisol, tetrahydrocortisone, cortisol and cortisone in one chromatographic step which takes only about one hour

A method for the determination of neutral steroid glucuronides in human urine is described. In most cases, a 10-ml sample of urine was chromatographed on Amberlite XAD-2, then on Amberlyst 15, hydrolysed with Ketodase® and the liberated steroids fractionated on silicic acid columns. The steroids were identified and the specificity of the quantitative determinations verified by gas-liquid chromatography and gas chromatography-mass spectrometry. Values for the excretion of fourteen steroid glucuronides during the follicular and luteal phases of the menstrual cycle are presented. The steroids measured were androsterone, etiocholanolone, dehydroepiandrosterone, 5α-androstane-3α,17α-diol, 5α-androstane-3β,17α-diol, 11β-hydroxyandrosterone, 11 β-hydroxyetiocholanolone, 3α, 16α-dihydroxy-5-androsten-17-one, 5-androstene-3β,16α,17α-triol, 5-androstene-3β,16α,17β-triol, 5β-pregnane-3α,20α-diol, 5β-pregnane-3α,17α,20α-triol, 5-pregnene-3β,17α,20α-triol and tetrahydrocortisone. Statistically significant increases in the excretion of 5β-pregnane-3α,20α-diol, 5β-pregnane-3α,17α,20α-triol and 5-pregnene-3β,17α,20α-triol were observed during the luteal phase. Correlation was demonstrated between the change from the follicular phase to the luteal phase of the menstrual cycle in the excretion ratios of glucuronide-conjugated androsterone /etiocholanolone, 11β-hydroxyandrosterone / 11β-hydroxyetiocholanolone, C 19 steroid diols/11β-hydroxylated 17-ketosteroids and an inverse correlation was observed with the changes in the excretion of C 19 O 2 17-ketosteroids and 5β-pregnane-3α,20α-diol+5β-pregnane-3α,17α,20α-triol. Trivial and systematic nomenclature of steroids: androsterone, 3α-hydroxy-5α-androstan-17-one; etiocholanolone, 3α-hydroxy-5β-androstan-17-one; dehydroepiandrosterone, 3β-hydroxy-5-androsten -i7-one; 11β-hydroxyandrosterone, 3α,11β-dihydroxy-5α-androstan-17-one, 11β-hydroxyetiocholanolone, 3α,11β-dihydroxy-5β-androstan-17-one; progesterone, 4-pregnene-3,20dione; pregnanediol, 5β-pregnane-3α,20α-diol; 17α-hydroxyprogesterone, 17α-hydroxy-4-pregnene 3,20 dione; pregnane-triol, 5β-pregnane-3α,17α,20α-triol; tetrahydrocortisone, 3α,17α,21-tri-hydroxy-5β -pregnane-11, 20-dione.

Several steroids of the pregn-4-ene-21-yl sulfate series such as cortisol, cortisone and corticosterone sulfates were previously quantified in the urine of newborn infants, and their output found to be quantitatively comparable to that of tetrahydrocortisone glucuronide. To determine the possible origin of these conjugates, their excretion was measured and compared in nonpregnant and pregnant women at midterm, term and following parturition. Double isotope assays were employed to characterize and quantify all urinary steroids, the main ones being the sulfates of cortisol, cortisone, corticosterone, 11-dehydrocorticosterone and the glucuronides of tetrahydrocortisone and tetrahydrocortisol. At term, one observed a marked increase of the urinary excretion of these conjugates above values obtained for nonpregnant subjects, while following parturition their urinary output fell within the range observed for nonpregnant subjects. There was no conspicuous difference in the urinary excretion of the glucu...

The Chromatographic behavior of 17-ketosteroids and 17-hydroxycorticosteroids normally present in human urine was studied on a column of Sephadex LH-20 equilibrated with 99% 1-butanol; 99% 1-butanol was also used as eluent. Most of the steroids were eluted in order of their increasing polarity and 17-ketosteroids were separated into two groups, namely, C19O2-17-ketosteroid and C19O3-17-ketosteroid. Tetrahydrocortisone and tetrahydrocortisol were completely separated from each other, but cortisone and cortisol were eluted between the former. This method was applied to the analysis of urinary steroids, and the efficiency of separation and the purity of steroids eluted in four main peaks was studied by other chromatographic systems.

o,p′-DDD was administered orally in doses of 6–9 g daily to 5 patients with untreated Cushing's syndrome and to 8 patients with Addison's disease who received constant doses of exogenous cortisol. Sequential measurements of plasma and urinary 17-hydroxycorticosteroids, cortisol secretion rates, plasma cortisol disappearance rates, and serum transcortin levels were performed before and during treatment with o,p′-DDD. There was invariably a prompt 50–80% decrease in urinary 17-OHCS, regardless of whether the patient's source of cortisol was endogenous or exogenous. Plasma 17-OHCS levels and cortisol secretion rates, however, did not fall. Evaluation of the pattern of cortisol metabolites excreted in the urine of these patients revealed that o,p′-DDD altered the extra-adrenal metabolism of cortisol so as to decrease the proportion of cortisol excreted as tetrahydrocortisol and tetrahydrocortisone and to increase the proportion excreted as 6β-hydroxycortisol.

A new reagent system with p-hydrazinobenzenesulfonic acid in phosphoric acid for the determination of urinary 17-hydroxycorticosteroids is described. Use of this reagent results in increased sensitivity of detection of compounds possessing a dihydroxyacetone side chain while it retains the same specificity as obtained with the Porter-Silber reagent. The reaction conditions employed with the new reagent allow the assay to be performed within 4 to 8 hours after hydrolysis of the samples of urine.

In summary, we have demonstrated that: (a) Infusions of epinephrine, norepinephrine, and phenylephrine in 5 per cent glucose for 7 to 10 hours have little effect on, or decrease, urinary aldosterone excretion. (b) Angiotensin II infusions markedly decrease sodium excretion and the Na/K ratio and increase the excretion of aldosterone; of its ring-A reduced metabolite, pregnane-3-α,18,21-triol,11,20-dione; and, to a much lesser degree, of cortisol and tetrahydrocortisone in all normal subjects studied. (c) In patients with benign essential hypertension, infusions of angiotensin also stimulate urinary aldosterone excretion hut have a completely opposite effect on electrolytes-that of increasing sodium output and the Na/K ratio. This basic difference in response to angiotensin points to a fundamental problem to be solved for a better understanding of the disease. It is felt that the relative or absolute excess of aldosterone over progesterone secretion may be the important adrenal disturbance among the basic factors involved in the pathogenesis of arterial hypertension. This disturbance is definitely linked with angiotensin and sodium regulation.

β-Cortolone (3α,17α,20β,21-tetrahydroxypregnane-11-one) was found as a regularly occurring metabolite in the urine of 20 young and elderly subjects of both sexes. The range of excretion values for young subjects was 0.3 to 2.9 mg. per twenty-four hours, and that for elderly subjects was 0.4 to 1.0 mg. per twenty-four hours. When expressed as a function of creatinine excretion, β-cortolone excretion was not significantly altered by age. It was, however, significantly correlated (r = 0.91) and proportional (20.1 per cent ± 1.5) to the urinary excretion of tetrahydrocortisol, allotetrahydrocortisol and tetrahydrocortisone.

Urinary steroid excretion of 6β-OH-cortisol (6β-OH-F), tetrahydrocortisone (THE), l6α-OH-pregnenolone (16α-0H-PG) and 16a-OH-dehydroepiandrosterone (16α-OH-DHA) was determined in 23 full-term normal newborn infants, 8 normally grown premature and 8 intra-uterine growth retarded (IUGR) newborns. The method used measured free, glucuronide- and sulfate-conjugated steroids. Labeled internal standards were used for each steroid measured. The excretions were expressed in relation to urinary creatinine levels. Steroid excretion values in the normally grown full-term and premature groups were quite similar. On comparison of steroid excretion values in the IUGR and normal full-term groups by the Mann-Whitney (two-tailed) test, there were no significant differences in 6β-OH-F and THE excretions but the excretions of 16α-0H-PG and 16α-0H-DHA were significantly less (p < 0.02 and p < 0.01, respectively) in the IUGR group. The ratio 6β-OH-F + THE/16α-OH-PG + 16α-OH-DHA was significantly greater in the IUGR infants tha...

A new technique is reported for the determination of cortisol metabolites. This technique measures a higher percentage of the metabolites of cortisol than the acid phenyl hydrazine (Porter-Silber) procedure and determines them with greater specificity than the 17-ketogenic (Norymberski) method. The cortisol metabolites—tetrahydrocortisone, tetrahydrocortisol, the 20-epimeric cortolones, the 20-epimeric cortols, and their 5α-isomers are extracted after hydrolysis with β-glucuronidase and converted to the corresponding cortols, which are subsequently oxidized to a mixture of 11β-hydroxyetiocholanolone and small amounts of 11β-hydroxyandrosterone. This final product is then measured by the Zimmermann reaction. The application of this method to the determination of urinary cortisol metabolites of normal subjects and of adrenalectomized patients is described. Metabolites of other steroids that might contribute to the values obtained by this technique have also been investigated.

Excretion of aldosterone, pregnanetriol, pregnanediol, 17-ketosteroids, tetrahydrocortisone (THE; 3α,17,21-trihydroxy-5β-pregnane-11,20-dione) and tetrahydro-11-deoxycortisol (THS; 3α,17,21-trihydroxy-5β-pregnan-20-one) was measured in 13 patients with congenital adrenal hyperplasia. An attempt was made to correlate the steroid excretion pattern with the tendency to lose salt. Nine of the patients maintained normal levels of serum sodium and excreted normal or increased amounts of aldosterone. Most of them excreted increased amounts of aldosterone when maintained on a low-salt diet. Four of the 13 patients were unable to maintain normal levels of serum sodium; one excreted no detectable aldosterone and 3 excreted essentially normal amounts. The salt losers also excreted very small quantities of THE. There appeared to be a good correlation between the tendency to lose salt and the ability of the patient to increase urinary aldosterone excretion in response to salt depletion or to the administration of adre...

Levels of free and conjugated 17-hydroxycorticosteroids (17-OHCS) in plasma were measured after intravenous infusions of cortisol, tetrahydrocortisone (Tetra E) and adrenocorticotropic hormone (ACTH) in subjects wi h hyperthyroidism and hypothyroidism. After administration of cortisol to hyperthyroid patients, the levels of free 17-OHCS decayed more rapidly than normal, while those of conjugated 17-OHCS rose faster and to higher values than in normal subjects. The opposite occurred in hypothyroid patients. Conjugation of administered Tetra E was increased in hyperthyroid patients but was normal in those with hypothyroidism. Plasma concentrations of free 17-OHCS were influenced more by the rate of removal from plasma than by the adrenal response to ACTH in both types of subjects. Accordingly the level of free 17-OHCS after ACTH was a misleading index of adrenocortical function, as the rise was greatest in hypothyroid patients and least in those with hyperthyroidism. The level of conjugated 17-OHCS after in...

Cortisol homeostasis is implicated in hypertension and metabolic syndrome. Two enzymes modulate cortisol availability; 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) preferentially converts inactive cortisone to cortisol, whereas 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) converts cortisol to cortisone. In contrast, 5α and 5β reductases inactivate cortisol by conversion to its tetrahydrometabolites: tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone. A subtle local increase in cortisol can be detected by measuring 24-h urine metabolites, LC–MS/MS being the reference method. The 11β-HSD2 activity is assessed based on the cortisol/cortisone ratio, and the 11β-HSD1 activity on the (tetrahydrocortisol + allo-tetrahydrocortisol)/tetrahydrocortisone ratio. To better understand hypertension and/or metabolic syndrome pathogenesis a method for simultaneous determination of cortisol, cortisone, tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone was developed and validated in an LC coupled with the new detector AB Sciex QTrap® 4500 tandem mass spectrometer. The steroids were extracted from 1 mL urine, using cortisol-D4 as internal standard. The quantification range was 0.1–120 ng/mL for cortisol and cortisone, and 1–120 ng/mL for tetrahydrometabolites, with >89 % recovery for all analytes. The coefficient of variation and accuracy was

The adult male and female baboon has been shown to excrete relatively large amounts of polar corticosteroids [l-3] . Following the administration of radiolabelled cortisol to female baboons (Papio papio), only 11% of the radioactivity recovered in the urine was accounted for by the metabolites tetrahydrocortisol (THF) and tetrahydrocortisone (THE), while 29% was associated with more polar metabolites [2]. Although these compounds were not identified, it was suggested that they might be hydroxylated derivatives of THF [2]. During a detailed investigation of urinary steroid conjugation by the baboon (Papio hamadryas), quantitatively one of the major steroids exreted was found to be more polar than THF [ I] . Several milligrams of this compound were isolated from a large pool of urine and using gas chromatography-mass spectrometry (GC-MS) identified as 3a,6&1 l&l 7o,21pentahydroxy-5/3-pregnan-20-one (6&hydroxy-THF) ]41. In view of the importance of 6&hydroxylation in this animal, preliminary studies were made to investigate the pathway leading to the formation of 6/3-hydroxy-THF.

Role of HSD11B2 polymorphisms in essential hypertension and the diuretic response to thiazides. Background The renal 11beta-hydroxysteroid dehydrogenase type 2 (11beta HSD2) enzyme inactivates 11-hydroxy steroids in the kidney, thereby protecting the nonselective mineralocorticoid (MR) receptor from occupation by glucocorticoids. Loss-of-function mutations in the gene encoding 11beta HSD2 (HSD11B2) result in overstimulation of the MR and cause salt-sensitive hypertension. Methods We have investigated the role of HSD11B2 in hypertension in 377 genetically homogeneous essential hypertensives from North Sardinia. Results Thirty of these patients displayed increased urinary cortisol metabolite ratios (greater than or equal to 2) (tetrahydrocortisol [THF]+allotetrahydrocortisol [aTHF]/tetrahydrocortisone [THE]) reflecting a mild reduction in 11beta HSD2 activity. No mutations in HSD11B2 were detected in these patients. All 377 patients were genotyped for a CA repeat microsatellite in intron 1 of HSD11B2 and a G534A polymorphism in exon 3 of HSD11B2. CA repeat length was associated with the (THF+aTHF)/THE ratio, which in turn was significantly related to PRA levels. No associations were found between the G354A polymorphism and the other parameters. There were no differences in blood pressure (BP) levels between HSD11B2 genotypes, but in a subgroup of 91 patients that underwent diuretic therapy, CA repeat length was strongly associated with the BP response to hydrochlorothiazide. Conclusion This study highlights the role of this HSD11B2 polymorphism in sodium handling and is consistent with a role in the BP response to thiazide diuretics.

THE-3-G was added to H2O and 56 urines, and the recovered THE was measured by the Porter-Silber method. The recovery from H2O was quantitative (98 ± 2%), but highly variable from urine, ranging from 35 to 109%. The necessity of the proper standard in analysis of urinary steroid glucuronides was demonstrated. The presence in urine of endogenous inhibitors to β-glucuronidase was confirmed.

White New Zealand rabbits were immunized with 21-succinate-bovine serum albumin conjugates of 20β-cortol and 20α-cortolone. The resulting 20β-cortol antiserum shows 100% crossreaction with 20β-cortol, 26% with 20α-cortol and with tetrahydrocortisol and 52% with tetrahydrocortisol-21-glucosiduronate. The 20α-cortolone antiserum shows 100% crossreaction with 20α-cortolone, tetrahydrocortisone and tetrahydrocortisone-21-glucosiduronate and 60% with 20β-cortolone. We describe the application of both antisera for radioimmunoassays in human urine.

The following findings were obtained which bear on the mechanisms concerned in the rise of plasma free 17-hydroxycorticoid (17-OHCS) levels during pregnancy: 1) Cortisol accounted for approximately 90 per cent of the plasma free 17-OHCS in pregnant women during the third trimester. 2) No cortisol (F), cortisone (E), tetrahydrocortisol (THF) or tetrahydrocortisone (THE) were detected in the urine of 2 adrenalectomized pregnant patients maintained on 1 mg. of 9-alpha-fluorohydrocortisone daily during the third trimester. 3) When ACTH was administered intravenously to a woman with intact adrenals during pregnancy and the postpartum period, there was a greater response during pregnancy as determined by the excretion of F+E and the rise in the level of plasma free 17-OHCS, but no greater response as determined by the excretion of F+E+THF+THE. 4) In a normal woman followed throughout pregnancy there was a marked rise in plasma free 17-OHCS levels but little change in urinary total 17-OHCS excretion. 5) When cor...

In 37 patients with paroxysmal disorders of the cardiac rhythm cortisol metabolism was studied. Investigations showed an increased excretion of 17-OCS at the time of paroxysmal arrhythmia. It was found to be the highest in patients without any organic lesions of the myocardium and also in cases of microfocal infarctions. The cortisol excretion proved greatest in the hours of vasovagal attacks. The latter were attended by the most pronounced disorders of the cortisol metabolism, particularly significant in patients with ischemic heart disease accompanied by congestive failure of circulation.

It was demonstrated that oral administration of 7β,17α-dimethyltestosterone (Calusterone) reversibly alters cortisol production and metabolism in women with advanced breast cancer. There was a 30% decrease in cortisol production rate, a 50% decrease in transformation to tetrahydrocortisone (THE) glucuronide, a 50% prolongation of plasma cortisol half-life, and decreased conversion to C-19 metabolites. The plasma cortisol concentrations (means and 24-hour profiles) remained unchanged by Calusterone despite the reduction in adrenocortical function. We have concluded that the beneficial effect of Calusterone in breast cancer is probably not due to its effects on cortisol production.

The site of omeprazole inhibition of adrenal steroidogenesis has been sought in vivo by analyzing the patterns of urinary steroid metabolite excretion after 6 days of treatment with placebo/omeprazole. Excretion rates of androsterone, aetiocholanolone, dehydroepiandrosterone, 11 beta hydroxyandrosterone, tetrahydrocortisone, tetrahydrocortisol and alpha cortolone were reduced, indicating a block at an early step in steroidogenesis, possibly cholesterol side-chain cleavage. In vitro studies have confirmed this finding by measuring conversion of added precursors to cortisol in isolated bovine adrenocortical cells. Cortisol synthesis from added 20 alpha hydroxycholesterol was inhibited by 83% in the presence of 100 micrograms omeprazole/ml. Conversion from pregnenolone and progesterone and their 17 alpha hydroxylated derivatives was inhibited by 20-40% whereas cortisol production from added 11 deoxycortisol was not affected. These data suggest that omeprazole primarily inhibits cholesterol cleavage and does not inhibit 3 beta hydroxysteroid dehydrogenase, 17 alpha hydroxylase or 11 beta hydroxylation; 21 hydroxylase activity may be marginally attenuated.

The urinary excretion of 17-hydroxycorticosteroids and the relations between the glucocorticoids in urine and their precursors as well as between 17-hydroxycorticoids and 17-hydroxycorticoids and 17-dehydroxycorticosteroids was measured in two subjects before and after 8 days flight in spaceship "Soyuz-22". During a readaptation period after the space flight activation of the glucocorticoid function of adrenals was observed which was accompanied by signs of stress and relative deficiency of 11-hydroxylation in glucocorticoid synthesis. The assumptions on possible causes of observed changes are discussed.

Evaluating plasma levels of cortisol and corticosterone after ACTH-stimulation, and the urinary metabolites tetrahydrocortisone and tetrahydrocortisol in asthmatic children it could be demonstrated, that in most cases there was no suppression of adrenal function following treatment over 6 to 18 months with a daily dose of 200 to 300 microgram beclomethasone dipropionate. Since in a few patients suppression was found, an ACTH stimulation after a six months treatment is recommended.

Normal infants exhibited circadian rhythmicity of plasma F concentration. Infants from 2.1 to 3.2 months of age had CSR significantly higher than those of older infants. THF/THE urinary excretion ratios increased with age. The 17OHCS excretion was higher in the younger infants. Urinary excretions of free corticoids and cortisol were similar in all age groups. In marasmus, plasma F concentrations in the morning and evening were significantly elevated. Normal diurnal variation returned following therapy. CSR and 17OHCS excretions were not different from age controls, but were significantly lower than size controls, THF/THE ratios, urinary excretion of free corticoids and cortisol were normal. In marasmic kwashiorkor, plasma F concentrations were significantly elevated in the morning and evening. There was a suggestive decrease with therapy. CSR was low before and after treatment. THF/THE ratios, urinary 17OHCS excretion, and urinary free corticoids and cortisol were not significantly different from infants matched for size or patients with marasmus.

The following examinations, including the estimation of urinary neutral steroid metabolites, plasma cortisol, the percentage of unbound-cortisol, cortisol production rate, plasma adrenocorticotrophin (ACTH), dexamethasone suppression test, ACTH stimulation test, metopirone test, lysine-vasopressin (LVP) test and insulin tolerance test, were conducted in 16 patients with Cushing's syndrome for the presence of hypercoticism and for identifying the cause of this syndrome. Of these tests, the measurements of plasma cortisol late in the day and single dose dexamethasone suppression test were most useful for the diagnosis of hypercorticism because of their reliability and simplicity. Urinary 17-KGS, THF/THE ratio, cortisol production rate and low dose dexamethasone suppression test were also useful, whereas insulin test and LVP test were less valuable for this purpose. For the identification of the causes of this syndrome, lysine vasopressin test and metopirone test were most reliable, and plasma ACTH was also useful for this purpose, whereas insulin test and ACTH stimulation test were less valuable.

The urinary steroids excreted by an infant with a salt-wasting syndrome due to a suspected defect in the 18-oxidation of corticosterone have been analysed by gas chromatography-mass spectrometry. The excretion of tetrahydroaldosterone was low (3.5 μg/24 h) whilst the excretion of 3α, 11β,21-trihydroxy-5α-pregnan-20-one (allo-tetrahydrocorticosterone) and other corticosterone metabolites was high (total about 2 mg/24 h). The excretion of cortisol metabolites was apparently normal for age (total about 2 mg/24 h) but 3α,11β,17α,21-tetrahydroxy-5α-pregnan-20-one (allo-tetrahydrocortisol) rather than tetrahydrocortisone, was the major component of the group. The excretion of an 18-hydroxycorticosterone metabolite 3α,18,21-trihydroxy-5β-pregnane-1 1,20-dione (18-hydroxytetrahydroCompound A) was higher than normal for infants of this age (between 50 and 200 μg/24 h), suggesting that the defect was in 18-hydroxysteroid dehydrogenase rather than 18-hydroxylase. In addition, 18-hydroxytetrahydrocorticosterone, another metabolite of 18-hydroxycorticosterone was tentatively identified and it was found that the rate of excretion of this compound was of similar magnitude to 18-hydroxytetrahydroCompound A. The salt balance of the infant has been successfully controlled by salt administration (77 mEq./24 h) and treatment with Fludrocortisone (0.5 mg/day).

The finding that urine cortisol excretion was increased in patients with hypokalaemic hypertension induced by licorice addiction led to this study on the effect of licorice in normal subjects. Thirteen normal volunteers ate either 100 or 200 g licorice for 1-4 weeks and assessment of pituitary-adrenal function was made before, during, and 1 week after cessation of licorice ingestion. Urine cortisol excretion more than doubled in 10 of 13 subjects (mean, 33.8 +/- 15.6 SD before and 83.3 +/- 56 SD micrograms/24 h at 1 week after commencing licorice) and excretion rates similar to those observed in Cushing's syndrome were seen in 7 subjects (range, 91-226, compared to normal range of 11-82 micrograms/24 h). Urine cortisol excretion remained significantly elevated (P less than 0.01) above control levels for at least 1 week after licorice was withdrawn. Despite these increases, urinary steroid metabolite (tetrahydrocortisol, tetrahydrocortisone, tetrahydro-11-deoxycortisol, 17-ketogenicsteroids, and 17-ketosteroids) excretion was not affected, plasma cortisol and ACTH values were unchanged, and normal 0800-1600-h diurnal variation of plasma cortisol was maintained. The direct intraadrenal infusion of the active mineralocorticoid component of licorice, glycyrrhetinic acid, in two sheep with autotransplanted adrenal glands failed to stimulate cortisol secretion acutely. It is concluded from these studies that the licorice-induced changes in cortisol excretion are not a result of adrenocoritcal stimulation but more likely represent a change in the renal handling of cortisol.

To predict 11beta-hydroxylase deficiency congenital adrenal hyperplasia antenatally, studies were performed in urines and amniotic fluids from 2 pregnant women who had previously given birth to affected infants and whose present pregnancies also resulted in infants with the disease. Urinary tetrahydro-11-deoxycortisol [pregnane-3alpha, 17alpha, 21-triol-20-one (THS)] was abnormally elevated in the first, second, and third trimesters (maximal values, 3.5 and 0.9 mg/24 h, respectively) but was undetectable after delivery in these mothers, in 15 normal pregnancies (10--40 weeks of gestation), and in 6 heterozygote parents. Amniotic fluid levels of THS, tetrahydrocortisol [pregnane-3alpha, 11beta, 17alpha, 21-tetra-o1-20-one (THF)], tetrahydrocortisone [pregnane-3alpha, 17alpha, 21-triol-11, 20-dione (THE)] measured by RIA at 18 weeks of gestation in the first mother and at 40 weeks in the second revealed 12.5- and 8.4-fold increases in THS, respectively, but normal THF and THE levels compared to mean levels in normal pregnancies. The THS to THF plus THE ratio, which was constant throughout pregnancy in 125 normal women (mean +/- SD, 0.63 +/- 0.34) despite the variable levels of these metabolites, was significantly elevated in both patients (4.4 and 3.8, respectively). These studies indicate that prenatal diagnosis of 11beta-hydroxylase deficiency congenital adrenal hyperplasia based on hormonal measurements is feasible.

In response to an albumin-hydrocortisone hemisuccinate conjugate, antisteroid hormone antibodies have been produced in rabbits and sheep. Evaluation via competitive protein binding (radioimmunoassay) yielded the specificities of the antibodies to steroids. Steroids most closely related to the hapten bound to the antibodies, namely: hydrocortisone, cortisone, prednisolone, hydrocortisone sodiumsuccinate, THE and THF; in contrast, progesterone, 17 β-oestradiol, testosterone, cholesterol, and aldosterone failed to bind. Antigenicity and binding related to the C-17 α-hydroxyl and also the side groups on the D, C, B and A rings of the steroids. Least important were the changes in molecular structure or side groups of the A-ring. Finally, radioimmunoassay proved a sensitive method of evaluating antihormone antibodies.

The metabolism of iv administered 14C-cortisol (F) by mature female baboons was compared with that reported for humans. Approximately 60% of 14C was recovered in urine within 24 hr and 80% within 120 hr after injection. The distribution of urinary 14C was 12.8% unconjugated, 50.3% glucuronoside, and 7.5% sulfate (H2SO4-e,thyl acetate) with 21% unextractable with ethyl acetate. Metabolites were isolated by paper chromatography and purified by crystallization and derivative formation. More than 50% of unconjugated and sulfate fractions were more polar than the cortols with a significant portion of the former even more polar than 6β-hydroxy-F. Unconjugated F plus cortisone [ratio < 1] represented < 2% urinary 14C. Tetrahydro-F (THF) and tetrahydrocortisone (THE) [ratio < 1], constituting 20% of the glucuronoside fraction, were the principal C21 metabolites isolated. Thirty-five percent of the glucuronoside fraction was less polar than cortisone indicating extensive side-chain cleavage. The daily production r...

6 alpha-Hydroxy metabolites of cortisol were determined in the urine of pregnant (36-40 weeks of gestation) and nonpregnant women and in amniotic fluid from nearly fullterm pregnant women because relatively large amounts of these compounds are excreted in the urine of 2-day-old infants (200 micrograms/day). The corticosteroids analyzed by high pressure liquid chromatography and gas chromatography-mass spectrometry were 6 alpha-hydroxy derivatives of (allo)tetrahydrocortisone (3 alpha, 17 alpha, 21-trihydroxy-5 epsilon-pregnan-11,20-dione), (allo)tetrahydrocortisol (3 alpha, 11 beta, 17 alpha, 21-tetrahydroxy-5 epsilon-pregnan-20-one), and alpha- and beta-cortolone (3 alpha, 17 alpha, 20 epsilon, 21-tetrahydroxy-5 beta-pregnan-11-one). All of these compounds were found in the urine samples from both groups of women and in the amniotic fluid samples in contrast to those found in the urine samples from the neonates where 6 alpha-hydroxy compounds of (allo)tetrahydrocortisol and allotetrahydrocortisone were not positively identified because of insufficient yields. The pregnant women excreted significantly larger amounts of 6 alpha-hydroxy metabolites of cortisol (approximately 600 micrograms/day) than the control women (approximately 90 micrograms/day), and the rate of urinary excretion of these 6 alpha-hydroxy compounds was 7.82 and 1.30 micrograms/kg . day, respectively, for these groups of women compared to 54.3 micrograms/kg . day for the neonates. The precursors of these metabolites within the fetal body originated largely from the maternal circulation, and, therefore, the 6 alpha-hydroxy metabolites of cortisol excreted by the mother refer mainly to fetal metabolism and to a lesser extent, to the fetal secretion of cortisol.

The kinetics of 3alpha-hydroxysteroid : NAD oxidoreductase (EC 1.1.1.50) from Pseudomonas testosteroni (ATCC 11996) have been investigated. The kinetic analysis based on initial activity measurements and product inhibition studies, indicates that the addition of substrate to the enzyme and the release of products from it, follows an obligatory order (ordered bi bi mechanism). The ability of the enzyme to utilize the thionicotinamide analogue of NAD (sNAD) as cofactor has been investigated using various 3alpha-hydroxysteroids from both the C19, C21, and C24 series. The results show that the reaction velocity with sNAD as the cofactor is generally lower than with NAD. The decrease, however, varies considerably, being negligible with some steroids such as litocholic acid and deoxycholic acid and very pronounced with other such as tetrahydrocortisol and tetrahydrocortisone. The introduction of an 11beta-hydroxy or an 11-oxo group into the steroid molecule significantly reduces the ability of the enzyme to attack the 3alpha-hydroxy group. No such effect could be seen when the 11-hydroxy group was in the alpha-position. The results also indicate that, whereas NAD can serve as cofactor for both the monomeric and the dimeric forms of the enzyme, sNAD only acts as cofactor for the monomeric form. Thus sNAD is a valuable tool for the study of the reversible, concentration-depenedent monomeric-dimeric transition of the 3alpha-hydroxysteroid dehydrogenase.

The metabolism of deuterated cortisol (9,12,12,-2H)cortisol, 2H3-F) was compared to that of radioactive cortisol (3H2-F) and natural cortisol, when these three compounds were administered simultaneously to an adrenalectomized piglet. The relative isotope dilution of tritium was determined from the specific activities of the main urinary neutral cortisol metabolites, tetrahydrocortisone (THE) and tetrahydrocortisol (THF), normalized to that of the cortisol mixture administered. To obtain a comparison of the isotope dilution of deuterium in the metabolites THE and THF to that in the cortisol mixture, the three steroids were converted to the common oxidation product 11-oxo-aetiocholanolone, and derivatized to the methoxime-tert-butyl-dimethylsilyl ether. The relative 2H-isotope dilution then was measured by gas chromatography/mass spectrometry. It was found that the specific activity of THE in the cumulative urine collections was similar to that of the cortisol mixture administered; the two-day value was, however, less. The specific activity of THF was slightly but significantly smaller than 1 (approximately 0.9) at all times. The relative 2H-isotope dilution in THE was slightly but significantly larger than one (approximately 1.1) at all times, whereas that in the THF was larger than 1.0 at 9 and 32 h or equal to 1.0 at 20 and 47 h of urine collection. When comparing the metabolism of the two tracer cortisol species the quotient of the 3H- and the 2H-isotope dilutions in THE and THF was smaller than 1.0. It can be concluded that (2H3)cortisol may be used for the determination of the cortisol production rate.

Urinary excretion of 17-ketosteroids was measured during dexamethasone substitution followed by ACTH (Synacthen Depot®, 1 mg daily) in 10 non-virilized adrenalectomized women. No significant changes could be demonstrated indicating ovarian androgenic dependence of ACTH in non-virilized females.

The THF/THE ratio of the radioactive metabolites derived from iv cortisol-4-14C was compared in 20 normal subjects and 20 patients with miscellaneous diseases. None of the patients had elevated cortisol production, which is known to raise the THF/THE ratio. In normal subjects, the mean ratio was 0.65, with 95% confidence limits of 0.41 and 0.89. The patients showed a very wide range of values, from 0.34 to 2.08; the mean value was 1.03, 58% higher than normal (p < 0.001), and about ⅔ of the patients had values higher than the upper 95% confidence limit of normal. The total excretion of THF + THE was the same in patients as in normals, so that the elevated THF/THE ratio in patients was due to increased THF precisely matched by decreased THE. This abnormality represents a second type of “nonspecific illness effect” on steroid metabolism. The previously described abnormality in androgen metabolism (decreased recovery of androsterone + etiocholanolone from tracers of androgens) was believed to be due...

Four children with 11 beta-hydroxysteroid dehydrogenase deficiency are described. All patients had severe hypertension, hypokalaemia, and low plasma aldosterone and renin activities. Two of the patients were siblings and two were unrelated. The most noticeable biochemical feature of these individuals was the extremely low excretion of cortisol metabolites containing an 11-carbonyl group compared to the excretion of the 11 beta-hydroxyl containing metabolites. Although this condition is readily diagnosed in affected individuals by urinary steroid analysis, carriers of the defect do not differ from normal in their urinary steroids. Both parents of the affected siblings had normal 11-oxo-steroid/11 beta-hydroxysteroid ratios under baseline conditions and the lesions could not be revealed by ACTH administration.

SUMMARY The quantities of nine corticosteroids in 24 h urine samples collected by pregnant women (nine with normal foetuses and nine with anencephalic foetuses) were measured after hydrolysis with β-glucuronidase and separation by paper chromatography. The excretion (μmol/24 h, mean ± s.d.) of pregnanetriol (0·85 ± 0·17), 3α,17α-dihydroxy-5β-pregnan-20-one 17α-hydroxypregnanolone, 0·55 ± 0·17), 3α,17α,21-trihydroxy-5β-pregnan-20-one (tetrahydro-11-deoxycortisol, 0·17 ± 0·14) and tetrahydrocorticosterone (0·65 ± 0·26) by women with an anencephalic foetus was significantly lower (P < 0·01 or No evidence was obtained therefore to indicate the secretion of cortisol or deoxycorticosterone by the foetal zone of the adrenal of the undisturbed human foetus in late gestation.

During continuous infusion of 3H-cortisol in the circulation of the guinea-pig mother or fetus, radioactive metabolites appear in both maternal and fetal blood. These cortisol-derived compounds were identified principally as cortisone, tetrahydrocortisol (THF) and tetrahydrocortisone (THE). There were unidentified others in low quantities. The cortisone of the maternal plasma is 100% maternal in origin since that of the fetal plasma is 50% fetal in origin between days 62 and 66 and increased thereafter. An identical profile was noted for THF. THE seemed to be synthetized in the fetal guinea-pig and was transferred to the mother in increasing amounts near term. Liver concentrations of cortisol were higher than those of plasma in the mother. Maternal liver appeared to be the main organ of cortisol metabolism in the mother-fetus unit, but maternal adrenal may contribute to this metabolism.

THE 3-G was added to H2O and 56 urines, and the recovered THE was measured by the Porter-Silber method. The recovery from H2O was quantitative (98 +/- 2%), but highly variables from urine, ranging from 35 to 100%. The necessity of the proper standard in analysis of urinary steroid glucuronides was demonstrated. The presence in urine of endogenous inhibitors to beta-glucuronidase was confirmed.

Highly purified preparations of the 3alpha-hydroxysteroid:NAD- oxidoreductase (E.C.1.1.1.50) from Pseudomonas testosteroni (ATCC 11996) which consist of two major isoenzymes, with traces of a third, have been split into two enzymatically inactive polypeptides A and B by the use of sodium dodecylsulphate, urea and guanidinium hydrochloride. Both polypeptides have a molecular weight of 25,000 +/- 2,500 as shown by thin-layer gel chromatography and ultracentrifugations. They differ, however, in charge as shown by electrophoresis on cellulose acetate strips in the presence of 8 M urea. Each of the isoenzymes, have molecular weight of about 50,000 and thus consist of two subunits. The presence of the three isoenzymes may be explained by the following combinations of the subunits, AA, AB and BB. Close to 100% of the original activity towards the three substrates, androsterone tetrahydrocortisone and desoxycholate could be restored within 24 h when the inactivated enzyme was diluted in order to remove the effect of the denaturant.

A new method has been devised to extract 17-hydroxycorticosteroids (17OH-CS) from a hydrolyzed urine using an EXTUBE fiber column. This extraction was combined with a modified Porter-Silber color reaction to quantitate total urinary 17OH-CS. The new method takes only half the time required in older methods. Recovery of tetrahydrocortisone in the new method was improved to 85--95% compared with 70% in the previous method. Between-run precision (C.V.%) was concentration dependent in both methods and was improved to 5--7% compared with 10--18% in the previous method. Evidence was obtained to demonstrate that the new methodology would provide the same information and interpretation as the older, more cumbersome method.

A method for gas-liquid chromatographic separation and quantitation of urinary steroid metabolites is described. A normal reference range has been established and classic examples of urinary steroid profiles of the major steroid disorders are given.

In late pregnancy the mean excretions of tetrahydrocorticosterone, tetrahydro-11-deoxycortisol, pregnanetriol and 17-hydroxy-pregnanolone by women pregnant with an anencephalic fetus were significantly lower than the excretion of these steroids by women pregnant with a normal fetus. In contrast, there was no significant difference between these groups of subjects in the excretion of tetrahydrocortisol, tetrahydrocortisone, cortolone (20α + 20β), cortol (20α + 20β) and tetrahydrodeoxycorticosterone. From this evidence it is concluded that the fetal zone of the human fetal adrenal near term contributes to the secretion of corticosterone and 17-hydroxyprogesterone or their precursors, but not to the secretion of cortisol (or its precursors). Radioactive cortisol was formed during incubation of radioactive progesterone and pregnenolone with adrenal tissue (largely definitive zone) from newborn anencephalic infants. In contrast, the incorporation of [ 14 C]-acetate into 4-en-3-oxo steroids by adrenal tissue from fetuses of eighteen weeks gestation could not be demonstrated. The evidence presented suggests that any synthesis of cortisol by the human fetal adrenal occurs largely in the definitive zone.

We describe a ratio gas-chromatographic method for determination of cortisol production rate by measuring the isotope dilution of urinary cortisol metabolites. The method was calibrated by analyzing [3H]tetrahydrocortisol and [3H]tetrahydrocortisone of known specific activities. Results are reasonably well reproducible, the coefficients of variation ranging from 8-15% of allotetrahydrocortisol/tetrahydrocortisol and from 9-19% for tetrahydrocortisone. Correlation coefficients were 0.966 and 0.998 for tetrahydrocortisone and allotetrahydrocortisol/tetrahydrocortisol, respectively, when the method was compared with a method involving thin-layer chromatography and colorimetry. Only one chromatographic step is needed for both purification and quantitation, thus time and effort are saved.

The 24 hours urines of six two days old fullterm newborn infants were investigated for polar corticosteroids. 6alpha-hydroxy-tetrahydrocortisone, 6alpha-hydroxy-20alpha-cortolone and 6alpha-hydroxy-20beta-cortolone were identified by gas chromatographic-mass spectrometric comparison of the urinary steroids to compounds synthesized previously. These 6alpha-hydroxylated corticosteroids as well as seven other polar corticosteroids were quantified by gas chromatography or mass fragmentography. It was shown that the newly identified steroids constituted a quantitatively important part of the neonatal urinary corticosteroids. The unconjugated- and glucuronic acid conjugated steroids were quantified separately. It was found that the extent of glucuronoconjugation decreased with increasing polarity of the steroid moiety.

[4-14C] Cortisol was administered intramuscularly to one adult female macaque monkey, MF3 (Macaca fascicularis). To adult female macaque monkey, MF4, [4-14C]corticosterone was administered intramuscularly. Urine samples were collected and the metabolites excreted identified using gas chromatography, radio-gas chromatography and gas chromatography-mass spectrometry. The principal metabolites of cortisol were identified as glucuronide conjugates of 11-oxygenated-17-oxosteroids. The excretion of tetrahydrocortisol and tetrahydrocortisone relative to the other corticosteroid metabolites was low compared with that of man. Two compounds, 3β-cortol and 3β-cortolone not normally present in human urine were identified in the urine from this species. The principal metabolites of corticosterone were glucuronide conjugates of hexahydroCompound A and hexahydrocorticosterone. Two unidentified radioactive compounds were also present.