Clinical and metabolic findings in a 6-year-old boy with a Leydig cell tumour.

AIM: To analyse the urinary steroid metabolome in a boy who had true precocious puberty after a Leydig cell tumour. METHOD: Case report and detailed description of clinical and metabolic findings in a 7-year-old-boy with a Leydig cell tumour. RESULTS: Before surgery, the urinary steroid metabolome showed an activation of an alternative route to gonadal androgens independent of dehydroepiandrosterone (DHEA). After surgery, the boy entered true precocious puberty. Under leuprolide acetate treatment, clinical and laboratory findings normalized. CONCLUSION: Central precocious puberty after precocious pseudopuberty may be more common than expected and should be considered in children with persistent or recurrent symptoms after initial treatment of precocious pseudopuberty. Patients with a Leydig cell tumour seem to reactivate the so-called 'back door pathway' of androgen production, which is independent of the classical route via DHEA.

Histories of depression, allopregnanolone responses to stress, and premenstrual symptoms in women.

Twenty-six women meeting DSM criteria for premenstrual dysphoric disorder (PMDD) and 39 non-PMDD controls were tested for allopregnanolone (ALLO) responses to mental stress. Approximately half of each group had a history of depression (DEP) (14 PMDD, 17 non-PMDD), though all were free of current psychiatric illness. ALLO was sampled in response to venipuncture stress, after an extended baseline, and again 30 and 60 min following the onset of mental stressors. All women with prior DEP, regardless of PMDD status, showed a blunted ALLO stress response at 30 and 60 min (p < 0.05), and also failed to show the expected decrease from venipuncture to baseline rest (p = 0.08) compared to women with no prior DEP. Women with prior DEP did not show the expected correlation between progesterone and ALLO (r = 0.16) that was seen in those with no prior DEP (r = 0.37, p < 0.05). ALLO levels at extended baseline and blunted ALLO reactivity predicted more severe premenstrual symptoms, but only in PMDD women with prior DEP (p values <0.05). These results suggest that a history of DEP is associated with a failure of ALLO to be appropriately responsive to challenge, with alterations in the conversion of progesterone to ALLO, and confirm prior reports linking ALLO to symptoms in PMDD, but only in PMDD women with histories of DEP.

The implications of 7-dehydrosterol-7-reductase deficiency (Smith-Lemli-Opitz syndrome) to neurosteroid production.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive, multiple malformation/mental retardation syndrome with an estimated incidence among individuals of European ancestry of 1 in 20000 to 1 in 30000. It is caused by inactivity of the enzyme 7-dehydrosterol-delta(7)-reductase, which catalyses the terminal transformation in cholesterol synthesis. Patients show not only an increased level of 7-dehydrocholesterol in blood and tissues, but also increased 8-dehydrocholesterol because of the presence of an active delta(8)-delta(7) isomerase. A major consequence of these biochemical abnormalities is the alteration of normal embryonic and fetal somatic development causing postnatal abnormalities of growth, learning, language and behavior. While deficient cholesterol during early development is the primary cause of central nervous system (CNS) abnormalities and retardation, we questioned whether neurosteroids could also be involved since they can have a profound influence on behavioral characteristics. Disordered neurosteroidogenesis would be expected in SLOS and could be caused by a deficiency in classical neurosteroid synthesis secondary to cholesterol deficiency, or by synthesis from 7- and 8-dehydrocholesterol of novel neurosteroids with delta(7) or delta(8) unsaturation which may have altered activity compared with conventional neurosteroids. In particular we sought analogues of dehydroepiandrosterone sulfate, pregnenolone sulfate and the pregnanolone epimers. We targeted urine from post-pubertal females, as this type of sample would be most likely to yield identifiable amounts of the pregnanolone metabolites of progesterone. Analysis by GC/MS of urinary steroids excreted by post-pubertal females confirmed the presence of neurosteroid-like compounds in SLOS patient's urine. Even though the new neuroactive steroids identified were unlikely to have been formed in the brain, it is likely that mechanisms for their synthesis are operable in this organ.

Patterns of urinary and fecal steroid excretion during the ovarian cycle and pregnancy in the African elephant (Loxodonta africana).

The aims of the present study were to (i) determine the relative abundance of the 5alpha-reduced progestins 5alpha-pregnane-3-ol-20-one (5alpha-P-3OH) and 5alpha-dihydroprogesterone (5alpha-DHP) and progesterone (P4) in African elephant feces and to establish improved fecal progestin assays for monitoring ovarian function; and (ii) describe longitudinal profiles of urinary and fecal progestin and estrogen metabolites during pregnancy. Matched urine and fecal samples were collected weekly from six adult females throughout 18 nonfertile cycles and two complete pregnancies (89 and 93 weeks duration). Fecal samples were lyophilized and extracted with 80% methanol in water and immunoreactive 5alpha-P-3OH, 5alpha-DHP, and P4 and (for pregnant females only) estrone (E1) and estradiol (E2) determined by enzyme immunoassay. Urine samples were hydrolyzed, ether-extracted, and assayed for 5alpha-P-3OH, E1, and E2. HPLC cochromatography of fecal extracts with various radioactive progestin tracers confirmed the presence of large amounts of both 5-reduced progestins (5alpha-P-3OH > 5alpha-DHP) but not of P4. 5-Reduced progestins (but not P4) were excreted in a cyclic pattern and levels were significantly correlated with urinary 5alpha-P-3OH. Fecal 5alpha-P-3OH showed the more pronounced and consistent luteal-phase elevation and a better correspondence to urine with respect to timing of the luteal-phase rise. Fecal and urinary 5-reduced progestins increased gradually during early pregnancy to maximum values around week 40-45. Levels gradually declined during the second half of pregnancy, reaching baseline values 2 days before parturition. Urinary estrogens did not show any cyclic pattern during the preconception period and levels remained low during the first 30 weeks of gestation. Thereafter, there was a rapid 10- to 20-fold increase to maximum values at mid-pregnancy, followed by a gradual decline to birth. There was no mid-pregnancy elevation in fecal estrogens, but there was a modest increase in E1 during the second half of gestation.

Screening of eltanolone metabolites in dog urine by anion-exchange/reversed-phase liquid chromatography and mass spectrometry.

Strong anion-exchange (SAX) chromatography and reversed-phase liquid chromatography (RPLC) followed by different mass spectrometric techniques for the separation and identification of conjugated and unconjugated 14C-labelled eltanolone (5beta-Pregnan-3alpha-ol-20-one) metabolites in biological fluids are presented. Conjugates of estradiol were used as model compounds for the development of a SAX based group separation of neutral steroids, glucuronides, sulfates and di-conjugated steroids. The usefulness of the technique is demonstrated by the analysis of 14C-labelled eltanolone metabolites in dog urine. The analytical SAX column used prior to RPLC improved the capacity to separate the metabolites from each other and from endogenous components, compared to a single reversed-phase system. Liquid chromatography negative ion electrospray-mass spectrometry (LC-ESI-MS) was used for the molecular mass determination of conjugated eltanolone metabolites. Unconjugated metabolites and hydrolysed conjugates were identified using gas chromatography-mass spectrometry with an electron impact ion source (GC-MS) after trimethylsilyl (TMS) derivatization. An unexpected finding in dog urine was the diglucuronide formation of eltanolone (presumably after enolisation of its carbonyl group).

Metabolism of 5 alpha-dihydroprogesterone in women and men: 3 beta- and 3 alpha-,6 alpha-dihydroxy-5 alpha-pregnan-20-ones are major urinary metabolites.

The metabolism of 5 alpha-dihydroprogesterone (5 alpha-DHP) in women and men was evaluated by defining the pattern and identity of selected metabolites excreted in urine after the iv infusion of radiolabeled 5 alpha-DHP. Virtually all of the radioactivity in urine (approximately 37% of the administered dose) was excreted within 72 h. Quantitatively, the 2 major urinary metabolites of 5 alpha-DHP in each of 13 studies conducted in 7 women and 2 men were 3 beta,6 alpha-dihydroxy-5 alpha-pregnan-20-one and 5 alpha-pregnane-3 alpha,20 alpha-diol, which could be extracted after beta-glucuronidase, but not solvolysis, treatment of the urine. Radiolabeled 3 alpha,6 alpha dihydroxy-5 alpha-pregnan-20-one (glucuronoside), in lesser amounts, also was identified in the urine of each subject. The 3 alpha/beta, 6 alpha-dihydroxy-5 alpha-pregnan-20-ones arise through specific extrahepatic pathways of progesterone/5 alpha-DHP metabolism. These metabolites are not the products of the enzyme reaction catalyzed by the cytochrome P450 steroid 6 alpha-hydroxylase of human liver (and other tissues), which affects the 6 alpha-hydroxylation of C19- and C21-delta 4-3-ketosteroids (e.g., progesterone, testosterone, and cortisol), but does not act upon 5 alpha-reduced steroids. Moreover, the steroid 5 alpha-reductases do not act upon 6 alpha-hydroxy-delta 4-3-ketosteroids. In addition, the 6 alpha-hydroxylation of 5 alpha-reduced-3 alpha/beta-hydroxysteroids is not demonstrable in adult liver tissue. Rather, the formation of 6 alpha-hydroxylated-5 alpha-pregnane-3 alpha/beta-ol-20-ones is indicative of an extrahepatic pathway of progesterone metabolism, viz. progesterone-->5 alpha-DHP-->5 alpha-pregnan-3 beta/alpha-ol-20-one(s)-->3 beta/alpha,6 alpha-dihydroxy-5 alpha-pregnan-20-one(s), in which 5 alpha-pregnan-3 alpha/beta-ol-20-ones are metabolized by an enzyme(s) that catalyzes the 6 alpha-hydroxylation of saturated substrates. There are important differences among mammalian species in the enzymes that catalyze the C-6-hydroxylation of 5 alpha-reduced C19- and C(21)-3 beta/alpha-hydroxysteroids, but in all species studied, these enzymatic reactions are the final steps in the extrahepatic inactivation of 5 alpha-reduced bioactive metabolites of progesterone (or testosterone).

Identification of 20 alpha-hydroxy-5 alpha-pregnan-3-one and 20 beta-hydroxy-5 alpha-pregnan-3-one in human pregnancy urine.

20 beta-Hydroxy-5 alpha-pregnan-3-one and 20 alpha-hydroxy-5 alpha-pregnan-3-one were isolated and identified from a pool of urine collected from women in the third trimester of pregnancy. Following isolation by Sephadex LH-20 and HPLC, the identity of each compound was established by comparison of GC-MS data for the methyloxime-trimethylsilyl ethers with those for authentic standards.

Detection of late onset steroid 21-hydroxylase deficiency by capillary gas chromatographic profiling of urinary steroids in children and adolescents.

Patients suffering from late onset 21-hydroxylase deficiency (LO-CAH) excreted only slightly higher amounts of 17-hydroxypregnanolone (17-OH-PO), pregnanetriol (PT) and 11-oxo-pregnanetriol (11-O-PT) than age-matched healthy controls. To discriminate between LO-CAH and virilization of unknown origin and precocious pubarche, we calculated the following ratios: (1) pregnanetriol to tetrahydrocortisone (PT/THE), (2) the sum of 17-OH-PO, PT and 11-O-PT (OHP-M) to the sum of THE, tetrahydrocortisol (THF) and allotetrahydrocortisol (a-THF) (C-M) and (3) 11-O-PT to C-M. The following patients were studied: 9 patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency-non-salt losing (CAH-NSL), never treated; 8 patients with CAH (NSL/SL: 3/5) off treatment; 10 patients with LO-CAH; 11 patients with virilization of unknown origin (prepubertal/pubertal: 5/6) and 9 patients with precocious pubarche. Healthy individuals and obligatory heterozygote carriers of comparable ages served as controls. LO-CAH showed increased ratios (median (range] of PT/THE: 2.27, (1.15-9.09), OHP-M/C-M: 2.30, (1.24-8.15), and 11-O-PT/C-M: 0.24, (0.13-1.23) compared to healthy individuals and heterozygous carriers: PT/THE 0.28, (0.03-0.57), OHP-M/C-M 0.23, (0.06-0.46) and 11-O-PT/C-M less than 0.01, (less than 0.01-0.06), respectively. The calculation of ratios, rather than absolute amounts seems to allow the detection of LO-CAH in a single spontaneously voided urine specimen. The clinical and measurable hormonal manifestations of LO-CAH occur at the same time.

Embryotoxic effects of thalidomide derivatives in the non-human primate Callithrix jacchus. II. Elucidation of the susceptible period and of the variability of embryonic stages.

Data are presented on the normal intra- and inter-litter variability of the embryonic stages in the common marmoset (Callithrix jacchus) on day 56 of pregnancy. The embryology of the marmoset is described with respect to the susceptible period for the teratogenic action of thalidomide.

Identification of the main gestagen metabolite in marmoset (Callithrix jacchus) urine by NMR and MS spectroscopy.

Hydroxypregnanolone is the most abundant progesterone metabolite in the urine of marmoset monkeys (Callithrix jacchus). The substance is excreted as conjugate. The concentration of this steroid may be monitored by high performance, thin layer chromatography and postchromatographic derivatization. Hydroxypregnanolone was purified and subsequently identified by NMR spectroscopy and gas chromatography/mass spectroscopy. The exact chemical structure is 5 alpha-pregnane-3 alpha, 7 alpha-diol-20-one.

Determination of ovulation and pregnancy in the marmoset (Callithrix jacchus) by monitoring of urinary hydroxypregnanolone excretion.

In Callithrix jacchus the urinary excretion of hydroxypregnanolone (5 alpha-pregnane-3 alpha, 7 alpha-diol-20-one) shows a luteal rise during the ovulatory cycle. This progesterone metabolite can therefore be used as an indication of ovulation, implantation, and subsequently for the persistence of the pregnancy. The excretion can be monitored by high performance thin-layer chromatography (HPTLC) and postchromatographic derivatization after enzymatic hydrolysis of the conjugate. Profiles of urinary hydroxypregnanolone levels are presented and correlated with luteinizing hormone excretion. The method is noninvasive and therefore suited for long-term studies in these monkeys.

Late-onset 21-hydroxylase deficiency: reliable diagnosis by steroid analysis of random urine collections.

The feasibility of performing steroid analysis by capillary gas chromatography on random urine samples for the detection of mild late-onset 21-hydroxylase deficiency was evaluated. Comparisons were made of basal excretions of androgen and 17 alpha-hydroxyprogesterone metabolites with plasma levels (basal and stimulated) of 17 alpha-hydroxyprogesterone and testosterone in six patients with the disorder. The following steroid metabolite excretion ratios were determined for normal controls and affected individuals. 1) 17 alpha-hydroxypregnanolone/tetrahydrocortisone + tetrahydrocortisol + 5 alpha-tetrahydrocortisol (cortisol metabolites) (normal 0.017-0.10, affected 0.17-0.42); 2) pregnanetriol/cortisol metabolites (normal 0.03-0.15, affected 0.17-0.99); 3) pregnanetriolone/cortisol metabolites (normal 0.02-0.014, affected 0.08-0.20); 4) androsterone + etiocholanolone/cortisol metabolites (normal 0.26-1.02, affected 0.34-1.47). Among the 21-deoxy steroid ratios, there was no overlap between affected and unaffected individuals. Two of six affected individuals had androsterone + etiocholanolone/cortisol metabolite ratios in the normal range. This method provides excellent discrimination between normal and affected individuals, precluding the need for an ACTH-stimulation test. It is anticipated that it will be increasingly used for diagnosis of the condition.

[An analysis and identification of urinary steroids in normal people and patients with 21-hydroxylase deficiency and urinary excretion patterns].

Urinary steroids in normal males and females, whose ages ranged from 7 to 73, and 5 patients with 21-hydroxylase deficiency, 2 twin cases and a singleton case, were analyzed in some detail by gas-liquid chromatography (GCL) and our newly developed pretreatment procedure. Thereby, 40 steroids were found in the urine and two of them, pregnanetetrol -20 beta and pregnenetetrol -20 beta, which have so far not been reported in biological reports, were identified in the patients' urine. The differences in the steroid synthesis, as for sex and age, were confirmed by the results. Although the ratio, androsterone (An)/etiocholanolone (Et) was not so characteristic of sex as has been reported, a ratio: 11 beta-HO-An/11 beta-HO-Et was more characteristic of sex. That is, normal adult females had ratios below 5.1, and normal adult males were divided into two groups: one with a ratio below 5.1 and the other. The patients excreted not only large quantities of steroids lacking the hydroxyl group at C-21 position, which were never found in any of the urine from the normals, but also significant amounts of 21-HO-steroids that might explain steroid synthesis through by-pass ways or efficiencies of synthesis in these cases. The excretion and the ratios differed to an appreciable degree among the cases but not so greatly between the twin cases. This suggests that the detailing urinary steroid excretion represents a proper genetic expression of the enzyme activities on the biosynthesis and the metabolisms of steroids.

Metabolism of deoxycorticosterone and deoxycorticosterone sulfate in men and women.

In this investigation we found that little of intravenously infused [14C]deoxycorticosterone (DOC) was converted to [14C]DOC-SO4 that entered plasma. Moreover little of intravenously infused [3H]DOC-SO4 was metabolized by way of DOC except by intestinal bacterial enzymes. However evidence was obtained that plasma DOC is converted to DOC-SO4 in liver, but little of the DOC-SO4 formed in liver escapes into blood; rather the DOC-SO4 enters bile and in the intestine is converted, in part, to progesterone (or metabolites thereof) by the action of bacterial enzymes. The estimated intrahepatic fractional conversion of DOC to DOC-SO4 was significantly greater in premenopausal women (0.72 +/- 0.118, mean +/- SEM) than in men (0.28 +/- 0.036, P less than 0.005).

The diagnosis of 21-hydroxylase deficiency in a prematurely born infant on the basis of the urinary steroid excretion pattern.

The urine of a 6-day-old prematurely born female infant (birth weight 1060 g) suspected of having a 21-OH-deficiency showed no steroid abnormalities on capillary GLC analysis. Using GC-MS tetrahydrocortisone (THE) and also 3 alpha, 17 alpha-dihydroxy-5 beta-pregnane-20-one (17-OH-Polone) were absent, but two androstanetriolone peaks were observed. In the urine collected on day 9 THE was absent, but a large amount of 3 alpha, 11 beta-dihydroxy-5-alpha-androstane-17-one (11-HA) was found by GC-MS to be contaminated by a small amount of 17-OH-Polone. The next urine specimen collected on the 22nd day while the child received cortisol therapeutically showed the characteristic steroid profile for the diagnosis 21-OH deficiency, large peaks of 17-OH-Polone, pregnanetriol (P3) and 11-keto-pregnanetriol (11-keto-P3). Over the next few weeks two other compounds were found to have been excreted in relatively large amounts, 3 xi, 16 xi, 17 xi, 20 xi-pregnanetetrol (16-OH-P3) and surprisingly also a 21-hydroxylated compound, namely 3 xi, 20 alpha, 21-trihydroxy-5-pregnene. These same two compounds were also found in the urine of another infant with suspected 21-OH deficiency. The urinary steroid excretion patterns characteristic for 21-OH deficiency are dependent on the maturity and age of the infant. In the prematurely born infant androstanetriolones appear in the urine before 17-OH-Polone. The occurrence of these different steroid excretion patterns is tentatively explained.

Dissociation of chorioadenoma destruens from hydatidiform mole by urinary steroid analysis.

Dissociation between hydatidiform mole (HM) and chorioadenoma destruens (CA) was attempted by gas chromatographic analysis of urinary steroids. In the chromatogram, fraction 2 (F2) including four pregnane steroids (17-hydroxypregnanolone, pregnanolone, pregnanediol, and pregnanetriol) reflected the biologic activity of molar human chorionic gonadotropin (hCG), and the ratio of F2 to fraction 1 (F1) (17-ketosteroids) was found useful in monitoring secondary growth of trophoblastic tumor: Urinary log F2/F1 after curettage declined quickly and unidirectionally in HM, but the same parameter exhibited a temporal rise in CA at 11 to 20 days after curettage because of the molar hCG from residual tumor tissue. The resolution between HM and CA at that stage (11 to 20 curettage days) was of the order of diagnostic use and superior to that with immunoreacive hCG. The data obtained are discussed in the light of pathophysiology of CA.

Steroid profiles in hydatidiform mole associated with theca-lutein cysts.

Neutral steroids in urine were determined quantitatively with gaschromatography on capillary columns in a case of benign hydatidiform mole associated with bilateral theca-lutein cysts. A remarkable finding was the very high levels of 17-hydroxypregnanolone and pregnanetriol, which continued to rise until the 15th day after molar evacuation.

Corticosteroid production by the human foetus: evidence from analysis of urine from women pregnant with a normal or an anencephalic foetus.

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 beta-glucuronidase and separation by paper chromatography. The excretion (mumol/24 h, mean +/- S.D.) of prenanetriol (0-85 +/- 0-17), 3 alpha, 17 alpha-dihydroxy-5 beta-pregnan-20-one (17 alpha-hydroxypregnanolone, 0-55 +/- 0-17), 3 alpha, 17 alpha, 21-trihydroxy-5 beta-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 less than 0-01 or less than 0-05) than the excretion of these compounds by women with a normal foetus (pregnanetriol, 2-42 +/- 0-62; 17 alpha-hydroxypregnanolone, 2-72 +/- 0-69; tetrahydro-11-deoxycortisol, 0-56 +/- 0-37; tetrahydrocorticosterone, 1-95 +/- 0-94). These differences suggest that the adrenal of the normal foetus contributes to the quantity of pregnanetriol, 17alpha-hydroxypregnanolone, tetrahydro-11-deoxycortisol and tetrahydrocorticosterone in maternal urine. The excretion of tetrahydrocortisol, tetrahydrocortisone, tetrahydrodeoxycorticosterone, cortol and cortolone were similar in both groups of subjects. 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.