Impact of Gestational and Postmenstrual Age on Excretion of Fetal Zone Steroids in Preterm Infants Determined by Gas Chromatography-Mass Spectrometry.

CONTEXT: Fetal zone steroids (FZSs) are excreted in high concentrations in preterm infants. Experimental data suggest protective effects of FZSs in models of neonatal disease. OBJECTIVE: We aimed to characterize the postnatal FZS metabolome of well preterm and term infants. METHODS: Twenty-four-hour urinary FZS excretion rates were determined in early preterm (<30 weeks' gestation), preterm (30-36 weeks), and term (>37 weeks) infants. Pregnenolone and 17-OH-pregnenolone metabolites (n = 5), and dehydroepiandrosterone sulfate and metabolites (n = 12) were measured by gas chromatography mass spectrometry. Postnatal concentrations of FZSs were compared with already published prenatal concentrations in amniotic fluid. RESULTS: Excretion rates of total FZSs and most of the single metabolites were highest in early preterm infants. In this group, excretion rates approach those of term infants at term equivalent postmenstrual age. Preterm infants of 30-36 weeks had more than half lower median excretion rates of FZSs than early preterm infants at the same time of postmenstrual age. Postnatal concentrations of FZSs were partly more than 100-fold higher in all gestational age groups than prenatal concentrations in amniotic fluid at midgestation. CONCLUSION: The excretion rates of FZSs as a proxy of the involution of the fetal zone of the most immature preterm infants approached those of term infants at term equivalent. In contrast, the fetal zone in more mature preterm infants undergoes more rapid involution. These data in exclusively well neonates can serve as a basis to investigate the effects of illness on the FZS metabolome in future studies.

Detection of Urine Metabolites in a Rat Model of Chronic Fatigue Syndrome before and after Exercise.

Purpose. The aim of the present study was to elucidate the metabolic mechanisms associated with chronic fatigue syndrome (CFS) via an analysis of urine metabolites prior to and following exercise in a rat model. Methods. A rat model of CFS was established using restraint-stress, forced exercise, and crowded and noisy environments over a period of 4 weeks. Behavioral experiments were conducted in order to evaluate the model. Urine metabolites were analyzed via gas chromatography-mass spectrometry (GC-MS) in combination with multivariate statistical analysis before and after exercise. Results. A total of 20 metabolites were detected in CFS rats before and after exercise. Three metabolic pathways (TCA cycle; alanine, aspartate, and glutamate metabolism; steroid hormone biosynthesis) were significantly impacted before and after exercise, while sphingolipid metabolism alone exhibited significant alterations after exercise only. Conclusion. In addition to metabolic disturbances involving some energy substances, alterations in steroid hormone biosynthesis and sphingolipid metabolism were detected in CFS rats. Sphingosine and 21-hydroxypregnenolone may be key biomarkers of CFS, potentially offering evidence in support of immune dysfunction and hypothalamic-pituitary-adrenal (HPA) axis hypoactivity in patients with CFS.

Modified-Release and Conventional Glucocorticoids and Diurnal Androgen Excretion in Congenital Adrenal Hyperplasia.

Context: The classic androgen synthesis pathway proceeds via dehydroepiandrosterone, androstenedione, and testosterone to 5α-dihydrotestosterone. However, 5α-dihydrotestosterone synthesis can also be achieved by an alternative pathway originating from 17α-hydroxyprogesterone (17OHP), which accumulates in congenital adrenal hyperplasia (CAH). Similarly, recent work has highlighted androstenedione-derived 11-oxygenated 19-carbon steroids as active androgens, and in CAH, androstenedione is generated directly from 17OHP. The exact contribution of alternative pathway activity to androgen excess in CAH and its response to glucocorticoid (GC) therapy is unknown. Objective: We sought to quantify classic and alternative pathway-mediated androgen synthesis in CAH, their diurnal variation, and their response to conventional GC therapy and modified-release hydrocortisone. Methods: We used urinary steroid metabolome profiling by gas chromatography-mass spectrometry for 24-hour steroid excretion analysis, studying the impact of conventional GCs (hydrocortisone, prednisolone, and dexamethasone) in 55 adults with CAH and 60 controls. We studied diurnal variation in steroid excretion by comparing 8-hourly collections (23:00-7:00, 7:00-15:00, and 15:00-23:00) in 16 patients with CAH taking conventional GCs and during 6 months of treatment with modified-release hydrocortisone, Chronocort. Results: Patients with CAH taking conventional GCs showed low excretion of classic pathway androgen metabolites but excess excretion of the alternative pathway signature metabolites 3α,5α-17-hydroxypregnanolone and 11ß-hydroxyandrosterone. Chronocort reduced 17OHP and alternative pathway metabolite excretion to near-normal levels more consistently than other GC preparations. Conclusions: Alternative pathway-mediated androgen synthesis significantly contributes to androgen excess in CAH. Chronocort therapy appears superior to conventional GC therapy in controlling androgen synthesis via alternative pathways through attenuation of their major substrate, 17OHP.

Clinicopathological features, biochemical and molecular markers in 5 patients with adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a very rare malignant tumor with poor prognosis. To gain insight into the pathogenic significance of ACC, we studied clinicopathological features and gene expression profile in ACC. We analyzed five ACC cases (two men and three women) with the median age of 45-year-old who underwent adrenalectomy at our institute. Endocrine studies revealed that two cases had subclinical Cushing's syndrome (SCS) and one with concomitant estrogen-secreting tumor, while the rest of three cases had non-functioning tumors. Analysis of urinary steroids profile by gas chromatography/mass spectrometry showed increased metabolites of corticosteroid precursors, such as 17-OH pregnenolone, 17-OH progesterone, dehydroepiandorosterone (DHEA), and 11-deoxycortisol in all five cases. The pathological diagnosis of ACC was based on Weiss's criteria with its score ≥ 3. The mean size of the resected tumors was 87 mm and Ki67/MIB1 labeling index, a proliferative marker, was 3-27%. Immunohistochemical analysis revealed a disorganized expression of several steroidogenic enzymes, such as 3ß-hydroxysteroid dehydrogenase, 17α-hydroxylase, and DHEA-sulfotransferase. Among several genes determined by RT-PCR, insulin-like growth factor (IGF)-II mRNA was consistently and abundantly expressed in all 5 tumor tissues. Postoperatively, two cases with SCS developed local recurrence and liver metastasis. The present study suggests that the disorganized expression of steroidogenic enzymes and the overexpression of IGF-II by the tumor are hallmarks of ACC, which could be used as biochemical and molecular markers for ACC.

Urinary steroids from a newborn human infant. Identification of 2 alpha-hydroxy-4-pregnene-3,20-dione, 3 beta,15 beta-dihydroxy-5-pregnen-20-one and 3 beta,15 alpha-dihydroxy-5-pregnen-20-one.

Urinary steroids from healthy newborn human infants were analyzed by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. The identification of 2 alpha-hydroxy-4-pregnene-3,20-dione and the characterization of its 2 beta-isomer is recorded here for the first time. Mass spectrometric evidence supporting the identification of 3 beta,15 beta-dihydroxy-5-pregnen-20-one and 3 beta,15 alpha-dihydroxy-5-pregnen-20-one is also presented. Furthermore, the following 15-hydroxylated steroids were also found and identified: 3 beta,15 epsilon,16 epsilon-trihydroxy-5-androsten-17-one, 5-androstene-3 beta,15 alpha,16 alpha,17 beta-tetrol, 3 beta,15 beta,17-trihydroxy-5-pregnen-20-one and 5-pregnene-3 beta,15 epsilon,17,20 epsilon-tetrol. The origin of these 2- and 15-hydroxylated urinary steroids is discussed in relation to current knowledge of 4-pregnene-3,20-dione and 3 beta-hydroxy-5-pregnen-20-one metabolism during the human perinatal period.

Urinary excretion of 17-hydroxypregnanolones in patients with different forms of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency.

To improve diagnostic criteria in different (classical salt-wasting (SW), classical simple virilizing (SV) and non classical late onset (LO)) forms of congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency, we investigated the urinary excretion of 17-hydroxypregnanolones (17OH-PO(5 beta) and (5 alpha)), 15 beta-hydroxypregnanolone(15 beta OH-PO), pregnanetriol(PT) and 11-oxo-pregnanetriol (11-O-PT) compared to hydrocortisone metabolities. During the 1st month of life newborn infants with CAH-SW excreted from barely detectable to very large amounts of 17OH-PO(5 beta), 15 beta OH-PO and PT, and, in 12 of 14 cases, also 11-O-PT in their urines. From the 1st to the 28th day of life, cortisol metabolites were virtually absent in urines of CAH-SW infants. This was in contrast of 36 healthy newborn infants. We measured the excretion of 17OH-PO(5 alpha) in children with CAH of whom 19 patients with CAH-SV had a median 17OH-PO(5 alpha) excretion of 1110 micrograms/day (range: 152-5515). In 21 patients with CAH-LO, median excretion of 17OH-PO(5 alpha) was 294 micrograms/day (range: 66-1273). Besides the conventional metabolites of 17-hydroxyprogesterone (17OH-PO(5 beta), PT and 11-O-PT), no 17OH-PO(5 alpha) was detected in the urines of 14 patients with precocious pubarche, in 14 patients with virilization of unknown origin and in 94 healthy children of comparable age. The ratio of 17OH-PO(5 alpha) to tetrahydrocortisone (THE) discriminated between CAH-SV and CAH-LO from the 1st to the 18th year of age.(ABSTRACT TRUNCATED AT 250 WORDS)

A paradox: elevated 21-hydroxypregnenolone production in newborns with 21-hydroxylase deficiency.

21-Hydroxypregnenolone and its metabolite 5-pregnene-3 beta, 20 alpha 21-triol have been measured in the sulfate fraction of neonatal urine. These two steroids are the major two 21-hydroxylated 5-pregnenes produced by neonates and are almost exclusively excreted as disulfates. The excretions of these steroids by normal infants and infants with 21-hydroxylase deficiency were compared. In addition to measurement of the absolute excretion, the excretion relative to the total 3 beta-hydroxy-5-ene output was also determined. The results show that 21-hydroxypregnenolone excretion is highly elevated in 21-hydroxylase deficiency (affected, mean 887 micrograms/24 h, range 453-1431 micrograms/24 h; normal, mean 117 micrograms/24 h, range 17-263 micrograms/24 h), but when compared to excretion of other delta 5 steroids the excretion is slightly low [(21-hydroxypregnenolone + 5-pregnene-3 beta, 20 alpha, 21-triol)/total 3-beta-hydroxy-5-ene steroids, 2.9% affected; 3.6% normal]. This difference was not statistically significant. There is thus no evidence that the 21-hydroxylase acting on pregnenolone is deficient in congenital adrenal hyperplasia. The explanation of the normal activity of "pregnenolone 21-hydroxylase," although not clearly defined, is probably associated with two recent findings by other workers: (a) that the human fetus has an active 21-hydroxylase distinct from the adrenal enzyme and (b) that a 21-hydroxylase structurally very different from the adrenal enzyme, with high activity towards pregnenolone (but no activity towards 17-hydroxyprogesterone), has been isolated from rabbit hepatic microsomes.

Placental steroid sulfatase deficiency: biochemical diagnosis and clinical review.

Twenty-three cases of placental steroid sulfatase deficiency are reported. All children were boys who later acquired ichthyosis of the recessive X-linked type. The steroid sulfatase deficiency was present in placental tissue, umbilical cord leucocytes, and cultured skin fibroblasts of affected boys. An antepartum diagnosis can be obtained either by detecting the enzyme deficiency in cultured amniotic fluid cells or by finding an elevated total excretion of androstenetriol , 16 alpha-hydroxy-dehydroepiandrosterone, and 16 alpha-hydroxy-pregnenolone in maternal third-trimester urine. Vaginal delivery was accomplished in 16 patients (70%). No conspicuous pregnancy complications or neonatal problems were noted. However, birth weights tended to be relatively low. Intervention is unnecessary unless other obstetric indications require it. The incidence of this disorder appears to be approximately one per 2000 male births.

Timing of ovulation and implantation in the common marmoset, Callithrix jacchus, by monitoring of estrogens and 6 beta-hydroxypregnanolone in urine.

Urinary estrogen and 6 beta-hydroxypregnanolone excretions have been determined in female marmosets by means of HPTLC. The levels observed in cycling females during implantation and abortion are reported. Hormone analysis revealed a characteristic pattern showing cyclic fluctuations of estrogens and progesterone. Estrogen metabolites increase during the follicular phase up to 1 microgram/ml, the progesterone metabolite level rises to 1.2 micrograms/ml urine during the luteal phase. Maximum values of both are superimposed in many cases. Ovulatory cycles can be monitored by following estrogen and progesterone metabolites in urine samples. Thus ovulation and the onset of pregnancy can be estimated with a precision of +/- 24 h (or less).

Increased urinary excretion of total 16 alpha-hydroxypregnenolone in newborn infants with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Urinary excretion of total 16 alpha-hydroxypregnenolone (16 alpha-OH-P'O), pregnanetriol (PT), and 11-oxopregnanetriol (11-O-PT) were determined by capillary gas chromatography in 32 healthy neonates and three newborn infants with congenital adrenal hyperplasia (CAH) during the first 4 weeks of life. In the 2nd and 3rd week of life, only the 16 alpha-OH-P'O excretion was pathognomonically elevated in infants with 21-hydroxylase deficiency. The values amounted to 1023, 1611 (age 1--2 weeks), and 2955 micrograms/day (3 weeks of life) compared to much lower levels in healthy peers (2nd week: mean 243, range 0--520 micrograms/day; 3rd week; mean 515, range 66--1541 micrograms/day).

Isolation fo 15alpha-hydroxypregnenolone and 15alpha-hydroxydehydroisoandrosterone from human pregnancy urine.

15alpha-Hydroxydehydroisoandrosterone and 15alpha-hydroxypregnenolone were isolated from hydrolyzed extracts of human late pregnancy urine and identified by means of the isotope dilution technique. In two separate determinations the excretion rate of 15alpha-hydroxydehydroisoandrosterone was found to be 1.7 and 3.2 microgram per day while that of 15alpha-hydroxypregnenolone was 1.7 and 2.9 microgram per day. It is postulated that 15alpha-hydroxydehydroisoandrosterone might serve as a precursor of 15alpha-hydroxylated estrogens already isolated from late pregnancy urine. Similarly, 15alpha-hydroxypregnenolone might be an endogenous precursor of 15alpha-hydroxyprogesterone.

[Urinary excretion of 3beta-hydroxy-delta5-steroids in the newborn infant and the effect of ACTH administration (author's transl)].

The urinary 3beta-hydroxy-delta5-steroids excreted during the period of the first week by newborn infants were analyzed by means of gas chromatography and gas chromatography-mass spectrometry to study the changes in the amount of these steroids. The changes in the amount of each steroid after the administration of ACTH were also studied. The results are summarized as follows: 1) Seven kinds of 3beta-hydroxy-delta5-steroids were identified in the urine of the newborn infants. 17alpha, 21-dihydroxypregnenolone was newly identified. 2) The daily changes in the amount of each of these steroids differed. Generally, the amount of the C-19 steroids increased day by day and that of the C-21 steroids decreased during the period of our observation. 3) After the administration of ACTH, the amount of these urinary steroids excreted increased at nearly the same rate.

Significance of 3alpha, 17 alpha-dihydroxy-5beta-pregnane-20-one in the diagnosis of congenital adrenal hyperplasia.

The 3alpha-17alpha-dihydroxy-5beta-pregnane-20-one (17-hydroxy-pregnanolone) excretion was found by gas chromatography to be as high as the pregnanetriol in urine of 4 virilising CAH female and 2 male patients. According to our results the significance of the determination of 17-hydroxy-pregnanolone is equal to that of the pregnanetriol in the diagnosis of adrenal hyperplasia. As the 17-hydroxy-pregnanolone is demonstrable after mild acid hydrolysis, too, it can be applied independently from the pregnanetriol determination. In five out of the six patients we found the excretion of the pregnanediol to be high, too. Its value reached, irrespective of sex, the characteristic quantity of the woman's luteal phase, so it undoubtedly originated from the adrenal cortex. We dealt shortly with the explanation of high pregnanediol excretion.

Studies on steroids in urine of the male newborn.

Four different methods of isolation and purification were utilized to study steroids in urine of male newborns which was collected during the first 5 days of life. These methods included celite column, ion exchange column and thin-layer chromatography, solvolysis and enzyme hydrolysis with beta-glucuronidase and aryl sulfatase. Procedural losses were evaluated by using radioactive internal standards. Final quantitation of each steroid was achieved by comparison of its chromatographic and quantitative behavior with the respective standard steroids on various gas-liquid chromatography systems, either as parent compound or as trimethylsilyl ether derivative. The following steroids were found in the amounts indicated: progesterone, 2.1 mug/1 (pool I), 4.6 mug/1 (pool III); pregnanediol, 625.0 mug/1 (pool IIa), 605.0 mug/1 (pool IIb glucuronide), 25.4 mug/1 (pool IIb sulfate), 4.2 mug/1 (pool IIb free), 729.0 mug/1 (pool III); 16alpha-hydroxyprogesterone, 713.0 mug/1 (pool III), 16alpha-hydroxypregnenolone, 14,000.0 mug/1 (pool III); 16alpha-hydroxydehydroepiandrosterone, 2,350.0 mug/1 (pool III); 16-dehydroprogesterone, 155.0 mug/1 (pool I), 21.2 mug/1 (pool IIb glucuronide), 97.5 mug/1 (pool IIb sulfate), 5.3 mug/1 (pool III); 16-dehydropregnenolone, 382.0 mug/1 (pool I), 1,380 mug/1 (pool IIb glucuronide), 172.0 mug/1 (pool IIb sulfate), 174.0 mug/1 (pool III); 16-dehydropregnanolone, 8.3 mug/1 (pool I), 239.0 mug/1 (pool IIb sulfate). Pregnenolone, pregnanolone, 17alpha-hydroxyprogesterone and 17alpha-hydroxypregnenolone could not be detected. The results support the concept that the steroid patterns of urine of the newborn and amniotic fluid are very similar and that the amniotic fluid steroid content is mainly dependent on fetal urinary steroid excretion. The data on delta16-C21-steroids are discussed.