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.

Gas Chromatography-Mass Spectrometry Analysis of Urinary Steroid Metabolomics for Detection of Early Signs of Adrenal Neoplasm Malignancy in Patients with Cushing's Syndrome.

The metabolomics of urinary steroids was studied by gas chromatography-mass spectrometry in 25 patients with Cushing's syndrome without malignant potential and in 12 patients with malignant potential of adrenal neoplasms (Weiss score 1-3). Patients with adrenocortical adenoma (N=24) constituted the control group. In patients with Cushing's syndrome and malignant potential, increased urinary excretion of 16-oxo-androstendiol, tetrahydro-11-deoxycortisol, and 16-hydroxypregnendiol, which had 100% specificity and sensitivity >90% for the diagnosis of malignant potential. Additionally, non-classical 5-ene-pregnenes (16-OHpregnenolone, 21-OH-pregnenolone, 3ß,16,20-pregnentriol, and 3ß,17,20-pregnentriol) were identified. The revealed changes in the metabolomics of steroids can be early signs of malignant potential in patients with Cushing's syndrome. In patients with malignant potential, three signs of reduced activity of 11ß-hydroxysteroid dehydrogenase type 2 were detected and in patients without malignant potential, one sign was found. In patients with and without malignant potential, three signs increased activity of 5ß-reductase were found.

Quantifying endogenous androgens, estrogens, pregnenolone and progesterone metabolites in human urine by gas chromatography tandem mass spectrometry.

A method for the quantitation of 22 urinary steroids (androgens, estrogens and the main pregnenolone and progesterone metabolites) by means of gas chromatography tandem mass spectrometry using a triple quadrupole analyzer has been developed. Two different enzymatic hydrolysis protocols were investigated; one capable of releasing steroids present as both sulfates and glucuronides (total fraction), and another with ß-glucuronidase activity only. After selecting adequate internal standards and choosing the optimal instrumental parameters, i.e. chromatographic separation and ion transition conditions, the method was fully validated using both hydrolysis protocols. The method was shown to be linear (r >0.99) in the range of endogenous concentrations for all studied steroids with extraction recoveries higher than 80%. The use of labeled internal standards allowed for both a correct quantification and the evaluation of the rate of deconjugation for sulfates and glucuronides in every sample. In general, the sensitivity of the method was suitable for the detection of the endogenous levels, with limits of quantification ranging from 0.1 to 20ng/mL. Accuracies ranging from 80% to 120%, and relative standard deviations below 25% in intra- and inter- assay experiments were found for most of the analytes. The applicability of the validated method was tested by quantifying twenty-two metabolites in 24-h urine samples collected from healthy individuals. The ranges for the excretion of steroids in the total and glucuronide fractions obtained with the new method were compared with those available in the literature. By comparing the figures in both fractions, an estimation of the percentage that the sulfation represents for each steroid was also calculated. The presence of side enzymatic activities and the utility of the method for clinical studies as well as for doping control analysis is discussed.

Urinary cysteinyl progestogens: Occurrence and origin.

The presence of two cysteinyl progestogens, 16-cysteinyl-progesterone (16-Cys-Prog) and 16-cysteinyl-pregnenolone (16-Cys-Preg), in human urine is described for the first time. Their occurrence was unequivocally confirmed by comparison with synthesized material by using mass spectrometric detectors. Several experiments were performed in order to clarify their origin. The adrenal origin of both 16-Cys-Prog and 16-Cys-Preg can be inferred from the increase in their concentrations after ACTH stimulatory test, together with their circadian variation similar to the one observed for cortisol. Moreover, the notable increase in excretions of 16-Cys-Prog during the luteal phase of the menstrual cycle points towards an ovarian production for this progestogen. However, the analysis of samples during the course of two pregnancies revealed that, in spite of the large amounts of progesterone produced during gestation, the human placenta lacks the capacity to make 16-Cys-Prog. The adrenal and ovarian origin has been further indicated by the absence of both metabolites in samples collected from a subject with bilateral adrenalectomy and hypogonadotrophyic hypogonadism. Regarding liver action, in vitro studies with hepatocytes and progesterone indicate that, although the liver is able to metabolize progesterone to 6-dehydroprogesterone, it has not the enzymatic machinery for the generation of 16-dehydroprogesterone. Taken together, these results open the possibility for a noninvasive test for the simultaneous evaluation of progesterone biosynthesis in different organs.

Investigations on changes in ¹³C/¹²C ratios of endogenous urinary steroids after pregnenolone administration.

For the detection of possible misuse of naturally occurring anabolic androgenic steroids like testosterone (T), anti-doping laboratories use a combination of two techniques. One is molecular steroid profiling to evaluate urinary steroid concentrations and normal diagnostic ratios. The other is isotope ratio mass spectrometry (IRMS), in which the ¹³C/¹²C ratios of target analytes like T are compared to the ¹³C/¹²C ratios of endogenous reference compounds (ERCs). The ¹³C/¹²C of the most commonly used ERC, pregnanediol (5ß-pregnane-3α,20α-diol, PD), can be influenced by administration of pregnenolone (3ß-hydroxy-pregn-5-en-20-one, PREG). Therefore PREG administration bears the potential to circumvent IRMS testing for doping control samples. In order to investigate the influence of PREG on PD and on other urinary excreted steroids, administration studies with oral and transdermal application of PREG were carried out. The influence of PREG administration on concentrations and ¹³C/¹²C ratios of all investigated target analytes was negligible. Only PD and 5ß-pregnan-3α-ol-20-one (3aP) showed significant depletion in both their glucuronidated and sulfated steroids. The results suggest that appropriate alternative ERCs are: 11ß-hydroxy-androsterone/etiocholanolone, 5ß-pregnane-3α,17,20α-triol, pregn-5-ene-3ß,17,20α-triol and cholesterol. Due to its properties to disguise the misuse of anabolic steroids by influencing the ¹³C/¹²C ratio of PD, PREG should be considered to be added to the World Anti-Doping Agency (WADA) list of prohibited substances as a masking agent.

Metabolomics approach to anabolic steroid urine profiling of bovines treated with prohormones.

In livestock production, illegal use of natural steroids is hard to prove because metabolites are either unknown or not significantly above highly fluctuating endogenous levels. In this work we outlined for the first time a metabolomics based strategy for anabolic steroid urine profiling. Urine profiles of controls and bovines treated with the prohormones dehydroepiandrosterone (DHEA) and pregnenolone were analyzed with ultraperformance liquid chromatography in combination with time-of-flight accurate mass spectrometry (UPLC-TOFMS). The obtained full scan urinary profiles were compared using sophisticated preprocessing and alignment software (MetAlign) and multivariate statistics, revealing hundreds of mass signals which were differential between untreated control and prohormone-treated animals. Moreover, statistical testing of the individual accurate mass signals showed that several mass peak loadings could be used as biomarkers for DHEA and pregnenolone abuse. In addition, accurate mass derived elemental composition analysis and verification by standards or Orbitrap mass spectrometry demonstrated that the observed differential masses are most likely steroid phase I and glucuronide metabolites excreted as a direct result from the DHEA and pregnenolone administration, thus underlining the relevance of the findings from this untargeted metabolomics approach. It is envisaged that this approach can be used as a holistic screening tool for anabolic steroid abuse in bovines and possibly in sports doping as well.

Urinary marker of oral pregnenolone administration.

Pregnenolone (PREG) can potentially be abused by athletes to maintain an equilibration of the steroidal environment after sex steroids administrations. Five men volunteers orally ingested 50 mg PREG to determine optimal urinary markers for detection of this steroid. Our findings show that ingestion of PREG has no significant effects on the testosterone/epitestosterone (T/E) and testosterone/luteinizing hormone (T/LH) ratios, whereas variable changes on the carbon isotopic values of three T metabolites: androsterone, etiocholanolone, 5beta-androstane-3alpha,17beta-diol (5beta-androstanediol) together with 16(5alpha)-androsten-3alpha-ol (androstenol) and 5beta-pregnane-3alpha,20alpha-diol (pregnanediol) have been observed. The difference between the carbon isotopic values (delta13C-values) of androstenol and pregnanediol is potentially the most reliable marker of exogenous PREG administration in males. For all subjects, the differences differ by 3.0 per thousand or more over a period of about 10 h and for both of them the detection window for positivity is extended over 40 h.

A retrospective study of increased plasma progestagen concentrations in compromised neonatal foals.

Plasma progestagen concentrations were measured daily by radioimmunoassay (RIA) in 35 sick foals for the duration of their illness. The foals were divided into three groups on the basis of time to stand after birth. Foals were given intensive care treatment according to the severity of their illness. Plasma and urine concentrations of pregnenolone (P5) and pregnenediol (P5 beta beta) were measured by gas chromatography--mass spectrometry; plasma cortisol concentrations were measured by RIA and the foals' renal and respiratory status were assessed by creatinine clearance ratios and arterial oxygen concentrations respectively. Five patterns of plasma progestagen concentrations were identified; in general, values increased when the foal's clinical condition deteriorated and decreased as the foal improved. Median progestagen concentrations decreased over the first three days post partum in Group 1 foals but remained elevated in foals from Groups 2 and 3. Similar changes were observed in plasma P5 and P5 beta beta concentrations. Plasma cortisol concentrations were highest in foals from Groups 2 and 3 (P < 0.01) compared with foals from Group 1. Regardless of foal group, mean cortisol concentrations were highest (P < 0.001) in those foals treated with adrenocorticotrophic hormone compared with those treated with dexamethasone or with neither drug. There was no relationship (r2 = 0.21) between plasma cortisol and progestagen concentrations. Results from renal clearance, steroid conjugation and respiratory status suggest that these factors did not play a significant role in elevating progestagen concentrations in sick foals. It is hypothesized that there may be a relationship between adrenal stimulation and an enzyme block resulting in overproduction of P5 and P5 beta beta in the sick neonatal foal.

Metabolism of oestradiol-17 beta and progesterone in the white rhinoceros (Ceratotherium simum simum).

14C-Labelled oestradiol-17 beta and progesterone (50 mu Ci each) were injected i.v. into an adult female white rhinoceros and all urine and faeces collected separately over the next 4 days. The total recovery of injected label was 61%, 25% being present in the urine and 36% in the faeces. Of the radioactivity recovered, 69% was excreted on Day 2 of the collection period. Repeated extraction of samples obtained on Day 2 showed that, of the radioactivity in faeces, 92.4% was associated with unconjugated steroids whereas in the urine the proportion of conjugated and unconjugated steroids were similar (41.2% and 51.4% respectively). After phenolic separation of urinary steroids, HPLC followed by derivatization and recrystallization techniques identified progesterone as the major component of the unconjugated portion with 4-pregnen-20 alpha-ol-3-one as the principal metabolite in the conjugated fraction. Pregnanediol was not present. Oestrone appeared to be the most abundant oestrogen metabolite with smaller but significant amounts of oestradiol-17 beta and oestradiol-17 alpha in the unconjugated and conjugated fractions respectively. Small amounts of progesterone were found in the faecal extract in which the radioactivity consisted mainly of oestradiol-17 alpha and oestradiol-17 beta. The results have established the major excreted metabolites of oestradiol-17 beta and progesterone in the white rhinoceros and the development of more appropriate assay methods for monitoring ovarian function in African rhinoceroses should now be possible.

Identification of new steroids in patients with 17 alpha-hydroxylase deficiency by capillary gas chromatography/mass spectrometry.

Urines of two children with 17 alpha-hydroxylase deficiency contained a number of 5-pregnane- and pregnenediols, -triols and -tetrols with a hydroxy or oxo group in position 11 of the steroid ring. They are formed mainly from progesterone via 11-hydroxyprogesterone, pregnanolone and corticosterone, respectively, or from pregnenolone. Three metabolites not previously described, 16-hydroxypregnenolone, 6,21-dihydroxypregnanediol and 6-hydroxytetrahydrocorticosterone, were identified.

Effect of trilostan on steroid excretion in man: compensated inhibition of 3 beta-hydroxysteroid dehydrogenase.

Trilostan (240, 360 and 480 mg/day administered p.o. to healthy men) induced an increase (P less than 0.05) in the urinary excretion rates of DHEA, androstenediol and pregnenolone but failed to influence the excretion rates of cortisol, aldosterone, and of the four glucocorticoid metabolites, THF, allo-THF, THE and allo-THE. A rise in plasma renin concentrations was seen in the initial phase of the trial. Administration of dexamethasone in addition to trilostan suppressed plasma and urinary concentrations of cortisol and the excretion rates of all estimated steroid metabolites but did not modify the relative abundance in the excretion rates of DHEA, androstenediol and pregnenolone. These results confirm that trilostan interferes with the activity of 3 beta-hydroxysteroid dehydrogenase in man. However, under physiological conditions production of cortisol and aldosterone is kept at a constant level, most likely by compensatory stimulation of the secretion of ACTH and renin.

Steroid spectrum in human urine as revealed by gas chromatography. III. Identification and quantitation of pregnenediol at different stages of bodily development.

Gas chromatographic analysis of steroids using Sp-2100 stationary phase revealed that all urine samples from 34 healthy children (aged 4-14 years) and from 35 adults contained 5-pregnene-3 beta, 20 alpha-diol, which was also identified by GC-MS. The extent of pregnenediol excretion continuously increased from prepuberty to adult age. The observed increase in pregnenediol excretion during prepuberty points to an enhanced biosynthetic activity of the adrenal gland in the years preceding sexual maturation.

Measurement of urinary steroid production rates using stable-isotopes and GC-MS.

The urinary production rate of pregnenolone has been determined for a male subject using 7,7-d2-pregnenolone as an isotopic tracer.

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.