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.

New oxymesterone metabolites in human by gas chromatography-tandem mass spectrometry and their application for doping control.

Oxymesterone (17α-methyl-4, 17ß-dihydroxy-androst-4-ene-3-one) is one of the anabolic androgenic steroids (AAS) banned by the World Anti-Doping Agency (WADA). The biotransformation of oxymesterone is performed in vitro by human heptocytes and human urinary metabolic profiles are investigated after single dose of 20 mg to two adult males as well. Cell cultures and urine samples were hydrolyzed by ß-glucuronidase, extracted, and reacted with N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide (NH4 I), and dithioerythritol. After derivatization, a gas chromatography triple quadruple tandem mass spectrometry (GC-MS/MS) using full scan and MS/MS modes was applied. The total ion chromatographs of the blank and the positive samples are compared, and 7 new metabolites were found. In addition to the well-known 17-epioxymesterone, oxymesterone is metabolized by 4-ene-reduction, 3-keto-reduction, 11ß-hydroxylation, and 16ξ-hydroxylation. Based on the behavior of the MS/MS results of product ion and precursor ion modes, a GC-MS/MS method has been developed monitoring these metabolites. The structures of metabolite 2 and 4 are tentatively identified as 17α-methyl-3ß, 17ß-dihydroxy-5α-androstane-4-one and 17α-methyl-3α, 4ξ, 17ß-trihydroxy-5α-androstane, respectively. Detection of oxymesterone using new metabolites M2 and M4 can extend the detection window up to 4 days since the parent steroid was not detectable. Copyright © 2015 John Wiley & Sons, Ltd.

Soy intake and urinary sex hormone levels in preschool Japanese children.

The authors investigated whether soy intake is associated with sex steroid levels in Japanese children. This cross-sectional study was conducted in autumn 2006. Subjects were substantially healthy preschoolers, 230 boys and 198 girls, aged 3-6 years. Dietary data, including soy intake, were assessed using 3-day dietary records. Each child's dietary intake was controlled for total energy intake using the Willett method (Nutritional Epidemiology. Oxford, United Kingdom: Oxford University Press; 1990:245-271). Urinary estrone, estradiol, testosterone, and 5-androstene-3ß,17α diol levels measured using liquid chromatography-electrospray ionization tandem mass spectrometry, and urinary dehydroepiandrosterone level measured with a radioimmunoassay, were adjusted for urinary creatinine levels. In the analysis of covariance for sex steroids after adjustments for age and body mass index, soy intake was significantly negatively related to estrone and estradiol in boys and positively related to testosterone and 5-androstene-3ß,17α diol in girls. Isoflavone had a significant tendency to be negatively associated with estradiol in boys and to be positively associated with testosterone in girls. Total energy intake was not associated with any sex steroids in boys or girls. These results suggest that soy intake might affect the secretion or metabolism of sex steroids in childhood and that the effects might differ by sex.

Prepubertal healthy children's urinary androstenediol predicts diaphyseal bone strength in late puberty.

CONTEXT: During the physiological process of adrenarche, the adrenal glands of healthy children secrete increasing amounts of weak androgenic steroids partly metabolized to potent sex steroids. OBJECTIVE: The aim of the study was to examine whether adrenal androgen metabolite excretion rates before the onset of puberty may be prospectively associated with late-pubertal diaphyseal bone strength. SETTING: We conducted the study in an auxological and metabolic child nutrition research facility. STUDY POPULATION AND DESIGN: The sample included 45 healthy adolescents who underwent proximal forearm bone and muscle area measurements by peripheral quantitative computed tomography at the age of 16 yr (SD 1.5) and who had collected a 24-h urine sample 8 yr earlier, allowing to quantify the prepubertal urine metabolome. Prepubertal hormonal predictors quantified by gas chromatography-mass spectrometry were: dehydroepiandrosterone, its 16-hydroxylated downstream metabolites, 5-androstene-3beta,17beta-diol (androstenediol), sums of total androgen and glucocorticoid metabolites, cortisol, and 6beta-hydroxycortisol. MAIN OUTCOMES: Proximal forearm radius was measured. RESULTS: Of all prepubertal hormones analyzed, only sex- and age-specific androstenediol levels significantly predicted pubertal stage-, height-, and muscularity-adjusted diaphyseal bone modeling (periosteal circumference, beta = 0.67, P = 0.002; cortical area, beta = 2.15, P = 0.02), bone mineral content (beta = 2.2; P = 0.04), and polar strength strain index (beta = 12.2; P = 0.002). Androstenediol explained 5-10% of the late-pubertal diaphyseal radius variability. CONCLUSIONS: Our prospective profiling of urinary steroid metabolites in 24-h urine samples collected before puberty suggests that androstenediol is an early predictor of the diaphyseal bone strength in late puberty. This predominantly peripheral conversion product of adrenarchal dehydroepiandrosterone by 17beta-hydroxysteroid dehydrogenase may hence be involved in a sustained improvement of radial bone accretion during growth.

Over-the-counter anabolic steroids 4-androsten-3,17-dione; 4-androsten-3beta,17beta-diol; and 19-nor-4-androsten-3,17-dione: excretion studies in men.

Since the appearance of 4-androsten-3,17-dione (I) as a nutritional supplement in early 1997, we have frequently observed a characteristic deterioration of endogenous steroid profiles in athletes' urine in routine anabolic steroid testing in which concentrations of major endogenous urinary steroids and testosterone exceed normal. Human excretion studies are performed with I and newer, over-the-counter "supplements" 4-androsten-3beta,17beta-diol (II) and 19-nor-4-androsten-3,17-dione (III). Endogenous urinary steroids affected by I and II are androsterone, etiocholanolone, their hydroxylated derivatives 5alpha- and 5beta-androstan-3alpha,17beta-diols, testosterone, and epitestosterone. Their concentrations briefly increase by one to two orders of magnitude and return to normal 24 h after oral administration of I and II. The average male may test positive for testosterone because testosterone concentration rises faster than that of epitestosterone, causing the testosterone/epitestosterone (T/E) ratio to rise above the positive cutoff of 6:1. A remarkable distinction in excretion patterns was observed in eastern Asian men, for whom I and II did not affect urinary concentrations of testosterone and did not increase the T/E ratio. First-pass metabolism deactivates most of the orally administered drugs I and II, rapidly converting them into inactive androsterone and etiocholanolone. Drug II is a more effective testosterone booster because of its different metabolic pathway. After the use of III, a precursor of the potent anabolic nandrolone, high concentrations of norandrosterone and noretiocholanolone appear in urine, similar to nandrolone. These are detectable in urine for 7-10 days after a single oral dose of III (50 mg).

Estimation by gas chromatography-mass spectrometry with selected ion monitoring of urinary excretion rates of 3 alpha-androstanediol during/after i.v. administration of 13C-labelled testosterone in man.

To study in vivo the conversion of testosterone (T) into its metabolites, dihydro-testosterone (DHT) and 5 alpha-androstane-3 alpha, 17 beta diol (3 alpha-Diol) the urinary excretion rates of these steroids were determined by mass spectrometry in 6 healthy men during/after the i.v. infusion (t = 4 h) of 20 mg [13C]testosterone. In addition, plasma concentrations of T, DHT and 3 alpha-Diol were determined by radioimmunoassay. During steady state conditions at the end of the 4-h infusion of [13C]T the increase in the plasma concentrations of T from, basal, 405 +/- 140 ng/dl to 4205 +/- 804 ng/dl was paralleled by an increase in the plasma concentrations of DHT to 106.4 +/- 62.5 ng/dl) (basal: 30.8 +/- 21.8 ng/dl), and of 3 alpha-Diol to 32.2 +/- 12.5 ng/dl (basal: 12.5 +/- 13.9 ng/dl). Plasma concentrations of T, DHT and 3 alpha-Diol then returned to basal concentrations within 24 hours. Using mass-spectrometry we found a cumulative renal excretion of 13C-labelled T of 15.6 +/- 9.6 micrograms/24 h, equivalent to 0.08 +/- 0.05% of the infused amount (20 mg) of [13C]T. Whereas urinary excretion of [13C]DHT was below the level of detection by mass-spectrometry the cumulative excretion of [13C]3 alpha-Diol was 67.7 +/- 19.9 micrograms/24 hours which is equivalent to 0.3 +/- 0.1% of the infused dose of 13C-labelled testosterone. These data suggest that the determination of urinary 3 alpha-Diol by mass-spectrometry during/after the infusion of stable-labelled testosterone represents an alternative to the use of radioactive label for turnover studies.

[Androgen-producing adrenal adenoma in an 18 year-old woman: diagnosis by gas-chromatographic steroid analysis, histology and postoperative course]. / Androgen-produzierendes Nebennierenadenom bei einer 18jährigen Patientin: Diagnostik mittels gaschromatographischer Steroidanalyse, Histologie und postoperativer Verlauf.

Gas-chromatographic analysis of the urinary steroids in an 18 year-old girl with primary amenorrhoea and hirsutism revealed markedly elevated excretion of androsterone, etiocholanolone, dehydroepiandrosterone, and androstendiole, both under basal conditions and after oral dexamethasone (0.5 mg q.i.d. for three days). Adrenal scintigraphy revealed the presence of a tumour of the right adrenal gland, which was subsequently removed by unilateral adrenalectomy. Histologically, the tumour showed marked anisocytosis, but since there was no evidence of capsular or angioinvasion or of mitotic activity, it was classified as an adrenocortical adenoma. Postoperatively the patient showed regression of the virilizing syndrome and normal menstrual bleeding. Urinary steroid excretion has remained normal for four years after adrenalectomy. Androgen-producing tumours must be considered as a possible cause of hirsutism in young females and should, thus, be excluded in each case. Analysis of urinary steroids by gas chromatography is a valuable tool in accomplishing this task.

Prenatal diagnosis of placental sulphatase deficiency.

A gas chromatographic procedure for the quantitation of neutral steroid sulphates in maternal urine and its application to a suspected case of placental sulphatase deficiency is described. Low levels of oestriol coincident with elevated 16-hydroxylated metabolites of dehydroepiandrosterone in the maternal urine are shown to occur in this particular condition, and thus provide a convenient differentiation from fetal adrenal hypoplasia before birth.

The C19-mineralocorticoids in hypertension.

The excretion rates of the C19-mineralcorticoids, 16beta-hydroxy-DHEA and 16-oxo-androstenediol, were measured in subjects with low-renin essential hypertension and toxemia of pregnancy. C19-mineralocorticoid excretion in low-renin essential hypertension ranged from 70-790 microgram per day. No significant difference in 16beta-hydroxy-DHEA and 16-oxo-androstenediol excretion was found between these subjects and normal controls. Subjects with toxemia of pregnancy excreted between 350 and 2500 microgram per day of these steroids. There was no significant difference between toxemic and normal pregnancy. Thus, 16beta-hydroxy - DHEA and 16-oxo-androstenediol probably do not play an important role in either low-renin essential hypertension or toxemia of pregnancy.

Urinary metabolites of 2-formyl-17 alpha-methylandrosta-1,4-diene-11alpha, 17beta-dihydroxy-3-one in the rat.

The principal urinary metabolites of 2-formyl-17alpha-methylandrosta-1,4-diene-11alpha,17beta-dihydroxy-3-one (for myldienolone) are 2-carboxy- and 2-hydroxymethyl-17 alpha-methylandrosta-11alpha,17beta-dihydroxy-3-one, whose structures were confirmed by comparing them with synthesized samples.