Free Steroid Fraction Research Articles

Uptake and metabolism of water-borne progesterone by the mussel, Mytilus spp. (Mollusca)

Previous studies have shown that mussels can pick up 17β-estradiol [E2] and testosterone [T] from water, metabolize them and conjugate them to fatty acids (esterification), leading to their accumulation in tissue. A key requirement for the esterification process is that a steroid must have a ‘reactive’ hydroxyl group to conjugate to a fatty acid (which in T, and probably E2, is the β-hydroxyl group on carbon 17). Progesterone (P) lacks any hydroxyl groups and theoretically cannot be esterified and hence should not accumulate in mussels in the same way as E2 or T. However, it is already known that mussels have an enzyme that can achieve 5α-reduction of the A ring of T and P and that there is also another reductase that can transform the 3-oxo group of the 5α-reduced A ring of T into a hydroxyl group. We hypothesized that, although intact P cannot be directly esterified, it might nevertheless be transformed into metabolites that can. To test this hypothesis, we investigated the rate and capacity of uptake, metabolism and potential depuration of tritiated P by the common mussel, Mytilus spp. We found that tritiated P was taken up from water at a similar rate to E2 and T (mean clearance rate 49mL−1 animal−1h−1) and that, as found with the other steroids, the rate of uptake could not be saturated by the addition of non-radioactive steroid (even at 7.6μgL−1). We found that up to 66% of the radioactivity that was taken up was present in the ester fraction, suggesting that hydroxylation of the P must indeed have occurred. We then definitively identified two metabolites in the ester fraction: 5α-pregnane-3β,20β-diol and 3β-hydroxy-5α-pregnan-20-one. These same two steroids were also present in the free steroid fraction. Intact P was not detected in either of the fractions. When undergoing depuration (under semi-static conditions), the radioactivity in the ester fractions remained at the same concentration in the animals for at least 10 days. Our findings suggest that the lack of reactive hydroxyl groups on P does not preclude it from being taken up, metabolized and subsequently stored. Many questions remain, not least of which is why, when P seems to be so rapidly metabolized, two previous studies on mussels have reported concentrations of up to 30ngg−1 wet weight of P in their flesh.

Uptake and metabolism of sulphated steroids by the blood-brain barrier in the adult male rat.

Little is known about the origin of the neuroactive steroids dehydroepiandrosterone sulphate (DHEAS) and pregnenolone sulphate (PregS) in the brain or of their subsequent metabolism. Using rat brain perfusion in situ, we have found 3H‐PregS to enter more rapidly than 3H‐DHEAS and both to undergo extensive (> 50%) desulphation within 0.5 min of uptake. Enzyme activity for the steroid sulphatase catalysing this deconjugation was enriched in the capillary fraction of the blood–brain barrier and its mRNA expressed in cultures of rat brain endothelial cells and astrocytes. Although permeability measurements suggested a net efflux, addition of the efflux inhibitors GF120918 and/or MK571 to the perfusate reduced rather than enhanced the uptake of 3H‐DHEAS and 3H‐PregS; a further reduction was seen upon the addition of unlabelled steroid sulphate, suggesting a saturable uptake transporter. Analysis of brain fractions after 0.5 min perfusion with the 3H‐steroid sulphates showed no further metabolism of PregS beyond the liberation of free steroid pregnenolone. By contrast, DHEAS underwent 17‐hydroxylation to form androstenediol in both the steroid sulphate and the free steroid fractions, with some additional formation of androstenedione in the latter. Our results indicate a gain of free steroid from circulating steroid sulphates as hormone precursors at the blood–brain barrier, with implications for ageing, neurogenesis, neuronal survival, learning and memory.

Comments on ‘Low serum sex hormone binding globulin is associated with nonalcoholic fatty liver disease in type 2 diabetic patients’

Comments on ‘Low serum sex hormone binding globulin is associated with nonalcoholic fatty liver disease in type 2 diabetic patients’

Gas chromatography–mass spectrometry profiling of steroids in urine of patients with acute intermittent porphyria

ObjectiveAcute intermittent porphyria (AIP) is an autosomal dominant disease that results from a deficiency of hydroxymethylbilane synthase, the third enzyme of the heme biosynthetic pathway. AIP carriers may present acute neurovisceral attacks with hepatic overproduction of heme-precursors. In some patients, remission of the acute symptoms leads to long-term hepatic metabolic abnormalities. In this study, gas chromatography–mass spectrometry (GC/MS) was used to investigate urinary steroid metabolome of AIP patients. Design and methodsSteroid profiling in urine was performed in a group of AIP patients with biochemically active disease (n=22) and healthy controls (n=20). Five asymptomatic AIP family carriers were also studied. Commonly used ratios for the evaluation of disturbances in the steroid metabolism were calculated. ResultsWe found that etiocholanolone/androsterone and tetrahydrocortisol/5α-tetrahydrocortisol (THF/5α-THF) metabolic ratios were significantly increased in the urine of AIP patients compared to controls (2.3±0.3 vs 0.8±0.1; p<0.001 and 2.9±0.7 vs 0.9±0.1; p<0.01). The (THF+5α-THF)/tetrahydrocortisone ratio was reduced among the AIP patients (p<0.01). Quantification of the steroid absolute concentrations showed that these variations were due to a decrease of the 5α metabolites. Other ratios, like cortisol/cortisone and 6β-hydroxycortisol/cortisol in the free steroid fraction did not show differences between patients and controls. All ratios were normal among the family carriers. ConclusionA significant number of AIP patients present a basal decrease of steroid 5α-reductase activity in the liver. The deficiency may be related to malnutrition and hepatic energy misbalance associated with active AIP. Urinary steroid profiling by GC/MS may be a valuable tool to assess hepatic metabolome in AIP.

Identification of Neuroactive Steroids and Their Precursors and Metabolites in Adult Male Rat Brain

Steroids in the brain arise both from local synthesis and from peripheral sources and have a variety of effects on neuronal function. However, there is little direct chemical evidence for the range of steroids present in brain or of the pathways for their synthesis and inactivation. This information is a prerequisite for understanding the regulation and function of brain steroids. After extraction from adult male rat brain, we have fractionated free steroids and their sulfate esters and then converted them to heptafluorobutyrate or methyloxime-trimethylsilyl ether derivatives for unequivocal identification and assay by gas chromatography analysis and selected ion monitoring mass spectrometry. In the free steroid fraction, corticosterone, 3alpha,5alpha-tetrahydrodeoxycorticosterone, testosterone, and dehydroepiandrosterone were found in the absence of detectable precursors usually found in endocrine glands, indicating peripheral sources and/or alternative synthetic pathways in brain. Conversely, the potent neuroactive steroid 3alpha,5alpha-tetrahydroprogesterone (allopregnanolone) was found in the presence of its precursors pregnenolone, progesterone, and 5alpha-dihydroprogesterone. Furthermore, the presence of 3beta-, 11beta-, 17alpha-, and 20alpha-hydroxylated metabolites of 3alpha,5alpha-tetrahydroprogesterone implicated possible inactivation pathways for this steroid. The 20alpha-reduced metabolites could also be found for pregnenolone, progesterone, and 5alpha-dihydroprogesterone, introducing a possible regulatory diversion from the production of 3alpha,5alpha-tetrahydroprogesterone. In the steroid sulfate fraction, dehydroepiandrostrone sulfate was identified but not pregnenolone sulfate. Although pharmacologically active, identification of the latter appears to be an earlier methodological artifact, and the compound is thus of doubtful physiological significance in the adult brain. Our results provide a basis for elucidating the origins and regulation of brain steroids.

Salivary progesterone for the assessment of the ovarian function in the capuchin monkey (Cebus apella)

Salivary and plasma progesterone were measured in normally cycling (n=10) and castrated (n=4) femaleCebus monkeys (Cebus apella). During the follicular phase, progesterone levels in saliva ranged between 0.05 and 1.40 ng/ml and in the luteal phase they increased to between 0.22 and 4.70 ng/ml. These values represented on average 6.5 and 3.2% of those values measured in plasma, for the follicular and luteal phases, respectively. The regression analysis of the steroid concentrations in both fluids showed a highly significant correlation (r=0.8985,n=180,P<0.0001). Ovariectomized monkeys had consistently low salivary (0.37±0.02 ng/ml) and plasma (4.70±0.25 ng/ml) progesterone, showing a low, but significnat, correlation coefficient (r=0.2592,n=58,P=0.047). The ratio of plasma/salivary progesterone was significantly higher in the luteal phase (31.09±1.65) than in the follicular phase (23.06±2.26) and in castrated monkeys (16.00±1.38). The free fraction of progesterone constituted 5.3±0.2% of the total plasma progesterone during the follicular phase and 3.3±0.1% during the luteal phase. Ovariectomized monkeys showed a significantly higher percentage of free progesterone in plasma (7.7±0.1%). In contrast, free progesterone made up 64.4 and 70.9% of the total salivary progesterone for the follicular and luteal phases, respectively. The proportion of free progesterone in castrated animals was within the range observed in cycling animals. We suggest that the levels of progesterone in the saliva of capuchin monkey follow a pattern similar to that for plasma progesterone, reflecting the free steroid fraction. Thus, the measurement of such steroid in saliva may offer a valuable alternative to plasma determinations for the assessment of the ovarian function inCebus and probably other New World monkey species.

Studies on anabolic steroids. VII Analysis of urinary metabolites of formebolone in man by gas chromatography—mass spectrometry

The metabolism of formebolone in man is described. After Sep-Pak C 18 extraction, the free, conjugated and acidic steroids were separated. After enzymatic hydrolysis of the conjugates, the neutral steroids were identified by GC—MS. Two metabolites identified as 2-hydroxymethyl-11α-17β-dihydroxy-17α-methylandrost-1,4-diene-3-one and 11α-hydroxymethyltestosterone were isolated from both the free and conjugated steroid fractions. Ah acidic metabolite identified as 11α,17β-dihydroxy-17α-methyl-3-oxo-androsta-1,4,-diene-2-carboxylic acid was isolated from the free steroid fraction. This acidic steroid was stable when derivatized as the methyl ester—TMS ether derivative but its TMS ester—TMS ether derivative progressively degraded to yield the 17-mono and 11,17-di-O-TMS ether derivatives of 11α-hydroxymethandienone. The GC—MS features of formebolone metabolites are presented and its routes of biotransformation are discussed.

Studies on anabolic steroids—4. Identification of new urinary metabolites of methenolone acetate (primobolan®) in human by gas chromatography/mass spectrometry

The metabolism of methenolone acetate (17β-acetoxy-1-methyl-5α-androst-1-en-3-one), a synthetic anabolic steroid, has been investigated in man. After oral administration of a 50 mg dose of the steroid to two male volunteers, twelve metabolites were detected in urine either in the glucuronide, sulfate or free steroid fractions. Methenolone, the parent steroid was detected in urine until 90 h after administration. Its cumulative urinary excretion accounted for 1.63% of the ingested dose. With the exception of 3α-hydroxy-1-methylen-5α-androstan-17-one, the major biotransformation product of methonolone acetate, metabolites were excreted in urine at lower levels, through minor metabolic routes. Most of methenolone acetate metabolites were isolated from the glucuronic acid fraction, namely methenolone, 3α-hydroxy-1-methylen-5α-androstan-17-one, 3α-hydroxy-1α-methyl-5α-androstan-17-one, 17-epimethenolone, 3α,6β-dihydroxy-1-methylen-5α-androstan-17-one, 2ξ-hydroxy-1-methylen-5α-androstan-3,17-dione, 6β-hydroxy-1-methyl-5α-androst-1-en-3,17-dione, 16α-hydroxy-1-methyl-5α-androst-1-en-3,17-dione and 3α,16α-dihydroxy-1-methyl-5α-androst-1-en-17-one. Interestingly, the metabolites detected in the sulfate fraction were isomeric steroids bearing a 16α- or a 16β-hydroxyl group, whereas 1-methyl-5α-androst-1-en-3,17-dione was the sole metabolite isolated from the free steroid fraction. Steroids identity was assigned on the basis of the mass spectral features of their TMS ether, TMS enol-TMS ether, MO-TMS, and d 9-TMS ether derivatives and by comparison with reference and structurally related steroids. The data indicated that methanolone acetate was metabolized into several compounds resulting from oxidation of the 17-hydroxyl group and reduction of A-ring substituents, with or without concomitant hydroxylation at the C 6 and C 16 positions.

Comparison of DHT-binding and monoclonal immunoradiometric methods for the measurement of sex hormone-binding globulin

Sex hormone-binding globulin (SHBG) is conventionally measured by saturation of binding sites with 5cu-dihydrotestosterone (DHT) of known specific activity followed by separation of the bound and free steroid fractions. The techniques used for separation cf these fractions vary and the reference ranges obtained for normal subjects are not in agreement (1, 2 and refs. cited therein]. More recently, antibodies raised against purified SHBG have been used in radioimmunologic and immunodiffusion assays [2-71. Although some antisera contained immunoglobulin G and transferrin antibodies [5,8-lo], several workers have produced monospecific antisera [3,5,7]. A partially characterized murine monoclonal antibody has been reported [ll]. These antibody-based assays are not without problems. Highly purified SHBG may progressively lose binding capacity [3] and immunological activity [2] and can be extensively damaged by radioiodination [4]. Recently, a commercial kit using a radiolabelled monoclonal antibody to SHBG which recognizes a discrete determinant on the molecule has been produced. We have compared this kit with the commonly used binding assay, using ammonium sulfate to separate bound from free steroid.

Plasma sex hormone-binding globulin levels decrease during the second decade of life irrespective of pubertal status.

The plasma concentrations of sex hormone-binding globulin (SHBG) and sex steroids determine the nonprotein bound or free steroid fraction, which probably exerts the biological activity of sex steroids. Androgens lower and estrogens raise SHBG levels. The established pubertal fall in SHBG levels occurring in men has been attributed to rising androgen levels. In this study we examined the relationship between plasma SHBG and androgens in four men with untreated isolated gonadotropin deficiency and in two siblings with complete androgen insensitivity. In patients with untreated isolated gonadotropin deficiency there was a highly significant inverse correlation between SHBG levels and age (r = -0.9, P less than 0.001), although testosterone levels did not rise and there was no relationship between SHBG levels and testosterone, androstenedione, or dehydroepiandrosterone-sulfate. Two 46 XY siblings, who were phenotypic females, with complete androgen insensitivity had a marked decline in SHBG levels from 28.0 ng/ml at 9 yr to 17.1 ng/ml at 13 yr and from 15.2 ng/ml at 12 yr to 8.1 ng/ml at 16 yr, respectively. These observations indicate that the fall in SHBG levels during the second decade of life occurs irrespective of androgen activity and is under the control of other unidentified influences.

The application of rate dialysis to the determination of free steroids in plasma

Rate dialysis is used to obtain the free steroid fraction in undiluted plasma at 37°C. The free steroid fraction is determined from the rate at which a small amount of tritiated steroid diffuses from plasma on one side of a semipermeable membrane into an identical plasma sample on the other side which lacks radioactive steroid. The method may be generally applicable to steroids since the cell permeability constant, which is a function of the volume of the dialysis cell and the area and diffusion properties of the membrane, was similar for seven steroids tested. The method requires only 0.3 ml of plasma, is simple and economical to perform, and enables up to 120 determinations to be made in one day. The free fractions of cortisol, progesterone, and estradiol-17β were measured in plasma pooled from pregnant and non-pregnant women and pregnant and lactating sows. The results were compared with those obtained for the same plasma pools by centrifugal ultrafiltration.

Hypersecretion of a new corticosteroid, 18-hydroxycortisol in two types of adrenocortical hypertension.

The most abundant substance in the urinary free steroid fraction of patients with primary aldosteronism has been identified as 18-hydroxycortisol. 18-hydroxycortisol is very likely an adrenocortical secretory product rather than a peripheral metabolite, since it is abundantly synthesized by aldosteronoma tissue slices. The biogenesis of 18-hydroxycortisol takes place from cortisol rather than from 18-hydroxycorticosterone; that is, 17 alpha-precedes 18-hydroxylation. Cortisol 18-hydroxylation appears to be unrelated to the two other types of adrenocortical hydroxylation at this position. The pathway is present to a small extent in the normal human adrenal cortex and is only moderately stimulated by ACTH. Cortisol 18-hydroxylation is markedly accentuated in two circumstances: in the aldosteronoma cell where its presence may serve to distinguish tumor from bilateral hyperplasia and in ACTH-stimulatable hyperaldosteronism where it represents the first qualitative steroid biochemical abnormality to be demonstrated and as such may be useful in diagnosis and genetic studies. The possible contribution of 18-hydroxycortisol to the severity of the clinical manifestations of mineralocorticoid excess in these two types of aldosteronism remains to be explored.

Glucosiduronidation and esterification of androsterone by human breast tumors [formula omitted][formula omitted

The metabolism of 3H-androsterone was studied in homogenates (fortified with uridine 5'-diphosphoglucuronic acid and andenosine 3'-phosphate 5'-phosphosulfate) of eighteen breast tumors, one muscle underlying the primary breast carcinoma and metastatic axillary lymph nodes from a patient with suspected primary breast cancer. The major metabolites identified were less polar than androsterone. On saponification these lipoidal derivatives afforded androsterone as the only product (3 to 48%). Unmetabolized androsterone and lesser quantities of epiandrosterone, 5α-androstane-3α,17β-diol and 5α-androstane-3,17-dione comprised the free steroid fraction. Androsterone glucosiduronate was isolated (0.17–4.1%) from eight breast tumor homogenates and from the node tissue incubation (17%). There was no apparent correlation between glucuronyltransferase activity and histopathology or estrogen receptor content.

Androgen biosynthesis by testes of the goldfish Carassius auratus in vitro: The effect of temperature on the formation of steroid glucuronides

Testes of the goldfish, Carassius auratus, were incubated with [ 3H]testosterone, [ 3H]-androstenetrione, and [ 3H]pregnenolone at a range of temperatures from 1 to 46°. With testosterone as substrate, high yields of the glucuronides of testosterone and 11-ketotestosterone were obtained between 26 and 31°, but formation of free 11-ketotestosterone was favored by lower temperatures (11–26°). When androstenetrione was used as substrate, the sole metabolites were 11-ketotestosterone and its glucuronide which were obtained in maximal yields of 26–31 and 31–36°, respectively. With pregnenolone as substrate, the optimal temperature for formation of glucuronides of testosterone and 11-ketotestosterone was 31–36°. Pregnenolone glucuronide was formed in significant quantities only above 41° indicating a substrate specificity of the glucuronyl transferase enzyme. We were unable to isolate either testosterone or its 11-oxygenated derivatives from the free steroid fraction, the major components of which were pregnenolone and an unidentified hydroxylated derivative of 4-pregnene-3, 11-dione which showed an optimum temperature for formation of 26–36°.

Deficiency of Cortisol 11??-Ketoreductase ??? A New Metabolic Defect

A deficiency of cortisol 11β-ketoreductase was observed in 2 patients with the syndrome of apparent mineralocorticoid excess and no evidence of oversecretion of any known steroid hormone. The syndrome is characterized by hypertension, hypokalemia and suppressed renin and ACTH despite low secretion of aldosterone, cortisol and other adrenocortical steroids. Excretion of 5α-reduced cortisol (DHF) is increased in the urinary free steroid fraction. The 11-ketoreductase deficiency was demonstrated by a markedly elevated ratio of urinary THF/THE (6-10). In normal children the THF/THE ratio is usually less than 1. The deficiency was further proven by demonstrating an inability to form tritiated water after infusion of 11α3H cortisol. In normal subjects and in the unaffected mother of a patient, 65-80% of 11α3H cortisol appeared as tritiated water. Speculation: In these patients the 11-ketoreductase deficiency produces an impairment of the metabolism of cortisol to cortisone, resulting in a prolonged cortisol half-life, suppression of ACTH and normal serum cortisol concentration. The enzyme defect protects the patient from adrenal insufficiency despite low cortisol secretion and may contribute to hypertension and hyporeninemia because of formation of excess DHF which has mineralocorticoid activity.

318 DEFICIENCY OF CORTISOL 11-β-KETOREDUCTASE — METABOLIC DEFECT

A deficiency of cortisol 11-β-ketoreductase was observed in 2 patients with the syndrome of apparent mineralocorticoid excess and no evidence of oversecretion of any known steroid hormone (JCEM 44:924, 1977). The syndrome is characterized by hypertension, hypokalemia and suppressed renin and ACTH despite low secretion of aldosterone, cortisol and other adrenocortical steroids. Excretion of 5-α-reduced cortisol (DHF) is increased in the urinary free steroid fraction (JCEM 44:799, 1977). The 11 ketoreductase deficiency was demonstrated by a markedly elevated ratio of urinary THF/THE (6-10) in the resting state during ACTH and hydrocortisone treatment. In normal children the THF/THE ratio is usually less than 1. The deficiency was further proven by demonstrating an inability to form tritiated water after infusion of 11α3H cortisol. In normal subjects and in the unaffected mother of a patient, 65-80% of 11α3H cortisol appeared as tritiated water. Speculation: In these patients the 11 ketoreductase deficiency produces an impairment of the metabolism of cortisol to cortisone, resulting in a prolonged cortisol half-life, suppression of ACTH and normal serum cortisol concentration. The enzyme defect protects the patient from adrenal insufficiency despite low cortisol secretion and may contribute to hypertension and hyporeninemia because of formation of excess DHF which has mineralocorticoid activity.

Die Messung von freiem Cortisol, freiem Testosteron und freiem Östradiol-17β; Vergleich fünf verschiedener Methoden

5 methods were used to measure plasma levels of free cortisol testosterone and estradiol 17-beta in plasma samples at 37 degrees C taken from persons 20-40 years of age. Some of the subjects were women who used oral contraceptives and women in the second trimester of pregnancy. The results of these methods were compared to standard values to determine their relative suitability and accuracy. The methods used were: equilibrium dialysis of undiluted plasma; equilibrium dialysis of 1.5 diluted plasma; equilibrium analysis of undiluted plasma with graphic determination of the free steroid fraction; zonal chromatography; and centrifugal filtration. All 3 equilibrium dialysis methods showed practically identical hormone values with the graphical determination deviating the most. The centrifugal filtration methods showed the greatest deviation. Zonal chromatography gave the most accurate results and also registered very low levels of hormone concentration but it requires larger plasma samples making it impractical. Equilibrium dialysis with undiluted plasma is nearly as accurate and is recommended for its practicability. (Summary in ENG)

Steroid metabolism in human and boar testis tissue. Steroid concentrations and the position of the sulfate group in steroid sulfates

The steroid composition of and steroid conjugation in human cadaver and boar testes were investigated by analyzing the endogenous steroids. Gas-liquid chromatography and gas chromatography-mass spectrometry were used to identify the steroids and to determine the position of the sulfate group in sulfate conjugates. For the latter purpose, the steroids were first acetylated and subsequently solvolyzed and converted to trimethylsilyl ethers. In addition to the compounds previously identified as endogenous components, human testis was also found to contain progesterone, 17 α -hydroxyprogesterone and 20 α -hydroxy-4-pregnen-3-one in the free steroid fraction and 3 β -hydroxy-17 α -5-pregnen-20-one in the monosulfate fraction.

Free and sulphate-conjugated 16-unsaturated C 19 steroids in human testis tissue

Free and monosulphated 5α-androst-16-en-3α-ol, 5α-androst-16-en-3β-ol and 5,16-androstadien-3β-ol were identified and quantified in human cadaver testis tissue. Free and monosulphated steroid fractions were isolated by Sephadex LH-20 chromatography. Further separation and purification of the 16-unsaturated C 19 steroids was achieved using silicic acid and hydroxyalkoxypropyl Sephadex chromatography. The steroids were finally identified by gas-liquid chromatography and gas chromatography-mass spectrometry. In the free steroid fraction the relative abundance of the 16-unsaturated C 19 steroids was 5α-androst-16-en-3β-ol > 5,16-androstadien-3β-ol > 5α-androst-16-en -3α-ol and in the monosulphate fraction 5,16-androstadien-3β-ol > 5α-androst-16-en-3β-ol > 5a-androst-16-en-3α-ol.

Excretion of steroid hormones in adults steroids in urine from adults

Gas chromatography-mass spectrometry has been used to study the excretion pattern of neutral steroids in urine from adult female and male subjects. The compounds were isolated as free steroids, steroid “glucuronides”, monosulphates and disulphates. The major part, 72% (female) and 76% (male) of the steroids isolated occurred as “glucuronides”, whereas the free, monosulphate and disulphate fractions contained 19% (female) and 12% (male), 6% (female) and 9% (male), 3% (female) and 3 % (male) of the steroids isolated, respectively. The free steroid fractions contained 10 steroids, and the glucuronide, monosulphate and disulphate fractions 17, 18 and 7 steroids, respectively. Of the total steroids identified 23 were C 19 steroids and 13 were C 21 steroids. 9 steroids were found only in urine from female subjects and 3 only in urine from male subjects. The following 11 compounds have not previously been found in urine from normal, non-pregnant adult subjects: 5-androstene-3α,17α-diol, 3α-hydroxy-5β, 17α (and 17β)-pregnan-20-one, 3α(and 3β), 18-dihydroxy-5α-androstan-17-one, 5α-androstane-3α, 15α,17β-triol, 5α(and 5β)-pregnane-3α,16α,20α-triol, 5-pregnene-3α, 16α, 20α-triol, 5α-pregnane-3β, 11β,17α,20β-tetrol and 3α, 16β-dihydroxy-5α-androstan-17-one. The results are discussed in relation to present knowledge of excretion of steroids in humans.