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1.  Studies on steroid fever II. Pyrogenic and anti-pyrogenic activity in vitro of some endogenous steroids of man 
Journal of Clinical Investigation  1970;49(12):2418-2426.
The pyrogenic properties of some C-19 and C-21 steroids were examined by in vitro incubation of human blood leukocytes with serum-buffer solutions of the steroids and injection of the 18-hr supernatants into rabbits. In previous studies this method demonstrated release of leukocyte endogenous pyrogen by etiocholanolone. With two exceptions, steroids known to cause fever in man, such as 11β-OH etiocholanolone and 3α-hydroxy-5β-pregnane-20-one were also pyrogenic in vitro. All steroids tested which are nonpyrogenic in man, such as androsterone, 3β-OH etiocholanolone, and 3α, 17α-dihydroxy-5β-pregnan-20-one were also nonpyrogenic in vitro. Solubility in aqueous solution did not correlate with pyrogenic capacity.
Inhibition of pyrogen release from human leukocytes in vitro by hydrocortisone and estradiol was demonstrated. Hydrocortisone-treated leukocytes released less pyrogen than did normal leukocytes when stimulated either by etiocholanolone or by phagocytosis of heat-killed staphylococci. On the other hand, estradiol-treated blood leukocytes and mononuclear cells showed significant suppression of pyrogen release when phagocytosis, but not etiocholanolone, was used as the stimulus. When blood cells were incubated with progesterone, greater than normal amounts of pyrogen were released following phagocytosis, and the inhibiting effect of estradiol could be partially reversed. Neither estradiol nor hydrocortisone appeared to act on rabbit leukocytes.
These studies indicate that a variety of naturally-occurring steroids may alter pyrogen release from leukocytes. Alterations in steroid balance in man may influence normal temperature regulation and contribute to clinical fevers.
PMCID: PMC322743  PMID: 5480865
2.  Studies on steroid fever 
Journal of Clinical Investigation  1968;47(1):107-117.
When a serum-buffer solution of etiocholanolone is incubated with human blood leukocytes in vitro, a pyrogen is released. Like endogenous pyrogen of leukocyte origin, this pyrogen produces prompt monophasic fevers in rabbits, does not induce fever tolerance when given daily, and is inactivated by trypsin. In many respects, the characteristics of the in vitro reaction resemble experimental steroid-induced fever. For example, release of pyrogen varies directly with the concentration of steroid. 4-8 hr of contact between steroid and leukocyte is required for activation of the cell. Rabbit leukocytes are not activated by etiocholanolone. Finally, androsterone, the 5α-isomer of etiocholanolone, does not induce pyrogen release in vitro. These studies suggest that experimental steroid fever in man may be mediated by an endogenous pyrogen released from leukocytes.
PMCID: PMC297152  PMID: 16695933
3.  Glucuronidation of the steroid enantiomers ent-17β-estradiol, ent-androsterone and ent-etiocholanolone by the human UDP-glucuronosyltransferases 
Steroids enantiomers are interesting compounds for detailed exploration of drug metabolizing enzymes, such as the UDP-glucuronosyltransferases (UGTs). We have now studied the glucuronidation of the enantiomers of estradiol, androsterone and etiocholanolone by the 19 human UGTs of subfamilies 1A, 2A and 2B. The results reveal that the pattern of human UGTs of subfamily 2B that glucuronidate ent-17β-estradiol, particularly 2B15 and 2B17, resembles the glucuronidation of epiestradiol (17α-estradiol) rather than 17β-estradiol, the main physiological estrogen. The UGTs of subfamilies 1A and 2A exhibit higher degree of regioselectivity than enantioselectivity in the conjugation of these estradiols, regardless of whether the activity is primarily toward the non-chiral site, 3-OH (UGT1A1, UGT1A3, UGT1A7, UGT1A8 and, above all, UGT1A10), or the 17-OH (UGT1A4). In the cases of etiocholanolone and androsterone, glucuronidation of the ent-androgens, like the conjugation of the natural androgens, is mainly catalyzed by UGTs of subfamilies 2A and 2B. Nevertheless, the glucuronidation of ent-etiocholanolone and ent-androsterone by both UGT2B7 and UGT2B17 differ considerably from their respective activity toward the corresponding endogenous androgens, whereas UGT2A1-catalyzed conjugation is much less affected by the stereochemistry differences. Kinetic analyses reveal that the Km value of UGT2A1 for ent-estradiol is much higher than the corresponding value in the other two high activity enzymes, UGT1A10 and UGT2B7. Taken together, the results highlight large enantioselectivity differences between individual UGTs, particularly those of subfamily 2B.
PMCID: PMC3234363  PMID: 21899827
4.  Detection of Synthetic Testosterone Use by Novel Comprehensive Two-Dimensional Gas Chromatography Combustion Isotope Ratio Mass Spectrometry (GC×GCC-IRMS) 
Analytical chemistry  2011;83(18):7158-7165.
We report the first demonstration of Comprehensive Two-dimensional Gas Chromatography Combustion Isotope Ratio Mass Spectrometry (GC×GCC-IRMS) for the analysis of urinary steroids to detect illicit synthetic testosterone use, of interest in sport doping. GC coupled to IRMS (GCC-IRMS) is currently used to measure the carbon isotope ratios (CIR, δ13C) of urinary steroids in anti-doping efforts; however, extensive cleanup of urine extracts is required prior to analysis to enable baseline separation of target steroids. With its greater separation capabilities, GC×GC has the potential to reduce sample preparation requirements and enable CIR analysis of minimally processed urine extracts. Challenges addressed include on-line reactors with minimized dimensions to retain narrow peaks shapes, baseline separation of peaks in some cases, and reconstruction of isotopic information from sliced steroid chromatographic peaks. Difficulties remaining include long-term robustness of on-line reactors and urine matrix effects that preclude baseline separation and isotopic analysis of low concentration and trace components. In this work, steroids were extracted, acetylated, and analyzed using a refined, home-built GC×GCC-IRMS system. 11-hydroxy-androsterone (11OHA) and 11-ketoetiocolanolone (11KE) were chosen as endogenous reference compounds (ERC) because of their satisfactory signal intensity, and their CIR was compared to target compounds (TC) androsterone (A) and etiocholanolone (E). Separately, a GC×GC-qMS system was used to measure testosterone (T)/EpiT concentration ratios. Urinary extracts of urine pooled from professional athletes, and urine from one individual that received testosterone gel (T-gel) and one individual that received testosterone injections (T-shot) were analyzed. The average precisions of δ13C and Δδ13C measurements were SD(δ13C) approximately ± 1‰ (n=11). The T-shot sample resulted in a positive for T use with a T/EpiT ratio of > 9 and CIR measurements of Δδ13C > 5‰, both fulfilling World Anti-Doping Agency criteria. These data show for the first time that synthetic steroid use is detectable by GC×GCC-IRMS without need for extensive urine cleanup.
PMCID: PMC3176586  PMID: 21846122
5.  The Interaction between Steroid Hormones and Lipid Monolayers on Water 
The Journal of General Physiology  1971;58(6):650-666.
The interaction of progesterone, testosterone, androsterone, and etiocholanolone with insoluble lipid films (cholesterol and saturated hydrocarbons containing either alcohol, ester, acetamide, phosphate, amine, or carboxyl groups) was studied. In addition to surface pressure and surface potential measurements of the surface films, radioactive tracers were used to measure the concentration of adsorbed steroid in the lipid films. In general, steroids form mixed films with the insoluble lipid films. Compression of the insoluble lipid films to their most condensed state leads to complete ejection of adsorbed steroid from the surface in all cases except with the amine, for which a small amount of steroid is still retained in the surface. Interactions between the steroids and insoluble lipids are primarily due to van der Waals or dispersion forces; there were no significant contributions from dipole-dipole interactions (except possibly with the amine). Specific interactions between cholesterol and the soluble steroids were not observed. Evidence suggests that low steroid concentrations influence structure of lipid films by altering the hydration layer in the surface film. In contrast to a specific site of action, it is proposed that steroid hormones initiate structural changes in a variety of biological sites; this model of steroid action is consistent with the ubiquity of many steroid hormones.
PMCID: PMC2226044  PMID: 5120392
6.  Recurrent fever of unknown etiology: failure to demonstrate association between fever and plasma unconjugated etiocholanolone 
Journal of Clinical Investigation  1969;48(3):558-563.
A sensitive method for determination of plasma unconjugated etiocholanolone by double-isotope-derivative dilution has been described. The mean values for normal subjects was 0.038±0.003 (SEM) μg/100 ml.
40 patients, 20 with familial Mediterranean fever and 20 with other diseases characterized by recurrent fever were studied. The over-all mean concentration of plasma unconjugated etiocholanolone for the patients (febrile or afebrile) was 0.101 ±0.012 μg/100 ml, significantly above that of normals. Mean plasma values for the patients while they were febrile did not differ from the mean values when they were afebrile. It is suggested that the concentration of plasma unconjugated etiocholanolone is not related to fever in these patients.
PMCID: PMC535721  PMID: 4886315
7.  Comparison of agents producing a neutrophilic leukocytosis in man. Hydrocortisone, prednisone, endotoxin, and etiocholanolone. 
Journal of Clinical Investigation  1975;56(4):808-813.
To study the potential application of glucocorticosteroid administration for the measurement of the bone marrow neutrophil reserve response, blood neutrophil count changes were measured in normal subjects after the administration of intravenous hydrocortisone (25, 50, 100, 200, and 400 mg) and oral prednisone (5, 10, 20, 40, and 80 mg). The upper three doses of both steroids increased the blood neutrophil count by approximately 4,000 cells/mm3. The neutrophilia occurring after hydrocortisone (200 mg) and/or prednisone (40 mg) was compared with that observed after endotoxin (0.8 ng/kg) and etiocholanolone (0.1 mg/kg) in 14 normal subjects, 7 patients with Wegener's granulomatosis on cyclophosphamide therapy and 10 patients with chronic idiopathic neutropenia. The normal responses (mean increase of blood neutrophils/mm3 above base line +/- 1 SEM) were: hydrocortisone 4,220 +/- 320, prednisone 4,610 +/- 360, endotoxin 6,060 +/- 880, and etiocholanolone 3,780 +/- 440. In the patient studies, etiocholanolone gave the smallest mean responses, but, in general, the results were similar for all agents. These data indicate that these glucocorticosteroids can be used as equivalent agents to endotoxin and etiocholanolone for measuring the neutrophil reserve response.
PMCID: PMC301935  PMID: 1159089
8.  Anticonvulsant Activity of Androsterone and Etiocholanolone 
Epilepsia  2005;46(6):819-827.
Men with epilepsy often have sexual or reproductive abnormalities that are attributed to alterations in androgen levels, including subnormal free testosterone. Levels of the major metabolites of testosterone – androsterone (5α-androstan-3α-ol-17-one; 5α, 3α-A), a neurosteroid that acts as a positive allosteric modulator of GABAA receptors, and its 5β-epimer etiocholanolone (5β-androstan-3α-ol-17-one; 5β, 3α-A) – may also be reduced in epilepsy. 5α 3α-A has been found in adult brain and both metabolites, which can also be derived from androstenedione, are present in substantial quantities in serum along with their glucuronide and sulfate conjugates. This study sought to determine whether these endogenous steroid metabolites can protect against seizures.
The anticonvulsant activity of 5α 3α-A and 5β, 3α-A was investigated in electrical and chemoconvulsant seizure models in mice. The steroids were also examined for activity against extracellularly-recorded epileptiform discharges in the CA3 region of the rat hippocampal slice induced by perfusion with 55 μM 4-aminopyridine (4-AP).
Intraperitoneal injection of 5α, 3α-A protected mice in a dose-dependent fashion from seizures in the following models (ED50, dose in mg/kg protecting 50% of animals): 6 Hz electrical stimulation (29.1), pentylenetetrazol (43.5), pilocarpine (105), 4-AP (215), and maximal electroshock (224). 5β, 3α-A was also active in the 6 Hz and pentylenetetrazol models, but was less potent (ED50 values, 76.9 and 139 mg/kg, respectively), whereas epiandrosterone (5α,3β-A) was inactive (ED50, ≤300 mg/kg). 5α, 3α-A (10–100 μM) also inhibited epileptiform discharges in a concentration-dependent fashion in the in vitro slice model, whereas 5β, 3α-A was active but of lower potency and 5α, 3β-A was inactive.
5α, 3α-A and 5β, 3α-A have anticonvulsant properties. Although of low potency, the steroids are present in high abundance and could represent endogenous modulators of seizure susceptibility.
PMCID: PMC1181535  PMID: 15946323
Androsterone; Etiocholanolone; Epiandrosterone; Pentylenetetrazol; Pilocarpine; 4-Aminopyridine; 6-Hz model; Seizure; Mouse
9.  Synthesis of 19-Trideuterated ent-Testosterone and the GABAA Receptor Potentiators ent-Androsterone and ent-Etiocholanolone 
19-Trideuteromethyl enantiomers of androgens namely ent-testosterone, ent-androsterone and ent-etiocholanolone were prepared by total synthesis. The isotope labeling at the carbon-19 angular methyl group was achieved by using deuterated methyl iodide (99.5% d3) for introduction of C-19 before closure of the steroid A-ring. This method yields 19,19,19-trideuterated steroids without increasing the number of steps involved in the total synthesis of ent-androgens. Analysis by mass spectrometry showed no loss of deuterium during incorporation of C-19 into ent-testosterone. The availability of the compounds will enable these ent-androgens to be distinguished by mass spectrometry from their natural enantiomers in future pharmacokinetic and metabolic studies.
PMCID: PMC2613317  PMID: 19122784
ent-testosterone-19,19,19-d3; trideuterium label; ent-androsterone-19,19,19-d3; ent-etiocholanolone-19,19,19-d3; GABAA receptor modulators
10.  Androsteroneetiocholanolone Ratios in Male Homosexuals 
British Medical Journal  1973;3(5873):207-210.
Analyses of 24-hour specimens of urine from healthy adult males for androsterone and etiocholanolone produced values which, when calculated as discriminant scores, discriminated between heterosexual and exclusively homosexual individuals. This confirms a previous study.
No significant differences were found between heterosexuals and homosexuals in parental ages, secondary sex characteristics, genitalia, anthropometry, 17-ketosteroids, and 17-ketogenic steroids.
A significant difference was found between the heterosexual group and homosexual group in the number of homosexual relatives in the immediate and extended families.
PMCID: PMC1586258  PMID: 4718836
11.  Serum metabolomic profile and potential biomarkers for severity of fibrosis in nonalcoholic fatty liver disease 
Journal of Gastroenterology  2013;48(12):1392-1400.
Biomarker for usefulness in diagnosing advanced fibrosis in nonalcoholic fatty liver disease (NAFLD) is expected. In order to discover novel biomarkers for NAFLD and its pathogenesis, we performed matabolomics screening.
(1) The initial cohort was 44 NAFLD patients. (2) This validation cohort was 105 NAFLD patients, 26 primary biliary cirrhosis (PBC) patients, and 48 healthy controls. Using capillary electrophoresis and liquid chromatography with mass spectrometry, we analyzed low molecular weight metabolites in these groups.
1. In the initial cohort, we found 28 metabolites associated with advanced fibrosis. Among them, 4 sulfated steroids showed the greatest difference. A decrease of dehydroepiandrosterone sulfate (DHEA-S) and 5α-androstan-3β ol-17-one sulfate (etiocholanolone-S) was observed with the progression of fibrosis. Furthermore, 16 hydroxydehydroepiandrosterone sulfate (16-OH-DHEA-S) increased with the progression of fibrosis. 2. In the validation cohort, the decrease of DHEA-S and etiocholanolone-S, as well as the increase of 16-OH-DHEA-S, with the progression of fibrosis was confirmed. The 16-OH-DHEA-S/DHEA-S ratio and 16-OH-DHEA-S/etiocholanolone-S ratio were even more strongly associated with the grade of fibrosis. Among PBC patients, 16-OH-DHEA-S tended to be higher in stages 3 and 4 than in stages 1 and 2. However, levels of DHEA-S, etiocholanolone-S, and the two ratios were not associated with the stage of PBC.
Several metabolic products were found to be biomarkers of fibrosis in NAFLD and could also be useful for diagnosis of this condition. Our findings suggested disturbance of hormone metabolism in NAFLD and might lead to the development of new therapy.
Electronic supplementary material
The online version of this article (doi:10.1007/s00535-013-0766-5) contains supplementary material, which is available to authorized users.
PMCID: PMC3889284  PMID: 23478936
12.  Effect of oxytetracycline administration on intestinal metabolism of oestrogens and on plasma sex hormones in healthy men. 
Gut  1987;28(4):439-445.
The effect of oxytetracycline (1 g/day for five days) on the enterohepatic recycling of oestrogens and on plasma sex hormone concentrations was assessed in healthy men. Plasma oestrone (E1), oestradiol-17 beta (E2), 4-androstenedione (A), 5 alpha-dihydrotestosterone (5 alpha-DHT), total and free testosterone (T and free T), binding capacity of sex hormone binding globulin, luteinizing hormone, dehydroepiandrosterone-sulphate, urinary total E1, E2, and oestriol (E3), and oestriol-3-glucuronide (E3-3G) and faecal unconjugated and conjugated E1, E2, and E3 were measured by radioimmunoassay (RIA). Treatment with the antibiotic significantly increased the excretion of faecal conjugated oestrogens, which parallelled a decrease in urinary oestrogen excretion, especially of E3. The effect on urinary E3 could be explained almost entirely by the simultaneous decrease of urinary E3-3G concentrations. In urine and faeces the E2/E3 and E1 + E2/E3 ratios increased, probably because of the diminished reductive metabolism of oestrogens in the gut. No significant effects on plasma unconjugated oestrogen concentrations were observed. Moreover, in the present study oxytetracycline had no remarkable effect on plasma total, or free T concentrations, nor on other plasma hormones measured. Our results suggest that enterohepatic recycling and intestinal metabolism of oestrogens may be significant in men. The mechanism of action of antibiotics on oestrogen metabolism probably involves decreased hydrolysis by beta-glucuronidase of oestrogen conjugates by the intestinal contents, diminishing the reabsorption of aglycones of oestrogen conjugates and resulting in faecal loss of the steroids.
PMCID: PMC1432818  PMID: 3034744
13.  Characterization of 17α-hydroxysteroid dehydrogenase activity (17α-HSD) and its involvement in the biosynthesis of epitestosterone 
BMC Biochemistry  2005;6:12.
Epi-testosterone (epiT) is the 17α-epimer of testosterone. It has been found at similar level as testosterone in human biological fluids. This steroid has thus been used as a natural internal standard for assessing testosterone abuse in sports. EpiT has been also shown to accumulate in mammary cyst fluid and in human prostate. It was found to possess antiandrogenic activity as well as neuroprotective effects. So far, the exact pathway leading to the formation of epiT has not been elucidated.
In this report, we describe the isolation and characterization of the enzyme 17α-hydroxysteroid dehydrogenase. The name is given according to its most potent activity. Using cells stably expressing the enzyme, we show that 17α-HSD catalyzes efficienty the transformation of 4-androstenedione (4-dione), dehydroepiandrosterone (DHEA), 5α-androstane-3,17-dione (5α-dione) and androsterone (ADT) into their corresponding 17α-hydroxy-steroids : epiT, 5-androstene-3β,17α-diol (epi5diol), 5α-androstane-17α-ol-3-one (epiDHT) and 5α-androstane-3α,17α-diol (epi3α-diol), respectively. Similar to other members of the aldo-keto reductase family that possess the ability to reduce the keto-group into hydroxyl-group at different position on the steroid nucleus, 17α-HSD could also catalyze the transformation of DHT, 5α-dione, and 5α-pregnane-3,20-dione (DHP) into 3α-diol, ADT and 5α-pregnane-3α-ol-20-one (allopregnanolone) through its less potent 3α-HSD activity. We also have over-expressed the 17α-HSD in Escherichia coli and have purified it by affinity chromatography. The purified enzyme exhibits the same catalytic properties that have been observed with cultured HEK-293 stably transfected cells. Using quantitative Realtime-PCR to study tissue distribution of this enzyme in the mouse, we observed that it is expressed at very high levels in the kidney.
The present study permits to clarify the biosynthesis pathway of epiT. It also offers the opportunity to study gene regulation and function of this enzyme. Further study in human will allow a better comprehension about the use of epiT in drug abuse testing; it will also help to clarify the importance of its accumulation in breast cyst fluid and prostate, as well as its potential role as natural antiandrogen.
PMCID: PMC1185520  PMID: 16018803
14.  Use of Steroid Profiles in Determining the Cause of Adrenal Insufficiency 
Steroids  2006;72(1):71-84.
A cortisol response to adrenocorticotropin injection is the standard test for diagnosing adrenal insufficiency. Multiple steroid hormones can now be accurately measured by tandem mass spectrometry in a single sample. The study objective was to determine whether a steroid profile, created by simultaneous measurement of ten steroid hormones by tandem mass spectrometry, would help determine the cause of adrenal insufficiency.
A ten-steroid profile was measured by tandem mass spectrometry during the performance of a standard high dose cortrosyn stimulation test. The steroids were measured at baseline, and 30 and 60 minutes following synthetic adrenocorticotropin injection. Adrenal insufficiency was defined as a peak cortisol level of less than 20 μg/dL. Testing was conducted in the General Clinical Research Center of a University Medical Center. Normal volunteers, patients suspected of having adrenal insufficiency, and patients with known adrenal insufficiency participated.
Our results showed that adrenal insufficiency of any cause was adequately diagnosed using the response of 11-deoxycortisol, dehydroepiandrosterone, or these analytes combined in a two-steroid profile. A three-steroid profile yielded a test with 100% accuracy for discriminating primary adrenal insufficiency from normal status. Primary adrenal insufficiency was well separated from secondary adrenal insufficiency using only a single aldosterone value. 11-deoxycortisol, dehydroepiandrosterone, and a two-steroid profile each provided fair discrimination between secondary adrenal insufficiency and normal status.
We conclude that stimulated levels of aldosterone, 11-deoxycortisol, dehydroepiandrosterone, and a two- or three-steroid profile provided additional discrimination between states of adrenal sufficiency and insufficiency. It is proposed that a steroid profile measuring cortisol, aldosterone, 11-deoxycortisol, and dehydroepiandrosterone would potentially improve the ability to determine the cause of adrenal insufficiency.
PMCID: PMC1952234  PMID: 17157339
adrenal insufficiency; steroid profile; tandem mass spectrometry
15.  Marked elevation of adrenal steroids, especially androgens, in saliva of prepubertal autistic children 
Autism is diagnosed on the basis of behavioral manifestations, but its biomarkers are not well defined. A strong gender bias typifying autism (it is 4–5 times more prevalent in males) suggests involvement of steroid hormones in autism pathobiology. In order to evaluate the potential roles of such hormones in autism, we compared the salivary levels of 22 steroids in prepubertal autistic male and female children from two age groups (3–4 and 7–9 years old) with those in healthy controls. The steroids were analyzed using gas chromatography–mass spectrometry and radioimmunoassay. Statistical analysis (ANOVA) revealed that autistic children had significantly higher salivary concentrations of many steroid hormones (both C21 and C19) than control children. These anomalies were more prominent in older autistic children and in boys. The levels of androgens (androstenediol, dehydroepiandrosterone, androsterone and their polar conjugates) were especially increased, indicative of precocious adrenarche and predictive of early puberty. The concentrations of the steroid precursor, pregnenolone, and of several pregnanolones were also higher in autistic than in healthy children, but cortisol levels were not different. Some steroids, whose levels are raised in autism (allopregnanolone, androsterone, pregnenolone, dehydroepiandrosterone and their sulfate conjugates) are neuroactive and modulate GABA, glutamate, and opioid neurotransmission, affecting brain development and functioning. These steroids may contribute to autism pathobiology and symptoms such as elevated anxiety, sleep disturbances, sensory deficits, and stereotypies among others. We suggest that salivary levels of selected steroids may serve as biomarkers of autism pathology useful for monitoring the progress of therapy.
Electronic supplementary material
The online version of this article (doi:10.1007/s00787-013-0472-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4042015  PMID: 24043498
Autism; Children; Saliva; Steroids; Neurosteroids
16.  Prenatal Diagnosis of Congenital Adrenal Hyperplasia Caused by P450 Oxidoreductase Deficiency 
Mutations in the electron donor enzyme P450 oxidoreductase (POR) result in congenital adrenal hyperplasia with apparent combined 17α-hydroxylase/17,20 lyase and 21-hydroxylase deficiencies, also termed P450 oxidoreductase deficiency (PORD). Major clinical features present in PORD are disordered sex development in affected individuals of both sexes, glucocorticoid deficiency, and multiple skeletal malformations.
The objective of the study was to establish a noninvasive approach to prenatal diagnosis of PORD including assessment of malformation severity to facilitate optimized prenatal diagnosis and timely treatment.
We analyzed 20 pregnancies with children homozygous or compound heterozygous for disease-causing POR mutations and 1 pregnancy with a child carrying a heterozygous POR mutation by recording clinical and biochemical presentations and fetal ultrasound findings. In 4 of the pregnancies (3 homozygous and 1 heterozygous for disease-causing POR mutations), prenatal analysis of steroid metabolite excretion in maternal urine was carried out by gas chromatography/mass spectrometry during gestational weeks 11–23.
Pregnancy complications in our cohort included maternal virilization (6 of 20) with onset in the second trimester. Seven pregnant women presented with low unconjugated estriol at prenatal screening (triple or quadruple antenatal screening test). Overt dysmorphic features were noted in 19 of the 20 babies at birth but observed in only 5 by prenatal ultrasound. These 5 had the most severe malformation phenotypes and poor outcome, whereas the other babies showed normal development. Steroid profiling of maternal urine revealed significantly increased steroids of fetal origin, namely the pregnenolone metabolite epiallopregnanediol and the androgen metabolite androsterone, with concomitant low values for estriol. Diagnostic steroid ratios conclusively indicated PORD as early as gestational week 12. In the heterozygous pregnancy, steroid ratios were only slightly elevated and estriol excretion was normal.
Prenatal diagnosis in PORD is readily established via urinary steroid metabolite analysis of maternal urine. Visible malformations at prenatal ultrasound predict a severe malformation phenotype.
PMCID: PMC3708032  PMID: 23365120
17.  Characterization of Nonpolar Lipids and Selected Steroids by Using Laser-Induced Acoustic Desorption/Chemical Ionization, Atmospheric Pressure Chemical Ionization, and Electrospray Ionization Mass Spectrometry† 
Laser-induced acoustic desorption (LIAD) combined with ClMn(H2O)+ chemical ionization (CI) was tested for the analysis of nonpolar lipids and selected steroids in a Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR). The nonpolar lipids studied, cholesterol, 5α-cholestane, cholesta-3,5-diene, squalene, and β-carotene, were found to solely form the desired water replacement product (adduct-H2O) with the ClMn(H2O)+ ions. The steroids, androsterone, dehydroepiandrosterone (DHEA), estrone, estradiol, and estriol, also form abundant adduct-H2O ions, but less abundant adduct-2H2O ions were also observed. Neither (+)APCI nor (+)ESI can ionize the saturated hydrocarbon lipid, cholestane. APCI successfully ionizes the unsaturated hydrocarbon lipids to form exclusively the intact protonated analytes. However, it causes extensive fragmentation for cholesterol and the steroids. The worst case is cholesterol that does not produce any stable protonated molecules. On the other hand, ESI cannot ionize any of the hydrocarbon analytes, saturated or unsaturated. However, ESI can be used to protonate the oxygen-containing analytes with substantially less fragmentation than for APCI in all cases except for cholesterol and estrone. In conclusion, LIAD/ClMn(H2O)+ chemical ionization is superior over APCI and ESI for the mass spectrometric characterization of underivatized nonpolar lipids and steroids.
PMCID: PMC3081587  PMID: 21528012
LIAD; Chemical ionization; Nonpolar lipids; Steroids; APCI; ESI; Aquachloromanganese ion; FT-ICR; Linear quadrupole ion trap
18.  Synthesis and In vitro cytotoxic activity evaluation of (E)-16-(substituted benzylidene) derivatives of dehydroepiandrosterone 
Background and the purpose of the study
Modified androsterone derivatives are class of steroidal compounds with potential anticancer properties. Various steroidal derivatives containing substitution at position 16 have shown diversified pharmacological activities. In the present study, a new series of cytotoxic 16-(substituted benzylidene) derivatives of dehydroepiandrosterone (DHEA) were synthesized and evaluated against three different cancer cell lines.
The cytotoxic 16-(substituted benzylidene) derivatives of DHEA were synthesized via aldol condensation of DHEA with corresponding benzaldehyde derivatives. The cytotoxic activity of synthesized derivatives was evaluated against three different cancer cells including KB, T47D and SK-N-MC cell lines by MTT reduction colorimetric assay.
The results indicated that 16-(substituted benzylidene) derivatives of DHEA could be served as a potent anti-cancer agent. The 3-cholro benzylidene derivatives of DHEA was the most potent synthesized derivative especially against KB and T47D cell lines (IC50 values were 0.6 and 1.7 μM; respectively).
The cytotoxic potential of novel benzylidene derivatives of DHEA is mainly attributed to the position and nature of the substituted group on the benzylidene pendant.
PMCID: PMC3673839  PMID: 23635011
Synthesis; Dehydroepiandrosterone (DHEA); MTT assay; Cytotoxic activity
19.  Differential Effects of Ethanol on Serum GABAergic 3α,5α/3α,5β Neuroactive Steroids in Mice, Rats, Cynomolgus Monkeys and Humans 
Acute ethanol administration increases plasma and brain levels of progesterone and deoxycorticosterone-derived neuroactive steroids (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP) and (3α,5α)-3,21-dihydroxypregnan-20-one (3α,5α-THDOC) in rats. However, little is known about ethanol effects on GABAergic neuroactive steroids in mice, non-human primates or humans. We investigated the effects of ethanol on plasma levels of 3α,5α- and 3α,5β-reduced GABAergic neuroactive steroids derived from progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone using gas chromatography-mass spectrometry.
Serum levels of GABAergic neuroactive steroids and pregnenolone were measured in male rats, C57BL/6J and DBA/2J mice, cynomolgus monkeys and humans following ethanol administration. Rats and mice were injected with ethanol (0.8 – 2.0 g/kg), cynomolgus monkeys received ethanol (1.5 g/kg) intragastrically and healthy men consumed a beverage containing 0.8 g/kg ethanol. Steroids were measured after 60 minutes in all species and also after 120 minutes in monkeys and humans.
Ethanol administration to rats increased levels of 3α,5α-THP, 3α,5α-THDOC and pregnenolone at the doses of 1.5 g/kg (+228, +134 and +860%, respectively, p<0.001) and 2.0 g/kg (+399, +174 and +1125%, respectively, p<0.001), but not at the dose of 0.8 g/kg. Ethanol did not alter levels of the other neuroactive steroids. In contrast, C57BL/6J mice exhibited a 27% decrease in serum 3α,5α-THP levels (p<0.01), while DBA/2J mice showed no significant effect of ethanol, although both mouse strains exhibited substantial increases in precursor steroids. Ethanol did not alter any of the neuroactive steroids in cynomolgus monkeys at doses comparable to those studied in rats. Finally, no effect of ethanol (0.8 g/kg) was observed in men.
These studies show clear species differences among rats, mice and cynomolgus monkeys in the effects of ethanol administration on circulating neuroactive steroids. Rats are unique in their pronounced elevation of GABAergic neuroactive steroids, while this effect was not observed in mice or cynomolgus monkeys at comparable ethanol doses.
PMCID: PMC2858248  PMID: 20028362
GABAergic Neuroactive Steroids; Ethanol; C57BL/6J and DBA/2J Mice; Non-Human Primates; Humans
20.  Targeted Androgen Pathway Suppression in Localized Prostate Cancer: A Pilot Study 
Journal of Clinical Oncology  2013;32(3):229-237.
Ligand-mediated activation of the androgen receptor (AR) is critical for prostate cancer (PCa) survival and proliferation. The failure to completely ablate tissue androgens may limit suppression of PCa growth. We evaluated combinations of CYP17A and 5-α-reductase inhibitors for reducing prostate androgen levels, AR signaling, and PCa volumes.
Patients and Methods
Thirty-five men with intermediate/high-risk clinically localized PCa were randomly assigned to goserelin combined with dutasteride (ZD), bicalutamide and dutasteride (ZBD), or bicalutamide, dutasteride, and ketoconazole (ZBDK) for 3 months before prostatectomy. Controls included patients receiving combined androgen blockade with luteinizing hormone-releasing hormone agonist and bicalutamide. The primary outcome measure was tissue dihydrotestosterone (DHT) concentration.
Prostate DHT levels were substantially lower in all experimental arms (0.02 to 0.04 ng/g v 0.92 ng/g in controls; P < .001). The ZBDK group demonstrated the greatest percentage decline in serum testosterone, androsterone, and dehydroepiandrosterone sulfate (P < .05 for all). Staining for AR and the androgen-regulated genes prostate-specific antigen and TMPRSS2 was strongly suppressed in benign glands and moderately in malignant glands (P < .05 for all). Two patients had pathologic complete response, and nine had ≤ 0.2 cm3 of residual tumor (defined as a near-complete response), with the largest numbers of complete and near-complete responses in the ZBDK group.
Addition of androgen synthesis inhibitors lowers prostate androgens below that achieved with standard therapy, but significant AR signaling remains. Tissue-based analysis of steroids and AR signaling is critical to informing the search for optimal local and systemic control of high-risk prostate cancer.
PMCID: PMC3887479  PMID: 24323034
21.  Human leukocytic pyrogen test for detection of pyrogenic material in growth hormone produced by recombinant Escherichia coli. 
Journal of Clinical Microbiology  1984;20(3):323-329.
Human growth hormone is biosynthetically produced in recombinant strains of Escherichia coli as methionyl human growth hormone (met-hGH). When purified from the bacterial culture, met-hGH is biologically active in established assays for growth hormone. Therefore, a phase I trial of met-hGH was carried out in healthy human adults; during the first trial, however, signs, symptoms, and clinical laboratory tests characteristic of an acute-phase response to pyrogenic agents was observed. Prior testing of the met-hGH preparation used in the phase I trial did not reveal evidence of toxicity, and the U.S. Pharmacopeial Convention rabbit pyrogen test, as well as the Limulus amoebocyte lysate (LAL) test, had not detected significant levels of exogenous pyrogens or endotoxin. In addition, standard inhibition studies with added endotoxin showed no inhibition by the LAL test. When this preparation of met-hGH was incubated with human blood mononuclear cells, leukocytic pyrogen (LP) was released into the supernatant medium, suggesting that the preparation contained pyrogenic material. Various lots of met-hGH based on different purification and formulating methods were tested by the human LP assay for contaminating pyrogens. The results of these tests aided in the identification of procedures for met-hGH preparations which did not induce LP in vitro. Thus, subsequent lots of met-hGH which had passed the LP test were used in repeat clinical studies, and no inflammatory or pyrogenic reactions were observed. When the LP test was used, experiments revealed that the original lot of met-hGH was contaminated with endotoxin which had not been detected in the LAL or rabbit pyrogen tests. Lyophilization in glycine-phosphate buffer had resulted in a 10- to 20-fold reduction of endotoxin reactivity in the LAL test and the U.S. Pharmacopeial Convention rabbit pyrogen test. These data provide a probable explanation for the negative result from the LAL and rabbit pyrogen test in the initial lot of met-hGH which induced acute-phase reactions. In addition, these studies demonstrate that the release of LP from human cells is a reliable indicator of the presence of materials that are pyrogenic for humans.
PMCID: PMC271322  PMID: 6386853
22.  Glucocorticoid-Binding Proteins in Human Acute Lymphoblastic Leukemic Blast Cells 
Journal of Clinical Investigation  1973;52(7):1715-1725.
The first known step in steroid hormone action is the association of the steroid with specific cytoplasmic steroid-binding proteins (SBP). Using a competitive binding assay, we detected, quantified, and partially characterized such a SBP in cytosol from glucocorticoid-sensitive human lymphoblastic leukemic blasts. The affinity of steroids for the SBP was directly related to their known killing potency. For example, steroids without glucocorticoid effect such as androstenedione, etiocholanolone, and tetrahydrocortisol were unable to displace radiolabeled dexamethasone from the SBP in the binding reaction. The dose-response curve for in vitro inhibition of [3H]thymidine uptake in leukemic blasts correlated closely with the binding affinity of glucocorticoids to the SBP, providing additional support for an essential physiologic role for SBP in steroid action. SBP activity was either greatly diminished or absent in glucocorticoid-resistant cells. Six patients who intially had SBP in their blasts and were responsive to combinations of drugs including glucocorticoids no longer had SBP activity detectable at a time when they no longer responded to combinations of drugs including glucocorticoids. In vitro [3H]thymidine uptake was not inhibited by steroids in leukemic blast cells lacking SBP activity. Other patients who had received some antileukemic therapy including glucocorticoids and who still had SBP in their leukemic blasts, were still responsive to drug combinations that included glucocorticoids. This appears to be the first study demonstrating glucocorticoid receptors in a human tissue.
PMCID: PMC302447  PMID: 4352461
23.  Integration of clinical data with a genome-scale metabolic model of the human adipocyte 
Combining large-scale immunohistochemical analysis and proteomics data, 7340 gene products are identified in human adipocytes. Based on this data, a genome-scale metabolic model is reconstructed and used to integrate clinical and transcriptome data from lean and obese subjects.
We simulated the metabolic differences between the individuals with different body mass indexes (BMIs) using transcriptome and fluxome data.An increase in the metabolic activity around androsterone, ganglioside GM2 and degradation products of heparan sulfate and keratan sulfate, and a decrease in mitochondrial metabolic activities are found in obese subjects compared with lean subjects.We simulated the change in lipid droplet (LD) size and found that lean subjects have large dynamic changes in LD formation compared with obese subjects.Besides enabling patient stratification, our study allows the identification of novel therapeutic targets for obesity.
We evaluated the presence/absence of proteins encoded by 14 077 genes in adipocytes obtained from different tissue samples using immunohistochemistry. By combining this with previously published adipocyte-specific proteome data, we identified proteins associated with 7340 genes in human adipocytes. This information was used to reconstruct a comprehensive and functional genome-scale metabolic model of adipocyte metabolism. The resulting metabolic model, iAdipocytes1809, enables mechanistic insights into adipocyte metabolism on a genome-wide level, and can serve as a scaffold for integration of omics data to understand the genotype–phenotype relationship in obese subjects. By integrating human transcriptome and fluxome data, we found an increase in the metabolic activity around androsterone, ganglioside GM2 and degradation products of heparan sulfate and keratan sulfate, and a decrease in mitochondrial metabolic activities in obese subjects compared with lean subjects. Our study hereby shows a path to identify new therapeutic targets for treating obesity through combination of high throughput patient data and metabolic modeling.
PMCID: PMC3619940  PMID: 23511207
adipocyte; flux balance analysis; genome-scale metabolic model; obesity; proteome
24.  Revalidation and rationale for high pKa values of unconjugated bilirubin 
BMC Biochemistry  2007;8:7.
Our prior solvent partition analysis, published in 1992, yielded pKa values for unconjugated bilirubin of about 8.1 and 8.4, but these results have been challenged and studies by other methods have suggested pKa values below 5.0.
We repeated our published solvent partition studies, using 14C-unconjugated bilirubin highly purified by extraction of residual labeled impurities from CHCl3 into an aqueous buffer, pH 7.0. Partition ratios at six pH values from 5.0 to 9.0 were determined by radioassay and compared with our prior values obtained by diazo assay.
At pH values ranging from 4.8 to 9.2, stable aqueous/chloroform 14C-partition ratios did not differ significantly from our published partition ratios based on diazo assay.
These results support the high pKa values of unconjugated bilirubin, above 8.0, derived from our earlier solvent partition study. In both studies, our measurements were based on the rapid analysis of clearly under-saturated solutions of highly-purified bilirubin over a wide pH range, using properly purified and preserved solvents. No previous direct estimate of the aqueous pKa values of unconjugated bilirubin meets all these preconditions. Three theoretical factors acting in combination, each related to the unique, extensive internal H-bonding of the -COOH groups, are proposed to support high pKa values of unconjugated bilirubin in water: a) donation of an H-bond from the -OH moiety of the -COOH group, which is broken on ionization; b) hindered solvation of the -COO- group after ionization; and c) restricted rotation of the -COO- and -COOH groups. Our findings and rationale rebut methodological and theoretical criticisms leveled against our prior work. High pKa values for unconjugated bilirubin dictate that: a) bilirubin diacid, which readily diffuses across membranes and can cause neurotoxicity, is the dominant unbound bilirubin species of unconjugated bilirubin in plasma at physiological pH; b) at the near-neutral pH range of gallbladder bile, the monoanion is the major unconjugated bilirubin anion present, concordant with the finding that the calcium bilirubinate precipitated in gallstones is the monoanion salt. Our conclusions are thus relevant to understanding bilirubin-induced neurological disease in severely jaundiced neonates and the precipitation of calcium bilirubinate salts in gallstones.
PMCID: PMC1877803  PMID: 17475001
The Journal of Experimental Medicine  1974;139(6):1369-1381.
Human monocytes and neutrophils were separated from buffy coats of blood obtained from normal donors. Following incubation with heat-killed staphylococci, monocyte preparations contained 20 times more pyrogenic activity in the supernatant media than did supernates from an equal number of neutrophils. During purification of these pyrogens it was discovered that these cell preparations each produced a distinct and different pyrogen. The pyrogen obtained from neutrophils had a mol wt of 15,000 following Sephadex G-75 gel filtration, an isoelectric point of 6.9, and could be precipitated and recovered from 50% ethanol at –10°C. In contrast, the pyrogen derived from monocyte preparations had a mol wt of 38,000, an isoelectric point of 5.1, and was destroyed in cold ethanol. Both molecules were unaffected by viral neuraminidase but biologically destroyed at 80°C for 20 min and with trypsin at pH 8.0. The febrile peak produced by partially purified neutrophil pyrogen occurred at 40 min while that from monocytes was at 60 min. In addition, monocyte pyrogen produced more sustained fevers for the same peak elevation as neutrophil pyrogen. These studies demonstrate for the first time two chemically and biologically distinctive pyrogens derived from circulating human white blood cells and have important implications for our understanding of the pathogenesis of fever in man.
PMCID: PMC2139679  PMID: 4829934

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