Detection of 5α-androst-2-en-17-one and variants: Identification of main urinary metabolites in human urine samples by GC-MS and NMR.

Two steroids were identified in a supplement named D-2 following the detection of unknown compounds during the routine testing of an athlete's sample. The main glucuroconjugated metabolites were isolated from this urine by high performance liquid chromatography (HPLC) following enzymatic hydrolysis and identified by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) analyses as being 2α-hydroxy-5α-androst-3-en-17-one (M1) and 2ß,3α-dihydroxy-5α-androstan-17-one (M2). A third metabolite, 3α,4ß-dihydroxy-5α-androstan-17-one (M3) was also detected, however in lower amounts. The precursor steroids, 5α-androst-2-en-17-one (1) and 5α-androst-3-en-17-one (2) were present in the first D-2 products offered on the Internet. Later, the corresponding 17-hydroxyl compounds were offered as such or as esters (acetate, cypionate) in different relative ratios. Both M2 and M3 were synthesized from the trans-diaxial hydrolysis of the corresponding 2α,3α- and 3α,4α-epoxides (3). These were excreted in the hours following the controlled administration of the commercial product called D-2 R to a male volunteer and were also produced from the incubation of 1 and 2 with S9 liver fractions. Some preparations contain predominantly the alkene in C-2 and, therefore, an efficient detection method must include both primary metabolites M1 and M2. The latter was found equally in the fractions extracted following the enzymatic hydrolysis with ß-glucuronidase and the chemical solvolysis, which may ease its identification. Copyright © 2016 John Wiley & Sons, Ltd.

Comparing older and younger Japanese primiparae: fatigue, depression and biomarkers of stress.

This cohort study of primiparae was conducted to answer the following questions: Do older (≧ 35 years) and younger (20-29 years) Japanese primiparous mothers differ when comparing biomarkers of stress and measures of fatigue and depression? Are there changes in fatigue, depression and stress biomarkers when comparing older and younger mothers during the postpartum period? The Postnatal Accumulated Fatigue Scale and the Edinburgh Postnatal Depression Scale were administered in a time-series method four times: shortly after birth and monthly afterwards. Assays to measure biomarkers of stress, urinary 17-ketosteroids, urinary 17-hydroxycorticosteroids and salivary chromogranin-A, were collected shortly after delivery and at 1 month postpartum in both groups and a third time in older mothers at the 4th month. Statistical testing showed very little difference in fatigue, depression or stress biomarkers between older and younger mothers shortly after birth or 1 month later. Accumulated fatigue and depression scores of older mothers were highest 1 month after delivery. Additional cohort studies are required to characterize physical/psychological well-being of older Japanese primiparae.

[Effect of chronic sensorineural hearing loss on several indicators of immune and endocrine systems of 7-11 year-old children].

We investigated the effects of chronic bilateral sensorineural hearing loss of III-IV degree on the performance of interleukins, immunoglobulins serum and saliva, the functional activity of granulocyte-monocyte cell immunity, evaluated the activity of the hypothalamic-pituitary-adrenal system in children aged 7-11 years. It was found that due to stress activation of the sympathetic-adrenal system the function of granulocytes and monocytes is suppressed, with a predominance of production of anti-inflammatory interleukins. This leads to the dominance of T-helper type 2. Products granulocytes and T-helper type-2 anti-inflammatory interleukins IL-4, IL-5, IL-10, IL-13 leads to the activation of B-cells. Thus, in children 7-11 years of age with congenital bilateral sensorineural hearing loss is a decrease of non-specific humoral immunity dominated type of immune response to increased levels of IgG.

Monitoring of therapy in congenital adrenal hyperplasia.

BACKGROUND: Congenital adrenal hyperplasia is a group of disorders caused by defects in the adrenal steroidogenic pathways. In its most common form, 21-hydroxylase deficiency, patients develop varying degrees of glucocorticoid and mineralocorticoid deficiency as well as androgen excess. Therapy is guided by monitoring clinical parameters as well as adrenal hormone and metabolite concentrations. CONTENT: We review the evidence for clinical and biochemical parameters used in monitoring therapy for congenital adrenal hyperplasia. We discuss the utility of 24-h urine collections for pregnanetriol and 17-ketosteroids as well as serum measurements of 17-hydroxyprogesterone, androstenedione, and testosterone. In addition, we examine the added value of daily hormonal profiles obtained from salivary or blood-spot samples and discuss the limitations of the various assays. SUMMARY: Clinical parameters such as growth velocity and bone age remain the gold standard for monitoring the adequacy of therapy in congenital adrenal hyperplasia. The use of 24-h urine collections for pregnanetriol and 17-ketosteroid may offer an integrated view of adrenal hormone production but target concentrations must be better defined. Random serum hormone measurements are of little value and fluctuate with time of day and timing relative to glucocorticoid administration. Assays of daily hormonal profiles from saliva or blood spots offer a more detailed assessment of therapeutic control, although salivary assays have variable quality.

Unique potentiometric detection systems for HPLC determination of some steroids in human urine.

Isocratic HPLC with potentiometric detection is used for the determination of some 17-ketosteroids (17-KS), e.g., androsterone, dehydroepiandrosterone and estrone, and their respective sulfated conjugates (17-KSS). Glassy carbon or composite electrodes containing a mixture of graphite and poly(vinyl chloride), PVC, were used as substrate electrodes. These substrates were covered either by montmorillonite or potassium tetrakis(p-chlorophenyl) borate containing PVC-based rubber phase membranes. The neutral 17-KS compounds were derivatized with Girard's reagent P (GP) to obtain cationic pyridinium acetohydrazones prior to the HPLC/potentiometric detection assay. No side reactions were observed, and the GP itself was not interfering. The method yielded accurate and reproducible results and was applicable to samples containing down to micromolar concentrations. Next, the 17-KSS compounds, acting as anionic charged molecules, were determined directly in human urine samples with the HPLC/potentiometry combination without preliminary derivatization. For this purpose, a new anion-sensitive potentiometric electrode was developed using a macrocyclic polyamine containing, PVC-based, rubber phase membrane. The three 17-KSS compounds were also determined accurately down to micromolar concentrations. Especially, the main androgen metabolites as dehydroepiandrosterone sulfate and androsterone sulfate could be selectively determined with a developed potentiometric sensor in human urine samples without time-consuming cleanup and preconcentration step.

Xenobiotic action on steroid hormone synthesis and sulfonation the example of lead and polychlorinated biphenyls.

OBJECTIVES: In the present study, the metabolism of steroid hormones has been investigated to determine whether and how xenobiotics like lead (Pb) and polychlorinated biphenyls (PCBs) interfere with steroid hormone biotransformation in humans. METHODS: Three groups of subjects were tested for concentration of urinary total steroids, 17-ketosteroids (n = 5), pregnane derivates (n = 6), 17-hydroxycorticosteroids (n = 11) and their sulfonated compounds: 14 workers exposed to lead, with a mean Pb blood concentration (PbB) of 29.21 microg/dl; 15 subjects exposed to PCBs, with a mean PCB blood concentration (PCBB) of 61.69 microg/l; a control group (n = 25). RESULTS: The urinary concentrations of 17-ketosteroids and 17-hydroxycorticosteroids were significantly lower in the PCB-exposed groups. There were significantly fewer sulfonated 17-hydroxycorticosteroids in the subjects exposed to PCBs as compared to the controls, while the percentage of sulfonated steroids was lower for both 17-ketosteroids and 17-hydroxycorticosteroids in the PCB-exposed subjects, but only for the 17-hydroxycorticosteroids in the group of subjects exposed to Pb (P < 0.05). Pregnane derivate urinary concentrations did not differ between the three groups. CONCLUSION: Our results suggest that PCBs and Pb act on steroid hormone metabolism with different effects and only partially using the same hormone pathways; they may cause changes in endogenous hormone homeostasis and interfere with the xenobiotic phase II of detoxification. PCBs interfere on a larger number of steroids and cause more significant effects than Pb. It is likely that different mechanisms are involved in steroid hormone metabolism interference.

Dual examinations for identification of urine as being of human origin and for DNA-typing from small stains of human urine.

Concurrent methods for identification of urine as being of human origin, and for DNA-typing from small stains of human urine were examined. A urine stain was extracted with phosphate-buffered saline (PBS), and the extract was filtered using a Centricon-100 device. The filtrate was subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) for identification of human urine and a DNA-typing sample was obtained by dialfiltration of the residue using a DNA purification kit. After the purified residue was treated with an AmpflSTR Profiler PCR amplification kit, the DNA-types were analyzed by capillary electrophoresis using a Genetic Analyzer. It was possible to identify a urine stain as being of human origin, and complete DNA profiles could be successfully obtained from a urine stain which had been created by 50 microL of female urine. Serial analyses of urine stains found at a crime scene provide effective information for forensic investigation. This method is recommended for stain identification and for DNA-typing from a urine stain.

[Stress effects of 72-hour sleep deprivation].

Stress effects of 3-d (72-hr) continuous vigilance were studied for renal excretion of 17-oxycorticoids (17-KS), electrolytes (potassium and sodium), and liquid. The investigation involved 12 male subjects aged 23 to 36. Baseline parameters displayed diurnal variations with comparatively high day and comparatively low night values. During the 72-hr vigilance, significant and consistent (statistically fiducial) intensification of 17-KS and potassium excretion occurred in the night-time, i.e., in the period of naturally low values, but only in the first two sleepless nights. As for sodium and liquid excretion, stress-related reduction in these parameters was more consistent and distinct in the day-time, i.e., in the period of naturally high values, rather than in the nighttime. Therefore, the two factors that should be accounted for by investigators of continuous vigilance (and, maybe, other stresses) are the anticipated direction of a stress-reaction and baseline diurnal variation of parameters under study. If stress is expected to increase a parameter, measurements should be done when the parameter is naturally low and, vice versa, if stress is known to reduce a parameter, then measurement should be planned in the period when it is naturally high. If direction of stress-related changes is predictable, investigations can be performed on a twenty-four hour basis.

Stress barometer at diagnoses in children with school non-attendance.

BACKGROUND: School non-attendance is a major social problem in Japan. Many children stop attending school for a variety of reasons. The authors previously reported stress barometer values for healthy Japanese children. In this study, the authors examined the stress barometer values of children with school non-attendance. METHODS: The authors measured stress barometer values, that is, urinary 17-hydroxycorticosteroids (17-OHCS) and 17-ketosteroid sulfates (17-KS-S) in 65 children (40 girls and 25 boys; 7-15 years of age) with school non-attendance, except for pervasive developmental disorder and mental retardation, who attended the outpatient department of Dokkyo University School of Medicine Hospital, Tochigi, Japan, during the past 4 years. RESULTS: A total of 24 (36.9%) of the 65 children had urinary 17-OHCS values above 2SD, and 14 (21.5%) had urinary 17-OHCS below 2SD. In total, 10 (15.4%) children had urinary 17-KS-S values above 2SD, and four (6.2%) had urinary 17-KS-S below 2SD. Five (7.7%) children had urinary 17-KS-S/17-OHCS values above 2SD, and 10 (15.4%) had urinary 17-KS-S/17-OHCS below 2SD. CONCLUSION: The stress barometer values appear to be clinically useful for evaluating objectively whether children with school non-attendance have emotional stress.

Relationship between fluvoxamine and stress barometer for nocturnal enuresis.

BACKGROUND: It has been reported recently that fluvoxamine (a selective serotonin reuptake inhibitor) is effective and safe for children with monosymptomatic nocturnal enuresis (MNE). However, the exact mechanism by which fluvoxamine is beneficial in the treatment of MNE remains unknown. One possibility is that it controls emotional stress. METHODS: We divided children with MNE into primary MNE (n = 40) and secondary MNE (n = 7). We measured urinary 17-hydroxycorticosteroids (17-OHCS) and 17-ketosteroid sulfates (17-KS-S) as a stress barometer in children with MNE to evaluate adaptation to emotional stress before and during fluvoxamine treatment. We initially administered fluvoxamine at a dose of 25 mg at bedtime. If patients remained incontinent after 3 weeks, we increased the dose to 50 mg. RESULTS: Fluvoxamine was effective in 26 of 28 children (93%) with primary MNE and an abnormality of the stress barometer and in six of six children (100%) with secondary MNE and an abnormality of the stress barometer. Fluvoxamine was effective in only six of 12 children (50%) with primary MNE and normality of the stress barometer and was not effective in one child with secondary MNE and normality of the stress barometer. CONCLUSIONS: The stress barometer is useful clinically for evaluating the therapeutic effect of fluvoxamine for children with MNE.

Micro-analytical identification, by NMR, of elevated steroid in urine of Cushing's syndrome patients.

We previously reported that the daily urinary unidentified ketosteroid glucuronide (US-G) level in patients with Cushing's syndrome was much higher than that in the healthy subjects. Furthermore, urine samples from patients with Cushing's syndrome, including those with pituitary adenoma and adrenal adenoma, yielded almost the same high excretion levels, despite the different sites of the adenomas. We extracted US obtained by hydrolysis of US-G in urine of patients with Cushing's syndrome, purified it, and analyzed its chemical structure. Molecular weight and molecular formula were analyzed by MS spectrometry, and the chemical structure was analyzed by NMR spectrometry, utilizing small quantities of refined US. The substance has a molecular weight of 304 Da, a molecular formula of C19H28O3, and its chemical structure is 3alpha,11beta-dihydroxyandrost-4-en-17-one.

[A study of logotherapy for chronic low back pain patients].

DHEA-S (dehydroepiandrosterone sulfate) has many roles in human body as comprehensive vital power, whose metabolite is urine 17-KS-S (abbreviated S), having function of anti-cortisol. The metabolite of cortisol is urine 17-OHCS (abbreviated OH). DHEA-S is produced not only in adrenal glands but in brain. In order to examine the effects of logotherapy, urine S and OH were examined. Subjects were chronic low back pain patients treated by loxoprofen sodium (NSAID). In Group 1 (n = 11) logotherapy was not added, but in Group 2 (n = 10) it was added. Before the treatment, both groups showed low S and high OH. After 3 weeks, Group 2 showed higher S and lower OH than Group 1. After 18 weeks, 4 cases (40%) were relapsed in Group 2, and 10 (90.9%) were in Group 1 (p < 0.05). Group 2 was divided into 2 groups; relapsed group (n = 4) and non-relapsed group (n = 6). S, OH, S/OH were examined between 2 groups of Group 2 and Group 1. Relapsed group of Group 2 and Group 1 showed lower S and higher OH than non-relapsed group of Group 2. [Discussion] Logotherapy is a method to activate comprehensive human vital power. This is the mechanism through stimulating and activating human brain function.

[A study of psychoneuroendocrinological effects of music therapy].

The objective evaluation of Psychoneuroendocrinological effects of music is performed in listening and performing. Subjects were classified into 3 groups; a control group and performance/listening groups voluntarily in each 10 subjects. Urine 17-KS-S (abbreviated S) and 17-OHCS (abbreviated OH) were measured before (phase I), after the experiments (phase II) and on the following morning (phase III). They enjoyed listening to their favorite music by stereo sets or performing favorite music instruments. No directions were done on the quality of music. OH showed decreasing effects in both control and performance/listening groups. S showed significant increase in listening group. From a rise of S/OH a wear and tear by stress and a skewness of balance of restoration were corrected. Music is considered that it contributed to relaxation from stress and a wear and tear by the stress and correction of balance of restoration. The fact that the efficacy persisted until the morning of the following day indicates the efficacy persists for a relatively long period of time.

Identification of human urine stains by HPLC analysis of 17-ketosteroid conjugates.

A new method for identifying human urine stains utilizing high-performance liquid chromatographic (HPLC) analysis of five major 17-ketosteroid conjugates: dehydroepiandrosterone sulfate, etiocholanolone sulfate, etiocholanolone glucuronide, androsterone sulfate, and androsterone glucuronide was examined. Samples of urine stains were extracted with borate buffer solution (pH 9.3) and the extracts were applied onto a Sep-Pak tC18 cartridge. The analytes were eluted from the cartridge with methanol. The eluates were prelabeled with 2,4-dinitrophenylhydrazine in trichloroacetic acid-benzene solution and were separated by HPLC on a reversed-phase ODS column using a mobile phase of 80% methanol in a buffer consisting of 25 mM sodium acetate in 2% acetic acid. The eluates were monitored by a spectrophotometer at 380 nm. While all five 17-ketosteroid conjugates were clearly detected in the human urine stain samples, traces of only some of these conjugates were detected in the animal samples. Therefore, the presence of all five 17-ketosteroid conjugates indicated human specificity. In addition to the above finding, the properties of those five 17-ketosteroid conjugates were confirmed by electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS).

Impact of feeding ethanolic extracts of Achyranthes aspera Linn. on reproductive functions in male rats.

Feeding 50% ethanolic extract of A. aspera to male rats resulted in reduced sperm counts, weight of epididymis, serum level of testosterone and testicular activity of 3beta-hydroxysteroid dehydrogenase, while motility of the sperm and activity of the HMG CoA reductase were not affected. Cholesterol level in the testis, incorporation of labelled acetate into cholesterol, 17-ketosteroids in urine and hepatic and fecal bile acids were increased. The results suggest that ethanolic extract of A. aspera caused reproductive toxicity in male rats and the action may be by suppressing the synthesis of androgen.