Seasonal changes in the levels of 11-oxotestosterone and testosterone in the serum of male salmon, Salmo salar L., and their relationship to growth and maturation cycle

Levels of 11-oxotestosterone (17 β-hydroxyandrost-4-ene-3, 11-dione) and testosterone in the blood serum of individually marked adult male Atlantic salmon held in captivity, were measured by radioimmunoassay at approximately monthly intervals for periods of up to 18 months. In addition to peak concentrations of both hormones shown by all the maturing fish at the time to full sexual maturation during October and November, a majority of maturing fish also showed a significant elevation of 11-oxotestosterone during the early months of the year. The possible involvement of this early elevation of 11-oxotestosterone in controlling the mitotic multiplication of spermatogonia is discussed. Weight and length increases expressed as specific values G_W and G_L and weight to length relationships for the maturing males for each sampling period are presented and compared with those of non-maturing fish.     

A radio-gas chromatographic method for the determination of 11-oxotestosterone in fish plasma: its use in confirming estimations by radioimmunoassay

A radio-gas chromatographic method has been devised for the estimation of 11-oxotestosterone (17beta-hydroxyandrost-4-ene-3, 11-dione) in fish plasma samples which provides an independent means of validating the radioimmunoassay described earlier. Estimates of the concentration of 11-oxotestosterone in a sample of male rainbow trout plasma by radio-gas chromatography using peak height and peak weight measurements were 6.9 microgram/100 ml and 7.4 microgram/100 ml respectively, in good agreement with that of 7.1 microgram/100 ml determined by radioimmunoassay.     

Simplified ultraviolet liquid chromatographic method for determination of sertindole, dehydrosertindole and norsertindole, in human plasma

An ultra-violet high-performance liquid chromatographic method was developed for the determination of sertindole, an atypical antipsychotic drug and its main metabolites dehydrosertindole and norsertindole, in human plasma. With a small sample volume, after a single-step liquid-liquid extraction, the compounds were separated on a reversed-phase XTerra RP(18) column, eluted with 45% of acetonitrile and 55% of ammonium acetate buffer (0.05 M, adjusted pH 8) and detected at 256 nm within 11 min. This method shows a good linearity for plasma concentration between 5-100 ng/ml and 100-1000 ng/ml, a good precision (inter and intra day CV < 11%) and a good inter-assay accuracy (bias < 11%). The limit of quantification concentration was 5 ng/ml. The absolute recovery of sertindole was higher than 99%. This rapid and sensitive method could be used for therapeutic drug monitoring as well as for overdose management.     

Validation of radioimmunoassay systems for the measurement of 11-keto- and 11 beta-hydroxytestosterone in teleost blood

Highly specific antisera for 11-keto- and 11 beta-hydroxytestosterone have been raised in sheep. Assay systems for the simultaneous measurement of 11-ketotestosterone, 11 beta-hydroxytestosterone, testosterone, progesterone and estradiol-17 beta were validated for Ictalurus nebulosus plasma and Carassius auratus serum. In males of both species 11-ketotestosterone and testosterone were the major steroids detected. In females, testosterone and estradiol-17 beta were the predominant steroids measured. Data from samples taken at different stages of the annual cycle suggest that seasonal fluctuations in gonadal steroid secretion occur in I. nebulosus and C. auratus.     

Sample preparation and liquid chromatography-tandem mass spectrometry for multiple steroids in mammalian and avian circulation

Blood samples from wild mammals and birds are often limited in volume, allowing researchers to quantify only one or two steroids from a single sample by immunoassays. In addition, wildlife serum or plasma samples are often lipemic, necessitating stringent sample preparation. Here, we validated sample preparation for simultaneous liquid chromatography--tandem mass spectrometry (LC-MS/MS) quantitation of cortisol, corticosterone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), 17β-estradiol, progesterone, 17α-hydroxyprogesterone and testosterone from diverse mammalian (7 species) and avian (5 species) samples. Using 100 μL of serum or plasma, we quantified (signal-to-noise (S/N) ratio ≥ 10) 4-7 steroids depending on the species and sample, without derivatization. Steroids were extracted from serum or plasma using automated solid-phase extraction where samples were loaded onto C18 columns, washed with water and hexane, and then eluted with ethyl acetate. Quantitation by LC-MS/MS was done in positive ion, multiple reaction-monitoring (MRM) mode with an atmospheric pressure chemical ionization (APCI) source and heated nebulizer (500°C). Deuterated steroids served as internal standards and run time was 15 minutes. Extraction recoveries were 87-101% for the 8 analytes, and all intra- and inter-run CVs were ≤ 8.25%. This quantitation method yields good recoveries with variable lipid-content samples, avoids antibody cross-reactivity issues, and delivers results for multiple steroids. Thus, this method can enrich datasets by providing simultaneous quantitation of multiple steroids, and allow researchers to reimagine the hypotheses that could be tested with their volume-limited, lipemic, wildlife samples.     

Plasma testosterone values in patients with somatosexual development disturbances from 11 years old to adulthood]

The testosterone plasma level was determined in 5 groups: 1. in 69 normal juveniles and 85 fertile males at the age of 11 to 45 years, 2. in 42 patients with hypospadia or epispadia aged 11 to 25 years, 3. in 72 males with unilateral cryptorchidism at the age of 11 to 45 years, 4. in 83 males with bilateral cryptorchidism aged 11 to 45 years and 5. in 106 patients with Klinefelter's syndrome at the age of 16 to 45 years. A pubertal increase of the testosterone plasma level was found to begin in subjects with cryptorchidism or Klinefelter's syndrome at a similar age as in the control group. However, as early as at the age of 13 to 14 years decreased testosterone values were found in the patients as compared to normal juveniles. Between 19 and 20 years, the plasma testosterone level was significantly decreased in all patient-groups as compared to the controls of similar age. In adulthood, plasma testosterone concentrations in the patient groups were observed to be 4 to 6 ng/ml without significant age-dependent changes, which are characteristic of normospermic males. Different degrees of clinical symptoms indicating androgen deficiency found in various patient groups despite similar androgen levels in adulthood suggest a different responsiveness of their target organs to androgens.     

Androgens and male mating behavior in rough-skinned newts, Taricha granulosa

Cyproterone acetate was administered either orally or intraperitoneally to intact, adult male newts, Taricha granulosa. The number of males that exhibited the courtship behavior of clasping when tested with nuptial females was not altered by the antiandrogen treatments. In males which were unresponsive to nuptial females, the occurrence of clasping was not evoked by injections for 4 days of testosterone, dihydrotestosterone, or 11-ketotestosterone. Further, the incidence of clasping was not significantly elevated by injections of prolactin and/or testosterone for 30 days. The effect of sexual activity on testosterone and dihydrotestosterone levels in male newts was determined by radioimmunoassay of plasma collected from males which were: (1) isolated from females; (2) allowed to clasp a female for 2 min; or (3) allowed to clasp a female for 1 hr. The testosterone and dihydrotestosterone levels were unchanged during this period of clasping. In February and again in June, plasma androgen concentrations were measured in males which differed in their propensity to initiate courtship when paired with females. Androgen levels were similar for males that clasped a female and males that never attempted to clasp a female. Plasma androgen levels in the male newt are apparently not correlated with sexual responsiveness.     

Immunoaffinity purification of 11-dehydro-thromboxane B2 from human urine and plasma for quantitative analysis by radioimmunoassay

11-Dehydro-thromboxane B2 is now considered to be a reliable parameter of thromboxane A2 formation in vivo. An immunoaffinity purification method was developed for radioimmunoassay of this compound contained in human urine and plasma. Monoclonal anti-11-dehydro-thromboxane B2 antibody was prepared and coupled to BrCN-activated Sepharose 4B. Human urine or plasma was applied to a disposable column of the immobilized antibody. After the column was washed with water, 11-dehydro-thromboxane B2 was eluted with methanol/water (95/5) with a recovery of more than 90%. The purified extract was subjected to a radioimmunoassay utilizing 11-[3H]dehydro-thromboxane B2 methyl ester and the monoclonal anti-11-dehydro-thromboxane B2 antibody. The detection range of the assay was 10-600 fmol (IC50 = 90 fmol). The cross-reactivities of the antibody with thromboxane B2, 2,3-dinor-thromboxane B2, and other arachidonate metabolites were less than 0.05%. These compounds were efficiently separated from 11-dehydro-thromboxane B2 by the immunoaffinity purification. This procedure also allowed the separation of 11-dehydro-thromboxane B2 from unidentified urinary and plasma substances which interfered with the radioimmunoassay. Validity of the results obtained by the radioimmunoassay was confirmed by GC/MS employing selected ion monitoring for quantification.     

A simple method for the assay of eight steroids in small volumes of plasma

A simple method is described for the simultaneous radioligand assay of four Δ⁵-3β-hydroxysteroids adjacent to one another on the biosynthetic pathway (pregnenolone [1], 17α-hydroxypregnenolone, dehydroepiandroste rone and 5-androsterone-3β,17β-diol), and their four Δ⁴-3keto products (progesterone, 17α-hydroxyprogesterone, 4-androstene-3, 17-dione and testosterone). Two plasma aliquots are extracted and fractionated each for four steroids and individual corrections are made for losses. For fractionation, maximum use is made of the high resolution and reproducibility of celite minicolumns, using propylene glycol as stationary phase, and a discontinuous gradient of ethyl acetate in iso-octane as mobile phase. The fractions are then assayed in the appropriate radioligand end-assay system. Each assay was finally validated by demonstrating coincidence of peaks of immuno- and radioactive steroid In extracts of female plasma. Results in pre-pubertal girls and women in the follicular phase of the menstrual cycle suggest that the major change in adrenal steroid production at puberty may be an increase in 17,20-desmolase activity. There appears to be little reversal of this change in adrenal function after ovariectomy.     

Reduced activity of androgen biosynthesis in the testes of rats with analbuminemia

Steroid metabolism in Nagase Analbuminemia Rats (NAR), a mutant strain established from Sprague-Dawley rats, was studied. NAR are characterized by lack of serum albumin and hyperlipidemia. Total testosterone concentration in the serum of NAR was lower than that of normal rats, while the serum free testosterone, LH and FSH concentrations were similar. The half lives of 14C-labeled testosterone administered intravenously in NAR and normal Sprague-Dawley (SD) rats were 4.4 and 4.1 min, respectively. The plasma clearance rates of testosterone in NAR and normal rats were 34.7 and 39.1 ml/min per kg body weight. On Sephadex G-100 chromatography, a mixture of [3H]testosterone and normal rat serum gave two protein peaks eluted in the void volume and the albumin fraction, and the radioactivity was eluted all in the albumin fraction. In contrast, a mixture of [3H]testosterone and NAR serum gave a single protein peak eluted in the void volume and the radioactivity was mainly eluted with this protein peak. The association constants of testosterone to NAR and normal rat sera were 1.25 and 2.24 X 10(4) M-1. Enzyme activities related to the synthesis of testosterone by the testicular microsomal fractions of NAR and normal rats were examined. The activities of 3 beta-hydroxysteroid dehydrogenase, 5-ene-4-ene isomerase, 17 alpha-hydroxylase, C-17-C-20 lyase and 17 beta-hydroxysteroid dehydrogenase were lower in NAR than in normal rats. The activity for synthesis of testosterone from pregnenolone by the testicular microsomal fraction of NAR was about 40% of that of normal rats. These findings indicate that the low serum concentration of testosterone in NAR is mainly attributable to decreased biosynthesis of testosterone in the testes.     

Effect of neonatal injections of estradiol, testosterone and cryproterone acetate on plasma and testicular testosterone and on the genital system in adult male mice]

On day old male mice received a single injection of oestradiol benzoate, testosterone propionate or cyproterone acetate in order to study their action on testicular development, particularly testosterone secretion. Oestrogenization of newborn males leads, when the animals mature, to a high proportion or cryptorchidism, to atrophy of testes and seminal vesicles, and inhibition of spermatogenesis. Testosterone levels were reduced in the plasma. Testosterone propionate produced moderate reduction of testicular weight but spermatogenesis was not impaired. Plasma testosterone level was reduced. Cyproterone acetate increased significantly testicular testosterone level.     

Serum steroid hormones including 11-ketotestosterone, 11-ketoandrostenedione, and dihydroprogesterone in juvenile and adult bonnethead sharks, Sphyrna tiburo.

Previous studies in the placental viviparous bonnethead shark, Sphyrna tiburo, have correlated 17 beta-estradiol, progesterone, testosterone, and dihydrotestosterone with reproductive events in both males and females. However, several key reproductive events, including implantation, maintenance of pregnancy, and parturition, did not correlate with these four steroid hormones. Therefore, the present study investigated three steroid hormones, 11-ketotestosterone, 11-ketoandrostenedione, and dihydroprogesterone, which have demonstrably important roles in the reproductive cycles of teleosts. It was hypothesized that one or more of these three hormones would correlate with specific reproductive events in S. tiburo. Concurrently, developmental (growth and/or maturation) analyses of these three steroids plus 17 beta-estradiol, progesterone, testosterone, and dihydrotestosterone were investigated in juvenile bonnethead sharks. Serum dihydroprogesterone concentrations were highest in mature females and 11-ketotestosterone concentrations were highest in mature males. In mature females, 11-ketoandrostenedione levels were elevated from the time of mating, through six months of sperm storage and another four months of gestation. At parturition concentrations became significantly lower and remained lower until mating occurred again in another two to three months. Serum 11-ketotestosterone concentrations were the highest at implantation though not significant. In mature males, significantly elevated serum levels of dihydroprogesterone occurred in April and May, near the start of annual testicular development. During growth in males, testosterone and dihydrotestosterone increased progressively and in females, testosterone increased progressively. At maturity in males, significant increases occurred in testosterone and 11-ketotestosterone concentrations while, in females, dihydroprogesterone, 11-ketotestosterone, 17 beta-estradiol, progesterone, testosterone, and dihydrotestosterone concentrations increased. This study shows that although testosterone may be the primary androgen in the bonnethead shark, other derived androgens may have important functions in growth, maturation, and reproduction. J. Exp. Zool. 284:595-603, 1999.     

Seasonal variations in plasma concentrations of 11-ketotestosterone and testosterone in male rainbow trout, Salmo gairdnerii Richardson

Measurements were made of plasma 11-ketotestosterone and testosterone in control and sex-reversed male rainbow trout, before and during their first spawning season (winter 1978/1979). Plasma concentrations of both androgens (in both groups) were 2–3 ng ml^-1 in January 1978 (apart from some precocious spawners), rose slowly to 9–11 ng ml^-1 in April and July and then increased rapidly to 100–150 ng ml^-1 in November. From this time, testosterone levels declined but those of 11-ketotestosterone continued to rise to a peak of 260 ng ml^-1 in February 1979. No significant differences in hormone levels were found between control male and masculinized female fish.     

Determination of testosterone concentrations in rat plasma using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry combined with ethyl oxime and acetyl ester derivatization

A quantitative method for measuring testosterone (T) concentrations in rat plasma was developed using ethyl oxime and acetyl ester derivatization and liquid chromatography-atmosphere pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS). The method utilizes a solid phase extraction with Varian Bond Elut C18, a derivatization process to form testosterone ethoxime acetate and LC-APCI-MS/MS with a reversed phase LC and a C8 column. This method is capable of detecting testosterone concentrations as low as 0.2 ng/ml in a 0.05 ml sample of rat plasma. This method can be used as a sensitive chromatography-based assay for small sample volumes of rat blood.     

Decreasing the level of ethyl acetate in ethanolic fermentation broths of Escherichia coli KO11 by expression of Pseudomonas putida estZ esterase

During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced. With ethanologenic Escherichia coli strain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter(-1). Although the E. coli genome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity. A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change. This method allowed identification of Pseudomonas putida NRRL B-18435 as a source of this activity and the cloning of a new esterase gene, estZ. Recombinant EstZ esterase was purified to near homogeneity and characterized. It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine. As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate. The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer. By using alpha-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40 degrees C. The Km and Vmax for alpha-naphthyl acetate were 18 microM and 48.1 micromol. min(-1). mg of protein(-1), respectively. Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 micromol. min(-1). mg of protein(-1)), followed by ethyl acetate (66 micromol. min(-1). mg of protein(-1)). Expression of estZ in E. coli KO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter(-1).     

Hydroxylation of testosterone at carbons 1, 2, 6, 7, 15 and 16 by the hepatic microsomal fraction from adult female C57BL/6J mice.

The metabolism of a mixture of [4-14C]- and [7 beta-2H]testosterone by the hepatic microsomal fraction from adult femal C57BL/6J mice has been investigated. The following metabolites were identified by their mass spectra and by their retention times on gas chromatography on one or two phases: 1epsilon-, 2beta-, 6alpha-, 6beta-, 7alpha-, 15alpha-, 15beta-, 16alpha- and 16beta-hydroxytestosterone; 6alpha-, 6beta- and 7alpha-hydroxy-4-androstene-3,17-dione; and 4-androstene-3,17-dione. A compound tentatively identified as 6- or 7-oxotestosterone was also isolated. 17beta-Hydroxy-4,6-androstadien-3-one, 17beta-hydroxy-1,4-androstadien-3-one and 4,6-androstadiene-3,17-dione were identified but are considered to arise non-enzymatically from 7alpha-hydroxytestosterone, 1epsilon-hydroxytestosterone and 7alpha-hydroxy-4-androstene-3,17-dione, respectively.     

Isolation and identification of 15-beta-hydroxy cyproterone acetate as a new metabolite of cyproterone acetate in dog, monkey and man.

15-beta-hydroxy cyproterone acetate could be identified by thin layer chromatography, mass spectrum, NMR and IR spectrum as a major unconjugated metabolite of cyproterone acetate in plasma and urine of dog, monkey and man. This metabolite has been found to interferein the Mattingly method for the determination of 11-hydroxy-corticosteroids which suggests, that this method is inadequate in controling the adrenal function of subjects treated with cyproterone acetate.     

Isolation and characterization of proteoheparan sulfate from plasma membranes of an ascites hepatoma, AH 66

A proteoglycan was isolated from plasma membranes prepared from AH 66 cells by the following procedure. The plasma membranes were isolated from cells according to the method devised by Funakoshi and Yamashina (1976) J. Biochem. 80, 1185-1193), then the membranes were made lipid-free. The lipid-free membranes were solubilized with 5 mM sodium phosphate buffer, pH 7.0, containing 0.5% sodium dodecyl sulfate (SDS), then the solution was fractionated on a Sepharose CL 6B column. The proteoglycan eluted near the void volume fraction was further purified by repeated precipitation with cetylpyridinium chloride (CPC). The proteoglycan isolated was homogeneous on electrophoresis on a cellulose acetate strip and was identified as proteoheparan sulfate. The preparation contained 10.6% protein, its amino acid composition being characterized by high contents of glutamic acid, aspartic acid, proline, glycine, threonine, and serine.     

11-ketotestosterone synchronously induces oocyte development and silvering-related changes in the Japanese eel, Anguilla japonica

To evaluate the effects of sex steroids on silvering in the Japanese eel, Anguilla japonica, the development of oocytes, eye size, digestive tract, and swim bladder were studied in relation to observations of the profiles of plasma levels of sex steroids (estradiol 17β, E2; testosterone, T; 11-ketotestosterone; 11-KT) during silvering for each sex and by administrating 11-KT to yellow eels. All steroids examined in the study increased in female eels after silvering had begun, whereas in males, only 11-KT increased significantly, and no statistical differences were found in plasma levels of E2 and T between eels in both developmental stages. 11-KT appeared to induce the early stage of oocyte growth, enlargement of the eyes, degeneration of the digestive tract and the development of the swim bladder. This suggested that 11-KT synchronously accelerates early development of the ovaries and the morphological changes, possibly in adaption to oceanic migration, and that 11-KT is one of the most important factors in early stages of development in the Japanese eel, as it appears to be in other anguillid eels.     

The effect of artificial photoperiodicity and antiandrogen treatment on the antler growth and plasma levels of LH, FSH, testosterone, prolactin and alkaline phosphatase in the male white-tailed deer

1. Artificial extension of day-length in adult male white-tailed deer during the autumn induced: (a) premature casting of antlers, early onset of the new antler growth and out of season mineralization, (b) early elevation of plasma levels of prolactin, LH, FSH, testosterone and alkaline phosphatase and (c) out of season hair molt. 2. Intramuscular administration of the antiandrogen cyproterone acetate immediately after velvet shedding induced: (a) dramatic reduction of testosterone levels in plasma, (b) premature casting in bucks with fully mineralized antlers and (c) renewal of bone rebuilding activity in incompletely mineralized antlers which resulted in blockage of casting.