Enzyme immunoassay of serum testosterone.

An enzyme immunoassay of serum testosterone using the testosterone-glucoamylase complex is described. Testosterone was estimated by the enzyme immunoassay after extraction with hexan: ether (4:1) for serum from men and additional thin layer chromatographic step for serum from women. The within and between assay errors, measured as the coefficient of variation were 11.1 percent (n=8) and 12.0 percent (n=12). The sensitivity of this assay was 0.25 ng. The mean testosterone concentration (+/- SD) in 19 normal men and 4 normal cycling women were 5.3 +/- 1.8 and 0.52 +/- 0.12 ng/ml, respectively. The level of testosterone found by the present assay compared favorably with those obtained by other methods.     

Determination of testosterone in saliva and blow of bottlenose dolphins (Tursiops truncatus) using liquid chromatography-mass spectrometry

A rapid, accurate and reproducible assay utilising high performance liquid chromatography-mass spectrometry (LC-MS) has been developed and validated for determining testosterone concentrations in saliva and blow of bottlenose dolphins. Sample preparation used solid phase extraction with specific preconditioning of cartridges. Analytes were eluted with 100% acetonitrile, dried under nitrogen and stored at -80 degrees C. Samples were reconstituted in 60% acetonitrile for LC-MS analysis. Chromatographic separation was achieved with an Alltech Macrosphere C8 stainless steel analytical column (2.1 mm x 150 mm i.d., 5 microm particle size, 300 angstroms pore size) using a 55% mobile phase B isocratic method (mobile phase A = 0.5% acetic acid; mobile phase B = 0.5% acetic acid, 90% acetonitrile). Samples were analysed in SIM at m/z 289.20 (testosterone mw 288.40) and a positive ion ESI. The limit of quantification was 0.5 ng/ml with a limit of detection of 0.2 ng/ml. The concentration curve was linear from 0.5 to 50 ng/ml (y = 0.01x + 0.0045, r(2) = 0.959, r = 0.979, p < 0.001). The R.S.D.s of intra- and inter-batch precision were less than 15% for saliva and 11% blow. Recovery of the assay for saliva was 93.0 +/- 7.9% (50 ng/ml) and 91.5 +/- 3.72% (1 ng/ml), and for blow was 83.3 +/- 6.8% (50 ng/ml) and 85.8 +/- 4.6% (1 ng/ml). Recovery of the internal standard in saliva was 73.0 +/- 14.2% and in blow was 78.63 +/- 4.29. The described assay was used to determine the presence of endogenous testosterone in saliva (9.73-23 ng/ml, n = 10) and blow (14.71-86.20 ng/ml, n = 11) samples of captive bottlenose dolphins.     

Characterization of male killer whale (Orcinus orca) sexual maturation and reproductive seasonality

Longitudinal serum testosterone concentrations (n=10 males) and semen production (n=2 males) in killer whales were evaluated to: (1) characterize fluctuations in serum testosterone concentrations with respect to reproductive maturity and season; (2) compare morphologic changes to estimated age of sexual maturity, based on changes in serum testosterone concentrations; and (3) evaluate seasonal changes in sperm production. Classification of reproductive status and age class was based on differences (P < 0.05) in serum testosterone concentrations according to age; juvenile males ranged from 1 to 7 years (mean+/-S.D. testosterone, 0.13+/-0.20 ng/mL), pubertal males from 8 to 12 years (2.88+/-3.20 ng/mL), and sexually mature animals were 13 years and older (5.57+/-2.90 ng/mL). For captive-born males, serum testosterone concentrations, total body length and height to width ratio of the dorsal fin were 0.7+/-0.7 ng/mL, 495.6+/-17.5 cm and 1.14+/-0.13c m, respectively, at puberty; at sexual maturity, these end points were 6.0+/-3.3 ng/mL, 548+/-20 cm and 1.36+/-0.1cm. Serum testosterone concentrations were higher (P<0.05) from March to June than from December to February in pubertal animals (4.2+/-3.4 ng/mL versus 1.4+/-2.6 ng/mL) and than from September to December in sexually mature animals (7.2+/-3.3 ng/mL versus 4.0+/-2.0 ng/mL). Ejaculates (n = 90) collected from two males had similar (P > 0.05) sperm concentrations across all months. These data represent the first comprehensive study on male testosterone concentrations during and after sexual maturation, and on reproductive seasonality in the killer whale.     

Effect of testosterone on n-methyl-d, l-aspartate-induced growth hormone secretion in male swine

Previous research from our laboratory demonstrated that n-methyl-d, l-aspartate (NMA), a potent agonist of glutamate, increased growth hormone (GH) secretion in barrows and boars. To determine if testosterone modulates NMA-induced GH secretion, Poland China x Yorkshire swine were challenged with NMA in a model that compared GH responses in boars with those of barrows or barrows treated with testosterone propionate (TP). Boars and barrows weighing 112.6+/-1.4 kg (mean +/- SE) were fitted with indwelling jugular vein catheters. Barrows (n = 16) were given i.m. injections of TP (25 mg in corn oil) twice daily from d 0 to d 6. Boars (n = 16) and control barrows (n = 15) received twice daily injections of corn oil. On d 6, blood was sampled every 15 min for 4 h. Two h after sampling began, all animals received an i.v. injection of NMA at a dose of 2.5 mg/kg body weight. Mean testosterone concentrations (ng/ml serum) were similar (P > .1) for boars (8.1+/-0.8) and barrows receiving TP (7.3+/-0.3), but were greater in both cases (P < .05) than for barrows receiving corn oil (.2+/-.01). Prior to NMA injections, mean GH concentrations were similar (P > .1) among groups and averaged 2.7+/-.2 ng/ml serum across treatments. Serum concentrations of GH after NMA increased (P < .05) similarly among groups and averaged 6.3+/-0.3 ng/ml across treatments during the 2-h period after injection. These results were not supportive of a role for testosterone as a modulator of NMA-induced GH secretion in male swine.     

Gonadal dysfunction in the spontaneously diabetic BB rat: alterations of testes morphology, serum testosterone and LH

Serum testosterone, luteinizing hormone (LH), testicular histology and ultrastructure were examined in 91 spontaneously diabetic BB, semi-starved, and control Wistar rats. Between 80-120 days of age serum testosterone was decreased (1.67 +/- .25 vs. 2.95 +/- .48 ng/ml; P less than .05) in the BB rats compared to controls but not different from semi-starved rats. LH values were similar in control and BB rats (49.4 +/- 10.9 vs. 46.8 +/- 6.2 ng/ml). Abnormal lipid droplets were noted within Leydig cells at this period. From 121-150 days of age serum testosterone was lower in BB (1.38 +/- .23 vs. 3.42 +/- .45 vs. 2.94 +/- .81 ng/ml; P less than .05) than controls or semi-starved rats. Serum LH was not significantly higher in controls than in BB rats (63.2 +/- 7.4 vs. 36.6 +/- 12 ng/ml; P = NS). Between 151-200 days of age, there was further lipid accumulation in Leydig cells in the BB rat and occasional epithelial disorganization. After 200 days, serum testosterone decreased (P less than .05) to similar levels in both control and BB rats (1.42 +/- .87 vs. 1.22 +/- .25; P = NS) and was similar in BB rats after 250 days (1.02 +/- .2 ng/ml). After 250 days of age Leydig cell morphology appeared relatively normal but marked alterations were apparent in Sertoli cells, germ cells and morphology of the tubule wall.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low-volume,high-sensitivity assay for cadmium in blood and urine using conventional atomic absorption spectrophotometry

An assay for cadmium in whole blood and urine using deuterium background-correction electrothermal atomic absorption spectroscopy (D(2)-ETAAS) was developed. Cadmium (in a 1- to 2-ml sample) was bound to 15 mg anion-exchange resin, interfering ions were removed in a 2-ml Bio-Spin column, and cadmium was extracted into 100 microl 1M nitric acid for analysis. Cadmium in the sample extract was concentrated 7-fold for blood and 10-fold for urine over the starting material. These steps produced cadmium atomic absorption traces with high signal to background ratios and allowed analysis against aqueous standards. At approximately 0.1 ng Cd/ml, mean intra- and interassay coefficients of variation were 11-12%. Cadmium recovery for 0.1 to 0.6 ng added cadmium was 107+/-4% for blood and 94+/-4% for urine (mean+/-SE, n=3). The mean detection limit (mean + 3 x SD of blank) was 0.008 ng/ml for blood and 0.003 ng/ml for urine. Samples from "unexposed" animals including humans ranged from 0.051+/-0.000 to 0.229+/-0.035 ng/ml. Values were approximately 10-fold lower than those obtained by the method of Stoeppler and Brandt using Zeeman background-correction ETAAS. This new high-sensitivity, low-volume assay will be useful for epidemiological studies, even those involving children, and will provide a means to help determine the contribution of cadmium to disease incidence in the general population.     

Serum bisphenol a concentrations showed gender differences, possibly linked to androgen levels

To investigate human exposure to bisphenol A (BPA), a widely used endocrine disruptor, we measured serum BPA concentrations and analyzed the interrelation of BPA with sex-related hormones. BPA was detected in all human sera by a novel enzyme-linked immunosorbent assay. Serum BPA concentrations were significantly higher in normal men (1.49 +/- 0.11 ng/ml; P < 0.01) and in women with polycystic ovary syndrome (1.04 +/- 0.10 ng/ml; P < 0.05) compared with normal women (0.64 +/- 0.10 ng/ml). There were significant positive correlations between serum BPA and total testosterone (r = 0.595, P < 0.001) and free testosterone (r = 0.609, P < 0.001) concentrations in all subjects and likewise between serum BPA and total testosterone (r = 0.559, P < 0.01) and free testosterone (r = 0.598, P < 0.001) concentrations in all female subjects, but not between serum BPA and other sex-related hormone concentrations in any group. These findings showed that there are gender differences in serum BPA concentrations, possibly due to differences in the androgen-related metabolism of BPA.     

Pancreatic B-cell function in non-insulin-dependent diabetes mellitus during successive periods of sulfonylurea and insulin treatment: serum C-peptide response to glucagon and urine C-peptide excretion

Serum C-peptide responses to glucagon and daily urine C-peptide excretion in successive periods of different treatment in two groups of patients with non-insulin-dependent diabetes mellitus (NIDDM) (mean interval between two tests less than 1 month) were compared. In group A patients (n = 8), the glycemic control was improved after transferring the treatment from sulfonylurea (SU) to insulin (fasting plasma glucose: SU: 192 +/- 47, insulin: 127 +/- 21 mg/dl, mean +/- S.D., p less than 0.01). Fasting serum C-peptide immunoreactivity (CPR) was significantly lower at the period of insulin treatment (SU: 1.93 +/- 1.01, insulin: 1.47 +/- 0.79 ng/ml, p less than 0.05), but there was no difference in the increase in serum CPR (maximal--fasting) (delta serum CPR) during glucagon stimulation in the two periods of treatment (SU: 1.70 +/- 0.72, insulin: 1.47 +/- 0.98 ng/ml). In group B patients (n = 7), there was no significant difference in glycemic control after transferring the treatment from insulin to SU (fasting plasma glucose: insulin: 127 +/- 24, SU: 103 +/- 13 mg/dl). Fasting serum CPR was significantly lower during the period of insulin treatment (insulin: 1.39 +/- 0.64, SU: 2.21 +/- 0.86 ng/ml, p less than 0.025), but delta serum CPR during glucagon stimulation still showed no significant difference between the two periods (insulin: 1.97 +/- 1.16, SU: 2.33 +/- 1.57 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Reproductive hormone secretion and testicular growth in bull calves actively immunized against testosterone and oestradiol-17 beta

Groups of bull calves received a primary immunization against testosterone (Group T; N = 7) or oestradiol-17 beta (Group E; N = 9) at 3 months of age and booster injections on four occasions at approximately 2 month intervals. Controls (Group C, N = 7) were immunized against human serum albumin alone using the same protocol. Immunity was achieved against both steroids as judged by the secondary antisteroid antibody titres in Group T (730 +/- 231; reciprocal of titre) and Group E (12,205 +/- 4366) bulls; however, peak antibody titres generally declined with successive booster injections. Mean plasma concentrations of LH, FSH and testosterone during the period from 3 to 10 months of age were higher (P less than 0.05) in Group T bulls than in Groups C and E. Group T bulls had larger testes compared with controls from 6 months of age onwards. At castration at 14 months of age, testes of Group T bulls were heavier (P less than 0.05) than those of Groups C and E (179 +/- 13, 145 +/- 8 and 147 +/- 6 g, respectively). At 10 months of age, there were no differences among treatment groups in LH responses to LHRH, but the testosterone responses were greater (P less than 0.05) in bulls in Group T (26.2 +/- 4.9 ng/ml) and Group E (16.6 +/- 1.8 ng/ml) compared with those in Group C (6.9 +/- 0.6 ng/ml). Testosterone responses to hCG determined at 13 months of age were also greater (P less than 0.05) in Groups T and E relative to controls. At 14 months of age daily sperm production rates per bull (X 10(-9)) were higher (P less than 0.10) in Group T bulls (2.2 +/- 0.1) than those in Groups C (1.6 +/- 0.2) and E (1.6 +/- 0.1). These results indicate that early immunity against testosterone is associated with increased gonadotrophin secretion and accelerated growth of the testes in prepubertal bulls. Also, chronic immunity against testosterone or oestradiol-17 beta enhances the steroidogenic response of bull testes to gonadotrophic stimulation. If the above responses observed in young bulls are shown to be sustained, then immunity against gonadal steroids early in life may confer some reproductive advantage in mature animals.     

Enzyme immunoassay for testosterone and androstenedione in culture medium from rabbit oocytes matured in vitro

Two enzyme immunoassays (EIAs) were validated to determine testosterone and androstenedione levels in culture medium (Brackett's medium with or without the addition of IGF-I, hormone and serum-free), without previous extraction, from rabbit oocytes matured in vitro. Polyclonal testosterone (C917), and androstenedione (C9111) antibodies were raised in rabbits using testosterone 3-carboxymethyloxime:BSA, and androstenedione 3-carboxymethyloxime:BSA. Horseradish peroxidase was used as label, conjugated to testosterone 3-carboxymethyloxime, and to androstenedione 6-hemisuccinate. Standard dose response curves covered a range between 0 and 1 ng/well. The low detection limits of the technique were 11.43 pg/ml for testosterone, and 2.32 pg/ml for androstenedione. Intra- and inter-assay coefficient of variation percentages were < 6.4 and < 7.1 for testosterone, and < 5.1 and < 6.3 for androstenedione, respectively (n= 10). The recovery rate of known testosterone or androstenedione concentrations added to pools of culture maturation medium samples averaged 97.58 +/- 2.11%, and 95.73 +/- 1.59%, respectively. Compared with RIA, EIA values were in close agreement for testosterone (n= 15, r= 0.96, P< 0.001), and androstenedione (n= 15, r= 0.94, P< 0.001). Culture medium samples were obtained at the end of oocyte in vitro maturation (14-16 h). Mean +/- SE culture maturation medium concentrations (ng/ml) were 1.80 +/- 0.09 and 0.52 +/- 0.01 for testosterone, and 1.70 +/- 0.04 and 0.24 +/- 0.01 for androstenedione in both the oocytes with and without cumulus cells, respectively. We concluded that our EIA is a highly sensitive and specific assay that provides a rapid, simple, inexpensive and nonradiometric alternative to RIA for determining testosterone and androstenedione concentrations in oocyte maturation culture medium.     

Progesterone is a mediator in the ovulatory process of the in vitro-perfused rat ovary

The role of steroids in the ovulatory process of the rat was explored in an in vitro perfusion system. Immature rat ovaries were primed with pregnant mare's serum gonadotropin (20 IU) and perfused in a recirculating perfusion system for up to 20 h. Unstimulated ovaries did not ovulate whereas the addition of luteinizing hormone (LH; 0.1 micrograms/ml) plus 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) resulted in 13.6 +/- 1.0 ovulations per treated ovary. Addition of an inhibitor of 3 beta-hydroxysteroid dehydrogenase (Compound A; 10 micrograms/ml) significantly (p less than 0.01) decreased the number of ovulations after LH plus IBMX stimulation (1.6 +/- 0.8 ovulations per treated ovary). This inhibition was reversed by the addition of progesterone, with 6.6 +/- 2.1 ovulations at approximately 100 ng/ml progesterone in the perfusion medium and 15.2 +/- 3.4 ovulations at approximately 3000 ng/ml progesterone. The addition of testosterone (10 micrograms/ml) did not reverse the inhibition of ovulations by Compound A. High levels of progesterone in the perfusion medium (greater than 3000 ng/ml) did not significantly (p greater than 0.05) increase the number of ovulations after stimulation with LH plus IBMX (20.2 +/- 4.8 ovulations), and progesterone (greater than 3000 ng/ml) was not by itself able to induce ovulations. Addition of LH plus IBMX resulted in a marked increase in the levels of progesterone, testosterone, and estradiol in the perfusion medium. The production of these steroids was almost completely inhibited by the addition of Compound A, and the levels of testosterone and estradiol were restored by the addition of high concentrations of progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estradiol-induced blockade of ovulation in the cow: effects on luteinizing hormone release and follicular fluid steroids

Administration of 10 mg estradiol valerate (EV) to nonlactating Holstein cows on Days 16 of the estrous cycle prevented ovulation in 7 of 8 cows for 14 days post-injection. In these 7 cows, the timing of luteolysis and the luteinizing hormone (LH) surge was variable but within the normal range. At 14 days post-treatment, each of these cows had a large (greater than 10 mm) follicle, with 558 +/- 98 ng/ml estradiol-17 beta, 120 +/- 31 ng/ml testosterone, and 31 +/- 2 ng/ml progesterone in follicular fluid (means +/- SE). A second group of animals was then either treated with EV as before (n = 22), or not injected (control, n = 17) and ovariectomized on either Day 17, Day 18.5, Day 20, or Day 21.5 (24, 60, 96, or 132 h post-EV). Treatment with EV did not influence the timing of luteolysis, but surges of LH occurred earlier (59 +/- 8 h post-EV vs. 100 +/- 11 h in controls). The interval from luteolysis to LH peak was reduced from 44 +/- 6 h (controls) to 6.9 +/- 1.5 h (treated). Histologically, the largest follicle in controls tended to be atretic before luteolysis, but nonatretic afterwards, whereas the largest follicle in treated animals always tended to be atretic. Nonatretic follicles contained high concentrations of estradiol (408 +/- 59 ng/ml) and moderate amounts of testosterone (107 +/- 33 ng/ml) and progesterone (101 +/- 21 ng/ml), whereas atretic follicles contained low concentrations of estradiol (8 +/- 4 ng/ml) and testosterone (12 +/- 4 ng/ml), and either low (56 +/- 24 ng/ml) or very high (602 +/- 344 ng/ml) concentrations of progesterone. This study suggests that EV prevents ovulation by inducing atresia of the potential preovulatory follicle, which is replaced by a healthy large follicle by 14 days post-treatment.     

Testicular development and maturation of the hypothalamic-pituitary-testicular axis in the male tammar, Macropus eugenii

Testicular growth and maturation of the hypothalamic-pituitary-testicular axis were assessed in male tammars from 12 to 25 months of age to establish the time of sexual maturity. The testicular dimensions and body weights of 20 male tammars, approximately 12 months of age at the beginning of the study, were measured monthly for 1 year. Groups of 3 animals were castrated at 13, 19 and 25 months of age and their testes sectioned for histological examination. Testicular volume increased between 12 and 24 months of age and was highly correlated with body weight (r = 0.91). In the 13-month group the seminiferous tubules were closed with few mitotic figures. Spermatogenesis had begun in 2 of the 19-month animals. All stages of spermatogenesis were present in the other 19-month male, and in all of the 25-month males. Basal FSH concentrations increased with the age of the animal (21.0 +/- 32.48, 94.40 +/- 55.18 and 193.05 +/- 40.21 ng/ml (mean +/- s.d.) at 19, 20 and 25 months respectively) while basal LH concentrations were similar at 20 months and 25 months (0.43 +/- 0.18 and 0.58 +/- 0.25 ng/ml respectively). Basal testosterone concentrations were also similar 0.11 +/- 0.04, 0.35 +/- 0.16 and 0.22 +/- 0.10 ng/ml in 13-, 19- and 25-month-old animals. LHRH injection in tammars at 13, 19 and 25 months of age induced release of both LH and testosterone 10-30 min after injection. The hormone concentrations increased in both magnitude and duration with increasing age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amplified androstenedione enzymeimmunoassay for the diagnosis of cryptorchidism in the male horse: comparison with testosterone and estrone sulphate methods

An amplified enzymeimmunoassay (EIA) was validated for androstenedione in the serum of male horses. We will use the assay as a tool for the diagnosis of equine cryptorchidism. We will compare androstenedione EIA to the currently used methods (testosterone and estrone sulphate determinations). The study was conducted on 115 horses of pure Spanish and Arabian breeds, that included 30 geldings, 60 bilateral cryptorchids and 25 stallions. Androstenedione standard curve covered a range between 0 and 1 ng per well. Low detection limit was 1.54 pg/ml. Intra- and inter-assay coefficients of variation (CV%) were <8.2 and <9.3, respectively (n=10). Recovery rate of known androstenedione concentrations averaged from 96.62+/-2.69 to 97.63+/-1.87%. Androstenedione mean+/-S.E. serum concentrations were 10.52+/-1.36 ng/ml in stallions (n=25), 0.51+/-0.04 ng/ml in cryptorchids (n=60), and 0.03+/-0.01 ng/ml in geldings (n=30). Diagnostic validation parameters in basal samples showed for estrone sulphate the lower positive predictive value (0.85) with the higher number of false positives, and lower specificity (0.84). Testosterone showed the higher number of false negatives with a negative predictive value of 0.85, and lower sensitivity (0.85). Among the three hormones evaluated, androstenedione presented the best results with the smaller number of horses diagnosed as false positives (0.93) or negatives (0.91). This technique also resulted in higher sensitivity, specificity and efficiency over the other two methods assayed. We concluded that our amplified EIA is a highly sensitive and specific assay that provides a rapid, simple, and inexpensive alternative to other methods.     

Serum androstenedione and testosterone concentrations during pregnancy and nonpregnant cycles in dogs

Concentrations of testosterone and of androstenedione were determined by radioimmunoassay in serum samples collected every 2-5 days throughout the periovulatory and luteal phases of the ovarian cycles of pregnant and nonpregnant beagle bitches. Testosterone levels were consistently lower than those of androstenedione, reached peaks of 29 +/- 4 ng/dl near the time of the preovulatory luteinizing hormone peak, and were reduced to near the limits of detection (less than or equal to 5-10 ng/dl) throughout the luteal phase. Androstenedione levels reached preovulatory peaks of 73 +/- 13 ng/dl, were 54 +/- 7 ng/ml during early estrus, increased (P less than 0.05) to early luteal phase peaks of 76 +/- 8 ng/dl between Days 6 and 18, and then declined to 41 +/- 5 ng/dl by Day 35-40 in both pregnant (n = 8) and nonpregnant (n = 4) bitches. Subsequent protracted increases in androstenedione occurred in 4 of 8 pregnancies but in none of the nonpregnant bitches. From Days 42 to 64 the differences in mean levels between pregnant (45 +/- 2 ng/ml) and nonpregnant (32 +/- 3 ng/ml) bitches was not significant (P greater than 0.05). At parturition androstenedione levels fell (P less than 0.05) abruptly from 39 +/- 7 to 13 +/- 3 ng/dl. These results suggest that, in the bitch, androstenedione is the major circulating androgen during the follicular and luteal phases and that patterns of androstenedione levels during the luteal phase parallel those reported for progesterone in pregnant and nonpregnant bitches, including maintenance of elevated levels throughout gestation and an abrupt decline at parturition.     

Systemic levels contribute significantly to increased intraocular IGF-I, IGF-II and IGF-BP3 [correction of IFG-BP3] in proliferative diabetic retinopathy.

Increased intraocular levels of angiogenic growth factors such as insulin-like growth factor I (IGF-I) have been demonstrated in proliferative diabetic retinopathy (PDR). It is unclear whether increased leakage of the blood retina barrier or local synthesis primarily determine intraocular levels of IGFs in man, which is of special interest regarding possible therapeutic options with somatostatin analogues in PDR. This is the first study investigating parallelly serum and vitreous levels of IGF-I/II, IGF-BP3 and the liver-derived permeability marker albumin to determine in vivo the amount of circulation-derived intraocular IGFs. A control group without retinal proliferation and patients with PDR were compared. Levels of IGF-I/II, IGF-BP3 and albumin were determined by immunological methods. Vitreous levels of albumin were 2.2-fold elevated in patients with PDR (254.1 +/- 37.2mg/dl; n = 27; p = 0.0027) compared to controls (115.7 +/- 36.2mg/dl; n =10), whereas serum levels were slightly decreased in diabetes patients (5049 +/- 196 mg/dl vs. 4330 +/- 186 mg/dl; p = 0.0283). This was comparable to an increase of IGF-I/11 and IGF-BP3 in vitreous from PDR patients (IGF-I: 2.3 +/- 1.1 ng/ml p = 0.005. IGF-II: 37.9 +/- 4.9 ng/ml; p = 0.0003. IGF-BP3: 97.9 +/- 26.9 ng/ml; p = 0.0001; n = 34) compared to controls (IGF-I: 0.7 +/- 0.1 ng/ml. IGF-II: 21.3 +/- 4.2 ng/ml. IGF-BP3: 31.3 +/- 4.9 ng/ml: n = 19). Serum levels did not differ significantly among the groups regarding IGF-I, II and IGF-BP3. Intraocular albumin and IGF-I levels calculated as percentage of the respective serum levels correlated significantly (r = 0.42; p = 0.012). This study demonstrates that influx of IGF-I, II and IGF-BP3 in PDR quantitatively parallels influx of the liver derived serum protein albumin suggesting that leakage of the blood retina barrier and serum levels of IGF primarily determine intravitreal IGF levels rather than local synthesis. Suppression of systemic IGF levels by new, highly effective somatostatin-analogues therefore provides a promising approach to prevent PDR.     

Highly sensitive quantification of 7alpha-hydroxy-4-cholesten-3-one in human serum by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of serum 7alpha-hydroxy-4-cholesten-3-one (C4), which has been used as a biomarker for bile acid biosynthesis. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). C4 was extracted from human serum (2-50 mul) by a salting-out procedure, derivatized into the picolinoyl ester (C4-7alpha-picolinate), and then purified using a disposable C(18) cartridge. The resulting picolinoyl ester derivative of C4 was quantified by LC-MS/MS using the electrospray ionization mode. The detection limit of the C4 picolinoyl ester was found to be 100 fg (signal-to-noise ratio = 10), which was approximately 1,000 times more sensitive than the detection limit of C4 with a conventional HPLC-ultraviolet method. The relative standard deviations between sample preparations and between measurements by our method were calculated to be 5.7% and 3.9%, respectively, by one-way layout analysis. The recovery experiments were performed using serum spiked with 20.0-60.0 ng/ml C4 and were validated by a polynomial equation. The results showed that the estimated concentration with 95% confidence limit was 23.1 +/- 2.8 ng/ml, which coincided completely with the observed X(0) +/- SD = 23.3 +/- 1.0 ng/ml with a mean recovery of 93.4%. This method provides highly reliable and reproducible results for the quantification of C4, especially in small volumes of blood samples.     

Reversible inhibition of testicular androgen secretion by 3-, 5- and 6-month controlled-release microsphere formulations of the LH-RH agonist [D-Trp6, des-Gly-NH10(2)] LH-RH ethylamide in the dog

This study examines the effect of treatment with controlled-release poly(DL-lactide-coglycolide) microsphere formulations of the LH-RH agonist [D-Trp6, des-Gly-NH10(2)]-LH-RH ethylamide (LH-RH-A) designed to release about 100 or 200 micrograms of the peptide per day for 3, 5 or 6 months in male dogs. Plasma levels of testosterone and LH-RH-A were measured at 2-day intervals. After the first injection of the 100-micrograms/day formulation, plasma testosterone increased from 1.6 +/- 0.2 to 3.5 +/- 0.6 ng/ml for 5-7 days before decreasing and remaining at 0.05 +/- 0.008 ng/ml for approximately 150 days (5 months). After two months of recovery, microspheres designed to release 100 micrograms for 6 months of LH-RH agonist per day were then injected. Plasma testosterone levels showed an elevation from 1.5 +/- 0.5 to 4.7 +/- 2.0 ng/ml during the first few days before gradually decreasing to castration levels for 200 days (6 months). One month later, plasma testosterone had returned to normal levels. When microspheres designed to deliver an average of 200 micrograms per day of the peptide for 3 months were injected in another series of animals, castration levels of plasma testosterone were maintained for 95 days with a progressive increase to normal values at later time intervals. The animals of the first series of experiments were then sacrificed after 4 months of recovery following maintenance of plasma testosterone at castration levels for a total period of 11 months. The testes, prostate and pituitary gland were kept for histological examination which was completely normal in all tissues. The efficacy and excellent tolerance of the controlled-release form of LH-RH-A as inhibitor of the pituitary-gonadal axis strongly support the use of such long-term controlled-release formulations of LH-RH agonists for the treatment of sex steroid sensitive diseases.     

Relationship of serum testosterone concentrations to mate preferences in rams

This study examined systemic testosterone concentrations in rams that were classified according to their sexual behavior and partner preference as either female-oriented (FOR), male-oriented (MOR), or asexual (NOR). For this purpose, we measured testosterone concentrations under three separate conditions: in conscious rams during the nonbreeding season (June) and breeding season (November), and in anesthetized rams during the breeding season. Basal testosterone concentrations in conscious rams were not different among the three groups (P > 0.05) in either season. However, when rams were anesthetized, mean systemic concentrations of testosterone in FORs (mean +/- SEM, 13.9 +/- 7.4 ng/ml serum) were greater (P < 0.05) than in NORs (0.9 +/- 0.1 ng/ml), but not in MORs (2.2 +/- 6.2 ng/ml), whereas testosterone concentrations were not different between MORs and NORs (P > 0.05). Concentrations of testosterone in the spermatic vein of FORs (127 +/- 66 ng/ml) were greater (P < 0.05) than in MORs (41 +/- 10 ng/ml) and NORs (19 +/- 7 ng/ml). Serum LH concentrations were not different. Cortisol was higher (P < 0.05) in anesthetized MORs (25.1 +/- 4.2 ng/ml) and NORs (27.2 +/- 4.4 ng/ml) than in FORs (10.9 +/- 1.8 ng/ml). These results demonstrate that circulating testosterone concentrations are related to sexual behavior only when rams are bled under anesthesia. Thus, differences in basal androgen concentrations in adulthood cannot be responsible for expression of male-oriented preferences or low libido in sheep. Instead, functional differences must exist between the brains of rams that differ in sexual preference expression.     

Radioimmunoassay of 5-androstene-3 beta,17 beta-diol in plasma and in breast cyst fluid

A simple and reliable radioimmunoassay for the determination of 5-androstene-3 beta, 17 beta-diol in peripheral plasma and in breast cyst fluid, after a chromatography on Celite microcolumn has been described and evaluated. The antiserum used was raised in rabbits injected with dehydroepiandrosterone-15 alpha-(O-carboxymethyl)-bovine serum albumin. In men below 40 years of age the levels ranged from 0.85 to 2.80 ng/ml (mean +/- SEM: 1.52 +/- 0.11; n = 24) and from 0.50 to 2.20 ng/ml (mean +/- SEM: 0.93 +/- 0.09; n = 20) in men aged between 41 and 62 years. The mean level was significantly different (P less than 0.001) between the 2 groups. A significant correlation (r = -0.56; P less than 0.01) was demonstrated between age and all male levels. In females the mean plasma level was in the follicular phase: 0.81 +/- 0.07 ng/ml (range: 0.40-1.50; n = 17; age: 19-41 years) and in the luteal phase: 0.83 +/- 0.05 ng/ml (range: 0.40-1.30; n = 29; age: 18-43 years). No cyclical change and no correlation with age could be evidenced. A significant difference (P less than 0.001) was shown between females and the young male group. In breast cyst fluid the levels ranged from 0.05 to 13.70 ng/ml (mean +/- SEM: 2.36 +/- 0.86; n = 20) whereas the sulfate concentrations ranged from 75 to 7500 ng/ml (mean +/- SEM: 1891 +/- 565; n = 15), thus demonstrating very wide inter-individual variations.