Age-related changes in 11β-hydroxyandrostenedione concentration in normal and osteoporotic women

The secretion of dehydroepiandrosterone (DHEA) and its sulfate is known to decline gradually with advancing age. Furthermore DHEA is known to be significantly lower in osteoporotic subjects than in normals. Recently 11β-hydroxyandrostenedione (11-OHA) has been proposed as an important indicator of the adrenal source of hormone excess in different hyperandrogenic states. In the present study we measured 11-OHA in 224 normal women aged 20–79 yr and 130 osteoporotic women aged 40–79 yr. RIA of 11-OHA was performed with highly specific antiserum raised in rabbits.

The mean 11-OHA serum concentration was 2.20±0.90 ng/ml in normal women and 1.75±0.58 ng/ml in osteoporotic women. In contrast to DHEA there was no age-related decrease in 11-OHA serum concentrations in normal and osteoporotic women. Osteoporotic subjects showed statistically significantly lower 11-OHA serum concentrations than normal women. Therefore low serum 11-OHA might represent a further risk factor for osteoporosis.     

Profiles of LH, FSH and progesterone in postpartum dairy cows and their relationship to the commencement of cyclic functions

In 25, 3-to-13 year old, dairy cows (Braunvieh and Hoehenfleckyieh) FSH, LH and progesterone plasma profiles were determined by RIA. Blood was sampled at 6-hour intervals from parturition to 40–78 days postpartum, and the results correlated with the commencement of cyclic functions. For FSH, generally basic values were recorded, without characteristic features associating any values with the onset of cyclic ovarian activities or the occurrence of the first heat. LH profiles varied greatly between individuals with regard to the onset of elevations, regularity of patterns and peak values. The first preovulatory LH peak was recorded 17.3±9.8 days (range 4–46) postpartum. The first heat occurred on day 28.4±16 (range 6–55) postpartum, indicating that 13/23 cows ovulated without behavioral estrus, as reproductive cycles were re-established. Peak LH values increased with progressive cycles (1st peak 5.7±4.8 ng/ml; 2nd peak 11.8±8.7 ng/ml; 3rd peak 13.5±9.9 ng/ml plasma). Progesterone values also showed great variations in the profile of their first postpartum elevation. In 13/25 cows the first cycle was shortened (13.1±2.9 days), prolonged in 3 animals (34±4 days) and normal in 7 cows (20.4±1.9 days). Heat, preovulatory LH peak and progesterone profile were normal in all animals on subsequent cycles. Two animals did not start cycling.     

Radioimmunoassay of plasma androsterone

A method is described and evaluated for the determination of androsterone in peripheral venous plasma (1.0 ml) from men and women. The procedure involves addition of labelled internal standard and extraction with diethyl ether. Aliquots (10 %) are removed for radioimmunoassay. An antiserum to androsterone-17-carboxymethyl oxime-bovine serum albumin and tritiated androsterone complete the system. The practical systematic errors have been determined by replicate analyses. The range of values (mean ± S.D.) in plasma from 40 healthy men are 54 ± 32 ng/100 ml, and the corresponding values for women 46 ± 28.     

Determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A highly sensitive HPLC method with automated column switching was developed for the simultaneous determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in plasma samples from man, Cynomolgus monkey, rabbit, rat and mouse. Plasma (0.4 ml) was deproteinated by adding ethanol (1.5 ml) containing the internal standard acitretin. After centrifugation, 1.4 ml of the supernatant were directly injected onto the precolumn packed with LiChrospher 100 RP-18 (5 μm). 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase during injection and 1% ammonium acetate and 2% acetic acid-ethanol (102:4, v/v) was added, on-line, to decrease the elution strength of the injection solution. After backflush purging of the precolumn, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm. Two coupled Superspher 100 RP-18 endcapped columns (both 250×4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v/v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The method was linear in the range 0.3–100 ng/ml, at least, with a quantification limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2%–94.4% and the mean inter-assay precision was 2.8%–3.2% (range 0.3–100 ng/ml). Similar results were obtained for animal plasma. The analytes were found to be stable in the plasma of all investigated species stored at −20°C for 4.3 months and at −80°C for 9 months, at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 human and 1000 animal plasma samples from clinical and toxicokinetic studies. Endogenous levels determined in control patients and pregnant women were similar to published data from volunteers.     

Effects of insulin-like growth factor I (IGF-I) on enzymatic activity in human adrenocortical cells. Interactions with ACTH

Cells obtained from 6 adult human adrenals or adrenal fragments were cultured in serum-free synthetic medium (McCoy's) in order to study the isolated effects of IGF-I on steroidogenesis and its interactions with ACTH. After addition of peptide, changes in the activities of steroidogenic enzymes were assessed by measuring certain steroids in the spent medium. These included pregnenolone, 17-hydroxypregnenolone (17-OH-Preg), dehydroepiandrosterone (DHA), 17-hydroxyprogesterone (17-OH-P), androstenedione (AD), 11-deoxycortisol and glucocorticoids (chiefly cortisol and its immediate precursors, 11-deoxycortisol and 17-OH-P) and cortisol itself.

The steroid responses obtained with repeated doses of IGF-I (40 ng/ml ≈ 10⁻⁹ M), added at 0, 48 and 72 h, over 4 days' culture were quite different from those obtained with repeated doses of ACTH (0.25 ng/ml ≈ 10⁻¹⁰ M). All the steroids measured increased with time of culture under the influence of ACTH and, apart from pregnenolone which peaked, tended to reach a plateau. With IGF-I, by contrast, DHA, AD, 11-deoxycortisol and glucocorticoid production increased initially, then decreased progressively, whereas pregnenolone, 17-OH-Preg and 17-OH-P production was either absent or negative.

Cumulative steroid production over 4 days reached similar levels in response to a single dose of IGF-I and/or ACTH, with two major exceptions: pregnenolone dropped significantly with IGF-I [46% ± 6 (SEM) as opposed to 93% ± 11 with ACTH, P < 0.005, N = 5], as did 17-OH-P (48% ± 11 vs 113% ± 8 with ACTH, P < 0.001, N = 6). Increased formation of down-stream metabolites (DHA, AD, 11-deoxycortisol and glucocorticoids) would suggest that IGF-I induced stimulation of the 17-, 21- and 11β-hydroxylases.

The responses to ACTH stimulation of cells which 4 days previously had been pre-treated with an initial and single dose of IGF-I and/or ACTH emphasized the impact of IGF-I on the 3-hydroxylation steps in cortisol biosynthesis. Compared with ACTH pre-treatment, the effects of which faded in the long term, pre-treatment with IGF-I resulted in a significantly increased steroidogenic response (P between < 0.05 and < 0.01). With the single exception of pregnenolone (43% ± 4.7), production of all the metabolites was amplified: 17-OH-Preg: 348% ± 88; DHA: 643% ± 127; 17-OH-P: 193% ± 36; AD: 725% ± 200; 11-deoxycortisol: 573% ± 110; cortisol: 1000%.

Our findings strongly suggest that IGF-I plays a major rôle in the regulation of steroidogenesis by promoting and maintaining enzymatic activity (17, 21- and 11β-hydroxylases) via which the function of ACTH is achieved, viz., biosynthesis of cortisol.     

Twice single doses of 100,000 IU of vitamin D in winter is adequate and safe for prevention of vitamin D deficiency in healthy children from Ushuaia, Tierra Del Fuego, Argentina

In order to improve vitamin D status of children from Ushuaia (55°S), at the South of Argentina, double supplementation with 100.000 IU of vitamin D was administered at the beginning of winter (March 2004), and 3 months later during winter (June 2004). In 2004, serum 25-hydroxyvitamin D (25OHD) was measured before the first supplementation, a month after, and 3 months after receiving the second supplementation (March, April and September). We studied 18 healthy children from Ushuaia, age (mean ± S.D.) 7.3 ± 4.4 years old (range 1.2–14.6), seven girls and 11 boys. Before treatment, serum 25OHD was 29.3 ± 5.9 ng/ml. It increased significantly 1 month after the first supplementation (April): 35.3 ± 4.4 ng/ml (p < 0.001), and decreased significantly 3 months after the second supplementation: 22.4 ± 4.6 ng/ml (September (p < 0.001). No child was neither deficient (<10 ng/ml) nor insufficient (10–15 ng/ml) of vitamin D. On April, a month after the first supplementation, no children had vitamin D intoxication levels (>50 ng/ml). These results disclosed that to prevent vitamin D deficiency for children at zones of risk at the south of our country, double supplementation of 100,000 IU of vitamin D during autumn and winter, would be adequate and safe.     

The effect of follicle-stimulating hormone on follicular development, granulosa cell apoptosis and steroidogenesis and its mediation by insulin-like growth factor-I in the goat ovary

The effect of FSH on goat follicular development, granulosa cell apoptosis and steroidogenesis and its mediation by insulin-like growth factor (IGF)-I were studied through both in vivo and in vitro experiments. The FSH treatment was begun on Day 9 after estrus and consisted of injections twice a day for 3 days in decreasing doses (7.5–7.5–5.0–5.0–2.5–2.5 mg). Does in both treatment and control groups were slaughtered for ovaries on Day 12. Granulosa cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Expression of IGF-I and IGF-II mRNA was determined by RT–PCR, while concentrations of progesterone (P4), estradiol (E2), IGF-I and IGF-II were measured by radioimmunoassay (RIA). Following parameters increased significantly (P<0.05) after the FSH treatment: follicle number (5.0±1.5 versus 9.0±2.0 per ovary), the level of E2 (0.1±0.1 ng/ml versus 0.7±0.2 ng/ml), the E2/P4 ratio (0.7±0.4 versus 4.7±3.0) and the concentrations of IGF-I (0.5±0.2 ng/ml versus 119.4±15.1 ng/ml) and IGF-II (0.12±0.03 ng/ml versus 40.9±18.7 ng/ml) in follicular fluid of the medium sized (3–5 mm) follicles and in the ovarian cortex the relative quantity of IGF-I mRNA (0.37±0.17 versus 0.90±0.12 Max OD). In contrast, the ratio of apoptotic granulosa cells in these follicles was reduced significantly (0.53±0.1 versus 0.10±0.01, P<0.05). In large (>5 mm) follicles, however, only the follicle number (2.3±0.7 versus 7.0±1.5 per ovary) and the level of IGF-I (38.4±11.0 ng/ml versus 87.3±13.9 ng/ml) increased significantly (P<0.05), whereas other values did not change. In vitro culture of granulosa cells showed that FSH significantly (P<0.05) enhanced IGF-I production (12.7±2.1 ng/ml versus 26.±21.9 ng/ml) by these cells, and both FSH and IGF-I reduced the ratios of apoptotic cells (from 0.7±0.07 to 0.3±0.1 and 0.2±0.04, respectively) and the effect was additive when both were used together. H89, the PKA pathway inhibitor, blocked the effect of FSH on granulosa cell apoptosis and IGF-I production in vitro. These results indicated that FSH mainly enhanced the development of medium sized follicles in the goat by suppressing the apoptosis of granulosa cells via increasing production of IGF-I and steroids, possibly through the PKA pathway.     

Simple and rapid determination of irinotecan and its metabolite SN-38 in plasma by high-performance liquid-chromatography: application to clinical pharmacokinetic studies

Irinotecan (CPT-11) is an anticancer agent widely employed in the treatment of colorectal carcinoma. A simple, rapid and sensitive high-performance liquid chromatographic method for the simultaneous determination of CPT-11 and its metabolite SN-38 in plasma, and their preliminary clinical pharmacokinetics are described. Both deproteinisation of plasma specimens (100 μl) and addition of the internal standard, camptothecin (CPT), are achieved by incorporating to samples 100 μl of a solution of CPT (1 μg/ml) in acetonitrile–1 mM orthophosphoric acid (90:10); 200 μl of this acidified acetonitrile solution, drug-free, is also added to accomplish complete deproteinisation: this procedure reduces sample preparation time to a minimum. After deproteinisation, samples are treated with potassium dihydrogenphosphate (0.1 M) and injected into a Nucleosil C₁₈ (5 μm, 250×4.0 mm) column. Mobile phase consists of potassium dihydrogenphosphate (0.1 M)–acetonitrile (67:33), at a flow-rate of 1 ml/min. CPT-11, SN-38 and CPT are detected by fluorescence with excitation wavelength set at 228 nm and emission wavelengths of CPT-11, SN-38 and CPT fixed, respectively, at 450, 543 and 433 nm. The limits of quantitation for CPT-11 and SN-38 are 1.0 and 0.5 ng/ml, respectively. This method shows good precision: the within day relative standard deviation (RSD) for CPT-11 (1–10 000 ng/ml) is 5.17% (range 2.15–8.27%) and for SN-38 (0.5–400 ng/ml) is 4.33% (1.32–7.78%); the between-day RSDs for CPT-11 and SN-38, in the previously described ranges, are 6.82% (5.03–10.8%) and 4.94% (2.09–9.30%), respectively. Using this assay, plasma pharmacokinetics of CPT-11, SN-38 and its glucuronidated form, SN-38G, have been determined in one patient receiving 200 mg/m² of CPT-11 as a 90 min intravenous infusion. The peak plasma concentration of CPT-11 at the end of the infusion is 3800 ng/ml. Plasma decay is biphasic with a terminal half-life of 11.6 h. The volume of distribution at steady state (V_ss) is 203 l/m², and the total body clearance (Cl) is 14.8 l/h·m². The maximum concentrations of SN-38 and SN-38G reach 28.9 and 151 ng/ml, respectively.     

Modified method of nalbuphine determination in plasma: validation and application to pharmacokinetics of the rectal route

A solid-phase extraction (SPE) procedure was developed for the quantification of nalbuphine in a small volume (500 μl) of human plasma with subsequent assay by high-performance liquid chromatography (HPLC) and electrochemical detection using 6-monoacetylmorphine as internal standard. Plasma was extracted using Bond Elute certified extraction columns (LCR: 10 ml, 130 mg) after conditioning with methanol and 0.2 M Tris buffer (pH 8). Elution was performed with a CH₂Cl₂-isopropanol-NH₄OH (79:20:, v/v). The organic phase was evaporated to dryness and resuspended in HPLC mobile phase containing 2% isopropanol. Linearity was assessed over the 5–100 ng/ml concentration range and a straight line passing through the origin was obtained. Experiments with spiked plasma samples resulted in recoveries of 95±5.4% and 98±6.2% for nalbuphine and 6-monoacetylmorphine, respectively. The optimal pH conditions for the SPE were found at pH 8. The intra-day coefficients of variation (C.V.) for 5, 40, and 100 ng/ml were 5.3, 3.0 and 2.3% (n=8) and the inter-day C.V.s were 7.7, 3.2 and 3.5% (n=10), respectively. The detection limit for 500 μl plasma sample was 0.02 ng/ml and the limit of quantification 0.1 ng/ml (C.V.=12.4%). The ease of the proposed method of analysis, as well as its high accuracy and sensitivity allow its application to pharmacokinetic studies. A preliminary kinetic profile of nalbuphine after rectal administration in a pediatric patient is presented.     

Study of plasma aldosterone in normal pregnancy, in pre-eclamptic women and in cord plasma of newborns.

A radioimmunoassay without chromatography was used for the determination of plasma aldosterone in pregnancy. The mean values (+/- S.D.) of aldosterone concentration increased consistently from 23.2 +/- 5.3 ng/100 ml (n = 14) during the first trimester to 37.2 +/- 10.6 ng/100 ml (n = 17) during the second trimester and 64.0 +/- 18.8 ng/100 ml (n = 29) during the third trimester of pregnancy. The highest values were found at delivery (71.9 +/- 14.2 ng/100 ml; n = 21) and in the cord plasma of newborns (83.4 +/- 14.9 ng/100 ml; n = 21). Significantly lower plasma aldosterone values were found in the plasma of pre-eclamptic women during the third trimester of pregnancy (41.9 +/- 21.3 ng/100 ml; n = 11).     

Plasma selenium levels in healthy blood bank donors in the central-eastern part of Belgium

Graphite furnace atomic absorption spectrometry, with Zeeman background correction and after improved matrix modification, was used to measure the plasma selenium content of healthy blood bank donors in the central part of Belgium.

The mean plasma selenium concentration of 80 men and 80 women was 79.7±4.4 ng/mL with a range of 55.0–117.4 ng/mL.

There was no gender difference observed. Plasma selenium level was significantly highest for the adult group, aged 45–64 years, compared to the others, except the young adults (18–24 years).

The mean plasma selenium concentration measured corresponded well with literature data for Belgium. The obtained values were found to be in the medium range, compared with recent literature values for the European countries.     

Involvement of hypothalamic somatostatin in glucagon-induced suppression of growth hormone secretion in conscious rats

The role of central glucagon in regulating GH secretion was studied in conscious male rats with chronic indwelling intra-atrial and intracerebro-ventricular (ICV) cannulae. Repeated blood sampling every 20 min from 1000 hr to 1700 hr showed two major GH bursts occurring at regular intervals (3.6±0.1 hr) around 1200 hr and 1540 hr. The ICV (lateral ventricle) injection of glucagon (10 μg/rat) at 1100 hr inhibited spontaneous GH secretion, and the mean (±SE) plasma GH levels from 1120 hr to 1700 hr were lower than those in controls injected ICV with the vehicle solution only (31.9±7.8 ng/ml vs. 157.1±13.4 ng/ml, p<0.01). The GH bursts did not appear until 5 hr after the injection. The intravenous (IV) injection of glucagon (10 μg/rat) did not change plasma GH levels or the occurrence of spontaneous GH bursts. The glucagon-induced suppression of GH release was attenuated when anti-somatostatin serum (ASS), but not normal rabbit serum (NRS), was given IV in a volume of 0.25 ml immediately before the ICV injection of glucagon (10 μg/rat) (mean GH levels at 1120–1700 hr: ASS+glucagon, 133.6±26.7 ng/ml vs. NRS+glucagon, 30.5±7.4 ng/ml, p<0.01). These findings suggest that central glucagon may play an inhibitory role in regulating GH secretion by stimulating SRIF release from the hypothalamus in the rat.     

Determination of molsidomine in plasma by high-performance liquid column chromatography

A high-performance liquid chromatographic method is described for the analysis of the anti-anginal compound 5-ethoxycarbonyl-3-morpholinosydnonimine (Molsidomine) in human and dog plasma. The drug was extracted from plasma into chloroform and the analysis was carried out on a reversed-phase column, the column effluent being monitored by UV absorption at 312 nm. The method is sensitive (2 ± 0.3 ng/ml) and specific. The method was applied to a study in which human volunteers received an aqueous solution of the drug and then, on a separate occasion, a tablet formulation. Peak plasma levels of 20–30 ng/ml (tablet) and 10–19 ng/ml (aqueous solution) were obtained following a 2-mg oral dose.     

Determination of 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine in plasma and whole blood by high-performance liquid chromatography

A selective and sensitive HPLC assay for the quantitative determination of a new antifilarial drug, 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine (CDR 101) is described. After extraction from plasma and blood, CDR 101 was analysed using a C₁₈ Nucleosil ODS column (250×4.6 mm, 5 μm particle size) and mobile phase of acetonitrile-0.05 M ammonium acetate adjusted to pH 3.0, with UV detection at 318 nm. The mean recoveries of CDR 101 in plasma and blood over a concentration range of 25–500 ng/ml were 95.5±2.01% and 83.3±1.87%, respectively. The within-day and day-to-day coefficient of variations for plasma were 3.23-6.21% and 2.59-9.90%, respectively, those for blood were 2.59-5.92% and 2.89-6.82%, respectively. The minimum detectable concentration for CDR 101 was 1 ng/ml in plasma and 2.5 ng/ml in whole blood. This method was found to be suitable for clinical pharmacokinetic studies.     

Selective determination of sultopride in human plasma using high-performance liquid chromatography with ultraviolet detection and particle beam mass spectrometry

We developed a sensitive and selective method for determining levels of sultopride, a neuroleptic drug of the substituted benzamide, in human plasma using high-performance liquid chromatography (HPLC) combined with UV detection and particle beam mass spectrometry (PBMS). Sutopride was extracted with tert.-butylmethyl ether using a salting-out technique. Tiapride served as an internal standard (I.S.). Sutopride and I.S. were separated by HPLC on a silica column with a mobile phase of acetonitrile-0.1 M ammonium acetate (94:6, v/v). The calibration curves were linear over the concentration range from 5 to 1000 ng/ml by HPLC with UV detection and from 10 to 1000 ng/ml with PBMS detection. The limit of quantitation was 5 ng/ml with UV detection and 10 ng/ml with PBMS detection. The absolute recovery was 92% and the within-day coefficients of variation were 2.9–7.1% at plasma concentrations from 50 to 500 ng/ml, determined by HPLC with UV detection. Using this method, we measured the plasma concentrations of sultopride with replicate analyses in four hospitalized patients and steady-state plasma levels were determined to be 161.6±30.8, 321.1±93.7, 726.5±143.1 and 1273.6±211.2 ng/ml, respectively.     

Effects of moderate short-term potassium depletion in normal humans the role of prostaglandins

The role of prostaglandins (PG) in the effects of potassium (K⁺)depletion was studied in six normal women. A mean K⁺-deficit of 220 mEq was induced with and without concomitant treatment with indomethacin (150 mg/day). Mean serum K⁺ concentration decreased from 4.2 ± (S.E.) 0.1 to 3.2 ± 0.1 mEq/L without indomethacin and from 4.1 ± 0.1 to 3.2 ± 0.1 mEq/L with indomethacin. “Supine” and “upright” plasma renin activity (PRA) and plasma norepinephrine concentration (NE) were unaltered by K⁺ -depletion alone but decreased with indomethacin. Plasma aldosterone (PA) was suppressed during K⁺-depletion (control: 7.2 ± 2.6 ng/dl supine, 19.3 ± 8.1 ng/dl upright; K⁺-depletion: 2.6 ± 0.3 ng/dl supine, 5.5 ± 1.3 ng/dl upright) and was paralleled by a decrease in urinary aldosterone. K⁺-depletion decreased urinary PGE₂ from 667 ± 133 to 343 ± 60 ng/day (P < 0.025) without a change in PGF₂. The dose of exogenous angiotensin II (A II) which increased diastolic blood pressure by 20 mm Hg (pressor dose) was 7.1 ± 1.4 ng/kg/min during control and increased to 11.0 ± 0.7 ng/kg/min during K⁺-depletion (P < 0.05). Indomethacin increased the sensitivity to A II both during control (pressor dose: 4.9 ± 0.6 ng/kg/min) and K⁺- depletion (pressor dose: 6.0 ± 1.0 ng/kg/min). These results indicate that in healthy subjects, moderate short-term K⁺-depletion does not affect PRA or NE but decreases production of aldosterone and PGE₂ by the kidney. The changes in vascular sensitivity to exogenous A II during K⁺-depletion and indomethacin and the decreases in plasma NE and PRA during indomethacin may be explained by changes in vascular vasodilator PG.     

Sensitive assay for midazolam and its metabolite 1′-hydroxymidazolam in human plasma by capillary high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method is described for the quantification of midazolam and 1′-hydroxymidazolam in human plasma. Sample (1 ml plasma) preparation involved a simple solvent extraction step with a recovery of approximately 90% for both compounds. An aliquot of the dissolved residue was injected onto a 3 μm capillary C₁₈ column (150 mm×0.8 mm I.D.). A gradient elution was used. The initial mobile phase composition (phosphate buffer–acetonitrile, 65:35) was maintained during 16 min and was then changed linearly during a 1-min period to phosphate buffer–acetonitrile, 40:60. The flow-rate of the mobile phase was 16 μl/min and the eluate was monitored by UV detection. The limits of quantification for midazolam and 1′-hydroxymidazolam were 1 ng/ml and 0.5 ng/ml, respectively. The applicability of the method was demonstrated by studying the pharmacokinetics of midazolam, and its major metabolite 1′-hydroxymidazolam, in human volunteers following i.v. bolus administration of a subtherapeutic midazolam dose (40 μg/kg).     

Radioimmunoassay of plasma 20 alpha-dihydroprogesterone

A radioimmunological method is described and evaluated for the determination of 20α-dihydroprogesterone (20α-hydroxypregn-4-en-3-one) in peripheral venous plasma (0.1 – 2.O ml) of men and women. The antiserum is highly specific. The concentration (mean ± S.D., ng/ml plasma) in a group of healthy men was 0.19 ± 0.08; in women in follicular phase, 0.28 ± 0.11; in luteal phase, 2. 19 ± 1.19; and during pregnancy (24th week to term) in the range 8.44– 40.94.     

Effect of atrial natriuretic factor on plasma vasopressin in conscious rats

An intravenous (IV) bolus injection (10 μg) of synthetic rat atrial natriuretic factor [ANF (Arg 101-Tyr 126)] into normal conscious Sprague-Dawley rats produced a significant decrease of plasma arginine vasopressin (AVP) while 1-, 2-, and 5-μg doses exerted no such effect. Mean arterial blood pressure (MAP) was lowered about 15 mmHg by an IV 10 μg bolus injection of ANF. When plasma AVP rose significantly in rats exposed to such osmotic stimuli as 600 mM NaCl and 900 mM mannitol intraperitoneally (IP), subsequent IV injection of ANF (10 μg) markedly depressed this parameter. Lower doses of ANF were ineffective against 600 mM NaCl IP. The significant elevation of plasma AVP levels by hypertonic sucrose 900 mM IP was not modified by ANF (10 μg). Blood pressure remained unchanged after IP administration of various osmotic stimuli, except mannitol, and in all these experiments an IV bolus of ANF exerted a lowering effect on MAP. Seventy-two hr water deprivation (mixed osmotic and volume stimulus) resulted in elevated plasma AVP levels which were unaffected by an IV bolus injection of ANF at doses of 0.06–10 μg. Immunoreactive ANF (IR-ANF) rose in plasma to 39.3±13 ng/ml 1 min after an IV bolus injection of 10 μg ANF, dropping to 1.01±0.2 ng/ml after 5 min and to 0.32±0.01 ng/ml after 10 min (when ANF and AVP interactions were studied), but still remained approximately six times higher than in control rats. These results suggest that, in the conscious rat, only pharmacological levels of ANF observed after an IV bolus infusion may influence both resting and osmotically-stimulated AVP levels.     

Localization and structural characterization of an oligosaccharide O-linked to bovine PDC-109 Quantitation of the glycoprotein in seminal plasma and on the surface of ejaculated and capacitated spermatozoa

PDC-109 (13 kDa) is the most abundant component, and the major heparin-binding protein, of bovine (Bos taurus) seminal plasma. Here, we show that PDC-109 contains a single O-linked oligosaccharide (NeuNAc(2–6)-Galβ(1–3)-GalNAc-) attached to Thr¹¹. Immunoquantitation of PDC-109 indicates that its concentration in seminal plasma is 15–20 mg/ml. Though PDC-109 is not present on epididymal sperm, ejaculated spermatozoa on average are coated with (9.5 ± 0.3) × 10⁶ molecules of PDC-109/cell. This value remained constant in swim-up sperm and decreased to (7.7 ± 0.4) × 10⁶/spermatozoon after incubation for 24 h in capacitation medium at 39°C. These data substantiate the hypothesis that PDC-109 may be one of the seminal plasma components that enhance the fertilizing capacity of bull spermatozoa upon interaction with heparin-like glycosaminoglycans present in the female genital tract.