Studies to confirm the source of 11ß-hydroxyandrostenedione

In a longitudinal study of 82 children we found a gradual rise in median plasma concentrations of 11ß-hydroxyandrostenedione (11ß-OH-A4) from 2.5 to 6.4 nmol/1 during childhood which was similar in both sexes. This could reflect changes in adrenal function during the adrenarche and sexual maturation. Plasma concentrations of 11ß-OH-A4 in adults follow the patterns of cortisol secretion. In patients with diseases of the adrenal cortex, the plasma concentrations of 11ß-OH-A4 were consistent with the pathology of each condition. In women with polycystic ovaries (PCO) undergoing gonadotrophic stimulation for in vitro fertilization and embryo transfer, 11ß-OH-A4 (median = 3.8 nmol/l), testosterone and androstenedione, were raised when compared to women with normal ovaries (11ß-OH-A4 median = 2.6 nmol/l). Follicular fluid has concentrations of 11ß-OH-A4 six to twelve times greater than plasma levels and in women with PCO, 11ß-OH-A4 concentrations were lower than in women with normal ovaries, which is consistent with an inhibition of ovarian 11ß-hydroxylase. Granulosa cells in vitro demonstrated the production of 11ß-OH-A4 by side chain cleavage of cortisol. These data support an adrenal source for 11ß-OH-A4 but the raised plasma concentrations in women with polycystic ovary syndrome (PCOS) may reflect the excess androgen output from the ovary. 11ß-OH-A4 may therefore be an additional marker for ovarian dysfunction.     

Optimisation and validation of a sensitive high-performance liquid chromatography assay for routine measurement of pyridoxal 5-phosphate in human plasma and red cells using pre-column semicarbazide derivatisation

There are few studies in which direct measurement of vitamin B6 status in both plasma and red cells has been assessed. The aims of the present study were to evaluate the use of a simple, robust HPLC method of direct pyridoxal 5'-phosphate (PLP) measurement in plasma and red cells and to assess its use in establishing reference ranges in a healthy population. A reverse phase HPLC method with pre-column derivatisation using semicarbazide for the simultaneous measurement of PLP, its degradation product, 4-pyridoxic acid (PA) and pyridoxal (PL) in plasma and red cells was developed. Pre-column derivatisation, reverse phase chromatography and detection procedures were optimised. The recovery, precision, linearity and sensitivity of the assay for plasma and red cell PLP, PA and PL was established. The recovery of PLP was greater than 95% for both plasma and red cell samples. The Intra and Inter batch imprecision for PLP was less than 6% and 7%, respectively. The method for PLP was linear up to at least 1000 nmol/l and the detection limit was 2.1 nmol/l (limit of quantification; 5.8 nmol/l). Accuracy of PLP measurements in plasma were acceptable, showing a mean bias of 4.5% from the mean value of laboratories (N=34) participating in an external quality assurance scheme. Geometric mean (95% reference intervals) for plasma and red cell PLP in the healthy subjects (N=126) were 56 (21-138) nmol/l and 410 (250-680) pmol/g Hb, respectively. There was a strong positive correlation (r(2)=0.81) between plasma and red cell PLP levels in the reference population. The HPLC method described was found to be suitable for the routine measurement of PLP in both plasma and red cells.     

Chromatographic system for the simultaneous measurement of plasma 18-hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone by radioimmunoassay: reference data for neonates and infants and its application in aldosterone-synthase deficiency

A new chromatographic system for the steroid precursor separation and a sensitive radioimmunoassay system for the subsequent measurement of 18-hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone has been developed. 18-Hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone were extracted with methylene chloride and separated from cross-reacting steroids by Sephadex LH-20 column chromatography. Anti-18-hydroxy-11-deoxycorticosterone and anti-18-hydroxycorticosterone antibodies raised in rabbits were used. The lower detection limit of the assay is 0.03 nmol/l and 0.128 nmol/l for 18-hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone, respectively. Normal values for this assay in 128 healthy neonates and infants aged 0-5 months were established as a basis for the early hormonal diagnosis of aldosterone synthase deficiency types I and II. Its application for the diagnosis of aldosterone synthase deficiency is demonstrated in two patients with homozygous mutation/deletion in the encoding CYP11B2 gene.     

Ovarian morphology and the concentration of steroids, and of gonadotrophins during the breeding season in ewes actively immunized against oestradiol-17 beta or oestrone

Ewes were actively immunized against oestrone-6-(O-carboxymethyl)-oxime-bovine serum albumin, 17 beta-oestradiol-6-(O-carboxymethyl)oxime-bovine serum albumin or bovine serum albumin (controls). All 4 control ewes, 1 of 5 oestradiol-immunized ewes and 1 of 5 oestrone-immunized ewes had regular oestrous cycles. The other animals displayed oestrus irregularly or remained anoestrous. The plasma concentrations of LH and, to a lesser degree, FSH were increased relative to those in control ewes on Days 11-12 after oestrus or a similar total period after progestagen treatment in ewes not showing oestrus. The ovaries were examined and jugular venous blood, ovarian venous blood and follicular fluid were collected at laparotomy on Days 9-10 of the oestrous cycle. The ovaries of immunized ewes were heavier than those of control ewes. There were no CL in 5 of the immunized ewes but in the other 5 there were more CL than in the control ewes. Ovaries from 4 of 5 oestrone-immunized ewes contained luteinized follicles, while ovaries from 4 of 5 oestradiol-immunized ewes contained very large follicles with a degenerated granulosa and a hyperplastic theca interna. Both types of follicles produced progesterone, detectable in ovarian venous plasma and production of other steroids, particularly androstenedione, was also increased. The steroid-binding capacity of plasma was increased in the immunized ewes. The binding capacity of follicular fluid for oestradiol-17 beta and oestrone was similar to that of jugular venous plasma from the same ewes. These results suggest that immunization against oestrogens disrupts reproductive function by interfering with the feedback mechanisms controlling gonadotrophin secretion.     

Steroid determinations in human ovarian follicular fluid using reversed phase liquid chromatography

A method is presented, based on high performance liquid chromatography (HPLC) with u.v. absorbance detection, to simultaneously analyse all major unconjugated steroids in ovarian follicular fluids. The total analysis time is only 30 min. The use of a 3 mm i.d. column allows us to obtain detection limits for 3-oxo-4-ene steroids of 2 ng/ml. Calibration curves are linear in the 10-20,000 ng range per injection. Excellent agreement is obtained with the results using a previously published gaschromatography method.     

Changes of several adrenal delta 4-steroids measured by HPLC-UV spectrometry in neonatal patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

We have developed an easy and rapid method of reverse-phase high-performance liquid chromatography (HPLC)-UV spectrometry for measuring adrenal delta 4-steroids. Three female neonates with adrenal 21-hydroxylase deficiency (2 salt-losers and 1 simple virilizer), two of whom were recalled by neonatal mass-screening for congenital adrenal hyperplasia (CAH), were diagnosed using this method. Changes of several adrenal steroids were examined in these patients before and after treatment with hydrocortisone. Before treatment, the cortisone and cortisol peaks were very low and those of 17 alpha-hydroxyprogesterone (17-OHP) and 21-deoxycortisol (21-DOF) were high in all 3 patients (17-OHP: 79.9-997 nmol/l, 21-DOF: 83.7-324 nmol/l). The androstenedione peak was also high in 2 of them. A peak produced by 21-deoxycortisone, which is a product of oxidation of 21-DOF at the C-11 position, was also detected in all cases (14.5-297 nmol/l). After treatment, all of these abnormally elevated delta 4-steroids decreased or disappeared. This new method is thought to be valuable for the rapid diagnosis of CAH, and especially for use in neonatal mass-screening for CAH.     

Simple method for determination of terbutaline plasma concentration by high-performance liquid chromatography

A method is described in which low nanomolar concentrations of terbutaline in plasma can be quantitated by use of a standard isocratic high-performance liquid chromatography system with electrochemical detection. Samples were prepared for injection by solid-phase extraction and preserved from degradation by addition of glutathione. Terbutaline and internal standard metaproterenol were resolved from plasma constituents on a single C₁₈ column by ion-pairing chromatography. The method is precise and accurate for measurement of freebase concentrations as low as 4.4 nmol/l (1 ng/ml).     

Follicular activity and hormonal secretory profile in vicuna (Vicugna vicugna)

The objective of the present study was to characterize ovarian activity in non-mated vicunas, relating ovarian structures (evaluated by transrectal ultrasonography, daily for 30 days) to changes in plasma concentrations of estradiol-17beta and progesterone. Ovarian follicular activity occurred in waves, characterized by the follicle emergence, growth and regression. The mean duration of follicular waves was 7.2+/-0.5 days (mean+/-S.E.M.), with a range of 4-11 days. The follicular growth phase averaged 3.0+/-0.2 days, the static phase 1.4+/-0.1, the regression phase 2.9+/-0.3 days, and the inter-wave interval was 4.2+/-0.3 days. The mean growth rate during the growing phase was 1.8+/-0.1mm/day, while the duration of the interval from 6mm to maximum diameter was 1.4+/-0.1 days. The mean maximum diameter of the dominant follicle was 8.4+/-0.3mm (range: 6.2-11.2) and mean diameter of the largest subordinate follicle was 5.4+/-0.1mm. There was an inverse relationship between the size of the largest follicle and the total number of follicles (r=-0.21, P=0.002). Follicle activity alternated between ovaries in 77% of the waves, with 40% of dominant follicles present in the left ovary and 60% in the right ovary. Plasma estradiol-17beta concentrations also had a wave-like pattern, varying between 12.0 and 62.8 pmol/l. Plasma progesterone concentrations remained below 5.0 nmol/l and there was no ultrasonographic evidence of ovulation during the study.     

Effects of weaning on concentrations of inhibin in follicular fluid and plasma of sows.

Changes in plasma and follicular fluid concentrations of inhibin were examined in sows after weaning at 28-32 days post partum. From 0 to 48 h after weaning, inhibin concentrations were 200-300 times higher in follicular fluid from small (less than 4 mm) and medium-large (greater than or equal to 4 mm) follicles than in ovarian venous plasma. Inhibin concentrations increased in follicular fluid from medium-large follicles at 24 and 48 h after weaning; concentrations in ovarian venous plasma were positively correlated with the number of medium-large follicles (r = 0.40) and with ovarian venous plasma concentrations of oestradiol (r = 0.61). Blood samples were collected for 30 days from sows (n = 6) that exhibited oestrus within 5 days after weaning and from sows (n = 5) that remained anoestrous for 11 days after weaning. Plasma inhibin concentrations rose in oestrous and anoestrous sows by 12 h and continued to rise for 60 h after weaning. Plasma inhibin concentrations rose further and were higher at 3.5-4.5 days after weaning in oestrous sows than in sows that remained anoestrous. After oestrus, plasma inhibin concentrations declined. At weaning, plasma concentrations of follicle-stimulating hormone (FSH) were higher in sows that subsequently exhibited oestrus than in sows that remained anoestrous. After weaning, plasma concentrations of FSH declined in both groups, reached a nadir at 2.5 days, and increased gradually in anoestrous sows; oestrous sows exhibited an FSH surge at oestrus. Plasma FSH returned to preweaning concentrations in both groups of sows at Days 7-8.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum 5-androstene-3 beta,17 beta-diol sulphate and 5 alpha-androstane-3 alpha,17 beta-diol sulphate in hirsute females with polycystic ovarian disease

Serum sulphates of 5-androstene-3 beta,17 beta-diol (5-ADIOL-S), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-DIOL-S) and dehydroepiandrosterone (DHEA-S), unconjugated androstene-dione (AD) and testosterone (T), sex hormone binding globulin (SHBG), free androgen index (FAI), 17 alpha-hydroxyprogesterone (17OHP), luteinising hormone (LH) and follicle stimulating hormone (FSH) were measured by specific radioimmunoassay in 28 hirsute women with polycystic ovarian disease (PCO) and in normal women (n = 73). Mean levels of steroids measured were significantly elevated, and SHBG significantly depressed, in the women with PCO with values (mean +/- SE) for 5-ADIOL-S (516 +/- 51 vs 267 +/- 10 nmol/l), 3 alpha-DIOL-S (130 +/- 9 vs 52 +/- 2 nmol/l), DHEA-S (7.3 +/- 0.5 vs 4.4 +/- 0.2 mumol/l), AD (11.3 +/- 1.1 vs 3.4 +/- 0.2 nmol/l), T (3.3 +/- 0.2 vs 1.5 +/- 0.1 nmol/l) and 17OHP (5.1 +/- 0.8 vs 2.8 +/- 0.2 nmol/l). SHBG levels were 31 +/- 2.9 vs 65 +/- 2.5 nmol/l, and the free androgen index [100 x T (nmol/l) divided by (SHBG nmol/l)] was 12.5 +/- 1.4 vs 2.4 +/- 0.1. The mean LH to FSH ratio was also elevated at 2.8 +/- 0.3. These studies suggest that the measurement of 5-ADIOL-S and DHEA-S may indicate adrenal gland involvement in PCO while 3 alpha-DIOL-S appears to be a reflection of peripheral androgen metabolism. A comprehensive biochemical profile of PCO should thus include the analysis of these sulphoconjugates as well as unconjugated steroids.     

Monitoring ovarian function in scimitar-horned oryx (Oryx dammah) by measurement of fecal 20α-progestagen metabolites

The feasibility of monitoring ovarian function in scimitar-horned oryx (Oryx dammah) by measurement of fecal 20α-progestagens was investigated. Fecal samples were collected daily or on alternate days over a 4–11 month period from five oryx during natural (n = 4) or synthetic PGF_2α (cloprostenol)-controlled (n = 1) cycles. Of the four oryx undergoing natural cycles, three had regular access to a vasectomised male, and mating dates were recorded. Ultrasonography was used to monitor changes in reproductive tract morphology in the female administered with cloprostenol. Neutral steroids were extracted from feces with methanol:petroleum ether (2:1 v/v) after first removing phenolic steroids with potassium hydroxide (1 M). The concentration of 20α-progestagens in the methanol phase was measured by enzymeimmunoassay. Excretion of 20α-progestagens in all females followed a cyclic pattern corresponding to the follicular and luteal phases of the ovarian cycle. Concentrations of fecal 20α-progestagens were on average twentyfold greater during the luteal phase compared with the follicular phase. Mean (±SD) ovarian cycle length, based on fecal progestagen profiles, was 24.4 ± 2.2 days with mean (±SD) luteal and follicular phase lengths of 18.7 ± 2.8 and 5.7 ± 1.6 days, respectively. Mating by a vasectomized male occurred when 20α-progestagen concentrations were still elevated or declining. Similarly, fecal progestagens did not return to follicular phase concentrations for 4–5 days after administration of cloprostenol, and a 4 day delay was observed between ovulation, as visualized by ultrasound scanning, and a rise in fecal 20α-progestagens. These data suggest a time lag of approximately 4 days between reproductive events and changes in fecal 20α-progestagen concentrations. We conclude that measurement of immunoreactive 20α-progestagen concentrations in feces has limited application for predicting ovulation or accurately timing inseminations because of delay in steroid excretion, but will enable noninvasive monitoring of ovarian cycles in scimitar-horned oryx for fertility assessment and for determining the outcome of artificial insemination programs. © 1995 Wiley-Liss, Inc.     

Follicular fluid lipoproteins in the mare: Evaluation of HDL transfer from plasma to follicular fluid

Using a density gradient ultracentrifugal procedure, we have separated equine plasma and follicular fluid high-density lipoproteins (HDL). The density distribution of the follicular fluid HDL was clearly displaced towards the highest densities in comparison with that of plasma HDL. Similarly, an analysis of size distributions showed a decrease in follicular fluid HDL diameters (4.2 to 9.2 nm) compared to plasma HDL (5.5 to 9.5 nm). HDL were isolated into three subfractions on the basis of the disposition of the Sudan Black stained bands in the centrifuge tubes. Concentrations of each subfraction were clearly lower in the follicular fluid, and the relative percentages with regard to the plasma equivalents were inversely proportional to the molecular weights (23.8% for HDL-1, 49.9% for HDL-2 and 63.7% for HDL-3). The cholesterol/phospholipid molar ratio and the esterified/free cholesterol molar ratio were clearly increased in the follicular HDL-2 and HDL-3 subfractions. The apolipoprotein distribution in follicular fluid HDL was very close to that in plasma HDL. LCAT activity measured in human as well as equine samples was weaker in follicular fluid compared to plasma in both species (4.0 nmol of free cholesterol esterified per h per ml vs. 24 nmol per h per ml). Theoretical concentrations of follicular fluid HDL were calculated assuming that the HDL particles would be merely a filtration product undergoing no detectable metabolic modifications. Biochemical measurements showed that the lightest particules (HDL-1) were less numerous than suggested by the theoretical calculation. Thus, although follicular fluid HDL appear to be a filtration product of plasma HDL, they undergo metabolic transformations that we suggest may be linked to hormonal synthesis and reverse cholesterol transport.     

Levels of androgen conjugates and oestrone sulphate in patients with breast cysts

Plasma and cyst fluid were obtained from patients with palpable breast cysts and analysed for androgen conjugates and oestrone sulphate content by radioimmunoassay. Concentrations of androgen conjugates in cyst fluids varied from 15.6 to 475.5 mumols/l. These levels were much greater than those in plasma (1.3-5.2 mumols/l) and there was no association between values in cyst aspirates and plasmas obtained from the same individuals. Levels of oestrone sulphate in breast cyst fluids (1.5-744.0 nmol/l) were also generally in excess of those in plasma (2.0-59.9 nmol/l) and again no relationship was evident between concentrations in cyst fluid and the circulation. Neither was there a relationship between levels of androgen conjugate and oestrone sulphate in plasma. In contrast, a highly significant correlation (P less than 0.001) was identified between the androgen conjugate and oestrone sulphate content of cyst fluids. Levels of both androgen conjugates and oestrone sulphate were also significantly different in groups of cysts subdivided according to electrolyte classification, cysts with low Na+:K+ ratios having higher steroid concentrations than those with high Na+:K+ ratios. The biological significance of the relationship between the two conjugates in cyst fluids remains unclear but it is suggested that the accumulation of these steroids involves a common mechanism.     

The effect of ACTH on the plasma concentrations of the "hypertensinogenic" steroids, 17 alpha-hydroxyprogesterone and 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one in sheep

The plasma concentrations of 17 alpha-hydroxyprogesterone (17 alpha OHP) and 17 a'20 alpha-dihydroxy-4-pregnen-3-one (17 alpha 20 alpha OHP) have been measured in sheep during 5 days of ACTH administration at 20 micrograms/kg/day a rate of infusion known to produce hypertension. Five days of ACTH administration produced a progressive increase in plasma 17OHP from 0.45 +/- 0.12 to 128.9 +/- 28.4 nmol/l and in 17 alpha 20 alpha OHP from 0.54 +/- 0.15 to 73.1 +/- 7.2 nmol/l. Calculation of the blood production rate of both steroids during ACTH treatment confirms that the rates of infusion of 17OHP (3.0 mumol/h) and 17 alpha 20 alpha OHP (1.5 mumol/h) used to produce hypertension, when infused together with the other major ovine adrenocortical steroids, produced plasma concentrations in the range as found following administration at a rate to increase blood pressure.     

Presence of immunoreactive atrial natriuretic peptide in follicular fluid, ovary and ovarian perfusates

Immunoreactive atrial natriuretic peptide (ir-ANP) was measured in the follicular fluid of pig ovarian follicle, and rabbit ovarian homogenates and perfusates using a specific radioimmunoassay (RIA). Serial dilution curves made with the extracts of follicular fluid, ovarian homogenates and perfusates using SepPak C18 cartridges were parallel with the RIA standard curve. On gel filtration chromatography and reverse phase HPLC, all extracted materials showed high and low molecular weight forms of ir-ANP. The amount of ir-ANP in rabbit ovary was 40.70 +/- 0.39 pg/mg and that in follicular fluid of pig ovarian follicle was 18.88 +/- 2.49 pg/ml.     

Effect of exogenous progesterone and eCG treatment on ovarian follicular dynamics in vicunas (Vicugna vicugna)

The aim of the present study was two-fold. First, to evaluate the effect of exogenous progesterone on ovarian follicular dynamics in order to assess its ability to synchronize ovarian activity in the vicuna. Secondly, to evaluate the ovarian response to the treatment with eCG through the observation of the structures developed in the ovaries. Follicular dynamics was monitored daily by transrectal ultrasonography in 12 adult, non-pregnant vicunas. Plasma progesterone and estradiol-17beta concentrations were measured in blood samples collected daily. In experiment 1, intravaginal devices containing 0.33g of progesterone were inserted into the vagina and kept in place for 5 days (treatment group, n = 8). After progesterone withdrawal, five animals were further monitored in order to evaluate the efficacy of the CIDR to synchronize the emergence of a dominant follicle. In experiment 2, four females received 750IU of eCG IM. Two were previously monitored ultrasonographically to confirm the absence of a dominant follicle at the beginning of the superstimulatory treatment (group A). The other two animals had a CIDR inserted into the vagina for 5 days and the superstimulatory treatment was applied 24h after device withdrawal (group B). Females from both groups were surgically explored 96 h after eCG injection; the ovaries were exposed and the number of newly formed structures produced by each ovary was counted. Peak progesterone concentrations (25.9 +/- 5.29 nmol l(-1), mean +/- S.E.M.) were attained on day 1 after device insertion, remained high until the day of device withdrawal (9.7 +/- 1.98 nmol l(-1)) and decreased to 5.5 +/- 1.13 nmol l(-1) the day after. There was no follicle development to the state of dominance after device insertion. Moreover, mean follicle diameter steadily decreased after insertion of the device until the minimum mean value (1.85 +/- 0.17 mm) was recorded on day 5 (P = 0.006). Similarly, plasma concentrations of estradiol-17beta remained below 35 pmol l(-1) during the period of progesterone treatment in all animals and the mean estradiol-17beta declined with the lowest value (22.1 +/- 2.19 pmol l(-1)) being recorded on day 4 after device insertion. After superstimulation of follicular development with eCG, the total number of follicles that developed was 33 in group A and 58 in group B and the mean number of newly developed ovarian structures per female was 22.75 +/- 4.26. In conclusion, progesterone released by the CIDR exerts a negative effect on ovarian follicular development and function suggesting intravaginal devices could be used to synchronize the beginning of follicular waves during a superstimulatory treatment. There was also a tendency for greater ovarian follicular development when the animals were previously treated with progesterone.     

Estrogens, androgens, and progestins in follicular fluid from preovulatory follicles: identification and quantification by gas chromatography/mass spectrometry associated with stable isotope dilution

The synthesis and use of stable isotope-labeled analogs of various steroids have made it possible to undertake a study of follicular fluid (FF) aspirated from mature and preovulatory follicles. Our previous results have been brought together here in order to review quantitative work done by gas chromatography-mass spectrometry. A positive gradient between peripheral plasma and FF concentrations of a steroid suggests the possibility of ovarian biosynthesis. This is particularly relevant to the catecholestrogens, 19-norsteroids, and some corticosteroids.     

Determination of melarsoprol in biological fluids by high-performance liquid chromatography and characterisation of two stereoisomers by nuclear magnetic resonance spectroscopy

The analysis of melarsoprol in whole blood, plasma, urine and cerebrospinal fluid is described. Extraction was made with a mixture of chloroform and acetonitrile followed by back-extraction into phosphoric acid. A reversed-phase liquid chromatography system with ultraviolet detection was used. The relative standard deviation was 1% at concentrations around 10 μmol/l and 3–6% at the lower limit of determination (9 nmol/l in plasma, 93 nmol/l in whole blood, 45 nmol/l in urine and 10 nmol/l in cerebrospinal fluid). Melarsoprol is not a stable compound and samples to be stored for longer periods of time should be kept at −70°C. Plasma samples can be stored at −20°C for upt to 2 months. Chromatography showed that melarsoprol contains two components. Using nuclear magnetic resonance spectroscopy the two components were shown to be diastereomers which slowly equilibrate by inversion of the configuration at the As atom.     

Inhibition of gonadotropin sensitive adenylate cyclase by ovarian follicular fluid.

Follicular fluid obtained from medium or large bovine ovarian follicles inhibited ovarian luteinizing hormone/human chorionic gonadotropin sensitive adenylate cyclase in a dose-dependent manner (I₅₀ = 3 mg follicular fluid protein/ml). The inhibitory activity was excluded by Sephadex G-10 and was fully retained following treatment with charcoal. Fluoride-stimulated enzyme activity was not inhibited. Binding of ¹²⁵I human chorionic gonadotropin to ovarian plasma membranes was only slightly reduced by the follicular fluid. The post-microsomal supernatant of homogenates from ovaries of immature (27-day-old) rats collected 24–36 h after treatment with 15 i.u. of pregnant mare serum gonadotropin also inhibited luteinizing hormone-sensitive adenylate cyclase. The extent of this inhibition seemed to decline with follicular maturation. The possibility is raised that ovarian sulfated glycosaminoglycans are responsible for the observed inhibition of adenylate cyclase.     

Validation of the measurement of low concentrations of 5-hydroxytryptamine in plasma using high performance liquid chromatography

A sensitive and rapid assay is described for the measurement of low concentrations of 5-hydroxytryptamine (5-HT) present in human platelet-depleted plasma (PDP) using reverse-phase high performance liquid chromatography (HPLC) with fluorimetric detection. With an analysis time of 12 min, this method is particularly useful for large-scale clinical trials investigating small differences in PDP 5-HT concentrations in conditions such as functional gastrointestinal disorders (FGID). The limit of detection and quantification were 1 and 3 nmol/l, respectively, and the calibration curve linear between 1 and 1000 nmol/l. The within-day and between-day precision were 4.3 and <13.6%, respectively.