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.     

Protein binding of the contraceptive steroids gestodene, 3-keto-desogestrel and ethinylestradiol in human serum

The protein binding of ethinylestradiol (EE2), gestodene (GEST) and 3-keto-desogestrel (KDG) has been determined by ultrafiltration in the serum of women who had either taken a gestodene (n = 37) or desogestrel (n = 28) containing oral contraceptive for a time period of at least 3 months. GEST and KDG were analyzed in individual serum pools whereas EE2 was repeatedly measured in two serum pools, each one representing one treatment group. The respective free fractions of the three steroids were 0.6 +/- 0.1% (GEST), 2.5 +/- 0.2% (KDG), 1.7 +/- 0.6% (EE2, in the gestodene-group) and 1.5 +/- 0.2% (EE2, in the desogestrel-group). EE2 was exclusively bound to albumin, whereas GEST and KDG were also bound to sex-hormone-binding globulin (SHBG). The distribution of the two progestins over the serum binding proteins was determined after heat-treatment of serum samples. For GEST, the contribution of albumin and SHBG was 24.1 +/- 9.1 and 75.3 +/- 9.1%, respectively and for KDG it was 65.9 +/- 11.9 and 31.6 +/- 12.0%, respectively. SHBG and corticosteroid-binding globulin (CBG) concentrations were measured in the serum samples obtained from both treatment groups. In the gestodene-group 180 +/- 61 nmol/l (SHBG) and 89 +/- 13 mg/l (CBG) were measured, the corresponding values in the desogestrel-group were 226 +/- 64 nmol/l (SHBG) and 93 +/- 14 mg/l (CBG). SHBG concentrations were correlated with the total concentration of GEST and its free fraction and a positive (r = 0.395) and negative (r = -0.491) correlation respectively was found. Only a weak negative correlation (r = -0.291) was found for SHBG and the free fraction of KDG in the serum. These data demonstrate that the three contraceptive steroids EE2, GEST and KDG were all bound extensively to serum proteins, however, with pronounced differences concerning their distribution over the various binding proteins.     

A radioimmunoassay for plasma dehydroepiandrosterone sulphate incorporating placental steroid sulphatase as a hydrolysing reagent

We describe a method for the measurement of plasma dehydroepiandrosterone sulphate (DHAS) which incorporates a Triton X-100 solubilised preparation of human placental steroid sulphatase as a hydrolysing agent and a direct radioimmunoassay of liberated DHA using a specific antiserum. The hydrolysis procedure is carried out at 50 degrees C for 1 h and an assay run can be completed in 4 h. As determined by the method, plasma concentrations of DHAS in 32 normal adult men (ages 23-58 yr) had a mean value +/- SD of 5.5 +/- 1.89 mumol/l. For 30 normal adult cyclic women (ages 22-35 yr) the mean plasma concentration of DHAS +/- SD was 3.1 +/- 1.35 mumol/l which was significantly lower (P less than 0.01) than found for men. Plasma DHAS concentration were also measured in 50 hirsute female patients. The mean value +/- SD was 5.03 +/- 2.52 mumol/l which was significantly higher (P less than 0.01) than the value for the normal female group. Some 42% of the hirsute patients had DHAS concentrations above the upper 95% probability limit of the normal range for premenopausal women.     

The effects of sex hormone binding globulin (SHBG) on testosterone transport into the cerebrospinal fluid.

The movement of testosterone (T) from blood across the blood-brain barrier (BBB) is thought to reflect the combined effects of T's lipid solubility and the presence of circulating binding proteins for T such as albumin or sex hormone binding globulin (SHBG). Since the adult rat lacks a circulating specific high affinity sex steroid binding protein, examination of the disappearance from serum and uptake into cerebrospinal fluid (CSF) of [3H]T before and after SHBG or albumin infusion should provide insight into the function of these two proteins with respect T transport. Three groups of adult male Sprague-Dawley rats were cannulated at the femoral vein and cisterna magna. In a control group (n = 8), [3H]T was given as an intravenous bolus beginning at time zero; multiple serum and CSF collections were assayed for counts per min (cpm) during the subsequent 45 min. Data from these animals were then compared to those seen in animals that received either purified human SHBG (hSHBG) (n = 7) or human albumin (hALB) (n = 6) 10 min prior to the [3H]T infusion. High performance liquid chromatography was used to monitor the metabolic fate of the steroid infusate at the end of each study period. Infusion of hSHBG increased serum concentrations from undetectable to 93.8 nM/l (mean +/- SEM, n = 6). Administration of hALB significantly increased (25.0 +/- 1.2 g/l at baseline, 33.4 +/- 1.6 g/l post-infusion, mean +/- SEM, P less than 0.03, n = 5) the circulating albumin concentration. Comparison of data from each group of animals demonstrated that (1) following an i.v. injection of radiolabeled T, the initial decline in serum [3H]T was significantly reduced (P less than 0.03) in the presence of hSHBG, (2) hALB did not affect the movement of [3H]T out of serum, (3) the time to peak appearance of [3H]T in the CSF was significantly delayed (P less than 0.02) by the presence of circulating hSHBG, and (4) the net quantity of [3H]T found in the cSF under steady-state conditions was not affected by serum SHBG or albumin levels. This study demonstrates that high-affinity steroid binding proteins do modulate the transport of sex steroids across the BBB. Specifically, SHBG delays the clearance of T from serum and slows the rate of T uptake into the CSF during non-equilibrium conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serum 5-androstene-3 beta,17 beta-diol sulphate, sex hormone binding globulin and free androgen index in girls with premature adrenarche

Serum sex hormone binding globulin (SHBG), testosterone (T), DHEA sulphate (DHEA-S), androstenedione (AD) and delta 5-androstene-3 beta,17 beta-diol sulphate (5-ADIOL-S) levels were measured by specific radioimmunoassay in 16 girls presenting with premature adrenarche (PA) and in 14 normal girls. Mean levels of steroids measured were elevated, and SHBG significantly depressed, in the girls with PA, with values (mean +/- SE) for DHEA-S (1.73 +/- 0.17 vs 0.25 +/- 0.06 mumol/l), 5-ADIOL-S (104 +/- 8 vs 31 +/- 4 nmol/l), AD (0.89 +/- 0.06 vs 0.62 +/- 0.04 nmol/l), and T (0.49 +/- 0.03 vs 0.23 +/- 0.06 nmol/l). SHBG levels were 68 +/- 6 vs 108 +/- 5 nmol/l, and the free androgen index [100 x T (nmol/l) divided by SHBG (nmol/l)] was 0.89 +/- 0.17 vs 0.22 +/- 0.01. These studies show that SHBG is depressed in girls with premature adrenarche; with the increased testosterone levels, this results in a markedly elevated free androgen index, a measure of testosterone which is bioavailable to target tissue. This may be compounded by the elevated levels of 5-ADIOL-S in girls with PA since its role may be as a prohormone for more potent androgens (testosterone, 5 alpha-dihydrotestosterone) in target tissues such as pubic skin.     

Oxytocin in the cerebrospinal fluid and plasma of pregnant and nonpregnant subjects

The oxytocin concentration in the cerebrospinal fluid (CSF) and plasma of pregnant women at term with and without labor pain were measured by radioimmunoassay and compared with those of non-pregnant women of matched age. The oxytocin concentrations in the CSF were 4.9 +/- 4.1 microU/ml (mean +/- S.D.) in pregnant women with labor pain, 4.1 +/- 2.4 microU/ml in those without labor pain and 4.0 +/- 2.8 microU/ml in nonpregnant women, and the oxytocin concentrations in the plasma of these subjects were 45.2 +/- 19.6, 17.1 +/- 22.2 and 7.0 +/- 5.3 microU/ml, respectively. Thus the oxytocin level in the CSF did not change appreciably even when the level in the plasma was raised in the pregnant women with labor pain. These findings suggest that oxytocin does not penetrate the blood-brain barrier, and that oxytocin in the CFS has little or no central role in parturition in women.     

Age, insulin, SHBG and sex steroids exert secondary influence on plasma leptin level in women

AIM: As the link between body fat and leptin is well known, the aim of the study was to seek for secondary regulators of plasma leptin level. PATIENTS: 86 women (mean: age 47.0+/-14.3 years; estradiol 50.0+/-60.6 ng/l; FSH 52.4+/-42.9 IU/l; BMI 26.9+/-5.9) divided into three groups according to their BMI. Group A: 39 normal weight women (mean: age 44.4+/-16.0 years; estradiol 69.6+/-79.8 ng/l; FSH 50.4+/-47.7 IU/l; BMI 22.9+/-1.3). Group B: 27 overweighted women (mean: age 55.0+/-6.4 years; estradiol 25.1+/-17.2 ng/l; FSH 75.6+/-26.3 IU/l; BMI 27.7+/-1.6). Group C: 21 obese women with mean: age 48.7+/-12.2 years; estradiol 36.9+/-44.0 ng/l; FSH 42.3+/-36.6 IU/l and BMI 34.6+/-4.9. METHODS: Standard clinical evaluation and hormone evaluation (LH, FSH, prolactin, estradiol, leptin, insulin-like growth factor-I (IGF-I), human growth hormone (hGH), insulin-like growth factor binding protein-3 (IGFBP-3), insulin, dihydroepiandrosterone sulphate (DHEAS), sex hormone binding globin (SHBG) and testosterone were done in basic condition which levels of were measured by RIA kits. Statistical analysis. Shapiro-Wilk test, Mann-Whitney-Wilcoxon u test, Spearman rank correlation coefficient and stepwise multiple regression: p values of 0.05 or less were considered as significant. RESULTS: Taking all women into account (n=86) the plasma leptin level correlated directly with age (r=0.32; p<0.02), body mass (r=0.60; p<0.001), BMI (r=0.71; p<0.001) as well as inversely with estradiol (r=-0.21; p<0.05), IGF-I (r=-0.24; p<0.05), SHBG (r=-0.34; p<0.01) and DHEAS (r=-0.30; p<0.01). However only in the group B leptin/age relation remained (r=0.40; p<0.05) after the division according to BMI. In the group B the leptin /DHEAS (r=-0.40; p<0.05) and leptin/PRL (r=0.51; p<0.05) links were also present. In the group C the leptin/SHGB relation (r=-0.56; p<0.02) only remained and an association between insulin and leptin was found (r=0.48; p<0.05). The body mass and BMI relation to age were again present only in all 86 women (r=0.30; p<0.002: r=0.36; p<0.001 resp.). Having split the women into groups, these links either disappeared or became inverse (rC=-0.39; p<0.05). Taking into consideration age/leptin relation in all women, the division according to the menopausal status revealed the direct relation in premenopausal women (n=29; r=0.43; p<0.02) and a reverse one in postmenopausal women (n=38; r=-0.32; p<0.05). The plasma leptin level was the highest (p<0.001) in group C (23.2+/-10.4 microg/l) and the lowest was found in the group A (8.9+/-4.1 microg/l). That corresponded with the differences in mean body mass index and mean body mass. The stepwise multiple regression revealed that body mass index accounted for 31% (p<0.001) and plasma SHBG level accounted for 17.7% (p<0.02) of plasma leptin variance in all women. In the group A body mass and age together accounted for 61% (p<0.01) and estradiol alone accounted for 44% (p<0.02) of plasma leptin variance. In the group B insulin alone accounted for 39% (p<0.05) and together with testosterone accounted for 46% (p<0.05) of plasma leptin variance. Finally in obese women none of the evaluated parameters significantly accounted for leptin variance. CONCLUSION: The results presented in this paper confirmed the strong influence of body fat mass on serum leptin concentration. However insulin, SHBG, sex steroids as well as age may also exert secondary influence on plasma leptin level in certain groups of women.     

Biophysical and biochemical characteristics of bactrian camel semen collected by artificial vagina

Seminal characteristics were investigated in Bactrian camel in this study. Semen samples from ten mature Bactrian camel bulls were collected using a modified bovine artificial vagina. The biophysical parameters including volume, color, sperm concentration and fast forward progressive motility, percentage of live sperm and the biochemical parameters including osmolarity, pH, glucose, calcium, phosphorus, chloride, triglycerides, phospholipids, total protein, albumin and non-protein nitrogen concentrations in seminal plasma were measured. The mean time for semen collection was 5.3 +/- 0.29 min. The volume of semen varies from 1.2 to 26 (8.2 +/- 0.7 mls). The majority of semen samples (83.6%) were milky in color and consistency. The average osmolarity of semen was 316.1 +/- 1.48 mOsm/kg H(2)O. The pH of semen was slightly alkaline (7.4 +/- 0.03). The mean concentration of spermatozoa was 414.8 +/- 25.04 x 10(6)cells/ml. The fast forward progressive motility of spermatozoa was 62.4 +/- 1.57%. The percentage of live spermatozoa was 85.6 +/- 1.15. Seminal plasma concentration of glucose was 35.8 +/- 0.9 mg/dl. Non-protein nitrogen, total protein and albumin were 32.5 +/- 2.5, 2200 +/- 100 and 1100 +/- 100mg/dl, respectively. The average concentrations of phospholipids and triglycerides in seminal plasma were 36.4 +/- 2.1 and 101.6 +/- 5.5mg/dl, respectively. The concentrations of calcium, phosphorus and chloride were 8.2 +/- 0.1, 2.9 +/- 1.7 mg/dl and 97.9 +/- 2.9 mEq./l, respectively.     

High serum circulating telopeptide type I in multinodular goiter.

Recently, concentrations of serum carboxy-terminal-1-telopeptide (ICTP), a marker of bone collagen resorption, were found to be more sensitive than sex hormone-binding globulin (SHBG) in identifying peripheral overexposure to thyroid hormones in exogenous subclinical hyperthyroidism. The aim of the present study was to assess serum ICTP and SHBG in multinodular goiter with (pretoxic goiter) or without biochemical evidence of endogenous subclinical hyperthyroidism. Forty-five women affected by multinodular goiter were enrolled in this study. They were subdivided into two groups: group 1, consisting of 27 patients affected by pretoxic goiter; group 2, consisting of 18 patients affected by non toxic goiter; group 3, consisting of thirty-six euthyroid women matched with the other groups for age and lifestyle. In group 1, serum ICTP (mean +/- SD: 5.8 +/- 2.9 microg/l) concentrations were significantly higher when compared either to group 2 (3.6 +/- 1.2 microg/l; p < 0.02) or controls (2.7 +/- 0.7 microg/l; p < 0.0001); serum ICTP concentrations were also slightly but significantly higher in patients of group 2 compared to controls (p < 0.003). In contrast, mean serum SHBG concentrations did not show any difference among the three groups. No significant correlation was found between serum TSH and ICTP concentrations, while a weak positive correlation (p < 0.05) was only found between serum FT 3 and ICTP concentrations when data from the two patient groups were analyzed together. Moreover, when we subdivided patients into pre- and postmenopausal patients, we observed that SHBG but not ICTP serum concentrations were influenced by estrogenic status. In summary, the measurement of serum ICTP seems to be more suitable than SHBG for identifying those with a higher degree of peripheral thyroid hormone exposure in women affected by endogenous subclinical hyperthyroidism.     

Comparison of serum ethinyl estradiol, sex-hormone-binding globulin, corticoid-binding globulin and cortisol levels in women using two low-dose combined oral contraceptives

The study included 69 women taking a desogestrel (n = 30)- or gestodene (n = 39)-containing low-dose combined oral contraceptive for at least 3 months. Group size was calculated to detect a difference in mean values of 80% of 1 standard deviation (alpha = 0.05, beta = 0.1). Seven serum samples were obtained up to 4 h, and 1 sample 24 h, after drug intake on 1 day between the 10th and the 21st day of the cycle. The concentrations of sex-hormone-binding globulin (SHBG), corticoid-binding globulin (CBG) and cortisol were measured in a 0- to 4-hour serum pool by radioimmunoassay. Ethinyl estradiol (EE2) levels were analyzed in single and pooled samples using anti-EE2-6 beta-carboxymethyloxime-bovine serum albumin antiserum. The area under the curves (AUC) up to 4 and 24 h and Cmax and tmax were evaluated. Statistical analysis (analysis of covariance) did not reveal a dependence of values on duration of treatment or day of cycle. Both treatments resulted in almost identical values for all parameters evaluated. The mean levels of SHBG, CBG and cortisol were in the range of 186-226 nmol/l, 89-93 mg/l and 280-281 micrograms/l, respectively. Mean maximum EE2 levels of 106-129 pg/ml were found 1.6-1.8 h after pill intake and AUC0-4 h accounted for 329-374 pg.h.ml-1. The recently reported differences in serum EE2 and CBG levels between two groups of 11 women each treated with desogestrel- and gestodene-containing pills, respectively, could not be confirmed.     

Retention of estradiol negative feedback relationship to LH predicts ovulation in response to caloric restriction and weight loss in obese patients with polycystic ovary syndrome

The present study tests the hypothesis that specific endocrine, metabolic, and anthropometric features distinguish obese women with polycystic ovary syndrome (PCOS) who resume ovulation in response to calorie restriction and weight loss from those who do not. Fifteen obese (body mass index 39 +/- 7 kg/m(2)) hyperandrogenemic oligoovulatory patients undertook a very low calorie diet (VLCD), wherein each lost > or =10% of body weight over a mean of 6.25 mo. Body fat distribution was quantitated by magnetic resonance imaging. Hormones were measured in the morning at baseline, after 1 wk of VLCD, and after 10% weight loss. To monitor LH release, blood was sampled for 24 h at 10-min intervals before intervention and after 7 days of VLCD. Responders were defined a priori as individuals exhibiting two or more ovulatory cycles in the course of intervention, as corroborated by serum progesterone concentrations > or =18 nmol/l followed by vaginal bleeding. At baseline, responders had a higher sex hormone-binding globulin (SHBG) concentration but were otherwise indistinguishable from nonresponders. Body weight, the size of body fat depots, and plasma insulin levels declined to a similar extent in responders and nonresponders. Also, SHBG increased, and the free testosterone index decreased comparably. However, responders exhibited a significant decline of circulating estradiol concentrations (from 191 +/- 82 to 158 +/- 77 pmol/l, means +/- SD, P = 0.037) and a concurrent increase in LH secretion (from 104 +/- 42 to 140 +/- 5 U.l(-1).day(-1), P = 0.006) in response to 7 days of VLCD, whereas neither parameter changed significantly in nonresponders. We infer that evidence of retention of estradiol-dependent negative feedback on LH secretion may forecast follicle maturation and ovulation in obese patients with PCOS under dietary restriction.     

Effects of pregnancy and labor on oxytocin levels in human plasma and cerebrospinal fluid

In order to investigate the significance of oxytocin in pregnancy and labor, oxytocin concentrations in plasma and cerebrospinal fluid (CSF) were determined using the specific radioimmunoassay. Plasma and CSF samples were obtained from 23 pregnant women (11 pre labor, 12 in labor), 15 nonpregnant women and 4 men at spinal puncture for anesthesia. In males and nongravidas, CSF levels of oxytocin were significantly higher than plasma levels. Plasma levels in pregnant patients pre or in labor were significantly higher than those in nongravidas. No significant difference between CSF levels in prelabor gravidas (mean +/- SE, 9.7 +/- 1.5 mu u/ml) and nongravidas (10.1 +/- 1.2 mu u/ml) was found. However, CSF levels in gravidas in labor (18.6 +/- 2.3 micromicrons/ml were significantly higher than the levels in prelabor gravidas. These results strongly suggest that oxytocin levels in human plasma and CSF are controlled by different mechanisms and that the increased oxytocin could have some specific central actions.     

Steroid dynamics in the rabbit

Male rabbits were infused at a constant rate with 3H-androstenedione/14C-estrone (n = 5) or 3H-testosterone/14C-estradiol-17 beta (n = 3) for 3 1/2 hr and blood samples were obtained over the last hour and analyzed for radioactivity as androstenedione (A), testosterone (T), estrone (E1), estradiol-17 beta (E2 beta) and estradiol-17 alpha (E2 alpha). The mean value for the metabolic clearance rate of androstenedione (MCRA) was 85 +/- 10 l/day/kg, which was significantly greater than the mean MCRE1 59 +/- 10 l/day/kg. MCRT, 42 +/- 8 l/day/kg, and MCRE2 beta, 45 +/- 9 l/day/kg were not different. The conversion ratio of androstenedione to testosterone (CRA,T) was greater than CRT,A but for the estrogens, CRE2 beta, E1 was greater than CRE1,E2 beta. CRE2 beta, E2 alpha was greater than CRE1,E2 alpha. The overall aromatization of androstenedione to estrone, the fraction of 3H-androstenedione infused into the blood and measured as 3H-estrone in blood [( rho]A,E1BB) was 0.0005 +/- 0.0001 and for [rho]T,E2 beta BB was 0.0012 +/- 0.0006. In the rabbit both sex hormone binding globulin (SHBG) and albumin binding may effect the MCRs, and peripheral aromatization of androgens occurs to a far lesser degree than in humans and primates.     

Oxidase activity of ceruloplasmin and concentrations of copper and zinc in serum in chronic arterial occlusion of the lower limbs.

The presence of ischaemic tissue excites an inflammatory reaction and synthesis of acute phase proteins (APhPs). Ceruloplasmin (Cp) protein binds 90% of the copper in plasma and it is one of the positive APhPs, and its concentration increases in infection, inflammation or necrosis. The study presents the relationship of the oxidase activity of Cp and concentrations of Cu and Zn in serum of men with different degrees of ischaemia of the lower limbs. The subjects were 32 men with chronic arterial occlusion (AO) of the lower limbs. The oxidase activity of Cp was measured in serum with o-dianisidine as a substrate. Concentrations of Cu and Zn were determined by using atomic absorption spectrometry. The mean activity of Cp in serum in AO (173 +/- 69.2 U/l) was higher as compared with the control group (123.7 +/- 28.6 U/l), and in men with critical ischaemia (> or = 194.8 U/l) than in men with a moderate level of ischaemia (109.3 +/- 31.6 U/l). The mean concentrations of Cu and Zn in serum were found to be higher in AO (22.2 +/- 4.2 and 19.1 +/- 6.9 mumol/l, respectively) than in the control group (16.3 +/- 1.8 and 15.2 +/- 2.3 mumol/l), and in men with critical ischaemia (> or = 22.2 and 19.1 mumol/l) than in men with a moderate level of ischaemia (18.5 +/- 3.3 and 14.5 +/- 4.3 mumol/l). Significant positive correlation coefficients were calculated for the activities of Cp and concentrations of Cu in the control group (r = 0.86) and the AO group (r = 0.76), and low, but significant, correlations for Cp and Zn in the AO group (r = 0.66). The increase in the oxidase activity of Cp and concentration of Cu in serum in ischaemia is caused by the acute phase response. The relationship of Zn concentration and Cp activity in ischaemia is indirect and needs further study.     

Carrier-Mediated Transport of Chloride Across the Blood-Brain Barrier

36Cl concentrations in each of eight brain regions and in cisternal cerebrospinal fluid (CSF) were determined 30 min after the intravenous injection of 36Cl in dialyzed-nephrectomized rats with plasma Cl concentrations between 14 and 120 mumol X ml-1. CSF 36Cl exceeded 36Cl concentrations in brain extracellular fluid. The calculated blood-to-brain transfer constants for Cl, kCl, ranged from 1.8 X 10(-5) S-1 at the parietal cortex to 3.8 X 10(-5) S-1 at the thalamus-hypothalamus. kCl fell by 42-62% when mean plasma [Cl] was elevated from 16 to 114 mumol X ml-1. Brain uptake of [14C]mannitol or of 22Na was independent of plasma [Cl], but 22Na influx into CSF fell when plasma [Cl] was reduced. Cl flux into brain and CSF could be represented by Michaelis-Menten saturation kinetics, where, for the parietal cortex, Km = 43 mumol X ml-1 and Vmax = 2.5 X 10(-3) mumol X S-1 X g-1, and for CSF Km = 68 mumol X ml-1. At least 80% of 36Cl influx into the parietal cortex was calculated to occur at the cerebrovascular endothelium, whereas the remainder was derived from tracer that first entered CSF. The CSF contribution was greater at brain regions adjacent to cerebral ventricles. The results show that Cl transport at the cerebrovascular endothelium as well as at the choroid plexus epithelium is a saturable concentration-dependent process, and that the CSF is a significant intermediate pathway for Cl passage from blood to brain.     

Alterations in sex steroids and gonadotropins in post-menopausal women subsequent to long-term mifepristone administration

Long-term administration of progesterone antagonists (PAs) and progesterone receptor modulators (PRMs) has been proposed as a novel hormonal therapy for various hormone dependent maladies. We studied the long-term endocrine effects of mifepristone on the kinetics of estradiol (E(2)) and its precursors, and on gonadotropin levels in five postmenopausal women treated for unresectable meningioma with mifepristone [200 mg/day] for at least 15 months. Serum samples were analyzed for LH, FSH and SHBG with fluoroimmunoassay; androstenedione (A), testosterone (T), estrone (E(1)) and E(2) were measured with radioimmunoassay (RIA). Serum levels of mifepristone were measured using both RIA and high performance-liquid chromatography (HPLC). Serum levels (mean +/- SD) of LH and FSH were suppressed from pretreatment values of 32 +/- 16 and 65 +/- 30 IU/l to 13 +/- 7 and 33 +/- 16 IU/l at 6 months (P < 0.05), respectively. Serum (mean +/- SD) A, T, E(1), and E(2) were increased from initial values of 6.9 +/- 0.9 nmol/l, 1.2 +/- 0.3 nmol/l, 77 +/- 25 pmol/l, and 29 +/- 14 pmol/l to 6 month values of 13.1 +/- 5.6 nmol/l, 1.8 +/- 0.6 nmol/l, 178 +/- 60 pmol/l, and 45 +/- 22 pmol/l (n.s.). The correlation coefficients between the levels of A, T, E(1), and E(2) were statistically significant, whereas the ratios of T/A, E(1)/A, E(2)/E(1), and E(2)/T remained unchanged. The levels of SHBG remained stable, and ranged from 48 +/- 10 to 65 +/- 9 nmol/l (mean +/- SD). Thus, prolonged mifepristone treatment marginally increased the serum levels of A, T, E(1) and E(2). These effects of mifepristone are likely due to its antiglucocorticoid effect and thus increased secretion of adrenal A. Serum levels of LH and FSH declined. The serum levels of gonadotropins and those of T, E(1) and E(2) were inversely, yet significantly, correlated. Therefore the decrease in LH and FSH might reflect the slightly increased levels of T, E(1) and E(2). However, the lack of change in SHBG and the low E(2) levels suggest that enhanced systemic estrogen effects are unlikely during long-term mifepristone treatment.     

The role of serum fructosamine as a screening test for gestational diabetes mellitus

The serum fructosamine concentration indicates the degree of glycation of serum proteins, particularly albumin, and reflects an average blood glucose level over the previous 1-3 weeks. Serum fructosamine, glycated haemoglobin (HbA1c), total serum protein, serum albumin, fasting plasma glucose and oral glucose tolerance test (OGTT) have been measured in 127 healthy control subjects, 102 type 1 and 152 type 2 diabetes mellitus patients and 106 nondiabetic pregnant women. Fructosamine concentration of 2.24 +/- 0.16 and 3.21 +/- 0.41 mmol/l (mean +/- S.D.) has been found in control subjects and diabetics respectively (P less than 0.001). During the second trimester a significantly lower fructosamine level (1.92 +/- 0.21 mmol/l) has been found in pregnant women, most likely due to the low serum albumin concentration (31.35 +/- 3.97 g/l). None of them had a fructosamine level above the normal limit of 2.55 mmol/l. On the other hand, 12 pregnant women showed a disturbed OGTT with normal fructosamine. If the serum fructosamine concentration was adjusted for 40 g/l albumin, then a mean fructosamine of 2.16 +/- 0.24 mmol/l was found in patients with gestational diabetes. Our results show that serum fructosamine has a similar diagnostic value as HbA1c for non-pregnant adults, but neither can replace OGTT for the diagnosis of gestational diabetes.     

Inverse relationships between steroid concentration and volume in preovulatory follicles of the golden hamster

In order to investigate variations in the microenvironment of oocytes within a cohort of maturing follicles the follicular volumes as well as the intrafollicular concentrations of oestradiol (E2) and progesterone (P) were measured in the golden hamster. At 10 h before ovulation the follicular volumes varied from 0.009 to 0.037 mm3 (mean +/- SD: 0.0187 +/- 0.0071 mm3; n = 36). Large follicles (greater than 0.025 mm3; n = 8) contained statistically significantly lower E2 and P levels (30.1 +/- 10.4 and 517 +/- 113 mumol/l, respectively) than the medium sized group (less than 0.025 and greater than 0.015 mm3; n =20): 46.9 +/- 16.0 (P less than 0.02) and 919 +/- 264 (P less than 0.0001) mumol/l, respectively. Small follicles (less than 0.015 mm3) showed the highest steroid levels: 97.0 +/- 33.3 and 1590 +/- 517 mumol/l for E2 and P (P less than 0.001 versus the medium sized group values). Correlation coefficients for the steroid concentrations and the follicular volumes appeared to be -0.674 for E2 and -0.612 for P (P less than 0.001). At the time studied a positive correlation between E2 and P concentrations in the follicles was found: r = 0.655 (P less than 0.001). The mean ratios of intrafollicular over serum steroid concentrations appeared to be approx 36 x 10(3) in the case of E2 and about 17 x 10(3) in the case of P. These results clearly show that there is an inverse relationship between follicular volume and intrafollicular steroid concentrations. The presence of a fine regulatory mechanism for a collective maturation of follicles is hypothesized.     

Measurement of free and bound fractions of extracellular ATP in biological solutions using bioluminescence.

Measurement of extracellular ATP in biological solutions is complicated by protein-binding and rapid enzymatic degradation. We hypothesized that the concentration of extracellular ATP could be determined luminometrically by limiting degradation and measuring the free and protein-bound fractions. ATP was added (a) at constant concentration to solutions containing varying albumin concentrations; (b) at varying concentrations to a physiological albumin solution (4 gm/dL); (c) at varying concentrations to plasma. After centrifugation, a fraction of each supernatant was heated. ATP in heated and unheated samples was measured luminometrically. Blood was drawn into saline or an ATP-stabilizing solution and endogenous plasma ATP measured. ATP-albumin binding was a linear function of albumin concentration (3.5% ATP bound at 100 micromol/L to 33.2% ATP bound at 1000 micromol/L) but independent of ATP concentration (29.3%, 10-1000 nmol/L ATP in 602 micromol/L albumin). Heating released the majority of bound ATP from albumin-containing solutions (94.8 +/- 1.7%) and plasma (97.6 +/- 5.1%). Total endogenous plasma ATP comprised 93 +/- 27 nmol/L (free) and 150 +/- 40 nmol/L (total fraction). Without stabilizing solution, degradation of free endogenous plasma ATP occurred. Within a physiological range (10-1000 nmol/L), ATP binds albumin independently of ATP concentration. Heating releases bound ATP, enabling accurate luminometric measurement of total extracellular ATP (free and bound) in biological samples.     

Metoclopramide increases plasma but not cerebrospinal fluid vasopressin levels in man: study in hydrocephalic patients.

Arginine vasopressin (AVP) concentrations were determined in plasma and in cerebrospinal fluid (CSF) in 8 adult male patients suffering from hydrocephalus of various etiologies, before and after intravenous administration of 10 mg metoclopramide. Metoclopramide was able to increase the plasma (2.6 +/- 0.2 ng/l in basal conditions and 6.1 +/- 0.6 ng/l at 30 min) but not the CSF AVP levels. The results suggest that the neurons which secrete AVP into the CSF may be functionally different from those secreting into the peripheral circulation.