Determination of prostaglandin F2α and E2 contents in human cerebrospinal fluid by the radioisotope dilution method

Prostaglandin F_2α and E₂ contents in human cerebrospinal fluid were determined by the radioisotope dilution method. The mean values of PGF_2α and PGE₂ of men were 9.8±0.87 ng/ml and 6.5±1.39 ng/ml, respectively. Those of women were 8.3±1.4 ng/ml and 6.9±1.72 ng/ml, respectively. The correlation between age and PG was significantly with PGE₂ of men and with PGF_2α of women.     

Evaluation of progesterone and 20-oxo-progestagens in the plasma of Asian (Elephas maximus) and African (Loxodonta africana) elephants

The corpus luteum of African elephants produces high amounts of 5α-reduced progesterone metabolites (5α-pregnane-3,20-dione and 5-α-pregnane-3α-ol-20-one), whereas progesterone itself is quantitatively less important, and plasma levels of progesterone during the estrous cycle in elephants are considerably lower than those of other mammals. The objective of this study was to compare the concentration of progesterone in plasma of Asian and African elephants as determined by specific progesterone assays with those of total immunoreactive progestagens containing a 20-oxo-group (20-oxo-P). These metabolites were determined by an enzyme immunoassay using an antibody against 5-α-pregnane-3β-ol-20-one, 3HS:BSA. Plasma of non-pregnant Asian (n = 4) and African (n = 4) elephants was collected at weekly intervals for periods of 8–15 months and at random intervals during pregnancy in one Asian elephant. High-performance liquid chromatography separation of plasma samples of both species demonstrated that in the 20-oxo-P assay, 5α-pregnane-3,20-dione makes up ˜60% of the total immunoreactive material. The progesterone and 20-oxo-P values during the estrous cycle showed a parallel pattern and were significantly correlated (P < 0.001; Asian: r = 0.80; y = 3.76 × –0.10; African: r = 0.75; y = 2.66 × –0.08). Progesterone and 20-oxo-P values in Asian and African elephants were <0.15 ng/ml during the follicular phase (weeks –4 to 0) of the estrous cycle; progesterone values during the luteal phase (weeks 2–9) were 0.60 ± 0.03 and 0.53 ± 0.03 ng/ml, and the 20-oxo-P values were 2.19 ± 0.16 and 1.48 ± 0.12 ng/ml, respectively. The 20-oxo-P values of the pregnant animal, although slightly higher, were comparable to those of non-pregnant elephants during the luteal phase. Total immunoreactive 20-oxo-P values are about three times higher than those of progesterone during the luteal phase, and 5α-pregnane-3,20-dione is the major immunoreactive 20-oxo-P in the plasma of Asian and African elephants. Zoo Biol 16:403–413, 1997. © 1997 Wiley-Liss, Inc.     

Simultaneous radioimmunoassay of 5α-amdrostane-3α, 17β-diol and 5α-androstane-3β, 17β-diol unconjugated and conjugated in human serum

The simultaneous determinations of both 3α and 3β epimers of 5α-androstane-3, l7β-diol as their glucuronides, sulfates and in their unconjugated forms are described. The diol estimation is carried out by radioimmunoassay with two specific immune sera after purification of the serum by use of chromatography on Sephadex LH-20. The values obtained (mean ± S.D.) in pg/ml for the unconjugated 3α and 3β epimers were, respectively, 267 ± 67 and 816 ± 76 for men; 114 ± 33 and 515 ± 177 for women; 142 ± 77 and 779 ± 200 for hirsute women. Among the conjugates, the most important were the sulfoconjugates, their rates being, respectively, (men ± S.D. in ng/ml) 41.3 ± 9.5 and 103 ± 40 for men; 12.4 ± 3.1 and 51.2 ± 14.9 for women and 36 ± 22 and 72 ± 36 for hirsute women. Differences in the conjugation of both epimers were also noticed.     

Radioimmunoassay for etiocholanolone

A sensitive and reliable radioiinmunoassay for serum unconjugated etiocholanolone (3α-hydroxy-5β-androstan-17-one) is reported. The antiserum was obtained from rabbits by immunization of etiocholanolone-17-(O-carboxy-methyl) oxime [CMO] -bovine serum albumin [BSA]. Two ml of serum with ³H-etiocholanolone added for recovery was extracted with ether, and etiocholanolone was separated from cross-reacting steroids by Sephadex LH-20 column chromatography.The mean recovery after extraction and chromatography was 80.7 ± 6.8(S.D.)%. The sensitivity of the assay was less than 40 pg. The intraassay and interassay coefficients of variation were 9.2% and 10.9%, respectively. The mean of serum unconjugated etiocholanolone concentration determined by the present method was 0.39 ± 0.10 (S.D.) ng/ml (n = 50) in normal men and 0.36 ± 0.08 (S.D.) ng/ml (n = 20) in women in the follicular phase of the menstrual cycle.     

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.     

Simultaneous radioimmunoassay of testosterone and dihydrotestosterone

A radioimmunoassay, which simultaneously measures both testosterone (T) and dihydrotestosterone (DHT) in the same serum sample, is presented. Celite column chromatography is employed to separate T from DHT, and these two steroids from other potentially cross-reacting and interfering steroids. The normal values for men, women in the follicular phase, women in the luteal phase, ovariectomized and adrenal ectomized women, post-menopausal women and ovariectomized women for T are 5, 140 ± 1190 pg/ml, 307 ± 97 pg/ml, 285 ± 46 pg/ml, undetectable (<5 pg/ml), 262 ±47 pg/ml and 199 ±44 pg/ml; and for DHT 470 ± 165 pg/ml, 160 ±45 pg/ml, 147 ±44 pg/ml, undetectable (<5 pg/ml), 168 ± 27 pg/ml, 94 ± 15 pg/ml. The maximum sensitivity of the method was 10 pg/ml for T and 14.3 pg/ml for DHT when 1 ml was extracted. The blank in most assays was undetectable, but rarely exceeded 10 pg.     

Prostaglandin F2α concentration in genital tract secretions of dairy bulls

Prostaglandin F_2α (PGF_2α) concentrations in genital tract secretions of conscious dairy bulls were determined by radioimmunoassay procedures and compared with peripheral blood plasma levels. The mean (± SD) PGF_2α concentration of coccygeal venous blood plasma from four bulls was 0.14 ± 0.05 ng/ml. Values for rete testis fluid and seminal plasma were the same, namely 0.17 ± 0.01 ng/ml (n = 5) and 0.17 ± 0.02 ng/ml (n = 4), respectively. However, the PGF_2α level in cauda epididymal plasma was 1.61 ± 0.41 ng/ml, or over 8 to 10 times (P < 0.01) the concentration of any other fluid studied.Added PGF_2α had no effect on the endogenous oxygen consumption of washed cauda epididymal spermatozoa or on the oxidative and glycolytic activities of washed ejaculated spermatozoa in vitro. No evidence was obtained suggesting that the prostaglandin may interact with the stimulatory effect of added testosterone or phosphatidylinositol (PI) on the motility, respiration or glucose uptake of ejaculated spermatozoa.     

Solid phase method for measurement of the binding capacity of testosterone-estradiol binding globulin in human serum

A solid phase method for measuring the binding capacity of serum testosterone-estradiol binding globulin (TeBG) is described and compared with other methods. TeBG, a glycoprotein, is adsorbed from serum or plasma onto a solid phase matrix of concanavalin-A, a carbohydrate-specific adsorbent. The TeBG binding capacity is determined by Scatchard analysis of the binding of radioactive testosterone at physiologic pH, in standard test tubes, and without the addition of albumin. Transcortin binding of testosterone is inhibited by the addition of cortisol.The levels of TeBG binding capacity determined with this solid phase method showed an excellent correlation with levels determined by procedures using equilibrium dialysis (with added cortisol) or ammonium sulphate precipitation. The serum TeBG binding capacity was 0.798±0.064 (mean±SE) μg/100 mL in men (n=32), 1.06±0.13 in women (n=10), 2.18±0.19 in women taking oral contraceptives (n=4), 6.2±2.9 in hyperthyroid women (n=2), and 11.6±3.1 in pregnant women (n=5). The serum TeBG binding capacity determined in heparinized plasma did not differ from that determined in serum. The within-assay variation is 9.6% and the between-assay variation is 11.2%.This solid phase method for measurement of serum TeBG binding capacity is simple, precise, and reproducible, and gives values which correlate well with those determined by other methods.     

Antiserum to 5alpha-dihydrotestosterone: production, characterization and use in radioimmunoassay

5α-Dihydrotestosterone (5α-DHT) was rendered antigenic by covalent attachment to bovine serum albumin (BSA) through position 1 of the steroid. Nucleophilic attack by β-mercaptopropionic acid on the 1,2-dehydro derivative of 5α-DHT yielded the corresponding 1α-thioether alkanoic acid which was coupled to bovine serum albumin by use of the carbodiimide reagent. The method should be generally applicable to 3-oxosteroids. Immunization of rabbits with 5α-DHT-1α-carboxyethyl-thioether-BSA gave rise to antisera of high affinity for 5α-DHT (K_a= 1.4 × 10⁹ 1/mol) that showed little cross reaction with 17β-hydroxy-5β-androstan-3-one (3%), and with a variety of 17-oxoandrostane compounds (≤0.5%). However the serum cross-reacted significantly with testosterone (10%) and with 5α-androstene-3α, 17β-diol (16%). A radioimmunoassay procedure for the determination of 5α-DHT in plasma is described. Chromatographic purification of the plasma extracts proved necessary for obtaining valid results. The plasma level of 5α-DHT(pg/ml; ean ± S.D.) was 364±79 (n = 7) in normal human adult males and 188 ± 62 (n = 5) in normal non-pregnant women.     

The concentration of 6-keto-PGF1α and TXB2 in plasma samples from patients with benign and malignant tumours of the breast

Peripheral plasma concentrations of 6-keto-PGF_1α and TXB₂ were measured in patients with benign and malignant tumours of the breast, in patients with nongynecological disease,a nd in healthy female controls. The values were significantly higher in female patients with maligants tumours of the breast than in healthy controls (146 ± 28 vs 13 ± 2.5 pg/ml for 6-keto-PGF_1α p<0.01 and 78 ± 17 vs 11 ± 2 pg/ml for TXB₂, p<0.01). Benign tumours of the breast were also associated with significantly raised plasma levels of 6-keto-PGF_1α and TXB₂ compared to normal controls (52 ± 5 vs 13 ± 2.5 pg/ml for 6-keto-PGF_1α, p < 0.01 and 26 ± 5 vs 11 ± 2 pg/ml for TXB₂, p < 0.05). The high levels of 6-keto-PGF_1α and TXB₂ were not found to be correlated with clinical and histopathological data. The surgical removal of the primary tumour has apparently no effect on the plasma concentration of 6-keto-PGF_1α and TXB₂ over a follow-up period of 9 days after operation. The lack of alterations in the ratio of TXB₂: 6-keto-PGF_1α in the cancer patients and other subjects studied before and after surgery is indicative of the regulatory power of metabolic systems to preserve the homeostatic balance.     

Competitive protein binding assay of progesterone without chromatography

A competitive protein binding assay for the measurement of progesterone in human plasma without chromatographic separation of steroids and recovery evaluation in individual samples is described. It is based on the specificity of the progesterone binding plasma protein (PBP) of the pregnant guinea pig. A dried petroleum ether extract of plasma was incubated with ³H-progesterone and 1600 fold diluted pregnant guinea pig plasma. Bound radioactivity was measured with a dextran coated charcoal suspension technique. Plasma progesterone concentration was obtained by comparison with a standard curve and correction for extraction separately measured for each batch of petroleum ether. The sensitivity was 100 pg. Recovery experiments for progesterone and competing steroids added to plasma respectively showed the accuracy and the specificity of the method. However comparison of the results from assays with and without chromatographic separation of steroids, showed that in the latter-case the specificity was good only for plasmas containing more than 1ng/ml of progesterone. Concentration of progesterone in plasma from men was 0.46±0.14 ng/ml (mean ± S.D) and from post menopausal women 0.30± 0.13 ng/ml.Between days 1 and 13 and days 16 and 22 of the normal menstrualcycle the concentrations were respectively 0.81 ± 0.38 and 12.50 ± 2.96 ng/ml. The variations of the progesterone concentration during pregnancy are also shown.     

Mass fragmentographic determination of prostaglandin F2α in human and rabbit urine

Analysis of prostaglandin F_2α (PGF_2α) in urine is a useful indicator of renal prostaglandin synthesis. A mass fragmentographic method for PGF_2α analysis in human urine was developed using [3,3,4,4-²H₄]PGF_2α as an internal standard and carrier. PGF_2α was extracted from urine (20 ml) with chloroform, purified by preparative thin-layer chromatography and converted to the methyl ester trimethylsilyl ether before analysis by gas chromatograph—mass spectrometry. The specificity of the urine analysis was demonstrated by retention time and the use of two pairs of fragments m/e 494/498 and 513/517 with the same results. The coefficient of variation for duplicate analysis averaged 12.6%, n = 17. Urine from recumbent women contained 4.9 ± 2.6 (S.D.) ng/ml or 4.1 ± 1.0 ng PGF_2α per mg creatinine (n = 10) with little diurnal variation. Male urine contained 5.0 ± 2.7 (S.D.) ng/ml or 3.7 ± 2.1 ng/mg creatinine (n = 10). Similar concentrations were found in boys and in girls. These observations indicate that urinary PGF_2α originates from the kidneys with little contribution from the male accessory sexual glands. This method can also be applied to analysis of PGF_2α in rabbit urine.     

Simultaneous radioimmunoassay of testosterone,dihydrotestosterone, 5α-androstane-3α, 17β-diol and 5α-androstane-3β, 17β-diol in the plasma of adult male rats

A single thin layer chromatography and three antibodies were used for the specific radioimmunoassay of four androgens in pooled rat plasma (Sprague-Dawley adult males). The following values were found (pg/ml ± SD). Testosterone : 3, 138 ± 173; dihydrotestosterone : 374 ± 20; 5α-androstane-3α 17β-diol : 284 ± 24; 5α-androstane-3β, 17β-diol : 223 ± 11.     

Effect of acute ethanol intake on thromboxane and prostacyclin in human

To investigate the effects of acute ethanol administration on the production of proaggregatory thromboxane A₂ (TxA₂) and anti-aggregatory prostacyclin (PGI₂), ethanol (1.5 g/kilogram body weight) was given to eight healthy nonsmoking men, and the stable metabolites thromboxane B₂ (TxB₂) and 6-keto-prostaglandin F_1α (6-keto-PGF_1α), respectively, measured by radioimmunoassay from serial blood samples before drinking and during the ensuing 18 hours. Each subject was studied as his own control on another occasion when only an equivalent volume of water was given. Serum TxB₂ level decreased (p < 0.01) from 206 + 31 ng/ml (mean) ± S.E. to 167² ± 24 and 161 ± 23 ng/ml (two and four hours after beginning of the drinking, respectively) concomitantly with the attainment of maximal blood ethanol concentrations (about 120 mg/100 ml), whereas no changes occurred in plasma 6-keto-PGF_1α concentrations. Our results may provide an explanation for known effects of ethanol on platelet aggregation. They also raise speculation whether TxA₂-inhibition and the antiatherogenic effect of alcohol intake are somehow related.     

Effect of ACTH and CRH on Plasma Levels of Cortisol and Prostaglandin F<Subscript>2α</Subscript> Metabolite in Cycling Gilts and Castrated Boars

The present study was designed to evaluate the effects of synthetic ACTH (1–24, tetracosactid) and porcine CRH on the plasma levels of cortisol and PGF_2α metabolite in cycling gilts (n = 3) and castrated boars (n = 3). The experiments were designed as crossover studies for each gender separately. Each animal received, during three consecutive days; 1) ACTH (Synacthen^® Depot) at a dose of 10 μg/kg body weight in 5 ml physiological saline, 2) porcine CRH at a dose 0.6 μg/kg body weight in 5 ml physiological saline or 3) physiological saline (5 ml). The test substances were administered via an indwelling jugular cannula in randomized order according to a Latin square. The administration of ACTH to cycling gilts resulted in concomitant elevations of cortisol and PGF_2α metabolite with peak levels reached at 70.0 ± 10.0 and 33.3 ± 6.7 min, respectively. Similarly, the administration of ACTH to castrated boars resulted in concomitant elevation of cortisol and PGF_2α metabolite with peak levels reached at 60.0 ± 0.0 and 20.0 ± 0.0 min, respectively. Cortisol peaked at 20 min after administration of CRH in both cycling gilts and castrated boars with maximum levels of 149.3 ± 16.5 nmol/1 and 138.3 ± 10.1 nmol/1, respectively. It can be concluded that administration of synthetic ACTH (tetracosactid) to pigs caused a concomitant elevation of cortisol and PGF_2α metabolite levels in both cycling gilts as well as castrated boars. The administration of CRH to pigs resulted in an elevation of cortisol levels in both cycling gilts and castrated boars. Conversely, PGF_2α metabolite levels were not influenced by the administration of CRH either in cycling gilts or in castrated boars.     

Effects of prostaglandin on estrous cycles,behavior and blood progesterone levels of American Saddlebred mares

Twelve American Saddlebred mares ranging in weight from 365 to 450 kg were given intramuscular injections of 2.5, 5.0 and 7.5 mg of Prostaglandin (PGF_2α) on day 6 of diestrus a mean length of control estrus and diestrus were 6.5 ± .6, 16.9 ± 1.0 days, respectively. The 2.5, 5.0 and 7.5 mg PGF_2α doses significantly (P < .01) shortened the length of the treatment diestrus to 10.8 ± 1.8, 9.9 ± .7 and 9.9 ± .7, respectively. The 2.5 mg dose was 90% effective in shortening the duration of diestrus while doses of 5.0 and 7.5 mg were 100% effective. No effects were noted on the mean length of estrus or diestrus following treatment. Peripheral plasma progesterone concentrations were measured by radioimmunoassay to determine the luteolytic effect of PGF_2α. As compared to the non-treatment estrous cycles, all three treatments caused a significant (P < .01) decline in peripheral plasma progesterone concentrations 24 and 48 hr after treatment. The 2.5 mg PGF_2α dose caused a drop in progesterone from 7.7 ± .4 on day 6 to 2.6 ± 1.0 and 2.1 ± .9 ng/ml 24 and 48 hr later, respectively. Similarly, 5.0 mg lowered the progesterone level from 7.7 ± .3 to 1.6 ± .6 and 1.5 ± .5 ng/ml, and the 7.5 mg dose lowered the progesterone level 7.5 ± .3 to 1.2 ± .2 and 1.3 ± .3 PGF_2α. Abdominal cramps were noted in some mares after treatment. The incidence and severity of these reactions increased with the dose of PGF_2α.     

On the mechanism of midtrimester abortions induced by the prostaglandin “impact”

Using Csapo's technique a single dose of 24.3±1.1mg PG F_2α had been delivered intraamniotically to 20 sedated 15.9±0.6 weeks pregnant patients, to provoke a “PG Impact” (PGI), a consequent progesterone (P) withdrawal and a conversion of the pharmacologically refractory normal pregnant uterus into a reactive organ. The side effects were occasional and acceptable and no further PG F_2α treatment was needed except in 4 cases (5–10mg). Only after the Oxytocin Test showed that the uterus is becoming reactive was 50mU/min oxytocin infused i.v., to facilitate the evolution of IUP to 93±3mm Hg and thus promote clinical progress. All the 20 patients aborted both the fetus and the placenta in 16.5±2.1 hours, but 8 women retained small placental residues to be removed by curettage. The Csapo Score was high, 92±2.As early as 3 hours after PGI, the plasma P levels already decreased significantly. They continued to decline throughout the IAT and reach a 72% withdrawal when the fetus was aborted. Fifteen patients, whose P-withdrawal was rapid aborted before the mean IAT, while those 5 women whose P-withdrawal was slow aborted after this time. Thus, the rate of P-withdrawal was directly, while parity and gestational age indirectly related to the IAT. Studies are in progress to elucidate further the abortifacient action of PG F_2α and through this knowledge promote predictable therapy.     

Determination of 15-keto-13,14-dihydro-metabolites of PGE2 and PGF2α in plasma using high performance liquid chromatography and gas chromatography-mass spectrometry

A method is described for the measurement of 15-keto-13,14-dihydrometabolites of PGE₂ and PGF_2α in peripheral human plasma. This involves purification by high performance liquid chromatography followed by determination of levels by combined gas chromatography-mass spectrometry using tetradeuterated analogs of the metabolites as internal standards. The levels of these metabolites in plasma are considered to be a more reasonable index of the entry of PGE₂ and PGF_2α into peripheral blood than are the levels of the corresponding primary prostaglandins. The endogenous levels of 15-keto-13,14-dihydro-PGE₂ and 15-keto-13,14-dihydro-PGF_2α found in peripheral plasma are 33 ± 10 pg/ml (SD; n=6) and 40 ± 16 pg/ml (SD; n=6), respectively.     

A radioimmunoassay of androstenedione

A dextran-coated charcoal radioimmunoassay for androstenedione (4-androsten-3, 17-dione) is reported which uses an anti-testosterone antiserum raised in sheep, against a testosterone-17-hemisuccinate-Bovine Serum Albumin conjugate. It is more sensitive and rapid than previously published double dilution, gas chromatographic and competitive protein binding assays. Androstenedione is separated from cross-reacting Steroids by Celite column chromatography. The intra-assay and interassay coefficients of variation were 10.7 and 11.6 per cent, respectively. Using this method serum androstenedione in men was 1.15 ± 0.35 ng/ml; in women, 1.41 ± 0.30 ng/ml; in post-menopausal women, 0.88 ± 0.34 ng/ml; in ovariectomized women, 0.67 ± 0.17 ng/ml; and in ovariectomized-adrenalectomized women, 0.14 ± 0.05 ng/ml. The blank of the method was usually 4 to 5 pg, but ranged between 0 and 12 pg. The sensitivity of the standard curve was 8 pg.     

Relationships between the amount of saliva and medications in elderly individuals

Gerodontology 2010; doi: 10.1111/j.1741‐2358.2009.00358.x
Relationships between the amount of saliva and medications in elderly individuals Objective: To investigate medications that are related to volume of saliva in the elderly. Background data: In the elderly, many cases of mouth dryness may represent side effects of medication. Materials and methods: The volume of unstimulated saliva was measured for 30 s (cotton roll test), and with stimulation for 3 min (gum test) in 368 subjects 79–80 years old (177 men, 191 women). Medications were investigated using subject’s medication notebooks. Results: Mean volumes of unstimulated and stimulated saliva were 0.14 ± 0.13 and 4.30 ± 2.54 ml respectively. Significant differences were seen between gender and mean volume of saliva. The volume of unstimulated saliva was 0.16 ± 0.15 ml for men and 0.11 ± 0.10 ml for women. The volume of stimulated saliva was 4.99 ± 2.67 ml for men and 3.67 ± 2.25 ml for women. The percentage of subjects taking medication was 64.7% (238/368). Mean number of medications was 2.08 ± 2.26, with no significant difference with gender (2.01 ± 2.37 for men, 2.16 ± 2.16 for women). In a stepwise multiple regression analysis with volume of saliva as the objective variable and number of drugs by category as explanatory variables, significant explanatory variables in addition to gender and number of medications were blood‐coagulating agents, Ca antagonists and peptic ulcer drugs for volume of unstimulated saliva, and diabetes medications and peptic ulcer drugs for volume of stimulated saliva. Conclusion: These findings suggest that differences exist between gender in volume of saliva for elderly individuals, and that the volume of saliva is affected by the number and type of medications.