Harvey S. Schiller

A filter assay for the corticosteroid binding globulin of human serum

A filter assay for corticosteroid-binding globulin (CBG or transcortin) in human serum was developed using DEAE-cellulose filter paper discs to bind the [3H]-cortisol-CBG complex. The CBG binding capacity is determined by incubating diluted serum samples with a saturating concentration of [3H]-cortisol in the presence and absence of 100-fold molar excess of radioinert cortisol and quantitating the amount of [3H]-cortisol remaining specifically bound to the filter. After washing filters with buffer to remove unbound and non-specifically bound (albumin bound) steroid, 82% of this complex is retained on the filter. The specificity of this assay is demonstrated by the ability of various steroids, known to bind to CBG, to compete for [3H]-cortisol. Endogenous steroids, which would otherwise dilute the specific activity of the [3H]-cortisol, are removed from the undiluted serum by charcoal treatment (50 mg/ml serum). The assay is linear with serum dilutions of 1:20 or greater. The procedure is particularly useful at high serum dilutions where the sensitivity depends only upon the specific radioactivity of the steroid. The calculated concentrations of CBG in serum from men and pregnant and non-pregnant women are 37 mg/1, 70 mg/1 and 37 mg/1, respectively; these values agree with those reported in the literature obtained by other methods. The equilibrium association constant of the cortisol-CBG complex as determined by the filter assay is 3.5 ± 0.3 × 10−9M−1 at 4°C which is slightly higher than the reported values determined by equilibrium dialysis. This new method is reproducible (CV 2.0–4.9%), rapid, accurate, and requires less than 0.5 ml serum. The procedure is not only useful for clinical investigation, but also for biochemical characterization of the protein.

Sex steroid binding protein in the plasma of Macaca nemestrina

A sex steroid binding protein (nSBP) has been identified in the plasma of Macaco nemestrina. The protein specifically binds 5α-androstane-17β-ol,3-one (KA(male) = 1.87 ± 1.05 × 109 M−1, KA(female) = 2.04 ± 0.88 × 109 M−1, KA(pregnancy) = 2.21 ± 0.59 × 109 M−1; 4°, 10 mM Tris, pH 7.4), testosterone (KA = 4.10 ± 2.50 × 108 M−1, 4°, 10 mM Tris, pH 7.4), and estradiol-17β (2.41 ± 1.34 × 108 M−1, 4°, 10 mM Tris, pH 7.4). The steroids bind to the protein at the same site. Progesterone, cortisol, estrone, estriol, and diethylstilbestrol have relatively low affinity for the binding site. Specificity studies also suggest that 5α-androstane derivatives with a 3α-hydroxyl show little affinity compared with those having 3β-hydroxyl. Electrophoresis in 5% polyacrylamide gels yields one active molecular species with RF 0.32. The rate constant of dissociation of the DHT-nSBP complex at 4° and pH 7.4 is 0.013 min−1 with a half-time of 52.8 min. The binding capacity of nSBP expressed in μg 5α-androstane-17β-ol,3-one bound/100ml plasma for males (5 samples) and females (7 samples) is 5.62 ± 1.24 (S.D.) and 11.07 ± 1.85 (S.D.), respectively (P < 0.001). These results show that nSBP and human SBP are quite similar in their binding and structural properties and therefore establish Macaca nemestrina as a good animal model to study the physiological role of SBP.

A radioimmunoassay of plasma estradiol-17β with the use of polyethylene glycol to separate free and antibody-bound hormone

Abstract A radioimmunoassay for plasma estradiol-17β was developed using polyethylene glycol to separate free from antibody-bound hormone. Specificity for estradiol-17β was achieved by a modified celite microcolunm procedure in which estradiol was.separated from interfering estrogens, including estrone. Using trace 3 H-estradiol to monitor procedural losses, the method was shown to be sensitive and accurate. Intra- and inter-assay coefficient of variation of the method was 8.7 and 10.6%, respectively. Polyethylene glycol used for antibody precipitation appears to be a generally applicable method for steroid hormone radioimmunoassays. The simplicity, precision and rapid analysis, coupled with its lack of time dependence and ease in automation, makes this a convenient and practical method.

Effect of ergot alkaloids on serum prolactin in non-psychotic organic brain syndrome of the elderly

A compound of ergot alkaloids (Hydergine-Sandoz) or placebo was given to sixty elderly nursing home patients with non-psychotic organic brain syndrome. Subsequent to 12 weeks of treatment, the mean serum prolactin of the drug was significantly lower than that of the placebo group. No correlation was found between changes in prolactin and changes in behavior using Sandoz Assessment of Clinical Status Rating Form-Geriatric [SCAG]. It may be that a higher dose of Hydergine with an accompanying greater drop in prolactin would be required to observe this effect.

A simple method for studying steroid-antibody interaction under conditions of thermodynamic equilibrium

Abstract We compare the classic method of equilibrium dialysis for achieving thermodynamic equilibrium with a method in which polyethylene glycol is used to separate antibody-bound and unbound steroid. Antisera to both polar and nonpolar steroids were studied. Comparable Scatchard plots were obtained by the two methods. Equilibrium constants of association of 10 9 to 10 10 liters/mol of the different antisera were determined by both methods. The described method should simplify the study of steroid-antibody interaction. In addition, this method will permit investigators to characterize quickly and conveniently the antisera to be used for sensitive radioimmunoassays.

Simple Routine Method for Enzymatic Iodination of Peptide Hormones

Abstract A simple enzymatic method for iodinating peptide hormones usable in radioimmunoassays is evaluated. We iodinated human LH, human FSH, human and rat prolactin multiple times over the past year using similar conditions. In every instance we obtained an iodinated protein which had little damage and high specific activity and was useful in the respective radioimmunoassay. We recommend this method of iodination for laboratories continually performing multiple peptide hormone radioimmunoassays because of the methods simplicity, consistency and wide versatility. In addition, radio-iodination to desired specific activity is possible with this method.

Perturbations of the human menstrual cycle by oxymetholone

The luteolytic activity of oxymetholone, and anabolic steroid, has been evaluated in 10 women. Administration early in the follicular phase of the cycle inhibited ovulation and prolonged the duration of the cycles in 2 of 3 subjects, but treatment beginning on Day 10 (3 subjects) did not prevent ovulation, although subsequent plasma progesterone concentrations were reduced. Treatment after ovulation (4 subjects) suppressed progesterone levels by 50 to 80 per cent and shortened cycle length by 6 to 8 days. Side effects were weight gain and bromosulfophthalein retention. The most likely mechanisms producing these perturbations are the inhibition of luteinizing hormone release early in the cycle and, later, inhibition of progesterone biosynthesis.

A Simplified Method for the Separation of Steroid Hormones and Its Application to Radioligand Assays

Abstract A simplified method for separating the major progestogens, estrogens and androgens in plasma was developed for use in both competitive protein binding assays (CPBA) and radioimmunoassays (RIA). The method, based on liquid-liquid partition chromatography, utilizes microcolumns consisting of plastic disposable syringes packed with celite:ethylene glycol. The method was applied to the plasma measurement of progesterone by CPBA and estradiol and testosterone by RIA.

Effect of prostaglandins on fatty acid metabolism in lung

Abstract This is the first report of the effect of prostaglandins on the biochemical pathways for fatty acid synthesis. PGE2 and PGF2α inhibited fatty acid elongation in a lung microsomal fraction. Neither prostaglandin affected the de novo, or soluble, system for fatty acid synthesis (i.e. acetyl CoA carboxylase or fatty acid synthetase). The results also suggest that the initial inhibition of fatty acid synthesis leads to a decrease in free fatty acids available for esterification into phospholipids. The site and possible mechanisms of inhibition are discussed.

A rapid specific radioimmunoassay for unconjugated estriol in plasma

A rapid, non-chromatographic radioimmunossaay for unconjugated estriol in pregnancy plasma has been developed which utilizes a commonly available antiestrogen antisera. Estradiol-17beta and estrone demonstrate 135% relative cross-reactivity with our antiserum, as compared with 100% for estriol. Specificity is achieved by purification of estriol with solvent partitioning using benzene: petroleum ether (1:1). The results obtained using this method are similar to a radioimmunoassay utilizing a highly specific, but commercially unavailable, antiestriol antiserum. The method is precise, with coefficients of variation ranging from 3.0 to 8.2%.