A competitive protein binding assay for allopurinol and oxipurinol

A competitive protein binding assay for allopurinol or oxipurinol has been developed based on the tight binding of these drugs to reduced xanthine oxidase. Free drug is separated from that bound to xanthine oxidase by absorption with dextran-albumin coated charcoal. This assay can detect as little as 0.1 μm allopurinol or oxipurinol in water, serum, plasma, or urine. Competitive analogs such as hypoxanthine, xanthine, and uric acid require concentrations 100- to 1000-fold greater than those of allopurinol or oxipurinol to cause significant interference with the assay. This assay is simple and rapid with the ability to assay 20–30 samples within 2 h. Measurement of oxipurinol levels in clinical samples shows good correlation with published results using more complex analytical techniques.     

Determination of serum testosterone and androstanediol by competitive protein binding

A method for the simultaneous measurement of serum testosterone (T) and androstanediol (Ad) utilizing aluminum oxide thin layer chromatography and competitive protein binding analysis is presented. The method separates not only T from Ad, but also the androstanediol isomers. The primary Ad found was 5α-androstane-3α, 17β diol. Levels of both Steroids were determined in normal adults and children, and in a variety of endocrine disorders. The average $\text{T}\text{Ad}$ ratio was higher in female children than other controls except adult males. However, results were too variable and number of patients insufficient to draw definite conclusions as to the value of determination of this ratio in patients with androgen disorders.     

A sensitive competitive protein binding assay for vitamin D in plasma

A sensitive protein binding assay for vitamin D is described. The vitamin D3 was extracted from plasma with diethyl ether and methylene chloride. The lipid extract was purified in Sephadex LH-20 followed by Lipidex 5000 and finally by high pressure liquid chromatography on a Zorbax Sil column (0.79 x 25 cm) developed in 0.25:99.75 isopropanol: methylene chloride. The vitamin D fraction was collected and quantitated by competitive protein binding assay with a 1/50,000 dilution of sheep plasma in 0.05 M potassium phosphate buffer (pH 7.5) containing 0.01% gelatin. [H3]-25-Hydroxyvitamin D3 was used as a radioactive tracer in the assay. We found that under these conditions, sheep plasma had equal affinity for vitamin D2 and vitamin D3 and could detect as little as 0.1 ng of vitamin D. When rat, cow, or human plasma was substituted for the sheep plasma, the decline in sensitivity to vitamin D2 was fivefold to tenfold. With this assay, we found excellent agreement (r = 0.98) between the results obtained by competitive protein binding analysis and direct U.V. absorbance analysis by high pressure liquid chromatography.     

Colorimetric assay for free and bound L-fucose

A novel, rapid, and reliable colorimetric method for measuring L-fucose has been developed. This method utilizes NADH formed from the interaction of L-fucose with fucose dehydrogenase and NAD to generate color in a reaction involving CuSO4 and neocuproine. NADH reduces Cu2+ to Cu1+ and the latter interacts with neocuproine to yield a complex with a maximal absorption at 455 nm. The reaction of NADH with copper-neocuproine is immediate and under the conditions of the assay the color formed remains stable for at least 2 h. When the assay is used to determine levels of L-fucose, the absorbance is found to be linearly proportional to exogenously added fucose concentrations from 16 to 179 nmol with resulting molar extinction coefficient of 13,660. Using this procedure, L-fucose released by acid hydrolysis from porcine submaxillary mucin, and by alpha-L-fucosidase from p-nitrophenyl-alpha-L-fucopyranoside, was quantitated.     

Simple reproducible competitive radioligand assay for plasma protein binding of aldosterone

A simple reliable and specific binding assay for the estimation of plasma (or serum) aldosterone-binding globulins (ABGs) is described. The method is based on the determination of the aldosterone-binding capacity of diluted plasma (with water 1:5), and relating it to the total aldosterone concentration in the same sample. The method distinguishes between the heat-labile and heat-stable ABG, the binding to the latter being determined following heating of plasma at 60 degrees C for 25 min. The binding to the heat-labile protein is determined by subtraction of the value for the binding to the heat-stable protein from the binding of the nonheated diluted plasma. Free aldosterone is separated from the bound fraction by adsorption of the former to dextran-coated charcoal. Two different concentrations of [3H]-aldosterone are used throughout the assay. Data obtained by incorporating chromatographic separation into the cross-reactivity procedure using several steroids are presented as evidence for the specificity of the method. This chromatography separates plasma ABG from corticosteroid-binding globulin. In 316 male or female controls, ABG capacity was 9.7 +/- 0.38% (SE)-range 2-17. Samples in females were taken during the first 8 days from the onset of menstruation. Higher ABG-binding capacity (p less than 0.001) was found during pregnancy.     

An enzyme-linked immunosorbent assay for fetal steroid binding protein of human serum

The binding characteristics and quantitation of the recently reported fetal steroid binding protein (FSBP) cannot be determined on unpurified samples; an immunoassay was therefore desirable. The protein was purified to homogeneity in order to raise a highly specific polyclonal antibody. An enzyme-linked immunosorbent assay applicable to unpurified samples was developed. Intra- and inter-assay coefficients of variation are 8.0% and 9.2% respectively; there is a sensitivity of 30 fmol FSBP per well, and there is no cross-reactivity with other binding proteins. Results obtained with the assay correlate with the more complex ligand binding assay (r = 0.85; p less than 0.02). Measurement of sera showed that FSBP levels are higher in women than in men (51.2 +/- 10.62 nM; 41.2 +/- 13.65 respectively; p less than 0.05) and are elevated in cirrhotic women (66.4 +/- 18.67; p less than 0.05) and in males with hepatocellular carcinoma (62.2 +/- 13.05; p less than 0.002). Use of the enzyme-linked immunosorbent assay confirmed the identity of FSBP separate from sex hormone binding globulin.     

Contraceptive steroid treatment affects steroid binding proteins and the percentage of free 17 beta-estradiol and testosterone in the cynomolgus monkey (Macaca fascicularis)

The levels of steroid binding globulins were characterized in cynomolgus monkeys that were treated with contraceptive steroid preparations delivered either by intravaginal rings (CVR) or orally (OC) in the diet. Levonorgestrel (dNG) was the bioactive progestin and the estrogen was either 17 beta-estradiol (E2) in the CVR treatment or ethinyl estradiol (EE) in the OC treatment. Both contraceptive treatments lowered sex hormone binding globulin (SHBG) levels below those observed in males (P less than 0.05) and normal females (P less than 0.01). Corticosteroid binding globulin (CBG) was elevated (P less than 0.01) in the OC treatment, demonstrating the potency of EE. The distribution of E2 and testosterone (T) between binding to SHBG or albumin and the unbound fraction was calculated after the determination of the percentage of free steroid by centrifugal ultrafiltration. Both contraceptive treatments increased the percentage of free T and E2 (P less than 0.01) in the subset of monkeys that were evaluated, but the percentage bound to SHBG and albumin were different only for the CVR group (P less than 0.05). Decreased total T concentrations in the treatment groups offset any increase in free T concentrations associated with an increase in the percentage of free T. The differences in the distribution of binding to SHBG associated with these contraceptive steroid treatments was influenced more by the reduction in the binding capacity of SHBG than by the displacement of E2 and T from SHBG by dNG.     

Immunoenzyme method for differential assay of free and bound ABA

We performed the immunochemical study of two solid-phase competitive ELISA systems differing in their specificity toward free and bound ABA. A possible application of these systems for the quantification of natural ABA forms without their preliminary separation and purification in a single sample of plant material was demonstrated.     

Measurement of a progestational and antiandrogenic compound by a competitive protein binding assay

Medrogestone (MDG), viz. 6,17-dimethy1-4,6-pregnadiene-3,20-dione (Colprone®) is a compound with progestational and antiandrogenic properties. A simple method based on competitive protein binding is described for the determination of piasma MDG concentration. The method involves extraction with petroleum ether of a small volume of human plasma (50–200 μ1) containing an internal standard of 3_H-progesterone for the correction of procedural losses. Sample MDG is used to displace ³H-progesterone bound to pregnant quinea pig serum. Free and bound ³H-progesterone are separated by the use of Florisil. The precision, accuracy, reproducibility and specificity of the assay were evaluated and shown to be satisfactory for the measurement of MDG in human male plasma. The concentration of MDG in blood after oral administration was determined in male dogs and humans. The compound was rapidly absorbed; partially micronized MDG produced higher blood levels than the non-micronized compound.     

Fluorescence investigation of the sex steroid binding protein of rabbit serum: steroid binding and subunit dissociation

The relationship between steroid binding and protein subunit interactions of rabbit sex steroid binding protein (rSBP) has been studied by steady-state and time-resolved fluorescence spectroscopy. The high-affinity (Ka approximately 10(8) M-1 at 4 degrees C), fluorescent estrogen d-1,3,5(10),6,8-estrapentaene-3,17 beta-diol [dihydroequilenin (DHE)] was used as a fluorescent probe of the steroid-binding site. Perturbation of the binding site with guanidinium chloride (Gdm.Cl) was monitored by changes in the steady-state fluorescence anisotropy of DHE as well as by changes in fluorescence quenching of DHE with acrylamide. The results of acrylamide quenching at 11 degrees C show that, while between 0 and 1 M Gdm.Cl the steroid-binding site is completely shielded from bulk solvent, there is decreased DHE binding. To study the subunit-subunit interactions, rSBP was covalently labeled with dansyl chloride in the presence of saturating 5 alpha-dihydrotestosterone (DHT), which yielded a dansyl-conjugated protein that retained full steroid-binding activity. The protein subunit perturbation was monitored by changes in the steady-state fluorescence anisotropy of the dansyl group. At 11 degrees C, the dansyl anisotropy perturbation, reflecting changes in global and segmental motions of the dimer protein, occurs at concentrations of Gdm.Cl above 1 M. The Gdm.Cl titration in the presence of steroids with equilibrium association constants less than 10(8) M-1 shows a plateau near 3 M Gdm.Cl at 11 degrees C; at this Gdm.Cl concentration, no DHE is bound. No plateau is observed at 21 degrees C. At higher Gdm.Cl concentrations, the dansyl fluorescence anisotropy decreases further and shows no steroid dependence. Recovery of steroid-binding activity (assayed by saturation binding with [3H]DHT), under renaturation conditions, is dependent on both steroid concentration and affinity. Both unlabeled and dansyl-labeled protein recovery the same amount of activity, and according to fluorescence anisotropy, dansyl-labeled rSBP re-forms a dimer upon dilution below 1 M or removal of Gdm.Cl. From the steroid requirement for recovery of steroid-binding activity, it appears that a conformational template is required for the dimeric protein to re-form a steroid-binding site with native-like properties.     

Sex steroid hormone binding globulin levels and free 17 beta-estradiol and testosterone in cynomolgus monkeys during different reproductive states

The effect of differences in sex hormone binding globulin (SHBG) levels associated with reproductive status on free 17 beta-estradiol (E2) and testosterone (T) was characterized in cynomolgus monkeys. Cycling female cynomolgus monkeys had higher SHBG levels than males, pregnant and lactating females (P less than 0.05). Ovariectomized females were not different than control females, suggesting no hypoestrogenic effect. The percentage of free T was elevated in pregnant animals (P less than 0.05) compared to normal males and females, but the percentage of free E2 was similar between these groups. Although a gender difference in the percentages of free E2 and T was not detected, there was a gender difference in the free T and E2 concentrations due to endogenous secretion. Increased free E2 concentrations during pregnancy were the result of endogenous secretion rather than the decreased binding capacity of SHBG; the increased percentage of free T during pregnancy significantly increased free T concentrations. These data suggest that the gender difference in SHBG levels in cynomolgus monkeys is due to androgenic influences and that estrogens have minimal influence. Furthermore, the decrease in SHBG levels during pregnancy and lactation may not be entirely dependent on these androgenic influences.