Competitive Protein-binding Technique Research Articles

Routine clinical determination of testosterone in plasma by competitive-protein-binding

A method is described for the determination of testosterone in peripheral female plasma, based on a competitive protein-binding technique. The procedure includes extraction, precipitation of fatty material in cold methanol, and chromatography on partially deactivated neutral alumina. Sex steroid-binding plasma protein (SBP) is derived from late twin pregnancy plasma. The precision and practicability of the method make it suitable for clinical use. The sensitivity is 11 ng/100ml of plasma in a duplicate determination. The method eliminates most of the steroids which interfere with the measurement of testosterone in plasma, and the results are comparable to those obtained with highly specific procedures. The mean plasma testosterone found in normal females is: 48 ng/100 ml (range 24–73) and in normal males: 740 ng/100 ml (range 430–960). Values are reported for a number of subjects with various diseases.

PLASMA LEVELS OF 17α-HYDROXYPROGESTERONE RELATED TO PROGESTERONE AND 17β-OESTRADIOL LEVELS DURING NORMAL MENSTRUAL CYCLES IN WOMEN

A sensitive and simple method for assay of 17α-hydroxyprogesterone based on competitive protein binding technique has been developed. The steroid was isolated by liquid-gel chromatography on a hydrophobic Sephadex derivative. A mixture of n-hexane/2-methylpropan-2-oI 9:1 was used as eluent in 300 × 5 sized columns. The minimum detection limit was 0.1 ng per ml plasma. Only 0.5 ml of plasma was required for the analysis. Progesterone was assayed by competitive protein binding subsequent to purification in the same system as used for 17α-hydroxyprogesterone. 17β-Oestradiol was measured using a radioimmunoassay (Johansson, this congress).

PLASMA LEVELS OF PROGESTERONE IN EARLY PREGNANCY AFTER REMOVAL OF THE FOETOPLACENTAL UNIT, AND FOLLOWING REMOVAL OF CORPUS LUTEUM

Plasma levels of progesterone were measured by a competitive protein binding technique during the first trimester of human pregnancy.

DIURNAL SERUM THYROXINE LEVELS

A competitive protein-binding technique was used to monitor the levels of thyroxine in the serum of males during the course of a 24-hour period at the end of a normal working week. A modification of the method by Murphy (1965) was used and the accuracy was enhanced by including a recovery determination for each serum specimen.

Rat serum corticosterone assay: Use of a radiosteroid assay method

Corticosteroid binding globulin (CBG) of human plasma saturated with tritiated corticosterone was used to measure the concentration of rat serum corticosterone using the competitive proteinbinding technique described by Murphy (1967). Linear standard curves were obtained and a series of corticosterone determinations performed on normal rats. The recovery rate was greater than 90% when known amounts of corticosterone were added to rat serum during the deproteinization step of the procedure.

Effect of phenobarbital, hypophysectomy and x-irradiation on preovulatory progesterone levels in the cyclic hamster.

Progesterone levels were measured at proestrus in intact, phenobarbital (sodium phenobarbital)-treated, hypophysectomized (ℏ) and irradiated hamsters by the competitive protein binding technique. Progesterone content and concentration in the ovary increased dramatically between 2:30 and 3:00 PM and the highest levels were reached at 8 PM. The concentration of follicular progesterone paralleled but was only one half of the total ovarian value. Plasma progestei-one was highest at 6 PM and did not decrease significantly until 12 midnight. Phenobarbital (phen) or ℏ at 1 PM completely blocked ovarian progesterone synthesis at 4 PM. LH but not FSH restored progesterone secretion in ℏ or phen-treated animals. LH significantly increased ovarian progesterone within 15 min in ℏ animals. Hamsters ℏ at 4 PM (after the initial rise in progesterone) continued to show progesterone at control levels for at least 4 hr. Therefore, the continued presence of LH does not appear to be necessary for sustained progesterone secre...

Determination of total and free serum thyroxine in thyroid diseases.

ABSTRACT The free thyroxine fraction in serum was determined by means of a rapid method based on the uptake of radioactive thyroxine by Sephadex (T4U). The corresponding values for percentage free thyroxine (PFT4) were obtained by simultaneous determination of the dialysable fraction. A good linear correlation was established between the two methods. Serum total thyroxine (T4) was determined by the competitive protein-binding technique. The concentration of the absolute free thyroxine (AFT4) was calculated. Of the three laboratory parameters tested, AFT4 showed the highest discrimination in both hyper- and hypothyroidism. The »free thyroxine index«, FT4I, which is a product of T4 and T4U, proved equally useful. Good results were also obtained with T4, whereas PFT4 showed the largest overlap with the normal values. PFT4 is important, however, for the calculation of AFT4. The rapid method for determination of the free thyroxine fraction described in this paper is suitable for routine use in the clinical laboratory.

Relationship between blood levels of luteinizing hormone and testosterone in bulls, and the effects of sexual stimulation.

SUMMARY Luteinizing hormone (LH) and testosterone were measured in the peripheral plasma of two bulls by radioimmunoassay and competitive protein binding techniques. Samples were collected from an indwelling jugular catheter once an hour for 24 h, and then at more frequent intervals after a number of experimental procedures. Each bull showed its own characteristic pattern of cyclic LH changes, with 5–10 peaks during 24 h that were apparently unrelated to daylight, feeding or sleep. Each LH peak was associated with a testosterone peak; the LH concentrations ranged from 5 to 50 ng/ml, and those of testosterone from 2 to 20 ng/ml. Sexual stimulation, such as the sight of a cow, or 'teasing', or on one occasion the act of ejaculation itself, caused an immediate release of a large amount of LH. If the testosterone levels were low at the time, the LH peak was followed by a testosterone peak. But when the testosterone levels were high at the time of LH discharge, the testis seemed to be unable to respond any further. An intravenous injection of 500 i.u. human chorionic gonadotrophin was associated with LH release and caused the testosterone levels to rise to maximal values of 22 ng/ml within 1½ h. It is concluded that the cyclical pattern of LH release is due to some inherent central rhythm, and that each transient LH peak results in transient maximal stimulation of testicular testosterone secretion.

The site of progesterone production in early pregnancy.

ABSTRACT The disappearance of progesterone* and HCG from the plasma was measured following therapeutic abortions in 17 patients from the seventh to the sixteenth week of pregnancy. Between the seventh and the twelfth week of gestation the evacuation of the uterine contents was performed by vacuum aspiration, and from the thirteenth week onwards abdominal hysterotomy was performed. Plasma progesterone was assayed using a competitive protein binding technique, while plasma HCG was determined by a radioimmunosorbent assay. After the evacuation of the uterine contents in the early pregnancy cases (weeks 7–8) the plasma levels of progesterone remained elevated much longer than in the later stages of pregnancy (weeks 12–16), thus suggesting that the corpus luteum graviditatis was the main source of progesterone in the first group, while in the last group the placenta was the main source of progesterone production. When comparing the earlier and later cases of pregnancy, the disappearance rate of plasma HCG remained essentially unaltered. From the results obtained, it seems likely that the transition from the ovarian to the placental production of progesterone occurs during the ninth to eleventh weeks of pregnancy.

Plasma levels of progesterone after vaginal, rectal, or intramuscular administration of progesterone

Plasma levels of progesterone were determined after vaginal, rectal, or intramuscular administration of 10 to 100 mg. of progesterone to 35 female volunteers. Progesterone was assayed by a competitive protein-binding technique. The absorption was very rapid by all three routes of administration, usually resulting in peak plasma levels of progesterone within the first 8 hr. after administration. The plasma levels remained elevated for longer periods of time than would be expected from the rapid rate of disappearance described for progesterone. Plasma levels corresponding to those encountered during the luteal phase of the menstrual cycle were attained with an intramuscular injection of 25 mg. of progesterone in oil or four times this dose by vaginal or rectal administration, while an intramuscular dose of 100 mg. resulted in a mean peak level corresponding to mid-pregnancy plasma levels of progesterone.

Progesterone levels in plasma during the oestrous cycle of the sow measured by a rapid competitive protein binding technique.

An adaptation of the technique described by Johansson (1969), whereby progesterone was measured by a rapid competitive protein binding technique, has been used to determine the progesterone levels in the peripheral plasma of the cow (Edqvist, Ekman, Gustafsson & Åström, 1970). The purpose of this paper is to report the usefulness of the same technique for the measurement of progesterone levels in the peripheral blood plasma of the sow during the oestrus cycle. Two sows of the Swedish Landrace Breed, fed on a commercial pig diet with unlimited water supply, were used. Sexual receptivity was checked twice a day with a boar. The first day of receptivity was considered to be the first day of the oestrous cycle. About 1 ml of blood was collected daily from an ear vein into

Progesterone levels in the bovine peripheral plasma measured by the competitive protein binding technique.

SummaryThe competitive protein binding technique for measurement of progesterone has been applied for progesterone in venous blood plasma of the cow. Chicken plasma was used as binding protein. Data are presented on the progesterone level in blood plasma of the cow during the oestrus cycle, after enucleation of the corpus luteum, after ovariectomy and during pregnancy.ZusammenfassungBestimmung des Progesterongehaltes im Venenblut des Rindes mit der kompetitiven Protein‐BindungsmethodeFür die quantitative Progesteronbestimmung im venösen Plasma von Kühen wurde die Methode der kompetitiven Proteinbindung angewandt.Als bindendes Protein wurde Hühnerplasma verwendet. Die vorgelegten Daten betreffen den Progesterongehalt während des Brunstzyklus', nach Enukleation des Gelbkörpers, nach Ovarektomie und während der Trächtigkeit.RésuméDétermination de la teneur en progestérone du sang veineux bovin à l'aide de la méthode de fixation compétitive des protéinesOn emploie la méthode de fixation compétitive des protéines pour une détermination quantitative de la progestérone dans le plasma veineux de vaches.Comme protéine de fixation, on emploie du plasma aviaire. Les résultats cités donnent le taux de progestérone pendant le cycle oestral, après énucléation du corps jaune, après ovariectomie et pendant la gestation.ResumenDeterminación del contenido de progesterona en la sangre venosa de vacas con la técnica de fijación competitiva de proteínasPara la valoración cuantitativa de la progesterona en plasma venoso de vacas se utilizó la técnica de la fijación proteica competitiva.Como proteína fijadora se empleó plasma de gallinas. Se presentan datos sobre el contenido de progesterona durante el ciclo estral, tras enucleación del cuerpo amarillo, tras ovariectomía y durante la gestación.

Assay of aldosterone by competitive protein binding.

ABSTRACT We have applied a competitive protein binding technique to the assay of aldosterone, utilizing the aldosterone binding protein (ABP) in kidney as the specific receptor molecule. Intracellular ABP was obtained from the homogenization of adrenalectomized rat kidneys in 0.25 M sucrose. The 10 000 × g supernatant contained the greatest binding affinity for aldosterone and was used without further modification. 1,2-3H-aldosterone was used as the tracer. After maximum binding, which occurred after 5 hours of incubation at 0–4°C, the aldosterone-ABP complex was stable for several hours and could be separated from unbound aldosterone by gel filtration using a 1 × 15 cm Biogel P-10® column. However, since the aldosterone-ABP complex was relatively easily dissociated by warm temperatures, all procedures had to be conducted at 4°C. Competitive inhibition of aldosterone binding to ABP occurred with 9-alpha fluorocortisol, cortisol, and progesterone. No inhibition of binding was found with oestradiol. Using this in vitro binding system, 1.05 × 10−12 and 2.10 × 10–12 mol aldosterone were assayed with coefficients of variation of 13.4% and 13.3%, respectively. Because cortisol effectively competes with aldosterone for ABP, application of this technique to biological fluids must be preceded by separation of aldosterone from cortisol. However, when this separation was achieved with one-step descending paper chromatography, our blank value was high and variable. This is apparently because some material which eluted from the paper interfered with the association of aldosterone and ABP. Therefore, this sensitive assay for aldosterone cannot be applied to biological specimens until the problem of the non-specific blank is circumvented.

The Prepubertal Androgenic Response to ACTH

The following studies were undertaken to explore in detail the androgenic secretory capabilities of the prepubertal adrenal. Five 6–11-year-old children with active rhcumatic carditis, otherwise non-toxic, were given ACTH gel 2.2–2.9 μ/kg i.m. daily for one week as initial therapy. Plasma C 19 steroids and indices of testosterone binding protein concentration and unbound androgens were determined by competitive protein binding techniques developed in this laboratory. Mean urinary corcoids rose from a mean (±SD) of 1.1±1.2 to 15.7±4.3 mg/day and 17-KS from 0.84±0.69 to 3.4±0.98 mg/day. Bascline mean plasma concentrations (±SEM) were: testosterone (T) 6.7±2.4 (range 1.6±15.0), androstenedione (Δ) 15.2±4.4 (range 2.7–29.6), and dehydroepiandrosterone (D) 71.6±6.2 (range 39.8–1124) ng%. Following ACTH, these were increased in each: T–22.3±3.9, Δ–149±22, and D–347±95 ng%. D-sulfate rose in each from 13.5±3.2 (range 2.3–19.8) to 62.8±24 μg%. These changes were accompanied by a fall in testosterone binding protein concentration in four. There was a concurrent rise in unbound androgen levels in all subjects as a consequence of ACTH. This steroidogenic pattern is characterized by a disproportionate increase in Δ concentration, the values sometimes exceeding those of the normal adult. Evidence, thus, has been gained that the adrenal cortex of the prepubertal to chronic ACTH administration.

Quantitation of Plasma Testosterone by Improved Competitive Protein-Binding Technique

Abstract A method is described for quantitation of testosterone in plasma from females and males. The principal operations of extraction, chromatographic fractionation, and competitive protein-binding assay can be completed for eight duplicate samples in a single day. Experimental data used in the development of the test and the rationale for the specific operations and conditions are presented. The specificity of the method was established by comparing plasma testosterone concentrations determined by the double isotope derivative technique. Concentrations are reported for normal female and male subjects, and for patients with hirsutism, adrenal hyperplasia, Klinefelter’s syndrome, and other endocrine conditions.

The simultaneous estimation of plasma cortisol and transcortin binding characteristics by a competitive protein binding technique

A slight modification of the competitive protein binding assay of Murphy (13) allows the simultaneous determination of plasma cortisol, transcortin binding capacity (P t) and apparent transcortin-cortisol association constant (K t). P t and K t may he derived from the standard curve data, usino the plasma under investiqation as the binding agent. The method is simple, reproducible and relatively rapid. The values obtained for P t and K t agree well with those found using other techniques. Many of the difficulties inherent in other methods used to study transcortin hindino characteristics are obviated. The authors stress the critical temperature dependence of values derived for K t.

Progesterone levels in peripheral plasma during the luteal phase of the normal human menstrual cycle measured by a rapid competitive protein binding technique.

ABSTRACT The competitive protein binding technique for the measurement of progesterone has been further simplified for clinical use. Only 0.25 ml of plasma is needed for the determination during the luteal phase. One technician can assay 20 samples in one day with good precision and accuracy. The progesterone concentration in 20 normal human menstrual cycles was studied. During the follicular phase a mean concentration of 0.32 ± 0.25 (s) ng/ml plasma was found. At the mid-cycle peak of the total oestrogens the progesterone concentration started to rise and reached a plateau between 10–20 ng/ml 4 to 6 days later. From the plateau level, the concentration fell rapidly to less than 1 ng/ml at the onset of menstrual bleeding. The mean luteal phase lasted for 14.15 ± 1.05 (s) days.

Plasma levels of progesterone in pregnancy measured by a rapid competitive protein binding technique.

ABSTRACT Plasma levels of progesterone* were measured during normal human pregnancy by a sensitive and rapid competitive protein binding technique. During the first half of gestation 440 determinations in 321 women and in the second half of gestation, 209 determinations in 160 women were performed. The average plasma level of progesterone in the 5th week of gestation was 24.8 ± 7.3 (s) ng/ml which is above the normal range of the luteal plateau of the menstrual cycle (10–20 ng/ml). Between the 5th and the 9th week of gestation the plasma concentration decreased significantly. From the 9th to the 32nd week of gestation the plasma level of progesterone increased from 16.7 ± 7.4 (s) to 125.2 ± 37.9 (s) ng/ml. During the last 8 weeks of gestation the plasma levels of progesterone did not show any significant rise. One woman was followed up from the day of ovulation to the 8th week of gestation. An increase in progesterone levels was observed about 10 days after ovulation. Morning samples of progesterone, which were taken before the 20th week of gestation, when the woman was still in bed, were found to be somewhat higher than levels found during the remainder of the day. Progesterone concentrations in a few abnormal pregnancies are also reported.

Measurement of estradiol-17β in plasma by competitive protein-binding radioassay

A competitive protein-binding technique has been developed for the measurement of free estradiol-17β in the peripheral plasma of women or ovarian vein plasma of sheep. The method utilizes a soluble macromolecular fraction from the uteri of ovariectomized ewes which specifically binds estradiol-17β. The mean peripheral plasma level of estradiol-17β in normal women was found to be 18 ng/100 ml in the follicular phase and 47 ng/100 ml in the luteal phase. In four estrous ewes the level of estradiol-17β in the ovarian venous plasma was 39–90 ng/100 ml, and the secretion rate into the ovarian vein was 0.9–2.7 μg/24 hr. The results are in agreement with those obtained by more elaborate methods involving fluorimetry, gas chromatography, or double isotope derivatives.

A clinically useful method for plasma testosterone determination

A simple method has been developed for clinically measuring plasma testosterone. Its sensitivity allows one to measure normal female levels of plasma, and the results are comparable to those obtained by double isotope derivative methods. This procedure requires paper chromatography of a methylene chloride extract of plasma and final determination by the competitive protein-binding technique, using salt precipitation of the bound fraction. By this method, the normal range for plasma testosterone was 26 to 108 mμg/100 ml in females and 273 to 1211 mμg/100 ml in males.