Competitive Protein-binding Technique Research Articles

Changing Trends in Thyroid Practice: Understanding Nodular Thyroid Disease

To describe changes that have occurred between 1952 and 2002 in the evaluation and management of nodular thyroid disease. A 30-year personal experience, institutional contributions, and the related published literature on evaluation of thyroid function and evolving strategies for management of thyroid nodules are reviewed. Triiodothyronine (T(3)) was discovered in 1952, and measurement of plasma thyroxine by a competitive protein-binding technique became available in the 1960s. Late during that decade, the first radioimmunoassay for thyroid-stimulating hormone (TSH) was described, modified, and then used in clinical practice until the mid-1980s, when the more sensitive TSH assays became widely available. T(3) determination by radioimmunoassay was introduced early in the 1970s. Currently, sensitive thyroid function tests can detect early disease. In the general population, thyroid nodules have a prevalence higher than 50% after age 65 years, affecting more than 100 million people in the United States. Two important developments influenced thyroid nodule evaluation and management-- fine-needle aspiration (FNA) biopsy and ultrasonography. Because FNA biopsy has emerged as the most accurate test for nodule diagnosis, it has decreased the need for scanning and for thyroidectomy and thereby is likely to reduce health-care costs by more than $500 million annually in the United States. Thyroid ultrasonography is the imaging method of choice for evaluation of thyroid gland structure. Management of cytologically benign thyroid nodules remains controversial. TSH seems to be only one of many factors in pathologic thyroid growth. FNA, because of its diagnostic accuracy, should be the initial procedure used in nodule evaluation.

Single luteal phase serum progesterone assay as an indicator of ovulation

The availability of a rapid, accurate serum progesterone assay would alleviate many of the problems of patient inconvenience and discomfort encountered with the classical methods of ovulation detection. With the use of a competitive protein binding technique, by which a single technician can assay 30 or more samples for serum progesterone in one day, a range of daily follicular and luteal phase values was ascertained in a group of normally menstruating women. After a normal luteal phase range was established, single luteal phase serum progesterone sampling was performed in 51 infertile women with regular menses and 35 oligomenorrheic women undergoing clomiphene therapy. In the follicular phase of the cycle, progesterone levels were consistently less than 2 ng. per milliliter. Between 11 and 4 days prior to the onset of menses, in presumptively ovulatory cycles, serum progesterone levels were always 3 ng. per milliliter or greater. Progesterone values in this range were always accompanied by a secretory endometrium and, thus, can be considered presumptive evidence of ovulation.

Production of 1,25 Dihydroxyvitamin D3 by Perfused Pig Skin

Keratinocytes produce large amounts of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) in vitro. 1,25(OH)2D3 is detectable in anephric humans and pigs and can be increased to near-normal levels by vitamin D or 250HD, indicating an extrarenal source. To determine whether the skin is one of these extrarenal sources, we perfused isolated flaps of porcine skin for 8 h with 250HD3 in serum-free medium at 1 ml/min, collecting the venous effluent as 15-min samples. The samples were extracted and the vitamin D metabolites purified by high-performance liquid chromatography and assayed by competitive protein-binding techniques. Production of 1,25(OH)2D3 continued for the duration of the perfusion, tending to increase in the last 2 hours. The amount of 1,25(OH)2D3 produced varied both with time in the same pig skin and between pig skins; maximum production of 1,25(OH)2D3 in these experiments was 8 pg/min. 24,25(OH)2D3 production was higher than 1,25(OH)2D3 production, reaching a maximum rate of 180 pg/min. Considering that the production rate of 1,25(OH)2D3 in humans is 1.25 ng/min and that a 48-cm2 skin flap represents 1/350 the surface area of a human, and assuming that human and pig skin make 1,25(OH)2D3 at comparable rates, one can determine that the skin has the potential to maintain near-normal levels of 1,25(OH)2D3 in the absence of kidneys when provided with adequate substrate.

Sequential changes in plasma progesterone, total oestrogens and corticosteroids in the cow throughout pregnancy and around parturition

Blood samples were taken thrice weekly for 2 weeks from 33 pregnant cows in nine groups, 1 month apart, and twice daily for 8 days commencing 1 week before parturition from eight cows. Plasma progesterone and total oestrogens were quantified by validated radioimmunoassays and total corticosteroids by a competitive protein binding technique. During the first 3 months of pregnancy, no remarkable changes were observed for the three hormones studied. From the fourth month on, concentrations of progesterone and total corticosteroids decreased significantly ( P<0·05) and remained fairly constant until the ninth month. Values of total oestrogens increased significantly ( P<0·05) from the fourth to the sixth month and thereafter remained unchanged. In the periparturient cows, progesterone levels decreased linearly ( P<0·01) during the 3 days preceding delivery. Concentrations of total oestrogens rose sharply from day −5 and peaked at calving. Values of total corticosteroids increased dramatically on the sixth day and also at 24 hours before parturition. These results suggest that, contrary to earlier studies, plasma corticosteroids could have a primary key role for triggering parturition in cows.

Stimulation of cyclic adenosine 3′,5′-monophosphate formation in rabbit choroid plexus by β-receptor agonists and vasoactive intestinal polypeptide

The formation of cyclic adenosine 3',5'-monophosphate (cAMP) was determined in blood-free choroid plexus homogenates from all ventricles of rabbit using a competitive protein binding technique. Previous stimulation of the intact plexus tissue in vitro with sympathomimetic drugs or vasoactive intestinal polypeptide (VIP) leads to increased local synthesis of cAMP. Tests with selective beta-receptor agonists and antagonists suggested that beta 1-receptors predominate, which is consistent with studies of sympathomimetic effects on cerebrospinal fluid production in vivo.

Endocrine Effects of Heat Stress in Boars

The purpose of this study was to describe the temporal changes in peripheral plasma levels of testosterone and Cortisol in boars during and after heat stress. A total of 8 boars were utilized, 4 of them were exposed to 35°C, for 100 h in a climatic room, and 4 served as controls and were kept at 20 °C for 100 h in the climatic room. Blood samples were obtained via permanent vein catheters 3 times daily from 5 days before heat stress until 20 days after termination of heat stress. Testestorone levels were determined by radioimmunoassay and Cortisol by a competitive protein binding technique. For both hormones the pre-exposure levels were similar in both groups of boars. The control boars had significantly higher testosterone levels, while being in the climatic room, than during any other period. The experimental boars had slightly increased testosterone levels during the first day of heat stress and thereafter continuously decreased levels. In the control boars the testosterone levels returned to pre-exposure levels immediately after removal from the climatic room, whereas in the experimental boars the testosterone levels were dramatically increased during the first 5 days after exposure. The differences in Cortisol levels, between the 2 groups of boars were restricted to the period spent in the climatic room. During this period the experimental boars had significantly higher Cortisol levels.

Relationships between circulating and intracellular thyroid hormones: physiological and clinical implications.

THYROXINE (T4), measured as protein-bound iodine, was the first hormone to be quantitated in human serum. As a consequence, it was possible to demonstrate clear relationships between circulating T4 concentrations and many of the physiological manifestations of thyroid hormone excess or deficiency. In 1952, 3,5,3′-triiodothyronine (T3) was identified in human plasma and metabolic studies in man soon led to the realization that this hormone was 3–4 times as potent metabolically as T4 (1–4). At that time, the question was first raised as to whether T4 or T3 was the “active” thyroid hormone (3). This question became even more pertinent when it was suggested first in 1955 (5) and shown conclusively in 1970 that T4 could be converted to T3 by peripheral human tissues (6, 7). These studies were made possible by the development of methods for quantitating T3 in human serum, initially by competitive protein binding techniques, followed soon thereafter by specific and sensitive RIAs.

Trimethoprim interferes with serum methotrexate assay by the competitive protein binding technique.

Administration of Bactrim (a combination of trimethoprim and sulfamethoxazole) to a patient who also was receiving methotrexate caused a significant increase in apparent plasma methotrexate concentrations as determined by competitive protein binding assay with use of dihydrofolate reductase (EC 1.5.1.3) from Lactobacillus casei as the binding protein. This spurious increase was caused by trimethoprim in the patient's plasma. A plasma trimethoprim concentration of 0.1 mg/L inhibited binding of radiolabeled methotrexate to dihydrofolate reductase by 50%. In contrast, radioimmunoassay for methotrexate was not affected by concomitant administration of trimethoprim. The competitive protein binding assay for methotrexate should not be used in patients being treated with Bactrim or Septra (a similar combination). However, the L. casei competitive protein binding assay technique can be used to assay plasma trimethoprim concentrations with sensitivity to 0.02 mg of trimethoprim per liter.

Glucocorticoid concentrations, corticosteroid binding protein characteristics and reproduction performance of sows and gilts subjected to applied stress during mid-gestation.

Two experiments were conducted to evaluate the effects of applied heat (greater than or equal to 28 C) and crowding (less than or equal to .9 m2 floor space/animal) stress for 50 days during mid-gestation on glucocorticoid concentrations, corticosteroid binding globulin (CBG) characteristics and reproductive performance of 20 sows (Exp. 1) and 23 gilts (Exp. 2). Blood as collected via puncture of the anterior vena cava on days -1 and 0 before initiation of treatment and on days 1, 2, 12, 13, 48, 29 and 1, 5, 9 +/- l, 19 +/- 1, 50, 63 +/- 3, 90 +/- 1 after initiation of treatment for sows and gilts. respectively. Plasma glucocorticoids and CBG characteristics were determined by competitive protein binding techniques. Overall mean glucocorticoid concentrations for eight sows and 12 gilts subjected to stress were lower (P less than .05) than those of control animals, CBG binding capacities (CBC) were lower (P less than .05) in treated sows and gilts bled on days 12 and 9 +/- 1 after initiation of stress, respectively. Overall mean CBC and affinity (Ka) of cortisol to bind to the CBG molecule were lowered (P less than .05) in stressed gilts. Other than a reduction (P less than .05) in gestation length observed in stressed crossbred sows, reproductive performance was not affected by stress. When infrequent blood samplings are employed, measurement of CBC may serve as a more precise indicator of a physiological stress response than glucocorticoid concentration.

Steroid immunoassay in clinical chemistry

Abstract Immunochemical methods for the assay of steroid hormones first commenced in the form of radioimmunoassay in 1969. This assay has been extensively used during the last ten years, transforming the field of clinical chemistry. Steroid radioimmunoassay can no longer be regarded today as a pure research tool, it is emerging as an indispensible practical aid to clinical chemistry, the assay of steroid hormones being of great value in both routine clinical diagnosis and treatment. The present communication describes research recently pursued in our laboratory, made in order to obtain a clearer understanding of the fundamental principles involved in steroid immunoassay, and to cut down the cost of the assays. A new approach for the correct assessment of specificity is given in detail, and the question of homology and heterology in these types of assay discussed. Our latest research in the development of non-isotope immunoassays is presented with details of conjugation, purification of enzyme labels for enzyme-immunoassay. Immuno-enzymatic assay, fluoroimmunoassay and immunofluorimetric assays developed in London are described, together with a new competitive protein-binding technique for the measurement of femtogram [10-15g] amounts of steroids.

Effect of altered thyroid states on liver and kidney uptake of 109Cd in rats

Hypothyroid and hyperthyroid states were induced in rats by drug treatment with propylthiouracil and thyroxine, respectively. The thyroid states were verified by measuring serum thyroxine using a commercial competitive protein-binding technique. A single 109Cd ip injection was then given to three groups: hypo-, eu-, and hyperthyroid. The uptake of 109Cd in liver and kidneys was measured at times ranging from 1 to 120 hr. On a per gram basis, 109Cd uptake in livers and kidneys of hypothyroid rats was significantly greater than that of eu- and hyperthyroid rats, even though there was a dwarfing effect in the hypothyroid group. The transfer of 109Cd from the liver to the kidneys was not affected by the altered thyroid states.

CIRCADIAN RHYTHMS OF PLASMA CORTICOSTERONE BINDING ACTIVITY IN THE RAT AND THE MOUSE

Corticosterone binding activity (CBA) and corticosteroids were measured by competitive protein binding techniques in plasma samples drawn from rats and mice at different times of day. Circacian rhythms of plasma CBA were found in both rats (onset of 14-h daily photoperiod: 0600) and mice (onset of 14-h daily photoperiod: 0700). The plasma CBA rhythm was bimodal in rats with peaks at 1000 and 1800 and unimodal in mice with peak level from 0100 to 0500. Circadian rhythms of plasma corticosteroid concentration with peaks during the late afternoon were confirmed in both rats and mice. The plasma CBA rhythms appear to be related to the circadian rhythms of both locomotor activity and plasma corticosteroid concentration.

The measurement of thyroxine in urine using a competitive protein binding technique

The competitive protein binding assay used for the measurement of thyroxine in blood serum was modified for the measurement of thyroxine in urine. Samples of urine of 1 to 5 ml volume containing 0 to 5 ng/ml could be assayed with 100% recovery, and above this range, up to 10 ng, recoveries were higher due to non parallelism with the standard curves. Tests carried out using porcine serum albumin indicate that results obtained by the method are not likely to be affected by proteinuria. The cross reaction with triiodothyronine was 25%. Analysis of urine samples stored at 25°C gave higher values than those stored at +4°C or — 20°C over similar periods. These increases at 25°C were of the same magnitude as those obtained by acid hydrolysis of urinary thyroxine conjugates.

Radioassay of serum vitamin B12 using a competitive protein binding technique

A method has been developed for the radiassay of serum vitamin B12 using a competitive binding technique. By this method, vitamin B12 levels as low as 50 pg per ml of serum can be estimated in test samples. Egg yolk has been used as a test binding protein, which has several advantages over serum or plasma protein binding agents, as described in this paper. This binder is quite suitable and reliable for the assay of vitamin B12.

Effect of stress on the profile of plasma steroids in baboons (Papio hamadryas).

Adult baboons (5 males and 5 females) were exposed to immobilization stress by being strapped to a table in a horizontal position for 2 h. In females the experiment was performed during both the follicular and luteal phase. Peripheral blood was withdrawn at frequent intervals, the first sample just before immobilization, and the last one 3 days later. A number of steroids were measured in blood plasma samples by radioimmunoassay (17-hydroxypregnenolone, 17-hydroxyprogesterone, pregnenolone, testosterone, dihydrotestosterone, progesterone, 20alpha-dihydroprogesterone, oestone, oestradiol) or competitive protein binding (cortisol) techniques. The cortisol levels exhibited a marked increase in both sexes. This increase was observed already during the immobilization and lasted for approximately 24 h. A similar, even more pronounced increase was seen in 17-hydroxypregnenolone, 17-hydroxyprogesterone and pregnenolone levels. A marked, long-lasting (72 h) decrease of testosterone and dihydrotestosterone levels was a consistent finding in male baboons. This was not observed in the females which, on the other hand, exhibited a marked decrease (duration 48 h) of progesterone and 20alpha-dihydroprogesterone levels during the luteal phase, and a significant decrease (duration greater than 24 h) of oestradiol and oestrone concentrations during the follicular phase. It is concluded that stress has a marked inhibitory action on gonadal function both in male and female baboons. In females and inhibition of steroidogenetic function is exerted both on the ovarian follicles and on the corpus luteum.

Clinical Pharmacokinetics of Prednisone and Prednisolone

Prednisone and prednisolone are used in a wide variety of diseases. The two compounds are metabolically interconvertible; prednisolone is assumed to be the pharmacologically active species. Prednisone and prednisolone plasma concentrations are commonly determined by either radioimmunoassay or competitive protein binding techniques. No relationship has been demonstrated between prednisolone plasma concentration (unbound or total concentration) and clinical response; alternate day dosage regimens with fluctuating plasma concentrations being considered generally to be as effective with less side effects than a daily dosage regimen. Both drugs are rapidly absorbed after oral administration, reaching peak plasma concentrations after 1 to 3 hours. In general, plasma half-lives for prednisone are slightly longer (3.4 to 3.8h) than for prednisolone (2.1 to 3.5h). Either drug can be prescribed in most situations. On average, however, the bioavailability of prednisolone after oral prednisone is approximately 80% of that after prednisolone. A wide intersubject variation in prednisolone concentration is evident after both drugs, which may suggest impaired drug absorption in some individuals. Prednisolone shows dose dependent pharmacokinetics; an increase in dose leading to an increase in volume of distribution and plasma clearance. This can be explained in terms of the non-linear binding of the drug to plasma proteins. The degree of binding will determine the distribution and clearance of free (i.e. pharmacologically active) drug. Reduced doses are recommended in patients with hypoalbuminaemia. Prednisolone pharmacokinetics are also dependent on age; the half-life being shorter in children. Liver disease prolongs the prednisolone half-life and, due to the frequently associated hypoalbuminaemia, also increases the percentage of unbound drug. It has been recommended by some that prednisolone rather than prednisone is the drug of choice in active liver disease owing to the poor conversion of prednisone to prednisolone in some patients. However, the reduced plasma concentration of prednisolone in such patients is compensated for by delayed clearance. Thus, there is little advantage of one preparation over the other. Pharmacokinetic drug interactions have been reported, particularly accelerated clearance of prednisolone due to enzyme inducing agents such as barbiturates, phenytoin, rifampicin and other corticosteroids. On the other hand, oral contraceptives have been reported to increase the half-life and to decrease distribution volume and clearance of prednisolone, due to the increased levels of corticosteroid binding globulin (transcortin).

18-Hydroxy-11-deoxycorticosterone in Hypertensive Uremic Patients during Hemodialysis

The present study was carried out in 25 hypertensive uremic patients on regular 4 h dialysis, 3 times a week. Plasma 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), aldosterone (PA) and corticosteroids were determined by radioimmunoassay and competitive protein binding technique before and at the end of the 1st, 2nd and 3rd hour of hemodialysis. Plasma 18-HD-DOC was normal before dialysis and did not change significantly during hemodialysis, whereas body fluids and electolytes decreased progressively. No correlation was observed between blood pressure and 18-OH-DOC during dialysis. 18-OH-DOC did not correlate with PA which decreases progressively during hemodialysis and was correlated to plasma corticosteroids only at the 3rd hour of dialysis, probably on account of the enhanced influence of ACTH on the adrenal cortex.

CIRCANNUAL AND CIRCADIAN RHYTHMS IN THE CONCENTRATION OF CORTICOSTERONE IN THE PLASMA OF THE EDIBLE FROG (RANA ESCULENTA L.)

For a period of 21 months between May 1974 and September 1976, circadian variations in the plasma concentration of corticosterone were studied by competitive protein-binding techniques in mature male and female edible frogs living in their natural environment. Blood samples were taken from 8 to 12 frogs six times daily and conventional and cosinor methods were used for statistical analysis. Circadian rhythms were not detected during February and March (time of hibernation). Circannual rhythms were detected in three parameters of the circadian rhythm. The mean concentration of corticosterone over a 24 h period (24 h mean) reached a peak on 1 May (between 15 April and 15 May; 95% limits of confidence); the annual mean value of the 24 h means was 1.97 +/- 0.25 (S.E.M.) microgram/100 ml, with an amplitude of 0.66 microgram/100 ml (0.53--0.79 microgram/100 ml; 95% limits of confidence). Circadian variations in the concentration of corticosterone were largest in May (peak of reproductive activity). The times at which the peak concentration of corticosterone occurred showed circannual variations: peak values were detected around 24.00 h in May, 19.00 h in July and 08.00 h in November. Both circadian and circannual variations have therefore been demonstrated in an endocrine function of an amphibian in its natural habitat.

Genetic variation in plasma thyroxine levels and minimal metabolic rates of the mouse, Mus musculus.

SUMMARYA study of inbred strains of the mouse using competitive protein-binding techniques revealed significant strain variation in the total plasma thyroxine levels and in the plasma-free thyroxine index. Measurements of minimal metabolic rates also showed strain variation, but the positive correlation which might be expected between the plasma-free thyroxine index and the minimal metabolic rate did not obtain. Possible explanations are discussed.

Ontogenic study of plasma 17alpha-hydroxyprogesterone in the human. I. Postnatal period: evidence for a transient ovarian activity in infancy.

Summary: Plasma concentrations of 17α-hydroxyprogesterone (OHP) were measured by a specific and sensitive radioimmunoassay in 348 blood specimens of male and female infants from birth to 2 years of age. Total corticoids levels were assayed by a competitive protein binding technique. OHP levels were higher in the cord than in the peripheral blood of both the mother and the newborn. At term OHP cord levels were higher in female than in male infants. Mean peripheral concentrations of OHP were 419 ± 217 in boys and 431 ± 175 ng/dl in girls in the first day of life and decreased during the first week of life to 70 ± 37.5 and 95 ± 39 ng/dl in male and female infants, respectively. Thereafter, a sex-linked difference in the pattern of OHP plasma concentrations was observed during infancy and, moreover, this dimorphism varied with age, values being higher in boys at 1–2 months but higher in girls at 6–9 months of age. Total corticoids values did not vary significantly with either sex or age. The pattern of OHP in boys closely parallels that which we described previously for testosterone and further documents the postnatal endocrine activity of the testis. The pattern of OHP in girls suggests that in human infant the ovaries exhibit a moderate and rhythmic endocrine activity which lasts at least until the end of the first year of life. Speculation: It has been shown that in human infant the testis has a marked and transient (for about 4 months) endocrine secretion (OHP and testosterone) after the postnatal activation of the hypothalamopituitary function. In contrast, in female infants, the ovaries exhibit a moderate, rhythmic, but more prolonged endocrine secretory activity (OHP and estradiol). This could result from a pulsatile secretion with great amplitudes but a low tonic gonadotrophin activity in infancy. It is speculated that in the ovary a more sustained luteinizing hormone (LH) (or LH + follicle-stimulating hormone (FSH)) stimulation could be necessary to achieve full maturation of its steroidogenic enzymes, in particular, of the aromatase.