Competitive Protein-binding Radioassay Research Articles

Development of Simple Method for the Determination of 5-Methyl Tetrahydrofolate in Dried Blood Spot by High-Performance Liquid Chromatography

Folate (Reduced) or folic acid (oxidized), is a water-soluble vitamin necessary for thymidylate synthase, methionine synthase, serine hydroxymethyltransferase, and folate deficiency associated with megaloblastic anemia, neural tube defect, and coronary heart disease. The available folate data is minimal due to a dearth of suitable, accurate, field-friendly blood collection and estimation methods. Conventional assays like microbiological and competitive protein-binding radioassay methods are used to estimate the total folate in serum and whole blood. In erythrocytes, folate exists mainly as 5-methyltetrahydrofolate polyglutamate and indicates the long-term storage of folate status. Therefore, the study aimed to develop a simple method for estimating 5-methyl tetrahydrofolate (5-MTHF) by high-performance liquid chromatography (HPLC) in dried blood spots (DBS) as an indicator of folate status. The present study results indicate standard 5-methyltetrahydrofolate with different concentrations with linear regression was showed R2 -0.98, and the minimum detection limit of a standard was 20 pg. The inter-and intra-assay variations were found to be less than 10%. A good correlation (R2 -0.964) between the DBS and blood and the recovery of added standard in the DBS sample was more than 80%. A minimum of three DBS punches equivalent to 20mL of blood was required for the analysis, and the competitive protein-binding radioassay showed slightly high folate compared to the HPLC method. The stability of 5-MTHF in DBS was also tested periodically. DBS technique and HPLC method are simple, sensitive, not expansive, and safe compared to the microbiological and competitive binding assay for assessing sub-clinical folate status in a field survey on a large population.

Competitive Protein-binding Radioassay of Thiamine in Simple Solutions and in Multivitamin Pharmaceuticals

A competitive inhibition radioassay of thiamine is described using a gel obtained by coupling a buckwheat-seed thiamine-binding protein to CNBr-activated Sepharose. The sample to be analysed is incubated with gel suspension and [14C]thiamine and after centrifugation the radioactivity of the supernatant is measured. The method is simple and specific, and applicable over a thiamine concentration range 0.5-5 microM with a coefficient of variation typically below 5%. The gel is reusable and stable for several months. Applicability of the method for direct determination of thiamine in multivitamin pharmaceuticals is demonstrated.

Localising individual myeloma cells to the myeloma ‘niche’ in bone using multiphoton microscopy

[1] Haddad JG, Chyu K. Competitive protein-binding radioassay for 25-hydroxy [2] Houghton LA, Vieth R. The case against ergocalciferol (vitamin D2) as a [3] Aramas LA, Hollis BW, Heaney RP. Vitamin D2 is much less than vitamin D3 in [4] Holick MF, Biancusso RM, Chen TC, et al. Vitamin D2 is as effective as vitamin [5] Bouillon R, Okamura WH, Norman AW. Structure–function relationships in the [6] Bouillion R, Auwerx J, Dekeyser, et al. Two direct (nonchromatographic) [7] Belsey R, DeLuca HF, Potts JT. A rapid assay 25-OH-vitamin D3 without [8] Dorantes LM, Arnaud SB, Arnaud CD. Importance of the isolation of 25[9] Leventis HC, Garrison L, Sibley M, et al. Underestimation of serum 25[10] Carter GD, Jones JC, Berry JL. The anomalous behaviour of exogenous 25-

Determination of Folate Vitamers in Human Serum by Stable-Isotope-Dilution Tandem Mass Spectrometry and Comparison with Radioassay and Microbiologic Assay

Current clinical methods for folate give different results and cannot measure the various forms of folate. We developed an isotope-dilution tandem mass spectrometric method coupled to liquid chromatography (LC/MS/MS) as a candidate reference method for 5-methyltetrahydrofolic acid (5MeTHF), 5-formyltetrahydrofolic acid (5FoTHF), and folic acid (FA) in human serum. We quantitatively isolated folates from 275 microL of serum with a phenyl solid-phase extraction cartridge, then detected and quantified them in stabilized serum extracts by positive-ion electrospray ionization LC/MS/MS. We used an isocratic mobile phase of acetic acid in organic solvent on a C(8) analytical column. (13)C-labeled folates were used as internal standards. Limits of detection in serum were 0.13 (5MeTHF), 0.05 (5FoTHF), and 0.07 (FA) nmol/L. Within- and between-run imprecision (CV) was <7% for 5MeTHF and <10% for 5FoTHF at concentrations >0.5 nmol/L, and <10% for FA at concentrations >2.0 nmol/L. Total folate (TFOL) concentrations determined by competitive protein binding radioassay were approximately 9% lower than results obtained with LC/MS/MS. The microbiologic assay gave approximately 15% higher TFOL results with FA calibrator and no difference with 5MeTHF calibrator. The mean (SD) [range] TFOL in 42 sera was 35.5 (17.8) [6.5-75.6] nmol/L. Thirty-two samples with TFOL <50 nmol/L had, on average, 93.3% 5MeTHF, 2.3% FA, and 4.4% 5FoTHF. Ten samples with TFOL >50 nmol/L had, on average, 81.7% 5MeTHF, 15.7% FA, and 2.5% 5FoTHF. This stable-isotope-dilution LC/MS/MS method can quantify 5MeTHF, 5FoTHF, and FA in serum. Currently used clinical assays agree with this candidate reference method.

Competitive protein-binding radioassay of thiamine in selected biological materials

A principle of competitive protein-binding radioassay is developed for thiamine determination in some biological samples. Thiamine in an assay sample competes with radiolabelled thiamine for Sepharose-immobilized buckwheat-seed thiamine-binding protein. A blank sample is prepared by destruction of theiamine in hot alkaline solution. Model studies show that the radioassay works in thiamine concentration range of 1–10 μM, in samples of moderate ionic strenght (up to 0.25 M NaCl) and is specific for thiamine in the presence of up tp 5-fold molar excess of thiamine phosphates. Thiamina phosphates can be determined but after hydrolysis with a suitable phosphatase enzyme (Taka-Diastase). Using this method, thiamine contents are successfully determined: (i) in spinach juice, directly, (ii) in cow's milk, after deproteinization, and (iii) in human urine, after desalting. Both the precision (C.V. less than 15%) and the recovery of thiamine supplements (82–100%, depending on thiamine pre-extraction method) are reasonable. Results of thiamine radioassay show a good correlation with control determinations by the standard thiochrome method.

Normal P300 in acute schizophrenics during a continuous performance test

micro and ultra-micro measurement of various steroids in body fluids by competitive protein-binding radioassay. J Clin Endocrinol Metab 27:973990. Myers ED (1984): Serial Dexamethasone Suppression Tests in male chronic schizophrenic patients. Am J Psychiatry 141904-905. Overall JE (1984): The Brief Psychiatric Rating Scale in psychopharmacology research. In Pichot P (ed), Pharmacopsychiatry. Basel: Karger, pp 6778.

The influence of male contact on plasma cortisol concentrations in the prepubertal gilt.

Large White X (Large White X Landrace) prepubertal gilts, 165 days of age, were fitted with indwelling venous catheters and housed in modified metabolism crates. After a period of acclimatization, frequent blood samples were taken at regular intervals before, during and after the 7 gilts were exposed to various degrees of contact with male pigs. The plasma samples were assayed for cortisol concentration using a competitive protein-binding radioassay. Significantly elevated concentrations of plasma cortisol (P less than 0.001) occurred only when full physical contact between the boar and the gilts was allowed. Boar exposure without full physical contact induced only minor changes in plasma cortisol concentrations of gilts. Plasma cortisol concentrations have been shown to constitute a reliable indicator of a stress response in pigs, and so the results of this study suggest that tactile stimulation from a male pig induces a stress response in the recipient prepubertal gilt. This stress response in the gilt may be involved in the stimulation of puberty onset by contact with a mature boar (i.e. the 'boar effect').

Urinary free cortisol secretion in habitually violent offenders.

Male violent offenders (n = 90) and residivious arsonists (n = 10) were investigated by urinary (24 h) free cortisol measurements at mental examination on a psychiatric department. The measurements were made with competitive protein-binding radioassay. Only among the habitually violent offenders with antisocial personality were the values low when compared with other violent offenders, antisocial personality without the habitually violent tendency, and male clinic personnel. Poor motivation already in school, truancy, attention deficit and undersocialized aggressive conduct disorder problems seemed to be connected with the low cortisol levels.

Are there antibodies against brain in sera from schizophrenic patients?: Review and prospectus

demiological Study of Recent Trends. Am J Psychiatry 135:754-756. Ostroff R, Giller E, Gonese K, El?ersole E, Harkness L, Mason J (1983): Neuroendocrine risk factors of suicidal behavior. Am J Psychiatry 139:13231325. Pierson-Murphy BE (1967): Some studies of protein binding steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein binding radioassay. J Clinical Endocrinol Metab 27:973-990. Pokorny AD (1983): Prediction of suicide in psychiatric patients. Arch Gen Psychiatry 40:249257. Robbins DR, Alessi NE, Yanchyshyn GW, Colfer MV (1983): The dexamethasone suppression test in psychiatrically hospitalized adolescents. J Am Acad Child Psychiatry 22 (5):467-469. Targum SD, Rosen L, Capodanno AE (1983): The dexamethasone suppression test in patients with unipolar depression. Am J Psychiatry 140:877879.

Assessment of the value of a competitive protein binding radioassay of folic acid in the detection of folic acid deficiency.

The diagnostic value of the Becton Dickinson Radioassay Kit (125I) for the the assay of red cell folate has been investigated. The assay was acceptable with regards to precision but was non-linear with changing packed cell volume. Sensitivity of the assay was satisfactory, with 24 of 25 folate deficient patients giving red cell folate values which fell below the reference range. Specificity of the assay in the detection of folate deficiency was less satisfactory. As with microbiological assays, a considerable proportion of vitamin B12 deficient patients had low red cell folate values. In addition, low concentrations were found in 12% of patients who were unlikely to be deficient in either vitamin B12 or folic acid.

The determination of maternal and foetal rat plasma corticosterone concentration in late pregnancy by competitive protein binding analysis

A procedure for the determination of corticosterone in 20 μl of rat plasma, in maternal and foetal late pregnancy with a protein binding radioassay, is described. The method is suitable in a range of 0.5–3 ng of corticosterone. An amount of 0.5 ng can be detected with a coefficient of variation for duplicate of 13%. This procedure seems to be specific for glucocorticosteroids. The comparison of the results obtained with fluorimetry and the competitive protein binding radioassay show the reliability of both methods although the protein binding radioassay is much more sensitive.

Morning Plasma Cortisol Levels in Infants Treated with Topical Fluorinated Glucocorticosteroids

In the article "Morning Plasma Cortisol Levels in Infants Treated with Topical Fluorinated Glucocorticosteroids" by Weston et al (Pediatrics 65:103, 1980) an error has been noted in the description of the method used for cortisol assay. Samples were assayed by the competitive-protein binding radioassay of Murphy, which permits measurement of cortisol in 0.1-ml aliquots of plasma (0.2 ml for a duplicate determination) with a coefficient of variation of 7%.1,2 The Spencer-Peet fluorometric method, noted incorrectly in the text, would require a much larger volume of plasma than that obtained from the infants.

Competitive protein-binding radioassay for retinoic acid

Rat testes cytosol treated by Blue Sepharose was employed in a simple and sensitive method for the determination of retinoic acid in the rat serum, liver, and intestine. The method permits the detection of as little as 3 ng of retinoic acid. The mean concentrations of retinoic acid in normal male rats were 33.5 ng/ml of serum, 624.9 ng/g wet wt of liver, and 444.3 ng/g of intestine.

Improved protein-binding radioassay for plasma cortisol.

We describe an improved competitive protein-binding radioassay for total plasma cortisol, which permits rapid analysis of many samples. Plasma, 0.1 mL, is directly applied to a Whatman 3MM filter-paper strip, which is extracted once with dichloromethane. The extract is evaporated and the residue is assayed directly by the micro-method of Murphy (J. Clin. Endocrinol. Metab. 27: 973, 1967). Comparison with the original Murphy method (ethanol extraction) yielded excellent agreement (r = 0.988). One hundred samples, in duplicate, can easily be assayed at a time.

Improved protein-binding radioassay for plasma cortisol.

Abstract We describe an improved competitive protein-binding radioassay for total plasma cortisol, which permits rapid analysis of many samples. Plasma, 0.1 mL, is directly applied to a Whatman 3MM filter-paper strip, which is extracted once with dichloromethane. The extract is evaporated and the residue is assayed directly by the micro-method of Murphy (J. Clin. Endocrinol. Metab. 27: 973, 1967). Comparison with the original Murphy method (ethanol extraction) yielded excellent agreement (r = 0.988). One hundred samples, in duplicate, can easily be assayed at a time.

Maturation of adrenal stress responsiveness in the rat.

Serum and adrenal corticosterone was measured by competitive protein-binding radioassay in rats subjected to saline injection, ACTH administration or ether fumes. Groups of rats were tested at 5, 7, 9, 11, 13, 15, 20 and 25 days of age and measurement of hormone level was made either before treatment, to obtain basal values, or 15 min after treatment. Furthermore, the time-course of corticosterone release after ether was determined in 9- and 15-day-old rats. Neonatal rats responded to ether exposure, ACTH administration or saline injection with a significant rise in serum and adrenal corticosterone concentration above basal levels as early as 5 days of age. By 9 days of age, response to stress was qualitatively the same as that of the 25-day-old rat. The time course of adrenal responsiveness to ether stress was similar in 9- and 15-day-old rats, both age groups showing significant increases in hormone concentration by 15 min. These results contradict the concept of the 'stress non-responsive period' which was promoted by previous studies based on the fluorometric analysis of corticosterone.

25-OHD 3-26,23-lactone: A metabolite of Vitamin D 3 that is 5 times more potent than 25-OHD 3 in the rat plasma competitive protein binding radioassay

A new metabolite of Vitamin D 3 (25-OHD 3-26,23-lactone) has been found in the plasma of Vitamin D 3-toxic pigs and cows. This metabolite is at least 5 times more potent than 25-OHD 3 in the displacement of [ 3H]-25-OHD 3 from rat plasma protein binding sites under short-term incubation. This metabolite co-migrates with 24,25-(OH) 2D 3 on Sephadex LH-20 columns developed in chloroform:hexane 65:35 and with 25,26-(OH) 2D 3 on Sephadex LH-20 columns developed in hexane:chloroform:methanol 9:1:1. The presence of 25-OHD 3-26,23-lactone represents a possible contaiminant in the assay of 24,25-(OH) 2D 3 or 25,26-(OH) 2D 3 if only Sephadex LH-20 is used for pre-assay purification. 25-OHD 3-26,23-lactone is, however, resolved from 24,25-(OH) 2D 3 by high pressure liquid chromatography (HPLC) using Zorbax Sil silicic acid columns developed in either isopropanol:hexane 8:92 or isopropanol:methylene chloride 2.5:96.5. We assayed for the presence of this new metabolite of Vitamin D 3 and found it to be present in normal pig plasma and undetectable in normal cow plasma. Concentrations were elevated to 10–20 ng/ml following massive injection of Vitamin D 3 to both species.

Serum concentrations of 24,25(OH) 2D in uremic children: A reflection of renal function

The mean serum concentration of 24,25(OH)2D determined by competitive protein-binding radioassay was significantly lower in ten uremic children maintained on hemodialysis (0.82 +/- 0.43[SD] ng/ml) than in ten patients with impaired renal function not requiring hemodialysis (1.30 +/- 0.54 ng/ml, P less than 0.05), or in 12 normal children (2.98 +/- 1.57 ng/ml, P less than 0.01). The serum levels of 250HD were similar in all groups. There were significant (P less than 0.01) positive correlations between the serum concentration of 24,25(OH)2D or the ratio 24,25(OH)2D/25OHD and the creatinine clearance. The serum concentration of 24,25(OH)2D was significantly decreased also in six anephric adults relative to normal adult values. The data indicate that production of 24,25(OH)2D is impaired in subjects with compromised renal function. Inasmuch as the major active metabolite of Vitamin D, i.e., 1,25(OH)2D, is requried for renal synthesis of 24,25(OH)2D measurement of the latter metabolite may provide a convenient method for assessment of renal vitamin D metabolism. The role of this metabolite in the pathogenesis of renal osteodystrophy remains speculative.

Determination of Corticosterone Concentration in Plasma of Turkeys Using Radioimmunoassay

A method of determining corticosterone concentration in turkey plasma was developed using radioimmunoassay. Compared to fluorometric analysis and competitive proteinbinding radioassay, this method had the following advantages: 1) results were consistent, 2) the method was simple and rapid to perform, 3) only 20 λ of plasma was required, 4) all procedures were performed using the same tube except for counting, and 5) unknown and standard samples were treated identically. The coefficient of variation with the 5 ng/ml standard sample between 14 sets of assays was 12.6% and the average coefficient of variation within duplicate assays of unknown samples randomly selected from each of the 14 series was 4%. Using this method, there was an increase in the average plasma corticosterone concentration in all groups of inoculated turkeys one day after inoculation of Pasteurella multocida which was significantly (P<.05) greater than that of the noninoculated groups.

Negligible effects of nonesterified fatty acids on serum thyroxine analysis by competitive protein-binding radioassay on sephadex and by radioimmunoassay.

Negligible effects of nonesterified fatty acids on serum thyroxine analysis by competitive protein-binding radioassay on sephadex and by radioimmunoassay.