The measurement of thrombin in clotting blood by radioimmunoassay

We have developed a radioimmunoassay for human thrombin using rabbit anti-human thrombin IgG. The assay can measure 2 ng thrombin/ml plasma, 500-fold more sensitive than clotting assays. Human prothrombin is less reactive in the assay than thrombin by at least four orders of magnitude, and there is no demonstrable cross-reactivity with human factor Xa, the clotting factor structurally most similar to thrombin. The assay does not detect thrombin bound to anthithrombin III. Using the assay, we have demonstrated that plasma from 20 normal subjects does not contain detectable thrombin. We measured thrombin generation in clotting blood in polypropylene tubes and observed that thrombin appears (approximately equal to 3 ng/ml) within 45 S-5 min after venipuncture. This material is thrombin, not intermediates of prothrombin activation, since it disappears after addition of heparin, which promotes thrombin antithrombin III complex formation. After a plateau of 2-10 min, there is further thrombin generation, which results in clotting after 15-27 min at a level of 40-50 ng thrombin/ml. The thrombin generated 9-25 min before clotting may activate factors V and VIII and stimulate platelet aggregation and release. In contrast, the cascade hypothesis assigns a role for thrombin only late in blood clotting. Radioimmunoassay of thrombin and other clotting factors will be useful for clinical and physiological studies of blood clotting especially since the assay seems specific for thrombin and is independent of other activities that affect bioassays.     

Current problems in the measurement of gastrin release. A reproducible measure of physiological gastrin release

The interpretation of gastrin release is confused because of variation in the technique of the radioimmuoassay of gastrin, the lack of a standard stimulus for the release of gastrin and diversity in the method used to express the results. These problems have been analysed (a) by examining the cross-reactivity of three gastrin antisera and using each of the antisera to measure basal gastrin levels in fasting normal subjects, duodenal ulcer and post-vagotomy patients; (b) by determining a satisfactory stimulus for gastrin release in normal subjects; (c) by examining the results to determine the best method of presenting the data. The different a ntisera used were found to give different levels of plasma gastrin in the same sample of plasma. This was not related to the cross reactivity of the antisera. An English breakfast was found to be the most satisfactory stimulus for the release of gastrin. The expression of the results of such a stimulus of gastrin release was affected least by assay variation when the incremental integrated gastrin response was used. It is concluded that the incremental integrated gastrin response to an English breakfast is a satisfactory method for exploring variations in gastrin release.     

A radioimmunoassay for measurement of 3,3'-L-diiodothyronine (T2)

A sensitive, specific and reproducible radioimmunoassay (RIA) for measurement of 3,3'-L-diiodothyoride (T2) in concentrated ethanol extracts of serum and amniotic fluid is described. The T2 binding antiserum was prepared aby immunization of rabbits with T2-human serum albumin conjugate. Of various thyroid hormone derivatives tested, only 3-L-monoiodothyonine cross-reacted significantly with T2 binding sites on the antiserum...     

Psychosocial measurement and specific hypotheses: A research note.

Administered the Health Locus of Control Scale to 115 mastectomy patients. A factor analysis resulted in 2 factors—Self-Blame and Fate. This scale was used as a treatment outcome measure to compare a group of 18 mastectomy patients who received counseling to a group of 18 mastectomy patients who did not receive counseling. Posttreatment group means in the locus of control, although significantly different, were accounted for by changes on the Fate subscale. The validity of this scale for populations characterized by different health problems is discussed. (5 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Evaluation of two commercial kits for serum gastrin assay, and comparison with a conventional radioimmunoassay procedure

We compared two gastrin radioimmunoassay kits ("Immutope" kit, Squibb & Co.; "Gastrin R.I.A." kit, Schwarz/Mann) to the conventional gastrin radioimmunoassay of Yalow and Berson [Gastroenterology 58, 1 (1970)] as run by us and by a second reference laboratory. Although both kits were found to effectively discriminate above-normal and normal values for serum gastrin, they significantly underestimated very high values (greater than 1500 ng/liter). The Schwarz/Mann kit clearly had a superior quality label (lower nonspecific binding and higher specific activity) and a shorter incubation time. However, the 90-min incubation period cited for their kit caused overestimation of gastrin values in the lower range (5-300 ng/liter), which could be corrected by prolonging the incubation to 24 h. The Squibb antibody had fairly good cross reactivity to all gastrin species tested; the Schwarz/Mann antibody had poor affinity for natural human gastrin G34-II. Good correspondence was found for sera run by both reference laboratories (y = 0.96x + 10, r = 0.997), and values obtained with the Schwarz/Mann kit correlated best (+ 0.815) with those from the conventional radioimmunoassay procedure.     

Production of hematopoietic regulatory factors in cultures of adult and fetal mouse organs: measurement by specific bioassays

Factor-specific cell line bioassays were used to monitor the production in vitro by adult and fetal mouse organs of GM-CSF, G-CSF, M-CSF, Multi-CSF (IL-3), IL-6 and leukemia inhibitory factor (LIF). No tissue was observed to produce Multi-CSF. Highest producers of the other regulators were lung, muscle, thymus, heart and bone shaft and all tissues producing detectable growth factors produced all five with the same rank order of activity. Adult tissues produced more GM-CSF than G-CSF and less M-CSF than either, no differences being observed between male, female and pregnant female tissues. In contrast, the pregnant uterus produced high levels of M-CSF as did the fetal membranes and tissues with only low GM-CSF and no G-CSF production. Pre-irradiation did not alter the pattern of regulator production by adult tissues. The intravenous injection of endotoxin elevated serum levels of GM-CSF, G-CSF, M-CSF and IL-6 but the dominant rise was in G-CSF levels. The data indicating that multiple organs produce the regulators monitored in a common rank order suggest some overall linkage in their production with major differences between adult and fetal tissues.     

Proscillaridin A immunoreactivity: its purification, transport in blood by a specific binding protein and its correlation with blood pressure

A material crossreacting with antibodies against the bufadienolide proscillaridin A and inhibiting the sodium pump was found in human blood plasma. The concentration of the material with a retention time similar to ouabain in a reversed phase HPLC correlated to systolic blood pressure and pulse pressure. Affinity purification of this compound from bovine adrenals resulted in the isolation of a compound with molecular mass of 600 Da that was not identical with ouabain. Consistent with the postulate that endogenous ouabain and proscillaridin A immunoreactivities may belong to a new class of cardiotonic steroid hormones, a protein of Mr = 60 kDa has been found in bovine serum by affinity-labeling with N-hydroxysuccimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate.     

Quantitation of cannabinoids in biological fluids by radioimmunoassay

A tritium based radioimmunoassay for delta9THC and its metabolites has been developed for the use of investigators studying the epidemiological, medical, clinical, and research aspects of cannabis use. The assay is sufficiently sensitive to detect cannabinoids in the urine of marijuana smokers for several days after their last exposure to the drug. The results obtained from a 28 day study indicate that the assay reflects the administration and removal of oral doses of THC. The specificity of the antisera, as determined in cross reactivity studies, allows not only the assay of metabolites in biological samples without interference from other drugs, but also the evaluation of extracts of other kinds of samples which may contain unmetabolized delta9THC. The technique of radioimmunoassay has many advantages over other methods of analysis. It is simple to perform and can be readily applied to the rapid analysis of large numbers of samples. It can be used in the direct analysis of physiological fluids and other biological samples which ordinarily have to be processed before other techniques can be applied. The method is non-destructive abd obviates the need to use radiolabelled drugs in man during metabolic and other studies. This radioimmunoassay has been designed with particular emphasis on ease of use by other investigators. We anticipate that it will prove useful to investigators and scientists for determining the absence, or presence and amount, of THC metabolite in a biological specimen, for epidemiologists in determining the full extent of cannabis use and to the medical/clinical community for establishing the minimum effective dose of delta9THC for each patient. The widespread application of a single method of analysis should also remove a great deal of the controversy surrounding marijuana studies performed to date.     

Clinical pharmacology of bruceantin by radioimmunoassay

During the phase I clinical trial of a new antitumor agent, bruceantin, the pharmacology was studied in 18 cancer patients. The drug was infused intravenously (IV) for 3 h at doses ranging from 1 to 3.6 mg/m2 per day for 5 days. The plasma drug disappearance curves were biphasic, with a fast initial half-life of less than 15 min. The second half-life (t1/2 beta) varied from 0.7 to 38 h among different patients and was not dose-related. The difference between the t1/2 beta on day 1 and that on day 5 was not significant. In patients with normal liver function, the mean plasma concentration at the end of infusion was 22 ng/ml, and the value of the area under the concentration X time curve (AUC) was 111 (ng/ml)h. In contrast, in patients with abnormal liver function the corresponding values were 115 ng/ml and 830 (ng/ml)h, respectively. In addition, these patients had a slower elimination half-life of 10.9 h and a decreased total clearance of 157 ml/min/m2, as compared with 2.6 h and 671 ml/min/m2, respectively, for the normal group. All these differences were statistically significant. Patients with abnormal liver function developed more severe toxicity, including fever, severe nausea, vomiting, and hypotension. Two patients with severe hepatic dysfunction received a reduced dose and developed no toxicity. These results demonstrated the importance of the effects of liver dysfunction on drug disposition and showed that the dosage should be reduced in patients with hepatic dysfunction.     

Highly specific radioimmunoassay for human insulin based on immune exclusion of all insulin precursors

We describe a rapid and simple insulin RIA in which proinsulin and conversion intermediates do not interfere. Three monoclonal antibodies (S1, S2, and S53) were selected for their specificity (directed, respectively, against the B10 region, the junction between A chain and C-peptide, and the junction between B chain and C-peptide), their affinity constant (approximately 10(10) L/mol), and their interactive properties in mixture. S2 and S53 were able to bind simultaneously to the same proinsulin molecule, whereas neither could bind simultaneously with S1. Preincubation of serum samples with an excess of S2 resulted in capture of proinsulin and conversion intermediates modified at the junction between B chain and C-peptide into immune complexes that no longer reacted with S1. Similarly, preincubation with S53 prevented proinsulin and conversion intermediates modified at the junction between A chain and C-peptide from reacting with S1. Preincubation with an excess of both S2 and S53 left insulin as the sole reactant with S1. Thus, separation of insulin precursors from insulin by mutually exclusive antibodies is feasible, and on the basis of this new principle, a highly specific RIA for insulin was designed. The detection limit was 11 pmol/L, and the inter- and intraassay coefficients of variation were 11% and 5%, respectively. The potential of the assay for use in clinical studies was verified by application to serum samples from control subjects and patients with diabetes or insulinoma.     

Renal blood flow: measurement in vivo with rapid spiral MR imaging

PURPOSE: To evaluate the use of stent-grafts for the percutaneous closure of arteriovenous fistulas that develop after cardiac catheterization. MATERIALS AND METHODS: From January 1994 to November 1997, 14 arteriovenous fistulas in 13 patients (eight men, five women; age range, 46-65 years; mean age, 53.5 years) were treated. Eleven fistulas were situated between the deep femoral artery and the common femoral vein, and three fistulas were between the superficial femoral artery and the common femoral vein. All fistulas were closed with stent-grafts positioned in the artery at the level of the fistula. RESULTS: The percutaneous treatment of arteriovenous fistulas was successful in all cases. The findings at angiography performed after the procedure demonstrated the closure of the fistulas and the correct positioning of the prostheses; veins were no longer visible. One complication occurred--a partial thrombosis of the common femoral vein at the puncture site after manual compression. CONCLUSION: On the basis of the preliminary data, the authors believe that the percutaneous closure of arteriovenous fistulas with stent-grafts is a safe and effective alternative to conventional surgery.