Amidolytic Method Research Articles

Comparison of three methods for the estimation of plasma antithrombin.

Plasma antithrombin levels were measured by clotting, immunological, and amidolytic methods on two groups of subjects: 20 normal individuals and nine patients studied serially post-operatively (hip replacement). The postoperative patients were observed for the emergence of deep-vein thrombosis using 125I-fibrinogen uptake measurements (FUT). The three methods gave similar ranges for the normal subjects, were reproducible (cv less than 5%), and detected early postoperative reduction of antithrombin levels. All three methods failed to show any significant differences in preoperative antithrombin levels between the positive and negative FUT groups. Correlation studies were performed on the pooled data from the normal and postoperative group (range 60-130% of normal; 100 samples). The best correlation (r = 0.75; P less than 0.01) was achieved with the chromogenic kit assay method versus the Mancini immunoassay technique. The thrombin agarose (total antithrombin) gel diffusion technique correlated less well with the chromogenic (r = 0.65; P less than 0.01) and Mancini immunoassay (r = 0.45; P less than 0.01) methods. It is concluded that the chromogenic kit method gives a rapid, reproducible, and specific measurement of antithrombin III. The thrombin agarose diffusion method, though not specific for antithrombin III, is a cheap and simple method to perform. The potential of the three methods for detecting the prethrombotic stage and early thrombosis is discussed.

Study on discrepancy between factor X activities assayed by amidolytic method and clotting method

The activity of factor X in the plasma of 123 patients with various diseases was measured by the clotting method and by the amidolytic method. Measurement of clotting activity of factor X was carried out by assay of the prothrombin time with factor X deficient plasma. Measurement of the amydolytic activity of factor X was made by end-point assay with S-2222 as the substrate.In 29 patients, a distinct discrepancy was observed between the clotting activity and amidolytic activity of factor X. This discrepancy was shown markedly in patients with chronic renal failure (CRF) and uncontrolled diabetes mellitus (DM). In the patients with CRF, the amidolytic activity was higher than the clotting activity of factor X. On the other hand, in the patients with DM, the clotting activity was higher than the amidolytic activity of factor X.The changes in activities of factor X by addition of each fraction of plasma from patients with CRF or DM were observed. Fraction of the patient plasma was carried out by ultrafiltration with membrane filter PM 10 and gel filtration with Bio-Gel A-15m. On the experiment by the addition of low molecular weight fraction (MW<104) and high molecular weight fraction (MW≈106) from the plasma of patient with CRF, the amidolytic activity of factor X in the control plasma of factor X deficient plasma was increased by the addition of each fraction. The clotting activity of factor X in the control plasma was decreased by the addition of middle molecular weight fraction from the plasma of patients with DM.Based on these results, it is suggested that an intensifying effect on the amidolytic activity of factor X in the plasma of patients with CRF existed in the low molecular weight fraction (MW<104) and high molecular weight fraction (MW≈106), and that the factor X in patients DM had a different charactor from that in plasma of normal subject.

Determination of antithrombin activity by an amidolytic and a clotting procedure.

Plasma antithrombin activity was measured using an amidolytic method (substrate Chromozym TH) and a clotting method. The mean antithrombin values found in 76 hospital outpatients were 9.4 micronmol/min/ml with the amidolytic procedure and 100.1% of antithrombin activity with the clotting procedure. The two methods correlate fairly well (r = 0.85, P less than 0.01) and show satisfactory reproducibility. Coefficients of variation of 5.9% and 8.8% were obtained respectively with the amidolytic and the clotting procedures. In the presence of very high levels of fibrinogen degradation products, falsely elevated antithrombin activity levels were observed with the clotting procedure but the amidolytic method is essentially unaffected. It is concluded that both methods are suitable for determining antithrombin activity but a well-standardised amidolytic procedure has advantages.

Automated Amidolytic Method for AT III Determination

Automated Amidolytic Method for AT III Determination

Antithrombin III II. Comparison of different substrates and thrombin preparations

Antithrombin III (AT-III) activity may be measured by amidolytic methods; the synthetic chromogenic substrates, Chromozym® TH, S 2238 and S 2160, which are available at present for the measurement of thrombin activity, are compared in the assay of AT-III activity using an automated method. In addition, AT-III activities from patient plasmas are also compared using bovine and human thrombin. After suitable modification of the assay system no significant differences were found using all three substrates when either human or bovine thrombin was used. Commercially available lyophilized control plasmas were tested for possible use as a standard plasma. These plasmas were not preferred as all showed a decreased level of AT-III activity compared to that of fresh frozen pool plasma

The Determination of Antithrombin III

At III levels measured with six methods in 36 people: 10 healthy controls, 10 women taking a progestagen Lynestrenol and 16 women taking a combined oestrogen-progestagen contraceptive pill. The reproducibility and the sensitivity of these methods as well as the correlation between methods were studied. The Hensen and Loeliger technique had a poor reproducibility and the results obtained with the Howie technique were not in good correlation with those obtained with other methods, especially in the group taking oral contraceptives. The two amidolytic methods using chromogenic substrates were found to be very accurate for antithrombin III activity determination. The rocket immunoelectrophoresis was more accurate and more practical than the radial immunodiffusion. With all methods--except the one of Howie--the 10 women taking progestagen Lynestrenol and the 16 taking combined oestrogen-progestagen had a low AT III and differed significantly from the 10 normals tested. No difference was observed between women taking progestagen only or an oestrogen-progestagen combination.

Automated antithrombin III assay with a centrifugal analyser.

The factor Xa inactivating function of antithrombin III is measured automatically by an amidolytic method, adapted to a centrifugal analyser. Plasma is diluted in buffer with heparin. In stage I, diluted plasma is incubated with excess factor Xa. Heparin accelerates the saturation of antithrombin with factor Xa. In stage II, remaining factor Xa is determined with the chromogenic substrate Bz-Ile-Glu-Gly-Arg-pNA. The precision of the present assay compares favourably with that of the clotting assays and immunoassay. There is a close correlation (r = 0.82) between the results obtained with this assay and the immunoassay of antithrombin III.

Inactivation of bovine and human thrombin and factor Xa by antithrombin III studied with amidolytic methods

Antithrombin III (At-III) purified from human plasma was incubated with thrombin (T), activated factor X (Xa) or mixtures of T and Xa. The amount of enzymes inactivated by the amount of At-III in one ml of normal plasma was determined. - At-III which had inactivated one enzyme, was incapable of inactivating the other. Inactivation of Xa and T by At-III was found to proceed like a second order reaction. The inactivation rate was highly dependent on the At-III concentration, the half-time values of the enzyme activities decreasing with increasing At-III concentration. With excess inhibitor, variations in the concentration of enzyme had little influence on the inactivation rate. Bovine Xa was more rapidly inactivated than bovine T. In contrast, human T was more rapidly inactivated than human Xa.

Anticoagulant activity of heparin: Assay of bovine, human and porcine preparations by amidolytic and clotting methods

The anticoagulant activities of different heparin preparations have been compared using two clotting (APTT and CaTT) and two amidolytic assays. The heparin samples analyzed included two heparin standards, heparin isolated from human mastocytoma tissue, four commercial heparin preparations and two heparin preparations (“high affinity heparin” and “low affinity heparin”) separated by affinity chromatography on immobilized antithrombin III. In each assay system, specific activities were expressed relative to the specific activity of the 3rd International standard (assigned to 100). The specific activities obtained ranged from 3 to 198. In all assay systems “high affinity heparin” had the highest and “low affinity heparin” the lowest specific activity. For each individual heparin preparation the variation in specific activity recorded with the various assay methods was relatively slight. However, the specific activities of the commercial heparins determined in the present study differed considerably from those reported by the manufacturers using pharmacopoeial methods. Exchanging purified antithrombin III with plasma in the amidolytic assay systems caused relatively small changes in specific activities, but suggested that plasma antiheparin activity was more effective in the heparin-antithrombin III-thrombin than heparin-antithrombin III-Xa reaction. In view of the reproducibility and simplicity of amidolytic methods, it is suggested that they are adapted for heparin standardization.

Antithrombin III, heparin cofactor and antifactor Xa in a clinical material

Antithrombin III (At-III) activity and heparin cofactor activity were measured by amidolytic methods, antifactor Xa by a clotting method and At-III protein concentration by immunoassay in plasma and serum from normals and patients. There was a strong positive correlation between At-III activity, heparin cofactor activity and At-III protein concentration (r=0.82 and 0.84 respectively) and a positive, but less strong correlation between antifactor Xa activity and At-III protein concentration (r=0.64) in plasma. Thus, At-III seems to be the main inhibitor even of factor Xa, but the results suggest that plasma contains additional inhibitor(s) of factor Xa. High antifactor Xa in three haemophilisca apparently reflected acquired inhibitors of factor Xa. This inhibitory activity was consumed during coagulation in vitro.

Antifactor Xa Activity Measured with Amidolytic Methods

Methods for the assay of antifactor Xa activity in the presence and absence of heparin are described. Diluted plasma is incubated with bovine, activated factor X (Xa) in stage I, and remaining Xa is measured with the chromogene substrate Bz-Ile-Glu-Gly-Arg-pNA in stage II. In the presence of heparin, the inactivation is completed in 30 sec, and this method measures total Xa-inactivating capacity in diluted plasma (Method I). In a clincal material, this capacity showed a strong positive correlation (r=0.85) to the thrombin-inactivating capacity of diluted heparinized plasma (heparin cofactor activity) and apparently reflects antithrombin III (At-III) concentration. In the absence of heparin, the inactivation of factor xa occurs slowly. With an incubation of 5 min, about 25% of Xa is inactivated, and this assay reflects initial inactivation of Xa (Method II). With this method, a positive, but less strong correlation to the thrombin-inactivating capacity was found (r=0.58), indicating that inhibitors different from At-III accounts for a minor part of the initial inactivation. Determinations in plasma, in which At-III was removed by immunoadsorption, indicated that At-III accounts for about 80% of the initial inactivation. The results of the assays are not significantly influenced by varying concentrations of fibrinogen, fibrinogen degradation products or heparin in the test plasma.

A simple amidolytic method for the determination of functional active antithrombin III.

A simple amidolytic method for the determination of the concentration of functionally active antithrombin III is described. Plasma is diluted with buffer containing EDTA and Polybrene. In stage I, diluted plasma is incubated with thrombin. EDTA retards fibrin polymerization, and plasma fibrinogen does not influence the assay. Polybrene makes the assay result independent of heparin. In stage II, remaining thrombin is determined with the chromogenic substrate benzoyl-Phe-Arg-p-NA. The method is simpler and has a higher accuracy than clotting methods. There is a close correlation between the results obtained with this assay and with immunoassay of antithrombin III.

A Simple Amidolytic Method for the Determination of Functionally Active Antithrombin III

A Simple Amidolytic Method for the Determination of Functionally Active Antithrombin III

Heparin cofactor activity measured with an amidolytic method

A two stage method for the determination of heparin cofactor activity is described. Plasma is diluted 1 30 in buffer containing heparin, and incubated with thrombin for 30 seconds (Stage I). Stage II is initiated by the simultaneous addition of polybrene and the chromogenic substrate Bz-Phe-Val-Arg-pNA. Polybrene neutralizes heparin, and the chromogenic substrate is digested by remaining thrombin. After 60 seconds, digestion is stopped by acetic acid, and quantitated by reading the optical density at 405 nm. There is a linear relation between optical density and the concentration of plasma or purified antithrombin III. The results of heparin cofactor activity determinations in a small clinical material is presented.