A photometric method of determining the activity of antithrombin III using a peptide coumarylamide substrate]

Toz-Gly-Pro-Arg-4-methylcoumaryl-7-amide is recommended for antithrombin-III (AT-III) photometry as a substrate. This substrate is cleaved by thrombin at 366 nm. AT-III photometry with this substrate is in accord with the requirements to methods used in clinical laboratory diagnosis, and the results of such measurement are in good correlation with the data of AT-III analysis with Chromozym-TH substrate. Use of photometry with methylcoumaryl substrates for measuring protease activities is more available than the traditional fluorometry.     

A micromethod of determining the activity of factor XIII in whole blood]

A simple, available, and sensitive micromodification of Sigg and Baluda's methods for measurements of Factor XIII activity is suggested with registration of the blood clot hemolysis degree in the urea. The method was tried in rabbits and guinea pigs during immunization against cholera. The results of Factor XIII activity measurements were found to be unrelated to blood fibrinogen level but they are in slight correlation with the blood fibrinolytic activity.     

An optimized micromethod for determining the catalytic activity of serum ribonuclease

An optimized assay is described for the catalytic activity determination of serum ribonuclease, using polycytidylic acid as substrate and measuring the released acid-soluble ultra-violet absorbing products. Recommended final reaction concentrations are 0.3 mmol/l polycytidylic acid, 200 mmol/l imidazole/HCl buffer, pH 7.0, and 50 mmol/l NaCl. Optimal concentrations for the precipitation procedure, guaranteeing sufficient precipitation and minimal decomposition of unreacted substrate, are 160 mmol/l perchloric acid and 4 mmol/l lanthanum nitrate. Coefficients of variation for the method (within series and between days) ranged from 2.2 to 7.9%. No sex-related differences of catalytic activity were observed. In 63 blood donors with normal values of serum creatinine, the upper limit of the reference intervals (99th percentile) was 33.7 kU/l.     

A micromethod of determining the Willebrand factor in blood]

The suggested method for Willebrand's factor detection differs from the known methods by a 2-3 times higher sensitivity and takes 10 times less blood. The technique is based on estimation of washed formalin-treated donor platelet ristomycin aggregation index by scintillating these cells in Laborskel automated scintillator in supernatants of 2 samples with 0.1% ristomycin solution after the tested platelet-free plasma is added to one of these samples. Willebrand's factor level is found on the calibration curve of standard dilutions of a mixture of platelet-free donor plasma. The method is sufficiently simple, operative, and sensitive; it permits the diagnosis of not only Willebrand's disease, but of the hyperaggregation syndromes associated with vascular endothelium involvement that occur in various conditions, as well; it helps monitoring the efficacy of substitute and antiaggregation therapy.     

Effect of protamine sulfate on antithrombin III activity

When protamine sulfate was added to heparinized plasma in vitro for neutralization of heparin, the activities on both thrombin and Xa known as heparin cofactor in antithrombin action were completely abolished. However, progressive activities on thrombin and Xa both recovered within 30 minutes after protamine sulfate addition. When equivalent heparin was again added, heparin cofactor activity was immediately restored. Based on the fact that protamine sulfate did not show any direct action on the antithrombin III molecule, the presence of AT III with progressive activity was considered to play an important role in the rebound phenomenon of heparin after heparin neutralization with protamine sulfate.     

A scheme for determining the correct activity of the kinetic angiotensin-converting enzyme

The absorbance difference measured when angiotensin-converting enzyme (EC 3.4.15.1) hydrolyzes the substrate N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine is the basis for measuring its activity. We show this difference to be instrument dependent, and describe a method for deriving it that is applicable to manual or automated procedures.     

A method of determining heparin in blood plasma based on its antithrombin activity]

The suggested method for measuring blood plasma heparin is based on heparin ability to enhance antithrombin activity of antithrombin III (AT-III), the major Xa and thrombin inhibitor. The method consists in measurement of blood plasma AT-III activity in the presence and absence of protamine sulfate that destroys the heparin--AT-III complex. Heparin content in U/ml is determined from the difference in the activities of heparin--AT-III complex and AT-III proper activity represented on the calibration curve. The method is sufficiently sensitive, it permits registration of heparin concentrations in a wide band (from 0.01 U/ml to 0.75 U/ml of plasma).     

Preservation of antithrombin III activity in stored whole blood

Antithrombin III (AT-III) is the major inhibitor of thrombin, Factor Xa, and other coagulation enzymes. Congenital and acquired deficiencies of AT-III are thought to contribute to thrombosis and disseminated intravascular coagulation. Because a recent report suggested reduced AT III in stored blood, we evaluated blood bank storage effects. Serial samples were taken from 6 units of whole blood drawn into citrate-phosphate-dextrose-adenine over 42 days, and assays for AT-III functional activity were performed on the same day. The values (mean +/- SD) were as follows: day 0,91.8 +/- 10.7 percent; day 2, 101.9 +/- 10.7 percent; day 8, 107.3 +/- 7.4 percent; day 15, 118.9 +/- 11.1 percent; day 22, 105.4 +/- 9.8 percent; day 35, 93.4 +/- 8.8 percent; and day 42, 97.4 +/- 7.5 percent. The rise from day 0 to day 15 was significant but presumably secondary to interassay variation because analysis of frozen aliquots showed no significant change when all samples from each unit were assayed in one batch. Immunoassay of AT-III also showed no change with storage. The results indicate AT-III retains functional activity in whole blood stored at 2 to 6 degrees C for 42 days, and AT-III replacement does not require fresh blood or fresh-frozen plasma. Low values may reflect individual donor differences or dilution of plasma by anticoagulant.     

A micromethod for determining lipoprotein cholesterol in capillary blood

To measure the total cholesterol (CS) and high-density lipoprotein cholesterol (HDLP CS), the blood collected from the finger is mixed with an equal volume of physiologic saline with EDTA and the mixture is collected into a polyethylene tube to separate the diluted plasma from the formed elements by centrifugation. Low- and very low-density lipoprotein CS are sedimented after addition of heparin and Mn2+ ions to diluted plasma. Comparison of the values of the total CS and HDLP CS in the capillary blood plasma and in the blood sera of 20 patients has shown no significant difference between the two methods. The reproducibility and accuracy of the micro- and macromethods, compared in examinations of the same pool of sera, did not differ. This permits the use of the suggested micromethod for the aforesaid measurements during screenings of the population.