Methodological and clinical observation of chromogenic substrate for plasma prekallikrein and prekallikrein assay

On the assay method by the use of chromogenic peptide substrate (Chromozym PK) consisting of Bz-Pro-Phe-Arg-pNA the quantitative observations on plasma kallikrein are performed methodologically and clinically, and the following results are obtained.1) The minimum incubation time to activate plasma prekallikrein with kaolin requires about 20 minutes at 0°C. However, the incubation time is shorten to 3 minutes at 25°C or 37°C following a rapid decrease of kallikrein activity in the reaction mixture.2) The standard curve of the PK assay using chromogenic substrate with 0.94mmol/l at the optimal pH (7.9) demonstrates a linear manner within 8 minutes incubation at 37°C.3) For the purpose of anticoagulant sodium citrate is better than other remedies including sodium oxalate, EDTA and heparin, and it is observed that heparin can not be used for this chromogenic assay because of producing turbid precipitate.4) The plasma stored at 4°C or -20°C are stable on the amidase assay for one week.5) When the pooled normal plasma is taken as 100 per cent, the lenear relationship between diluted plasma and ΔOD is obtained in the range to 6.25 per cent.6) Coefficients of variation is low, normal and high activities are calculated as 13.7%, 2.6%, and 0.95%, respectively.7) Normal values of plasma prekallikrein and kllikrein in Japanese adults are in the range of 38.5-73.3mU/ml and 0-3.55mU/ml, respectively.8) Although the levels of plasma prekallikrein and kallikrein in the majority of tested patients are in the normal range, one case with DIC shows extremely low concentration of plasma prekallikrein, and six cases with liver cirrhosis are distributed in the subnormal range.9) The plasma samples of 11 cases from 14 pregnant women show high levels (42-63mU/ml) of plasma kallikrein accompanying with lower levels of plasma prekallikrein.10) Furthermore, all plasma samples from 6 cases taking estrogen derivatives as oral contraceptive indicate high levels of plasma prekallikrein (84-110mU/ml), and this fact seems to be worthy to explain the tendency to thrombosis in such conditions created by the pills.