Measurement of thromboxane B 2 in human plasma or serum by radioimmunoassay

Abstract

A radioimmunoassay for thromboxane B 2 (TxB 2), a stable metabolite of thromboxane A 2, is described. The method consists of extraction of TxB 2 into ethyl acetate from acidified plasma or serum samples and saturation analysis using specific antibodies produced in rabbits against TxB 2-BSA conjugate. The 50 % displacement level of the standard curve was 19.1 ± 2.9 pg/tube (mean ± S.D., n = 19). The method blank was 3.4 ± 3.1 pg/ml (n = 15) and the assay sensitivity thus 9.6 pg/ml (mean blank + 2 S.D.). When 100 to 200 pg of TxB 2 were added to plasma, 96.2–103.6 % were recovered. The intra-assay coefficient of variation varied from 6.7 to 9.7 %, and the inter-assay coefficient of variation was 18.6 % (n = 10). The TxB 2 concentration in the plasma of 14 healthy subjects varied from 29.3 to 120.8 pg/ml with a mean ± S.D. of 70.1 ± 26.1 pg/ml, when the blood was collected into tubes containing acetylsalicylic acid (ASA), whereas significantly higher (p < 0.001) TxB 2 concentrations of 68.3 – 285.3 pg/ml with a mean ± S.D. of 151.8 ± 66.6 pg/ml were obtained from the same subjects in the plasma of blood which was collected into tubes containing no ASA. When blood samples from 10 subjects were allowed to clot at 0, +24 or +37°C for 60 min., the TxB 2 concentrations in the sera were 2053 ± 870 pg/ml, 4001 ± 1370 pg/ml and 178557 ± 54000 pg/ml, respectively. The TxB 2 levels in sera which were separated from blood samples incubated at +37°C, correlated significantly (p < 0.001) with the TxB 2 productions in platelet-rich-plasma (PRP) after an induced aggregation. Our results indicate 1) when TxB 2 is measured in plasma, the use of prostaglandin synthesis inhibitor in the collection tubes is necessary and 2) the measurement of TxB 2 in serum of blood which has been kept at +37°C for a strictly standardized period of time could replace the use of PRP in TxB 2 studies.