DEVELOPMENT AND REVIEW OF RADIOIMMUNOASSAY OF 12-S-HYDROXYHEPTADECATRIENOIC ACID

For move than 25 years 12-S-hydroxyheptadecatrienoic acid (HHT) has be en known to be a product of thromboxanesynthase (TX-Syn) when synthesi zed with thromboxane A(2) (TXA(2)). Although there are some hints that HHT has anti-aggregatory effects, to date, it has neither been shown to have any specific pathological relevance nor is there much informat ion about its physiological role. This review presents a summary of th e physicochemical properties of HHT, its chemical syn thesis, the impa ct of various biological systems on its enzymatic and non-enzymatic pr oduction and its physiological function and metabolization, as well as a survey of the most important methods for analyzing this unsaturated hydroxy-fatty acid. Due to the low antibody-raising potency expected in HHT no immunological system for HHT quantification has been develop ed so far. In our report we present the development and validation of a sensitive and reliable, competitive radioimmunoassay (RIA) suitable for the quantitative determination of HHT. HHT was produced by an enha nced enzymatic method using platelet-rich plasma (PRP). With an effect ive and modified liquid-liquid and solid-phase extraction method we we re able to produce highly purified HHT (97% purity by GC/MS) in sub-mi lligram ranges. These fractions were used for the synthesis of BSA-ant igen-conjugates and for immunization of rabbits. The tritiated tracer was synthesized using prostaglandin H synthase for the production of p rostaglandin H-2 (PGH(2)) followed by an aqueous reaction with Fe2+-so lution to rearrange PGH(2) to HHT. The dynamic range of the assay was from 30-400 pg/tube, with a sensitivity of approximately 40 pg/tube. T he evaluation of the assay was performed by a HPLC-RIA method as well as by correlation with a quantitative HPLC method and correlation with TXB2 concentrations in a blood coagulation study. The assay may be us eful for the quantification of HHT in several tissues and body fluids under various physiological conditions and may also help to understand the possible physiological role of HHT in biological processes.