An improved assay for urinary LTE4

Abstract

An improved method for the measurement of urinary LTE4 is described, based on the combined use of solid phase extraction (SPE)-HPLC-enzyme-immunoassay (EIA). This allows the use of homologous radioactive tracer in easily measurable amount for HPLC retention time evaluation and recovery estimation. Recovery linearly correlated with the total amount of LTE4 present in the extracted sample, indicating the existence of a carrier effect. Identification of immunoreactivity in HPLC fractions as LTE4 was based on parallel dilution assay and confirmed by the observable isotopic separation between tritium labeled LTE4 and immunoreactive LTE4. Critical selection of urine sample size, on the basis of creatinine content, together with efficient purification by SPE, resulted in total absence of aspecific immunoreactivity in fractions surrounding those associated with LTE4. Urinary LTE4 was measured in normal subjects and in cirrhotic patients, where an increased LTE4 excretion has been reported. The method described fulfils the criteria of specificity, sensitivity and accuracy necessary for a potential successful use in the study of sulfidopeptide leukotrienes formation in normal and pathological conditions.