Separation and quantitation of urinary trypsin inhibitors by reversed-phase high-performance liquid chromatography

Summary Human urines contain a family of trypsin inhibitors (UTIs) in small quantities, which seem to be involved in important biological processes. A procedure for separation and quantitative determination of such endogenous inhibitors in human urine has been developed. The urine sample is adjusted to pH 8.3 and percolated through a trypsin-Sepharose 4B column: the inhibitors are eluted with acid solution. The eluate (1000 l) is analysed by RP-HPLC with programmed elution and ultraviolet detection (200 nm). Three principal peaks have been evidenced: they are due to the elution of urinary trypsin inhibitors (UTIs) having apparent m.w. of ca. 6000, 72000, 18000 daltons, respectively. Characteristics of the procedure are: limited sample volume (ca. 200 ml) and recovery of the global inhibition activity (95%). For each UTI determination reproducibility and linearity ranges are reported.

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Trennung und Bestimmung von Trypsin-Inhibitoren im Harn durch Umkehrphasen-HPLC