Quantitation of urinary alpha 2u-globulin and albumin by reverse-phase high performance liquid chromatography.

A rapid, reproducible, and sensitive high-performance liquid chromatography (HPLC) method for the quantitation of alpha 2u-globulin, the major urinary protein excreted by adult male rats, and albumin has been developed. Total urinary proteins, isolated by a simple Sephadex G-25 gel filtration step, are separated and quantitated by reverse-phase HPLC on a C4 Macrosphere 300 column. The proteins are separated and eluted with a two-step gradient of acetonitrile in aqueous trifluoroacetic acid. Detection limits of 9 and 25 micrograms/mL of urine were established for albumin and alpha 2u-globulin, respectively. Quantitation of urinary excretion of the two proteins in young adult male and female rats and aging male rats showed that values obtained with this method compared favorably with values from previously developed immunological techniques. To quantitate total urinary protein excretion, we modified the Bradford protein assay to use rat urinary protein as standard. Given the established importance of alpha 2u-globulin in the development of male rat-specific nephrotoxicity and nephrocarcinogenicity, these methods should be useful for studying the renal handling of this protein under normal and nephrotoxic conditions.