Analysis of Urinary Proteins by Liquid Chromatography

Abstract

The analysis of urinary proteins has played an important role in characterizing physiological and pathophysiological processes in the urinary tract and in detecting protein abnormalities related to other organs, especially myelomatosis. Proteins in urine are measured for screening purposes, for diagnosis or monitoring of disease. Qualitative routine screening of random urine samples is carried out with reagent teststrips, although these fail to detect 'microalbuminuria' (albumin < 300 mg/24 h), tubular or pre-renal disease. Urinary proteins may be quantitated as total protein or single specific proteins, or differentiated by examining the pattern of proteins present. Total protein determination is based on turbidimetry, dye-binding or colour formation. 1 Turbidimetric methods rely on precipitation either by acid or benzethonium chloride. They require careful control of reaction conditions and suffer, in varying degree, from interferences due to co-precipitation of non-proteins and unequal sensitivity of proteins to these reagents. Dyebinding methods (Coomassie Blue 0250 or Pyrogallol Red) depend on an absorbance wavelengthshift of the dye when bound to protein. They are sensitive but, despite the addition of a detergent to equalize the response of albumin and globulins, some tubular and pre-renal proteinurias are underestimated. Finally, colour reactions (Folin-Lowry or Biuret methods) are susceptible to many interferences and require tedious separation stages. The Biuret method is used as a reference procedure for urinary protein; copper ions react with the peptide bond in alkaline solution, so there is an equal response per unit mass of protein.