A Biuret Method for Determination of Protein in Normal Urine

Abstract

Abstract A biuret method has been developed which provides quantitative measurements of protein in normal urine without interference from drugs or pigments. This method is intended for use in monitoring clinical trials of new drugs—to detect nephrotoxicity. Protein is precipitated from duplicate samples of urine by addition of cold ethanolic phosphotungstic acid. The protein precipitates are separated by centrifugation and washed with ethanol. Protein from one of the duplicate samples is dissolvel in biuret reagent. Protein from the second sample is dissolved in an alkaline tartrate reagent which is identical to the biuret reagent, excepting that copper sulfate has been omitted. After 20 min., the differential absorbance of the two samples is measured at 540 mµ. The limit of sensitivity for detection of protein in urine is 0.5 mg./100 ml. The coefficient of variation of replicate analyses of protein in normal urine is 4.2%. The recovery of protein added to urine averages 103 ± 3%. Analyses of urinary protein by the biuret procedure provide close correlation with measurements by an amido black staining method. Systematic search has failed to reveal interference from urinary pigments, compounds, or drugs which are normally or occasionally encountered in hospitalized patients. In 24-hr. collections of urine from 28 healthy adults, the protein concentrations averaged 6.2 mg./100 ml. (range 3.0-12.2), and the protein excretions averaged 77 mg./day (range 40-150).