Urinary Protein and Albumin Determinations by High-Performance Gel-Permeation Chromatography

Abstract

Sensitive high-performance gel-permeation chromatographic protein assay method was developed by using non-ionic detergent of Brij-58 which was UV transparent as compared to Nonidet P-40. A column (70 × 8.0 mm I.D.) packed with Develosil 100 Diol-5 (pore size 10 nm) was used for the protein determination. A commercially available column (300 × 8.0 mm I.D.) packed with Develosil 300 Diol-5 (pore size 30 nm) was used for the urinary albumin determination. Eluent used was a 0.1 M sodium phosphate buffer (pH 5.6) containing 0.3 M sodium chloride, 1% (v/v) Brij-58, 50% (v/v) glycerol. Flow-rates for protein assay and albumin assay were 1.0 and 0.5 ml/min, repectively. Proteins were eluted at the position of the exclusion limit in the case of protein assay (100 Diol-5). Albumin was eluted from 300 Diol-5 column as a symmetric peak. Bovine serum albumin (BSA) was used as an external standard for both protein and albumin assays. Analysis times were within four min for protein assay, and 32 min for albumin assay, respectively. Improved sensitive measurement of BSA at 5–20 ng level was achieved as compared to Nonidet P-40 by use of UV 210 nm. This method was successfully applied for various urine samples, such as healthy random urine of before and after sports, and the 24-h urines from insulin-dependent diabetes mellitus (IDDM) patients. Comparisons with clinical urinary protein and albumin tests were performed using IDDM urine. Three cases out of four tests for sports showed increased protein and albumin content after physical exercise. Thus, this HPLC method was proven to be applicable to the protein and albumin measurements in human urine.