Establishment And Evaluation Of High Performance Liquid Chromatography And SDS-PAGE For Microprotein In Urine

Objective1. To develop a rapid method of microalbuminuria detection using size-exclusion high performance liquid chromatography.2. To investigate the significance of the vertical plate discontinuous SDS-PAGE.Methods1.high-performance liquid chromatography:Using a new mobile phase, samples were injected onto an Agilent Zorbax GF-250size-exclusion chromatographic column. The method was evaluated and urine albumin of fifty-six diabetic patients was analyzed.2.SDS-PAGE:Urinary proteins of150patients were analyzed using the vertical plate discontinuous SDS-PAGE. The results were compared with the ones of the urinary protein semi-quantitative, U-Pr, MA, NAG and β2-MG.Results1.high-performance liquid chromatography:In the condition of mobile phase0.1%formic acid and acetonitrile, sample size20μl, flow rate1ml/min, detection wavelength205nm, retention time of albumin in human serum and urine was1.7min. The linear range extended to2000mg/L. The lower limit of measurement was2mg/L. The intra-assay CV and the inter-assay CV were, respectively,4.11%and4.31%(20mg/L),3.69%and3.22%(200mg/L),4.53%and4.63%(2000mg/L). Recovery rate were95.3%、98.1%、97.2%. Microalbuminuria was detected in30by high performance liquid chromatography and in15by immunoturbidimetry out of56patients with diabetic mellitus.2.SDS-PAGE:The patterns of electrophoresis could be classified into physiological,tubular, glomerular and mixed proteinuria; SDS-PAGE was more sensitive than the above-mentioned detection indexes.Conclusion1. A fast and high sensitivity method has been achieved for analyzing microalbuminuria. It can detect more microalbuminuria and be suitable for clinical routine measurement.2. SDS-PAGE is rapid and sensitive, has high accurate of diagnosis, It not only can estimate the kidney trauma’s position and degree but also has more application value for diagnosing early renal injury.