Establishment And Evaluation Of Two Methods For Microalbuminuria

Objective1. To develope a simple, sensitive and affordable spot test which allows quantification of microalbuminuria.2. To establish a simple and rapid method for the determination of microalbuminuria by size-exclusion high performance liquid chromatography (SE-HPLC).Methods1. PVDF-Coomassie Blue assay:Urine sample or standard was spotted on PVDF membrane and stained with Coomassie Blue R-250 dye. The colour intensity of the stained spot was quantified using a imaging system.2. SE-HPLC:200μL Urine sample or standard was vortex-mixed with 400μL 0.2mol/L Phosphate buffer (pH7.0).20μL treated sample was automatically sampled onto a Agilent Zorbax GF-250 (250 mm×4.6 mm i.d,4 um) column. The mobile phase consisted of 85:15 (v/v) 0.1% formic acid solution and acetonitrile. Chromatography was isocratically performed at a flow rate of 1 ml/min at 30℃. The detection was made at the wavelength of 205nm.The area under the peak of albumin was used to calculate a standard curve which was linear.Results 1. PVDF-Coomassie Blue assay:Within-run and between-run CVs of the assay were, respectively,9.1% and 10.0% (50 mg/L),5.6% and 9.6%(100 mg/L) and 5.0% and 7.4%(200 mg/L). The results for 63 samples by immunonephelometry and PVDF-Coomassie Blue assay were, respectively,179.30±187.01 mg/L and 237.63±157.81mg/L. A good correlation (γ2=0.8552) was obtained between PVDF-Coomassie Blue and immunonephelometric assay, but the results of PVDF-Coomassie Blue assay were higher than that of immunonephelometric assay (P=0.000). The lower limit of detection of PVDF-Coomassie Blue assay was 15 mg/L, and linear range was between 12.5 mg/L and 600 mg/L This method was positively interfered by Hb, bilirubin and IgG. The pH of the urine has no significant effect on the results. The PVDF membrane was much better than cellulose acetate membrane.2. SE-HPLC:The retention time of albumin was about 1.73 min. Within-run and between-run CVs of the assay were, respectively,3.98% and 4.05%(20 mg/L),3.55% and 3.60%(200 mg/L) and 4.65% and 4.74%(2000 mg/L). The average recovery was 99.2%. The mean±SD albumin concentration was 8.4±4.3 mg/L for immunonephelometry in Normoalbuminuria group versus 40.0±24.6 mg/L for HPLC (P=0.000; n=40).These values were 57.9±37.3 mg/L for immunonephelometry in Microalbuminuria group versus 260.2±138.8 mg/L for HPLC (P=0.000; n=30), and 1212.7±696.3 mg/L for immunonephelometry in Macroalbuminuria group versus 1127.9±622.1 mg/L for HPLC(P=0.001; n=30). The lower limit of detection of the assay was 2 mg/L, and the linear range was up to 2000 mg/LConclusion1. A new spot test was developed for microalbuminria by stained using Coomassie Blue R-250 dye. The assay is high sensitive, simple and extremely cheap, which makes it potentially suited for screening of microalbuminuria.2. A new method was established for microalbuminria by size-exclusion high performance liquid chromatography (SE-HPLC). The method is simple and rapid, and its precision, accuracy and sensitivity are satisfactory for clinical study.