Owen T M Chan1, David A Herold. 1. Department of Pathology, VA San Diego Healthcare System 92161, and University of California, San Diego School of Medicine, La Jolla, CA, USA. ochan@ucsd.edu
Abstract
BACKGROUND: Microalbuminuria is an important prognostic marker in diabetic nephropathy and cardiovascular disease. Initially, most commercial assays used immunoreactivity to quantify microalbuminuria; however, size-exclusion HPLC demonstrated the existence of nonimmunoreactive forms of albumin that may not be detected by immunoassay. Recent liquid chromatography tandem mass spectrometry analyses suggested that size-exclusion HPLC gave higher results attributable to other urine proteins coeluting with albumin. We describe an assay that measures total microalbuminuria (immunoreactive and nonimmunoreactive) without any discernable interference from other common urine proteins. METHODS: We used an automated chip electrophoresis system that utilized microfluidic separation technology and fluorescent sample detection. Each albumin specimen was mixed with the manufacturer's sample buffer in addition to a chicken albumin internal calibrator and then electrophoresed without additional reducing agents. RESULTS: With variable concentrations of bovine serum albumin normalized to a chicken albumin internal calibrator, the electrophoresis system was best fit with a polynomial (R2=0.9997; concentration range, 5-300 mg/L). The lower limit of detection was 5 mg/L. Interchip and intrachip variation studies conducted on patient urine demonstrated CVs of 3%-13%. The introduction of potentially interfering agents (i.e., molecular analytes, nonalbumin proteins) did not alter precision. Compared with immunoassay, the chip electrophoresis identified higher microalbuminuria concentrations in all urine samples. The method also clearly resolved the albumin peak from interfering proteins. CONCLUSIONS: Unlike immunoassay, chip electrophoresis can detect both immunoreactive and nonimmunoreactive forms of albumin. This system is a simple, robust method to quantify microalbuminuria with good sensitivity, precision, and accuracy.
BACKGROUND: Microalbuminuria is an important prognostic marker in diabetic nephropathy and cardiovascular disease. Initially, most commercial assays used immunoreactivity to quantify microalbuminuria; however, size-exclusion HPLC demonstrated the existence of nonimmunoreactive forms of albumin that may not be detected by immunoassay. Recent liquid chromatography tandem mass spectrometry analyses suggested that size-exclusion HPLC gave higher results attributable to other urine proteins coeluting with albumin. We describe an assay that measures total microalbuminuria (immunoreactive and nonimmunoreactive) without any discernable interference from other common urine proteins. METHODS: We used an automated chip electrophoresis system that utilized microfluidic separation technology and fluorescent sample detection. Each albumin specimen was mixed with the manufacturer's sample buffer in addition to a chicken albumin internal calibrator and then electrophoresed without additional reducing agents. RESULTS: With variable concentrations of bovine serum albumin normalized to a chicken albumin internal calibrator, the electrophoresis system was best fit with a polynomial (R2=0.9997; concentration range, 5-300 mg/L). The lower limit of detection was 5 mg/L. Interchip and intrachip variation studies conducted on patient urine demonstrated CVs of 3%-13%. The introduction of potentially interfering agents (i.e., molecular analytes, nonalbumin proteins) did not alter precision. Compared with immunoassay, the chip electrophoresis identified higher microalbuminuria concentrations in all urine samples. The method also clearly resolved the albumin peak from interfering proteins. CONCLUSIONS: Unlike immunoassay, chip electrophoresis can detect both immunoreactive and nonimmunoreactive forms of albumin. This system is a simple, robust method to quantify microalbuminuria with good sensitivity, precision, and accuracy.