Urinary albumin determination by gel-filtration high-performance liquid chromatography

To determine urinary albumin in a minute amount, a gel-filtration high-performance liquid chromatographic procedure was newly established. When urine of a normal rat was eluted isocratically at 0.6 ml/min by 100 mM Na sulfate in 20 mM Na, K-phosphate (pH 7.4), approximately 6 hrs for complete elution of urinary peak-forming substances was needed. Retention time of albumin was found to be 22.9 min. To shorten the analytic time, 100 mM Na sulfate in 20 mM Na, K-phosphate (pH 7.4) was first used during a 30 min period for separation of albumin. A mixture of acetonitrile/the above solvent = 3/7 (v/v) was then flushed to wash away the peak-forming substances. By this elution mode, the analytic time could be reduced to 3 hrs. When the validity of this procedure was tested, the detection limit of albumin was 0.04 microgram/injection, and a linearity was observed between 0.2 and 50 micrograms/injection. Rats then received single subcutaneous injections of puromycin aminonucleoside, which is a nephrotoxic agent. The plasma albumin concentrations fell at 5, 10 and 15 days after the administration, and the urinary excretions of albumin rose from the 1st day up to the 15th day. The results denoted that our procedure could be a good evaluative tool for nephrotoxicity studies where albuminuria was manifested.