Assay of creatinine using the peroxidase activity of copper-creatinine complexes

Objectives: It was our goal to develop a urine dipstick that could measure creatinine with a peroxidase reaction. The simultaneous measurement of albumin and creatinine permits the estimation of the 24-h albumin excretion, an important value in judging existing or likely development of renal failure. A highly sensitive dye-binding dipstick method for albumin exists, and a suitable dipstick for the assay for urine creatinine is described here. Methods: Copper-creatinine and iron-creatinine complexes have peroxidase activity. With 3,3′,5,5′-tetramethylbenzidine (TMB), and diisopropyl benzene dihydroperoxide (DBDH); the peroxidase activity of copper-creatinine and iron-creatinine complexes can be demonstrated. This reaction was used in the assay of urine creatinine either in solution or by a suitably impregnated urine dipstick. Results: Our method based on the peroxidase activity of the copper-creatinine complex has an analytical range for creatinine of 100 mg/L (0.884 mmol/L) to 3000 mg/L (26.52 mmol/L). The creatinine assay is free from most interfering compounds that may be present in urine. Hemoglobin is an interferent, and its effects can be reduced but not eliminated by the addition of 4-hydroxy-2-methyl quinoline. We do not recommend using the dipsticks when visible blood is present or if the dipstick blood test is positive. The copper-creatinine complex oxidizes ascorbic acid; however, we were able to modify the reaction conditions so that ascorbic acid at < 4.4 g/L does not interfere. We found good agreement on fresh urines between the creatinine dipstick results and those by a standard rate-Jaffe cuvet method for creatinine. Discussion: With the simultaneous measurement of creatinine and albumin in urine, the albumin/creatinine ratio can be determined effectively reducing or eliminating the occasional false-negative and false-positive result in those with dilute or concentrated urines, respectively. The dipstick test for these analytes permits the simple identification of individuals with possible albuminuria and could serve well in a point-of-care setting.