Evaluation of the IFCC reference method for alanine aminotransferase: spurious blank ALT activity due to contamination of D-alanine with L-alanine, and recommendations for a correction.

During an evaluation of the IFCC reference method for alanine aminotransferase (ALT, EC 2.6.1.2), we noted that the specimen blank activity reaction was markedly increased. Experience with five different lots of D-alanine from four commercial sources indicated that substantial and varying negative bias (up to -10%) could be introduced into the blank-corrected ALT activity, depending on the lot of D-alanine used. Although the IFCC procedure for ALT mentions the possibility of this L-alanine contamination, we believe that the degree of contamination in commercial reagents is underestimated. Analyzing the five lots of D-alanine for L-alanine, we found the magnitude of negative bias to be correlated directly with L-alanine contamination. Here, we describe a quick, sensitive assay based on coupled reactions of L-amino acid oxidase/peroxidase for quantifying L-alanine in the concentration range of 0-15 mmol/L without a sample-dilution step. Results by this alternative L-alanine assay agreed well with those recommended in the IFCC ALT procedure. Further examination suggested an even simpler solution to the L-alanine contamination problem, because we found no difference in the blank-corrected ALT activity determined in Tris HCl buffer, with or without D-alanine (free of L-alanine). We therefore propose that D-alanine be omitted from the IFCC reference ALT procedure.