B-016 Evaluation of pyridoxamine phosphate (PAMP) for AST/ALT reagents with recombinant human apoenzymes

Abstract

Background ApoAST/ALT, which have lost a coenzyme such as pyridoxal phosphate (PALP), are normally present at low levels in serum. To form the holoenzyme, IFCC recommends adding PALP into AST/ALT reagents. However, commercial reagents often do not use PALP due to its instability. In recent years, pyridoxamine phosphate (PAMP), a more stable coenzyme, has been marketed. However, the differences in holo formation ability and reagent stability between their coenzymes are not well understood. In this study, we focused on the preparation of PAMP and recombinant human ApoAST/ALT (rhApoAST/ALT), followed by fundamental investigations into their use as AST/ALT reagents. Methods PAMP was synthesized from pyridoxamine through an enzymatic reaction using pyridoxal kinase. The reaction product was purified to achieve a purity of >99% in HPLC. rhApoAST/ALT were expressed in Escherichia coli and purified with chromatography. AST/ALT assay was performed on a Hitachi 7180 automatic analyzer. PALP reagents for AST/ALT were designed to correspond with the final concentration of the IFCC method. PAMP reagents were prepared with PAMP instead of PALP. To evaluate the holo formation ability of PALP and PAMP reagents, we measured a dilution series of rhApoAST/ALT. To assess stability, the reagents were stored at 11°C for 3 months and then measured same. The reagents were subjected to content analysis by HPLC. Results Similar results were shown between coenzymes in the evaluation of holo formation ability, and high correlations were observed. As for the stability test, the values of PALP reagents decreased after 3 months, while the values of PAMP reagents did not. Content analysis showed that a spontaneous transamination occurred in PALP reagents. These results were observed at both AST and ALT reagents. Conclusions In AST/ALT reagents, both PAMP and PALP can activate apoenzymes. While PAMP is stable, PALP causes a decrease in reagent performance due to a spontaneous transamination.