DETERMINATION OF PYRIDOXAMINE 5'-PHOSPHATE IN HUMAN BLOOD-PLASMA

Pyridoxamine 5′-phosphate in 18 μl of human capillary blood plasma is determined by catalytic amplification using the apoenzyme of aspartate aminotransferase. Prior isolation from interfering substances is accomplished by employment of a cation exchange resin in batch operation. The procedure consists of the following stages. Stage I, denaturation of proteins. Trichloroacetic acid is used to precipitate plasma proteins and liberate any bound coenzyme. Dilute NaCl is added to expand the volume thus minimizing coenzyme entrapment in the precipitate. Stage II, isolation of the coenzyme. A sulfonated polystyrene ion exchange resin is used inside a centrifugal filter. Pyridoxamine 5′-phosphate in the supernatant from Stage I adsorbs to the resin. Pyridoxal 5′-phosphate, other organic phosphates, and Pi are removed by centrifugation. Rinsing with dilute NaBH4 destroys traces of pyridoxal 5′-phosphate and washes off residual inhibitors. Pyridoxamine 5′-phosphate is then desorbed with NaOH and Tris buffer and recovered by centrifugation. Stage III, reconstitution and assay. The desorbate from Stage II is incubated with excess apoenzyme. Specific activity of the reconstituted enzyme is measured. Interpolation from a standard curve relating enzyme specific activity and pyridoxamine 5′-phosphate concentration yields the plasma level of the cofactor. Approximately 3 h are required to carry out the procedure. Much of the coenzyme was found not to be assayable if plasma was refrigerated overnight or if whole blood was left standing at room temperature for a few hours. The degradation was arrested with freezing at -80°C. In a 13-day experiment involving a healthy subject, sharp rises of plasma pyridoxamine 5′-phosphate were found to occur in response to small doses of oral vitamin B6. This represents the first observation of significant flux of pyridoxamine 5′-phosphate in blood plasma. © 1991.