FLOW-INJECTION DETERMINATIONS OF CREATINE AND CREATININE USING PACKED-BED ENZYME REACTORS UNDER EQUILIBRIUM CONDITIONS

A flow injection system with immobilized enzyme reactors is used for s equential determinations of creatinine and creatine. The first reactor , creatinine amidohydrolase (CAH), catalyzed the hydrolysis of creatin ine to creatine which was later phosphorylated by adenosine-5'-triphos phate (ATP) in a second reactor containing creatine kinase (CK), pyruv ate kinase (PK) and 1-lactate dehydrogenase (LDH). The intermediate pr oduct, adenosine-5'-diphosphate (ADP), dephosphorylated lated phosphoe nol pyruvate (PEP) to pyruvate by PK catalysis. The LDH reaction conve rted pyruvate to L-lactate, oxidizing an equivalent quantity of reduce d nicotinamide adenine dinucleotide, NADH. The consumption of NADH was measured spectrophotometrically at 340 nm and it was linear in the ra nge 5 - 400 mu M creatinine or creatine. The sample throughput was 25 - 30 h(-1). The CAH-reactor was stable for at least five months and th e multienzyme reactor for three months when used daily and stored in i midazole buffer (pH 7.6) containing 2-mercaptoethanol and EDTA.