Flow Injection Determinations of Creatine and Creatinine Using Packed Bed Enzyme Reactors Under Equilibrium Conditions

Abstract

A flow injection system with immobilized enzyme reactors is used for sequential determinations of creatinine and creatine. The first reactor, creatinine amidohydrolase (CAH), catalyzed the hydrolysis of creatinine to creatine which was later phosphorylated by adenosine-5′-triphosphate (ATP) in a second reactor containing creatine kinase (CK), pyruvate kinase (PK) and 1-lactate dehydrogenase (LDH). The intermediate product, adenosine-5′-diphosphate (ADP), dephosphory-lated phosphoenol pyruvate (PEP) to pyruvate by PK catalysis. The LDH reaction converted pyruvate to L-lactate, oxidizing an equivalent quantity of reduced nicotinamide adenine dinucleotide, NADH. The consumption of NADH was measured spectrophotometrically at 340 nm and it was linear in the range 5 – 400 μM creatinine or creatine. The sample throughput was 25 – 30 h⁻¹. The CAH-reactor was stable for at least five months and the multienzyme reactor for three months when used daily and stored in imidazole buffer (pH 7.6) containing 2-mercaptoethanol and EDTA.

Key Words:

• Creatinine
• Creatine
• Determination
• Creatinine amidohydrolase
• Creatininase
• Creatine kinase
• Flow injection analysis
• Enzyme reactors