title: Enzymatic cycling method using creatine kinase to measure creatine by real-time detection.
abstract: We have developed a novel enzymatic cycling method that uses creatine kinase (CK) to measure creatine. The method takes advantage of the reversibility of the CK reaction in which the forward (creatine phosphate forming) and reverse reactions are catalyzed in the presence of an excess amount of ATP and IDP, respectively. Real-time detection was accomplished using ADP-dependent glucokinase (ADP-GK) together with glucose-6-phosphate dehydrogenase. ADP, one of the cycling reaction products, was distinguished from IDP by using the nucleotide selectivity of the ADP-GK. The increasing level of ADP was measured from the level of reduced NADP at 340 nm. The method is appropriate for an assay that requires high sensitivity because the rate of increase in absorbance at 340 nm is proportional to the amount of CK present in the reaction mix. We reasoned that the method with CK in combination with creatinine amidohydrolase could be used to assay creatinine, an important marker of kidney function. Our results confirmed the quantitative capability of the assay.
authors.affiliation: R&D Group, Diagnostics Department, Asahi Kasei Pharma Corporation, Izunokuni-shi, Shizuoka 410-2321, Japan. Electronic address: ueda.sc@om.asahi-kasei.co.jp.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Ueda
authors.affiliation: R&D Group, Diagnostics Department, Asahi Kasei Pharma Corporation, Izunokuni-shi, Shizuoka 410-2321, Japan.
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Sakasegawa
mesh_terms: D000818:Animals; D003401:Creatine / Q000032:analysis* / Q000378:metabolism; D003402:Creatine Kinase / Q000378:metabolism*; D006801:Humans; D011817:Rabbits; D013997:Time Factors
chemical_list: D003402:Creatine Kinase; D003401:Creatine
keywords: ADP-dependent glucokinase; Creatine; Creatine kinase; Enzymatic cycling; Real-time detection