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(12) United States Patent (10) Patent No.: US 6,867,012 B2 Kishimoto Et Al

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(12) United States Patent (10) Patent No.: US 6,867,012 B2 Kishimoto Et Al USOO6867O12B2 (12) United States Patent (10) Patent No.: US 6,867,012 B2 Kishimoto et al. (45) Date of Patent: Mar. 15, 2005 (54) DETERMINATION METHOD OF FOREIGN PATENT DOCUMENTS BIOLOGICAL COMPONENT AND REAGENT EP O 437 373 A 7/1991 KIT USED THEREFOR EP O 477 OO1 A 3/1992 JP P2001-17198 A 1/2001 (75) Inventors: Takahide Kishimoto, Tsuruga (JP); WO WO 99/47559 A 9/1999 Atsushi Sogabe, Tsuruga (JP); Shizuo WO WO OO/28071 A 5/2000 Hattori, Tsuruga (JP); Masanori Oka, Tsuruga (JP); Yoshihisa Kawamura, OTHER PUBLICATIONS Tsuruga (JP) van Dijken et al (Archives of microbiol, V.111(1-2), pp (73) Assignee: Toyo Boseki Kabushiki Kaisha, Osaka 77-83,(Dec. 1976)(Abstract Only).* (JP) (List continued on next page.) (*) Notice: Subject to any disclaimer, the term of this Primary Examiner Louise N. Leary patent is extended or adjusted under 35 (74) Attorney, Agent, or Firm-Kenyon & Kenyon U.S.C. 154(b) by 330 days. (57) ABSTRACT The present invention provides novel glutathione-dependent (21) Appl. No.: 09/998,130 formaldehyde dehydrogenase that makes possible quantita (22) Filed: Dec. 3, 2001 tive measurement of formaldehyde by cycling reaction, and a determination method of formaldehyde and biological (65) Prior Publication Data components, Such as creatinine, creatine, and homocysteine, US 2002/0119507 A1 Aug. 29, 2002 which produces formaldehyde as a reaction intermediate. In addition, the present invention provides a reagent kit for the (30) Foreign Application Priority Data above-mentioned determination method. The present inven tion provides a novel determination method of a homocys Dec. 5, 2000 (JP) ....................................... 2OOO-370445 teine using transferase utilizing homocysteine and other Mar. 29, 2001 (JP) ....................................... 2001-096724 compound as a pair of Substrates. Particularly, the present (51) Int. Cl. .................................................. C12Q 1/48 invention provides a determination method of homocysteine (52) U.S. Cl. ............................. 435/15; 435/26; 435/25; which includes bringing betaine-homocysteine methyltrans 435/28; 435/975 ferase and dimethylglycine oxidase into contact with a (58) Field of Search .............................. 435/15, 26, 25, Sample and measuring produced hydrogen peroxide or form 435/28,975 aldehyde. Moreover, the present invention provides novel dimethylglycine oxidase stable to thiol compound, which is (56) References Cited Suitably used for the measurement. The present invention provides a reagent kit used for any of the above-mentioned U.S. PATENT DOCUMENTS determination methods of homocysteine. 5,668,173 A 9/1997 Garrow ...................... 514/557 5.998,191 A 12/1999 Tan et al. ..................... 435/32 73 Claims, 2 Drawing Sheets 0.25 g 0.20 o St cy - 0.15 30, 10 O 0.05 0.00 O 100 200 300 400 500 formaldehyde concentration (M) US 6,867,012 B2 Page 2 OTHER PUBLICATIONS Lasse Uotila, et al., “A Steady-State-Kinetic Model for Mori et al., “Purification and Properties of Dimethylglycine Formaldehyde Dehydrogenase from Human Liver”, Bio Oxidase from Cylindrocarpon didymum M-1”, Agricultural chem. J., 177, 869-878, (1979). and Biological Chemistry, 44(6): 1383-1390 (1980). Bernard Vinet," An Enzymic Assay for the Specific Deter Meskys, R. et al., “Peculiarities of creatinline catabolism in mination of Methanol in Serum", Clin. Chem., 33(12), Some Arthrobacter Strains”, Biologija, 1:61-64 (1997). Kerr R. G. et al., “An Enzyme-Based Formaldehyde ASSay 2204–2208 (1987). and Its Utility in a Sponge Sterol Biosynthetic Pathway”, J. Yuzo Kayamori, et al., “A Sensitive Determination of Uric Nat. Prod., 62(1):201–202 (1999). Acid in Serum Using Uricase/Catalase/Formaldehyde Dehy Kiba N. et al., “Determination of nano-molar levels of drogenase Coupled with Formate Dehydrogenase', Clin. formaldehyde in drinking water using flow-injection System Biochem., 27(2), 93–97 (1994). with immobilized formaldehyde dehydrogenase after off-line Solid phase extraction', Analytica Chimica ACTA, Yutaka Teranishi, et al., “Catalase Activities of Hydrocar 378(1-3):169-175 (1999). bon-utilizing Candida Yeasts' Agric. Biol. Chem. 38(6), Van Ophem P. W. et al., “NAD-and co-substrate (GSH or 1213–1220 (1974). factor)-dependent formaldehyde dehydrogenases from methylotrophic microorganisms act as a class III alcohol Jacob W. Dubnoff, et al., “Dimethylthetin and dehyrogenase”, FEMS Microbiology Letters, 116(1):87–93 Dimethyl-3-Propiothetin in Methionine Synthesis”, J. Biol. (1994). Chem., 176, 789–796 (1948). Kato N. et al., “Formaldehyde Dehyrdogenase from Methy Raymond F. White, et al., “Betaine-Homocysteine Transm lotrophic Yeasts', Methods in Enzymology, 188:455-459 ethylase in Pseudomonas denitrificans, a Vitamin B Over (1990). M.S. Quesenberry, et al., “A Rapid Formaldehyde Assay producer”, J. Bacteriol., 113(1), 218–223, (1973). Using Purpald Reagent: Application under Periodation Con Timothy A. Garrow, “Purification, Kinetic Properties, and ditions”, Anal. Biochem., 234(1), 50–55 (1996). cDNA Cloning of Mammaliam Betaine-Homocysteine Kensa to Gijutsu, 27(8): 973-980 (1999). Methyltransferase” J. Biol. Chem., 271 (37), 22831–22838 Enzyme Handbook 9, 1.1.1.1, “Alcohol Dehydrogenase” (1996). Springer-Verlag, pp. 1-23, (1995). Masayoshi Yasuhara, et al., “A New Enzymatic Method to Ingo Neben, et al., “Studies on an Enzyme, S-Formylglu Determine Creatine', Clin. Clim. Acta, 122, 181-188 tathione Hydrolase, of the Dissimilatory Pathway of Metha (1982). nol in Candida Boidinii', Biochlm. Biophys. Acta, 614, O. Sugita, et al., “Reference values of Serum and urine 81-91 (1980). creatine, and of creatinine clearance by a new enzymatic method”, Ann. Clin. Biochem, 29, 523-528 (1992). Horst Schütte, et al., “Purification and Properties of Form Anders Andersson, et al., “Homocysteine and Other Thiols aldehyde Dehydrogenase and Formate Dehydrogenase from Determined in Plasma by HPLC and Thiol-Specific Post Candida Boidinii”, Eur: J. Biochem. 62(1), 151-150 (1976). column Derivatization', Clin. Chem., 39, 1590–1597, (1993). * cited by examiner U.S. Patent Mar. 15, 2005 Sheet 1 of 2 US 6,867,012 B2 F.G. 1 0.25 0, 2 O 0.15 0, 10 0. O 5 O O. O O 100 200 300 400 500 formaldehyde concentration (puM) FIG 2 0. O 6 0. O4 0, O 2 0.00 O O 20 30 40 50 formaldehyde concentration (lM) U.S. Patent Mar. 15, 2005 Sheet 2 of 2 US 6,867,012 B2 EIG 3 0.12 : 0. O 0.02 0.00 O 20 40 60 8O 00 Creatinine concentration (uM) FIG 4 0.8 0.6 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 O O 20 30 40 50 L-homocysteine concentration (pM) US 6,867,012 B2 1 2 DETERMINATION METHOD OF pound and thio-NADH compound. The representative BIOLOGICAL COMPONENT AND REAGENT enzyme of the alcohol dehydrogenase used here, which KIT USED THEREFOR catalyzes the following reaction, is the enzyme of EC1.1.1.1: alcohol-i-NAD(P)'' (Paldehyde--NAD(P)H TECHNICAL FIELD OF THE INVENTION D. Schomburg, D. Stephan Eds., Enzyme Handbook 9 The present invention relates to a convenient and highly (Springer-Verlag) States that this enzyme oxidizes not only Sensitive determination method, using glutathione methanol as a Substrate but also its product (formaldehyde). dependent formaldehyde dehydrogenase, of formaldehyde Since formaldehyde exists as a hydrate in an aqueous or a compound forming formaldehyde as a reaction Solution and as alcohol, it is a Substrate oxidized with intermediate, and a reagent kit therefor. More particularly, alcohol dehydrogenase allowing oxidation to acetic acid. the present invention relates to a determination method of a Thus, a highly sensitive determination of formaldehyde biological component, Such as creatinine, creatine, and using this enzyme is unattainable. In addition, Since an homocysteine and via formaldehyde as an intermediate, and aldehyde dehydrogenase, Such as glutathione-independent a reagent kit therefor. The present invention also relates to a 15 formaldehyde dehydrogenase, irreversibly oxidizes determination method of homocysteine using a transferase formaldehyde, it cannot be applied to a highly Sensitive utilizing homocysteine and other Substance as a pair of determination by a cycling reaction. Substrates and a reagent kit therefor. In the field of analytical Science, Some compounds are known to be measured via formaldehyde as an intermediate. BACKGROUND OF THE INVENTION Of these, useful is the determination of creatinine, creatine and homocysteine in clinical tests. Formaldehyde is known to be a cytotoxin rich in reactiv Creatinine is a major diagnostic marker of kidney func ity with protein, membrane, and DNA, which causes various tion in clinical tests, and determination of creatine is used for disorders by being inhaled or oral administration. Formal the analysis of disease State of muscular dystrophy, hyper dehyde contained in atmosphere, waste water, and food has 25 thyroidism. As the determination method therefor, the Jaffe been considered problematic in recent years, and a conve method is dominantly used, but this method is pointed out to nient method for accurately measuring formaldehyde has be insufficient in the Specificity. Recently, enzymatic meth been demanded. ods using creatinine amidohydrolase, creatine As a determination method of formaldehyde, a method amidinohydrolase, Sarcosine oxidase and peroxidase having comprising calorimetric analysis using Hanz reagent, CTA high Specificity are increasingly used, but it has been pointed reagent (J. Biol. Chem, 231, 813 (1958)), Purpald reagent out that these methods may be influenced by reducing (Anal. Biochem, 234(1), 50 (1996)) and an analysis method substances present in the body. There are reported some using a reagent containing phenylhydrazine, potassium enzymatic methods wherein glutathione-independent form ferricyanide, chloroform and methanol in combination are aldehyde dehydrogenase is used instead of peroxidase to known. These methods are associated with problems in that 35 analyze formaldehyde derived from Sarcosine oxidase reac they require complicated manipulation and a long time for tion by the above-mentioned method (Clin. Clim. Acta, 122, the determination, or use of harmful reagents.
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  • Sarcosine Oxidase (Ec 1.5.3.1)
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  • Purification and Properties of Dimethylglycine Oxidase from Cylindrocarpon Didymum M-1
    Agric. Biol. Chem., 44 (6), 1383•`1389, 1980 1383 Purification and Properties of Dimethylglycine Oxidase from Cylindrocarpon didymum M-1 Nobuhiro MORI,* Bunsei KAWAKAMI,Yoshiki TANI and Hideaki YAMADA Departmentof AgriculturalChemistry, Kyoto University,Kyoto 606, Japan ReceivedFebruary 5, 1980 Dimethylglycine oxidase was purified to homogeneity from the cell extract of Cylindro carpon didymum M-1, aerobically grown in medium containing betaine as the carbon source. The molecular weight of the enzyme was estimated to be 170,000 by the gel filtration method and 180,000 by the sedimentation velocity method. The enzyme exhibited an absorption spectrum characteristic of a flavoprotein with absorption maxima at 277, 345 and 450 run. The enzyme consisted of two identical subunits with a molecular weight of 82,000, and contained two mol of FAD per mol of enzyme. The flavin was shown to be covalently bound to the protein. The enzyme was inactivated by Ag+, Hg2+, Zn2+ and iodoacetate. The enzyme oxidized dimethylglycine but was inert toward choline, betaine, sarcosine and alkylamines. Km and Vmax values for dimethylglycine were 9.1mM and 1.22ƒÊmol/min/mg, respectively. The enzyme catalyzed the following reaction: Dimethylglycine+O2+H2O ?? sarcosine+form aldehyde+H2O2. It has been reported that dimethylglycine The present paper deals with the purifica and sarcosine were metabolized to sarcosine tion and some properties of dimethylglycine and glycine, respectively, by the oxidative oxidase from C. didymum M-1. demethylation reaction in liver mitochondria.1) Dimethylglycine dehydrogenase and sarco sine dehydrogenase have been partially purfied MATERIALS AND METHODS from liver mitochondria of rat2,3) and Rhesus Materials.
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