We attempted to develop a novel serum 1,5-anhydroglucitol (1,5-AG) assay kit using glucose 3-dehydrogenase (G3DH) from Halomonas sp. alpha-15 strain. The major advantages of this method are that the 1,5-AG detection requires a very small amount of G3DH, and the enzyme catalyzes a simple reaction. The analytical performances were acceptable for clinical use operated with a clinical automatic analyzer. The correlation with a commercial assay kit against sera of healthy volunteers was y=0.975x+0.008, r=0.993, Sylx=1.32 microg/mL. However, sham-negative specimens were observed in the validation of this method using specimens from hospital patients.

A novel enzymatic organic synthesis was reported, utilizing glucose-3-dehydrogenase (G3DH) and its regeneration via electrochemical methods. We combined the water-soluble G3DH prepared from a marine bacterium, Halomonas sp. alpha-15, and electron mediator with the electrode system in order to regenerate the enzyme. Using this system, the conversion of 1,5-anhydro-D-glucitol (1,5AG), a diabetes marker in human blood, was investigated. The final yield of the product, 3-keto anhydroglucitol (3-ketoAG), which was identified by 13C nuclear magnetic resonance, was 82% based on the initial amount of 1,5AG. The electrochemical yield of the reaction proceeded almost stoichiometrically. The electrochemical conversion rate of 1,5AG was 1.24 mmol/(L.h), and the electrochemical yield of 1,5AG consumption was 80%, whereas that for 3-ketoAG was 60%.

N-carbamoyl-beta-D-glucopyranosylamine (NCG) is a compound, which can chemically be synthesized by the condensation of glucose and urea by heating under acidic condition. In this study, we isolated and identified NCG and its anomer from human serum. This is the first study showing the occurrence and isolation of NCG from a natural source. The NCG level in human serum was estimated to be 71+/-33 microM using a glucose-3-dehydrogenase-based assay. Because NCG is not commercially used in foods or drugs, we conclude that the NCG isolated from human serum is synthesized from blood glucose and urea in vivo.

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A statistical investigation was carried out on the distribution of serum N-carbamoyl-beta-D-glucopyranosylamine (NCG) among various patient groups. The serum NCG levels of patients treated in the departments of hemodialysis (131 +/- microM), nephritic syndrome (47 +/- 54 microM), and diabetes mellitus (55 +/- 70 microM) were significantly higher (p < 0.01) than those in other internal disease patients (18 +/- 22 microM) and healthy volunteers (6 +/- 22 microM). The serum NCG level was greatly reduced by hemodialysis therapy, however a return to initial NCG levels was observed within about one week. These results indicate that a high serum NCG level is a feature of renal failure patients, and a relationship was demonstrated between hyperuremia and NCG formation and accumulation in blood.