Method of quantitative assay for 1,5-anhydroglucitol

Details Method of quantitative assay for 1,5-anhydroglucitol Method of quantitative assay for 1,5-anhydroglucitol

1994-04-19

2001-07-31

Leary, Louise N. (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving oxidoreductase

C435S004000, C435S026000, C435S014000, C435S028000, C435S283100

Reexamination Certificate

active

06268166

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method of enzymatic assay for 1,5-anhydroglucitol (hereafter referred to as “1,5-AG”), which is expected as a marker for diagnosis of diabetes, in a simple and rapid way. This method is also applicable to an automated analysis device.
BACKGROUND OF THE INVENTION
Prior Art Statement
1,5-AG is a compound which is present in the cerebrospinal fluid and plasma of humans. It is reported that its quantity is markedly reduced in plasma with certain diseases, particularly with diabetes (Yasuo Akanuma, Kazuyuki Tobe: Journal of Japanese Internal Medicine Association, 80, 1198-1204, 1991), 1,5-AG is expected to be as a marker for diagnosis of diabetes.
As assay for 1,5-AG, there are hitherto known a method based on gas chromatography (Yoshioka, Diabetes, 25, 1115-1118, 1982; hereafter referred to as “GC method”) and methods using enzymes (hereafter referred to as “enzymatic methods”) such as pyranose oxidase (hereafter abbreviated as PROD) or L-sorbose oxidase (Japanese Patent KOKAI (Laid-Open) No. 63-185397).
Serum or plasma collected from the patient with diabetes is mainly a specimen to be assayed for 1,5-AG. In blood from the patient with diabetes, its glucose concentration is higher than that of normal person. In blood from normal person, the glucose concentration is in the range of approximately 60 to 100 mg/dl, whereas in blood from the patient with diabetes, the glucose concentration is widely distributed in the range of 100 to 1000 mg/dl. On the other hand, the concentration of 1,5-AG in blood is in the range of 1.64 to 2.68 mg/dl for normal person but in the patient with diabetes its concentration is as extremely low as 0.18 to 0.21 mg/dl (Japanese Clinic, 47, 1089, extra issue, Immunological Inspection in Blood and Urinary Chemical Test over Wide Range; first volume, 439-442, Kawai). Therefore, the concentration of 1,5-AG in blood from the patient with diabetes becomes about 1/470 or less. In addition, glucose is structurally similar to 1,5-AG so that it is impossible to perform selective assay in the presence of 1,5-AG and glucose on the current technical level. It is thus essentially required to selectively remove glucose or pretreat specimen by adequately modifying the specimen.
In the GC method, the pretreatment requires removal of glucose and labeling of 1,5-AG which makes procedures complicate and involves analysis over long periods of time. For these reasons, it is difficult to assay a large number of specimens by the GC method. There are thus problems for applying the method to clinical assay.
In the enzymatic method, the pretreatment is performed by removing glucose using an ion exchange column or by modifying glucose through phosphorylation. The enzymatic method is accompanied by considerably complicated separation procedures when glucose is removed using an ion exchange column. Turning to glucose modification by phosphorylation, the optimum reaction conditions for phosphorylation including difference in the optimum pH differ from the optimum conditions for reactions of quantitative assay for 1,5-AG. Therefore, phosphorylation and assay for 1,5-AG must be carried out under different reaction conditions, respectively. In addition, adenosine-5′-triphosphate (hereafter referred to as “ATP”) used for phosphorylation has an inhibitory action against PROD. In view of concentration, there is a limit in adding ATP to the assay system for accelerating phosphorylation and hence, it was difficult to terminate phosphorylation rapidly. In any event, it is impossible to perform quantitative assay rapidly by the prior art methods. In particular, any of the prior art methods has not come to be applied to an automated analysis device widely used for various clinical tests.
SUMMARY OF THE INVENTION
An object of the present invention is to eliminate the foregoing shortcomings of the prior art methods of assay for 1,5-AG in a specimen described above and provide a method of quantitative assay for 1,5-AG in a simple and rapid manner without requiring separation procedures such as filtration, centrifugation, adsorption, etc.
Another object of the present invention is to provide a method of assay for 1,5-AG using an automated analysis device.
In order to solve the foregoing problems the present inventor has made extensive investigations and as a result, succeeded in solving the problems. That is, by selecting a more appropriate enzyme than PROD as an enzyme acting on 1,5-AG, the inhibitory reaction of PROD by ATP can be avoided and reaction conditions for phosphorylation can be harmonized with reaction conditions for quantitatively assaying 1,5-AG. The present invention has thus been accomplished.
A first aspect of the present invention is to provide a method of quantiative assay for 1,5-AG using as PROD the one derived from
Basidiomycetous fungi
No. 52.
A second aspect of the present invention is to provide a method of quantitative assay for 1,5-AG which comprises selectively removing sugars other than 1,5-AG in a specimen so as to leave 1,5-AG and quantitatively assaying 1,5-AG using PROD derived from
Basidiomycetous fungi
No. 52.
A third aspect of the present invention is to provide a method of quantitative assay for 1,5-AG which enables to perform the aforesaid selective removal of sugars other than 1,5-AG and quantitative assay for 1,5-AG subsequent thereto in a pH range of 6 to 9.
A fourth aspect of the present invention is to provide a method of quantitative assay for 1,5-AG in which comprises effecting the selective removal of sugars other than 1,5-AG using a large excess of ATP and then quantitatively assaying 1,5-AG.


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Fukumura, Yukihito et al., Interference of Maltose for the Determination of 1,5-Anhydroglucitol with Lana AG&egr; Kit, Rhinsho Kagku (Nippon Rinsho Kagaki), 21 (1992), pp. 43-48, month not available.*
Journal of Japanese Internal Medicine 80:1198-1204, 1991. Month not available.
Yoshioka et al.,J. Japan. Diab. Soc. 25(10):1115-1118, 1982. Abstract only. Month not available.
Kawai,Japanese Clinic 47:439-442, 1989. Month not available.
Izumi et al.,Agric. Biol. Chem. 54(3):799-801, 1990. Month not available.
Izumi et al; “Purification & Properties of Pyranose Oxidase from Basidiomycetous Fungus No. 52”; Agricultural and Biological Chemistry; 54(6), 1393-1399, 1990, month not available.

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