CA1286211C - Method of quantitative assay for 1,5-anhydroglucitol - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The present invention relates to a method of quantitative assay for 1,5-anhydroglucitol which comprises oxidizing 1,5-anhydroglucitol in an aqueous solution of a specimen in the presence of an electron acceptor to produce a compound represented by formula (1) below or a hydrate thereof represented by formula (2) below, and quantitatively determining 1,5-anhydroglucitol from either the consumption amount of electron acceptor consumed, or from the amount of the reduction product of said electron acceptor produced, or from an amount of oxidized product of 1,5-AG represented by formula (1) or formula (2), in the aqueous specimen solution.

Description

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METHOD OF QUANTITATIVE ASSAY FOR 1,5-ANHYDROGLUCITOL
The present invention relates to a method of quantitative assa~ for 1,5-anhydroglucitol (hereina~ter referred to as "1,5-AG"I which can be used as a marker in body fluids for the diagnosis of diabetes.
Background of the Invention 1,5-AG is a compound which is present in the cerebrospinal fluid and plasma of humans. It is reported that its quantity in plasma is reduced with certain diseases, particularly with diabetes. No enzymes are known to oxidize this 1,5--AG. The assay for 1,5-AG has been hitherto performed mainly by techniques based on gas chromatography (J. Biochem., 90, 157-162 (1981)).
However, the prior art method requires techniques for pretreatment of the specimens, and maintenance and control of analysis equipments to high degree. A simple method of assay for 1,5-AG is therefore desirable.
The present invention is based on novel findings that 1,5-AG is oxidized, in the presence of an electron acceptor, to produce a compound represented by formula (1) below:

HO ~ H

~ ( 1 ) HO ~ ~ H
H O

and this compound is easily hydrated in water to produce a compound represented by formula (2~ below:

~ H

HO ~ O~ H (2) H OH
Thus, the present invention relates to a method of quantitative assay for 1,5-anhydroglucitol which comprises oxidizing 1,5-anhydroglucitol in an aqueous solution of a specimen in the presence of an electron acceptor to produce a compound represented by formula (l) above, or a hydrate : thereof represented by formula (2) above, and then quantitatively determining 1,5-anhydroglucitol from either the amount of said electron acceptor consumed, from the amount of the reduction : product of said electron acceptor produced, or from an amount of an oxidized product of 1,5-AG represented formula (1), or formula (2) in said aqueous solution of a specimen.
In the following description, reference will be made to the drawings, in which:
Figure 1 is a curve showing the optimum pH of an enzyme used in the present invention;
Figure 2 is a curve showing the optimum temperature of the enzyme;
~; Figure 3 shows a calibration curve for the oxygen electrode method;
Figure 4 shows a calibration curve for the ferricyanide method;

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~ 8~X~l Figure 5 shows a calibration curve for the dichloro-phenol~indophenol method;
Figure 6 shows a calibration curve for the H2O2 colorimetry method;
Figure 7 shows a calibration curve for the H2O2 fluorometry method;
and Figure 8 shows a calibration curve for the H2O2 electrode method.

There is no particular limitation to the aqueous solution of specimen to be used in the present invention, as far as it is intended to measure the concentration of 1,5-AG.
Examples include cerebrospinal fluid, plasma, serum and urine or, a solution obtained by treating these specimens so as to readily measure the concentration of 1,5-AG.
Any electron acceptor is usable without any particular limitation as long as it participates in the oxidation of 1,5-AG. Examples of the electron acceptor include oxygen, phenazine methosulfate, dichlorophenol-indophenol; ferricyanide compounds such as potassium ferricyanide, sodium ferricyanide;
cytochrome C, etc., and coenzymes such as NAD , NADP , FAD, FMN, etc. The amount of the electron acceptor to be used is, for example, at least 1 ~m per Q in the aqueous solution of specimen, preferably approximately 3 ~m to 500 mM.
Examples of the reduction products of the electron acceptor include hydrogen peroxide, the reduction product of dichlorophenol-indophenol, ferrocyanide compounds, reduced ~286~

cytochrome C, NAD~, NADPH, reduced FMN, etc.
In oxidizing 1,5-AG to the compound represented by formula (1), an enzyme is generally utilized. This enzyme is an enzyme capable of oxidizing l,S-AG to the compound repre-sented by formula (1), hereafter referred to as "1,5-AG
oxidase". The enzyme having such an ability has been found by the present inventors for the first time,and is obtained from microorganisms producing it. Examples of such microorganisms include Pseudomonas sp. NK-85001 (FERM BP-1037, deposited in the Fermentation Research Institute), Pycnoporus coccineus IFO 4923 and IFO 6490, Coriolus consors IFO 9078, _ _ , . .. .
Coriolus versicolor (IFO 4937), DaedaleoDsis styracina (IFO
__ _ 4910), Gloeophyllum sepiarium (z-al~ NRRL 12S06), Pleurotus ostraetus (Z-64, NRRL 12507), etc. Of these microorganisms, , the microorganism belonging to the genus Pseudomonas is a novel strain isolated from the soil collected by the present inventors in Saitama Prefecture, Omiya-shi, Yoshino-cho, in June 1983. The bacteriological properties of this strain are as follows.
1. Mor~hology (cultured in bouillon-agar medium at 27C for 16 hours) (1) Size of cell: 0.7-0.8 x 1.0-1.7 um, rod z~

(2) Pleomorphism: not recognized (3) Motility: It possess polar flagella with motilityO

(4) Presence or absence of spore: not recognized (5) Gram staining: negative (6) Acid fast: negative 2. Growth condition in various media:
(1) Bouillon-agar plate culture: It forms lustrous, opaque and entire, circular colonies with brown-white color.
(2) Bouillon-agar slant culture: It diffuses and proliferates on the surface of medium to grow opaque and lustrous. The color is brown white.
(3) Bouillon l~quid culture: On the first day of culture, it gets turbid as a whole and cells precipitate at the bottom of a test tube on the 3rd day.
Pellicle is observed.
(4) Bouillon gelatin stab culture:
It grows only on the surface by culture at 20C.
No liquefication of gelatin by culture for 20 days.
(5) Litmus milk: no change 3. Physiological properties (cultured a-t 27C) (1) Reduction of nitrate: positive ~28~iZl~l (2) Denitration: positive (3) ~R Test: negative (4) VP Test: negative (5) Indole formation: negative (6) Formation of hydrogen sulfide: negative (7) Hydrolysis of starch: negative (8) Utilization of citric acid:
It utilizes citric acid in Christensen's and Simmon's mediabut not in Kosar's medium.

(9) Utilization of inorganic nitrogen source:
It utilizes ammonia but not nitrates.

10) Formation of pigment:
negative (11) Urease: positive (12) Oxidase: positive (13) Catalase: positive 14) Growth conditions: 10-37C
pH 7-8.5 (lS) Behavior to oxygen: aerobic (16~) O-F test: oxidative 17) Utilization of carbohydrates:
It utilizes glucose, glycerin, sodium succinate and sodium citrate but neither sodium acetate nor p-hydroxybenzoic acid.
(18) Formation of acids and gas from sugars:

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Formation Formation . of Acids of Gas L-Arabinose +
D-Xylose +
D-Grucose +
D-Fructose +
D-Galactose +
Glycerin +
Rhamnose +
D-Mannose Maltose Sucrose Lactose Trehalose D-Sorbitol - -D-Mannitol Inositol Raffinose - Starch (19) Resistance to sodium chloride Sodium chloride was added to basic medium composed of 10 g of tryptone and 1 liter of distilled,water at pH 7.0 in concentrations of 2%, 5~ and 7~, respectively. After inoculating a bacterial solution thereon, stationary culture was performed.
Growth was noted in media of 2% and 5~ but no growth was noted in the medium 7~.
(20) Phenyl pyruvate test: negative (21) Tyrosine solubility negative ,.. :.

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Based on the foregoing properties, the taxonomical properties of this strain were compared with the classifi-cation in Bergey's Mannual o~ Determinative Bacteriology, 8th edition (1974); this strain is a~in to Pseuùomonas stutzeri belonging to the genus Pseudomonas at page 220. However, the strain possesses properties that it does not hydrolyze starch and does not produce any acid from maltose and is thus different from Pseudomonas stutzerl in these points. From the foregoing reasons, the strain is given Pseudomonas sp.
NK-85001.
As media for culturing the above-described strain, there may be used media containing l,5-AG, inorganic nitrogen sources and inorganic salts. For the purpose of accelerating the growth, organic nutrient sources can be supplemented. As the inorganic nitrogen sources, there can be used ammonium sulfate, ammonium chloride, etc. and, salts of sodium, potassium, magnesium, calcium, iron, zinc, etc. can be used as the inorganic salts. As the organic nutrient sources there can be used peptone, Casamino acid, meat extract, corn steep liquor, yeast extract, etc.
It is preferred that culture be performed under aerobic conditions such as sha~ing, aerial agitation, etc. Incubation is performed at pH of 6 to 8 and temperatures of 25 to 35C.

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The 1,5-AG enzyme derived from the genus Pseudomonas which can be ùsed in the present invention is isolated by the following method. The enzyme is present in a membrane fraction of cells, consequently, the cells are isolated from the culture and destroyed in an appropriate buffer solution. The membrane fraction is collected Erom this treated solution.
To destroy the cells, physical methods such as by means of dyno mill, French press, ultrasonic wave, etc., chemical methods using Triton X-100,* EDTA, etc., or enzymatic methods using lysozyme, etc. car. be used singly or in combination. The membrane fraction can be obtained as a suspension in which the membrane fraction is separated from the cell wall components, nucleic acids, intracellular soluble proteins, etc. by utilizing a plurality of centrifuging steps.
Subsequently, active components are extracted with membrane fraction-solubilizing agents such as Triton X-100 (polyoxyethylene octyl phenyl ether), cholic acid, deoxy-cholic acid, etc. to obtain the 1,5-AG oxidase extract. From the extract, the 1,5-AG oxidase can be isolated utilizing methods generally used for purification of an enzyr~e such as polyethylene glycol fractionation, ammonium sulfate fractiona-tion, etc.

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Next, properties of the 1,5-AG enzyme derived from the genus Pseudomonas are described below.
1. Activity It oxidizes 1,5-AG to produce the compound of formula (1) described above.
2. Substrate specificity It specifically acts on :L,5-AG.
3. Optimum pH
pH 6 to 7~5 4. Optimum temperature 25 to 41C
5. Stable pH
6.5 to 8 The 1,5-AG oxidase derived from the genus Pycnoporus and the genus Coriolus which may be used in the present invention can be isolated by the following method.
This enzyme is present in the cytoplasm fraction in the cells so that the cells are separated from the culture and destroyed in an appropriate buffer solution and the cytoplasm fraction is obtained from this treated solution.
To destroy the cells, the cells are destroyed in a manner similar to the case or the genus Pseudomonas described above. The cytoplasm fraction can be separated as precipi-tates from the membrane fraction, cell wall components, etc.

~36~211 by centrufugation of a solution obtained after destruction of the cells.
Subsequentl~ the supernatant is fractionated in a conventional manner generally used for purification of enzyme, such as polyethylene glycol fractionation, ammonium sulfate fractionation, etc. to isolate the l,5-AG oxidase.
When an enzyme of higher purity is required, the enzyme can be purified, if necessary, by column chromatography such as gel ~iltration and ion exchange chromatograph~, etc.
conventionally used.
Next, properties of the 1,5-AG oxidase obtained from the microorganisms belonging to the genus Pycnoporus and the genus Coriolus are shown below.
1,5-AG Oxidase Derived from the Derived from the genus Pycoporus genus Coriolus (IFO 4923) Action It oxidizes 1,5-AG to produce the compound of formula (1) or (2) described above.

Substrate It oxdizes not only 1,5-AG but also glucose Specificity Optimum pH 5 - 6 5.0 - 5.5 Optimum temperature 37 - 55 35 ~ 55 Stable pH 4 - 9 4 ~ 9 ~2~6Z~L~

The 1,5-AG oxidase can be obtained from the micro-organisms belonging to the genus Daedaleopsis and the genus Pleurotus in a similar manner.
In the present invention, not only the isolated 1,5-AG
oxidase but also the cell-treated products such as the 1,5-AG
oxidase extract, the suspension of membrane fraction, etc.
can also be utilized. Further, they can be utillzed in the ~resence of carriers such as a resin, a membrane, etc. having an immobilized enzyme thereon.
Next, products produced in the reaction solution by adding 1,5-AG to the solution containing 1,5-~G oxidase such as 1,5-~G oxidase extract and the membrane fraction suspension are described below.
When about 2 mg/ml of 1,5 AG is added to a membrane fraction suspension tconcentration of protein, 10 mg/ml;
tris-hydrochloride buffer, 0.05 M; pH 7) from a microorganism belonging to the genus Pseudomonas and they are reacted at 30 C for 16 hours with shaking, 1,5-AG disappears but Substance (A) is produced and accumulates. This can be confirmed by TLC analysis. The reaction solution is spotted onto a silica gel plate and developed with a solvent of iso-PrOH:H2O (95:5) and then thoroughly dried. Anisaldehyde sulfate reagent is sprayed thereon and heated at 90 to 100C

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for 5 to 10 minutes, Substance (A) can be observed at Rf of about 0.4 as a blue spot.
The membrane fraction is removed by ultracentrifuga-tion from the reaction solution after the reaction is completed. The supernatant is freeze dried to give white powders. The white powders are dissolved in a small quantity of ethanol and lnsoluble matters are removed. A 2,4-dinitro~
phenylhydrazine-saturated ethanol solution and a trace amount of concentrated hydrochloric acid are added to the filtrate.
After heating in hot water, the mixture is then cooled and water is added thereto until it gets turbid. The mixture is then allowed to stand to give brown precipitates. The prcipitates are taken by filtration, recrystallized from ethanol-water and, if necessary, purified by silica gel chromatography to give yellow brown needles.
Physicochemical properties of the crystals are as follows.
1. Melting point: 192C
2. Molecular weight: 342 (mass spectrum) 3. Molecular formula: C12H14N4O8 Found by mass spectrum: 343 (M+H) Calcd. : 342, 272 4. UV spectrum max. nm (El cm (in methanol) 231 (416.2), 255sh (313.5), 280sh ~286X~ ~L

(17~.4), 364 (659.4) 5. IR spectrum IR spectrum of specimen is measured using the tablet method with KBr.

3 6 0 0 - 3 0 0 0 cm ( b~oad ~, ~ 6 22.

1 5 8 4. ~ 5 1 8. 3 5 0 4. ~ 4 I S.

I 3 3 3, ~ 2 7 3. ] 2 2 4. ~ I 3 7, ] 0 , 3. 3 0 5 0. 1 0 2 8. 9 9 3, 9 2 5. 8 7 8. 7 4 0 6. 13CNMR chemical shift The spectrum is measured in a DMSO-d6 solvent. The chemical shift is determined as a comparative value using 0 ppm as internal standard tetramethylsilane.Underthe experi-mental conditions, the signal of the DMSO-d6 solvent appears at 40.40 ppm. In conformity with the data in the mass spectrum, 12 carbons were observed.

6 1.9(t). 7 1.2~t~. 7 3.6(d). 7 8.4id).

o 2.3~d). ~ ] 6.2(d). ] 2 4.01d). ~ 3 ~.

(s). 1 3 ~.8(d). ~ 3 7.6(sJ. ~ 4 5.5(s).

] 5 3.2(s) .... ~

12~!3621,~1.

7. H NMR Spectrum:
The spectrum was measur~ed in DMSO-d6. Chemical shift was a comparative value with data obtained using tetramethyl-silane as internal standard o~ O ppm.

3.6 9 PPm ( I H. dd ). 4.J 4~4.1 5 ( 2H. ~Bq ). 4.6 2--4.6 5 ( 2H.
t, d )7 5.6 3--5.6 5 ( I H. d ), 7. 2 4 ( ] H, broad ), 7. 8 7--7. 9 0 ( IH7 d ), 8.3 3~8.3 7 ( I H, dd ), 8.8 6 ~8.8 7 ( I H. d ), 4. 6 2~4.6 5 ( I H, t--OH), 5.6 3~5.6 5 ( J H.
d--OH), 7.2 4 ( ~ H, br--~1H) From these data, the aforesaid yellow needles are assumed to have the following chemical formula (3):

O--~
O

HO~<OH /\'H NOz H ~3) H N--N H~l O-H H

~281~21.1 From this, Substance (A) was assumed to possess the aforesaid chemical formula (1).
Further the compound of formula t3) described above was also obtained by treating l,5-AG using the enzyme obtained from microorganisms belonging to the genus Pycno-porus, the genus Coriolus, the genus Daedaleopsis, the genus Pleourotus and the genus Gloeophyllum in a manner similar to the membrane fraction suspension of the microorganisms belonging to the genus Pseudomonas described above.
Next, to confirm the structure of the product, the compound of formula (1) was chemically synthesized. It has been found that the compound of formula (1) readily hydrates in the presence of water to form the compound of formula (2).
This hydrate was reacted in a manner similar to the case where 2,4-dinitrophenyhydrazine was reacted with the treated prodcut of 1,5-AG with the membrane fraction susoension of the microorganisms belonging to the genus Pseudomonas described above, whereby quite the same compound of formula (3) described above was obtained. From this, it is assumed that the product of the 1,5-AG oxidase or the compound of formula (1) would have been present in the form of the hydrate showed by ~ormula (2).
Comparison of the product obtained by the 1,5-AG

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oxidase and the hydrate chemically synthesized was performed by gas chromatography.
Both compounds were trimethylsilylated and analyzed by a column, where both were detected at the same retention time. Further, analysis of the fragment pattern o~ the peak compound by gas chromatography and mass spectrometry ~GC-MS) revealed that quite the same pat~ern was obtained.
From the foregoing, the above assumption is believed to be correct. Accordingly, it is thought that the enzyme or membrane fraction suspension used in the present invention would catalyze the following reaction:

HO--~ HO--~o H /H ~H , ~1 ~/H \ H (a) HO>~H HO f\o~H
H OH H

Further it is believed that the reaction in the present invention would follow the reaction equation (a) described above.

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Physicochemical properties of the chemcally synthe-sized hydrate of formula (2) are as follows.
1. Thermal analysis (in nitrogen flow) o Dehydration 86C(weight reduction corres-temperature ponding to 1 molecule ofwater was noted) o ~lelting point 63-74C
2. Molecular weight: 180 (mass spectrum) 3. Molecular formula: C~H1206 4. IR Spectrum c~mB : 3 4 o 0, 2 9 5 O, 2 8 7 5, ] O 9 0! ~ O 4 O, 8 4 0 5 ~3C~ O O M Hz) 0 : g 3 4 5(s), 8 1.4 7(~. 7 7.7 5(dl, 7 2.5 6~t), fi 9.8 5(~. 6 æo 3(~) 6. H--~ R ~ 4 0 0 ~Hz ) PPm 3-8 9 ( I H, dd), 3 7 5 ( ~ H
. .
d )~ 3.6 7 ~ ~ H, dd), 3.5 6 ( I H, d ), 3.4 5 ( ~ H. d ), 3.4 4 ( I H, t ), 3.4 0 ( I H. m~

7. Crystalline form: amorphous white powder.

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The compounds of formulaa (1) and (2) are novel compounds. Further the reaction in which 1,5-AG is dehydrogenated to produce the compound of formula (1) is also a novel reaction.
The method of measurement of 1,5-AG of the present invention is based on the above reaction. Utilizing the reaction progress or reaction products, variou.s measurements can be made and their contents are described below.
(1) Method based on consumption of oxygen In a sealed reactor are charged 1 ml of 0.05M
tris-hydrochloride buffer (pH 7.0), 20 yl of 30 mM phenazine-methosulfate and 0.3 ml of solution or suspension containing 1,5-~G oxidase. O~gen electrodes are inserted in the reaction mixture. While stirring the content of the reactor at 34C, 50 yl of a 1,5-AG solution is added thereto to initiate the reaction. The amount of oxygen consumed is measured with an oxygen monitor with the passage of time. Using 1,5-AG solutions having ~nown concentrations, a calibration curve is prepared and the concentration of 1,5-AG is calculated from the amount of oxygen consumed.
(2) Method utilizing color change of electron acceptor:
In a reactor are charged 0.7 ml of tris-hydrochloride buffer (0.05M, pH 7), 0.1 ml of a O.lM potassium ferricyanide solution, 0.1 ml of 1,5-~G o~idase obtained from the .

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microorganism belonging to the genus Pseudomonas or an extract thereof and 0.1 ml of a 1,5-AG solution. After reacting at 34C for 10 minutes, 0.5 ml of a ferric sulfate-dupanol reagent (5 g of ferric sulfate, 3 g of sodium laurylsulfate, 95 ml of 85~ phosphoric acid and 900 ml of distilled water) and 3.5 ml of distilled water are added to the mixture followed by allowing to stand for 10 minutes.
Then, absorbance is measured at 660 nm. Using 1,5-AG
solutions having known concentrations, a calibration curve is prepared and the concentration of l,~-AG is calculated from the absorbance of the specimen.
As electron acceptors, there can be used dichloro-phenol-indophenol, etc., in adcition to fe-ricyanides such as potassium ferricyanide, sodium ferricyanide, ammonium ferri-cyanide, etc.
(3) Method of detecting H2O2:
In a reactor are charged 0.3 ml of sodium phosphate buffer (1/15 M, pH 5.6), 0.5 ml of a hydrogen peroxide detecting solution containing substrate for horse radish peroxidase (e.g., 4 mM of 2,2'-azino-di-[3-ethylbenzo-thiazoline sulfonate (6)] (ABTS)) and 12 u/ml o~ horse radish peroxidase, O.1 ml of 1,~-AG o~idase and O.1 ml of a specimen solution containing l,S-AG. After reacting at 37C for 30 minutes, the reaction is stopped under ice ~LZ86Z~l cooling and the absorbance is measured at 405 nm. ~sing l,S-AG solutions having known concentrations, a calibration curve is prepared and the concentration of l,S-AG is calculated from the absorbance of the specimen.
As substrates for horse radish peroxidase, there can be utilized, in addition to ABTS, color-forming substrates such as 5-aminosalicylic acid, 4-aminoantipyrine and phenol, o-toluidine, etc., and fluorescent substrates such as p-hydroxyacetic acid, p-hydroxypropionic acid, etc.
Further for detecting H2O2 produced in the oxidation reaction of 1,5-AG, a method for directly measuring H2O2 using H2O2 electrodes, a method utilizing chemical lumines-cence generated by oxidation of lucigenine, aryl o~alates, etc. with H2O2 may also be utilized in addition to the above method.
(4) Method of quantitative assay for the compound of formula ~I) or the compound af formula (2):
; The membrane fraction suspension derived from bacteria of the genus Pseudomonas is added to a l,S-.~G solution followed by reacting at 30C for 16 hours. After completion of the reaction, the membrane fraction is removed by ultracentrifugation and the supernatant is feeze dried to give white powders. The powders are treated with a labeling agent for the carbonyl group or a protecting agent for the ...

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hydroxy group to effect the assay. In the case of using, e.g., 2,4-dinitrophenylhydrazine, as the labeling agent for the carbonyl group, the freeze dried powders are dissolved in a small quantity of ethanol and insoluble matters are removed; a saturated ethanol solution of 2,4-dinitro-phenylhydrazine and a trace amount of concentrated hydro-chloric acid are added to the filtrate followed by reacting in hot water with heating. By analysis of the product by means of reversed phase HPLC (liquid chromatography), the product of formula (1) can be detected. Further in the case of using trimethylsilylchloride (TMS) as the protecting agent for the hydroxy group, the freeze dried powders are dissolved in a small quantity of pyridine and TMS is then added to the solution. By stirring the mixture at room temperature, a compound wherein all hydroxy groups of the product of formula (2) are protected is obtained. By analysis of a part of the solution by gas chromatography, the compound of formula (2) can be quantitatively determined.

.
The reagent for analysis of the present invention is a reagent comprising at least the enzy~e of the present invention. Namely, its form is not limited but the enzyme may be a soluble enzyme of a solution, freeze d-ied, powdery or granular type. Further, the enzyme may be immobilized i.X~86Z~:l onto carriers of a membrane, gel, particulate, microcapsular, tubular or container type, in various manners. In addition to the enzyme of the present invention, the reagent may be supplemented with buffers such as liquid or powdery phosphate buffer, tris-hydrochloride buffer, acetate buffer, citrate buf~er, veronal buffer, etc.;
salts (sodium chloride, etc.), sugars that do not react with the enzyme of the present invention (sucrose, etc.);
polyvalent alcohols (glycerol, propylene glycol, sorbitol, etc.); coenzymes (FAD, etc.); and other appropriate stabi-lizers, surfactants, etc.
Upon analysis of 1,5-AG, the reagent for analysis described above is used so as to obtain a necessary enzyme activity depending upon the aforesaid various methods for detection. Further, an amount of the reagent appropriate for each of the detection methods may be previously sealed in a container such as a reagent bottle, ampoule, etc.

The ~it for analysis is composed of the aforesaid reagent for analysis comprising the enzyme of the present invention and reagents for detection which ar- reagents for detecting the reaction caused by the enzyme of the present invention. The reagent for detection refe~s to an electron aCcePtr per se that participates in the o~idation of l,5-AG

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accompanied by color formation or to reagents tha-t is necessary fo~ detection of one of the 1,5-.~G o~id~tion reaction products as an inde~ of the 1,5-AG o~idation. As e~amples of the latter, in the case of using hydrogen peroxide as the index, examples of the reagents for detection include combination of peroxidase or a peroxidase-like substance and its color forming substrate or color forming substrate and a coupler, combination of peroxidase or a peroxidase-li~e active substance and its fluorescent substrate, combination of peroxidase or a peroxidase-like active substance and a luminescence forming reagent, etc. Specific e~amples of these reagents are clear from the description entitled "Method 'or detecting H202" described above.
Further similarly in the case of using the com?ound or formula (l) or the compound of formula (2), the o:cidation products of 1,5-AG, as the index, reagents necessary for detection are combined with the reagent for analysis comprising the enzyme of the present invention and the combination can be constructed as the kit for analysis. The reagent for analysis comprising the enzyme of the present invention and the aforesaid reagents for detection may all be mixed together to form a single reagent; alternatively, in the case where mutually interfering components are present, each component may be separated so as to form an appropriate .

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combination. Further these components may be prepared in the form of a solution or powders. Furthermore, they may be incorporated into an appropriate support such as a film to prepare a test paper sheet or an analysis film.
The kit for analysis of the present invention may further contain, in addition to the combinations described above, pretreatment reagents for selectively removing con-taminants, standard reagents containing a definite amount of 1,5-AG, etc.
Preferred examples of the kit for analysis of the present invention include a kit for detecting l,5-AG by spectroscopic detection of the reduction product of a ferricyanide as an electron acceptor and a kit for analysis of 1,5-AG by spectroscopic detection of hydrogen peroxide.
In the case of the kit using -the ferricyanide as an electron acceptor, the enzyme is used more than 0.2 uni-ts/test and ferricyanide is used more than 5-fold moles, preferably more -than 10-fold moles of 1,5-AG and usua~ly 10 5 mol/test of potassium ferricyanide is used.
In the case of the kit for spectroscopically detecting hydrogen peroxide, the enzyme is used more than 0.2 units/test, peroxidase is used 1-10 unit/test and substrate for color for-mation is used more than 5-fold moles, preferably more than 10-fold moles of hydrogen peroxide produced in the o~idation of 1,5-AG and usually 5xlO 7 to 5xlO mols/test of ABTS is used as a substrate for color formation.

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Next, the effects of the present invention will be described beIow.
Test Example 1 tsubstrate specificity) In order to examine substrate specificity, the reaction was performed in the ferricyanide method described above using the 1,5-AG oxidase extract obtained in ~eference Example 1 later described except that the substra~e was replaced by several sugars and related sugar alcohols. As a result, the 1,5-AG oxidase produced by the microorganism belonging to the genus Pseudomonas shows high specificity to 1,5-AG as shown in Table 1.

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Table 1. Substrate Speci.ficity SubstrateRelative Reactivitv ~%) 1,5-AG 100 Glucose 6 Galactose 3 Sorbose 6 Xylose . 3 Sorbitol Mannitol 2 Xylitol 3 Arabitol 6 Erythritol 2 Glucosamine 3 Gluconic acid 2 Test Example 2 (optimum pH and temperature conditions) Using the extract obtained in Reference Example 1 described later, the optimum pH and the optimum temperature of the 1,5-AG oxidase produced by the microorganis~ belonging to the genus Pseudomonas in the conversion reaction of 1,5-AG
were examined to give the results shown in Eigures 1 and 2.
These figures reveal that the optimum pH and optimum temperature are approximately pH 6 to 7.5 and 25 to 41 C, ~L~862~l1 .

respectively.
Further in order to e~amine pH stability, the extract was added to phosphate buffer (pH 6 to 7) and tris-hydro-chloride buffer (pH 7.2 to 9) having different pH values.
Arter storing at 4C for 1 day, the conversion activity was examined; it was stable in the pH range of 6.5 to 8.
Test Example 3 (measurement of 1,5-AG) (1) Calibration curve by the oxygen electrode method The extract (protein concentration: 5 mg/ml) obtained in Reference Example 1 later described was used.
The following reaction solution was added to a reactor of an oxygen densitometer (Oxygraph manufactured by Guilsol Co. in America) and kept at 34C while agitating.

Tris-hydrochlor-ide buffer (0.05 M, pH 7) 1 ml - Solubilizing solution (protein content, ; 5 mg/ml) 0.3 ml 30 mM Phenazine metho-sulfate 20 yl After the reactor was stoppered and sealed, 50 yl of 1,5-AG solutions having known concentrations were injected into the reactor using a microsyringe and, a rate of oxygen consumed was recorded. As a result, a proportional relationship was noted between the l,5-AG concent-ation and * Trade Mark :.
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the rate of oxygen consumed, as shown in Figure 3.
(2J Calibration curve in the method using a ferricyanide as an electron acceptor The extract (protein content, 5 mg/ml) obtained in Reference Example 1 later described was used. A reaction solution having the following composition was reacted at 34C
for 10 minutes in a test tube.

Tris-hydrochloride (0.05 M, pH 8) 0.7 ml Potassium ferricyanide solution (0.1 M) 0.1 ml Extract 0.1 ml 1,5-AG Solution 0.1 ml (Distilled water was used for blank) After the reaction, 0.5 ml of ferric sulfate-dupanol reagent and 3.5 ml of distilled water were added to terminate the reaction. The system was allowed to stand for 10 minutes; the system was colored green. At this stage the absorbancy was measured at 660 nm. When tested using the 1,5-AG solutions having ~nown concentrations, a proportional relationship in absorbance was noted between the 1,5-AG concentration and the absorbance at 660 nm, as shown in Figure 4.
(3) Calibration curve in the method using dichlorophenol-~2~3621~L

indophenol (DCIP) as an electron acceptor The e.~tract (protein content, 5 mg/ml) obtained in Reference Example 1 later described was used. A reaction solution having the following composition was charged in a cell of a spectrophotometer and kept at 34C.

Tris-hydrochloride buffer (0.05 M, pH 8) 1.8 ml 1 mM DCIP 0.3 ml 10 mM KC~ 0.3 ml Extract 0.3 ml The 1,5-AG solution kept at 34C was char~ed in the cell and the absorbance at 600 nm was recorded while stirring, with the passage of time.
When tested with 1,5-AG having known concentrations, a proportiona~ relationship was noted between the 1,5-AG
concentration and the rate of change in the absorbance at 600 nm, i.e., the reduction rate of DCIP, as shown in Figure 5.
(4) Calibration curve in the method for detecting H2O2 by color formation The enzyme (enzyme activity, 3.2 u/ml) obtained in Reference Example 6 later described was used. A reaction solution having the following composition was reacted at 37C
for 2 hours in a test tube.

1~8~21~

Sodium phosphate buffer (1/15 M, pH 5.6) 0.3 ml Color forming reagent: 0.5 ml aforesaid phosphate buffer containing 4 mM ABTS
and 12 u/ml of peroxidase Enzyme 0.1 ml 1,5-AG Solution 0.1 ml (nistilled water was used for blank) After the reaction, the system was ice cooled to discontinue the reaction and the absorbance was measured at 405 nm. When tested using l,S-AG solution having known concentrations, a proportaional relationship was noted between the 1,5-AG concentration and the absorbance at 405 nm, as shown in Figure 6.
(5) Calibration curve in the method for detecting H202 by fluorescence The enzyme (enzyme ~ctivity, 1.5 ~/ml) obtained in Reference Example 6 later described was used. A reaction solution having the following composition was reacted at 37C
for 2 hours in a test tube.

Fluorescent reagent 0.2 ml sodium acetate buffer ~0.05 M, pH 5.0) containing 0.1~ of p-hydroxyphenylpropionic acid and 4 u/ml of peroxidase Enzyme 0.1 ml ., ~

i21~

1,5-AG Solution 0.1 ml (Distilled water was used for blank) After the reaction, 2.5 ml o~ sodium glycine buf~er (0.1 M, pH 10.3) was added to the system to terminate the reaction. A relative fluorescent intensity was r.~easured at excited wavelength of 315 nm and fluorescent wavelength of 450 nm.
When tested with l,S-~G solution having known concent-rations, a proportional relationship was noted between the 1,5-AG concentration and the relative fluorescent intensity, as shown in Figure 7~
(6) Calibration curve in the H2O2 electrode method In the column having immobilized thereto 1,5-AG
oxidase obtained in Reference Example 7 later described, a pump injector and H2O2 electrodes tIshikawa Seisakusho, L.d., BH type) were connected at the up stream to the do:m stream, respectively. The H2O2 electrodes were set with a hydrogen peroxide meter (Ishikawa Seisakusho, Ltd., Model AI-10006) and a recorder. The 1,5-AG immobilized column and the H2O2 electrodes were dipped in a water bath with thermostat set at 37C. Phosphate buffer (1/15 M, pH 5.6) was pumped throu~h at a rate of 1 ml/min to stabilize. Into the flow path, 50 ~1 of the 1,5-AG solution was injected and .

, '' ~ .
.
~ '' ' ` `

~621~

the peak area on the recorder produced by the oxidakion of 1,5-AG was measured. When tested with 1,5-AG solutions having known concentrations, a calibration curve as shown in Figure 8 was obtained between the 1,5-AG concentration and the peak area.
As is evident from the foregoing, 1,5-~G can be quantitatively determined in an extremely m~nner in accor-dance with the present invention.

Example 1 With respect to a specimen having the following composition, the 1,5-AG content was measured by 3 methods described below. As shown below, it was possible to measure 1,5-AG by the respective methods.

Composition of specimen solution:
1,5-AG 100 ~g/ml Glucose 1000 ~g/ml Sorbitol 1000 ~lg/ml MethodFound Value of 1,5-AG
Enzyme electrode method109 ~g/ml Ferricyanide method107 ~g/ml DCIP method 98 ~g/ml ~LZ~362~1 Example 2 To 0.4 ml of human serum was added 30 ~1 of a perchloric acid aqueous solution (60~ w/v). After shaking, the mixture was centrifuged and 0.2 ml of the resultant supernatant was passed through a pretreatment column packed with 0.8 ml of borate type strongly basic resin AG1-X-8 (manufactured by Bio-Rad Co., Ltd.), which was washed with 3 ml of water to give 3 ml of a passed liquid. ~f~er 3 ml of the column-passed liquid was concentrated to dryness, distilled water was added thereto to accurately adjust to 0.5 ml. The thus obtained specimen from which protein had been removed and to which the pretreatment had been subjected was measured by the 3 methods for detecting H2O2 shown in Test Example 3 described above. As shown below, it was possible to measure l,S-~G in serum by the respective methods. Each calibration curve was prepared by treating standard solutions containing ~nown concentrations of 1,5-AG in quite the same manner as described above and measuring by each method.

MethodFound Value( r ) of 1,5-AG in Serum Detection of color H22 28.0 ~g/ml Detection of fluorescence of H2227.6 ~g/ml H2O Electrode method 30.~ ~g/ml * Trade Mark .
; ~

~36Z~

(') The value found in the gas chrornatography was 28.5 ug/ml.
Reference Example 1 Harvest of membrane fraction having 1,5-AG oxidase derived from the microorganism belonging to the genus Pseudomonus In a Erlenmeyer's flask of 500 ml volume was charged 100 ml each of medium composed of 1~ of Casamino acid, 0.2~o of 1,5-AG, O-l~o of (NH4)2S04, O-l~o of K2HP04, o.l$ of NaCl, 0.02~o of MgS04 7H20, O.l~o of yeast extract and distilled water and adjusted to pH 7. After sterilizing the medium at 115C for 15 minutes, a platinum loop of the culture obtained by slant culture of Pseudomonas sp. NK-85001 [Ferm BP-1037 deposited in the Fermentation Research Institute] was inoculated in the mediurn followed by culturing at 30C for 16 hours on a rotary shaking culture machine (220 rpm).
The cells were separated from the culture solution by centrifugation and washed with tris-hyd:rochloride buffer (0.05 M, pH 7) to give a cell suspension of a 1/10 volume based on the amount of the starting solution. The cell suspension was cooled and destroyed with a French press to give the cell-destroyed suspension. The suspension was centrifuged for 10 minutes (10,000 x g).
After the precipitated cell walls were ~36~

removed, centrifugation was continued for further 1 hour (100,000 x g) to give the precipitates. The precipitates were washed with tris-hydrochloride buffer (0.05 M, pH 7) and suspended in the same buffer to give a membrane fraction suspension. Triton X-100 was added to the suspension in a concentration of 1% (w/v). After agitating at 4C for 1 hour, the insoluble matters were removed by centrifugation (100,000 x g) to give the 1,5-AG oxidase extract.
Reference Example 2 ~ While coling the active component-solubilized solu-tion obtained in Reference Example 1, ammonium sulfate powders were added thereto. The precipitated protein was separated by centrifugation (10,000 x g, 10 minutes) and the activity was measured by the ferricyanide method described in the specification. It was noted that the activity wa~ mainly recovered in the 40% ammonium sulfate-saturated fraction~

Specific Activity*

Membrane fraction suspension 0.23 Extract 0.47 40% Ammonium sulfate-saturated fraction 0.85 60% Ammonium sulfate-saturated fraction 0 80~ Ammonium sulfate-saturated fraction 0 -"` 3L;~8621~

* A value per 1 mg of protein calculated based on 1 unit of enzyme which is defined to be activity of reducing 2 ~ moles of ferricyanide for 10 minutes.
Reference Example 3 Harvest of 1,5-AG oxidase derived from Pycnoporus coccineus IFO 4923 In an Erlenmeyer's flask of a 500 ml volume was charged 100 ml each of medium composed of 0.3~ of 1,5-AG, 0.4~ of yeast extract, 0.5~ of rnalt extract and tap water.
After sterilization at 115C for 15 minutes, one platimum loop of the slant culture of Pycnoporus coccineus IFO 4923 was inoculated on the medium followed by culturing at 27C
for 6 days on a rotary shake culture machine (220 rpm). The cells were separated from the culture solution by centrifuga-tion and washed with sodium phosphate buffer (0.1 M, pH 6) to form a cell suspension of a 7.5-fold volume of the wet weight of the cells. The cell suspension was cooled and destroyed with a French press to give the cell-destroyed liquid. The liquid was centrifuged (10,000 x g) for 10 minutes under cooling. After the precipitated cell walls were removed, centrifugation was continued for further 1 hour (100,000 x g) to remove membrane fraction and obtain the cytoplasm supernatant. ~nder cooling, ammonium sulfate powders were added to the supernatant and the mixture was agitated to . , dissolve. The precipitated protein in this case was separated by centrifugation (10,000 x g, 10 minutes) and the activity of 1,5-AG oxidase was measured with each ammonium sulfate fraction by the method for detecting ~22 described in the specification (wherein a l,5-~G solution having a 1%
concentration was used) and it was noted that the activity was present mainly in the 40-60~ ammonium sulfate-saturated fraction. When one unit of enzyme is defined to be an amount that oxidizes 1,5-AG to produce 1 ~mole/min of H2O2, the specific activity of the ammonium sulfate fraction is 4Ø
Enzyme of 11 units are obtained per 1 g of the wet cells.
Reference Example 4 The strain was changed to Pycnoporus coccineus IFO
6490 in Reference Example 3 and cultured in medium having the same composition as in Reference Example 3 for 4 days. The same procedure for purification as in Reference Example 3 was performed to give 1,5-AG oxidase having a specific activity of 3.6.
eference Example 5 (Collection of 1,5-AG oxidase derived from coriolus consors IFO 9078) The strain was changed to coriolus consors IFO 9078 in Reference Example 3 and cultured in medium having the same composition as in Reference Example 3 for 10 days. The same procedure for purification as in Reference Example 3 was ., performed to give l,5-AG oxidase extract having a specific activity of 2.8.
Reference Example 6 (Harvest of highly pure enzyme) A solution of the 60% ammonium sulfate-saturated precipitate of 1,5-~G oxidase obtained in Reference Example 3 in distilled water was used. All of the procedures including chromatography using DEAE-Toyopearl* (manufactured by Toyo Soda Mfg. Co., Ltd.) for purification were performed under cooling at 4C. A 4100 units of enzyme/20 ml of the solution was dialyzed to a 100-fold volume of phosphate buffer (0.01 M, pH 6.0) and charged in DEAE-Toyopearl column (2.5 cm x 40 cm) equilibrated with the same buffer. Afte- thoroughly washing the column with phosphate buffer (0.01 M, pH 6.0), elution was performed with a density gradient of 0.01 M to 0.5 M using phosphate buffer. The active fraction was eluted in the concentrations between O.l ~ and 0.2 M and therefore, the active fractions were collected and concentrated using pl~ 10 ultrafiltration membrane (manufactured by Amicon Co., Ltd.) to give 6.5 ml of the enzyme solution (360 units/ml) showing the specific activity of 18.
Reference Example 7 (Production of immobilized column) In a conventional manner, 0.5 g of porous glass, CPG-10 (200/400 mesh, mean pore size of 500 .~, manufactured by Electronucleonic Co., Ltd.) was subjected to a cou?ling * Trade Mark -- ~28~2~1 treated using 0.5 g of '~aminopropyl-triethoxysilane followed by carboxylation with 0.5 g of succinic anhydride. The dried porous glass was treated with an excess of thionyl chloride in chloroform to convert the carboxyl groups into the acid chloride. To 1 g of the thus obtained acid chloridated porous glass was added 2.5 ml of the l,S-AG oxidase solution prepared in Reference Example 6 described above. While keeping pH at 6 to 7, the reaction was performed at 25C for 12 hours while mildly agitatiny to complete the condensation reaction. The obtained 1,5-AG oxidase-immobilized porous glass was filled up in a column (1 ml of a syringe) having an inner diameter of 2.3 mm and a length of 70 mm. Through the column 20 ml of phosphate buffer (1/15 M, pH 5.6) containing 1 M table salt was passed to remove the enzyme not bound covalently. Further the column was washed by passing phosphate buffer (1/15 M, pH 5.6) therethrough to give the 1,5-AG oxidase-immob~lized column.

'

Claims (10)

1. A method of quantitative assay for 1,5-anhydro-glucitol which comprises oxidizing 1,5-anhydroglucitol in an aqueous solution of a specimen in the presence of an electron acceptor to produce a compound represented by formula (1) below or a hydrate thereof represented by formula (2) below and quantitatively determining 1,5-anhydroglucitol from the consumption amount of said electron acceptor, from the production amount of the reduction product of said electron acceptor produced or from an amount of an oxidized product of 1,5-AG represented by formula (1) below or formula (2) below, in said aqueous solution of a specimen.

(1) (2)

2. The method of Claim 1 wherein said electron acceptor is oxygen, phenazine methosulfate, dichlorophenol-indophenol, a ferricyanide compound, cytochrome C, NAD+, NADP+, FAD or FMN.

Case FM-940

3. The method of Claim 1 wherein said oxidation is done in the presence of enzyme.

4. The method of Claim 3 wherein said enzyme is enzyme produced by microorganisms belonging to the genus Pseudomonas, the genus Pycnoporus, the genus Coriolus, the genus Daedaleopsis, the genus Pleurotus or the genus Gloeophyllum.

5. The method of Claim 1 wherein said reduction product of an electron acceptor is hydrogen peroxide, the reduction product of dichlorophenol-indophenol, a ferro-cyanide compound, reduction type of cytochrome C, NADH, NADPH
reduced type of FAD, or reduction type of FMN.

6. The method of Claim 1 wherein said quantitative assay is made based on the reduction product of said electron acceptor.

7. The method of Claim 6 wherein said reduction product of the electron acceptor is hydrogen peroxide.

8. The method of Claim 7 wherein a hydrogen peroxide-detecting solution containing horse raddish per-oxidase and a substrate thereof is used.

9. The method of Claim 8 wherein said said substrate of horse raddish peroxidase is 3,3',5,5'-tetramethyl-benzidine, o-phenylenediamine, 2,2'-azino-di-[3-ethylbenzo-thiazoline sulfonate], 5-aminosalicylic acid, 4-amino-antipyrine, phenol, o-toluidine, p-hydroxyacetic acid or p-hydroxypropionic acid.

10. A kit for the quantitative assay of 1,5-anhydroglucitol as claimed in claim 1, said kit comprising a reagent for analysis comprising an enzyme 1,5-anhydroglucitol oxidase, and a reagent for detecting the reaction caused by the enzyme.