JPH10108692A - Highly sensitive quantification of d-sorbitol and galactitol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly quantify the level of minute amounts of D-sorbitol or galactitol or their mixture in a sample such as of blood, tissue, body fluid, food or the like in a simple operation and in high sensitivity. SOLUTION: First, a dehydrogenase reactive to D-sorbitol and/or galactitol in the presence of NAD<+> is added to a sample to carry out a reaction. Secondly, the resultant NADH is phosphorylated by the use of NADH kinase to form NADPH. Subsequently, cycling reactions are conducted by using an enzyme or electron carrier capable of oxidizing the NADPH into NADP<+> and that capable of reducing NADP<+> into NADPH, and the change in substrate consumed or product formed in the cycling reactions is detected, thus quantifying the D-sorbitol and/or galactitol in high sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for quantifying D-sorbitol or galactitol, and more particularly to a method for diagnosing diabetes, galactosuria and the like, which is used as a marker for pathological conditions in minute tissues and trace amounts of D in body fluids. The present invention relates to a method for quantifying sorbitol or galactitol with high sensitivity.

[0002]

2. Description of the Related Art In recent years, it has been pointed out that the number of diabetic patients has been increasing in Japan due to changes in lifestyles, westernization of eating habits, aging of the population, and the like. It is said that the total number of diabetics is 6 million. In diagnosing or managing the complications due to diabetes, it is extremely important to grasp the concentration of a trace amount of D-sorbitol or galactitol in blood or the like.
As a method for measuring such a trace amount of D-sorbitol, for example, a protein is deproteinized from a whole blood or erythrocyte fraction, and D-sorbitol in the supernatant is reacted with sorbitol dehydrogenase (an enzyme derived from sheep liver) and NAD ⁺ to form a protein. Method for measuring NADH at an excitation wavelength of 365 nm and a fluorescence wavelength of 450 nm using a fluorometer (Clinical Chemistry, Vol. 25, 109-11)
4 (1996)).

[0003] D-galactose is known to be used by involving three kinds of enzymes in vivo.
When metabolism is abnormal, for example, due to genetic deficiency of this enzyme system, D-galactose accumulates in the body, is reduced to galactitol by aldose reductase, and when D-sorbitol is excessively accumulated. Similarly, disorders such as cataracts occur. Therefore, when diagnosing galactosuria, a genetic disease, it is also extremely important to measure the concentration of galactitol in tissues and body fluids. As a method for measuring galactitol, for example, a method in which a commonly used polyalcohol is oxidized using a periodate and the product is colored to perform colorimetric determination [Analytical Chemistry, No. 2]
6, 1092-1093 (1954)], a method by gas chromatography, a method in which galactitol in a body fluid is brought into contact with a bacterium capable of producing D-tagatose from galactitol in vitro, and the produced D-tagatose is measured. (See Japanese Patent Publication No. 5-32036).

However, in the D-sorbitol measurement method described above, the measurable photometer is limited to a fluorometer, and the influence of interfering substances (blank increasing substances) at the time of measurement must always be taken into account. . Further, since the sorbitol dehydrogenase used is an enzyme derived from sheep liver and reacts similarly to xylitol that may be present in the measurement sample, D-sorbitol can be accurately quantified with this method alone. I couldn't do that. In the galactitol measurement method, the problem of its specificity,
Because of the problems of interfering substances, the complexity of operation, and the instability of measured values due to the use of bacteria, any of these methods can be applied to automated analyzers mainly used in clinical settings. Was difficult.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a method for measuring the concentration of D-sorbitol or galactitol present in a sample which does not have the drawbacks of the prior art methods described above.
The object is to provide a method for quantifying a trace amount of D-sorbitol or galactitol or a mixture of both in a particularly small amount of a sample such as a tissue, a body fluid, and a food by a simple operation, quickly, and with high sensitivity. It is a thing.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, using enzymatic methods, D-sorbitol, galactitol, NAD ^{+ and the} like as coenzymes, A dehydrogenase that reacts with D-sorbitol and galactitol is added and reacted, and the reduced NA is reduced.
It has been found that by phosphorylating DH with NADH kinase or the like and guiding the generated NADPH to a cycling reaction, trace amounts of D-sorbitol and galactitol can be accurately measured, and based on this finding, the present invention has been completed. Reached. That is, the present invention provides a sample containing D-sorbitol and / or galactitol, which is reacted with a dehydrogenase that reacts with D-sorbitol and / or galactitol in the presence of NAD ⁺ , and reacts the obtained NADH with
An enzyme or an electron carrier that oxidizes the generated NADPH to NADP ⁺ by phosphorylation using an NADH kinase that hardly acts on NAD ⁺ and specifically acts on NADH, and the NADP ⁺
A cycling reaction is performed using an enzyme or an electron carrier that reduces to PH, and D-sorbitol and / or galactitol are quantified by detecting a change in a substrate consumed or a product generated in the cycling reaction. This is a highly sensitive method for determining D-sorbitol and / or galactitol. Hereinafter, the present invention will be described in detail.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION In order to explain the method of the present invention in detail, the outline of the present invention is shown by the following reaction formula 1 and explained according to it.

[0008]

Embedded image

Each symbol in the above reaction formula has the following meaning. E1: A dehydrogenase that acts on D-sorbitol and galactitol and uses NAD ⁺ or the like as a coenzyme. E2: NADH kinase that hardly acts on NAD ⁺ and specifically acts on NADH. E3: NADP ⁺ -dependent dehydrogenase. E4: NADP ⁺ and P using NADPH and S3 as substrates
An enzyme or an electron carrier that catalyzes the reaction for producing 3. S1: D-sorbitol and / or galactitol in the sample to be measured. S2: Substrate of E3 S3: Oxidized substrate of E4 P1: Reaction product of E1 P2: Reaction product of E3 P3: Reduced product of the reaction of E4 XTP: X = A: (Adenosine-5 ′ triphosphate ) = U: (uridine-5′triphosphate) = G: (guanosine-5′triphosphate) = C: (cytidine-5′triphosphate) = I: (inosine-5′triphosphate) = dT: (thymidine-5 'triphosphate) = dA: (2' deoxyadenosine-5 'triphosphate) = dU: (2' deoxyuridine-5 'triphosphate) = dG: (2' deoxyguanosine- 5 'triphosphate) = dC: (2' deoxycytidine-5 'triphosphate) = dI: (2' deoxyinosine-5 'triphosphate) XDP: Each of the above XTP "triphosphates" Same as the one replaced with “diphosphate”

First, in order to carry out the present invention, the aforementioned D
-For sorbitol and / or galactitol containing samples,
NAD ⁺ in the presence, and reacted by adding dehydrogenase which reacts to D- sorbitol and / or galactitol NAD ⁺ and as a coenzyme (E1 in Reaction Scheme), NADH
Is generated. The sample applicable to the method of the present invention may be any sample containing D-sorbitol and / or galactitol or both.
For example, blood, tissues, body fluids, foods, and the like containing a trace amount of D-sorbitol or galactitol are included. And, the present invention is particularly effective when the samples are small, for example, 0 to 5
It can be applied preferably when it contains a small amount of D-sorbitol or galactitol or both of them in the order of nmol.

The dehydrogenase (E1) includes NA
Any source may be used as long as it acts on D-sorbitol and / or galactitol as a coenzyme, such as D ^+, and includes sorbitol, iditol or galactitol dehydrogenase, for example, those derived from Pseudomonas sp. See JP-A-8-33482),
From sheep liver ("sorbitol dehydrogenase S-3376" manufactured by Sigma) and from Bacillus (Jo
urnal of Biological Chemi
try Vol. 267, No. 35, No. 24989-249
94, 1992). Among them, the dehydrogenase described in JP-A-8-33482 works well on D-sorbitol and galactitol and hardly acts on xylitol as described later, and therefore, xylitol mixed in the sample. Is particularly preferably used because it is hardly affected by the reaction, has a wide stable pH range, is stable even when the reaction condition is pH 9.0, and has excellent thermal stability. be able to. The present enzyme can be used in the reaction system at a concentration of usually about 0.1 to 1000 U / ml, preferably 0.5 to 50 U / ml. The dehydrogenase described in JP-A-8-33482 is, for example, Pseudomonas (Pse
udomonas) sp. KS-E1806 (FERM
P-14299) in a culture medium. The main physicochemical properties of the dehydrogenase thus obtained are as follows.

(1) Action: 1 ^{mo in} the presence of NAD ⁺
1 D-sorbitol is oxidized to produce 1 mol of D-fructose and NADH, and 1 mol of D-fructose is reduced by the reverse reaction of this reaction in the presence of NADH to produce 1 mol of D-sorbitol and NAD ^+. Generate (2) Substrate specificity: specifically acts on D-sorbitol and galactitol. Has little effect on xylitol. (3) Optimum pH and stable pH range: The optimum pH is around 10, and the stable pH range is 30 ° C. for 4 hours,
5.5 to 10.5. (4) Range of suitable working temperature: The range of suitable working temperature is around 50 ° C. with 100 mM Tris-HCl buffer (pH 9.0). (5) Deactivation conditions by pH, temperature, etc .: stable at pH 5.5 to 10.5 by treatment at 30 ° C. for 4 hours;
It is completely inactivated at 0 or less and at pH 11.0 or more. pH
At 7.0, it is stable up to around 40 ° C. after 30 minutes of treatment, and completely inactivated at pH 7.0 at 40 ° C. in 24 hours. (6) Inhibition: strongly inhibited by HgCl ₂ . (7) Molecular weight: about 64,000 ± 5,000 (gel filtration method)

Examples of the NAD ⁺ include commercially available β-NAD ⁺ (manufactured by Oriental Yeast Co., Ltd.) and Thi.
o-NAD ⁺ (manufactured by Oriental Yeast Co., Ltd.) or α-
NAD ⁺ (manufactured by Sigma) can be used, and they are generally used in the reaction system at a concentration of about 0.01 to 100 mM, preferably 0.1 to 1 mM. NADH generated as a result of the reaction is β-NADH, α-NADH or Thio-NADH.

Next, the obtained NADH is converted into a divalent metal ion using NADH kinase (E2 in the above reaction formula) which hardly acts on NAD ⁺ and specifically acts on NADH, and XTP @ X indicated by A is A (adenosine), U (uridine), G: (guanosine), C: (cytidine), I: (inosine), dT: (thymidine), d
A: phosphorylation in the presence of (deoxyadenosine), dU: (deoxyuridine), dG: (2 ′ deoxyguanosine), dC: (deoxycytidine), dI: (deoxyinosine)} to generate NADPH . The NADH kinase is not particularly limited as long as it hardly acts on NADH ⁺ and specifically acts on NADH. Examples thereof include NADH kinase described in JP-A-4-252182. In particular, it is suitable for practical use because it has thermal stability that can withstand practical use and does not significantly increase the blank value. The enzyme is used in a reaction system at a concentration of usually about 5 to 500 mU / ml, preferably 5 to 50 mU / ml. The NADH kinase is, for example, as described below, for example, Pichia membrane braniferaciens (Pichiam).
Embranaefaciens) obtained by culturing cells obtained by culturing YS27 (Microtechnical Laboratory No. 3208) in a medium. The main physicochemical properties of the NADH kinase thus obtained are as follows.

(1) Action and substrate specificity: Mg ²⁺ , Mn
NADH and XTP [where X is A (adenosine), U (uridine), G (guanosine), C (cytidine) in the presence of at least one ion of ²⁺ , Ca2 ⁺ and Co2 ^+. , I (inosine), dT (thymidine), dA (deoxyadenosine), dU (deoxyuridine), dG
(Deoxyguanosine), dC (deoxycytidine) or dI (deoxyinosine)] as a substrate to phosphorylate NADH, and NADPH and XDP (where X is
Catalyzes the reaction to produce Very specific for NADH and has no effect on NAD ⁺ . (2) Optimum pH and stable pH range: Optimum pH is determined by NAD
When H is used as a substrate, the pH is 8.0 to 9.0, and the stable pH range is pH 7.0 to 9.0. (3) Suitable operating temperature range: 30 to 45 ° C (4) Thermal stability: 35 ° C, 83% by treatment for 10 minutes, 4
It shows 60% residual activity at 0 ° C for 10 minutes and 30% residual activity at 45 ° C for 10 minutes. (5) Molecular weight: about 270,000 (gel filtration method)

Further, the divalent metal ions include:
Mg ²⁺ , Mn ²⁺ , Ca ²⁺ , Co ^{2+ and the} like. Of these, Mg ²⁺ is particularly preferably used. For example, in a reaction system, magnesium sulfate or magnesium chloride is generally used at a concentration of 0.5 to 500 mM, preferably 5 to 50 mM.

Next, the generated NADPH is
An enzyme or electron carrier that oxidizes PH to NADP ⁺ (E4 in the above reaction formula) and converts NADP ⁺ to NADPH
Enzyme or electron carrier (E in the above reaction formula)
The cycling reaction is performed using 3), and the amount of D-sorbitol and / or galactitol is quantified by detecting the amount of change in the substrate consumed or the product generated in the cycling reaction. Examples of the enzymes that oxidize NADPH include dehydrogenase, diaphorase, and NADPH.
(P) H oxidase, Meldora blue, 1-methoxy PMS (1-Methoxy-5) as an electron carrier
-Methylphenazinium methyl
sulfate) (manufactured by Dojindo Laboratories Inc.), PM
S and the like can be mentioned. As the amount of the enzyme used, for example, 1-methoxy PMS is usually 0.0005 to 50 m
M, preferably at a concentration of 0.01 to 0.5 mM. As the catalyst for reduction, NADP ⁺ -dependent dehydrogenase is particularly suitable. For example, glucose 6-phosphate dehydrogenase derived from yeast (manufactured by Oriental Yeast Co., Ltd.) is usually used at 0.01 to 1000 U / ml. , Preferably 1
It can be used at a concentration of １００100 U / ml. Also,
As the substance (S3 in the above reaction formula) serving as the substrate of E4, for example, WST-4 @ 2-Benzothiaz
oly-3- (4-carboxy-2-methox)
yphenyl) -5- [4- (2-sulfeth)
ylcarbamoyl) phenyl] -2H-te
trozolium｝ (manufactured by Dojindo Laboratories Inc.), which is usually used at a concentration of 0.01 to 100 mM, preferably 0.05 to 5 mM.

The temperature, pH,
Conditions such as a stabilizer and a metal ion are appropriately determined based on a known technique. That is, the reaction temperature is usually in the range of 25 to 40 ° C., and the pH condition is appropriately set in the range of, for example, pH 6 to 10, depending on the optimum pH and stable pH conditions of the target enzyme. PH for these purposes
Various buffers such as Good's buffer, phosphate buffer, and Tris-HCl buffer are used as buffers for maintaining the conditions. The reaction time is a time required for performing an enzyme reaction, and its range is not particularly limited.

Each enzyme and necessary reagents for carrying out the present invention may be stored in a single system or, if necessary, in a plurality of systems, as long as they do not interfere with the reaction, and may be stored as an aqueous solution, or may be stored in a dry powder form. May be. The amount of each of these enzymes and necessary reagents is determined, and this is used as an aqueous solution for quantitative determination. The amount of liquid used per test is not particularly limited, but usually about 5 μl to 5 ml is used. I just need.

[0020]

The present invention will be described in more detail with reference to the following Reference Examples and Examples, which do not limit the scope of the present invention in any way.

Reference Example 1 (Preparation of dehydrogenase) D-sorbitol 0.5% and polypeptone 2.0 were prepared in the same manner as described in Example 1 of JP-A-8-33482.
%, 0.5% yeast _{extract, KH 2 P0 4 0.01%,} K
_{2 HP0 4 0.01%, MgSO 4} · 7H 2 O0.01%
Then, 100 ml of a medium (pH 7.2) consisting of tap water and tap water was placed in a Sakaguchi Kolben and sterilized at 121 ° C. for 15 minutes. Pseudomonas sp. KS-
One platinum loop was inoculated from the stock slant of E1806 (FERM P-14299), and this was shaken at 30 ° C. for about 2 hours using a shaker.
Shaking culture was performed for 4 hours to obtain a seed culture solution. Next, 100 ml (for one Sakaguchi Corben) of the seed culture solution was inoculated into a 30-liter jar fermenter containing 20 l of a medium prepared and sterilized in the same manner as described above, and the number of rotations was 300 rpm and the air flow rate was 20
The cells were cultured at 30 ° C. for about 20 hours at 1 / min. After completion of the culture, cells were collected from 20 l of the culture solution using Asahi Kasei Microza (PW-303), and the cells were collected in a 20 mM phosphate buffer (pH 7.0).
After washing the cells in 5), the cells were suspended in about 5 l of the same buffer. Purification of this enzyme was performed by the following operation.

Step 1 (Preparation of Crude Enzyme Solution): 10 g of lysozyme and 1 g of Triton X-100 were added to the cell suspension.
0 ml and 0.55 M EDTA · 2Na (pH 8.0)
Add 500 ml, mix and leave at 20 ° C. overnight,
200 ml of a 5% aqueous protamine solution (pH 8.0) was added dropwise with stirring to remove nucleic acids. The supernatant was filtered using an ultrafiltration membrane containing 1 mM containing 50 mM potassium chloride.
It was dialyzed against 0 mM Tris-HCl buffer (pH 8.0) (hereinafter referred to as "buffer A"). Step 2 (DEAE-cellulose treatment): Approximately 9 kg of wet weight of DEAE-cellulose was added to the dialysate (about 10 l), mixed, and allowed to adsorb the enzyme, followed by buffer A containing 50 mM potassium chloride. Wash DEAE-cellulose, then buffer A containing 0.5M potassium chloride
The enzyme was eluted and ultra-concentrated. Step 3 (QAE-Sephadex A-50 treatment): 1000 ml of Q was added to the concentrate (1000 ml).
AE-Sephadex A-50 is added and mixed,
After adsorbing this enzyme, QAE-Sephadex A-50 was added to buffer A containing 0.05 M potassium chloride.
The enzyme was then eluted with buffer A containing 0.5 M potassium chloride, ultra-concentrated, and further concentrated at 40 mM
It was dialyzed against 20 mM acetic acid-sodium acetate buffer (pH 6.0) containing potassium chloride (hereinafter referred to as "buffer B"). Step 4 (QAE-Toyopearl 550c column chromatography): The concentrated dialysate (100 m
l) was adsorbed on a QAE-Toyopearl 550c column (2.5 × 33 cm), washed with buffer B containing 0.04 M potassium chloride, and then washed with 0.05 M
It was eluted with a buffer B containing ０．１0.1 M potassium chloride by the linear concentration gradient method, and the active fraction eluted with about 0.06 M potassium chloride was ultra-concentrated. Step 5 (Biogel A 1.5m 200-40
0 mesh column chromatography): Concentrate (about 2
ml) with Biogel A 1.5m 200-400
Mesh column (2.5 x 95 cm)
Gel filtration was performed using buffer A containing 1 M salt and 0.02% sodium azide, and the eluted active fraction (46
ml) was collected.

The fraction obtained by the above-mentioned purification procedure is SD
It is a purified sample of the enzyme determined to be homogeneous by S-polyacrylamide gel electrophoresis and has a total protein amount of 5.3.
mg, the total activity was 480 U, and the specific activity was 90 U / mg. Examination of the physicochemical properties of this enzyme obtained as a precautionary measure revealed that it was as described above.
This was consistent with the properties of sorbitol dehydrogenase shown in No. 33482.

The titer of the present enzyme was measured by the following method, and the amount of the enzyme that produced 1 μmol of NADH per minute was defined as 1 U. (Reagent preparation); 1 solution; Substrate solution Dissolve 20 g of D-sorbitol in distilled water and add 100 ml
And Liquid 2: 86 mg of substrate solution NAD ⁺ is dissolved in distilled water to make 4 ml. Solution 3: buffer solution After dissolving 6.05 g of trisaminomethane in distilled water,
The pH is adjusted to 9.0 with 4N hydrochloric acid, and then adjusted to 500 ml. (Measurement procedure); 1) 1 solution 0.5 ml, 2 solutions 0.05 ml, 3 solutions
Mix 2.4 ml and preincubate for 5 minutes at 37 ° C. 2) the solution pre-incubated for 5 minutes;
0.05 ml of the enzyme solution adjusted to 1-0.8 U / ml is mixed, and the amount of increase in sample absorbance per minute at 340 nm at 37 ° C. is measured. 3) Measurement of blank value is 0.5m of distilled water instead of 1 solution
1 was added, 0.05 ml of solution 2 and 2.4 ml of solution 3 were mixed and pre-incubated at 37 ° C. for 5 minutes,
0.05 ml of enzyme solution adjusted to 0.1-0.8 U / ml
And measure the increase in blank absorbance per minute at 340 nm at 37 ° C. (Calculation of titer); The titer was calculated by the following formula.

[0025]

(Equation 1)

Reference Example 2 (Preparation of NADH Kinase) In the same manner as described in Examples of JP-A-4-252182, Pichia membrane branifaciens (Pichia) was used.
Membrana efaciens) YS27 (Microtechnical Laboratory No. 3208) in a 500 ml Sakaguchi flask,
Glucose 2%, yeast extract 1%, polypeptone 1%,
Monopotassium hydrogen phosphate 0.9%, ammonium sulfate 0.
The cells were inoculated into 50 ml of medium A (pH 5.5) having a composition of 6%, 0.05% calcium chloride, and 0.05% magnesium sulfate, and cultured at 30 ° C. for 24 hours with shaking. The seed culture is inoculated into 20 l of medium A, cultured at 30 ° C. for 18 hours in a 30 l jar fermenter under aeration rate of 20 l / min and stirring speed of 300 rpm, and the culture is collected by centrifugation. As a result, 1406 g of cells were obtained. 0.5% glucose, 1% yeast extract, 1% polypeptone
%, Monopotassium hydrogen phosphate 0.9%, ammonium sulfate 0.6%, calcium chloride 0.05%, magnesium sulfate 0.05%, sodium succinate 2%, inoculate 20 l of medium B (pH 5.5) Then, with a jar fermenter of 30 l, aeration amount 20 l / min, stirring speed 300 rpm
After culturing at 30 ° C. for 6 hours under the conditions of m, the culture was collected by centrifugation to obtain 1,428 g of cells. This whole amount,
0.1 M saccharose, 0.5% Triton X-100,
Disperse in 50 mM phosphate buffer (pH 6.0) to make a total volume of 5 l, and add this to Dynomill (WA, Switzerland)
(Company B) to crush the glass beads. 5280 ml of the collected crushed liquid was subjected to centrifugation to remove the precipitate, and then subjected to buffer exchange using an ultrafiltration membrane (fraction molecular weight: 6,000) to give 0.05 M sodium chloride, 10 m
M phosphate buffer (pH 6.0). Next, 5260 ml of the enzyme solution was passed through a CM-Sephadex C-50 column (Pharmacia) pre-buffered with 0.05 M sodium chloride and 10 mM phosphate buffer (pH 6.0) to be adsorbed. After washing with 1 M sodium chloride and 10 mM phosphate buffer (pH 6.0), elution was performed with a concentration gradient of 0.1 to 0.4 M sodium chloride, and the active fraction was collected. 455 ml of the eluate was subjected to buffer exchange using an ultrafiltration membrane (fraction molecular weight: 6,000), and 10% ammonium sulfate, 5 mM Mg
Cl ₂ , 10 mM HEPPS buffer (pH 7.
5) As a solution, the solution was passed through a phenyl-Toyopearl 650 column (Tosoh Corporation) previously buffered with the same buffer and adsorbed, and 10% ammonium sulfate, 5 mM MgCl 2
_2. After washing with 10 mM HEPPS buffer (pH 7.5), elution was performed with a concentration gradient of ammonium sulfate concentration of 10% to 0%, and an active fraction was collected. Subsequently, 372 ml of this eluate was concentrated to 25 ml using an ultrafiltration device (fraction molecular weight 10,000) manufactured by Amicon, and 0.2 M ammonium sulfate, 5 mM MgCl ₂ , 10 mM
Gel filtration was performed using a Sephacryl S-300HR column (Pharmacia) buffered with mM HEPPS buffer (pH 7.5). The obtained active fraction was concentrated and lyophilized to obtain 117.3 mg (recovery rate: 34%) of this enzyme preparation. The specific activity of this sample is 102 mU / mg
Met. Examination of the physicochemical properties of this enzyme obtained as a precautionary measure revealed that the physicochemical properties were as described above, and were consistent with the properties of NADH kinase disclosed in JP-A-4-252182.

The titer of the present enzyme was measured by the following method, and the amount of the enzyme producing 1 μmol of NADPH per minute at 30 ° C. was defined as 1 U. (Substrate solution); 10 mM NADH 0.2 ml 0.5 M HEPPS buffer (pH 8.5) 0.1 ml 10 mM ATP 0.3 ml 0.1 M magnesium chloride 0.1 ml 2.0 M sodium acetate 0.1 ml distilled water 0.1 ml (Measurement procedure): Substrate solution containing NADH of the above composition
9 ml was preheated to 30 ° C., and 0.1 ml of the present enzyme solution having an appropriate concentration was added thereto, and reacted at 30 ° C. for 20 minutes.
Thereafter, the reaction was stopped by treatment at 100 ° C. for 2 minutes to remove denatured proteins, and then the amount of NADPH or NADP ⁺ produced by the reaction of each substrate was measured as follows. As a control, a mixture in which the reaction was stopped immediately after mixing the substrate solution with the present enzyme was used. That is, 10 mM D-glucose 6-disodium phosphate hydrate (manufactured by Oriental Yeast Co., Ltd.), 50 mM HEPPS buffer (pH 8.0),
0 mM magnesium chloride, 0.1% bovine serum albumin,
2.5 IU / ml glucose 6-phosphate dehydrogenase
Derived from yeast (Oriental Yeast Co., Ltd.), 5 IU / ml
Diaphorase derived from Custridium (Toyobo Co., Ltd.),
1 ml of a color developing solution having a composition of 250 μM 2,6-dichlorophenol indophenol was added and mixed, and the mixture was immediately introduced into a thermostatic cuvette of a Stercer III spectrophotometer manufactured by Guildford and reacted at 30 ° C. At this time, the change in absorbance at 600 nm was measured over time simultaneously with the reaction, and the difference (ΔOD600 nm / min) between the absorbance value after 1 minute and the absorbance value after 2 minutes was obtained as a measured value.
Using a NADPH solution of known concentration in advance, ΔOD60
A calibration curve between 0 nm / min and the NADPH concentration was prepared, and the amount of generated NADPH was calculated from the measured value.

Example 1 (Quantification of D-sorbitol) First, "reaction liquid 1" to "reaction liquid 5" having the following compositions were prepared for the purpose of quantifying a trace amount of D-sorbitol. <Reaction liquid 1 (pH 9.0)> 125 mM TAPS-sodium hydroxide 10 U / ml sorbitol dehydrogenase (derived from Pseudomonas) 0.625 mM β-NAD ⁺ (Oriental Yeast Co., Ltd.) 0.125% bovine serum albumin ( The sorbitol dehydrogenase used was the same as that obtained in Reference Example 1 described above. <Reaction liquid 2 (pH 8.0)> 100 mM TAPS-sodium hydroxide 400 mM ammonium sulfate 400 mM sodium acetate 6 mM ATP (manufactured by Oriental Yeast Co., Ltd.) 20 mM magnesium sulfate 0.1% bovine serum albumin (manufactured by Sigma) 20 mU / ml NADH kinase (Derived from Pichia membrane branifaciens) The NADH kinase used was the same as that in Reference Example 2 described above. <Reaction liquid 3 (pH 7.0)> 100 mM HEPES-sodium hydroxide 500 mM EDTA · 2Na <Reaction liquid 4 (pH 7.0)> 100 mM HEPES-sodium hydroxide 20 mM D-glucose 6-phosphate disodium hydrate ( 10 U / ml glucose 6-phosphate dehydrogenase yeast-derived (manufactured by Oriental Yeast Co., Ltd.) 0.2 mM 1-methoxy PMS (manufactured by Dojindo Laboratories) 0.2% bovine serum albumin (Sigma) <Reaction liquid 5 (pH 7.0)> 100 mM HEPES-sodium hydroxide 0.5 mM WST-4 (manufactured by Dojindo Laboratories) 0.5% Triton X-100

In 0.4 ml of "reaction liquid 1", add each concentration (0 to 0)
0.1 μl of D-sorbitol (50 μM) was added.
Incubation was performed at 7 ° C. for 20 minutes. To this reaction solution, 0.5 ml of “reaction solution 2” was added and mixed, and 37
Incubation was performed at 20 ° C. for 20 minutes. afterwards,
The reaction was stopped by adding 0.1 ml of "reaction liquid 3".
After stopping the reaction, 0.5 ml of “reaction solution 4” and 0.4 ml of “reaction solution 5” were added and mixed, respectively, and immediately introduced into a constant temperature cuvette of a Hitachi U-2000A type spectrophotometer, at 37 ° C. At the same time, the change in absorbance at 550 nm for 6 minutes from 4 minutes to 10 minutes after the start of measurement was measured. The reagent blank value was a value when distilled water was used instead of the D-sorbitol solution as a substrate.

The results are as shown in FIG.
A linear relationship (r) showing an extremely high correlation between the sorbitol concentration (x) and the value (y) of ΔOD550 nm / 6 min.
² = 0.999, y = 1.05 × 10 ⁻³ +4.71
× 10 ^-3 x) was obtained. From this, according to the present invention, a trace amount of D-sorbitol in a sample can be obtained by a simple operation.
It can be seen that quantification can be performed quickly and with high sensitivity.

Example 2 (Quantitative determination of galactitol) First, a "reaction solution 6" having the following composition was prepared in order to quantify a trace amount of galactitol. Further, “reaction liquid 2”, “reaction liquid 3”, “reaction liquid 4”, and “reaction liquid 5” used below are used.
Were prepared in the same manner as described in Example 1. <Reaction liquid 6 (pH 9.0)> 125 mM TAPS-sodium hydroxide 20 U / ml sorbitol dehydrogenase (derived from Pseudomonas sp.) 0.625 mM β-NAD ⁺ (manufactured by Oriental Yeast Co., Ltd.) 0.125% bovine serum albumin ( The sorbitol dehydrogenase used was the same as that obtained in Reference Example 1 described above.

To 0.4 ml of the above "reaction solution 6", 0.1 ml of galactitol of each concentration (0 to 40 .mu.M) was added and incubated at 37.degree. C. for 20 minutes.
To this reaction solution, 0.5 ml of “reaction solution 2” was added, mixed, and incubated at 37 ° C. for 20 minutes. Thereafter, 0.1 ml of "reaction liquid 3" was added to stop the reaction. After stopping the reaction, 0.5 ml of “reaction solution 4” and 0.4 ml of “reaction solution 5” were added and mixed, respectively, and immediately introduced into a constant temperature cuvette of a Hitachi U-2000A type spectrophotometer, at 37 ° C. At the same time, the change in absorbance at 550 nm for 6 minutes from 4 minutes to 10 minutes after the start of measurement was measured. The reagent blank value was a value when distilled water was used instead of the galactitol solution as the substrate.

The results are as shown in FIG. 2 and show a linear relationship (r) showing a very high correlation between the galactitol concentration (x) and the value (y) of ΔOD 550 nm / 6 min.
² = 0.999, y = −6 × 10 ⁻⁴ + 7.01 × 1
0 ^-3 x) was obtained. From this, according to the present invention,
It can be seen that a trace amount of galactitol in the sample can be quantified quickly and with high sensitivity by a simple operation.

Example 3 (Quantification of D-sorbitol in the presence of xylitol) The following experiment was carried out for the purpose of quantifying a small amount of D-sorbitol in the presence of xylitol. The “reaction liquid 1” to “reaction liquid 5” used below were prepared in the same manner as described in Example 1. "Reaction liquid 1" 0.4 m
1 to each concentration (0 to 5) containing 100 μM xylitol.
0.1 μM) of D-sorbitol solution,
Incubation was performed at 37 ° C. for 20 minutes. To this reaction solution, 0.5 ml of “reaction solution 2” was added and mixed to obtain 3
Incubation was performed at 7 ° C. for 20 minutes. Thereafter, 0.1 ml of "reaction liquid 3" was added to stop the reaction. After the reaction was stopped, 0.5 ml of "reaction solution 4" and 0.4 ml of "reaction solution 5" were added and mixed, respectively, and immediately introduced into a constant temperature cuvette of a Hitachi U-2000A spectrophotometer, at 37 ° C. At the same time, and the change in absorbance for 6 minutes from 4 minutes to 10 minutes after the start of measurement at 550 nm was measured. The reagent blank value was a value when distilled water was used instead of the substrate solution. Further, the amount of change in absorbance was measured in the same manner as described above, using D-sorbitol solutions at various concentrations (0 to 50 μM) in which xylitol was not present. The results are shown in FIG. In FIG. 3,
Solid circles (●-●) indicate the calibration curve of D-sorbitol concentration in the presence of 100 μM xylitol, and white circles (○-○) indicate the D-sorbitol concentration in the absence of 100 μM xylitol.
-Means a calibration curve of sorbitol concentration, respectively.

The results are as shown in FIG.
The difference between the D-sorbitol concentration in the presence of 0 μM xylitol and ΔOD 550 nm / 6 minutes is D-sorbitol.
A linear relationship (r ² = 1.000, y = 1.07 × 10 ⁻² +4) showing an extremely high correlation between the sorbitol concentration (x) and the value (y) of ΔOD 550 nm / 6 min.
87 × 10 ⁻³ x) was obtained. Further, the calibration curve of the D-sorbitol concentration when xylitol is present and the calibration curve of the D-sorbitol concentration without xylitol are extremely similar. From these facts, according to the present invention, Pseudomonas s having the above-mentioned physicochemical properties as a dehydrogenase that reacts with D-sorbitol and / or galactitol.
p. By using a dehydrogenase prepared from cells obtained by culturing KS-E1806 (FERM P-14299) in a medium, a small amount of D-sorbitol in a sample is hardly affected by xylitol. With simple operation,
It can be seen that quantification can be performed quickly and with high sensitivity.

[0036]

According to the method for quantifying D-sorbitol and / or galactitol of the present invention, the method for sensitizing D-sorbitol and / or galactitol is more sensitive than the conventional method for quantifying D-sorbitol and / or galactitol which has many problems in the detection method. In the visible part, quantification can be performed quickly and with high sensitivity by a simple operation using a general spectrophotometer, and it can be applied to automatic analysis. Furthermore, even when xylitol is present in the solution to be measured, a trace amount of the target substance in the sample can be accurately quantified without being substantially affected by the effect.
Therefore, trace amounts of D-sorbitol and galactitol contained in tissues, body fluids, foods, and the like that serve as markers for disease states in the diagnosis of diabetes, galactosuria, and the like can be quantified with high sensitivity. Is extremely significant in the field of industry.

[Brief description of the drawings]

FIG. 1 shows a calibration curve of the relationship between D-sorbitol concentration and ΔOD 550 nm / 6 minutes in Example 1.

FIG. 2 shows galactitol concentration and Δ in Example 2.
4 shows a calibration curve relating to OD of 550 nm / 6 minutes.

FIG. 3 shows D in the presence and absence of xylitol during the measurement of D-sorbitol concentration in Example 3.
-Shows a calibration curve of the relationship between sorbitol concentration and ΔOD 550 nm / 6 min.

Claims (1)

[Claims] 1. A sample containing D-sorbitol and / or galactitol is reacted with a dehydrogenase that reacts with D-sorbitol and / or galactitol in the presence of NAD ⁺ , and the resulting NADH is reacted with NAD ⁺ Phosphorylation using NADH kinase that acts little on NADH and specifically acts on NADH to generate NADPH, an enzyme or an electron carrier that oxidizes the generated NADPH to NADP ⁺ and reduces the NADP ⁺ to NADPH A cycling reaction using an enzyme or an electron carrier, and quantifying D-sorbitol and / or galactitol by detecting a change in a substrate consumed or a product produced in the cycling reaction. A sensitive method for the determination of sorbitol and / or galactitol.