WO1992021775A1

WO1992021775A1 - Highly sensitive determination of d-sorbitol or d-fructose and composition therefor

Info

Publication number: WO1992021775A1
Application number: PCT/JP1991/001788
Authority: WO
Inventors: Shigeru Ueda; Mamoru Takahashi; Hideo Misaki
Original assignee: Asahi Kasei Kogyo Kabushiki Kaisha
Priority date: 1991-05-29
Filing date: 1991-12-27
Publication date: 1992-12-10
Also published as: JP3034986B2; JPH04349897A

Abstract

Determination of D-sorbitol or D-fructose by reacting a specimen solution with a reagent containing 1) sorbitol dehydrogenase, 2) A1, and 3) B1 to form the cycle (I) and determining the amount of A2 formed or the amount of B1 consumed; and a composition containing the ingredients 1), 2), and 3) for use in the above determination method. The method and the composition make it possible to determine a small amount of a specimen solution conveniently with high sensitivity and are useful in the field of clinical inspection and food testing.

Description

Specification

Highly sensitive method and composition for the determination of D-sorbitol or D-fructose

The present invention relates to a highly sensitive method and a composition for quantitatively determining D-sorbitol or D-fructose in the fields of clinical biochemical testing, food testing, and the like.

Background art

In recent years, clinically inhibitors to the polyol metabolic pathway aldose reductase have been administered to patients with diabetic complications and have been effective. At this time, there was a significant correlation between the degree of decrease in the concentration of D-sorbitol in erythrocytes and the degree of improvement in clinical findings, and the concentration of D-sorbitol in peripheral neurons also decreased, indicating that Measurement of D-sorbitol helps to understand the dynamics of D-sorbitol in tissues related to diabetic complications (Japanese clinical study, 48, Diabetes, Vol. 426-431, 1990 extra edition) o

On the other hand, D-sorbitol in erythrocytes is converted to D-fructoses by sorbitol dehydrogenase in erythrocytes and released into serum, and therefore, as in the measurement of D-sorbitol in erythrocytes, Measurement of D-fructose may also be useful in assessing the pathology of diabetic complications.

In fact, it has been reported that serum D-fructose may be elevated due to diabetes (Clinical Studies, 47, 468, extra edition 1989).

The determination of D-sorbitol or D-fructose is also important in food analysis and is commercially available, for example, as a kit from Behringer Mannheim. In addition, D-fructose is known to be involved in kinetic energy in sperm, and is also useful in diagnosing azoospermia.

Gas Chromatography (GLC), High Performance Liquid Chromatography (HPLC), Enzyme, etc. are used as methods for measuring D-sorbitol, but the enzymatic method is the most practical because the former two methods are complicated. ing. In this method, the coenzyme NAD is converted to the reduced form using D sorbitol as a substrate and sorbitol dehydrogenase π-genase. The amount to be measured is measured by absorbance or fluorescence intensity.

D-fructose is measured by glucose-6-phosphate dehydrogenase and coenzyme NAD (P) after converting it to glucose-6-phosphate using hexokinase and phosphoglucose isomerase. However, since hexokinase also acts on Dr "dalcoses, a measurement method using fructokinase specific for D-fructose has been reported (Anal. Biochem., 54, P205, 1973). .

In general, when performing an analysis using an enzyme, the substance to be measured is converted into hydrogen peroxide or reduced NAD (P) that can be detected spectroscopically. At this time, the amount of the detectable substance is stoichiometrically equal to the target. At present, the most widely used method for measuring detectable substances uses spectroscopy instruments, but this method also has a limitation in sensitivity and has the drawback that it cannot be applied when the content of the measured substance is small. .

Therefore, when the content of the object to be measured is small or when the number of analytes is small, fluorescence analysis, luminescence analysis, and the like with higher sensitivity than the above-described spectroscopic analysis are performed. However, these methods are not very suitable for general-purpose examinations such as clinical examinations in terms of dissemination of equipment.

As another method for measuring a trace substance, a so-called enzyme cycling method is known in which, when the substance can be converted into an equal amount of a coenzyme or the like, two kinds of enzymes are used to amplify the coenzyme or the like. For example, there are NAD cycling, CoA cycling, ATP cycling and the like, but these are hardly practical in routine analysis such as clinical tests due to complicated operations.

If it is possible to improve the sensitivity of the above measurement, it is possible to reduce the amount of the sample required for the measurement as well as when the content of the measurement target is small. When used for an analyte, the effect of a coexisting substance on the measurement system can be reduced. In addition, it is possible to increase the number of items that can be tested with a limited amount of sample, and if the sample is human blood, it is possible to reduce the amount of blood to be collected. It can also reduce the psychological burden. As described above, increasing the detection sensitivity requires blood testing in clinical tests. This is an indispensable requirement in view of the necessity of using valuable samples such as liquids and the need to measure trace components.

As described above, the conventional methods for quantifying D-sorbitol or D-fructose are not yet satisfactory, and it has been desired to develop a simple and highly sensitive quantification method. An object of the present invention is to provide a simple method for quantifying D-sorbitol or D-fructose with high sensitivity, high accuracy and high accuracy.

Further, a second object of the present invention is to provide a composition which can be suitably used for the high-sensitivity quantification method of D-sorbitol or D-frug. .

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, one selected from the group consisting of zeo-NADs and zeo-NADPs (hereinafter, also referred to as chi-type NAD (P) s); Solpititol dehydrogenase and non-thio NAD (P), which act on one coenzyme selected from the group consisting of NADPs (^, also referred to as non-thio NAD (P)) By using two types of coenzymes, the absorption wavelengths of the reduced form of the thio-type NAD (P) and the reduced form of the non-thio-type NAD (P) are around 400 nm, respectively, when measuring the absorbance. The present inventors have found that it is possible to carry out an enzyme cycling reaction capable of avoiding congestion at the absorption wavelength of another substance by utilizing the difference from the vicinity of 340 nm, and to perform measurement with high sensitivity, thereby completing the present invention. Here, in carrying out the enzyme cycling reaction, only the change amount of one of the two coenzymes may be separately quantified. Disclosure of the invention

The present invention relates to a subject having at least one test component selected from the group consisting of D-sorbitol and D-fructose, the following components (1) to (3):

(1) One selected from the group consisting of thio-NADPs and thio-NADs and one selected from the group consisting of NADPs and D's as coenzymes, and D-fructoses using at least D-sorbitol as a substrate Sorbitol dehydrogenase in a reversible reaction producing

(2) A, And reacting it with the following reaction formula (I)

Ai

(Wherein represents thio NADPs, thio NADs, NADPs or NADs, A ₂ represents a reduced product of, and represents reduced NADP when A, is a thio NADP or thio NAD. Or reduced NADs, and when A! Is NADPs or NADs, it indicates reduced-thio NADPs or reduced-thio NADs, and B ₂ indicates the oxidized product of B,

The present invention relates to a method for quantifying D-sorbitol or D-fructose, which comprises forming a cycling reaction represented by the following formula, and measuring the amount of A ₂ or A changed by the reaction.

Further, the present invention relates to a composition for quantifying D-sorbitol or D-fructose, which comprises the components (1) to (3).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a drawing showing the results of the rate at 400 nm with respect to the amount of D-sorbitol in Example 1.

FIG. 2 is a drawing showing a result of a late access at 400 nm with respect to the amount of D-fructose in Example 2.

BEST MODE FOR CARRYING OUT THE INVENTION

The sorbitol dehydrogenase used in the present invention comprises at least D-sorbitol + NAD (P) ⁺ = D-fructoses + NAD (P) H + H ⁺

Which catalyzes a reaction selected from the group consisting of thioNADPs and thioNADs and one selected from the group consisting of NADPs and NADs Can also be used.

Specifically, the enzyme EC1.1.1.1 U is mentioned, and Bacillus subtilis (Bacillus subtilis) and in liver and semen of humans, sheep and mice. As a substrate, it is said to act on polyols with D-xylo (D-xylo-) and D-lipo (D-ribobo-) 'structures (Enzyme Handbook, page 5, Asakura Shoten, 1983 _C ) Regarding the enzyme derived from sheep liver marketed by Boehringer Mannheim, L-iditol has an activity of 100% against D-sorbitol.

96%, 85% for xylitol, 49% for ribitol, and 45% for lysitol, erythritol, D.-L-ararabitol, D-iditol, D-mannitol, and inositol Does not work.

In addition, the specificity for coenzymes works not only for NAD but also for NADP with a strength of about 1/10 to 1/100 (Biochomi ca Information, p79 Boenringer Mannheim Biochemicals,

1987).

It is known that enzymes derived from Bacillus subtilis act almost equally to NAD as well as to NAD, thiocetyl NAD, and deamino NAD (J. Biol. Chem., 239, P830- 838, 1964).

Enzymes of other origins can also be used as appropriate. Specificity for coenzymes NAD (P) and thio NAD (P) is reactive with the substrate D-sorbitol and D-fructo. Anything that can produce toose can be used, and its specificity can be confirmed using these coenzymes and substrates.

Further, in the present invention, and coenzyme B ₂ is Chio-NADP, Chio NAD, NADP compound, exhibits NAD, as the out Chio NADP or Chio NAD, e.g. Chio nicotinamidine de adenine dinucleotide Phosphate (thioNADP), chonicotinamide dohypoxanthine dinucleotide phosphate; and chonicotinamide-denine dinucleotide (thioNAD), chonicotinamide-hypoxanthine dinucleotide. Examples of NADPs or NADs include nicotinamide adenine dinucleotide phosphate (NADP), acetylviridine adenine dinucleotide phosphate (acetyl NADP), acetylviridine hypoxanthine dinucleotide phosphate, and nicotinamide hypoxanthine dinucleotide. Leo Tide phosphate (damino NADP); and nicotinamide adodenine dinucleotide (NAD), acetylpyridin adenine dinucleotide (acetyl NAD), acetyl pyridine hypoxanthine dinucleotide, nicotinamide hypoxanthine dinucleotide (damino NAD) Is mentioned. The reduced forms of these coenzymes are indicated as thio NADPHs, thio NADHs, MDPHs, and NADHs, respectively.

In the present invention, for and, for example, when is a NAD (P), it is necessary that is a NAD (P) H, and one zeo-type coenzyme is used in relation to and.

When the sorbitol dehydrogenase used for the quantification uses only (thio) NADs as coenzymes, the sorbitol dehydrogenase used uses only (thio) MDPs as coenzymes. Sorbitol dehydrogenase to be used may be (thio) NADs or

(Cho) When both NADPs are used as a coenzyme, they may be appropriately selected from the above-mentioned Cho-NADs and Cho-NADPs and the above-mentioned NADs and fiADPs.

By using the quantification method of the present invention, D-sorbitol or D-fructose originally contained in the sample can be measured. Substrates and their enzyme activities can also be measured. Furthermore, by using the quantification method of the present invention, the enzyme system comprising a single or a plurality of steps capable of linking with the enzyme system that migrates and produces D-sorbitol or D-fructose is used. Substrates and their enzyme activities can also be measured. These enzyme systems are not particularly limited, but include, for example, the following various reaction systems.

(1) Enzymatic reaction system between D-glucose and glucose reductase (EC 1.1.2.11).

D—Darcose + NAD (P) H → D—Sorbitol + NAD (P)

In this system, the amount of D-sorbitol produced and quantified can be used to determine the amount of D-glucose or to measure the activity of glucose reductase. (2) Enzyme reaction between D-mannose and mannose isomerase (BC 5.3.1.7) ₀ D-mannose → D-fructoose

In this system, quantification of D-mannose or activity of mannose isomerase can be performed by quantifying the amount of D-fructose produced in the chicks.

(3) Enzyme reaction system of D-fructoth-6-phosphate and glucose phosphatase (BC 3.1.3.1).

D—Fructose-6-phosphoric acid + H ₂₀ → D—Fructose + phosphoric acid

In this system, the amount of D-fructose produced and released is determined,

-It can be used for the determination of fructose-6-phosphoric acid or the activity of calcium phosphatase.

(4) Enzymatic reaction system between sucrose and "1-Darcosidase (BC 3.2.1.20) or; 5-fructosidase (BC 3.2.1.26).

Disk - scan + H ₂ 0 → D- Fourques toast + D- glucose

In this system, by determining the amount of D-fructose released and produced, it is possible to determine the amount of sucrose or the activity of α-darcosidase or monofructosidase.

(5) Enzymatic reaction system between raffinose and] 9-fructosidase (BC 3.2.1.26). Rafi North + H ₂ 0 → D- Fourques toast + menu Li Biot Ichisu

In this system, by determining the amount of D-fructose released and produced, it is possible to determine the amount of raffinose or the activity of fructosidase.

(6) Enzymatic reaction system of sucrose and inorganic phosphorus with sucrose phosphorylase (BC 2.4.1.7).

Sucrose + Inorganic Lin-D-Fructose + D-Glucose-1-Linic Acid In this system, the amount of free and formed D-fructose can be quantified to determine sucrose or inorganic phosphorus or sucrose phosphorylation The activity of the enzyme can be measured. (?) When sucrose is derived from an enzymatic reaction system with raffinose and one galactosidase (BC 3, 2.1.22).

Raffinose + H ₂ 0 → galactosyltransferase door - scan + sucrose

By quantifying the D-fructoses finally formed in this system, it is possible to quantify raffinose or measure the activity of galactosidase. In the composition for quantification of the present invention, the concentration of and is preferably from 0.02 to 100 mM, particularly preferably from 0.05 to 20 mM, and the amount of sorbitol dehydrogenase is ^{1 to} 1000 u / m £, especially 2 to 400 u / m. Although m is preferred, the amount can be appropriately determined depending on the type of the subject and the like, and a larger amount can be used. In the present invention, and the amount of ^ used is D-sorbitol or D in the subject.

-It is necessary that the amount is excessive compared to the total amount of fructoses and that the amount of sorbitol dehydrogenase is excessive compared to the _Kra value for each of _Β and ιι. 20 to: L0000-fold molar is preferred.

In addition, the above-mentioned quantification method of the present invention relates to the case where sorbitol dehydrogenase uses both (thio) NADs and (thio) NADPs as coenzymes. When a combination of thio-NADPs and NADs or NADPs is selected, a second dehydrogenase that does not act on D-sorbitol as the component (4) and forms the reaction of by exerting a substrate for said second dehydrogenase further, as described below reaction formula ([pi), and may allowed to form a cycling reaction by allowed to impart reaction system for reproduction of between B _2.

That is, a sample having one or more test components selected from the group consisting of D-sorbitol and D-fructoses is added to the following components (1) to (4)

(1) One selected from the group consisting of thio NADPs and thio NADs and one selected from the group consisting of NADPs and NADs are used as coenzymes, and D-fructose is obtained using at least D-sorbitol as a substrate. Sorbitol aldehyde forming a reversible reaction

T3 genease (2) A,

(3) or Z and B ₂

(4) A second dehydrogenase that does not act on D-sorbitol and forms a reaction of B ₂ → B, and a substrate of the dehydrogenase

The following reaction formula (Π)

D—Sorbit Fructose

(Π)

(Wherein represents thio NADPs, thio NADs, NADPs or NADs, A ₂ represents a reduced product of, and B) represents reduced NADP when thio NADPs or thio NADs Or reduced NADs, or, when NADPs or NADs, indicates reduced thio NADPs or reduced thio NADs, B ₂ indicates an oxidized product of, and indicates B ₂ Shows the oxygen reaction that produces as a coenzyme)

Can form a cycling reaction represented by

In this case, with respect to the second dehydrogenase, it is preferable to set conditions that do not substantially act in this assay system. For example, a second dehydrogenase that does not essentially use A as a coenzyme the combination of selecting, more second dehydrogenase σ Genaze the quantitative relationship of B ₂ are combined, and the like to select the conditions that do not substantially act a, the. When quantitative measures the amount of A ₂ generated by the reaction.

In the composition for quantification using the above component (4), the concentration of is preferably from 0.02 to 100 mM, particularly preferably from 0.05 to 20 mM, and the concentration of B ₂ or Z and ^ is preferably from 0.05 to 5000 juM, particularly Five~ The concentration of sorbitol dehydrogenase is preferably 1 to 1000 u /? ^, Particularly preferably ₂ to 400 uA ^, and the second dehydrogenase is 20 times the Km value (in mM) for B2 (u / mg). The unit may be adjusted so as to be over £ 1, for example, preferably 1 to 100 u / mg, and the substrate of the second dehydrogenase is preferably in excess, for example, 0.05 to 20 mM. These amounts can be appropriately determined depending on the type of the subject and the like, and larger amounts can be used.

That is, the second aldehyde σ-genase is supplementarily added for the regeneration of, and this makes it possible to reduce the amount of, and is particularly effective when is expensive. Further, the reaction may be performed using B ₂ or a mixture of and B ₂ instead of. In this case, the use amounts of Z and B ₂ are not particularly limited, but are generally 1/10 mol £ 1 or less, preferably 1/100 or less. As the second dehydrogenase and its substrate, for example, when B ₂ is a NAD or a thio NAD, alcohol dehydrogenase (EC 1, 1.1.1) and ethanol, L-glycerol-3-phosphate dehydrogenase ( EC 1.1.1.8) (from heron muscle) and L-glycerol-3-phosphate, glycemic acid aldehyde dehydrogenase (Ε C 1.1.1.12) (ゥ heron skeletal muscle, liver, yeast, E. coli) Origin) and D-glyceride aldehyde phosphate and phosphoric acid, glyceryl monohydrogenase (BC 1.1.1.6) (from E. Coli) and glycer D-le, lactate dehydrogenase (EC 1.1. 1.37) (porcine heart muscle, © shea myocardial origin) and L one malic acid, when B ₂ is a NADP or the Chio-NADP, glucose - 6 - phosphate dehydrogenase one peptidase (BC 1.1.1.49) (from yeast) And glucose-6-phosphate, dehydrogenase isocnate (BC 1.1.1.42) (Derived from yeast and porcine myocardium), isogenic acid, glyoxylate dehydrogenase (EC 1.2.1.17) (derived from Pseudomonas oxalat icus_), CoA, dalioxylic acid, and phosphodalconate π-genase (BC 1.1.1.44) (Derived from rat liver, brewer's yeast, E. coli) and 6-phospho-D-dalconic acid, glyceraldehyde aldehyde phosphate dehydrogenase (BC 1.2.1.13) (derived from plant chloroplasts) and D-glycerol Examples thereof include aldehyde-3-phosphoric acid and phosphoric acid.

Further, the quantification method of the present invention is characterized in that the sorbitol dehydrogenase is And both (co) NADPs as coenzymes, the combination of two coenzymes with the combination of thio NADs and NADs or NADPs, or the combination of thio NADPs with ΝΑΪ) or ΝΑDPs When selected, a third dehydrogenase that does not act on D-sorbitol as the component (5) on the subject and forms an As-Ai reaction is used, and the third dehydrogenase is further used. A cycling reaction can be formed by allowing a reaction system for the regeneration of A, as shown in the reaction formula (m) described below, to give a reaction system between A and ₂ as shown in the following reaction formula (m).

That is, a sample containing one or more test components selected from the group consisting of D-sorbitol and D-fructose is added to the following components (1) to (3) and (5)

(1) One selected from the group consisting of thio-NADPs and thio-NADs and one selected from the group consisting of NADPs and DNAs are used as coenzymes, and D-fructose is obtained using at least D-sorbitol as a substrate. Sorbitol aldehyde u ^ j- i-se

(2) or and A ₂

(3) B,

(5) A third dehydrogenase which does not act on D-sorbitol and forms a reaction of A ₂ → A, and a substrate of the dehydrogenase

And reacting it with the following reaction formula (m)

Third dehydrogenase

D—Solirebit—Fructose

(1)

(Wherein represents thio NADPs, thios, NADPs or NADs, A ₂ represents a reduced product of, and represents reduced NADPs when A i is a thio NADP or thio NAD. Or reduced NADs, and when A! Is NADPs or NADs, it indicates reduced thio NADPs or reduced thio NADs, and B ₂ indicates an oxidized product of B, -Ai is an enzyme reaction to produce the Ho酵containing the a ₂₎

Can form a cycling reaction represented by

In this case, with respect to the third dehydrogenase, it is preferable to set conditions that cannot substantially act in this measurement system. For example, a combination of selecting an enzyme that does not essentially use as a coenzyme, and A Due to the quantitative relationship of ₂ , the third dehydrogenase is substantially B! And a combination for selecting a condition that does not affect. When quantifying, measure the consumption of B.

In the composition for quantification of the present invention using this component (5), the concentration of is preferably from 0.02 to 100 mM, particularly preferably from 0.05 to 20 mM, and the concentration of A ₂ or Z and is preferably from 0.05 to 5000 M, The concentration of sorbitol dehydrogenase is preferably 1 to 1000 u / m, particularly preferably ₂ to 400 u / mg, and the third dehydrogenase is 20 times the Km value (in mM) for A2. (U / mg unit) It may be adjusted so as to be more than the above. 0.05 to 20πιΜ is preferred. These amounts can be appropriately determined depending on the type of the subject and the like, and larger amounts can be used.

That is, the tertiary dehydrogenase is supplementarily added for the regeneration of, and this makes it possible to reduce the amount of tertiary dehydrogenase used, and is particularly effective when is expensive. It may also be carried out reaction with a mixture of Alpha ₂ or Alpha! And Alpha ₂ instead of. In this case, although the amounts of または and Α ₂ are not particularly limited, they are generally 1/10 mol or less, preferably 1/100 ^ below. As the third dehydrogenase and its substrate, for example, when is a NAD or a thio NAD, alcohol dehydrogenase (EC 1.1.1.1) is referred to as alcohol dehydrogenase or glycerol dehydrogenase. (EC 1. 1.1.6) (derived from E. coli), dihydroxyacetone, L-glycerol-3-phosphate dehydrogenase (derived from rabbit heron) and dihydroxyacetonitrate, lingo Acid dehydrogenase (EC 1.1.1.17) (derived from porcine myocardium and oak myocardium) and ogiza π-acetic acid, glycealdehyde aldehyde phosphodehydrogenase (EC 1.1.1.1.12) When skeletal muscle, liver, yeast, and B. Coli) and 1,3-diphospho-D-glyceric acid are NADPs or thio-NADPs, glyceroaldehyde dehydrogenase (BC 1.2.1.13) ) (Derived from plant chloroplasts) and 1,3-diphospho-D-glyce Such as phosphate and the like.

Regarding the composition of the reaction solution, two coenzymes are appropriately selected in consideration of the relative activities among the various enzymes of the sorbitol dehydrogenase used, and then the optimal PH conditions for the forward reaction and the reverse reaction are determined by enzyme cycling. What is necessary is just to set so that the ring reaction proceeds efficiently. The enzymes used may be used alone or in combination of two or more.

In order to measure D-sorbitol or D-fructose in a subject by using the composition for quantification of the present invention thus prepared, the components (1) to (3), (1) to ( 4) Alternatively, 0.001 to 0.5 ml of the test sample is added to the composition containing (1) to (3) and (5), and the mixture is reacted at a temperature of about 37 t. few minutes to several tens of minutes between points, e.g., 3 minutes 1 minute after 4 minutes after, or the amount of amount or consumed in a ₂ generated in 3 minutes and after 5 minutes after 8 minutes, The change in absorbance based on each absorption wavelength Measurement. For example, if A ₂ is chio NADH and is NADH, the production of A ₂ is measured by increasing the absorbance around 400 nm or B! Is measured by the decrease in absorbance around 340 nm and compared with 值 when measured using known concentrations of D-sorbitol or D-fludatose, indicating that D-sorbitol or D-fructo in the sample solution The quantity can be obtained in real time.

Further, when D-sorbitol and D-fructose coexist in the subject, the total amount of these is determined by the quantification method of the present invention. The individual component amount in the case of a constant amount, after the pre-processing such as you erase the enzyme that acts only in advance either component of the subject, it Michibike the enzymatic cycling reaction. For example, pretreatment with hex 4: nase (EC 2.7.1.1.1) or fructokinase (EC 2.7.1.1.4) will result in D-fructoses being D-fructoses. -Since it is converted to 6-phosphate, only D-sorbitol can be quantified by subsequently performing the enzyme silicing reaction according to the present invention. Further, by subtracting the amount of D-sorbitol from the total amount of D-sorbitol and D-fructose, a quantitative value of only D-fructose can be calculated.

In addition, the quantification method of the present invention guides D sorbitol or D-fructoses in a sample to an enzyme cycling reaction, and is not easily affected by coexisting substances in the liquid. The blank measurement can be omitted, and a simple measurement by late access can be performed.

In the present invention, in the measurement of A ₂ or, the quantification can be performed by using other known measurement methods instead of the absorbance measurement.

Example

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto.

Example 1 Quantification of D-sorbitol

100 πιΜ Triethanolamine buffer (ρΗ8.5) 4 mM Chio NAD (Sigma)

0.2 m reduced NAD (Oriental Yeast)

500 u / m £ sorbitol-ludelogenase (manufactured by Sigma; derived from hidge liver) <Operation>

Take the reagent particulate base in Tsu Bok, 0, 10, 20, 30 , 40, 50 Μ of D Sol Bitoru solution (Wako Pure Chemical Industries, Ltd.) was 20 i ^ added respectively, _c reactions the reaction was initiated at 37 t The absorbance at 400 nm at 3 minutes and 6 minutes after the start was read to determine the difference. When the time when the concentration was 0 was used as the reagent blank, the value of the reagent blank was subtracted from each result, and the results are shown in FIG. As is clear from FIG. 1, the amount of change in absorbance relative to the amount of D sorbitol showed good linearity.

Example 2 Quantification of D-Fructose

Reagent>

100m triethanol buffer (pH 8.5)

4 m Chio NAD (Sigma)

0.2 m reduced NADP (Oriental Yeast)

625 u / i sorbitol dehydrogenase (manufactured by Sigma; derived from hidge liver)

Reagent 1 was placed in a cuvette, and 0, 20, 40, 60, and 80 D fructoses solutions (manufactured by Wako Pure Chemical Industries, Ltd.) were added at 40 i ^ and the reaction was started at 37 t. . The absorbance at 400 ηπι was read at 2 minutes and 7 minutes after the start of the reaction, and the difference was determined. When the concentration of 0 was used as a reagent blank, the value of the reagent blank was subtracted from each result, and the results are shown in FIG. As is evident from FIG. 2, the amount of change in absorbance with respect to the amount of D fructose showed good linearity.

Industrial applicability

Since the present invention uses coenzymes having different reduced absorption wavelengths, measurement errors do not occur, and the measurement sensitivity can be increased by combining enzyme cycling reactions. As a result, the use of a small amount of sample allows easy and accurate lb

It can quantify D-sorbitol or D-fructose with high sensitivity, and is useful in fields such as clinical biochemical tests and food tests.

Claims

The scope of the claims

1. For a sample containing at least one test component selected from the group consisting of D-sorbitol and D-fructos, the following components (1) to (3)

(1) one selected from the group consisting of thionicotinamide adodenine dinucleotide phosphophosphates (hereinafter referred to as lower NADPs) and thionicotinamide adodenine dinucleotides (hereinafter referred to as thio NADs); and nicotinamide adodenine. A co-enzyme comprising one selected from the group consisting of dinucleotide phosphates (hereinafter referred to as NADPs) and nicotinamide adenine dinucleotides (hereinafter referred to as NADs), and at least D-fructoses using at least D-sorbitol as a substrate Sorbitol dehydrogenase forming a reversible reaction

(2) After reacting the reagent containing A, the following reaction formula (I)

D—Sorbitol D—Fructose

(I)

(Where, A, represents a chio NADP, a chio, a NADP or a NAD, A ₂ represents a reduced product of B, and B! Represents a case where A! Is a chio NADP or a chi NAD. Represents reduced NADPs or reduced NADs, or represents reduced NADPs or NADs when represents NADPs or NADs, and B ₂ represents an oxidized product of B! )

Forming a cycling reaction represented by the formula, and measuring the amount of A ₂ or A changed by the reaction, characterized in that D-sorbitol or D-fructose

2. NADPs are nicotinamide amide adenine dinucleotide phosphate (NADP), acetyl pyridine adenine dinucleotide phosphate (acetyl NADP), acetyl pyridine hypoxanthine dinucleotide phosphate and nicotinamide hypoxanthine dinucleotide 2. The method of claim 1, wherein the method is selected from the group consisting of phosphate (damino NADP).

3. NADs are selected from the group consisting of nicotinamide adenine dinucleotide (NAD), acetyl pyridin adenine dinucleotide (acetyl NAD), acetyl viridine hypoxanthine dinucleotide and nicotinamide hypoxanthine dinucleotide (damino NAD). 2. The method for quantifying D-sorbitol or D-fructose according to claim 1, which is selected.

4. The D-sorbitol according to claim 1, wherein the thioNADPs are selected from the group consisting of thionicotinamide amide adenine dinucleotide phosphate (thioNADP) and thionicotinamide hypoxanthine dinucleotide phosphate. D—Fructose quantitation method.

5. The D-sorbitol or D-fruct according to claim 1, wherein the choNADs are selected from the group consisting of chonicotinamide amide adenine dinucleotide (choNAD) and chonicotinine-mide hypoxanthine dinucleotide. Tooth quantification method.

6. The following components (1) to (3)

(1) One selected from the group consisting of thio-NADPs and thio-NADs and one selected from the group consisting of NADPs and NADs are used as capture enzymes, and D-fructose is obtained using at least D-sorbitol as a substrate. Reversible sorbitol dehydrogenase formed

(2)

(3) B

A composition for the determination of D-sorbitol or D-fructoses, which comprises: