JPS63248397A - Method for determining d-mannose - Google Patents

Abstract

PURPOSE:To simply determine D-mannose useful in diagnosis of deep mycosis and remedy, etc., in high precision, by reacting a sample containing D-mannose with D-mannose isomerase and measuring the resultant D-fructose. CONSTITUTION:A sample containing D-mannose is reacted with D-mannose isomerase to afford D-fructose, which is then reacted with D-fructose dehydrogenase and oxidation type electron acceptor (e.g. potassium ferricyanide) in the presence of oxygen and production amount of 5-keto-D-fructose, reduction type electron acceptor or superoxide anion (O2<->) or decrease amount of the oxidation type electron acceptor or the oxygen in the above-mentioned reaction is measured to determine D-mannose. The decrease amount of the oxidation type electron acceptor can be measured from absorbance of 660nm.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for quantifying D-mannose, and particularly to a method for quantifying D-mannose using an enzymatic reaction and with high precision.

(Prior Art) In nature, D-mannose is contained in the cell walls of plants as a constituent of polysaccharides; and in the cell membranes of bacteria and animal cells as a constituent of lipopolysaccharides and glycoproteins. Although D-mannose rarely exists in a free state in nature, it is found in high concentrations in the serum of patients with deep mycosis, for example. Normally, the concentration of mannose in serum is 0.70±(1,02μ/dl), whereas in patients with C. communis infection, for example, mannose concentration is 2.8
~8.0 mg/a. Therefore, measuring D-mannose in samples such as serum is considered useful for diagnosing various diseases, especially the deep mycoses mentioned above.

As methods for quantifying D-mannose, methods using gas chromatography and methods using enzymes are known. Measurement by gas chromatography requires a complicated operation method and requires a long time for measurement, so that many specimens cannot be processed in a short period of time.

Because it requires special techniques and equipment, it cannot be said to be appropriate as a general clinical testing method. In the measurement method using an enzyme, first, hexokinase is added to five samples to convert D-mannose to D-mannose-6-phosphate, and then mannose phosphate isomerase is applied to convert isomerism to D-fructose-6-phosphate. to become This was treated with glucose phosphate isomerase to produce D-
It is converted into glucose-6-phosphate, and further subjected to the action of glucose-6-phosphate dehydrogenase in the presence of NAD+. Through this dehydrogenation reaction, NAD” becomes NAD
D-mannose can be measured indirectly by measuring the amount of N A D 11.

Such an enzymatic method is easier to operate than a gas chromatography method, and does not require special techniques or equipment, and can be easily quantified with a colorimeter. However, four types of enzymes are required for measurement, and the preparation of each enzyme is complicated. Furthermore, among the enzymes mentioned above, hexokinase is D-
Since it acts on glucose as well as mannose, a preliminary operation is required to remove glucose from the sample in advance.

Since many steps of operations are required in this way, there is also the drawback that the cost required for measurement is high.

(Problem to be solved by the invention) The present invention solves the above-mentioned conventional drawbacks.

The purpose is to accurately extract D-mannose.
The object of the present invention is to provide a simple and inexpensive method for quantitative determination.

(Means for Solving the Problems) The method for quantifying D-mannose of the present invention is characterized by allowing D-mannose isomerase to act on a sample containing D-mannose and measuring the produced D-fructose.

D-fructose produced by the above method of the present invention can be measured by any method. For example, the following two methods may be mentioned. The first method is to add D-fructose to the produced D-fructose.
Fructose dehydrogenase and an oxidized electron acceptor are allowed to act in the presence of oxygen, and the amount of 5-keto D-fructose produced, the amount of reduced electron acceptor produced, the amount of superoxide anion (oz") produced in the reaction,
This method measures the amount of decrease in the oxidized electron acceptor or the amount of oxygen. The second method is to treat the generated D-fructose with D-sorbitol dehydrogenase in the presence of NADH, convert the NADH into NAD'', and measure the amount of decrease in NADII in the reaction system.

D-mannose isomerase used in the method of the present invention may be of any origin as long as it is a D-mannose isomerase classified as EC number 5.3.1.7. For example, Pseudomonas, Xanthomonas,
Examples include D-mannose isomerase produced by microorganisms belonging to the genus Streptomyces and Mycobacterium. D-fructose dehydrogenase has EC number 1
．． Any origin of D-fructose dehydrogenase classified as 1.99.11 can be used. for example.

Examples include D-fructose dehydrogenase produced by acetic acid bacteria. D-sorbitol dehydrogenase is E
Any origin of D-sorbitol dehydrogenase classified as C number 1, 1.1.14 may be used. For example, microorganisms of the genus Bacillus; or sheep,
Examples include D-sorbitol dehydrogenase produced by animals such as rats.

To quantify D-mannose by the method of the present invention, D-
D-mannose isomerase is added to a sample containing mannose, and the enzyme isomerizes D-mannose to D-fructose. One molecule of D-fructose is produced from one molecule of D-mannose. D-mannose is quantified by measuring the D-fructose thus produced. The first method for measuring D-fructose involves adding D-fructose to D-fructose in the presence of oxidized receptors.
By allowing fructose dehydrogenase to act, 5-keto-lowfructose is produced from D-fructose according to the linear equation. At this time, the oxidized electron acceptor is reduced to produce a reduced electron acceptor.

D-711)-su + oxidized electron acceptor + reduced electron acceptor (1) As the electron acceptor, phenazine methosulfate (P
MS), 1-methoxy-5-methylphenazonium methyl sulfate, 9-dimethylaminobenzo-α-
Including phenazoxonium chloride.

Compounds such as potassium ferricyanide are also used. In such a system, the amount of 5-keto-lowfructose produced, the amount of decrease in oxidized electron acceptors, or the amount of reduced electron acceptors produced can be quantified. The above 5-keto D-
Methods for quantifying fructose include a method using paper chromatography or thin layer chromatography; and an enzymatic method using 5-keto D-fructose reductase. Methods for measuring the amount of decrease in oxidized electron acceptors include 1. For example, when potassium ferricyanide is used as the oxidized electron acceptor, its absorption (420 nm
), etc. can be used to measure the degree of decrease in

As a method for measuring the amount of the reduced electron acceptor produced, several methods can be adopted depending on the type of electron acceptor used. For example, among electron acceptors, phenazine methosulfate, 1-methoxy-5-methylphenazonium methylsulfe-1 and 9-dimethylaminobenzo-α-phenazoxonium chloride donate electrons to 0□. is possible, so the reaction of the following formula proceeds by acting with Ot in the system.

Reduced electron acceptor +0□ Oxidized electron acceptor +0□- (2) In such a system, reduced electron acceptor can be determined by measuring the amount of decrease in 0□ or the amount of superoxide anion (Oz-) produced. field, that is, D in equation (1)
- The amount of fructose is determined. The amount of decrease in O2 is, for example, oxygen! It can be quantified by polarity. Known methods for measuring superoxide anion (O□-) include a method of measuring the amount of reduction of cytochrome C; and a method of measuring chemiluminescence due to oxidation of luminol in the absence of an oxidation catalyst. In addition, a reducing agent or a reducing agent and its auxiliary agent are further added to the reaction system of formula (2) containing the above 0□- to convert 02- into hydrogen peroxide, and the hydrogen peroxide is measured. By doing so, 02- may be measured indirectly. Examples of the reducing agent used in the method for converting Oz- into H2O2 include metal salts, metal complexes, and proteins containing metals. For the measurement of H20□, a method using a peroxidase reaction is adopted.

For example, in the presence of peroxidase, the chromogen is oxidized and condensed with ++20□ to form a color, and the absorbance is measured (color method); in the presence of peroxidase, 11□02
p-hydroxyphenylacetic acid, homovanillic acid,
Examples include a method of measuring the fluorescence produced by oxidative condensation of leucodichlorofluorescein, etc. (fluorescence method); a method of measuring the luminescence produced by oxidizing luminol or isoluminol with 820□ in the presence of peroxidase (luminescence method). It will be done.

(Another method for measuring the reduced electron acceptor produced in Formula 11 is to donate electrons from the electron acceptor to a reduced color-forming dye (herein refers to a compound that has the property of developing color when reduced), There is a method to measure the pigment produced.

Reduced electron acceptor + reduced chromogenic dye oxidized electron acceptor ten dyes (including the amount of oxide anion produced and the amount of dye of formula (3) above), the amount of D-fructose by measuring the amount of decrease in oxidized electron acceptor or the amount of decrease in 02,
That is, the amount of D-mannose is measured.

A second method for measuring D-fructose is to measure D-fructose.
A method using sorbitol dehydrogenase is mentioned. According to this method, D-fructose produced from D-mannose by D-mannose isomerase is reacted with D-sorbitol dehydrogenase in the presence of N/IDII as shown in the following formula (4).

D-Sorbitol Ten NAD” (4,) 2 (
As shown in 4), one molecule of D-fructose is reduced to D-
One molecule of sorbitol becomes NAD) I One molecule becomes N
One molecule of AD' is produced. Therefore, by measuring the amount of decrease in N A D H in the system of equation (4), D-
Fructose, and thus indirectly D-mannose, is quantified. Since NADll itself has absorption at 340 nm, its abundance can be determined by measuring its absorbance. Furthermore, highly sensitive measurements are possible by converting NADll to other compounds. For example, phenazine methosulfate, 1-methoxy-5-
Methylphenazonium sulfate acts with a tetrazolium salt in the presence of an electron acceptor such as diaphorase,
There is a method in which the tetrazolium salt is reduced and converted into a formazan dye, and this formazan dye is measured. moreover,
Another method is to measure the fluorescence caused by resazurin.

In both the reaction system for isomerizing D-mannose to D-fructose and the system for measuring the resulting D-fructose, the enzymatic reaction is carried out.

It is usually carried out in a suitable buffer. The pl+ of the reaction solution is usually 4 to 10. Preferably it is in the range of 4.5 to 6.0. The concentration of the enzyme used is not particularly limited and is appropriately set depending on the reaction conditions and measurement time. usually.

D-mannose isomerase is 10-100 OLI/
ml, D-fructose dehydrogenase is 10
~100OU/mf.

and D-sorbitol dehydrogenase is 10-10
It is contained in the reaction solution at a rate of 00 U/mff1. The concentration of the electron acceptor is also not particularly limited, but is preferably 0.1 m
M or higher, and when using diaphorase, 5 usually 0
．． IU/mf or more. N A D II in the measurement system using the above D-sorbitol dehydrogenase
? The H degree may be such that the D-sorbitol dehydrogenase reaction proceeds smoothly and the overall enzymatic reaction is not inhibited, and is preferably 0.1 to 1 mM.

The reaction temperature is also not particularly limited, but is usually in the range of 20 to 40°C. The enzymatic reactions such as the isomerization of D-mannose to D-fructose, the reaction of converting D-fructose to 5-keto-0-fructose by D-fructose dehydrogenase, and the coloring reaction that leads to the production of pigments are the same. may be carried out simultaneously in two reaction systems.

In this way, D
According to the present invention, D-mannose can be easily quantified by isomerizing D-mannose to D-fructose. Two types including enzymes for quantifying D-fructose (p-mannose isomerase and D-fructose dehydrogenase; or D-mannose isomerase and D-sorbitol dehydrogenase)
D-mannose can be quantified using the following enzyme.

It has high accuracy and is easy to operate, so it can be suitably used in various clinical tests.

(Example) The present invention will be explained below using examples.

[Quantification of D-mannose]

D-mannose isomerase (1000/ml)
0.1 ml D-fructo-stecht U kenase (5001J/mff1)
0.1-Potassium ferricyanide? g'lfl (
O, 1M) 0.1mlm) Pine mild liquid (pH 5,0)
' After preheating 0.5 ml of the reaction solution with the above composition at 35°C, add a D-mannose solution (with a concentration of 1 to 10 cm).
0.2 ml was added and the reaction was carried out at the same temperature.

After 20 minutes, 9.5 ml of Few(SOn)s Duvanol solution was added to stop the enzyme reaction, and after standing for 20 minutes, 3.5 ml of distilled water was added and the absorbance at 660 nm was measured using a spectrophotometer. D-mannose concentration and O
D value (value after subtracting the blank; shown as Δ00660)
Figure 1 shows the relationship between From Figure 1, the concentration of D-mannose solution is 10μ/d! It can be seen that there is a linear relationship between the D-mannose concentration and the OD value up to this point, and that D-mannose can be quantified.

(Effects of the Invention) According to the present invention, a method for quantifying D-mannose with high accuracy without requiring complicated operations is provided. Since it does not require special techniques or equipment, it can be used in general clinical tests, and can particularly contribute to the diagnosis and treatment of deep mycoses.

-L- Training 91 households [W group] Figure 1 is a graph showing the relationship between D-mannose concentration and absorbance when D-mannose is determined by the method of the present invention.

that's all

Claims (1)

[Scope of Claims] 1. A method for quantifying D-mannose, which comprises allowing D-mannose isomerase to act on a sample containing D-mannose and measuring the produced D-fructose. 2. Let D-fructose dehydrogenase and an oxidized electron acceptor act on the generated D-fructose in the presence of oxygen, and determine the amount of 5-keto-D-fructose produced and the amount of reduced electron acceptor produced in this reaction. , the amount of superoxide anion (O_2^-) produced, the amount of decrease in the oxidized electron acceptor, or the amount of decrease in oxygen is measured. quantitative method. 3. In the presence of NADH to the generated D-fructose,
2. The method for quantifying D-mannose according to claim 1, which comprises activating D-sorbitol dehydrogenase and measuring the amount of decrease in NADH in the reaction system.