JP2001017198A - Measurement of homocycteine and reagent composition for measuring homocysteine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply and accurately measure the subject compound by making a transferase using homocysteine as one of substrates and another substrate act on a sample and analyzing a produced compound without using a chromatography or an immune reaction. SOLUTION: A transferase (e.g. betaine-homocysteine methyltransferase) using homocysteine as one of substrates and another kind of substrate (e.g. betaine) are made to act on a sample to obtain a compound produced by a cransferase reaction (e.g. dimethylglycine). The resultant dimethylglycine is then reacted with dimethylglycine oxidase or the dimethylglycine oxidase and sarcisube oxidase without using a chromatography or an antigen-antibody reaction to analyze the product. Thereby, the objective homocysteine attracting attention as a risk factor to heart diseases is simply and accurately measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for enzymatic determination of homocysteine and a reagent composition used therefor.

[0002]

2. Description of the Related Art Homocysteine is a metabolic intermediate amino acid in the metabolism of a sulfur-containing amino acid, methionine, to cysteine in a living body, and usually exists in a low concentration in the living body.
However, this metabolic pathway abnormality is directly linked to an increase in homocysteine level, and is considered to be one of clinically useful amino acids because it can serve as an indicator for various diseases. For example, homocystinuria, a congenital metabolic disorder, is an amino acid metabolic disorder resulting from a deletion in cystathionine β synthetase or methyltetrahydrofolate methyltransferase. More recently, elevated blood homocysteine levels have been linked to the development of atherosclerosis, and it is believed that elevated blood levels are associated with risk factors for heart or vascular disease. It has also been reported as an indicator for diabetes, alcoholism, liver and kidney diseases, neurological diseases and the like.

A widely known method for measuring homocysteine is chromatography.
For example, after reacting homocysteine with adenosine in the presence of S-adenosylhomocysteine hydrolase, the product, S-adenosylmethionine, is subjected to HPLC.
(Totani et al., Biochem. Soc.,
14 (6), 11712 (1988) and Shimizu et al., Biotchnol.
Appl. Biochem., 8, 153 (1986)). There is also a method of reacting homocysteine with a fluorophore and performing HPLC-fluorescence analysis (Refsum et al., Clin. Chem., 35 (9), 1921 (1981).
9)). Furthermore, homocysteine N ⁵ - methyl tetrahydrofolate presence, N ⁵ - methyl tetrahydrofolate - after conversion to methionine by the action of homocysteine methyltransferase, is reacted with a fluorophore, HPLC, a method of fluorescence analysis reported (Garras et al., Anal. Bioche
m., 199, 112 (1991)).

[0004] Japanese Patent Application Publication No. Hei 8-506478 discloses S-
A non-chromatographic quantification method using adenosyl homocysteine hydrolase has been described. In this method, homocysteine is reacted with adenosine, fluorescein-labeled S-adenosylhomocysteine, and S-adenosylhomocysteine hydrolase, and then S-adenosylhomocysteine present in the reaction solution is replaced with anti-S-adenosyl. The method using S-adenosylhomocysteine hydrolase is described in detail focusing on the method of capturing with a homocysteine antibody and performing a fluorescence polarization immunoassay.

[0005] International Publication WO98 / 077872 states that
A method is described in which homocysteine is allowed to act on homocysteine desulfurase, and the generated ammonia, α-ketobutyric acid, or hydrogen sulfide is used for measurement.

[0006]

As described above, various methods for measuring homocysteine have been reported, but none of them are satisfactory. Assay methods utilizing chromatography generally require sophisticated equipment. In addition, this method is expensive and time-consuming.
It is also known that the immunoassay method is complicated and time-consuming as compared with general biochemical reactions. Further, since homocysteine desulfurase acts on cysteine, O-acetylserine and methionine in addition to homocysteine, it is difficult to accurately quantify homocysteine.

[0007]

Means for Solving the Problems In view of the above circumstances, the present inventors have conducted intensive studies, and as a result, have been made to react a transferase using homocysteine as one of the substrates and another substrate of the enzyme, The present inventors have found that accurate measurement of homocysteine can be realized by analyzing a compound produced by an enzyme reaction without using chromatography or an antigen-antibody reaction, thereby completing the present invention.

That is, it has the following configuration. (1) A sample is reacted with a transferase having homocysteine as one of the substrates and another substrate of the enzyme, and the compound produced by the transferase reaction is reacted without using chromatography or antigen-antibody reaction. A method for measuring homocysteine, which comprises analyzing. (2) The method for measuring homocysteine according to (1), wherein the transferase using homocysteine as a substrate is betaine-homocysteine methyltransferase or homocysteine methyltransferase. (3) The method for measuring homocysteine according to (2), wherein the transferase using homocysteine as a substrate is betaine-homocysteine methyltransferase. (4) The method for measuring homocysteine according to any one of (1) to (3), wherein the compound produced by the transferase reaction is methionine. (5) The method for measuring homocysteine according to any one of (1) to (3), wherein the compound produced by the transferase reaction is dimethylglycine. (6) The method for measuring homocysteine according to (5), wherein dimethylglycine oxidase is allowed to act on dimethylglycine produced by the transferase reaction, and the product is analyzed. (7) dimethylglycine oxidase and sarcosine oxidase act on the produced dimethylglycine,
The method for measuring homocysteine according to (5), wherein the product is analyzed. (8) A reagent composition for measuring homocysteine, which comprises betaine-homocysteine methyltransferase or a homocysteine methyltransferase and a substrate of the transferase other than homocysteine. (9) The reagent composition for measuring homocysteine according to (8), which comprises a reagent for measuring methionine. (10) Contains a reagent for measuring dimethylglycine (8)
4. A reagent composition for measuring homocysteine according to claim 1. (11) The reagent composition for measuring homocysteine according to (8), comprising dimethylglycine oxidase.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The method for measuring homocysteine according to the present invention comprises the steps of: transferring a sample to a transferase using homocysteine as one of the substrates; Is analyzed without using chromatography or antigen-antibody reaction.

As an example of the transferase using homocysteine as a substrate used in the present invention, betaine-homocysteine methyltransferase (EC 2.1.1.
5), homocysteine methyltransferase (EC 2.1.
1.10) and N ⁵ - methyl tetrahydrofolate - homocysteine methyltransferase (EC 2.1.1.13)
Can be mentioned. Preferably, it is betaine-homocysteine methyltransferase.

Betaine-homocysteine methyltransferase is an enzyme that catalyzes the following reaction. Betaine + L-
Homocysteine → dimethylglycine + L-methionine

Betaine-homocysteine methyltransferase can be collected from mammals and bacteria of the genus Pseudomonas, such as those produced by genetically modified microorganisms in which these genes have been incorporated into other microorganisms. It may be. It also includes those genetically modified in nature. When using betaine-homocysteine methyltransferase, betaine is used as another substrate, which is commercially available at low cost in the form of hydrochloride. Dimethylglycine and methionine, which are products of the above enzyme reaction, can be measured by the following method.

Dimethylglycine is decomposed by dimethylglycine oxidase (EC 1.5.3.30) into sarcosine, formaldehyde and hydrogen peroxide, and the process is described as oxygen consumption, hydrogen peroxide sensor, or direct and peroxidase presence. Below, measurement using a redox indicator is possible. Formaldehyde produced is Ha
Ultraviolet measurement or visible part measurement is possible with nz reagent and NAD ⁺ -dependent formaldehyde dehydrogenase. Furthermore, sarcosine oxidase (EC 1.5.3.1)
Is additionally decomposed into glycine, formaldehyde, and hydrogen peroxide, and can be measured by doubling with sensitivity. Furthermore, sensitivity can be further improved by formaldehyde oxidase. Dimethylglycine oxidase was obtained from mammals, genus Cylindrocarpon and Achromobacter, and formaldehyde oxidase was obtained from cylindrocapon.
Although it can be obtained from the genus carpon) or the like, it may be produced from a genetically modified microorganism in which these are incorporated into other microorganisms, or may be those genetically modified in properties. Further, those having genetically modified properties may be used. Sarcosine oxidase and formaldehyde dehydrogenase are commercially available. In addition, dimethylglycine dehydrogenase (EC
1.5.9.2) decomposes dimethylglycine into sarcosine and formaldehyde in the presence of an electron acceptor.
The process can be measured using a redox indicator or by measuring formaldehyde. The use of sarcosine dehydrogenase (EC 1.5.99.1) doubles the sensitivity. Dimethylglycine dehydrogenase is from higher animals and sarcosine dehydrogenase is from Pseudomonas
Although they can be obtained from the genus nas), they may be those produced from genetically modified microorganisms in which these genes are incorporated into other microorganisms, or those genetically modified in their properties.

On the other hand, methionine is decomposed into corresponding keto acids, ammonia and hydrogen peroxide by methionine racemase and D-amino acid oxidase. Ammonia can be measured by a known colorimetric technique such as the indophenol method, and hydrogen peroxide can be measured by the method described above. Methionine racemase is Pseudomo
nas) and Streptococcus bacteria, and D-amino acid oxidase is commercially available.

[0015] Homocysteine methyltransferase is an enzyme that catalyzes the following reaction. L-homocysteine + S-adenosylmethionine → L-
Methionine + S-adenosylhomocysteine

Homocysteine methyltransferase can be collected from mammals, plants, and yeasts (Saccharomyces), and is produced from a recombinant microorganism in which these genes have been incorporated into another microorganism. Is also good. It also includes those genetically modified in nature.
When homocysteine methyltransferase is used, S-adenosylmethionine is used as another substrate, and a commercially available product is available. Methionine, a product of the above enzyme reaction, can be measured by the method described above.

N ⁵ -methyltetrahydrofolate-homocysteine methyltransferase is an enzyme that catalyzes the following reaction. L-homocysteine + N ⁵ -methyltetrahydrofolate → L-methionine + tetrahydrofolate

N ⁵ -methyltetrahydrofolate-homocysteine methyltransferase can be collected from mammals and Escherichia coli, and is produced from a recombinant microorganism in which these genes have been incorporated into other microorganisms. You may. It also includes those genetically modified in nature.
When this enzyme is used, N5-
Methyltetrahydrofolic acid is used, which is commercially available. The product methionine can be measured by the method described above, and tetrahydrofolate can be measured in the ultraviolet or visible region by NADP ⁺ -dependent tetrahydrofolate dehydrogenase. Incidentally, N ⁵ - methyl tetrahydrofolate - homocysteine methyltransferase higher animals, can be harvested from a plant.

When the sample to be measured is a biological sample, in particular, plasma or urine, it is also present as free homocysteine, but most of it is circulating protein such as albumin or thiol compound such as cysteine by disulfide bond and disulfide bond. It exists as a body. When the measurement target is total homocysteine, the sample is pretreated with a reducing agent,
It must be converted to free homocysteine.

Examples of the reducing agent used in this case include thiols, borohydrides, amalgam, phosphine and phosphothioate. Specific examples of thiols include borohydrides such as dithiothreitol, dithioerythritol, 2-mercaptoethanol, 2-mercaptomethylamine, cysteine, cystamine, cysteine thioglycolate, thioglycolic acid, reduced glutathione, and the like. And sodium amalgam as amalgam.

In the method for measuring homocysteine according to the present invention, a transferase using homocysteine as one of the substrates is used in the presence of a reducing agent which is a pretreatment solution, or the reducing agent is used before the enzyme is used. Is preferred. In many cases of the former, the transferase is unstable at a low pH at which the reducing agent is stable, for example, at pH 3, and therefore it is more preferable to store it in two reagent solutions until just before use. In this case, the reducing agent is stored in an acidic pH solution having a low interference ability, and the transferase is stored in a neutral solution having a sufficient buffering ability, and is mixed and used during sample processing. Of course, it is desirable to use a transferase that can be stored and used at an acidic pH where the reducing agent is stable.

The reagent for measuring a compound produced by the action of a transferase using homocysteine as one of the substrates is preferably stored together with the transferase or separated and stored, if necessary. If the measuring reagent is based on oxygen consumption, a hydrogen peroxide sensor, or the ultraviolet measurement of a dehydrogenase reaction, it can generally be used simultaneously with the reducing agent, but the measuring reagent can be used directly or in the presence of peroxidase. In the case of measurement using a reducing indicator or visible part measurement using a dehydrogenase, it is used separately from the reducing agent.

Buffers used for reagents containing a transferase using homocysteine as one of the substrates and reagents for measuring the products thereof include Tris-HCl buffer, phosphate buffer, borate buffer, GOOD Buffers and the like can be mentioned.
Tris-hydrochloric acid buffer and phosphate buffer vary easily with concentration and temperature, but have the advantage of being inexpensive. On the other hand, GO
MES, Bis-Tris, AD
A, PIPES, ACES, BES, MOPS, TE
S, HEPES, Tricine, Bicine, PO
PSO, TAPS, CHES, CAPS and the like are exemplified. Although expensive, they are particularly useful for liquid diagnostics. The pH of these buffers is preferably between 5.0 and 9.
It is adjusted according to the purpose of use in the range of 0.

Metal salts, proteins, amino acids, sugars, organic acids, surfactants and the like can be used as stabilizers for the enzymes contained in the above-mentioned test solution. Desirable proteins do not affect the enzyme activity, and include bovine serum albumin (BSA), egg albumin, gelatin and the like. As amino acids, glycylglycine, polylysine, and the like can be used in addition to common amino acids such as lysine, histidine, and glutamic acid, and those having high water solubility are desirable. As the saccharide, any of monosaccharide, disaccharide, oligosaccharide and polysaccharide can be used. Further, these derivatives can also be used. Specifically, glucose, fructose, galactose, mannose, xylose, sucrose, lactose, maltose, trehalose, maltotriose, maltosyl cyclodextrin, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, dextrin, Examples include amylose, glycogen, starch, inulin, glucosamine, inositol, mannitol, sorbitol, ribitol, deoxyglucose and the like. Examples of the organic acid include α-ketoglutaric acid, malic acid, fumaric acid, gluconic acid, cholic acid and deoxycholic acid. Nonionic surfactants are preferred. In addition, boric acid, borax, potassium chloride, sodium chloride, ammonium sulfate, glycerol, ficoll, EDTA, and EGTA can be used.

In a reagent solution containing a transferase using homocysteine as one of the substrates and a solution used as a reagent for measuring the product, a preservative, a surfactant and the like are particularly adversely affected on the performance of the reagent. You may add in the range which does not exist. Examples of preservatives include sodium azide, chelating agents, antibiotics, antibacterial agents, fungicides, and the like. Examples of the surfactant include a nonionic surfactant, a cationic surfactant, an anionic surfactant, an amphoteric surfactant, and the like. Triton X-100, CHAPS, potassium cholate and the like are preferable.

[0026]

The present invention will be described below in more detail with reference to examples.

Example 1 Obtaining dimethylglycine oxidase The activity of dimethylglycine oxidase was measured by the following measurement method. <Reagent> 50 mM Tris-HCl buffer (pH 7.5), 100
mM dimethylglycine, 0.5 mM 4-aminoantipyrine, 2 mM phenol, 0.05% Triton X-100, 5 U / ml peroxidase (manufactured by Toyobo)

<Measurement conditions> 3 ml of the above mixture was measured at 37 ° C.
After pre-warming for about 5 minutes, add 0.1 ml of enzyme solution,
After gentle mixing, water is used as a control and recorded for 5 minutes using a spectrophotometer controlled at 37 ° C., and the change in absorbance at 500 nm per minute is measured from the linear portion. In the blind test, distilled water is added to the reagent mixture instead of the enzyme solution, and the absorbance change is measured in the same manner. Under the above conditions, the amount of enzyme that produces 1 micromol of hydrogen peroxide per minute is 1 unit (U).
And

3% dimethylglycine, polypeptone
6%, yeast extract 0.2%, potassium phosphate 0.55%,
1.4% dipotassium hydrogen phosphate, magnesium sulfate 0.
60 ml of a medium (pH 7.0) containing 1% was transferred to a 500 ml Sakaguchi flask, and autoclaved at 121 ° C. for 15 minutes. As an inoculum, Arthrobacter globiformis IFO1
One platinum loop of the 2137 strain was inoculated and cultured at 30 ° C. for 24 hours to obtain a seed culture solution. Next, 6 L of the same medium was transferred to a 10 L jar fermenter, subjected to autoclaving at 121 ° C. for 15 minutes, allowed to cool, and transferred with 60 ml of a seed culture solution.
The cells were cultured at 00 rpm, aeration at 2 L / min, and 30 ° C. for 48 hours. The culture is collected by centrifugation, and 50 mM Tris-
The suspension was suspended in a hydrochloric acid buffer (pH 7.5). The mixture was treated with a French press and centrifuged to obtain a supernatant (enzyme solution).
The resulting enzyme solution was purified to a specific activity of 12 U / mg by ammonium sulfate fractionation, DEAE-Sepharose chromatography, phenyl Sepharose chromatography, and gel filtration using Sephadex G-200.

Example 2 Obtaining betaine-homocysteine methyltransferase The activity of betaine-homocysteine methyltransferase was measured by the following measurement method. <Reagent> 50 mM Tors-HCl buffer (pH 7.5), 100
mM betaine, 20 mM L-homocysteine, 0.
5 mM 4-aminoantipyrine, 2 mM phenol, 0.05% Triton X-100, 10 U / ml
Dimethylglycine oxidase, 5U / ml peroxidase (Toyobo)

<Measurement conditions> 3 ml of the above mixture was measured at 37 ° C.
After pre-warming for about 5 minutes, add 0.1 ml of enzyme solution,
After gentle mixing, water is recorded as a control with a spectrophotometer controlled at 37 ° C. for 5 minutes, and the change in absorbance at 500 nm per minute is measured from the linear portion. In the blind test, distilled water is added to the reagent mixture instead of the enzyme solution, and the absorbance change is measured in the same manner. The amount of the enzyme that produces 1 micromol of hydrogen peroxide per minute under the above conditions is defined as 1 unit (U).

The pig liver was homogenized in ice-cold 50 mM phosphate buffer (pH 7.5) containing 0.1 M NaCl, centrifuged, and then ultracentrifuged to obtain a supernatant (enzyme solution). Was. The obtained enzyme solution was subjected to ammonium sulfate fractionation, DE
It was purified to a specific activity of 0.1 U / mg by AE-Sepharose chromatography.

Example 3 Measurement of homocysteine standard sample 50 μl of the homocysteine standard solution was applied to 1 U / ml betaine-homocysteine methyltransferase, 10 U / ml dimethylglycine oxidase, 5 U / ml peroxidase, 40 mM betaine, 0.5 mM 4-amino. Antipyrine, 2 mM phenol, 0.05% Triton X-100, 50 mM Tris-HCl buffer (pH
Mix with 2.0 ml of the solution containing 7.5) and add
The reaction was performed at 7 ° C., and the absorbance at 500 nm was measured with a spectrophotometer. The results are as shown in FIG. 1. The amount of homocysteine showed excellent linearity in the range of 0 to 0.5 micromol, and quantification was possible.

[0034]

As described above, the method for measuring homocysteine of the present invention has a higher sensitivity for homocysteine than the conventional method for quantifying homocysteine using chromatography or antigen-antibody reaction. Quantitation has become possible. In particular, the determination of homocysteine in serum has attracted attention as a risk factor for heart disease, and the highly sensitive method for measuring homocysteine of the present invention is expected to be widely used as an inexpensive and simple method.

[Brief description of the drawings]

FIG. 1 is a diagram showing the quantitativeness of a method for measuring homocysteine in the present invention.

Claims (11)

[Claims] 1. A sample is reacted with a transferase having homocysteine as one of its substrates and another substrate of the enzyme, and the compound produced by the transferase reaction is subjected to chromatography or antigen-antibody reaction. A method for measuring homocysteine, characterized in that the analysis is performed without the use of homocysteine. 2. The method for measuring homocysteine according to claim 1, wherein the transferase using homocysteine as one of the substrates is betaine-homocysteine methyltransferase or homocysteine methyltransferase. 3. The method for measuring homocysteine according to claim 2, wherein the transferase using homocysteine as one of the substrates is betaine-homocysteine methyltransferase. 4. The method for measuring homocysteine according to claim 1, wherein the compound produced by the transferase reaction is methionine. 5. The method for measuring homocysteine according to claim 1, wherein the compound produced by the transferase reaction is dimethylglycine. 6. A method comprising reacting dimethylglycine oxidase with dimethylglycine produced by a transferase reaction,
The method for measuring homocysteine according to claim 5, wherein the product is analyzed. 7. The method for measuring homocysteine according to claim 5, wherein the produced dimethylglycine is reacted with dimethylglycine oxidase and sarcosine oxidase, and the product is analyzed. 8. A reagent composition for measuring homocysteine, which comprises betaine-homocysteine methyltransferase or a homocysteine methyltransferase and a substrate of the transferase other than homocysteine. 9. The reagent composition for measuring homocysteine according to claim 8, comprising a reagent for measuring methionine. 10. The reagent composition for measuring homocysteine according to claim 8, comprising a reagent for measuring dimethylglycine. 11. The reagent composition for measuring homocysteine according to claim 8, which comprises dimethylglycine oxidase.