Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2002-02-28
2004-02-03
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S023000, C435S024000, C435S025000, C435S968000, C435S975000
Reexamination Certificate
active
06686172
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for determining total homocysteine.
2. Description of the Prior Art
Homocysteine, which is one of the metabolic intermediates in methionine metabolism, is reported to have vascular endothelial cytotoxicity and to be one of the risk factors for arteriosclerotic diseases independent from the other risk factors. It also has been evident that in addition to serious hyperhomocysteinemia (homocystinuria) caused by deficiency of homocysteine metabolic enzymes, moderate hyperhomocysteinemia is caused by a decrease in the metabolic enzyme activity due to abnormality of genes, renal insufficiency, aging, smoking, lack of exercise or the like (Jacobsen, Clin. Chem. 44:8(B), 1833-1843, 1998). Furthermore, hyperhomocysteinemia is also reported to be improved by taking vitamin B6, folic acid or the like (JAMA 270: 2693-2698, 1993). Therefore, not only for neonatal mass screening, but also for prevention of adult arteriosclerotic diseases or detection of vitamin deficiency diseases, a simple method for treating a large number of specimens is in demand.
Most of homocysteine in blood (99%) is present in the form of oxidized disulfide compounds (such as complex with protein, homocystine, cysteine-homocysteine) (Jacobsen, Clin. Chem. 44:8(B), 1833-1843, 1998). “Total homocysteine” refers to the total amount of oxidized and reduced homocysteines, and in general, it is necessary to convert homocysteine in a sample to reduced homocysteine by a reducing agent in order to determine the total homocysteine.
High-performance liquid chromatography (HPLC) and immunoassay are usually employed to determine homocysteine. However, the HPLC apparatuses used in high-performance liquid chromatography are not commonly used in the clinical test, and it takes time, labor and cost to operate the apparatuses. In the immunoassay, although apparatuses are automated (Shipchandler, Clin. Chem., 41, 7, 991-994, 1995), determination is performed by combining a process for converting homocysteine to S-adenosyl-L-homocysteine by an enzyme reaction and a process for detecting it by an immunoassay, so that an apparatus used exclusively for this purpose is required.
Determination methods of homocysteine based on an immunoassay are proposed in Japanese Laid-Open Patent Publication (Tokuhyo) No. 9-512634 and (Tokkai) No. 10-114797. In the method disclosed in Japanese Laid-Open Patent Publication No. 9-512634, homocysteine is determined immunologically by chemically modifying the homocysteine to enhance the antigenicity, which requires a large number of processes and is complicated. Japanese Laid-Open Patent Publication No. 10-114797 discloses a method for determining homocysteine, but this method directly determines only the homocysteine bound to albumin, and does not determine the total amount of homocysteine. In this method, only about 70% of the entire homocysteine can be determined.
On the other hand, biochemical determination methods of homocysteine are disclosed in Japanese Patent No. 2870704, U.S. Pat. Nos. 5,998,191 and 5,885,767. The method disclosed in Japanese Patent No. 2870704 is characterized by allowing homocysteine in a sample that has been treated with a reducing agent to be in contact with adenosine and S-adenosyl-L-homocysteine hydrolase and evaluating the amount of adenosine in the residual mixture. However, in this method, an inhibitor of the S-adenosyl-L-homocysteine hydrolase is not used, and therefore it is necessary to perform determination in kinetic mode. Furthermore, this method has the problem that produced hydrogen peroxide cannot be led to a commonly used oxidative color-developing agent in the presence of a reducing agent that is used for a reduction process, which is an essential process for determining the total homocysteine. Therefore, an automatic analysis apparatus for general purposes cannot be used. However, these patent specifications fail to disclose any method to avoid these problems.
The methods disclosed in Japanese Laid-Open Patent Publication (Tokuhyo) No. 2000-502262, U.S. Pat. Nos. 5,998,191 and 5,885,767 are characterized by reacting homocysteine with homocysteine desulfurase, homocysteinase, or methionine-&ggr;-lyase to detect the produced hydrogen sulfide, ammonia, or 2-oxobutyric acid. However, these methods have problems such as: requiring a large number of processes; employing a lead ion, which is a harmful heavy metal, for the detection of the hydrogen sulfide; and being affected by cysteine and methionine, which are structural analogs to homocysteine and contained in a biological sample in a larger amount than that of homocysteine.
Thus, the conventional methods of determining homocysteine have problems such as requiring a special apparatus and complicated operation and having insufficient sensitivity and specificity, so that a method for determining a trace concentration of homocysteine rapidly, simply and with high sensitivity has not been established yet.
SUMMARY OF THE INVENTION
The present invention provides a novel method for determining homocysteine contained in a biological sample or the like rapidly, simply and with high sensitivity, and a kit for use in this determination method.
The inventors of the present invention succeeded in detecting or determining homocysteine by (i) oxidizing the residual homocysteine cosubstrate, the produced homocysteine-converting enzyme product or an enzyme reaction product thereof in the presence of an SH reagent to produce hydrogen peroxide and detecting or determining the produced hydrogen peroxide by color development using an oxidative color-developing agent or (ii) reacting the residual homocysteine cosubstrate, the produced homocysteine-converting enzyme product or an enzyme reaction product thereof with a D-amino acid converting enzyme to produce an oxo acid and/or ammonia and detecting or determining the produced oxo acid and/or ammonia. With the method for determining homocysteine of the present invention, homocysteine in a biological sample, in particular in body fluids such as blood and urine can be detected and determined rapidly and simply.
The present invention is directed to a method for detecting or determining homocysteine in a sample including:
(a) reducing the homocysteine in the sample by a thiol compound,
(b) reacting the reduced homocysteine with a homocysteine-converting enzyme and a homocysteine cosubstrate, thereby producing a homocysteine-converting enzyme product, and
(c) detecting or determining the residual homocysteine cosubstrate, the produced homocysteine-converting enzyme product or an enzyme reaction product thereof by: (i) oxidizing the residual homocysteine cosubstrate, the produced homocysteine-converting enzyme product or an enzyme reaction product thereof in the presence of an SH reagent to produce hydrogen peroxide and detecting or determining the produced hydrogen peroxide by color development using an oxidative color-developing agent or (ii) reacting the residual homocysteine cosubstrate, the produced homocysteine-converting enzyme product or an enzyme reaction product thereof with a D-amino acid converting enzyme to produce an oxo acid and/or ammonia and detecting or determining the produced oxo acid and/or ammonia.
In one preferable embodiment, the homocysteine-converting enzyme in the step (b) is S-adenosyl-L-homocysteine hydrolase, and the homocysteine cosubstrate in the steps (b) and (c) is adenosine.
In one preferable embodiment, the step (c) of detecting or determining the adenosine is a step of detecting or determining the adenosine by reacting the adenosine with adenosine deaminase, phosphoric acid, purine nucleoside phosphorylase, and xanthine oxidase to produce hydrogen peroxide and detecting or determining the produced hydrogen peroxide by color development using peroxidase and an oxidative color-developing agent.
In a more preferable embodiment, the step (c) includes further reacting the adenosine with uricase.
In another more preferable embodiment, the homocysteine-converting enzyme i
Fukuhara Mina
Matsuyama Naoto
Mizuno Koji
Takayama Masaharu
Azwell Inc.
Leary Louise N.
Webb Ziesenheim & Logsdon Orkin & Hanson, P.C.
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