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Methods and compositions for assaying homocysteine

a technology of homocysteine and composition, applied in the field of homocysteine detection, can solve the problems of chromatographic separation, time-consuming and cumbersome, and myocardial infarction,

Inactive Publication Date: 2006-08-03
GENERAL ATOMICS
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Problems solved by technology

These individuals suffer from thromboembolic complications at an early age, which result in stroke, myocardial infarction, renovascular hypertension, intermittent claudication, mesenteric ischemic, and pulmonary embolism.
In these assays, chromatographic separation, which is often time-consuming and cumbersome to perform, is a common key step of these methods.
More particularly, these methods require highly specialized and sophisticated equipment and well-trained analytic specialists.
The use of such equipment is generally not well-accepted in routine clinical laboratory practice.
Though immunoassays avoid a time-consuming chromatographic separation step and are amenable to automation, production of monoclonal antibody is expensive, somewhat unpredictable, and often requires secondary or even tertiary antibodies for detection.

Method used

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  • Methods and compositions for assaying homocysteine
  • Methods and compositions for assaying homocysteine
  • Methods and compositions for assaying homocysteine

Examples

Experimental program
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Effect test

example 1

GLDH—NADH Coupling to Detect NH4+ Generated by the Enzymatic Cycling using Purified SAM

[0092] In this study, the following coupled enzymatic cycling reactions are used:  Adenosine→Inosine+NH4+  (3)

NH4++α-Ketoglutarate+NAD(P)H→Glutamate+NAD(P)   (4)

[0093] In scheme (1), the reaction is catalyzed by a SAM-dependent homocysteine S-methyltransferase. In scheme (2), the reaction is catalyzed by a SAH hydrolase. In scheme (3), the reaction is catalyzed by an adenosine deaminase. In scheme (4), the reaction is catalyzed by a L-glutamate dehydrogenase. The NAD(P)+ is spectraphotometrically detected at 340 nm. A more deteiled descirption of the reagents used in this study is set forth in the following Tables 1 and 2.

TABLE 1Compositions of Reagent 1ChemicalReagent 1ConcentrationPotassium phosphate15mMNAD(P)H5mMGLDH2KU / LBSA1.2g / LAdenosine Deaminase50KU / LHomocysteine methyltransferase10KU / LDTT0.2mMα-ketoglutarate30mMSAM3mM

[0094]

TABLE 2Compositions of Reagent 2ChemicalsReagent 2Concentrati...

example 2

GLDH—NADH Coupling to Detect NH4+ Generated by the Enzymatic Cycling using SAM Concurrently Converted by SAM Synthase from ATP and Methionine

[0096] In this study, the following coupled enzymatic cycling reactions are used:  Adenosine→Inosine+NH4+  (4)

NH4++α-Ketoglutarate+NAD(P)H→Glutamate+NAD(P)   (5)

[0097] In scheme (1), the reaction is catalyzed by a SAM Synthase. In scheme (2), the reaction is catalyzed by a SAM-dependent homocysteine S-methyltransferase. In scheme (3), the reaction is catalyzed by a SAH hydrolase. In scheme (4), the reaction is catalyzed by an adenosine deaminase. In scheme (5), the reaction is catalyzed by a L-glutamate dehydrogenase. The NAD(P)+ is spectraphotometrically detected at 340 nm. A more deteiled descirption of the reagents used in this study is set forth in the following Tables 3 and 4.

TABLE 3Compositions of Reagent 3ChemicalReagent 3ConcentrationGood's buffer15mMNAD(P)H5mMGLDH2KU / LBSA1.2g / LTCEP0.2mMα-ketoglutarate30mMATP10mMMethionine5mMSAM S...

example 3

Adenosine Kinase-Pyruvate-Kinase-Lactate Dehydrogenase-NADH Coupling to Detect Adenosine Generated by the Enzymatic Cycling

[0100] In this study, the following coupled enzymatic cycling reactions are used:  Adenosine+ATP→ADP+AMP   (3)

ADP+PEP→Pyruvate+ATP   (4)

Pyruvate+NADH→Lactate+NAD   (5)

[0101] In scheme (1), the reaction is catalyzed by a SAM-dependent homocysteine S-methyltransferase. In scheme (2), the reaction is catalyzed by a SAH hydrolase. In scheme (3), the reaction is catalyzed by an adenosine kinase. In scheme (4), the reaction is catalyzed by a pyruvate kinase. In scheme (5), the reaction is catalyzed by a lactate dehydrogenase. The NAD(P)+ is spectraphotometrically detected at 340 nm. A more deteiled description of the reagents used in this study is set forth in the following Tables 5 and 6.

TABLE 5Compositions of Reagent 5ChemicalReagent 5ConcentrationPotassium phosphate15mMNADH5mMGLDH2KU / LBSA1.2g / LAdenosine Kinase10KU / LHomocysteine methyltransferase10KU / LDTT0.2m...

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Abstract

This invention relates generally to the field of homocysteine detection. In particular, the invention provides a method for determining homocysteine presence or concentration in samples, which method comprises: contacting a sample containing or suspected of containing Hcy with a Hcy co-substrate and a Hcy converting enzyme in a Hcy conversion reaction to form a Hcy conversion product and a Hcy co-substrate conversion product; and assessing the Hcy co-substrate conversion product to determine the presence, absence and / or amount of the Hcy in the sample. A kit for assaying homocysteine based on the same principle is also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the priority benefit of provisional application U.S. Ser. No. 60 / 486,865, filed Jul. 10, 2003, the contents of which are incorporated by reference in their entirety.TECHNICAL FIELD [0002] This invention relates generally to the field of homocysteine detection. In particular, the invention provides a method for determining homocysteine (Hcy) presence or concentration in samples in which homocysteine reacts with a Hcy co-substrate in a Hcy conversion reaction catalyzed by a Hcy converting enzyme to form a Hcy conversion product and a Hcy co-substrate conversion product, and the Hcy co-substrate conversion product is assessed to determine the presence and / or concentration of the Hcy in the sample. A kit for assaying homocysteine based on the same principle is also provided. BACKGROUND OF THE INVENTION [0003] Total concentration of homocysteine in body fluids, such as plasma or serum, is an important marker for disea...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/48C12Q1/37C12Q1/34G01N33/68
CPCC12Q1/34G01N33/6815C12Q1/485C12Q1/48
Inventor YUAN, CHONG-SHENGDATTA, ABHIJITDOU, CHAO
Owner GENERAL ATOMICS
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