Homosteine (Hcy) Assay Kit
Intended Use
Enzymatic homocysteine (Hcy) assay kit is intended for the in vitro quantitative determination of total L-homocysteine in serum or plasma. The homocysteine (Hcy) assay kit can assist in diagnosis and treatment of patients suspected of having hyperhomocysteinemia and homocystinuria. The Hcy assay kit is not intended for correlating B12 or folate with homocysteine levels.
Clinical Significance
Homocysteine (Hcy) is a thiol-containing amino acid produced by the intracellular demethylation of methionine. Total homocysteine (tHcy) represents the sum of all forms of Hcy including forms of oxidized, protein bound and free. Elevated level of tHcy has emerged as an important risk factor in the assessment of cardiovascular disease. Excess Hcy in the blood stream may cause injures to arterial vessels due to its irritant nature, and result in inflammation and plaque formation, which may eventually cause blockage of blood flow to the heart. Elevated tHcy levels are resulted from four major causes including:
a. genetic deficiencies in enzymes involved in Hcy metabolisms such as cystathionine beta synthase (CBS), methionine synthase (MS), and methylenetetrahydrofolate reductase (MTHFR);
b. nutritional deficiency in B vitamins such as B6, B12 and folate;
c. renal failure for effective amino acid clearance, and
d. drug interactions such as nitric oxide, methotrexate and phenytoin that interfere with Hcy metabolisms.
Elevated levels of tHcy are also linked with Alzheimer’s diseaseand Osteoporosis. Guidelines for tHcy determination in clinical laboratories have recently been established.
Assay Principle
Enzymatic homocysteine (tHcy) assay kit is based on a novel assay principle that assesses the co-substrate conversion product (a molecule that is not a substrate of the Hcy conversion enzyme, and does not contain any element from sample Hcy) instead of assessing co-substrate or Hcy conversion products of Hcy as described in the literature. In this assay, oxidized Hcy is first reduced to free Hcy which then reacts with a co-substrate, S-adenosylmethionine (SAM) catalyzed by a Hcy S-methyltransferase to form methionine (the Hcy conversion product of Hcy) and S-adenosylhomocysteine (SAH, the co-substrate conversion product). SAH is assessed by coupled enzyme reactions including SAH hydrolase, adenosine (Ado) deaminase and glutamate dehydrogenase, wherein SAH is hydrolyzed into adenosine (Ado) and Hcy by SAH hydrolase. The formed Hcy that is originated from the co-substrate SAM is cycled into the Hcy conversion reaction by Hcy S-methyltransferase. This forms a co-substrate conversion product based enzyme cycling reaction system with significant amplification of detection signals. The formed Ado is immediately hydrolyzed into Inosine and ammonia which reacts with glutamate dehydrogenase with concomitant conversions of NADH to NAD+. The concentration of Hcy in the sample is indirectly proportional to the amount of NADH converted to NAD+ (ΔA340nm).
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