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Abstracts of Oak Ridge Posters |
Diazyme Laboratories Division, San Diego, CA
aaddress correspondence to this author at: Diazyme Laboratories Division, General Atomics, 3550 General Atomics Court, San Diego, CA 92121; fax 858-455-4750, e-mail chong.yuan{at}diazyme.com or www.diazyme.com)
Homocysteine (HCY) is a thiol-containing amino acid produced by the intracellular demethylation of the essential amino acid methionine. Intracellular HCY enters either the transsulfuration pathway or the remethylation cycle. When the enzymatic reactions involved in the 2 metabolic pathways of intracellular HCY are impaired, because of either genetic defects in enzymes for HCY metabolism or the nutritional deficiency of vitamins such as folate, B12, and B6, HCY accumulates in the cells and is exported to the circulation (1)(2)(3)(4). Approximately 80% of circulating HCY in the blood is protein bound by disulfide linkage (5). The remaining unbound HCY combines by oxidation, either with itself to form the dimer homocystine or with cysteine to form the mixed disulfide cysteine-HCY. Only a small amount circulates as free HCY. Total HCY (tHCY) represents the sum of all forms of HCY, including forms of oxidized HCY, protein bound, and free.
Highly increased concentrations of tHCY are found in persons with homocystinuria, a rare genetic disorder most commonly caused by a deficiency of cystathionine ß-synthase. Typical clinical manifestations of patients with homocystinuria include mental retardation, early arteriosclerosis, and arterial and venous thromboembolism (4)(6). Other less severe genetic defects, including polymorphisms in genes encoding for enzymes that participate in HCY metabolism, such as methylenetetra-hydrofolate reductase (MTHFR) and methionine synthase, are also found in patients who have moderately increased plasma concentrations of HCY (7)(8).
Patients with chronic renal disease have high morbidity and mortality resulting from arteriosclerotic cardiovascular disease (CVD). Increased tHCY is a frequently observed finding in the blood of these patients. Initial investigation (9) suggested that the markedly increased plasma tHCY found in end-stage renal disease patients contributed independently to their very high incidence of fatal and nonfatal CVD outcomes. The increased concentrations of tHCY are the result mainly of impaired removal of HCY from the blood by the kidney and are independent of their vitamin concentrations.
In most US clinical laboratories, 15 µmol/L is the cutoff value for a normal concentration of HCY for adults. Each laboratory is recommended to establish a range of normal values for the population in their region.
Recently, we developed an enzymatic cycling assay for detection of HCY in human serum or plasma. The Diazyme enzymatic HCY assay is based on a cosubstrate conversion product cycling principle, as shown in Scheme 1
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Oxidized HCY is first reduced to free HCY, which then reacts with a cosubstrate, S-adenosylmethionine (SAM), catalyzed by an HCY S-methyltransferase to form methionine (the HCY conversion product of HCY) and S-adenosylhomocysteine (SAH, the cosubstrate conversion product, which does not contain any element from the sample homocysteine molecule). SAH is hydrolyzed into adenosine and HCY by SAH hydrolase. The formed HCY that originated from the cosubstrate SAM is cycled back to the HCY conversion reaction by HCY S-methyltransferase, producing an enzymatic cycling system with substantial amplification of detection signals. The formed adenosine is 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 proportional to the amount of NADH converted to NAD+ (
A340nm).
The Diazyme HCY enzymatic assay has been formulated into a 3-reagent, homogeneous liquid system. For analyzers capable of handing 3 reagents, the assay is ready for use. For analyzers capable of handling only 2 reagents, premixing of Reagent 1 and Reagent 2 is needed. The assay has been implemented on various automated analyzers, and samples can be analyzed in as little as 10 min.
We evaluated the precision of the Diazyme enzymatic HCY assay on a Cobas Mira analyzer, according to NCCLS guideline EP5-A. In the study, 3 specimens containing 7, 12, and 29.5 µmol/L homocysteine were tested with 2 runs per day and with duplicates over 20 working days. The results demonstrated that within-run imprecisions (CVs) were 2.2% for 7 µmol/L, 3.0% for 12 µmol/L, and 1.8% for 29.5 µmol/L HCY, respectively; and total imprecisions (CVs) were 4.1% for 7 µmol/L, 5.9% for 12 µmol/L, and 4.0% for 29.5 µmol/L HCY, respectively.
We determined the diagnostic accuracy of the assay by testing individual serum or plasma samples with our enzymatic cycling HCY assay and comparing that assay with both a commercial enzymatic HCY assay and a commercial HCY immunoassay. To ensure that the concentrations of HCY distributed across the reportable dynamic range, some HCY samples used for the study were enriched with a stock solution of HCY to targeted concentrations. Fig. 1
illustrates the correlation results with the Catch enzymatic HCY assay and the Bayer HCY immunoassay on varieties of clinical chemistry analyzers. From these results, we can see that the Diazyme enzymatic HCY assay correlated very well with both assays. It appears that the Diazyme enzymatic cycling HCY assay correlated slightly better with the Catch enzymatic assay than with the Bayer HCY immunoassay.
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To demonstrate the linearity of the Diazyme HCY enzymatic assay, a serum sample containing 7 µmol/L HCY was enriched with L-HCY stock solution to 71.2 µmol/L HCY. Next, a serial dilution of the serum sample containing 71.2 µmol/L HCY was made with 7 µmol/L HCY serum to the indicated concentrations. The serum sample containing 7 µmol/L HCY was diluted with saline in series, and the final dilution contained 3.1 µmol/L HCY. The serum samples, prepared as described, were tested on Cobas Mira using the Diazyme enzymatic assay. The result demonstrated that the assay has a linear range of at least 50 µmol/L HCY. In a separate study, we determined the limit of detection (LOD) of the Diazyme HCY enzymatic assay. A zero calibrator was tested with 12 replicates. The LOD, defined as the mean + 3 SD, was <1.5 µmol/L HCY.
We determined the extent of interference from the substances typically present in the serum by testing a 12.9 µmol/L HCY serum sample enriched with various concentrations of substances. We also determined the interference from common prescription and over-the-counter pharmaceuticals by testing 2 serum samples, containing 11.5 µmol/L and 42.7 µmol/L HCY, respectively, which were enriched with various concentrations of substances examined. The test results, with the respective concentration of each substance, are listed in Table 1
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In conclusion, we have developed an innovative enzymatic cycling assay for the in vitro quantitative determination of total L-HCY in serum or plasma, which has been adapted on automated chemistry analyzers. The assay uses 3 reagents and has been implemented on the Hitachi 917, Synchron CX-7, and Cobas Mira, testing human serum and plasma. Within-run imprecision (CV) was <3%, and total imprecision (CV) was <6% for the 3 concentrations of HCY controls tested. The study testing human serum and heparin-plasma samples demonstrated excellent correlation with another enzymatic HCY assay on various automated chemistry analyzers. The assay also correlated well with an HCY immunoassay. Limit of detection in serum is <1.5 µmol/L HCY in serum, and the assay is linear up to 50 µmol/L HCY. Interference is <10% with 1 mmol/L L-cysteine, 0.5 mmol/L glutathione, 100 µmol/L adenosine, 20 µmol/L cystathionine, and 500 µmol/L NH4Cl. A panel of commonly prescribed drugs did not contribute interference to the assay at the concentrations indicated.
Footnotes
1 these authors contributed equally to this work; 
References
The following articles in journals at HighWire Press have cited this article:
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  S. Rossi, G. Rossi, A. Giordano, and S. Paltrinieri Homocysteine measurement by an enzymatic method and potential role of homocysteine as a biomarker in dogs J Vet Diagn Invest, September 1, 2008; 20(5): 644 - 649. [Abstract] [Full Text] [PDF]
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