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Annals of Clinical & Laboratory Science 35:155-160 (2005)
© 2005 Association of Clinical Scientists

Enzymatic Assay of Homocysteine on Microtiter Plates or a TECAN Analyzer Using Crude Lysate Containing Recombinant Methionine {gamma}-Lyase

Err-Cheng Chan1, Pi-Yueh Chang2, Tsu-Lan Wu1 and James T. Wu3
1 School of Medical Technology, Chang Gung University, Tao-Yuan, Taiwan;2 Department of Pathology, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan;3 ARUP Laboratories, University of Utah Health Sciences Center, Salt Lake City, Utah

Address correspondence to James T. Wu, Ph.D., ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108, USA; tel 801 583 2787; fax 801 584 5207; e-mail wuj{at}aruplab.com.

An enzymatic assay for plasma homocysteine was developed that uses a crude lysate of E. coli containing the recombinant enzyme, methionine {gamma}-lyase. The assay uses a commercially available fluorophore and 96-well microtiter plates; it can be performed manually or with the TECAN automated analyzer. The CVs for within-run and between-run precision are <10%. Close correlation (r >0.9) was obtained between results by this enzymatic method vs a reference HPLC procedure. In a Chinese population, the concentration of plasma total homocysteine was found to be gender- and age-dependent. Mean concentrations of plasma total homocysteine increased with age and were higher in men than women. Serum homocysteine concentrations did not differ significantly from those in plasma, provided the whole blood specimens were kept at 4°C for 2 hr , or at room temperature for <45 min, between venepuncture and centrifugation.

Keywords: homocysteine, enzymatic assay, microtiter plate, TECAN analyzer, methionine {gamma}-lyase

Abbreviations: tHcy: the total concentration of free homocysteine and homocysteine disulfides in plasma or serum




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