Enzymatic assay of homocysteine on microtiter plates or a TECAN analyzer using crude lysate containing recombinant methionine gamma-lyase.
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 degrees C for 2 hr, or at room temperature for < 45 min, between venepuncture and centrifugation.
Chan
Err-Cheng
EC
School of Medical Technology, Chang Gung University, Tao Yuan, Taiwan.
Chang
Pi-Yueh
PY
Wu
Tsu-Lan
TL
Wu
James T
JT
D000328:Adult; D000367:Age Factors; D000368:Aged; D044466:Asian People; D013437:Carbon-Sulfur Lyases / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology; D005260:Female; D006710:Homocysteine / Q000097:blood*; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D008875:Middle Aged; D011994:Recombinant Proteins / Q000737:chemistry; D012737:Sex Factors
D007202:Indicators and Reagents; D011994:Recombinant Proteins; D006710:Homocysteine; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase