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Letters to the Editor |
1 A/C Diagnostics, LLC, and AntiCancer, Inc., 7917 Ostrow St. San Diego, CA 92111
aAuthor for correspondence. Fax 858-268-4175; e-mail all{at}anticancer.com.
To the Editor:
Various methods have been developed for plasma total homocysteine (tHCY) measurement, including a tHCY enzyme conversion immunoassay designed for the Abbott IMx analyzer (1), a microtiter plate tHCY enzymatic immunoassay assay (2), HPLC methods (3)(4)(5), and gas chromatography–mass spectrometry methods (6). We have described a single-enzyme tHCY assay (enzymatic tHCY assay) based on a highly specific recombinant form of L-homocysteine 
,
-lyase (rHCYase) (7)(8). We report here the application of this tHCY enzymatic assay on the Hitachi 912 automatic chemistry analyzer. The principle of the assay is that rHCYase produces H2S from tHCY and that the H2S is quantified by its reaction with N,N-dibutylphenylenediamine, which produces a chromophore.
The assay uses four reagents and thus is compatible with implementation on the Hitachi 912. Briefly, 30 µL of EDTA plasma was incubated in a dithiothreitol reduction reaction (1 mmol/L dithiothreitol and 2 mL/L Triton X-100 in 40 mmol/L sodium phosphate buffer, pH 8.3) for 1.5 min to release bound homocysteine. The rHCYase reaction (0.05 g/L in 40 mmol/L sodium phosphate buffer, pH 8.3, containing 20 µmol/L pyridoxal 5-phosphate) was then run for 3.5 min. The N,N-dibutylphenylenediamine (12.5 mmol/L in 0.75 mol/L H2SO4) was then added, and 5 min later, an oxidant, 5 mmol/L K3Fe(CN)6 (in 10 mmol/L sodium phosphate buffer, pH 7.6), was added. Five minutes after addition of the oxidant, the end-points were read at wavelengths of 700 and 660 nm. (Assay conditions are given in detail in the Data Supplement accompanying the online version of this letter at http://www.clinchem.org/content/vol49/issue6/.) Because the assay is based on an increase in absorbance over baseline, no blank without enzyme was used. The limit of quantification of the assay is 1.54 µmol/L, as determined by serial dilution of a pooled plasma calibrator (Serologicals Corp.). Ten replicates of each dilution were analyzed. The limit of quantification is defined as the lowest concentration measured with a CV <20%. The assay is linear to at least 80 µmol/L tHCY, as determined visually after measurement of various amounts of homocysteine in phosphate-buffered saline.
The within-run imprecision (CV) was 4.8% at 8.9 µmol/L tHCY, 3.0% at 14.9 µmol/L tHCY, and 4.5% at 25 µmol/L tHCY (n = 8). The between-assay CV over 10 days was 7.8%, 5.9%, and 4.9% at 8.8, 15, and 25 µmol/L, respectively. These imprecisions are within ranges reported for currently used assays, including the Food and Drug Administration-cleared Bio-Rad HPLC assay (9)(10).
We assayed 121 plasma samples with the tHCY enzymatic assay on the Hitachi 912 (y) and with the Bio-Rad HPLC tHCY assay (x) (9)(10). The regression equation was: y = 0.98x + 1.90 µmol/L (r = 0.977; Fig. 1A
). The mean (SD) difference between the tHCY enzymatic assay and the Bio-Rad HPLC tHCY assay (11)(12)(13) was -1.62 (2.33) µmol/L (Fig. 1B
). Differences were not significantly correlated with homocysteine concentration (Pearson r = 0.12; P = 0.185).
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Interference from L-CYS (0–200 µmol/L) was <10% at physiologic tHCY concentrations (0–200 µmol/L), and L-MET (0–200 µmol/L) showed no interference at these same tHCY concentrations.
References
The following articles in journals at HighWire Press have cited this article:
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  Q. Han, X. Sun, M. Xu, N. Zhang, L. Tang, Y. Tan, and R. M. Hoffman 3-Deazaadenosine, a Stabilizer of Whole-Blood Homocysteine Content, Does Not Interfere with the Single-Enzyme Homocysteine Assay while Totally Inhibiting the Enzyme Conversion Homocysteine Immunoassay Clin. Chem., September 1, 2004; 50(9): 1703 - 1704. [Full Text] [PDF]
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Q. Han, M. Xu, L. Tang, X. Sun, N. Zhang, X. Tan, X. Tan, Y. Tan, and R. M. Hoffman Homogeneous Enzymatic Colorimetric Assay for Total Cysteine Clin. Chem., July 1, 2004; 50(7): 1229 - 1231. [Full Text] [PDF]
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