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Letters to the Editor |
1 A/C Diagnostics LLC and AntiCancer, Inc., San Diego, CA
aAddress correspondence to this author at: AntiCancer, Inc., 7917 Ostrow St., San Diego, CA 92111. Fax 858-268-4175; e-mail all{at}anticancer.com.
To the Editor:
Plasma total homocysteine (tHcy) is a risk factor for cardiovascular disease and possibly other diseases (1). Release of homocysteine (Hcy) from erythrocytes into the plasma before measurement remains a problem. The erythrocyte continues to carry out methylation reactions at room temperature in whole blood, producing and exporting Hcy as an end product while the blood is waiting for processing or during delivery. Export of Hcy from erythrocytes into the plasma is time- and temperature-dependent (2). At room temperature, the increase in plasma tHcy is 
1.0 µmol · L–1 · h–1 (2). This corresponds to an 10% increase per hour in a typical sample containing 10 µmol/L tHcy. Therefore, at present, the accuracy of Hcy measurements is compromised even when the plasma is separated within 1 h of sample collection (2). Hill et al. (3) studied the effect of temperature on the stability of plasma tHcy over a 72-h time course in blood collected into evacuated tubes containing either EDTA or 3-deazaadenosine (3-DA) and found that 3-DA is an effective stabilizer of plasma Hcy content. However, because 3-DA prevents Hcy production through competitive inhibition of the enzyme S-adenosylhomocysteine hydrolase (SAHH), 3-DA interferes with popular assays of tHcy that are enzyme-conversion immunoassays based on SAHH (1)(2)(4).
We have previously developed a simple assay method for tHcy (5)(6) that uses a single and specific recombinant homocysteine 
,
-lyase (rHCYase), which produces the analyte H2S from Hcy. The single-enzyme tHcy assay has received 510(K) clearance. The purpose of this study is to compare the interference of 3-DA on SAHH and rHCYase.
To determine the interference of 3-DA on SAHH, we used S-adenosylhomocysteine (SAH) as a substrate at 50 µmol/L and 3-DA at 0, 50, 100, and 200 µmol/L in the assay buffer. Conversion of SAH to adenosine and Hcy was measured by its subsequent conversion to H2S by rHCYase with colorimetric measurement at 675 nm using N,N-dibutylphenylenediamine hydrochloride as the chromophore (5)(6). When 3-DA was added from 0 to 200 µmol/L, the remaining activity decreased from 100% to 3.3% (Fig. 1
). These results confirm the report of Woltersdorf et al. (4), who found highly significant interference by 3-DA, starting from 50 µmol/L, in the Abbott IMx Hcy assay, which is based on SAHH.
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To determine the interference of 3-DA directly on rHCYase, we used 50 µmol/L L-Hcy with 3-DA at 0, 50, 100, or 200 µmol/L in the assay buffer. At 3-DA concentrations ranging from 0 to 200 µmol/L, the relative activity showed almost no change (<4.5%), a striking contrast to the interference of 3-DA on SAHH (Fig. 1
). For 10 plasma samples with and without 100 µmol/L 3-DA, measured with the rHCYase-based tHcy assay (5)(6), the mean (SD) tHcy was 10.4 (2.2) µmol/L with 3-DA and 10.6 (2.3) µmol/L without 3-DA. The concentrations measured by a HPLC tHcy assay (5)(6) were 10.7 (2.1) µmol/L with 3-DA and 10.8 (2.0) µmol/L without 3-DA.
We conclude that the SAHH-based assay is completely interfered by 3-DA at the concentrations needed to stabilize tHcy in whole blood, whereas the rHCYase-based tHcy assay is unaffected (5)(6). Thus, the remaining technical problem for routine and widespread tHcy measurement, the long-term storage of whole blood, can be solved with the use of 3-DA and the rHCYase-based tHcy assay.
References
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) was carried out with 50 µmol/L L-Hcy; 0, 50, 100, or 200 µmol/L 3-DA; and 0.05 U of rHCYase as described. The resulting H2S was measured as described above.