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Letters to the Editor |
Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL
aAddress correspondence to this author at: Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294-3360, Fax 205-996-5775, E-mail tamurat{at}uab.edu
To the Editor:
We read with great interest the letter by Hübner et al. (1) on the stability of plasma sulfur-containing metabolites in the presence of anticoagulants (EDTA and citrate) and a homocysteine stabilizer. These authors observed time-dependent increases in plasma concentrations of homocysteine (Hcy)1 and similar increases in plasma S-adenosylhomocysteine (SAH) in EDTA- and citrate-containing whole blood after 24 h at room temperature. In discussing the mechanisms of these increases, they postulated that plasma Hcy is generated from SAH in erythrocytes by the SAH-hydrolase–catalyzed reaction, and Hcy and SAH leak into plasma from erythrocytes (1). They further noted that the inhibition of SAH hydrolase by citrate results in a smaller Hcy increase compared with EDTA, which they claimed does not inhibit this enzyme.
We recently reported the iron-dependent in vitro chemical formation of Hcy from methionine (Met), SAH, and cystathionine, where EDTA or citrate (both 5 mmol/L) acted as a chelator (2). Based on these findings, we propose that a portion of the Hcy increases reported by Hübner et al. (1) results from the iron-dependent conversion of Met and SAH to Hcy. Furthermore, we showed that iron chelated by EDTA catalyzes the conversion of Met to Hcy more efficiently than iron chelated by citrate, and this should also be true for SAH (2). Although the inhibition of SAH hydrolase would prevent increases in plasma Hcy, this factor alone may not completely account for the differences in plasma Hcy increases of approximately 12 and 3 µmol/L in EDTA- and citrate-treated blood, respectively. One would expect that plasma SAH would be higher in the citrate-treated samples than EDTA-treated samples, if SAH hydrolase is inhibited by citrate. This is not true, because plasma SAH concentrations are 70–90 µmol/L at 24 h in samples containing EDTA or citrate. Plasma Hcy increases in whole blood treated with EDTA and 3-deazaadenosine (inhibitor of SAH hydrolase) after incubation at room temperature and 37 °C (3). Ubbink et al.(4) also reported that EDTA-treated blood produced a marked increase in plasma Hcy compared with Na-fluoride–treated blood. Na-fluoride may inhibit SAH production or SAH hydrolase; however, Na-fluoride is not an iron chelator and should not mobilize protein-bound iron. The amount of K3 EDTA in evacuated tubes is about 4 mmol/L (1.8 g/L of blood), which is similar to the EDTA concentration (5 mmol/L) that we used in our in vitro study (2). Therefore, iron in both plasma and erythrocyte would be exposed to chelation, because EDTA concentration is higher than that of protein that binds iron. Both SAH and the larger pool of Met could react with iron chelated by EDTA, resulting in a larger-than-expected increase in plasma Hcy. Serum samples contain no iron chelator and a small amount of iron and should not show an increase in Hcy.
In addition to the mechanistic points presented here, Hübner et al. (1) reported that the 24-h in vitro increases in plasma Hcy concentrations in the presence of EDTA were only 0.1–9.5 µmol/L in patients with renal disease whose baseline plasma Hcy values were far higher than those of healthy subjects. In contrast, the increases in plasma Hcy in healthy subjects were much higher, range 8.7–23.6 µmol/L. These findings might be explained by the fact that blood iron concentrations in patients with renal disease are much lower than those in healthy subjects, since patients with chronic renal disease often have iron deficiency (5). The amount of iron that would be chelated by EDTA may be lower in these patients, resulting in a reduced production of Hcy from Met and SAH. It may be of interest to examine the correlation between 24-h Hcy increase and blood iron concentrations in these patients and healthy individuals.
Hübner et al. (1) noted that the proprietary material in PrimavetteTM affected the fluorescence polarization immunoassay of Hcy. Although we have not evaluated Primavette, it may be found to interfere with other laboratory analyses. We recommend that whole blood samples should be collected with citrate rather than EDTA as an anticoagulant and centrifuged as soon as possible. If this is not possible, whole blood samples should be kept refrigerated until plasma separation. Further studies are needed to elucidate the mechanism of Hcy increase.
Acknowledgments
Grant/Funding Support: None declared.
Financial Disclosures: None declared.
Footnotes
1 Nonstandard abbreviations: Hcy, homocysteine; SAH, S-adenosylhomocysteine; Met, methionine. 
References
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