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Technical Briefs |
1 Hôpital Erasme, Université Libre de Bruxelles, 808 route de Lennik, B1070 Brussels, Belgium;
aauthor for correspondence: fax 32-2-555-6655, e-mail fcotton{at}ulb.ac.be
Homocysteine (HCy) is an intermediate amino acid formed during the metabolism of methionine to cysteine. In the recent years, it has emerged as a risk factor for cardiovascular disease independent of known other factors (1)(2)(3). The first methods developed for total plasma HCy measurements were HPLC, with or without derivatization, and gas chromatography. Later, immunoassays were developed, allowing automation and widespread use of the marker in the medical world. Plasma HCy is influenced by various factors: genetics, diet, sex, age, ethnic group, drugs, and diseases. EDTA-, citrate-, or heparin-anticoagulated blood may be used, but plasma should be immediately separated from blood cells to avoid spurious increases (4). Addition of different substances has been suggested to reduce these spurious increases in HCy concentrations. Acidic citrate has been shown to be effective in some studies (5) but not in others (6). A specific inhibitor of S-adenosylhomocysteine hydrolase (3-deaza-adenosine) was successfully used, but unfortunately, it is not compatible with immunoassays (7). Fluoride appears to be the most interesting additive. Ubbink et al. (8) showed that it prevents in vitro increases for 2 h. Several studies have confirmed this effect (9), although others have not (10). In most studies, plasma HCy from blood collected in fluoride tubes has been lower than in EDTA tubes (10)(11)(12).
Our aim was to evaluate the protective effect of fluoride against spurious increases of in vitro HCy and to establish reference intervals for the AxSYM immunoassay (Abbott) for plasma HCy from blood collected into fluoride tubes.
The HCy assay was performed on the AxSYM analyzer according to the manufacturer’s instructions. Folic acid and vitamin B12 measurements were performed on the Elecsys analyzer (Roche Diagnostics) according to the manufacturer’s instructions.
For the study of a possible fluoride protective effect, blood was drawn from 22 individuals and collected in EDTA and fluoride-oxalate tubes (Terumo). Fluoride-oxalate tubes contained 6.75 mg of lyophilized sodium fluoride (54 mmol/L of blood). Plasma was separated immediately (centrifugation started within 2 min) and after 1, 2, and 5 h at room temperature. The HCy assay was performed on each aliquot. Two hundred and thirty women (mean age, 41 years; range, 24–59 years) and 126 men (mean age, 42 years; range, 23–63 years) who are workers in Erasme Hospital (Brussels) were included in the reference interval and vitamin correlation studies. The study was approved by the local ethics committee. For the establishment of plasma HCy reference values, fasting venous blood was drawn in fluoride-oxalate tubes transported on crushed ice to the laboratory, where plasma was immediately separated from blood cells and analyzed within 2 h. For vitamin B12 and folic acid measurements, serum was analyzed within 4 h after collection. Statistical analyses were performed with Analyze-It for Microsoft Excel, Ver. 1.62, software (Analyze-It Software). Correlations were computed with the Spearman formula. Reference values were defined as the 2.5th–97.5th percentile interval of plasma HCy in individuals with serum folic acid and vitamin B12 above the 2.5th percentile according to gender.
Shown in Table 1
are the mean HCy concentrations obtained for 22 individuals whose blood was collected in tubes containing either EDTA or fluoride-oxalate. After 1 h at room temperature before separation of plasma from red blood cells, HCy increased by 8.5% in EDTA tubes but by 2.5% in fluoride tubes. After 2 h, the changes were 21.1% in EDTA tubes and 6.5% in fluoride tubes, and after 5 h, they were 39.9% and 14.8%. Results obtained for samples collected in EDTA tubes were systematically higher than for samples collected in fluoride-oxalate tubes. Even when plasma was separated immediately after blood collection, concentrations showed a 8.7% difference (P <10-6, paired Student t-test).
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Shown in Fig. 1
is the distribution of HCy results in the population sample. It was nongaussian (Shapiro–Wilk test, P <0.0001) and right-tailed. The median (2.5th–97.5th percentiles) was 8.90 (4.78–16.61) µmol/L. Fifteen individuals had mild hyperhomocysteinemia (15–20 µmol/L), and 4 had hyperhomocysteinemia (>20 µmol/L). HCy was significantly higher in men (median, 9.8 µmol/L; 2.5th–97.5th percentiles, 5.8–17.0 µmol/L) than in women (median, 8.3 µmol/Ls; 2.5th–97.5th percentiles, 4.4–16.3 µmol/L; P <0.0001, Wilcoxon signed-rank test). The 2.5th–97.5th percentile interval for serum folic was 5.7–36.9 nmol/L for women and 2.9–11.1 nmol/L for men. The serum 2.5th–97.5th percentile interval for vitamin B12 was 146–640 pmol/L for women and 215–682 pmol/L for men. Plasma HCy correlated negatively with serum vitamin B12 (r = -0.13; P = 0.0145) and serum folic acid (r = -0.41, P <0.0001) but not with age (r = 0.09; P = 0.0758). For individuals with serum folic acid and vitamin B12 above the 2.5th percentile, the median (2.5th–97.5th percentiles) plasma HCy concentrations were 8.2 (4.4–15.6) µmol/L for women, 9.5 (5.8–16.5) µmol/L for men, and 8.8 (4.7–16.3) µmol/L for women and men together.
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Hyperhomocysteinemia is a well-documented cardiovascular risk factor (1)(2)(3). Because folic acid and vitamin B12 supplementation can return plasma HCy concentrations to values within the reference intervals (13)(14), accurate measurement of this analyte is desired. Preanalytical factors are important. It has been shown that HCy concentrations increase by 
10% per hour in unseparated whole blood, whereas at 0 °C, no significant increase in HCy occurs during the first hour (4)(11). Nevertheless, in a recent French study, only 16% of samples with HCy requested arrived at the laboratory within 1 h, and 28% of samples arrived at room temperature (15), showing a potential useful role for a preservative agent such as fluoride. Although several authors have described the protective effect of this additive (8)(9), in a recent study (16), plasma HCy increased by 9% after 1 h and 24% after 4 h in whole blood samples collected in EDTA tubes and stored at room temperature. In fluoride tubes, similar increases of 8–24% were observed. Hughes et al. (10) even suggested that the protective effect was only apparent. Our results did not confirm these data. In similar conditions, for fluoride tubes, we observed increases of 2.5% after 1 h, 6.5% after 2 h, and 14.8% after 5 h, whereas in EDTA tubes, we observed increases of 8.5% after 1 h, 21.1% after 2 h, and 40% after 5 h. This suggests that fluoride can be an essential additive, especially when plasma separation cannot be achieved quickly or when samples have not been kept at 0 °C. Like others, we found lower HCy concentrations in fluoride tubes than in EDTA tubes. Although some authors believe that these lower concentrations are caused by dilution of plasma as a result of dehydration of red cells in the presence of fluoride (10), our data showing a much lower rate of increase in fluoride tubes compared with EDTA tubes suggest that this is truly a protective effect of fluoride. Age, sex, vitamin B12, and especially folic acid status are known to be determinants of plasma HCy concentrations (1)(3)(16). Our results were in accordance with the literature. There was a statistically significant negative correlation between serum folate and plasma HCy and between serum vitamin B12 and plasma HCy, but the correlation between age and HCy did not reach statistical significance. We found significantly higher HCy concentrations in men than in women (medians, 9.8 vs 8.3 µmol/L) and reference values (4.7–16.3 µmol/L) similar to, although slightly higher than, those commonly recommended (5–15 µmol/L) (1)(16).
Acknowledgments
This study was financed in part by Abbott Diagnostics Belgium (Louvain-La-Neuve, Belgium).
References
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