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Technical Briefs |
1
Institute of Vascular Surgery and
2
Institute of Clinical Chemistry and Pathobiochemistry, Ismaninger Strasse 22, 81675 Munich, Germany;
Because of the increasing interest in routine clinical measurement of plasma total homocyst(e)ine (tHcy), it is necessary to simplify the critical preanalytical phase, especially the centrifugation step required immediately after blood collection to separate homocysteine-producing and -releasing blood cells from plasma (1)(2)(3)(4). To overcome this procedural problem, which leads to falsely increased tHcy results when sample transport is prolonged, we recently developed a blood collection system that stabilizes tHcy in lysed whole blood (lysHcy) for at least 2 days at room temperature without requiring a centrifugation step (5). Because of the dilution with plasma with intracellular liquid, the lysHcy concentrations measured in the lysate system are lower than the homocysteine concentrations measured in the tHcy system; therefore, the aim of the present study was to evaluate the measurement of lysHcy in patients with symptomatic arterial disease and healthy volunteers, using the determination of tHcy as a reference method. tHcy and lysHcy determinations were compared with multiple linear regression analysis, taking into account age, and the concentrations of creatinine, folate, and vitamin B12.
We studied 224 consecutive patients (139 men and 85 women; ages 35–93 years) admitted for surgical repair of arterial disease, including 73 patients with high-grade carotid stenosis as classified by ultrasound and angiography according to the criteria of the European Carotid Surgery Trialists' study group (6) and 27 male patients presented with abdominal aortic aneurysm with a maximum diameter exceeding 5 cm. Peripheral arterial occlusive disease (PAOD) was present in 124 patients, 59 of whom suffered from rest pain or gangrene. Healthy volunteers (33 males and 58 females; ages 8–82 years) were recruited mainly from employees of our hospital and reported no history of PAOD, heart disease, diabetes, thrombosis, cerebrovascular disease, or renal impairment. Their routine blood analyses, including hematological investigations, were within the appropriate health-related reference intervals. Of this group, 15% took occasional multivitamin supplements, and their nutritional histories were unremarkable. Informed consent was given by all individuals, and the study was approved by the ethics committee for our institution.
Venous blood for Hcy measurement was collected in tubes containing EDTA or in a specially prepared monovette containing a mixture of a lysing agent, EDTA, and citric acid (5). Serum was collected for measurement of vitamins and creatinine. All samples were taken in the morning, collected on ice, and transported to the laboratory within 60 min. After centrifugation of the EDTA blood, plasma was separated from blood cells and frozen at -30 °C. Lysed whole blood was frozen at -30 °C without any prior treatment.
The HPLC procedures for tHcy and lysHcy were similar (5) and based on the method of Vester and Rasmussen (7). After a reduction step with phosphine and subsequent protein precipitation, the sulfhydryl compounds were derivatized, separated on a RP-18 column (LiChrosphere 100, 5 µm particle size; Merck) and quantified by fluorescence detection (F1080; Merck). Serum creatinine was measured with the phenol-aminophenazone method (Hitachi 747; Boehringer Mannheim), and folate and vitamin B12 were measured with an immunoassay analyzer (Access; Beckman Coulter). The reference intervals were as follows: folate, 6.8–38.6 nmol/L; vitamin B12, 148–703 pmol/L; creatinine, 62–115 µmol/L for males and 44–97 µmol/L for females.
Results were expressed as means ± SD. Differences between groups were tested with the Wilcoxon–Mann–Whitney-U-test. Regression analyses were performed according to Passing and Bablok (8). All statistical calculations were performed with MedCalc® (MedCalc Software).
We found mean tHcy and lysHcy concentrations of 16.3 ± 7.8 and
9.3 ± 4.5 µmol/L for the 224 patients and 11.1 ± 3.5 and
6.5 ± 2.9 µmol/L for the 91 volunteers. As expected, the
differences in tHcy and lysHcy between patients and volunteers were
significant (P <0.005) in both systems (Fig. 1
). There were no significant differences in tHcy or lysHcy
concentrations between the patient subgroups (carotid stenosis,
abdominal aortic aneurysm, or PAOD). Folate did not differ
significantly (P = 0.16) between patients (15.4 ±
7.3 nmol/L) and volunteers (16.1 ± 6.4 nmol/L), whereas vitamin
B12 concentrations were slightly higher (246
± 151 vs 257 ± 87 pmol/L) in the volunteers (P
<0.05). Creatinine was significantly higher (85 ± 49 vs 69
± 13 µmol/L) in patients (P <0.005), who were
significantly older than volunteers, with a mean age of 66 ± 12
years compared with 41 ± 15 years.
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The lysHcy cutoff was 8.5 µmol/L and was calculated from the regression equation [c(lysHcy) = 0.59 + 0.53 c(tHcy)], obtained from linear regression analysis of all tHcy and lysHcy concentrations (n = 315) and the generally accepted tHcy cutoff concentration of 15.0 µmol/L (9).
The tHcy cutoff (15 µmol/L) was exceeded by 42.3% of the patients and 15.4% of the volunteers; the lysHcy cutoff (8.5 µmol/L) was exceeded by 46.8% of the patients and 13.2% of the volunteers. The areas under the ROC curves (AUC) were not significantly different (P = 0.66) for tHcy (AUC, 0.77; 95% confidence interval, 0.72–0.81) and lysHcy (AUC, 0.76; 95% confidence interval, 0.71–0.81).
Multiple linear regression analysis (Table 1
) showed that both tHcy and lysHcy correlated positively with
creatinine (P 
0.005) and age (P <0.005)
and correlated negatively with folate (P <0.005).
Differences in the regression coefficients for lysHcy and tHcy
reflected the different concentration ranges of these systems. Neither
lysHcy nor tHcy depended significantly on vitamin
B12 concentrations. Goodness of fit statistics
for tHcy and lysHcy were 0.249 and 0.250 for
r2.
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Several well-designed cross-sectional and case-control studies have clearly shown evidence that tHcy is a major independent risk factor for PAOD, cardiovascular morbidity, and death (10)(11)(12). The determination of tHcy has been shown to be sensitive to preanalytical handling (1)(2)(3)(4), and in daily routine, the determination of tHcy commonly lacks standardized preanalytical processing conditions. An accurate determination of tHcy is desirable for several reasons. Several studies of carotid and coronary atherosclerosis, myocardial infarction, and venous thrombosis indicate that there is a linear relationship between tHcy concentrations and risk, rather than a threshold value, and that tHcy is pathologically active even at concentrations below the currently discussed cutoff of 15 µmol/L (13)(14). Verhoef et al. (15) and Nygard et al. (12) found that increases of tHcy concentrations of up to 3 or 5 µmol/L produced odds ratios or mortality ratios between 1.35 and 1.9. The mean increase of tHcy in EDTA blood is approximately 10% per hour if blood cells are not separated after blood collection (7)(16). This corresponds diagnostically to an estimated 1.3-fold increase in risk for a patient if blood is left standing for 3 h, given an initial tHcy concentration of 10 µmol/L. To avoid this overestimation of risk, the aim of the present study was to establish the robust lysHcy method (5) for a safe and clinically reliable determination of tHcy for routine clinical use.
We did not use the ROC to derive a cutoff for two reasons: the
sensitivity and specificity for both tHcy and lysHcy are quite low
because of a significant overlap (Fig. 1
) of the respective
distributions, and the two groups are not comparable in age. The
association of tHcy and creatinine, described in 1992 by Chauveau et
al. (17), could be confirmed for both tHcy and lysHcy
determinations (Table 1
). In 13.8% of patients, creatinine
concentrations exceeded the respective cutoff concentrations for males
and females and were accompanied by increased tHcy (20.3 ± 7.8
µmol/L) and lysHcy (11.7 ± 4.4 µmol/L). Of this group, 74%
had tHcy concentrations >15 µmol/L and 70% had lysHcy
concentrations >8.5 µmol/L.
Creatinine concentrations were not increased in any of our volunteers.
Although the folate concentrations were similar in the patients and
volunteers, regression analysis showed that there was a close
relationship between folate and tHcy or lysHcy (Table 1
). The current
lack of commercialized blood collection systems for lysHcy
determination will be overcome in the near future: Bio-Rad Germany
currently is establishing a similar system that uses stabilized lysed
capillary blood instead of lysed venous blood.
In summary, our data show that the prevalence of increased lysHcy is increased in patients with systemic atherosclerosis. Because of the 2-day stability of lysHcy in our blood collection system and the good comparability of tHcy and lysHcy determinations, the latter is the more reliable indicator for atherosclerotic risk assessment in the clinical routine, especially if rapid sample transport from the patient to the laboratory is not guaranteed.
Acknowledgments
This work was supported by a grant from the Kommission Klinische Forschungsprojekte der Technischen Universität München. We thank B. Matthes and S. Kaspar for skillful technical assistance and the performance of measurements, M. Scholz for statistical advice, and P. Luppa and M. Page for valuable advice and critical review the manuscript.
Footnotes
Klinikum rechts der Isar der Technischen Universität München, * author for correspondence: fax 49-89-41404875
References
The following articles in journals at HighWire Press have cited this article:
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  D. M. Hill, L. J. Johnson, P. J. Burns, A. M. Neale, D. M. Harmening, and A. C. Kenney Effects of Temperature on Stability of Blood Homocysteine in Collection Tubes Containing 3-Deazaadenosine Clin. Chem., November 1, 2002; 48(11): 2017 - 2022. [Abstract] [Full Text] [PDF]
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 E. S Ford, S J. Smith, D. F Stroup, K. K Steinberg, P. W Mueller, and S. B Thacker Homocyst(e)ine and cardiovascular disease: a systematic review of the evidence with special emphasis on case-control studies and nested case-control studies Int. J. Epidemiol., February 1, 2002; 31(1): 59 - 70. [Abstract] [Full Text] [PDF]
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