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Technical Briefs |
1
Klinikum rechts der Isar, Institute for Clinical Chemistry and Pathobiochemistry and
2
Klinikum rechts der Isar, Institute for Vascular Surgery, Ismaninger Str. 22, 81675 Munich, Germany;
a author for correspondence: fax 49-89-41404875
Homocysteine (Hcy) is a cytotoxic ((1)), sulfur-containing amino acid that increasingly appears to be an independent risk factor for coronary artery disease, stroke, and peripheral occlusive arterial disease (POAD) ((2)(3)(4)) when present at high plasma concentrations. In addition to several physiological factors that can increase Hcy concentration in blood such as vitamin B6, B12, or folate ((5)) deficiency and renal insufficiency ((6)), there is an artificial increase of Hcy concentration in whole blood after blood collection. This severe preanalytical problem is due to the well-known, time-dependent release of Hcy from erythrocytes ((7)) in the isolated blood sample, even when stabilization is attempted with different additives such as EDTA, NaF, or heparin/NaF ((8)(9)(10)). The result of this release is a falsely increased plasma Hcy concentration, unless plasma is separated from blood cells immediately after blood collection. We describe a method to overcome this problem for making the routine determination of Hcy possible for institutions where sample transport is critical.
For this purpose, we used a specially prepared blood collecting system, where whole blood is lysed and Hcy generating and converting enzymes are inhibited at the time of venipuncture, resulting in a stabilization of Hcy for 2 days at room temperature. Preparation of the blood collecting system for Hcy determination in lysate was performed by adding Nonidet P40 (25 µL, pure), 50 µL of EDTA disodium salt dihydrate (75 g/L), and 25 µL of citric acid monohydrate (610 g/L) to a 2.7-mL EDTA-Monovette (EDTA = 1 g/L blood; Sarstedt), which was then used for blood collection. These concentrations of additives enabled sufficient stabilization and caused minimum dilution of the blood sample. After venipuncture, we recommend vigorously shaking the blood collecting system for 5 s to lyse blood cells. Complete lysis was checked microscopically in several samples.
EDTA blood was used for plasma Hcy determination. Centrifugation of blood at 3000g for 10 min and the separation of plasma from the cellular fraction were performed within 30–60 min of blood collection. Sample preparation of EDTA plasma and lysate was performed as described by Vester and Rasmussen ((11)). The HPLC method was also carried out according to their protocol after minor modification. HPLC measurements of plasma and lysate Hcy were performed by using a C18 reversed-phase 125–4 mm column (Merck LiChrosphere 100, 5-µm particle) with a guard column [RP-18 (5 µm) LiChrosphere 100, 4–4 mm], a gradient pump (Merck, L-6200), an autosampler (Merck, AS-2000), and a fluorescence detector (Merck, F1080). The gradient started with 100% A (acetate buffer, pH 4.2, 20 mL MeOH), which was decreased to 92% over 3 min and then to 88% over the next 2 min at a flow rate of 0.9 mL/min. After an additional 3 min, the system reached its initial conditions; the whole run required 15 min. Eluent B was methanol.
A calibration curve was plotted by using three different concentrations (5, 15, and 40 µmol/L) of homocysteine dissolved in 5 mmol/L EDTA (pH 7) and added to three lysate or plasma samples (standard addition). Stability measurements were performed by aliquoting blood immediately after venipuncture. These aliquots were stored for different time intervals (0 1, 2, 4, 6, 24, and 48 h) at room temperature. Then the samples were frozen at -30 °C. Measurements were performed in triplicate. The CV was <3%. Initial (0 h) lysate Hcy concentrations ranged from 4 to 32 µmol/L.
We determined Hcy concentrations in samples from 136 individuals presenting with carotid stenosis and symptomatic POAD, all of them classified as having 70–99% stenosis by ultrasound and angiography. A group of 39 apparently healthy individuals was recruited from hospital employees. They gave no history of POAD, heart disease, thrombosis, or cerebrovascular disease. Informed consent was given from all individuals and approved by our institution's responsible committee.
The comparison of lysate and plasma homocysteine concentrations was performed with regression analysis according to Passing and Bablok ((12)). The correlation coefficients were calculated with linear regression. Astute®, a statistics program running under Microsoft Excel® version 5.0, was used for statistical analyses.
With the above-mentioned reagent mixture, we succeeded in achieving a sufficient stabilization of lysate Hcy concentration over 48 h at room temperature, and citric acid was necessary to prevent a time-dependent decline of Hcy ranging from 10% to 20% during 48 h. Time-dependent deviations of lysate Hcy concentrations from the initial Hcy concentration ranged from -3.3% to 6.4% over 24 (n = 13) and from -6.1% to 9% over 48 h (n = 10).
To verify the suitability of lysate homocysteine measurements with
HPLC, we determined and compared Hcy concentrations measured in EDTA
plasma and lysed blood from 136 patients with POAD (Fig. 1
).
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Hcy concentrations in lysed blood are lower than in plasma, because of a lower intracellular Hcy concentration, but increase parallel to increasing plasma Hcy concentrations. The squared correlation coefficients resulting from linear regression of plasma and lysed whole blood Hcy concentrations were 0.93 (n = 92) in men and 0.90 (n = 44) in women. The slope of the correlation for women's samples was 0.60 [95% confidence interval (CI), 0.53 to 0.69] and the intercept -0.06 (95% CI, -1.3 to -0.9). For men, the slope was 0.6 (95% CI, 0.54–0.65), and the intercept was -0.38 (95% CI, -1.2 to 0.4). Although an accepted reference range has not yet been defined ((13)), we chose to use the commonly discussed upper plasma Hcy cutoff value for an adult population ((14)) of 15 µmol/L. Because there is general agreement that women have lower Hcy values than men ((15)), we used 15 µmol/L for men and 14 µmol/L for women to calculate the respective lysate Hcy cutoffs from the linear regression equation of the comparison of plasma and lysate Hcy concentrations. The result was 8.3 for women and 8.6 µmol/L for men, which fits well with the 95% CI of our reference group, which is 8.8 and 8.3 µmol/L for women and men, respectively. The mean lysate and plasma Hcy levels for women and men presenting with POAD were 9.0 and 9.5 µmol/L and 15.1 and 16.5 µmol/L, respectively. The women (n = 18) and men (n = 23) from our reference group showed mean values of 6.0 (4.4–10 µmol/L) and 5.9 µmol/L (3.7–9.6 µmol/L) in lysate and 10.3 (4.8–17.4 µmol/L) and 11.6 µmol/L (6.4–15.9 µmol/L) in plasma.
The approximate correction of lysate Hcy concentrations for hematocrit [c(Hcy)/1 - hematocrit] resulted in only a slight improvement of the squared correlation coefficient for the male patient group (0.96) and was slightly poorer in the female patient group (0.87). Thus, we conclude that correction for hematocrit for the interpretation of lysate Hcy values is not necessary for clinical purposes.
In conclusion, an improvement of inter- and intralaboratory comparability of the results and therefore a more reliable atherosclerotic risk assessment might be achieved with this method. The unproblematic handling of blood samples after venipuncture is beneficial for clinicians, general practitioners, and clinical chemists. For this reason, we hold the opinion that the determination of Hcy in lysate is clinically more relevant than the hitherto performed determination of Hcy in plasma. Nevertheless, to verify the above statement, it is necessary to perform prospective studies with a greater number of patients.
Acknowledgments
We are grateful to B. Rohland for skillful assistance and S. Powarzynski for providing us with patient samples. We also thank Dr. M. Page for reading the manuscript and Dr. P. Luppa for valuable advice.
References
The following articles in journals at HighWire Press have cited this article:
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  H. Refsum, A. D. Smith, P. M. Ueland, E. Nexo, R. Clarke, J. McPartlin, C. Johnston, F. Engbaek, J. Schneede, C. McPartlin, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion Clin. Chem., January 1, 2004; 50(1): 3 - 32. [Abstract] [Full Text] [PDF]
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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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K. Gempel, K.-D. Gerbitz, B. Casetta, and M. F. Bauer Rapid Determination of Total Homocysteine in Blood Spots by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry, Clin. Chem., January 1, 2000; 46(1): 122 - 123. [Full Text] [PDF]
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J.-F. Salazar, B. Herbeth, G. Siest, and P. Leroy Stability of Blood Homocysteine and Other Thiols: EDTA or Acidic Citrate? Clin. Chem., November 1, 1999; 45(11): 2016 - 2019. [Full Text] [PDF]
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R. Brandl, R. Probst, B. Muller, S. Powarzynski, P. C. Maurer, and D. Neumeier Evaluation of the Measurement of Lysate Homocysteine in Patients with Symptomatic Arterial Disease and in Healthy Volunteers Clin. Chem., May 1, 1999; 45(5): 699 - 702. [Full Text] [PDF]
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