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Influence of citrate and EDTA anticoagulants on plasma malondialdehyde concentrations estimated by high-performance liquid chromatography

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Abstract

Estimation of lipid peroxidation through MDA formation measured by assaying thiobarbituric acid (TBA) reactive products separated by HPLC remains the method of choice to study the development of oxidative stress in blood plasma. In this report we describe the influence of citrate and EDTA anticoagulants used for blood collection on estimation of MDA concentrations using HPLC analysis of MDA-TBA adducts. We analyzed a group of 40 blood donors (21 men and 19 women), median age 27 years, range 19–48 years. The mean MDA concentration in citrate plasma was 1.43±0.51 μmol/l (range: 0.61–2.57 μmol/l) and in EDTA plasma 0.36±0.10 μmol/l (range: 0.13–0.63 μmol/l). There was a significant difference in MDA mean concentration that we attribute to different antioxidant properties of anticoagulants used for blood collection. Consistency in the choice of anticoagulant is clearly extremely important.

Introduction

Malondialdehyde (MDA) is generally formed as a consequence of lipid peroxidation and/or of prostaglandin’s metabolism [1], [2]. MDA is reactive toward amino groups of proteins and nucleic acids, it has been inferred to have mutagenic and cytotoxic roles, and possibly to be a participant in the onset of atherosclerosis. When heated at acidic pH, MDA reacts with a number of nucleophiles to yield a variety of condensation/dehydration products [3]. The 2-thiobarbituric acid (TBA) yields at these conditions with MDA red fluorescent pigment (MDA-2TBA) widely used in clinical chemistry laboratories for the spectrophotometric or spectrofluorimetric measurement of MDA amounts [4].

Many factors (e.g., stimulus for and conditions of peroxidation) modulate MDA formation from a lipid [3]. TBA-test reagents and constituents have profound effects on the test response to the MDA. As pointed out by Cordova et al. [5], both the acidification and the boiling of plasma samples, essential procedures in TBA method, facilitate autoxidation of polyunsaturated fatty acids (PUFA). Some lipid may still be present in samples, even after removal by precipitation with proteins. Therefore, addition of antioxidant (butylated hydroxytoluene–BHT) to the reaction mixture is essential for inhibition of lipid oxidation without affecting the formation of the MDA-2TBA chromogen [6]. The TBA test is intrinsically non-specific for MDA; non-lipid-related materials as well as fatty peroxide-derived decomposition products other than MDA are TBA positive [7] which results in overestimating of MDA levels.

HPLC techniques were proposed for determining MDA in which condensation compound of MDA and TBA is separated from interfering substances by chromatography on a column of octadecyl silica gel [8], [9], [10], [11], [12], [13], [14], [15]. HPLC methods generally yield lower values than those based on direct measurements, and this would seem to reinforce their specificity.

Sample preparative steps constitute the other pitfalls responsible for the lack of specificity and the poor reproducibility of the technique in complex biological fluids and for the large variability of results among techniques. One of these preparative steps influencing MDA yields in the early stages of sample preparation is the choice of an anticoagulant for blood collection.

Lepage et al. [16] suggested blood collection under EDTA anticoagulant for plasma MDA (P-MDA) determination to prevent autoxidation since platelets are rich in lipoperoxides and appear to be the main source of plasma MDA. Wong et al. [13] used the same conditions for blood collection. They found that malondialdehyde concentrations were increased by 1.5 to 2 times after 1 h storage of serum or of plasma specimens collected with heparin or citrate and stored at 4°C. In addition, Knight et al. [17] compared MDA concentration in plasma from 230 male and 148 female adult blood donors. They showed that man had higher MDA concentrations in plasma when compared with women, older donors had higher values than younger, and plasma from EDTA-anticoagulated blood had significantly lower MDA concentrations than does plasma from blood treated with sodium citrate. Nielsen et al. [18] compared MDA concentrations in serum and plasma from 13 fasting volunteers (seven men and six women, ages 20–33 years). Plasma samples were collected in Venoject tubes with different anticoagulants. They used TBA derivatization of MDA in samples and HPLC analysis of MDA-2TBA. Comparable concentrations of MDA were found both in serum and plasma anticoagulated either by citrate or heparin and much lower concentrations of MDA were found in EDTA anticoagulated plasma (Table 1). In contrast, Wasowicz et al. [19] analyzed spectrofluorimetrically thiobarbituric acid-reactive substances (TBARS) in plasma collected in EDTA and estimated TBARS concentrations (in MDA equivalents) by 30% significantly higher than from serum and heparinized plasma. In this paper we examined the influence of anticoagulants used in blood collection on MDA concentrations estimated by HPLC of MDA-2TBA.

Section snippets

Chemicals and reagents

Methanol (LiChrosolv grade) was supplied by Merck (Darmstadt, Germany). Water was double glass-distilled and deionized on Milli-Q50 (Millipore, Vienna, Austria), 1,1,3,3-Tetraethoxypropane (TEP), and butylated hydroxytoluene (BHT) were supplied by Sigma–Aldrich (Prague, Czech Republic). All other chemicals were of analytical grade.

Sample preparation

Blood was collected in parallel by venipuncture into polypropylene tubes, first to EDTA, and subsequently to citrate anticoagulant. Final citrate concentration in

Results and discussion

The analytical conditions used for alkaline hydrolysis of bound MDA and for chromatographic analysis were the same for both EDTA and citrate plasma. Enhanced buffer concentration (100 mmol/l) used in mobile phase facilitated the column equilibration of sample containing 1.6 mol/l acetate buffer, pH 3 (final values). The chromatograms of MDA-2TBA obtained from MDA standard in water and either citrate or EDTA anticoagulated plasma are shown in Fig. 1. The limit of the MDA detection was estimated

Acknowledgments

This work was supported by grants 4809-3 and 4642-3 from IGA MZ (Prague, Czech Republic).

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