Improved method for plasma malondialdehyde measurement by high-performance liquid chromatography using methyl malondialdehyde as an internal standard

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Abstract

Measurement of malondialdehyde (MDA) is an important contribution to the assessment of oxidative stress. We report a sensitive and reproducible high-performance liquid chromatography (HPLC) method for measurement of plasma MDA in the assessment of lipid peroxidation. Using methyl malondialdehyde (Me-MDA) as an internal standard with reversed-phase HPLC and UV detection and derivatisation with 2,4 dinitrophenylhydrazine (DNPH), we obtained maximum MDA values with 60-min incubation of 10% plasma with 1 M NaOH at 60 °C. The dilution of the plasma and a longer incubation time in the alkaline hydrolysis step greatly improved recovery of MDA from its bound form. Ratios of peak height of MDA/Me-MDA were linear over a range of 0–100 μM with correlation coefficients >0.99. The recovery was 88.5%. Within and between run variations were <4 and <7%, respectively. The mean MDA value measured in 20 healthy volunteers was 13.8 μM (±1.32).

Introduction

Tissue damage induced by reactive oxygen species has been implicated in the pathogenesis of cardiovascular and renal disease, and diabetes [1], [2], [3]. The break down of lipid peroxides in the biological system produces a number of aldehydes, including 4-hydroxylnonenal (4HNE) and malondialdehyde (MDA) [4]. These aldehydes are relatively stable and have been shown to be cytotoxic and genotoxic by reacting with proteins and nucleic acids [5].

MDA is in many instances the most abundant individual aldehyde resulting from lipid peroxidation. The assay of MDA using thiobarbituric acid (TBA) is commonly employed in lipid peroxidation studies despite the fact that the TBA assay is not specific for MDA [2], [5], [6]. It has been suggested that an additional high-performance liquid chromatography separation step might improve the assay [2], [5], [7], [8], [9]. However, the assay is still hindered by the harsh conditions used in sample preparation: it involves heating at 96 °C for 1 h at low pH and this could create artefactual intermediates which may form identical MDA-TBA adducts [2], [10], [11]. In addition, the results of the TBA assay often vary widely in different experimental conditions [12], [13].

To overcome the shortcomings of the TBA assay, other means of MDA derivatisation, which could be carried out in milder conditions, have been used. These include diaminonaphthalene (DAN) in acidic medium at 37 °C [14], 1-methyl-2-phenylindole at 45 °C [15] and 2,4-dinitrophenylhydrazine (DNPH) at room temperature [10], [13]. More recently, methods using gas chromatography–mass spectrometry (GC–MS) [11], [16], [17] have also been proposed. These techniques are considered the most reliable. However, they require extensive sample preparation and GC–MS is not always readily available in clinical laboratories.

The use of a stable isotope-labeled internal standard has proved to be successful in the determination of MDA using MS methods. There is little information on a suitable internal standard for other methods. However, Claeson et al. [18] recently reported the potential use of methyl malondialdehyde (Me-MDA) as an internal standard in the determination of MDA in rat brain homogenates using different techniques, including HPLC. In the present study, we have established the use of methyl malondialdehyde (Me-MDA) as an internal standard for the determination of MDA in human plasma [18]. We used a modified HPLC method based on the derivatisation of MDA with 2,4-dinitrophenylhydrazine (DNPH) as reported by Pilz et al. [13]. The hydrazones formed readily at room temperature in mild acid conditions, are unique for a given aldehyde, and can be separated easily by HPLC.

Section snippets

Chemicals

1,1,3,3-Tetraethoxypropane (TEP, 97%), 2,4-dinitrophenylhydrazine (DNPH), 3-dimethylamino-2-methyl-2-propenal (DMP) and 2-thiobarbituric acid were from Sigma–Aldrich (Sigma–Aldrich Pty Ltd, Australia). The 2,4-dinitrophenylhydrazine was prepared as a 5 mM solution in 2 M hydrochloric acid. Hydrochloric acid, 36% was from Ajax (Ajax Chemicals, Australia). Lipid peroxidation (LPO) assay kit, Cat. No. 437634 was from Calbiochem (Calbiochem-Novabiochem Pty Ltd, Australia).

Sample collection

Peripheral blood samples

HPLC separation of the DNPH derivatives

Fig. 1 shows the chromatographic separation of DNPH-derived MDA and Me-MDA in water and plasma. In the reagent blank, both MDA and Me-MDA peaks are absent as seen in Fig. 1A. Fig. 1B shows the presence of endogenous MDA eluting at 12.5 min in a plasma sample. Fig. 1C shows the chromatogram when the same plasma sample was spiked with Me-MDA, which elutes at 22.5 min. As seen in the chromatogram, the MDA and Me-MDA peaks are well separated and symmetrical. Fig. 1D is the same sample as in C,

Discussion

Determination of MDA is commonly used for monitoring lipid peroxidation in biological samples. However, the estimation of MDA in plasma is difficult due to the complex matrix. The TBA method, although easy to use, is not specific and often gives results that are not reproducible [20]. The initial objective of our study was to develop a suitable assay for MDA detection, which could include an internal standard without the problems of the TBA assay. We preferred derivatisation of MDA with DNPH

Acknowledgments

We are grateful to Dr Xingli Wang for reviewing the manuscript and to Natalia Duarte and Paul Stathakis for technical assistance.

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