OVERVIEW OF LIPID PEROXIDATION: FROM LABORATORY TO CLINIC
Corresponding author: Dr Nitin K Gopaul, Department of Experimental Therapeutics, William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, UK. Tel: +44 20 7882 6000 (extn 5784); Fax: +44 20 7882 6016; E-mail: n.k.gopaul@mds.qmw.ac.uk
The chemistry involved in the spontaneous reaction of oxygen with lipids has been the subject of considerable interest since the time of Lavoisier. Free radical-mediated oxidation of the building blocks of lipids, polyunsaturated fatty acids (PUFAs), constitutes the basis of lipid peroxidation. Increased lipid peroxidation is associated with diabetes mellitus (Type I and II), several cardiovascular and respiratory diseases, as well as hepatic cirrhosis and rheumatoid arthritis.
The mechanism of lipid peroxidation can be divided into three stages: initiation, propagation and termination. Initiation normally involves hydrogen abstraction from a weakened C – H bond on carbon atoms adjacent to double bonds in a PUFA, forming a carbon-centred radical. Rearrangement to a conjugated diene followed by reaction with oxygen, produces a highly reactive peroxyl radical which simultaneously propagates the reaction by hydrogen abstraction from another PUFA and forms a lipid hydroperoxide. The hydroperoxide may be reduced to a hydroxy fatty acid or can undergo cyclisation to produce cyclic endoperoxides. These endoperoxides constitute a common starting point in the formation of several biologically active prostaglandins, thromboxanes and leukotrienes (via the cyclooxygenase and lipoxygenase pathways). Non-enzymatic pathways lead to the formation of compounds such as isoprostanes, aldehydes and alkanes, which can also have concentration-dependent signalling or cytotoxic effects in vivo. Formation of these end-products constitute the termination stage of lipid peroxidation. Since PUFAs can have a number of C – H sites susceptible to free radical attack, several end-products can be generated from each PUFA during lipid peroxidation.
Lipid peroxidation plays a major role in the development of atherosclerosis. More specifically, the oxidation of low density lipoprotein (LDL) has been implicated in lesion formation in the aorta. Native LDL can become oxidised in the arterial sub-endothelial space, converting to a minimally oxidised form that stimulates the synthesis of chemotactic and growth factors. Increased monocyte infiltration into the sub-endothelial space and further peroxidation of the lipoprotein generate a highly-oxidised LDL, which becomes pro-atherogenic and exerts significant cytotoxicity. These effects are associated with the increased formation of lipid hydroperoxides, aldehydes and cholesterol oxides. In turn, this can lead to endothelial dysfunction and accelerated atherogenesis. Interestingly, lipid-soluble antioxidants such as vitamin E can attenuate the oxidation of LDL and consequently can minimise or even reverse aortic lesion development and endothelial dysfunction, offering the possibility for intervention in the disease process.
Laboratories involved in lipid research routinely monitor lipid peroxidation by measuring the accumulation of products (conjugated dienes, hydroperoxides, aldehydes, aldehyde-protein adducts, alkanes) or the depletion of substrates (PUFAs, antioxidants). The ability to measure several oxidation products enables assessment at different stages of the oxidative pathway, providing detailed information of this dynamic process. Until recently, the thiobarbituric acid reactive substances (TBARS) test was probably the most utilised assay for measuring lipid peroxidation. Although this approach is still used in some parts of the food industry (for instance, in testing the oxidative susceptibility of cooking oils) and can provide a valuable laboratory measure of lipid peroxidation in relatively simple in vitro systems, its application to complex biological fluids such as human plasma has been limited – primarily because of its non-specificity and the potential confounding influence of oxidised lipids in food. Monitoring lipid peroxidation in clinical situations calls for the use of methods that are sensitive, precise and accurate, and are based on techniques which are robust and have proven inter-laboratory reproducibility. In this context, the measurement of isoprostanes, which are specific peroxidation products of PUFAs, is now recognised as the most reliable index for the assessment of lipid peroxidation in humans and has been used to evaluate the clinical efficiency of antioxidant treatment in several diseases.
Analysis of isoprostanes has been directed mostly towards measurement of the major F2-isoprostane, 8-epi-PGF2a , which is derived from arachidonic acid peroxidation. In humans, 8-epi-PGF2a can be measured in several biological fluids (plasma, urine, synovial fluid), using gas chromatography – mass spectrometry (GC-MS) or commercially available enzyme immunoassay (EIA). EIA provides a sensitive and fairly robust method for 8-epi-PGF2a measurement and because of its relatively low cost compared to GC-MS, is accessible to many more laboratories worldwide. However, issues concerning the reliability of the EIA measurements (mainly because of potential cross-reactivity with other isoprostanes, many of which remain to be identified) are still being resolved, and GC-MS is currently regarded as the 'gold standard'. In terms of its use as a biomarker of lipid peroxidation, 8-epi-PGF2a analysis by GC-MS conforms with the following criteria:
The measurement of lipid peroxidation has therefore expanded from its origins in the research laboratory to practical applications in human healthcare. Excessive lipid peroxidation due to increased oxidative stress associated with for instance, Type II diabetes, may contribute significantly to the accelerated progression of cardiovascular complications in this disease [1]. With Type II diabetes now affecting about 20% of Mauritian adults aged ≥ 30 years, monitoring the 'oxidant status' along with the routine lipid profile could provide an additional tool for evaluating progress during treatment.
[1] Mezzetti A, Cipollone F, Cuccurullo F (2000) Oxidative stress and cardiovascular complications in diabetes: isoprostanes as new markers on an old paradigm. Cardiovascular Research 47: 475-488.