Advances in methods for the determination of biologically relevant lipid peroxidation products

Abstract

Lipid peroxidation is recognized to be an important contributor to many chronic diseases, especially those of an inflammatory pathology. In addition to their value as markers of oxidative damage, lipid peroxidation products have also been shown to have a wide variety of biological and cell signalling effects. In view of this, accurate and sensitive methods for the measurement of lipid peroxidation products are essential. Although some assays have been described for many years, improvements in protocols are continually being reported and, with recent advances in instrumentation and technology, highly specialized and informative techniques are increasingly used. This article gives an overview of the most currently used methods and then addresses the recent advances in some specific approaches. The focus is on analysis of oxysterols, F₂-isoprostanes and oxidized phospholipids by gas chromatography or liquid chromatography mass spectrometry techniques and immunoassays for the detection of 4-hydroxynonenal.     

Oxysterol Regulation of Estrogen Receptor α-Mediated Gene Expression in a Transcriptional Activation Assay System Using HeLa Cells

In order to test the estrogenic activity of sterol oxidation products from cholesterol and phytosterols, an estrogen-dependent gene expression assay was performed in estrogen receptor α-stably transformed HeLa cells. The ranking of the estrogenic potency of these compounds was different: 17β-estradiol >> genistein >> β-epoxycholesterol = daidzein = cholestanetriol = 22(R)-hydroxycholesterol = 20(S)-hydroxycholesterol = sitostanetriol > campestanetriol = β-epoxysitosterol = 7β-hydroxycholesterol. These compounds were not estrogenic in estrogen receptor-negative HeLa cells.     

The effects of (22S,23S)-and (22R,23R)-3β-hydroxy-22,23-oxido-5α-ergost-8(14)-en-15-ones on lipid biosynthesis and metabolism of exogenous cholesterol in Hep G2 cells

Novel synthetic oxysterols (22S,23S)-3β-hydroxy-22,23-oxido-5α-ergost-8(14)-en-15-one (I) and (22R,23R)-3β-hydroxy-22,23-oxido-5α-ergost-8(14)-en-15-one (II) efficiently inhibited cholesterol biosynthesis in human hepatoma Hep G2 cells during short-term incubation in a serum free medium (IC₅₀ values of 1.9 ± 0.2 and 0.6 ± 0.2 μ M, respectively). Cultivation of Hep G2 cells in the presence of 5 μM concentration of either (I) or (II) resulted in significant reduction of cholesterol biosynthesis (52% and 57% from control), and also changes in biosynthesis of fatty acids, triglycerides, and cholesteryl esters. Compounds (I) and (II) stimulated transformation of exogenous cholesterol to polar products secreted into the culture medium (156 % and 175% of control) as it that was shown in experiments in Hep G2 cells prelabeled with [³H]cholesterol.     

Oxysterols: Genesis and basic functions

The structural peculiarities of the most widespread oxysterols, the products of oxidative transformations of cholesterol are discussed. The transformations proceed with the participation of enzymatic systems of the body or as a result of various nonenzymatic reactions. The pathways of their formation from cholesterol are also considered. The role of oxysterols in the maintenance of cholesterol homeostasis and in the development of atherosclerosis is reviewed. The possibility of using oxysterols as markers of pathological processes is demonstrated.     

Differential expression and function of ABCG1 and ABCG4 during development and aging

ABCG1 and ABCG4 are highly homologous members of the ATP binding cassette (ABC) transporter family that regulate cellular cholesterol homeostasis. In adult mice, ABCG1 is known to be expressed in numerous cell types and tissues, whereas ABCG4 expression is limited to the central nervous system (CNS). Here, we show significant differences in expression of these two transporters during development. Examination of β-galactosidase-stained tissue sections from Abcg1^−/−LacZ and Abcg4^−/−LacZ knockin mice shows that ABCG4 is highly but transiently expressed both in hematopoietic cells and in enterocytes during development. In contrast, ABCG1 is expressed in macrophages and in endothelial cells of both embryonic and adult liver. We also show that ABCG1 and ABCG4 are both expressed as early as E12.5 in the embryonic eye and developing CNS. Loss of both ABCG1 and ABCG4 results in accumulation in the retina and/or brain of oxysterols, in altered expression of liver X receptor and sterol-regulatory element binding protein-2 target genes, and in a stress response gene. Finally, behavioral tests show that Abcg4^−/− mice have a general deficit in associative fear memory. Together, these data indicate that loss of ABCG1 and/or ABCG4 from the CNS results in changes in metabolic pathways and in behavior.     

The liver antioxidant status of fattening lambs is improved by naringin dietary supplementation at 0.15% rates but not meat quality

Twenty Assaf lambs fed barley straw plus a concentrate alone (CONTROL group) or enriched with naringin (1.5 g/kg DM, NARINGIN group) were used to assess the effect of this polyphenolic compound on meat quality attributes. Serum samples were collected for 7 weeks, then the animals were slaughtered and the livers and longissimus thoracis et lumborum muscles extracted for analysis. Triacylglycerol levels in the serum samples tended to show (P = 0.087) lower average values for the NARINGIN group when compared with the CONTROL, but no differences were observed when the meat was analysed for the intramuscular fat content. Lower thiobarbituric acid-reactive substances procedure (TBARS) values (P < 0.001) in the liver of the NARINGIN group were detected, probably as a consequence of naringenin accumulation in this organ. No significant differences were observed in the meat samples concerning TBARS or colour evolution during refrigerated storage, as not enough naringenin would have reached the muscle. Independent of naringin administration, the low levels of the most atherogenic oxysterols must be highlighted as the most important quality score in the lamb meat samples studied.     

Oxidation as a crucial reaction for cholesterol to induce tissue degeneration: CD36 overexpression in human promonocytic cells treated with a biologically relevant oxysterol mixture

Oxidative stress, inflammation and altered cholesterol metabolism and levels are among the pathogenetic mechanisms of cognitive impairment that may accompany aging. Within the research area of hypercholesterolemia and age-related disease processes, the molecular mechanisms of cholesterol interaction with the inflammatory cells of the macrophage lineage are yet to be elucidated. We thus investigated the effect of both non-oxidized and oxidized cholesterol on monocytic cell differentiation and foam cell formation, as it occurs within vascular lesions during progression of atherosclerosis. In vitro experiments performed on human U937 promonocytic cells showed that a biologically representative mixture of oxysterols markedly stimulated CD36 expression and synthesis. In contrast, non-oxidized cholesterol did not exert any effect on CD36 mRNA and protein levels. Furthermore, the oxysterol-induced up-regulation of CD36 appeared to be based on the subsequent activation of protein kinase Cdelta (PKCdelta), extracellular signal-regulated kinase 1/2 (ERK1/2) and peroxisome proliferator-activated receptor gamma (PPARgamma). Cells overexpressing CD36 were indeed able to actively take up oxidized low-density lipoproteins, and become foam cells. The essential role of ERK pathway and CD36 receptor in oxysterol-induced foam cell formation was proved by the prevention of the latter event when monocytic cells were incubated in the presence of MEK1/2 selective inhibitor or anti-CD36 specific antibody. These experimental findings point to cholesterol oxidation as an essential reaction for this sterol to exert cellular stress and tissue damage in age-related diseases in which inflammation represents a main driving force.     

Brain sterol dysregulation in sporadic AD and MCI: relationship to heme oxygenase-1

The objective of this study was to ascertain the impact of aging and Alzheimer's disease (AD) on brain cholesterol (CH), CH precursors, and oxysterol homeostasis. Altered CH metabolism and up-regulation of heme oxygenase-1 (HO-1) are characteristic of AD-affected neural tissues. We recently determined that HO-1 over-expression suppresses total CH levels by augmenting liver X receptor-mediated CH efflux and enhances oxysterol formation in cultured astroglia. Lipids and proteins were extracted from postmortem human frontal cortex derived from subjects with sporadic AD, mild cognitive impairment (MCI), and no cognitive impairment ( n  = 17 per group) enrolled in the Religious Orders Study, an ongoing clinical-pathologic study of aging and AD. ELISA was used to quantify human HO-1 protein expression from brain tissue and gas chromatography–mass spectrometry to quantify total CH, CH precursors, and relevant oxysterols. The relationships of sterol/oxysterol levels to HO-1 protein expression and clinical/demographic variables were determined by multivariable regression and non-parametric statistical analyses. Decreased CH, increased oxysterol and increased CH precursors concentrations in the cortex correlated significantly with HO-1 levels in MCI and AD, but not no cognitive impairment. Specific oxysterols correlated with disease state, increasing neuropathological burden, neuropsychological impairment, and age. A model featuring compensated and de-compensated states of altered sterol homeostasis in MCI and AD is presented based on the current data set and our earlier in vitro work.     

Marked differences in cholesterol synthesis between neurons and glial cells from postnatal rats

Neurons have a high demand for cholesterol to develop and maintain membrane-rich structures like axons, dendrites and synapses, but it remains unclear, whether they can satisfy their need by costly de novo synthesis. To address this, we compared cholesterol synthesis in serum-free cultures of highly purified CNS neurons and glial cells from postnatal rats. We observed marked cell-specific differences: Compared with glial cells, neurons showed different profiles of biosynthetic enzymes, post-squalene precursors and cholesterol metabolites, and they produced cholesterol less efficiently, possibly because of very low levels of lanosterol-converting enzymes. Astrocytes responded to inhibition of cholesterol synthesis with a much stronger up-regulation of biosynthetic enzymes than neurons. Our results support the idea that neurons cannot produce cholesterol efficiently and that they depend on an external source of this lipid.     

Marked accumulation of 27-hydroxycholesterol in the brains of Alzheimer's patients with the Swedish APP 670/671 mutation

There is a significant flux of the neurotoxic oxysterol 27-hydroxycholesterol (27OHC) from the circulation across the blood-brain barrier. Because there is a correlation between 27OHC and cholesterol in the circulation and lipoprotein-bound cholesterol does not pass the blood-brain barrier, we have suggested that 27OHC may mediate the effects of hypercholesterolemia on the brain. We previously demonstrated a modest accumulation of 27OHC in brains of patients with sporadic Alzheimer's disease (AD), consistent with a role of 27OHC as a primary pathogenetic factor. We show here that there is a 4-fold accumulation of 27OHC in different regions of the cortexes of patients carrying the Swedish amyloid precursor protein (APPswe) 670/671 mutation. The brain levels of sitosterol and campesterol were not significantly different in the AD patients compared with the controls, suggesting that the blood-brain barrier was intact in the AD patients. We conclude that accumulation of 27OHC is likely to be secondary to neurodegeneration, possibly a result of reduced activity of CYP7B1, the neuronal enzyme responsible for metabolism of 27OHC. We discuss the possibility of a vicious circle in the brains of the patients with familial AD whereby neurodegenerative changes cause an accumulation of 27OHC that further accelerates neurodegeneration.