Neven Zarkovic

Pathological aspects of lipid peroxidation.

Abstract Lipid peroxidation (LPO) product accumulation in human tissues is a major cause of tissular and cellular dysfunction that plays a major role in ageing and most age-related and oxidative stress-related diseases. The current evidence for the implication of LPO in pathological processes is discussed in this review. New data and literature review are provided evaluating the role of LPO in the pathophysiology of ageing and classically oxidative stress-linked diseases, such as neurodegenerative diseases, diabetes and atherosclerosis (the main cause of cardiovascular complications). Striking evidences implicating LPO in foetal vascular dysfunction occurring in pre-eclampsia, in renal and liver diseases, as well as their role as cause and consequence to cancer development are addressed.

Clinical Relevance of Biomarkers of Oxidative Stress

Abstract Significance: Oxidative stress is considered to be an important component of various diseases. A vast number of methods have been developed and used in virtually all diseases to measure the extent and nature of oxidative stress, ranging from oxidation of DNA to proteins, lipids, and free amino acids. Recent Advances: An increased understanding of the biology behind diseases and redox biology has led to more specific and sensitive tools to measure oxidative stress markers, which are very diverse and sometimes very low in abundance. Critical Issues: The literature is very heterogeneous. It is often difficult to draw general conclusions on the significance of oxidative stress biomarkers, as only in a limited proportion of diseases have a range of different biomarkers been used, and different biomarkers have been used to study different diseases. In addition, biomarkers are often measured using nonspecific methods, while specific methodologies are often too sophisticated or laborious for routine clinical use. Future Directions: Several markers of oxidative stress still represent a viable biomarker opportunity for clinical use. However, positive findings with currently used biomarkers still need to be validated in larger sample sizes and compared with current clinical standards to establish them as clinical diagnostics. It is important to realize that oxidative stress is a nuanced phenomenon that is difficult to characterize, and one biomarker is not necessarily better than others. The vast diversity in oxidative stress between diseases and conditions has to be taken into account when selecting the most appropriate biomarker. Antioxid. Redox Signal. 23, 1144–1170.

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.

Redox Control of Microglial Function: Molecular Mechanisms and Functional Significance

Neurodegenerative diseases are characterized by chronic microglial over-activation and oxidative stress. It is now beginning to be recognized that reactive oxygen species (ROS) produced by either microglia or the surrounding environment not only impact neurons but also modulate microglial activity. In this review, we first analyze the hallmarks of pro-inflammatory and anti-inflammatory phenotypes of microglia and their regulation by ROS. Then, we consider the production of reactive oxygen and nitrogen species by NADPH oxidases and nitric oxide synthases and the new findings that also indicate an essential role of glutathione (γ-glutamyl-l-cysteinylglycine) in redox homeostasis of microglia. The effect of oxidant modification of macromolecules on signaling is analyzed at the level of oxidized lipid by-products and sulfhydryl modification of microglial proteins. Redox signaling has a profound impact on two transcription factors that modulate microglial fate, nuclear factor kappa-light-chain-enhancer of activated B cells, and nuclear factor (erythroid-derived 2)-like 2, master regulators of the pro-inflammatory and antioxidant responses of microglia, respectively. The relevance of these proteins in the modulation of microglial activity and the interplay between them will be evaluated. Finally, the relevance of ROS in altering blood brain barrier permeability is discussed. Recent examples of the importance of these findings in the onset or progression of neurodegenerative diseases are also discussed. This review should provide a profound insight into the role of redox homeostasis in microglial activity and help in the identification of new promising targets to control neuroinflammation through redox control of the brain.

Pathophysiological relevance of aldehydic protein modifications.

There is growing body of evidence that oxidative stress, i.e. excess in production of reactive oxygen species, can lead to covalent modification of proteins with bioactive aldehydes that are mostly produced under lipid peroxidation of polyunsaturated fatty acids. Thus generated reactive aldehydes are considered as second messengers of free radicals because they react with major bioactive macromolecules, in particular with various humoral and cellular proteins changing their structure and functions. Therefore, the aldehydic-protein adducts, in particular those involving 4-hydroxy-2-nonenal, malondialdehyde and acrolein can be valuable biomarkers of numerous pathophysiological processes. The development of immunochemical methods is increasing the possibilities to study such non-enzymatic protein modifications, on the one hand, while on the other hand the increase of knowledge on bioactivities of the aldehydes and their protein adducts might lead to better prevention, diagnosis and treatments of pathophysiological processes associated with lipid peroxidation and oxidative stress in general. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

Reactive aldehydes – second messengers of free radicals in diabetes mellitus

Abstract Elevated levels of pro-oxidants and various markers of oxidative tissue damage were found in diabetic patients, indicating involvement of oxidative stress in the pathogenesis of diabetes mellitus (DM). On one side, physiological levels of reactive oxygen species (ROS) play an important role in redox signaling of various cells, while on the other, excessive ROS production can jeopardize the integrity and physiological functions of cellular macromolecules, in particular proteins, thus contributing to the pathogenesis of DM. Reactive aldehydes, especially 4-hydroxynonenal (HNE), are considered as second messengers of free radicals that act both as signaling molecules and as cytotoxic products of lipid peroxidation causing long-lasting biological consequences, in particular by covalent modification of macromolecules. Accordingly, the HNE and related reactive aldehydes may play important roles in the pathophysiology of DM, both in the development of the disease and in its progression and complications due to the following: (i) exposure of cells to supraphysiological levels of 4-hydroxyalkenals, (ii) persistent and sustained generation of 4-hydroxyalkenals that progressively affect vulnerable cells that lack an efficient bioactive aldehyde neutralization system, (iii) altered redox signaling influenced by reactive aldehydes, in particular by HNE, and (iv) induction of extracellular generation of similar aldehydes under secondary pathological conditions, such as low-grade inflammation.

An inter-laboratory validation of methods of lipid peroxidation measurement in UVA-treated human plasma samples

Abstract Lipid peroxidation products like malondialdehyde, 4-hydroxynonenal and F2-isoprostanes are widely used as markers of oxidative stress in vitro and in vivo. This study reports the results of a multi-laboratory validation study by COST Action B35 to assess inter-laboratory and intra-laboratory variation in the measurement of lipid peroxidation. Human plasma samples were exposed to UVA irradiation at different doses (0, 15 J, 20 J), encoded and shipped to 15 laboratories, where analyses of malondialdehyde, 4-hydroxynonenal and isoprostanes were conducted. The results demonstrate a low within-day-variation and a good correlation of results observed on two different days. However, high coefficients of variation were observed between the laboratories. Malondialdehyde determined by HPLC was found to be the most sensitive and reproducible lipid peroxidation product in plasma upon UVA treatment. It is concluded that measurement of malondialdehyde by HPLC has good analytical validity for inter-laboratory studies on lipid peroxidation in human EDTA-plasma samples, although it is acknowledged that this may not translate to biological validity.

Adaptation to oxidative stress induced by polyunsaturated fatty acids in yeast.

To create a conditional system for molecular analysis of effects of polyunsaturated fatty acids (PUFA) on cellular physiology, we have constructed a strain of yeast (Saccharomyces cerevisiae) that functionally expresses, under defined conditions, the Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This strain produces up to 15% PUFA, exclusively under inducing conditions resulting in production of 4-hydroxy-2-nonenal, one of the major end products of n-6 polyunsaturated fatty acid peroxidation. The PUFA-producing yeast was initially more sensitive to oxidative stress than the wild-type strain. However, over extended time of cultivation it became more resistant to hydrogen peroxide indicating adaptation to endogenous oxidative stress caused by the presence of PUFA. Indeed, PUFA-producing strain showed an increased concentration of endogenous ROS, while initially increased hydrogen peroxide sensitivity was followed by an increase in catalase activity and adaptation to oxidative stress. The deletion mutants constructed to be defective in the catalase activity lost the ability to adapt to oxidative stress. These data demonstrate that the cellular synthesis of PUFA induces endogenous oxidative stress which is overcome by cellular adaptation based on the catalase activity.

Enzyme-linked immunosorbent assay for 4-hydroxynonenal–histidine conjugates

Highly reactive aldehyde 4-hydroxynonenal (HNE) is the final product of lipid peroxidation, known as a second messenger of free radicals and a signaling molecule. It forms protein conjugates involved in pathology of various diseases. To determine cellular HNE–protein conjugates we developed indirect ELISA based on well-known, monoclonal antibody against HNE–histidine (HNE–His) adducts. The method was calibrated using HNE–albumin conjugates as standards (R2 = 0.999) and validated on human osteosarcoma cell cultures (HOS). The ELISA showed good sensitivity (8.1 pmol HNE–His/mg of protein), precision ( ± 8% intra-assay and ± 12% inter-assay) and spiking recovery ( ± 9%). The assay revealed 60-fold increase of cellular HNE–His adducts upon copper-induced lipid peroxidation of HOS. The ELISA matched HNE-immunocytochemistry of HNE-treated HOS cells and quantified the increase of cellular HNE–His conjugates in parallel to the decrease of free HNE in culture medium. The ELISA was developed as ELISA Stress for severe lipid peroxidation and ELISA Fine for studies on HNE physiology.

Dietary polyunsaturated fatty acids and heme iron induce oxidative stress biomarkers and a cancer promoting environment in the colon of rats

The end products of polyunsaturated fatty acid (PUFA) peroxidation, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE), and isoprostanes (8-iso-PGF2α), are widely used as systemic lipid oxidation/oxidative stress biomarkers. However, some of these compounds have also a dietary origin. Thus, replacing dietary saturated fat by PUFAs would improve health but could also increase the formation of such compounds, especially in the case of a pro-oxidant/antioxidant imbalanced diet. Hence, the possible impact of dietary fatty acids and pro-oxidant compounds was studied in rats given diets allowing comparison of the effects of heme iron vs. ferric citrate and of ω-6- vs. ω-3-rich oil on the level of lipid peroxidation/oxidative stress biomarkers. Rats given a heme iron-rich diet without PUFA were used as controls. The results obtained have shown that MDA and the major urinary metabolite of HNE (the mercapturic acid of dihydroxynonane, DHN-MA) were highly dependent on the dietary factors tested, while 8-iso-PGF2α was modestly but significantly affected. Intestinal inflammation and tissue fatty acid composition were checked in parallel and could only explain the differences we observed to a limited extent. Thus, the differences in biomarkers were attributed to the formation of lipid oxidation compounds in food or during digestion, their intestinal absorption, and their excretion into urine. Moreover, fecal extracts from the rats fed the heme iron or fish oil diets were highly toxic for immortalized mouse colon cells. Such toxicity can eventually lead to promotion of colorectal carcinogenesis, supporting the epidemiological findings between red meat intake and colorectal cancer risk. Therefore, the analysis of these biomarkers of lipid peroxidation/oxidative stress in urine should be used with caution when dietary factors are not well controlled, while control of their possible dietary intake is needed also because of their pro-inflammatory, toxic, and even cocarcinogenic effects.