Covalent modification of DNA by α, β-unsaturated aldehydes derived from lipid peroxidation: Recent progress and challenges

Abstract

Oxidative stress-induced lipid peroxidation (LPO) has been associated with human physiology and pathophysiology. LPO generates an array of oxidation products and among them reactive lipid aldehydes have received intensive research attentions due to their roles in modulating functions of biomolecules through covalent modification. Thus, covalent modification of DNA by these reactive lipid electrophiles has been postulated to be partially responsible for the biological roles of LPO. In this review, we summarized recent progress and challenges in studying the roles of covalent modification of DNA including nuclear and mitochondrial DNA by reactive lipid metabolites from LPO. We focused on the novel mechanistic insights into generation of lipid aldehydes from cellular membranes especially mitochondria through LPO. Recent advances in the technological front using mass spectrometry have also been highlighted in the settings of studying DNA damage caused by LPO and its biological relevance.     

Advances and challenges in understanding the role of the lipid raft proteome in human health

ABSTRACT

Introduction: Phase separation as a biophysical principle drives the formation of liquid-ordered ‘lipid raft’ membrane microdomains in cellular membranes, including organelles. Given the critical role of cellular membranes in both compartmentalization and signaling, clarifying the roles of membrane microdomains and their mutual regulation of/by membrane proteins is important in understanding the fundamentals of biology, and has implications for health.

Areas covered: This article will consider the evidence for lateral membrane phase separation in model membranes and organellar membranes, critically evaluate the current methods for lipid raft proteomics and discuss the biomedical implications of lipid rafts.

Expert commentary: Lipid raft homeostasis is perturbed in numerous chronic conditions; hence, understanding the precise roles and regulation of the lipid raft proteome is important for health and medicine. The current technical challenges in performing lipid raft proteomics can be overcome through well-controlled experimental design and careful interpretation. Together with technical developments in mass spectrometry and microscopy, our understanding of lipid raft biology and function will improve through recognition of the similarity between organelle and plasma membrane lipid rafts and considered integration of published lipid raft proteomics data.     

Extraction of lipids from mammalian liver using nontoxic solvents

A simple method for extracting and purifying lipids from rat liver in a single step using nontoxic solvents is described. The method consists of homogenizing the puliverized tissue with a mixture of tricholotrifluoroethane (Cl₂CF-CClF₂) and isopropyl alcohol ($\text{1 : 1,}\text{v}\text{v}$). Just enough water is added to the lipid extract to produce a biphasic system. Pure lipid extract is obtained by isolating the lower layer from the aqueous upper phase which contains the non-lipid materials. The described method compares favourably with that of Folch et al., both quantitatively and qualitatively. The solvent system used also has the advantage of being less toxic than the widely used chloroform/methanol system, which makes it safer for prolonged use. The new method is simple, efficient and reproducible.     

Molecular mechanism of action of chlorogenic acid on erythrocyte and lipid membranes

Abstract

The high antioxidant capacity of chlorogenic acid (CGA) in respect to biological systems is commonly known, though the molecular mechanism underlying that activity is not known. The aim of the study was to determine that mechanism at the molecular and cell level, in particular with regard to the erythrocyte and the lipid phase of its membrane. The effect of CGA on erythrocytes and lipid membranes was studied using microscopic, spectrophotometric and electric methods. The biological activity of the acid was determined on the basis of changes in the physical parameters of the membrane, in particular its osmotic resistance and shapes of erythrocytes, polar head packing order and fluidity of erythrocyte membrane as well as capacity and resistivity of black lipid membrane (BLM). The study showed that CGA becomes localized mainly in the outer part of membrane, does not induce hemolysis or change the osmotic resistance of erythrocytes, and induces formation of echinocytes. The values of generalized polarization and fluorescence anisotropy indicate that CGA alters the hydrophilic region of the membrane, practically without changing the fluidity in the hydrophobic region. The assay of electric parameters showed that CGA causes decreased capacity and resistivity of black lipid membranes. The overall result is that CGA takes position mainly in the hydrophilic region of the membrane, modifying its properties. Such localization allows the acid to reduce free radicals in the immediate vicinity of the cell and hinders their diffusion into the membrane interior.     

Oxysterols in cap and core of human advanced atherosclerotic lesions

Objective: Different parts of the advanced atherosclerotic lesion have characteristic differences in lipid content, but the distribution of lipid oxidation products has not been reported. This study provides novel data on oxysterol and hydroxyoctadecadienoic acids quantification in core versus cap. It compares the lipid composition of core and cap to assess the topographical distribution of evidence of lipid oxidation.

Methods: Lipids and oxidised lipids were analysed by gas chromatography (GC) and GC-mass spectrometry (GC-MS) in samples of human atheromatous lipid core and fibrous cap of individual advanced atherosclerotic plaques (Stary, Type V) in necropsy samples.

Results: The total lipid was of course massively greater in the core than in the cap. The oxidation products, cholest-5-en-3β,26-diol (26-OH-CHOL) and cholest-5-en-3β,7β-diol (7β-OH-CHOL) were detected in all the samples. 26-OH-CHOL was more abundant in the core than in the cap when related both to wet weight and to cholesterol. 7β-OH-CHOL levels were significantly higher in the core than in the cap when related to wet weight but not when related to cholesterol. Because the processing included a sodium borohydride reduction step, the 7β-OH-CHOL detected could partly originate from 7-ketocholesterol or 7-hydroperoxy-cholesterol. Several isomeric hydroxyoctadecadienoic acids were detected in both core and cap, more in the cap when related to cholesterol content. Most of the components of the cap showed a high degree of cross-correlation on linear regression analysis, but cross-correlations were weaker for the core. The core samples contained a larger proportion of linoleate relative to oleate than the fibrous cap.

Conclusion: The findings suggest that the different lipid and oxidised lipid contents of cap and core may be due to variations in oxidative activity in different parts of the lesion.     

The influence of an antitumor lipid – erucylphosphocholine – on artificial lipid raft system modeled as Langmuir monolayer

Abstract

Outer layer of cellular membrane contains ordered domains enriched in cholesterol and sphingolipids, called ‘lipid rafts’, which play various biological roles, i.e., are involved in the induction of cell death by apoptosis. Recent studies have shown that these domains may constitute binding sites for selected drugs. For example alkylphosphocholines (APCs), which are new-generation antitumor agents characterized by high selectivity and broad spectrum of activity, are known to have their molecular targets located at cellular membrane and their selective accumulation in tumor cells has been hypothesized to be linked with the alternation of biophysical properties of lipid rafts. To get a deeper insight into this issue, interactions between representative APC: erucylphosphocholine, and artificial lipid raft system, modeled as Langmuir monolayer (composed of cholesterol and sphingomyelin mixed in 1:2 proportion) were investigated. The Langmuir monolayer experiments, based on recording surface pressure-area isotherms, were complemented with Brewster angle microscopy results, which enabled direct visualization of the monolayers structure. In addition, the investigated monolayers were transferred onto solid supports and studied with AFM. The interactions between model raft system and erucylphosphocholine were analyzed qualitatively (with mean molecular area values) as well as quantitatively (with ΔG^exc function). The obtained results indicate that erucylphosphocholine introduced to raft-mimicking model membrane causes fluidizing effect and weakens the interactions between cholesterol and sphingomyelin, which results in phase separation at high surface pressures. This leads to the redistribution of cholesterol molecules in model raft, which confirms the results observed in biological studies.     

Luminescence in the study of lipid metabolism

Using bioluminescence assays for glycerol, free fatty acids, β-hydroxybutyrate and lactate, we were able to perform complex studies of human energy and lipid metabolism both in serum samples in vivo and in isolated fat cells in vitro. These studies would have been impossible without reliable, specific and highly sensitive luminescence methods. Oxidatively modified low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. Adaptation of a chemiluminescence assay for lipid hydroperoxides to LDL isolated by specific precipitation from serum makes it possible to measure LDL oxidation in vivo. Cell dependent chemiluminescence was used to investigate whether receptor mediated endocytosis of LDL by macrophages leads to oxygen radical production in these cells. No activation of the membrane NAD(P)H oxidase was observed.     

用鞘磷脂作为标志分子鉴定脂筏

脂筏是质膜双层中富含鞘脂、胆固醇及特殊蛋白质的质膜微区.对其功能的研究,首先要对其进行分离和鉴定.常利用密度梯度超速离心将其分离,然后以脂筏中富含的神经节苷脂GM₁作为标志分子,利用荧光或生物素标记的霍乱毒素-B亚基进行亲和标记来鉴定脂筏.但这一鉴定方法操作复杂、费时、易对环境造成污染,所用关键试剂霍乱毒素不易获得,再加上一些组织GM₁含量甚微或不含GM₁,使其应用受到局限.为建立一个特异性高又对各种组织广泛适应的脂筏鉴定方法.对两种细胞系脂筏的脂类组分进行了分析.结果发现,可用鞘磷脂作为脂筏的特异性标志分子,采用高效薄层层析技术对脂筏进行鉴定.     

富油微藻的筛选及其产油能力的评价

【目的】筛选具有较快生长速率及较强产油能力的微藻,探究所获得微藻的生理生化性能及不同培养方式对其生物量、产油能力、碳消耗等生长特性的影响与藻种对pH的适应能力。【方法】通过磷酸香草醛测定法及尼罗红染色对微藻进行初筛复筛,通过设置光合自养、异养和混养等3种培养方式,并采用气质联用等方法,研究不同培养方式对所获微藻生长特性、所产油脂脂肪酸组成以及碳代谢等方面的影响。【结果】筛选出两株产油能力较强的藻株H、Z_8,其油脂产量分别可达1.14±0.05 g/L和1.33±0.10 g/L,经形态观察和分子生物学鉴定初步表明藻株H属布朗单针藻(Chlorolobion braunii)、藻株Z_8属链带藻(Desmodesmus intermedius)。构建了不同培养方式下微藻动力学模型,H、Z_8属于生长偶联型。当培养环境的pH处于6.0–9.0,对藻株H、Z_8的总脂量与生物量无明显差异(P0.05)。【结论】筛选获得的藻株H、Z_8中C16与C18脂肪酸占总脂肪酸的比率能达到90%以上。藻种在混养条件下生物量积累优于异养,但异养条件下更加有利于油脂的积累,且H、Z_8均具有较为宽泛的pH适应能力,是具有一定产业化应用潜力的优良产油藻株。     

Kukoamine A analogs with lipoxygenase inhibitory activity

Kukoamine A (KukA) is a spermine (SPM) conjugate with dihydrocaffeic acid (DHCA), with interesting biological activities. The four possible regioisomers of KukA, as well as a series of KukA analogs incorporating changes in either the SPM or the DHCA structural units, were evaluated for their antioxidant activity and their inhibitory activity on soybean lipoxygenase (LOX) and lipid peroxidation. The reducing properties of the compounds were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay and found to be in the range 5–97.5%. KukA significantly inhibits LOX with IC₅₀ 9.5?μM. All tested analogs inhibited lipid peroxidation in the range of 11–100%. The most potent compounds KukA and its analog 3, in which the DHCA units had been replaced by O,O9-dimethylcaffeic acid units, were studied for their anti-inflammatory activity in vivo on rat paw edema induced by carrageenan and found to be of comparable activity to indomethacin. The results of the biological tests are discussed in terms of structural characteristics.     

Trioxsalen derivatives with lipoxygenase inhibitory activity

Trioxsalen (TRX) is a 4,5′,8-trimethylated psoralen analog presenting interesting biological activities when irradiated with UVA light. A series of TRX derivatives, which where obtained by its chemical modification and incorporation of a variety of unsaturated functions at position 4′ of the psoralen ring-system, were evaluated for their antioxidant activity and their inhibitory activity on soybean lipoxygenase (LOX) and lipid peroxidation. The reducing properties of the compounds were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay and found to be very low, in the range 0–14%, with the exception of the hydroxamic acid 6 which showed almost identical activity to BHT. TRX derivative 3 significantly inhibited LOX, with IC₅₀ 9.4?μM. With the exception of TRX, all tested analogs inhibited lipid peroxidation in the range of 35–91%. The most potent compound, namely TRX derivative 3, was studied for its anti-inflammatory activity in vivo on rat paw edema induced by carrageenan, and was found to be of almost identical activity to indomethacin. The results of the biological tests are discussed in terms of structural characteristics.     

Resolving the kinetics of lipid,protein and peptide diffusion in membranes

Abstract

Recent developments in the understanding of molecular diffusion phenomena in membranes are reviewed. Both model bilayers and biological membranes are considered in respect of lateral diffusion, rotational diffusion and transverse diffusion (flip-flop). For model systems, particular attention is paid to recent data obtained using surface-specific techniques such as sum frequency generation vibrational spectroscopy on supported lipid bilayers, and fluorescence correlation spectroscopy on giant unilamellar vesicles, both of which have yielded new insights into the intrinsic rates of diffusion and the energetic barriers to processes such as lipid flip-flop. Advances in single-molecule and many-molecule fluorescence methodologies have enabled the observation of processes such as anomalous diffusion for some membrane species in biological membranes. These are discussed in terms of new models for the role of membrane interactions with the cytoskeleton, the effects of molecular crowding in membranes, and the formation of lipid rafts. The diffusion of peptides, proteins and lipids is considered, particularly in relation to the means by which antimicrobial peptide activity may be rationalized in terms of membrane poration and lipid flip-flop.     

An evaluation of lipid extraction techniques for interpretation of carbon and nitrogen isotope values in bottlenose dolphin (Tursiops truncatus) skin tissue

We studied the effects of two common chemical extraction techniques on bottlenose dolphin (Tursiops truncatus) skin tissues with the intent to develop a mathematical lipid correction for dolphin skin δ¹³C. One method employs a hot solvent mixture (chloroform and methanol) while the other method requires washing the samples with cold solvent followed by water. The water wash method resulted in significant alteration of tissue δ¹⁵N. We found no correlation between change in sample mass and C/N or between change in sample mass and the change in δ¹³C (Δδ¹³C) following lipid extraction. Although Δδ¹³C was positive following lipid extraction (mean = 1.6‰ and 1.2‰, for the two methods), there was no correlation between C/N and Δδ¹³C for either method. Cumulatively, these results prevented us from applying a mathematical lipid normalization. Based on our findings and consideration of previously reported results, we suggest that applying these extraction techniques to dolphin skin with C/N < 4.5 introduces greater uncertainty than is warranted. We recommend against lipid correction for dolphin skins with C/N < 4.5, but stress that the resulting uncertainty in δ¹³C needs to be accounted for when implementing isotope mixing models to assess diet or organic matter sources.     

Expanding the horizons of lipidomics. Towards fluxolipidomics

Abstract

This short review takes into consideration the status of lipidomics as issued from almost a decade of development. Because of the huge number of molecular species analyzed, there is a trend in subdividing lipidomics according to subdomains, in particular relating to the function of molecules. It is also pointed out that lipid imaging without the use of exogenous probes will help making relationships between molecular structures and the topography of lipid assemblies, especially in cellular compartments. Finally, a fluxomics approach is proposed for lipid molecular species, both in terms of compartments and biochemical metabolism. The example of fluxolipidomics of essential fatty acids toward their enzyme-dependent oxygenated metabolites and further toward their degradation products is developed.     

Serum RBP4 positively correlates with triglyceride level but not with BMI,fat mass and insulin resistance in healthy obese and non-obese individuals

Abstract

Purpose: Retinol binding protein 4 (RBP4) has recently been identified as an adipokine possibly involved in the development of impaired glucose metabolism. We aimed to test serum RBP4 in healthy non-obese individuals and in patients with well-characterized phenotype: obesity without confounding effects of diabetes, metabolic syndrome or dyslipidaemia. Additionally, we examined whether serum RBP4 is associated with anthropometric parameters, insulin resistance and blood lipid parameters.

Patients and methods: Twenty-eight patients with obesity and no co-morbidities and twenty-five age-matched lean controls were recruited. Anthropometric parameters, body composition, fasting blood lipid profile, RBP4, glucose and insulin were assessed and HOMA-IR was calculated.

Results: Mean concentration of RBP4 did not differ between studied groups (in obese patients was 33.93?±?4.46?µg/ml and 32.53?±?2.53?µg/ml in non-obese controls). RBP4 positively correlated with serum triglycerides in obese and non-obese individuals (r?=?0.74, p?=?0.03 and r?=?0.62, p?=?0.02, respectively) and did not show any significant associations with HOMA-IR, anthropometric and body composition parameters.

Conclusions: Excessive adiposity without co-morbidities is not associated with higher levels of circulating RBP4. Serum RBP4 cannot be considered as a direct predictive marker for impaired glucose metabolism. RBP4 possibly contributes to lipid metabolism.     

高内涵脂滴三维影像定量分析研究细胞内的脂质储存

摘要 目的：研究细胞内脂滴含量的变化对肥胖、糖尿病等代谢性疾病发生发展的影响。方法：建立高内涵脂滴三维成像和定量分析系统,获得脂滴三维动态表型参数,例如细胞内脂滴的总体积量、脂滴平均体积、单一细胞内脂滴平均数量等指标。选择HeLa、AML-12、COS-7和3T3-L1四种细胞系进行油酸、基因沉默、酶活性抑制剂的处理,量化处理后四种细胞内的脂滴数量与大小的表型差异。结果：在加入油酸情况下,细胞随油酸浓度增加而生成更多、更大的脂滴,但AML-12细胞只有展现增加脂滴数量的变化表型;在HeLa细胞中进行19种中性脂合成通路上关键基因的转录表达沉默,发现需要同时双敲降两种甘油三酯合成酶DGAT1和DGAT2才能显着降低细胞内脂滴总体积储存量,但在COS-7细胞中只需要单敲降DGAT1即可降低脂滴存量;进一步使用了DGAT1/2抑制剂处理四种细胞后,发现对抑制剂响应可区分为两类细胞分组（HeLa、AML-12与COS-7、3T3-L1）的脂滴存量表型差异,其原因是DGAT1和DGAT2的转录表达谱在这两类细胞分组中的不同。结论：建立了高内涵脂滴三维成像和定量分析系统,量化了四种细胞系的脂滴数量与大小的表型差异,揭示了细胞的脂滴脂储存方式与蛋白酶表达谱的关系。     

Combined use of periodic reaction field and coarse-grained molecular dynamics simulations. I. phospholipid monolayer systems

Abstract

A periodic reaction field based on a linear-combination-based isotropic periodic sum (LIPS) method was applied for coarse-grained molecular dynamics simulations of zwitterionic lipid systems. In phospholipid monolayer systems with various number of lipid molecules, the density profile, lipid orientation and surface tension were mainly calculated using the periodic reaction field and Ewald sum. The results from the periodic reaction field were almost equal to that from the Ewald sum. It is concluded that the periodic reaction field method has a great possibility to provide a high accuracy in determining coarse-grained zwitterionic lipid systems.     

Oxygen concentration dependence of lipid peroxidation and lipid-derived radical generation: Application of profluorescent nitroxide switch

Abstract

Lipid-derived radicals and peroxides are involved in the pathogenesis of oxidative stress diseases and, although lipid peroxide production is a required reaction between a lipid radical and molecular oxygen, a useful lipid radical detection method has remained tentative. Also, the effect of oxygen concentration on lipid peroxide production must be considered because of the hypoxic conditions in cancer and ischemic regions. In this study, the focus was on nitroxide reactivity, which allows spin trapping with carbon-centred radicals via radical–radical reactions and fluorophore quenching through interactions with nitroxide's unpaired electron. Thus, the aim here was to demonstrate a useful detection method for lipid-derived radicals as well as to clarify the effects of oxygen concentration on lipid peroxide production using profluorescent nitroxide. This latter compound reacted with lipid-derived radicals in a manner inversely dependent on oxygen concentration, resulting in fluorescence due to alkoxyamine formation and, conversely, lipid peroxide concentrations decreased with lower oxygen in the reaction system. Furthermore, nitroxide inhibited lipid peroxide production and stopped oxygen consumption in the same solution. These results suggested that the novel application of profluorescent nitroxide could directly and sensitively detect lipid-derived radicals and that radical and peroxide production were dependent on oxygen concentration.     

Lanosterol Analogs: Dual-Action Inhibitors of Cholesterol Biosynthesis

Abstract

Deoxyribonucleic acid is one of the most interesting and also the most complex of all biological macromolecules. Paradoxically, this complexity arises from the simplicity of its basic subunit structure. A large eukaryotic chromosome probably contains a single chain of DNA with a molecular weight in excess of 10¹¹ daltons and is composed of a linear permutation of the four basic deoxyribonucleotides. Until recently, this fact posed considerable problems for the biochemist interested in isolating specific fragments of chromosomes as the methods available were nonspecific in nature. This is no longer so; the discovery of site specific endodeoxyribonucleases (restriction endo-nucleases) has opened new routes to the analysis of DNA structure and function and promises a revolution in molecular biology. A new field of genetic engineering is already being pioneered, and significant advances in many areas have been facilitated by the availability of these enzymes.