Lipid peroxidation markers in children with anxiety disorders and their diagnostic implications

Abstract

Objective

Numerous factors, including genetic, neurobiological, neurochemical, and psychological factors, are thought to be involved in the development of anxiety disorders. The latest findings show that the pathophysiology of anxiety disorders might be associated with oxidative stress and lipid peroxidation; however, no studies have so far investigated lipid peroxidation markers in children with anxiety disorders. Serum levels of lipid hydroperoxide (LOOH) are a reliable marker of lipid peroxidation. Paraoxonase and arylesterase are two enzymes that protect against such peroxidation, and might also be diagnostic markers. In this study, we investigated whether there are associations between anxiety disorders and lipid peroxidation markers in children, and assessed the diagnostic performance of these markers.

Methods

The study group consisted of 37 patients (children and adolescents) with anxiety disorders. A control group, matched for age and gender, was composed of 36 healthy subjects. Venous blood samples were collected, and LOOH levels and paraoxonase and arylesterase activity were measured.

Results

LOOH levels were significantly higher in the anxiety disorders group than in the control group. There were no significant differences in paraoxonase or arylesterase activities between the patient and the control groups.

Discussion

Lipid peroxidation or oxidative damage might play a role in the aetiopathogenesis of anxiety disorders. LOOH may be a potential biological marker for anxiety disorders in children.     

Lipid peroxidation in erythrocytes

Erythrocytes might be expected to be highly susceptible to peroxidation. Their membranes are rich in polyunsaturated fatty acids; they are continuously exposed to high concentrations of oxygen; and they contain a powerful transition metal catalyst. In fact, autoxidation is held in check in vivo by extremely efficient protective antioxidant mechanisms. These involve cellular enzymes such as superoxide dismutase and glutathione peroxidase, as well as vitamin E; but they mainly reflect effective structural compartmentalisation. This review surveys mechanisms which lead to red cell lipid autoxidation and the role of haemoglobin in these processes. The influence of haemoglobinopathies, of lipid composition and of abnormalities in antioxidant mechanisms induced by exogenous oxidant stress is also considered.     

Lipid peroxidation in mitochondria

The present review article takes into consideration the most important aspects of lipid peroxidation in mitochondria. Firstly the various ways by which lipid peroxidation is induced and the relevant mechanisms are described and discussed. After examining the major effects of lipid peroxidation on mitochondrial enzymes and bioenergetic functions, some aspects of the pathophysiology of lipid peroxidation are considered in connection with maturation of reticulocytes, alternative oxidase of plant mitochondria, aging, and ischemia-reperfusion syndrome. The final part of the article is devoted to the regulation and control of lipid peroxidation in mitochondria with particular emphasis to the role of the respiratory substrates.     

Lipid peroxidation and changes in cytochrome c oxidase and xanthine oxidase activity in organophosphorus anticholinesterase induced myopathy

A possible role of radical oxygen species (ROS) initiated lipid peroxidation in diisopropylphosphorofluoridate (DFP)-induced muscle necrosis was investigated by quantifying muscle changes in F₂-isoprostanes, novel and extremely accurate markers of lipid peroxidation in vivo. A significant increase in F₂-isoprostanes of 56% was found in the diaphragm of rats 60 min after DFP-induced fasciculations. As possible source of ROS initiating lipid peroxidation, the cytocrome-c oxidase (Cyt-ox) and xanthine dehydrogenasexanthine oxidase (XD-XO) systems were investigated. Within 30 min of onset of fasciculations Cyt-ox activity was reduced by 50% from 0.526 to 0.263 μmol/mg prot/min and XO activity increased from 0.242 to 0.541 μmol/mg prot/min. Total XD-XO activity was unchanged, indicating a conversion from XD into XO. In rats pretreatment with the neuromuscular blocking agent d-tubocurarine, prevented DFP-induced fasciculations, increases in F₂-isoprostanes and changes in Cyt-ox or XD-XO. The decrease in Cyt-ox and increase in XO suggest that ROS are produced during DFP induced muscle fasciculations initiating lipid peroxidation and subsequent myopathy.     

Lipid peroxidation in rats administrated with mercuric chloride

Parenteral administration of mercuric chloride (HgCl₂) to rats enhanced lipid peroxidation in liver, kidney, lung, testis, and serum (but not in heart, spleen, or muscle), as measured by the thiobarbituric acid reaction for malondialdehyde (MDA) in fresh tissue homogenates and body fluids. After sc injection of HgCl₂ (5 mg/kg body wt), MDA concentrations in liver and kidney became significantly increased by 9 h and reached peak values at 24 h. Dose-response studies were carried out with male albino rats of the Fisher-344 strain (body wt 170–280 g) injected with 1, 3, 5 mg Hg/kg as HgCl₂ and sacrificed after 24 h. In time-response studies, animals were administered 5 mg Hg/kg as HgCl₂ and sacrificed after 3, 9, 18, 24, and 48 h. Studies in the authors' laboratory have shown that (1) concentrations of MDA are increased in targets (liver, kidney, lung, and testis) of HgCl₂-treated rats; (2) severity of hepatotoxicity and nephrotoxicity is generally consistent with the elevation of Hg and MDA concentrations, based upon the time-course and dose-effect relationships observed after administration of HgCl₂ to rats; and (3) concentrations of MDA are reduced in target tissues after pretreatment with antioxidants and chelators to HgCl₂-treated rats. The results of this study implicate that the lipid peroxidation is one of the molecular mechanisms for cell injury in acute HgCl₂ poisoning.     

Lipid peroxidation in cardiac insufficiency

Peroxidation of lipids was studied in patients with heart failure after coronary heart disease and acquired valvular diseases. Even at early stages of the heart failure an increase in the concentration of dien conjugates, malonic dialdehyde and intensification of the peroxidation of blood lipids under stimulation by bivalent iron have been revealed. These changes do not depend on reasons which caused heart failure.     

Lipid peroxidation in rat uterus

Lipid peroxidation in rat uterus has been studied using NADPH- and ascorbate-induced systems. Lipid peroxidation in rat uterus is low as compared to rat liver. Uterus is more sensitive to ascorbate-induced lipid peroxidation than that induced by NADPH. Uterus contains lower amounts of phospholipids and has a lesser degree of unsaturation in lipids. Co-factor studies show that Fe2+ is more important for ascorbate-induced lipid peroxidation. Endometrium is more sensitive to ascorbate-induced lipid peroxidation than myometrium. It also contains more total lipids and phospholipids besides having a higher degree of unsaturation in the lipids as compared to myometrium. Among the subcellular fractions, mitochondria are more prone to ascorbate-induced lipid peroxidation, whereas microsomes are more sensitive to NADPH-induced lipid peroxidation. Uteri from old rats (24 months) and pregnant rats are more resistant to lipid peroxidation than those from 3-month-old control rats. Uterus of pregnant rats contains more factors which inhibit lipid peroxidation and also has a lesser degree of unsaturation in lipids compared with uterus of control rats. The possible consequences of the resistance of uterus to lipid peroxidation, especially during pregnancy and senescence, are discussed.     

Lipid peroxidation and nutrition

Levels of conjugated dienes of fatty acids (first peroxidation product) in relation to their substrates and promotors (triacylglycerols, homocysteine, iron) as well as to their inhibitors (essential antioxidative vitamins) were assessed in a vegetarian group (n=24) and compared with subjects on a mixed diet (traditional nutrition, n=24). Positive significant linear correlation between conjugated dienes and triacylglycerols, homocysteine, iron as well as inverse relationship between conjugated dienes and vitamin E, vitamin C, beta-carotene were observed in pooled groups. Lipid peroxidation risk in vegetarians seems to be caused predominantly by hyperhomocysteinemia, whereas in a mixed diet group this was due to a higher supply of substrates or risk iron values. The incidence of only 8 % of risk conjugated diene values in vegetarians in contrast to 42 % in the group with traditional diet indicates that vegetarians have a better antioxidative status as a consequence of regular consumption of protective food.     

Autophagy in a mouse model of distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy

Distal myopathy with rimmed vacuoles (DMRV) or hereditary inclusion body myopathy (hIBM) is an autosomal recessive disorder clinically characterized by weakness that initially involves the distal muscles, although other muscles can be affected as well. Pathological hallmarks include the presence of rimmed vacuoles (RVs) and intracellular Congo red-positive depositions in vacuolated or nonvacuolated fibers. Mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, which encodes the rate-limiting enzyme in sialic acid biosynthesis, are causative of DMRV/hIBM. Recently, we have generated a mouse model (Gne(-/-)hGNEV572L-Tg) for this disease, and have shown that these mice exhibit hyposialylation and intracellular amyloid deposition before the characteristic RVs are detected, indicating that autophagy is a downstream phenomenon to hyposialylation and amyloid deposition in DMRV/hIBM.     

Lipid peroxidation and antioxidant status in patients with periodontitis

Our aim was to assess the degree of oxidative stress in patients with periodontitis by measuring their levels of thiobarbituric acid reactive substances (TBARS), enzymatic antioxidants (superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (GSHPx)), and non-enzymatic antioxidants (vitamins E and C, reduced glutathione (GSH)). This study was conducted on 25 adult chronic periodontitis sufferers who were patients in Rajah Muthiah Dental College and Hospital, Annamalai University. The levels of TBARS and non-enzymatic antioxidants, and the activities of enzymatic antioxidants in the patients' plasma, erythrocytes and gingival tissues were assayed using specific colorimetric methods. The periodontitis sufferers had a significantly higher TBARS level than the healthy subjects. In the plasma, erythrocytes, erythrocyte membranes and gingival tissues of the periodontitis sufferers, enzymatic antioxidant activities were found to be significantly higher, whereas the levels of non-enzymatic antioxidants were significantly lower (except for reduced glutathione in the gingival tissues) relative to the parameters found for healthy subjects. The disturbance in the endogenous antioxidant defense system due to over-production of lipid peroxidation products at inflammatory sites can be related to a higher level of oxidative stress in patients with periodontitis.     

Female transmitters of Duchenne's myopathy: detection by quantitative ultrasonography combined with blood creatine kinase

Duchenne muscular dystrophy (DMD) is a disease inherited in an X-linked recessive manner. Nothing is yet known about the gene defect involved. The problem of DMD carrier detection is very important and now without satisfactory solution. Presently the best method, for this detection, is the determination of the serum creatine-kinase (CK) level. However about 30% of DMD children's mothers have normal CK levels. In this study we associated CK determination and a new echographic method. This physical investigation is based on the muscle attenuation measurements through the evolution of the running spectral moments. The parameter expressed is the slope of the ultrasound attenuation signal (in decibels by centimetre and by megahertz; dB . cm-1 . MHz-1). We measured this parameter in vivo on the vastus medialis. The use of the chemical technique and of the physical one improves largely the detection of obligate carriers of Duchenne muscular dystrophy than when one of these methods is employed alone. One such combination recognizes 17 out of the 19 obligate carriers in our series. The difference of the results obtained by these two unrelated methods cannot be yet interpreted, but can reflect genetic heterogeneity in DMD and the inactivation of the X chromosomes.     

Lipid peroxidation in plumbagin administered rats

Plumbagin was administered to rats at a concentration of 1,2,4,8 and 16 mg per kg body weight. After 24 h lipid peroxide levels were found to decrease in subcellular fractions of liver. Plumbagin inhibited ascorbate and nicotinafde adenine dinucleotide phosphate (reduced) dependent lipid peroxidation but was without any effect on cumene hydroperoxide dependent lipid peroxidation. Injection of 16 mg of plumbagin per kg body weight was found to decrease liver total reduced glutathione and also fcrosomal glucose-6-phosphatase. The results are discussed with reference to the anti- and prooxidant properties of plumbagin.     

Lipid peroxidation in experimental Salmonella infection

The results of investigation of lipid peroxidation in experimental Salmonella infection in 21-day-old rabbits are analyzed. Salmonella infection was accompanied by activation of lipid peroxidation not only in enterocytes, but also in blood serum. An increase in the level of malonic dialdehyde leads to a decrease in the antioxidation capacity and in the peroxidation resistance of erythrocytic membranes. The severity of the pathological process was found related to the activity of lipid peroxidation.     

Lipid peroxidation in isolated hepatocytes.

Intracellular lipid peroxidation was initiated by the addition of ADP-complexed ferric iron to isolated rat hepatocytes and the reaction monitored by the thiobarbituric acid method or by measurement of the formation of conjugated dienes. Both the production of malondialdehyde (thiobarbituric-acid-reacting substances) and of conjugated dienes was dependent, on the ADP-Fe-3+ concentration in a dose-related fashion. Malondialdehyde formation stopped spontaneously within 20 min after the initiation of the reaction and the plateau reached was also related to the ADP-Fe-3+ concentration. Control experiments revealed that more than 90% of the malondialdehyde accumulating during the incubation period could be ascribed to intracellular production. The cellular NADPH/NADP+ ratio was always high and only slightly decreased upon ADP-Fe-3+-induced lipid peroxidation which, however, was associated with a marked decrease in the cellular glutathione concentration. The rate of accumulation of malondialdehyde as well as the final level reached during ADP-Fe-3+-initiated lipid peroxidation was increased by the addition of chloral hydrate. This apparent stimulatory effect could, however, be ascribed to the inhibition of the mitochondrial oxidation of the malondialdehyde formed during cellular lipid peroxidation, thus allowing more malondialdehyde to accumulate during the process. ADP-Fe-3+-induced cellular lipid peroxidation was associated with a decrease in the concentration of glutathione. Also, lowering of the intracellular glutathione level by the addition of diethyl maleate or by simply preincubating the hepatocytes (up to 50 min) promoted the ADP-Fe-3+ malondialdehyde production and formation of conjugated dienes. Furthermore, when cellular glutathione concentration had been lowered by preincubation of the hepatocytes, significant malondialdehyde production could be observed even at ADP-Fe-3+ concentrations which were too low to induce measurable lipid peroxidation in fresh hepatocytes. It is thus concluded that glutathione has an important role in the cell defence against lipid peroxidation and suggested that the isolated hepatocytes provide a suitable experimental model system for the characterization of this and other possible cellular defence mechanisms and how they are affected by the nutritional status of the donor animal.     

Lipid peroxidation products in biological tissues

Over the past twenty-years of lipid peroxidation research in this laboratory, considerable effort has gone into development of new methods, with emphasis on measurement of lipid-soluble fluorophores and the volatile hydrocarbons ethane and pentane. Application of these and other methods has been made to biological materials and living animals. Although the various methodologies used in lipid peroxidation research do not necessarily measure the same class of products, and although agreement of results is not always 100%, there is substantial documentation of good correlations between measurements; for example, of trace volatile hydrocarbons with thiobarbituric acid-reacting substances, of pentane production with dietary and/or tissue vitamin E content, and of pentane production with lipid-soluble fluorophores accumulated in spleen as a function of oxidant stress. Individual methodologies do have their inherent limitations. However, measurements of multiple products and their correlations have added significantly to the base of information on biological damage and protection by dietary antioxidants against nutritional and toxicological insults to tissues, cells, and macromolecules as a result of peroxidative and oxidative reactions.     

Lipid peroxidation in experimental allergic encephalomyelitis

Lipid peroxidation (LPO) in the brain and blood of guinea-pigs was studied during experimental allergic encephalomyelitis. The most pronounced activation of LPO in the brain occurred at the 7th day of sensitization with encephalolitogenic emulsion. It manifested by an increase in the content of diene conjugates and malonic dialdehyde, activation of catalase and reduction of superoxide dismutase activity. LPO activation in the blood occurred at the 3th-5th day of sensitization. It is assumed that LPO activation is caused by antigen-antibody reaction that occurs in the blood at the 3d day and in the brain at the 7th day of sensitization.     

Lipid peroxidation in systemic lupus erythematosus

Lipid peroxidation is an important process in oxygen toxicity. Free radicals inflict this damage by attacking polyunsaturated fatty acids, thus setting off a deleterious chain reaction that ultimately results in their disintegration into malondialdehye, 4 hydroxy-2-nonenal and other harmful by-products. Peroxidation of lipids has been implicated in several diseases including systemic lupus erythematosus (SLE). SLE is an autoimmune disorder with unknown aetiology, characterized by the presence of autoantibodies to self-antigens. There is a significant increase in the production of free radicals like superoxide and hydroxyl radicals in SLE. Indices of lipid peroxidation, like conjugated dienes, malondialdehyde, 8-isoprostaglandin F2 alpha are significantly elevated in SLE. Increased ceruloplasmin levels and decreased transferrin levels in the sera of SLE patients have also been described. The activities of the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase and the amounts of the antioxidant reduced glutathione are also significantly altered in this disease. In addition, there are significant changes in the essential fatty acid profile in the sera of those affected with the disease. In animal models of the disease, immunization of mice with peptides derived from autoantigens induces SLE like disease. Immunization with an oxidatively modified autoantigen led to the rapid development of autoimmunity compared to immunization with the unmodified autoantigen. Thus, oxidative damage appears to play an important role in SLE pathogenesis.