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Free Radicals Frederick Van Lente Section of Biochemistry, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 This review willdiscuss thedevelopments in the biomedical chemistry of free radicals and their reactive metabolites. The salient work that has appeared in the two years since the last review will be discussed here. Since direct observation of free-radicalreactions in human pathophysiology is prohibited technically at this time, most investigations center on the indirect assessment of radical reactions, particularly their end products. The literature in the past several years indicates, with one exception, a gradual consolidation of the understanding of free-radical processes in human metabolism and disease and the limitations in our abilitv to relate consequences of these reactions to the diagnosis and management of disease. Several excellent and pertinent review articles have appeared recently and may provide the reader with more indepthcoverage ofspecific topicsin thisareaofresearch. They includereviews of lipid peroxidation of low-densitylipoprotein (611, analytical techniques (821, the role of iron (83). and general mechanisms (84). Mechanisms. The propagationofradicalreactions beyond what is useful andadvantageow to humanphysiologyremains a focus for research. As was discussed in the previous review, transition metals, particularly iron, are thought to potentiate such reactions under appropriate conditions (B5). The potential formation of hydroxyl radical ('OH) by means of the Fenton reaction
-
+
Fe(I1) + H,O, Fe(II1) + 'OH OHhas substantiated the need for sequestration of iron in human physiolo beyond that proposed traditionally for prevention of iron-r&ciency anemia. It has become clear that ironmediated reactions with biological macromolecules such as protein are subtle (B6) and may involve heretofore unrecognized coparticipants in metal-catalyzed reactions. Therehas beenarecentfocusonthepotentialroleofmetal transport and storage proteins in free-radical-induced injury. Extensive in vitro studies have demonstrated the ability of ferritin (87) to promote such reactions by reductive mobilization of iron from its hydratedferricoxide core. Biologically relevant species reported to be capable of unloading ferritinderived Fe(I1) include superoxide anion (B8). Transferrin also represents a potential source of free, ionic iron, and its ability to promote oxygen-free radical injury has been demonstrated in an animal model (B9). This action w a ~ receptor-dependent and led apparently to endocytosis, acidification, and reduction of transferrin-associated Fe(II1) to Fe(I1). In an experimental model of iron overload, direct evidence for the in vivo production of the OH radical by indirect spin-trapping using DMSO and N-tert-butyl-8 phenylnitrone (BIO)sup orts thepossiblerolefor theFenton reaction when the metagolism of iron is aberrant. The implication of past and current studies of the negative impact of free or chelated iron has been emphasized by the recent report of an association between iron status and the risk of myocardial infarction in a population of Finnish men ( B l l ) . This has led to a renewed interest in the role of stored iron in the pathogenesis of ischemic heart disease (B12). This relationship is considered strengthened by the association of oxidative damage with ischemic-reperfusion injury and the demonstration of oxidative modification of lipoproteins, especially low-density lipoproteins. Oxidative Modification of LDL. Current theories of atherogenesis include plaque formation from foam cells that arise subsequent to the uptake of oxidatively modified lowdensity lipoprotein by macrophages via the scavenger receptor (BZ). The modification of LDL is thought to occur via a free-radical process. Serum LDL contributes about 60% of total serum cholesterol and contains significant polyunsaturated fatty acid, mainly linoleic and some arachidonic and docosahexanoic acids (BZ). Interestingly, a number of supposed, lipophilic antioxidantsarealso associatedwithLDL particles. These include a-tocopherol (Bljl),carotenes(BZ4), and ubiquinol-10 (B15)just to name a few of the prospective candidates. However, the exact contribution of the various 374R
ANALYTICAL CHEMISTRY. VOL. 65, NO. 12, JUNE 15. 1993
Fr.d.rickVanLenteisHeadofmeSection of Biochemistry. Department of Clinical Pathology. at The Cleveland Clinic Foun-
dation and cwrdinated the clinlcal section ofthisreview. He received hisB.A.degree in chemistry from Hope College in 1967 and his Ph.D. in biochemical sciencesfrom Princeton University in 1975. After completing hispostdoctoral studies, he has held positions as Chief Laboratory Scientist at Overlook Hospital. Summit, NJ. and Head, AutomatedIAcute Care section of the Department of Biochemistry, at The Cleveland Clinic Foundation. He also holds the j position of Clinical Professor in the Department of Chemistry at Cleveland State University.
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potential antioxidantsto protection of against LDLoxidation rene.in3 BDmewhat uncIea7 (B16). The importance of vitamin E relative to other antioxidants for protection of LDL has been demonstrated (B16)as has been its consumption during induced oxidation (813,817). As will be discussed consistently in this review, however, the overall dynamic of oxidation and reduction is unlikely to be as simplistic as in uitro experiments suggest. Some studies have supported, for example, the recyclingof LDL-associated vitamin E by vitamin C via direct reduction of chromanoxyl radicals (B18). An apparent synergy between a-tocopherol and @carotene has also been reported in studies of a membrane model (B19). Probucol (4,4'-(isopropylidenedithio)bis(2.6-di-tert-hutylphenol)) is a widely used, cholesterol-lowering drug that is now thought to also limit LDL oxidation (B7). This antioxidant effect may slow the progression of atherosclerosis by ita antioxidant as well as lipidlowering effects. This compound has now been shown to be a strong superoxide free-radical scavenger in vitro (B20). The extrapolation of in uitro andlor model systems to the complex redox status of serum is problematical. The lipid peroxidation status of human plasma is complicated by the fact that plasma is highly oxygenated ( ~ 0 2 1 0 mmHg) 0 and there is a need to avoid antifactual oxidation after sample collection. The latter consideration was discussed in some detail in the last review. The need for EDTA in sample handling has been aptly demonstrated (B21, B22). An excellent review of the comparison of thiobarbituric acidreactive substances in the sera of normal and atherosclerotic subjectscan be foundin the firstreference ( B l ) . Inonestudy, for example, 50 patients with ischemic heart disease had average serum TBARS values of 4.37 nmolImL compared with 3.65 nmol/L for normal controls (823). Additional studies of the TBARS content of lipoproteins have been recently extended by an analysis of variations in oxidative susceptibility of LDL subfractions (824). Oxidation rates were found to correlate with unesterified cholesterol content and not with a-tocopherol or ,%carotene. However, there is some question regarding the appropriateness of measuring oxidative susceptibilityversus oxidized components that could reflect oxidative stress in vivo (825). In addition to direct assessment of TBARS or malondialdehyde in plasma, immunoassays using antibodies raised against oxidatively modified LDL have been described and additional studies using autoantibodies baveappearedin thelast few years (B26,827). These studies failed to demonstrate reactivity against LDL isolated by routine means. Reactivity against oxidatively modified LDL has been found in elderly but not younger subjects (B26).Itshould benoted thatoxidizedLDLepitopes may only be present in atherosclerotic lesions in coronary vessels. The titer of autoantibodies to malondialdehydelysine has been reported to be an independent risk factor for the progression of carotid atherosclerosis (828). It can be anticipated that additional prospective studies will evaluate the usefulness of the determination of indexes of lipid oxidative modification and ita associated risk factors for assessing the role of ischemic heart disease (829). The report of the association of iron status with atherosclerotic risk previously discussed (BIZ)should stimulate this work.
CLINICAL CHEMISTRY
Table B-I. Reference Limits for Plasma TBARS n limits (pmollL) ref method subjects adults adults men women
adults
24 24 32 14 18 30
1.11 t 0.31 1.30 & 0.23 2.51 t 0.25 2.57 t 0.28 2.44 t 0.20 0.429 t 0.05 0.83 0.40
*
36 36 37 37 37 40 41
fluor
spec fluor fluor fluor
HPLC HPLC
It is interesting to note, however, that a similar independent risk for acute myocardial infarction has also been reported for increased serum copper by the same group of investigators (B30). This is particularly intriguing since copper (B31) is the standard moiety used to induce LDL oxidation in uitro. Another study found that decreased toenail selenium is associatedwith increased risk for myocardial infarction (B31). Paradoxically, erythrocyte glutathione peroxidase activities were increased in cases with acute myocardial infarction, although this has not been confirmed (B32). A more recent study evaluating the association of glutathione peroxidase activities with ischemicheart disease found significantlylower levels in plasma and platelets in subjects after myocardial infarction (B33). Statistically significant corresponding differences in plasma have not been consistently reported in patients with ischemic heart disease but most re orta demonstrate lower values (B31,B34). The association o&HD with decreased selenium and glutathione peroxidase is likely to vary by geographical region studied due to intake differences, as has been shown in epidemiologicalstudies in Finland (B35). Measurement of Oxidative Damage. Free-radicalinduced damage may be assessed by measuring the products of these distinctive reactions in various body fluids includin plasma, blood, urine, and breath. These include conj ate! dienes, hydrogen eroxide, lipid peroxides, cholester3 peroxides, alkanes, al&hydes and, in particular, malondialdehyde (B2). With the exception of malondialdehyde, there has not been an extensive expansion of the literature regarding the analytical techniques employed since the last review. Endogenous malondialdehyde is derived principally from fatty acids with three or more double bonds (B36) and, therefore, represents the degree of peroxidation of polyunsaturated fatty acids. The measurement employing 1,3diethyl-2-thiobarbituric acid has been recently optimized (B36). Plasma reference values var somewhat b method (see Table B-I). The results obtaineJfrom plasma lave been found to correlate significantly with triglycerides, total cholesterol, total fatty acids, and number of double bonds above three (B36). Reagents for this TBARS determination are now available in kit form and provide reasonably acceptable analytical performance (B37). A new method utilizing solvent extraction-flow injection analysis has also
butanol extraction with increased sensitivity and specificity (B39). Overall, the variation in results observed for plasma remains as can be seen in the range of values for healthy subjects shown in Table B-I. The apparent nonspecificity of reaction and variations in conditions and means of determining the TBARS adduct undoubtedly contribute to this situation. Ischemic-Reperfusion Inquiry. Since the last review, work has continued apace on the precise role of free-radical reactions in ischemic-reperfusion tissue damage. This focus is driven, in part, b the ossibility that scavengersof reactive free radicals couldrametorate the consequences of existent ischemic tissue. As was discussed previously (B42), the original hypothesis relating ischemia-relatedxanthine oxidase activity to reperfusion free-radical activity has given way to more recent studies implicating iron- or copper-induced reactions (B43). A comprehensive discussion of the animal model results is not possible, but the findin s remain somewhat contradictory. For example, recent stukes on the ability of metal modulation to reduce myocardial infarct size in dogs indicates that desferrioxamine can delay but not eliminate cellular necrosis (B44).
Of more potential use is the direct assessment of free-radical activity in atients with known ischemic cardiovascular disease. It {as been reported that atients with pacinginduced *transient” ischemia signalei by angina and ECG changesexhibited increased malondialdehydevalues in serum and a later increase in creatine kinase-MB activity (B45). These changes were observed in the absence of myocardial infarction. Increased superoxide formation and intracellular calcium concentrationsin polymorphonuclearleukocytes have been described in younger arteriosclerotic patients (B46). These changes were interpreted as an accelerated agin process. A study in a small group of patients indicated[ subnormal selenium and vitamin E concentrations in patients with angina pectoris (B47). Additional studies on free-radical activity in patients undergoing intervention or surgical procedures have also been reported. Patients undergoin single-vessel ercutaneous transluminal angioplasty (PTEA) were f o u n l not to have increased li id peroxidation in either aorta or coronary sinus blood 2 a n i 10 min after the last balloon inflation (B48). These results argue against free-radical generation durin this procedure. However, the same group also reported[ increased concentrationsof lipid peroxides in coronaryvenous blood immediately after one to five balloon inflations (B49). The increase in lipid peroxides did not correlate with degree of preexistin myocardial ischemia. Our own experience has shown that bfood biochemical markers are difficult to assess via catheterization due to the concomitant effects of anticoagulation. It is also not clear whether membrane or cellularassociated peroxidation products readily fiid entry into the vascular compartment during the time course of these studies. Further studies have also been performed on patients undergoing coronary artery bypass where the emphasis on free-radical damage anticipates efforts to improve postoperative cardiacperformance and outcome. It has been shown that SOD-inhibitablechemiluminescence,superoxide anion, and blood malondialdehyde increased significantly during cardiopulmonary bypass and also perio eratively (B50). Antioxidant enzyme activities in myocarial biopsy tissue obtained from patients with angina and tetralogy of Fallot demonstrated a strong correlation with preoperative arterial oxygen tensions in the latter group but not the former (B51). However, glutathione eroxidase activities were over 4-fold higher in atients uniergoing coronary bypass afta and h rtropiic obstructive cardiomyopathy (B51). Fhis study i E r a t e s that chronic ischemia may not have expected effecta. Increased glutathione activities may be a generalized effect of aging. It is difficult to interpret the findings of freeradical-effect studies without the use of rigid control groups due to the multifactorial nature of free-radical metabolism. An attempt to approach this situation by evaluating the efficacy of various cardioplegia solutions to prevent freeradical damage during myocardial ischemia did not find significant differences (B52). However, improved performance of heart transplants in rats was observed when combined use of superoxide dismutaae,catalase, and glucoeeinsulin-potassium preservation was utilized (B53). Cardiac performancewas assessed by measurement of left ventricular end diastolic pressure, rate of increase of left ventricular pressure, myocardial blood flow, coronary resistance, and tissue adenosine triphosphate content. Other Diseases. The hypothesis that free-radical oxidative stress, also iron-induced, is central to the pathogenesis of Parkinson’s disease continues to be investigated (B54). The environment of the substantia nigra, in particular, dopamine neurons, may be at substantial risk for damage by oxidation. Dopamine metabolism involves monoamine oxidase at autoxidation to form hydrogen peroxidase (B54). The presence of iron in this tissue has been substantiated, and more recent reporta have also indicated decreased tissue ferritin concentrations (B55). The role of iron has drawn attention in Parkinson’s disease as it has in others, and it has been shown that iron infusion induced a dose-related destruction of substantia ni a neurons (B56) while desferrioxamine ameliorated s i m i E degeneration induced by 6-hydroxydopamine (B57). The latter affect is also seen by treatment with vitamin E in rats (B58). These studies, however, are somewhat circumstantial at this point; it has been previously discussed how difficult the extrapolation to the complexity of chronic oxidative mechanisms. ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
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The direct assessment of free-radical-related parameters in patients with Parkinson’s disease has been less fruitful. Vitamin E a-tocopherol values are not altered relative to controls in brain tissue from Parkinsonian patients (Bs9). Glutathione peroxidase activity in erythrocytes from patients with advanced Parkinson’s disease have been reported to be significantlylower (16.5 f 3.5vs 23.7 f 6.8 mUnits) in patients mth advanced disease when com ared with control groups, and the activit correlated with t e duration of disease but not age (B60). tudiea on glutathione peroxidase must always consider the increasing activity associated with aging when evaluating atient populationsaged 50 and greater. The latter study failefto identifyan aasociationbetween enzyme activity and age (B60). The drug deprenyl has been shown to slow the pro ession of Parkinson’sdisease to levodopa dependency (B54).y h e affectof this drug on antioxidant studies in lasma or CSF has proven to be inconclusive (B61). Depreny failed to modulate levels of lipid eroxide, vitamin E, or glutathione (B61). The inherent mecEanism of dru action must be far more subtle than what can be detected $,y gross analysis of antioxidant status. Free-radical participation in tissue damage in peripheral ischemic events also continues to attract some attention. In the case of acute cerebral stroke, a mild1 significant higher serum vitamin A was associated with etter neurological recovery and decreased mortalit (B62). Interestingly, .vitamin E concentrations failed to iemonstrate an association with outcome. Therefore, it remains unclear whether the measurement of antioxidant status will be useful in this arena although investigators have yet to evaluate all potential antioxidant parameters. A randomized double-blind trial of the radical scavengers allopurinol and dimethyl sulfoxide in combination with pain medication for treatment of alcoholinduced chronic pancreatitis showed increased efficacy of this regimen when compared with pain medication alone (B63). This study lends support to the implication of free-radicalinduced damage in the etiology of pancreatitis. In subjects with alcoholic liver disease, comprehensive evaluation of freeradical scavenger parameters in erythrocytes and serum revealed what was termed an unbalanced antipxidant system (B64). This finding included reduced glutathione peroxidase activity and reduced glutathione in erythrocytes. AIso serum zinc and selenium concentrations were significantlylower in patients with cirrhosis (B64). Decreased plasma selenium concentrations have also been reported in patients with euthyroid sick syndrome ( B e ) . It is likely. that alterations in trace metal status will be compromised in chronically or severely ill patients. The dynamics of lung hysiology are such that pulmonary tissue is exposed normai?ly to considerable oxidative stress. The fluid coverin the alveolar epithelial surface contains antioxidants incluiin ceuroloplasm, vitamin C, and reduced lutathione (B66). I? is interesting that bronchoalveolar ravage fluid has been shown to be an inhibitor of lipid peroxidation and the protein component of this material is the major contributor to this effect (B66). The effect of oxidant/antioxidant imbalance should contribute to pulmonary dysfunction. In idiopathic pulmonary fibrosis, a severe, progressiveinterstitial lung disease, lung epitheliallin’ glutathione has been reported to be deficient and rep etion aerosol therapy has been shown to decrease release of superoxide anion by alveolar macrophaees 0367). These findings were interpreted as indicatingoxidative involvement. Smoke inhalation also represents a severe form of pulmonary oxidative stress, and free radicals from smoke itself and those resulting from inflammation probably contribute to subsequent tissue injury (B68). In response to the renewed general interest in free-radicalinduced pathology, a number of studies have focused on possible oxidative damage in exercise (B69), osteo- and rheumatoid arthritis (B70), regnancy-induced hypertension ( B 7 4 , ac uired immunodeiciency syndrome 03721, vitiligo (B73),an\ cystic fibrosis (B74). In summary, the ast several years have witnessed a continued interest in t i e laboratoryassessment of freeradicalassociated pathology as well as antioxidant/prooxidant damage. However, the development of laboratory assays that provide useful information regarding diagnosis and treatment of free-radical-associateddisease has not been forthcoming. Traditional assessments of nutriture that provides the basis
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