Biochemical Consequences of Lipid Peroxidation
Jeffrey Bland University of Puget Sound Tacoma, Washington 98416
Lipid peroxides formed by abherant oxidation reactions of unsaturated lipid material have been identified as important degradative biomolecules involved in the cellular aging process ( I ), in oxygen toxicity (2), in air pollution oxidant damage to cells (3), and a host of other degenerative hiochemical transformations. The mechanism of formation of these lipid peroxides and their biochemical role in observed changes in cellular proteins, enzymes, and nucleotides, lipofusion and ceroid pigment formation, cellular aging, membrane alteration, cross.!inking of collagen, and drug/chemical.induced patho. logies constitute the focus of this report. The Production and Biochemical Significance of Lipid Peroxides The formation of lipid peroxides involves the reaction of hiochemical oxidants with polyunsaturated lipids. This process as diagrammed in Figure 1 is most commonly mediated through free radical intermediates (4). Free radical intermediates occur in almost all biological systems ( 5 , 6 ) .T h e radicals can be produced by three types of processes: 1) thermal homolvsis of bonds. 2) one-electron redox reactions, 3) high energy;adiation, and photolysis (7). These three processes are illustrated below.
+ B. M+ + ROOH M2+ + .OR + -OH HzO 2H. + .OH A-B-A.
-
A
(1) (2)
(3)
A mt~sition
drogen peroxide which is produced by a variety of enzyme systems (8).I t is well known that transition metals catalyze the decomposition of peroxides. Many metalloenzymes have the capability of catalyzing one electron redox reactions such as the hemoglobin catalyzed formation of lipid peroxides (9). Radicals which can initiate peroxidation can also be produced bv radiolvsis or ahsorotion of liebt. The radiolvsis of water produces HlO' and a solwted el?( tnm as tht. primary ipwi(,i which then leads bv a series of reactions to the hvdroxvl radical (.OH) which has been shown to he capablebf initiating radiation damage and suhseauent peroxidation in hiopolv. . mrrs (10,. ~he'pr(rductiono i singlet oxygen and its cut^ w e n t addition to unsaturated lipids has also heen shown to produce peroxides (11 ). S h. S*
+
GPLO0
+o.
n L LOO.
\LO..O2 Chain Reaction
L.
+ LH
LOOH
+LH
+a .-LOO.
-
Branching Chain qyLOOH Carbonyls Figure 1 . Mechanism of free radical chain reaction leading la fany acid hydroperoxides.
-
Termination 2 R R. +ROT
-
R -R
ROOR
The formation of lipid peroxides is most commonly associated with regions of the hiochemical system which are abundant in polyunsaturated fatty acids phospholipids (12). Cellular membranes including mitochondria1 and microsomal membranes are composed of fatty acids with 2, 4, 5, and 6 double bonds which jlave been shbwn to be rapidly peroxidized via free radical mechanisms with increasing ease both in uitro and in uiuo as the unsaturation increases-(13). Lipid peroxidation has been shown to correlate with swelling and eventual lysis of the mitochondrion by disruption of its membrane (14). The same observations have been made with microsomes (15) and lvsosomes (16). It is amarent therefore that lipidperoxidation is ubiq&us withidthe cell and the potential effects of these oeroxides upon hiochemical structure and function is of great significance. Effects of Lipid Peroxides in Biochemical Systems Changes - in Enzvmes. Proteins. and Nucleotides
Lipid peroxides are known to degrade proteins by reacting with them to form products hv scission. protein-protein crws-linking, and co\.,alent bonding with th; lipid p&oxide I l i , 181.It hni ht:m demonsrrntrd i19) that radical production is the factor most responsible for amino acid residue alteration. Sulfhydryl moieties are inactivated rapidly by lipid peroxides (20). Recently (21) N-acetyl cysteine has been shown to react with linoleic acid hydroperoxide by the following scheme which involves transition metal catalysis
-
T h r insidious character of these free r d i c a l induced lipid peroxidations is thut the mechanism of formation moit frequently involves a chain-carrying propagation process. Conceivably then only a few free radicals need necessarily be produced within a biochemical system to induce considerable lipid peroxidation-associated damage. This can occur by the following general mechanism (11) Initiation RH + 0% R. + .OH2 Propagation R. + 0% ROY
-
ROT + RH
-
-
ROOH
+ R. Volume 55. Number 3, March 1978 1 151
-
Without the catalvst no significant reaction was observed: therefore the conclusion is that one principle route for the degradation of proteins by peroxides is via transition metal catalyzed free radical addition of sulfhydryls to the lipid peroxides. Changes in genetir material by reaction aith lipid peroxides have received runsidrrable atwntion recmtlv. Mutatwns mas in part be caused by the effects of lipid perokide-induced free radical reactions (22). Nucleic acids themselves have been noted to be converted into their hydroperoxides by irradiation in the presence of oxygen (23).Recently a molecular mechanism involving hydroperoxides has been proposed (24) to explain the observed radiation induced mutagenesis originally observed by Miiller (25).When H. influenzae transforming DNA was exposed to thymine hydroperoxide, which can he formed by DNA exposure to irradiation in the presence of oxygen and a transition metal, a conferred resistance to streptomycin was realized (26).I t was found that C U ~was + much more effective than Co2+, Mn2+, Fez+, or Zn2+ in enhancinz this mutazenic activitv. These results suggest that .. thvmine hydroperuxidr, in cooperation u,ith metal inns, prtducr.; changes in trnnslorniin:! DNA capable of 1t:;tding to mutations. 'The necessity of trace metals to rffecr this mut;tgenic activity suggests that free radiral intrrrnediates are i n wired. The onmoied model !Fie. 1 1 of radiation induced mutagenicity therefore involves the formulation of hydroperoxides of pyrimidines via radiation which interact with a transition metal to yield free radicals. These radicals react with other bases to alter a cell's genome. and imnart abasemispairing character to the ~ ~ i w h i iesults c h in mutations as shown in Figure 2. I t appears that the mutants produced by this process are substitution rather than frameshift, so thvmine hvdroperoxide reacts in a similar fashion to manv m ~ t a g e n i c a l k ~ l a t i nreagents g in arrordnn(:e with the &;tn,phtlc theory of mutagenicity (271.
--
R H + 0,
.OOH + thymine
T
-
R + -0OH thymine hydroperoxide (THP)
thymine
TOOH = thvmine hvdrooeroride
-
. .
Lipofuscin and Ceroid Pigment Formation As far back as 1894, Hodpe reported pigment accumulatiun in the cytoplasm of ncunms 01 senile indi\.iduals. This rlass of pigments termcd lipofuscin or reroid has charartrristic fluorearenre spectral maxima at 470 nm when ext.irt:(l at 3fi.i nm & I . Formation ot'thu .otcmenrs annenri to involve Der. oxidation of polyunsaturated lipids of subcellular membranes (29,301.T h e production of lipofuscin is a result of malonaldehyde, a major product of polyunsaturated lipid peroxidation (311, reacting with primary amino groups of amino acids and proteins in a cross-linking reaction (32). The Schiff base product t h u s produced has the formula RN=CHCH=CH-NHR, and can also he produced when maloualdrhyde undergues smilar reartions with ammo groups of nucleic arids 133) and phospholipids (3.1).The rollrctim of this fluoresrent molrcular debris has brtn ohservrd ro he associated with many pathological processes (35).Peroxidation of subcellular organelles such as mitochronia, microsomes, and lysosomes has been shown to lead to lipofuscin-like fluorescent pigments (36).A number of investigators have reported the appearance of pigment in response to various types of stress and injury, including cold shock (37),and chronic vitamin E deficiencv as to " (38). . . There has been a lone" stand in^ -auestion . whether the deposition of lipofuscin pigment constitutes the nrimarv event leading to the observed-~atholoeies. " . or whether it fortuitously occurs& a secondary process concomitant with the development of the pathology. Recent work of Tonna (39) has shown that lipofuscin granules were observed to accumulate in aging connective tissue. Coincidental with pigment accumulation, the cells revealed significant degenerative changes accompanying cessation of cell division and diminished cell numbers. Accumulation of lipofuscin in cells other than muscle, nervous, and adrenal cortical tissues suggests that the age pigment has a broader distribution than was previously realized and may he a primary etiologic factor in lipid peroxide induced pathologies such as progeria, amaurotic
-
152 1 Journal of Chemical Education
.
Suh~titvtionmutation
Figure 2. A schematic representation of
peroxide-induced mutations.
familial idiocy (40),and gangliosidosis such as Tay-Sachs disease (41). Free Radical Induced Aging Like most hiological phenomena there is ample evidence to support both genetic and environmental contributions to the promotion of aging (42).Evidence exists which implicates dietary caloric intake (431, ionizing radiation (44), crosslinking of macromolecules (451,biological clocks (46),errors waning immunoin DNA transcription and translation (47), logic vigor (48,49)and changes in hormone patterns (50,51) with the aging process. These theories represent those that are fundamental in expression a t the cellular or molecular level and those that are operative a t higher levels of organization, respectively. Of the primary aging theories, the free radical1 lipid peroxidation theory proposed by Harman (52) has amassed considerable evidence in its support. This theorv is based upon the premise that aging is'&ociated with the deleterious effects of free radicals on cell constituents. These free radicals arise through oxidation reactions and lipid peroxide intermediacy. Harman has shown (53)that increasing the amount andlor degree of unsaturation of dietary fat leads to decreased mean life-span. It was also found (54)that dietary fat reduced learning behavior when administered in the absence of biological antioxidants, presumably implicating the develooment of livid . .oeroxide induced damaee in the central nervous system and loss of pruper cellular functiun. Harman conrludes that f ~ r t ~which m can modifv l i ~ i dw:roxidation rates (i.e. dietary fats, biological antinxihark) h a r contribute to the age of onset of central nervous system changes characteristic senility. Recently, however, other workers (55,561 have suggested that alternative explanations such as selective enzyme induction or post-translational enzyme modifications can account for the observed data concerning the complicity of free radical damage with cellular aging. The resolution of the debate which surrounds the question as to the validity of the
Harman hypothesis therefore awaits the results of future investigations. The accumulation of ceroid and lipofuscin pigments in the heart and brain resulting from lipid peroxidation has been associated with the aging process for some time (57,581.Although the question as to whether age pigment deposition is a primary etiologic factor responsible for cellular aging remains to be unequivocally answered, there is ample evidence supporting its association with the aging process (59). Drug-Induced Lipid Peroxidations The work of Di Luzio (60)has indicated that the nrimarv event in the development of ethanol-induced fatty liver, as well as certain other types of chemical-induced liver injury such as carbon tetrachloride,is the formation of lipid peroxides at selective sub-cellular sites. Both in uiuo and in uitro tests indicate that drugs induce lipid peroxidation selectively in liver cells and not the brain (61).Recent work (62)has indicated that ethanol-induced fatty liver is a result of ethanol metabolites and not ethanol itself. The work of Sorrell, et al. (63) suggests that liver damage and lipid peroxidation are occurring by increased microsomal mixed oxidase enzyme activity. They demonstrated that phenobarbital increased hepatic lipid peroxidation of microsomal lipids resulting in the formation of malonaldehyde and lipofuscin pigments. This biochemical change was accompanied by fatty infiltration of the liver, and reduced liver function. This work in conjunction with that of Rechnagel, et al. (64)suggests that lipid peroxide induced pathologies can be a result of in uiuo oxidase activity when the cell is loaded with excessive lipid suhstrates or mixed oxidase function activating substances. This model is supported by the results of Pawar, et al. (65,66)in which they demonstrated that riboflavin is an essential cofactor necessarv for the activity of the hepatic microsomal mixed oxidase enzymes (67).Taylor and Tappel (68)have found that low levels of the dietary antioxidants and selenium resulted in increased microsomal induced lipid peroxidation after phenobarbital and carbon tetrachloride treatment. This peroxide-induced cellular damage can occur at sites away from the site of primary generation. Roders, et al. (69) have found that when lipid peroxidation is induced at one site within a cell, such as the cytochrome P450 locus, some product yet to be indentified can traverse a finite distance to effect damage at a removed site. Recently Lazarow (70)has shown that the three hypolipidemic drugs clofibrate, tihric acid, and Wy-14,643 all lower serum linid concentrations hv increasine the liver neroxidase activities. In accord with these ohservati&s, it has k e n shown (71) that the antitumor antibiotic adriamycan induces severe cardiac toxicity in mice due in part to the release of a toxic metabolite resulting from lipid peroxidation. Interestingly, this effort can be reduced by pre-treatment of the test animal with the free radical scaveneer tocooherol. Recent work (72) suggests that the liver solubie fractibn contains factors yet to be identified capable of inhibiting microsomal lipid peroxidation, therehy counteracting the peroxidative damage of essential microsomal phospholipids which if chemically altered would lead to impairment of the drug oxidizing machinery of the liver cell (73). Crosslinking of Collagen Collagen comprises about one third of all proteins within the body and i t represents the single most important structural protein (74).The structure of collagen can be destroyed through the formation of intermolecular bonds from adjacent collagen fibrils. It has been shown that this process occurs concomitant with the aging process (75),and results in the loss of flexibility in tendons and ligaments. The composition of collaeen is uniaue amone nroteins because it contains a hieh percentage of'hydrox&;oline which relegates it to tLe "pleated-sheet" conformation thereby bringing adjacent fibrils into proximity for the formation of intermolecular bonds (76).The formation of adventitious cross-links is associated
with lipid peroxidation (77).The formation of malonaldehyde via lipid peroxidation leads to areactive intermediate which can form aldimine cross-links between adjacent collagen fibrils. I t has been observed that professional divers are often affected by hone necrosis through a collagen degenerative process (78).When collagen is exposed to oxygen in uitro for several weeks, therehy increasing lipid peroxidation and collagen cross-links, the collagen is noted t , no ~ longer he soluble in hot water and its physical properties are enormously changed (79).Sobel bas suggested (80)that this accelerated collagen degradation by oxygen mediated cross-linkage is the factor responsible for the observed necrosis in divers. Membrane Alleration The damage to sub-cellular membranes has been discussed oreviouslv in this renort. Considerable evidence is accumuiating to demonstrate that the cell membrane itself is also subject to lipid peroxide induced changes. Many disease conditions are associated with red blood cells membranes which are extremely sensitive to oxidative processes including heta-thalessemia major (81), erythropoietic protoporphyria (82) and the most common genetic defect in man, glucose6-phosphate dehydrogenase deficiency (83).Patients afflicted with 6-thalessemia major show virtually every kind of membrane deformation abnormality that has been described. In addition, Stocks, et al. have found an increased susceptibility of thalessemic red blood cells to oxidant stress (84).Rachmilewitz, et al. (81)have found that &thalessemics showed a nearlv twofold increase in total red cell b i d s and a correspondingly larger amount of lipid per cell uhtch is susceptihlc to nwoxidation. After oxidant StrQS.5they find cunsidrrahle incrrases in malcmaldehyde nmcentrationa constitent u,ith a linia neruxidntim mechanism fur the ot~srr\.edred cell destr;ctik. Graziano, et al. (85)have recently found that the iron chelating drug 2,3-dihydroxyhenzoic acid (2,3-DHB) is an effective inhibitor of red cell membrane peroxidation in thalassemics. They postulate that this effect is due to the free radical trapping ability of 2,3-DHB thereby terminating chain radical oropaaation and subsequent peroxidation. Thalas. . semis is characterized by preci$tatio" of hemoglobin which provides significant amounts of radical promotion agent Fez+ to be available for initiation of lipid peroxidation. The compound 2,3-DHB presumably complexes this available iron, and renders it unavailable for radical initiation. Hemolysis is almost always found to he preceded by lipid neroxidation (86).Barker and Brin (87)have found that nekxidation of the red cell membrane of vitamin E deficient [ats proceeds in a similar manner to the peroxidation of miscellar phospholipids in uitro, and they suggest that peroxidation exposes both polar and nonpolar lipid sites in the red cell membrane resulting in its weakening. Bland, et al. (88)have found that red cell peroxidation can be initiated by photooxidation conditions. Exposure of red cells to light and oxygen leads to red cell hemolysis, and simultaneous formation of membrane cholesterol hydroperoxide from cholesterol. Cholesterol hydroperoxide when incorporated into the red cell . . membrane has been shown (89)to greatly increase its osmotic fragility by destabilizing the lipid bilayer through inappropriate Van Der Wads associations. The evidence accumulates therefore to support the contention the lipid peroxides formed in uiuo lead to a significant cell membrane fragility due to disruption of proper bilayer interactions.
Effects of Biochemical Antioxidants on Lipid Peroxidation I t can be concluded from the previous discussions that the initially formed, highly reactive lipid peroxides do not accumulate in living tissue, but generally are either converted through secondary reactions into substances which give positive thibarbitnric acid tests for peroxidizing substances (90, 91) or are trapped by biological antioxidants before they can participate in chain radical processes (92).Chow and Tappel (93) have postulated that hydroperoxides are detoxified by Volume 55, Number 3, March 1978 / 153
conversion to hydroxy analogs by glutathione mediated by the action of the selenium containing metallo-enzyme glutathione peroxidase. The role of a-tocopherol, vitamin E, in this peroxidation prevention process is to work with gluthatione peroxidase in the inhibition of chain reaction autooxidation as shown in Figure 3 (94,95). The first evidence for the inhibition of lipid peroxidation in uiuo by vitamin E was the work of Mengel (96) in which he stressed animals under hyperbaric oxveen ." conditions in the Dresence and absence of dietary vitamin E supplementation, and was able to demonstrate t h a t were much more the red hlood cells of the vitamin E group . resistmt to hemolvs~sunder these conditions than thtr nunvitamin K study group. Shimasak~and I'riwtt (971 have recently demonitrated that vitamin K isoxidized to tncoquinonr in the process u i prwenring a lipid red cell perox~dation. ('onsidrrahlr u.ork has Iwtm done nhich demonitrates thst . vitamin E is capable of preventing lipofuscin pigment formation, and in vitamin E deficient animals exposed to oxidation stress the rate of lipofuscin deposition is accelerated (98,99). Shires (110) has shown that amino acid incorporation in uitro by the cell protein assembly machinery is inhibited by lipid peroxidation of membranes, thereby leading to lipofuscin deposition. Impairment of protein synthesis is known to be associated with the cellular aging process (101); therefore biological antioxidants such as vitamin E have been intimated to decelerate the rate of free radical induced aging (102,103). Packer and Smith (104) had reported that inclusion of vitamin E in the culture medium for human diploid cells greatly prolongs their lifetime in uitro which seemed to support the free radical theory of aging; however they have recently been forced to report an inability to reproduce this result (105). Green (106) discusses the important relationship which exists between the increasing amount of tocopherol necessary to prevent lipid peroxide induced damage as the amount of the nolvunsaturated fattv acids increases in the diet. ~ e c e n tBland l ~ e t ;I. (107) have shown that photo-oxidation induced damage to red hlood cell memhrane lipids can be effectively prevented by inclusion of vitamin E in the medium, and that the active peroxidizing species in this photohemolysis process appears to be singlet oxygen (108). Complements to these biological antioxidants are the enzymes such as peroxidase, catalase, and superoxide dismutase (SOD) which effect the following conversions in the hiochemical protection against harmful lipid peroxidation
F g ~ r e3 lntwactmns among seleno~m, v tam n E, an0 the su fur n nhoo loan of lop d peroxndal on damage
ammo aclar
~
~
CH3
CH:,
~
R = C,& Figure 4. n-tocopherol oxidation.
5) Pcwxide decomposrra inriuda glutathione, wlfw nmm, a d s . relrnium nmpounds. asrorhntr, and SADH. G ) Superc,xidedi~murnse indurps antioxidat~oneffrcrs. Conclusion Appreciation of the hiochemical importance of lipid peroxidation and its associated relationship with free radical aging and biological antioxidant theory has proved most valuable in providing unity for a large body of biochemical and nutritional observations. The mode of production and importance of lipofuscin and ceroid pigments in biochemical function remains as an important area of future research. The production, control, and importance of free radicals and their subsequent intimacy in the formation of lipid peroxides stands as a continuing problem in the formulation of unified aging theory (1 13). What has become evident, however, is that lipid peroxidation which has been known to have important consequences a t the cellular and organismic level is now better understood a t the hiochemical level. As research into the implications of linid neroxides in hioloeical svstems moves ahead, long. standing questions concerning biologic aging, memhrane destructive Droeesses. and lipid related patholoaies will become much beker undersood. knd the hi&hemical consequences of lipid peroxidation more fully appreciated.
. .
Fridovich has demonstrated (109) that dismutases are enzymes which provide defensr against the potentially rytotuxic rmct~\.itiesof the sll~rroxidcradical. Herentlv it has been reported (110) that brain and liver SOD activities did not diminish with age in rats, but that mitochondrially localized SOD did diminish with age in the liver. This is consistent with the fact that lipid of mitochondrial . peroxidation . membranes occurs mnrt. rapidly as the age of the cell increases, and suggests that superoxide mav be a contrihutur to lipid neroxidc formation and its rul,seauent hiochemical effects. I t has also been found that an a-tocopherol model compound is oxidized bv suoeroxide anion (111) . . . sueeestine that vitamin E might also he important in preventing lipid peroxidation via su~eroxideanion as well. In conclusion as seen in Figure 5, there are a variety of in uiuo nhvsical and chemical routes available for the suppression ~
~~~
~
~
~
.
uu
Acknowledgment The author thanks Dr. Paul Scheuer and the University of Hawaii Chemistry Department for their support in the preparation of the manuscript. Literature Cited (I) Packer. L., Desmer. D. W., end Hesfh, R.L., in "Advances in Gerontological Research: (Editor: Strehler, B. L.),New York, Academic, 1967,vol.2, p. 77. 121 Cnldntoin.B.D..andBalchum.O.J..Pr~c.Soc Exofl. B i d Mod.. l26.356(1967).
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(4) Tappel,A.L.,Fed~roIion Roe., 32.1870 11973). (51 Isenberg, I., Physiol. R ~ i l d d 4R7 119fi4l ~,. (6) Prvor, W,A,. (Editor),"R e Radical. in Bialcw."vok.I and 11,Academic Presr, New
~~~~.~~
Ynrk 1976. ~
(7) Pryor, W.A.,Fed~rofionP~oc.. 32,1862119731. (8) Pryor, W. A.. "Free Rsdieals: MeGraw-Hill, New Yark. 1366. (9) Barber, A. A , and Bernhoim, F.,Aduon. Gmnrologied Re& 2,355 11967). 110) Rhaese, H. J.. Freeze, E., and Melzer. M. G., B i o r h ~ m .Biophya. Act.., 155, 491
,,wm
GollnVk,L..Advon.Pholochem., 6.1 (1968). 112) Walling, C., J. Amen C h s m Soc.. 91,7590 11969). (13) Flaisaher. S.. and Rouser, G.. J. Amer Oil Chrmlsla Soe.. 42,588 (1965). (14) Witting. L. A,, J A m w Oil Chamisls'Soc.,12.908 (1965). (15) Tappcl, A. L.,Fed~r~fion Proc.. 24.73 (19651. (16) Rohinaon, J. D., Arch. Biorh~m.Biophys., 112,170 (1965). (17) Tappol. A. L., Swant. P. L.. and Shih, S.. in "Lysosoma," (Edirara: derewk. A.V. and Csmcron, M. P.), Churchill, London, 1363. (18) Zidin,A.,andKarel, M.. J. FoodSrr., 34,160 (19691. (11)
1) Polyunsaturated mnmhmnes. ....-
lipids are in protected structures, mainly
2) Vitamin E reacts as a chain-breakingantioxidant. 3) Vitamin E reacts with peroxide induced products and lipid
soluble radicals. 4) Glutathione ~eroxidasesystem reduces peroxides. 154 / Journal of Chemical Education
S.,
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,,O?C9
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