[32] Chemistry of lipid peroxidation

[32] Chemistry of lipid peroxidation

[32] 273 CHEMISTRY OF L I P I D PEROXIDATION [32] C h e m i s t r y of Lipid Peroxidation By N E D A . PORTER Diene Fatty Acids Systematic study ...

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273

CHEMISTRY OF L I P I D PEROXIDATION

[32] C h e m i s t r y of Lipid Peroxidation By N E D A . PORTER

Diene Fatty Acids

Systematic study of the chemistry of fatty acid autoxidation was begun in the late 1940s. i The degradation of fats and oils in the air had long been known but it remained for Criegee 2 to provide evidence that hydroperoxides are the primary products of hydrocarbon oxidation before significant advances could be made in the study of autoxidation of natural substances. Subsequent investigation by Bolland and his collaborators 3 established the primary autoxidation products of methyl linoleate as hydroperoxides containing conjugated diene. A mechanism for autoxidation of dicne fatty acids as written by these workers is shown in Scheme I. Only propagation steps of autoxidation are considered in this chapter. kp L--OO.

+

~ R

L--OOH

R'

+ .,.,__~."~n"~n::~n__n, *. ,,. ,~. A.. ,.~

OO • I

R--CH:::CH'"CH:::CH:CH--R'

+ 02

.~ /CH-- C H : CH-- C H = C H - - R

R

'

(LO0") + "0 I 0 I R - CH----CH--CH---- CH-- C%H R'

(LO0.)

OOH l

OOH I

R--CH-- C H = C H - - C H =

CH--R'

R - - C H = CH-- C H = C H - - CH--R'

9

18

(LOOH)

(LOOH) SCHEME

I.

' For a review, see W. G. Lloyd, "Methods in Free Radical Chemistry" (E. S. Huyser, ed.), Voi. 4. Dekker, New York, 1973. 2 R. Criegee, H. Pilz, and H. Flugare, Ber. 72, 1799 (1939). 3 L. Bateman, Q. Rev. 8, 147 (1954); J. L. Bolland, Q. Rev. 3, I (1949).

METHODS IN ENZYMOLOGY, VOL. 105

Copyright © 1984by Academic Press, Inc. All rights of reproduction in any form reserved, [SBN 0-12-182005-X

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Initiation of biological oxidation is considered extensively elsewhere in this volume. The two products of linoleate autoxidation described are the 9 and 13 substituted diene hydroperoxides, LOOH. For a period of some 20 years little additional evidence concerning the chemical mechanism of fatty acid autoxidation was published. In 1967, Howard and Ingold4 reported the absolute rate constant for hydrogen atom transfer from linoleate to peroxy radicals (kp = 62 M-~ sec-l) and in the mid-1970s several research groups ~ began to study diene fatty acid autoxidation. Reexamination of the products of autoxidation of linoleic acid or linoleate esters revealed a more complex mixture than was originally proposed. Four major conjugated diene hydroperoxides are formed and may be purified by HPLC. These major products have been shown to be 1-4 (R = H, Me, or Et). HOO

HOO

H11C~(CH,),COOR

HnCB~~,~(CHI),COOR

1

2

OOH HnCs~-~(CH,),COOR 3

OOH HazC5~ ~ ' ~

(CH,),COOR

4

Two of the products have trans,cis-diene stereochemistry (1 and 3) while two of the products (2 and 4) have trans,trans-diene stereochemistry. In addition to these four major products (~97% of the product mixture) trace amounts of nonconjugated hydroperoxides have also been isolated and identified. It should be emphasized that these diene hydroperoxides are themselves unstable with respect to decomposition. Thermal- or metal-catalyzed decomposition of 1, for example, presumably leads to the alkoxy radical 5 which can be a source of pentyl radicals 6 (and ultimately pentane) or epoxides derived from radical 7. Product mixtures derived from linoleates after extensive oxidation are thus complex. 4 j. A. Howard and K: U. Ingold, Can. J. Chem. 45, 793 (1967). 5 E. N. Frankel, in "Fatty Acids" (E. H. Pryde, ed.), p. 353. AOCS, Champaign, Illinois, 1970. 6 N. A. Porter, B. A. Weber, H. Weenen, and J. A. Khan, J. Am. Chem. Soc. 102, 5597 (1980). 7 H. W.-S. Chan, G. Levett, and J. A. Matthew, Chem. Phys. Lipids 24, 245 (1979). 8 L. R. C. Barclay and K. U. Ingold, J. Am. Chem. Soc. 102, 7794 (1980).

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275

0

CaHII"

+ H~-~(CH2)TCOOR

6

HttC~CH2) TCOOR 5

-.... HnC~(CH2).,,COOR 7

The primary products that lead to this complex mixture are, however, the four simple hydroperoxides 1-4 and the chain propagation sequence shown in Scheme I accounts for products formed at C-13 (1 and 2) and C-9 (3 and 4) of iinoleic acid. Any mechanism that accounts for stereochemistry of the products must, however, be more complex than the original Bolland mechanism, and radical isomerization (Scheme II) has been proposed to account for formation of trans,cis- and trans,transdienes. The radical 8 serves as a precursor to the trans, cis products while 9 could lead to trans,trans products by oxygen addition and hydrogen abstraction propagation. A mechanism more consistent with experimental observations is presented in Scheme III. In this scheme, the radical 8 is isomerized to 9 by reversible oxygen additions. Loss of oxygen from conformer 10a leads to 8 while conformer 10b leads to radical 9, a source of trans,trans-diene products. The necessity for proposing Scheme III comes from the observation that the ratio of trans,cis/trans,trans products depends directly on the concentration of L-H or other hydrogen atom donors (R-H) present in the oxidation mixture. With high concentrations of L-H or R-H, conversion of 10 (a and b) to trans,cis products (1 and 3) competes effectively with loss of oxygen from 10b (ka) to give 9 (and consequently trans,trans

oI

$

SCHEME II.

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BIOLOGICALDAMAGEIMPOSEDBY 02

[32] 00.

R/=V="R

R/-~~R

+ o2

• (L')

(L -H)

- 02

8

R~

lOa

R _._/ -Oa(k#) ~-- R / ~ ~ b O c,*" +02 9

R

oog

L_H(R_H).w_R / ~ - - < R

lob

• 00

+ L.

.

(1

and 3)

HO0

R

~

R + L.

(2 and 4) SCHEMEIll. products). Thus, the distribution of autoxidation products depends directly on the concentration and effectiveness (relative kp values) of any H atom donors present in the reaction mixture. A more detailed discussion of this mechanism of diene fatty acid oxidation has been published elsewhere. 6 It should be noted that singlet oxygen oxidation of diene fatty acids leads to a different set of products than does free radical autoxidation. Singlet oxygen oxidation of linoleate leads to the trans,cis-dienes 1 and 3, none of the trans,trans-dienes 2 and 4, and significant amounts of nonconjugated dienes 11 and 12. These nonconjugated products are formed in only trace amounts in free radical chain autoxidation while they may be major products in singlet oxygen oxidation. HOO /~ ~ HttC" v

(CH.).COOR ,v

11

OOH H.C,, ff--~ / ~ V/

V

CHa)TCOOR

12

Triene and Tetraene Fatty Acids

Autoxidation of triene or tetraene fatty acids leads to a much more complex mixture of products than that formed in diene fatty acid autoxi-

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CHEMISTRY OF LIPID PEROXIDATION

dation. Arachidonic acid, 13, as an example, has three carbon centers flanked by two double bonds (C7, Cm, C~3) while linoleate has only one such carbon (Cj0. Six major hydroperoxide products are formed in arachidonic acid autoxidation and these products may be separated and analyzed by HPLC. 9 All six major products have trans,cis-diene stereochemistry and hydroperoxide substitution in these products is at C-5, C-8, 9

s

5

H

~

C

13

O

O

H

14 COOH

OOH 15

C-9, C-11, C-12, and C-15. Two of the products, 14 and 15, formed from hydrogen atom abstraction at C-11 are shown here and products formed by abstraction at C-7 (the 5 and 9 substituted hydroperoxides) and C-10 (8 and 12 substituted hydroperoxides) have analogous structures. Autoxidation of arachidonic acid in benzene or chlorobenzene (0.24 M) solution I° gives rise to 15-hydroperoxyeicosatetraenoic acid (15-HPETE) as the major product (40%) with 5-HPETE being the next most prevalent product hydroperoxide (27%). Other products are formed as follows (8-HPETE, 7%; 9-HPETE, 9%; 11-HPETE, 11%; 12-HPETE, 6%). Two factors, selective hydrogen abstraction and peroxy radical cyclization, are responsible for this unequal distribution of products. In solution, C-13 hydrogen atom is abstracted more readily than is a hydrogen atom at C-10 or C-7. The relative rates of H atom abstraction are C-7, 0.86; C-10, 0.70; C-13, 1.33. Products that derive from abstraction at C-13 (15-HPETE and I 1HPETE) are thus formed preferentially to products that derive from abstraction at C-7 and C-10. It is unclear as to why the C-13 hydrogens are more prone to abstraction than those at C-7 or C-10 but it appears that hydrogens near the tail of the molecule are generally more available for reaction, perhaps because of overall molecular conformation. A similar preference for 15-HPETE formation is observed in singlet oxygen oxida9 N. A. Porter, R. A. Wolf, E. M. Yarbro, and H. Weenen, Biochem. Biophys. Res. Commun. 89, 1058 (1979). m N. A. Porter, L. S. Lehman, B. A. Weber, and K. J. Smith, J. Am. Chem. Soc. 103, 6447 (1981).

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tion of arachidonic acid ~l and conformational preferences may be important here also in determining product distribution. From these data it may also be noted that arachidonic acid is oxidized about 2.9 times as fast as is linoleate. Peroxy radical cyclization is a second factor that determines product distribution in triene and tetraene oxidation. The peroxy radical leading to hydroperoxide 15-HPETE has only two mechanistic pathways available, hydrogen atom abstraction to propagate the chain and loss of oxygen to form a carbon radical (/3 scission). In addition to these two pathways, a peroxy radical leading to hydroperoxide 11-HPETE has a third alternative. In particular, peroxy radicals have been shown to add to double bonds at adjacent centers to form five and six membered ring products. ~2,13In the case of peroxy radicals derived from arachidonic acid a host of products may ultimately form from cyclization. Monocyclic peroxides like 16, serial cyclic products such as 17, and bicyclic endoperoxides (18) (Scheme IV) have all been isolated from triene or tetraene fatty acid systems after autoxidation. The products 16, 17, and 18 exist in many diastereomeric forms with sixteen pairs of enantiomers possible for compound 17 alone. It should also be noted that products analogous to but different from 16, 17, and 18 may be formed from four different peroxy radicals that have adjacent double bonds available for cyclization. Thus, peroxy radicals leading to 8-HPETE, 9-HPETE, I1-HPETE, and 12HPETE have competitive cyclization pathways available and cyclization products may form from these species. It is for this reason that the relative amounts of the 8-,9-, 11-, and 12-hydroperoxides are smaller than the 5- and 15-HPETEs formed in arachidonic acid oxidation. The 5- and 15peroxy radicals do not have a competitive cyclization pathway and are formed in undiminished yield. Comment should also be made about the fact that both trans,cis and trans,trans diene hydroperoxides are formed in linoleate oxidation while trans,cis-diene hydroperoxides always dominate in triene or tetraene oxidation. Hydroperoxide products with trans, trans-diene stereochemistry are formed by unimolecular peroxy radical/3 scission (ko) that competes with bimolecular hydrogen atom abstraction. The rate of/3 fragmentation has been found 6 to be on the order of 150 sec -~ while unimolecular cyclization occurs with a significantly greater rate constants, ~° kc - 800 see -~. Thus,/3 scission leading to trans,trans product never competes effectively with cyclization and/or hydrogen atom abstraction. ii N. A. Porter, J. Logan, and V. Kontoyiannidou, J. Org. Chem. 44, 3177 (1979). n N. A. Porter, M. O. Funk, D. W. Gilmore, R. Isaac, and J, R. Nixon, J. Am. Chem, Soc, 98, 6000 (1976). t3 E. D. Mihelich, J. Am, Chem. Soc. 102, 7141 (1980).

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279

15 + L"

~

COOR

LIo

O ~ - ~

COOR On

HO0

c°°R OOH

O~/'~COOR 16

17 0

,

,

"

~

COOR

OOH 18 SCHEME IV.

Products such as 17 and 18 probably account for a significant fraction of the mixture formed in triene and tetraene oxidation and it seems likely that these compounds form malondialdehyde upon decomposition. Malondialdehyde is, of course, detected in the thiobarbituric acid assay (TBA), a standard test of lipid peroxidation used by workers in the field.

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The TBA assay then is probably an assay of peroxy radical cyclization in lipid peroxidation. Membrane Oxidation

Membrane destruction by free radical autoxidation has been the subject of extensive investigation by biochemists over the past several years. While considerable effort has been directed toward developing an understanding of the steps involved in initiation of the free radical process and the inhibition of the reaction, until very recently little attention has been given to the target of oxidation, the phospholipid. Polyunsaturated diacyiglycerophosphatidylcholines (diacyI-GPC, lecithin) readily undergo autoxidation as multilamellar or unilamellar vesicles. A variety of initiators of peroxidation may be used (Fenton's reagent, xanthine oxidase, adventitious initiation) but the products of oxidation are identical regardless of the method of initiation. High-pressure liquid chromatography has been used to good advantage in the study of lecithin oxidation. 14,~5For example, autoxidation of dilinoleoyl lecithin (19) can be monitored directly by reverse-phase high-pressure liquid chromatography. Two series of products are formed, one series containing monooxidation products the other compounds resulting from dioxidation. The first series contains products of autoxidation of 19 in which one acyl chain (at either C-1 or C-2 of the glycerol moiety) has been oxidized.

_

_ O

ro-t :, L--O--P--O---'-. .N--

19

In fact, 16 different monooxidation products are formed from 19. Eight of the products derive from oxidation at the C-I acyl chain and eight from the C-2 acyl linoleate. Four of the eight products in each of the acyl chains are trans,cis-diene hydroperoxide products while the other four products have trans,trans-diene stereochemistry. Within the set of trans,cis or trans,trans products, half are formed at C-9 of linoleate with a 10,12conjugated diene unit, the other half are formed with hydroperoxide substitution at C-13 with a 9,1 l-diene functionality. Finally, hydroperoxide at C-9 or C-13 may have either R or S configuration. While these stereoiso~4C. G. Crawford, R. D. Plattner, D. J. Sessa, and J. J. Ruckis, Lipids 15, 91 (1980). 15 N. A. Porter, R. A. Wolf, and H. Weenen, Lipids 1$, 163 (1980).

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281

mers would be enantiomers in autoxidation of simple linoleate esters, this is not the case in the oxidation of phospholipid esters. Since natural lecithins have only the R configuration at the C-2 glycerol carbon, the possibility of having R and S configuration in a particular oxidation product leads to a mixture of diastereomers, not enantiomers. The 16 monooxidation products of 19 do not separate by reversephase HPLC. The products may be analyzed, however, by the sequence outlined here for one of the sixteen products, 20. Treatment of mixtures containing 20 with Ph3P leads to conversion to 21 by simple reduction of 20. Exhaustive methanolysis (KOH, MeOH) converts all hydroxylinoleate glycerolphosphatidylcholines such as 21 to simple methyl esters, ~

'

~

~

~

-

~

00

~

-

-

~

~

~

~

RO

T

O0

-O--P--O~

~N~

20 R = O H 21 R : H

KOH/CHsOH

_

oc~

HO

0 22

22, and these methyl esters may be separated and analyzed by normalphase HPLC. In this way, details of oxidation within the membrane bilayer may be assessed. Similar analytical methods may be used for a variety of lecithin molecular species containing linoleate, linolenate, arachidonate, palmitate, and stearate functional groups. In each case, oxidation products are analyzed by normal-phase HPLC analysis of simple methyl esters. Products of oxidation of phospholipids in model membranes such as multilamellar or unilamellar vesicles are thus similar to products formed from free fatty acid or methyl ester oxidation in the bulk phase. Details of the mechanism of oxidation in bulk phase and membrane phase also appear to be the same. The ratio of trans,cis/trans,trans-diene products formed reflects the hydrogen donating ability in the bilayer much as it does in bulk phase. As an example, consider oxidation of dilinoleate

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lecithin (di-LinGPC) vs 1-palmitic-2-1inoleic lecithin (1-P,2-LGPC) ~6 in multilamellar vesicles. At 37°, the ratio of trans,cis/trans,trans-diene hydroperoxide products is 1.28 ± 0.05 for Di-LinGPC while the same ratio is 0.63 ± 0.04:for oxidation of 1-P,2-LGPC. The concentration of hydrogen atom donors (linoleates) in the bilayer is relatively high for Di-LinGPC (two linoleates/lecithin) while I-P,2-LGPC has half the number of good donors in the bilayer. According to the mechanism presented in Scheme III, the trans,cis/trans,trans-diene ratio should depend directly on the number of H-atom donors available in the oxidation medium and the results of oxidation of Di-LinGPC and 1-P,2-L-GPC described here are consistent with that view. It thus appears that the mechanism for oxidation in a bilayer is qualitatively the same as oxidation in bulk phase. Simple diene hydroperoxides are formed from diene phospholipid esters and the mechanism for their formation in bulk phase and bilayers is the same. The phospholipid bilayer seems to provide a medium where autoxidation occurs in the normal way. It should also be noted that the distribution of phospholipid autoxidation products does not depend on method of initiation. For example, oxidation of iinoleate phospholipid bilayers initiated by xanthine oxidase/acetaldehyde at pH 7 gave a product distribution identical to that obtained from adventitious initiation.t6 Autoxidation of arachidonate phospholipid esters has also been investigated ~7and it appears that here also bilayer and bulk phase autoxidations take a similar course. In multilamellar vesicle oxidations of 1-stearic-2arachidonic-GPC (I-S,2-AGPC), the same six hydroperoxide products are formed as are found in oxidations of arachidonic acid in benzene. Furthermore, in bilayer, the distribution of products is the same as that found in bulk phase. Cyclic peroxides like those found in bulk phase autoxidations are also formed in the bilayer.

Summary The free radical chemistry of lipid peroxidation is complex. The classical mechanism of autoxidation involving a peroxy radical abstracting hydrogen atom from lipid and oxygen addition to the carbon radical thus formed must be modified to include (1) peroxy radical/3 fragmentation and (2) peroxy radical cyclization. A host of diene hydroperoxides, cyclic peroxides, bicyclic peroxides and epoxy alcohols may be formed in free fatty acid or phospholipid autoxidation. The distribution of products and the effects of hydrogen atom donors on product distribution are understandable by referring to a general scheme for autoxidation described in Scheme III and in Ref. 10. 16 N. A. Porter and L. S. Lehman, J. Am. Chem. Soc. 104, 4731 (1982). 17 H. Weenen and N. A. Porter, J. Am. Chem. Soc. 104, 5216 (1982).