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features of this model appear relevant to the mechanism of membrane insertion or translocation of other toxins. These features include initial interaction with the membrane via a receptor or more directly with the membrane via an electrostatic interaction, low pH conditions, a hydrophobic region accessible to the membrane and a transmembrane potential. Low pH conditions enhance initial membrane binding, expose and orientate hydrophobic regions and optimize the effect of membrane potential on translocation. Hydrophobic regions play a role in the initial insertion of the toxin into the membrane and if this region is sufficiently large, it may lead to the formation of stable pores. We would stress that the relative importance of the features we have outlined in this paper will vary from one system to another. For example, some toxins do not require a low pH medium for membrane insertion; in these cases other factors such as proteolytic cleavage, disulfide bridge reduction and the receptor may play an important role. In the near future crystal structures of a number of other toxins should become available and it will be of great interest to see how toxins
LIPID OXIDATION, leading to rancidity, has been recognized since antiquity as a problem in the storage of fats and oils. Characteristic changes associated with the oxidative deterioration of vegetable oils and animal fats include the development of unpleasant tastes and odours as well as changes in colour, viscosity, specific gravity and solubility. The mechanisms by which unsaturated lipids react with molecular oxygen to undergo 'autoxidation', or 'peroxidation' (as it is now more widely known) were established in the 1940s by Farmer and his collaborators working at the research laboratories of the British Rubber Producers Association (reviewed in Ref. 1). Peroxidation reactions have long been studied by food scientists, polymer chemists, and even J. M. C. Gutterldge is at the National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts. EN6 3QG, UK. B. Halllwell is at the Biochemistry Department, King's College London, The Strand, London WC2R 2LS, UK.
with little resemblance in structure cope with the problem of membrane transport at a molecular level.
References 1 Terwilliger, T. C., Weissman, L. and Eisenberg, D. (1982) Biophys. J. 37,353-361 2 Fussle, R., Bhakdi, S., Sziegoleit, A., TranumJensen, J., Kranz, T. and Wellensiek, H. J. (1981) J. Cell. Biol. 91, 83-94 3 Collier, R. J. (1975) Bacteriol. Rev, 39, 54-85 4 Zacks, S. I. and Sheft, M. F. (1971)in Neuropoisons, Their Pathological Actions (Simpson, L., ed.), Vol. 1, pp. 222-262, Plenum Press 5 Ramsay, G., Montgomery, D., Berger, D. and Freire, E. (1989) Biochemistry 28, 529-533 6 Morion, J., Lloubes, R., Varenne, S., Chartier, M. and Lazdunski, C. (19831 J. Mol. Biol. 170, 271-285 7 Gray, G. S. and Kehoe, M. (1984) Infect. Immun. 46, 615-618 8 Falmagne, P., Capiau, P., Lambotte, P., Zanen, J., Cabiaux, V. and Ruysschaert, J-M. (1985) Biochirn. Biophys. 4cta 827, 45-50 9 Montecucco, C., Tomasi, M. and Rappuoli, R. (1986) in Bacterial Proteins Toxins (Falmagne, P., Alouf, J. E., Fehrenbach, F. J., Jeljaszewicz, J. and Thelestam, M., eds), Zentralbl. Bakteriol. Suppl. 15, pp. 117-118, Gustav Fischer 10 Eisel, U., Jarausch, W., Goretzki, K., Henschen, A., Engels, J., Weller, U., Hudel, M., Habermann, E. and Niemann, H. (1986) EMBO J. 5, 2495-2502 11 Zalman, L. S. and Wisnieski, B. J. (1985) Infect. Immun. 50, 630-635 12 Bernheimer, A. W. and Rudy, B. (1986) Biochim. Biophys. Acta 864, 123-141
13 Bashford, C. L., Alder, G. M., Menestrina, G., Micklem, K. J., Murphy, J. J. and Pasternak, C. A. (1986) J. Biol. Chem. 261, 9300-9308 14 Ribi, H. 0., Ludwig, D. S,, Mercer, L., Schoolnik, G. K. and Komberg, R. D. (1988) Science 239, 1272-1276 15 Jiang, G-S., Solow, R. and Hu, V. W. (1989) J. Biol. Chem. 264, 13424-13429 16 Lazdunski, C., Baty, D., Geli, V., Cavard, D., Morion, J., LIoubes, R., Howard, P., Knibiehler, M., Chartier, M., Varenne, S., Frenette, M., Dasseux, J-L. and Pattus, F. (1988) Biochim. Biophys. Acta 947,445-464 17 Parker, M. W., Pattus, F., Tucker, A. D. and Tsemoglou, D. (1989) Nature 337, 93-96 18 Massotte, D., Dasseux, J-L., Sauve, P., Cyrklaff, M., Leonard, K. and Pattus, F. (1989) Biochemistry 28, 7713-7719 19 Slatin, S. L., Raymond, L. and Finkelstein, A. (1986) J. Membr. Biol. 92, 247-254 20 Williams, R. J. P., Wormald, M. R. and Cramer, W. A. (1989) Biophys. J. Abstr. 495 21 Olnes, S., Moskaug, J. 0., Stenmark, H. and Sandvig, K. (1988) Trends Biochem. Sci. 13. 265-269 22 Rees, B., Samama, J. P., Thierry, J. C., Gilibert, M., Fischer, J., Schweitz, H., Lazdunski, M. and Moras, D. (1987) Proc. Natl 4cad. Sci. USA 84, 3132-3136 23 Raymond, L., Slatin, S. L., Finkelstein, A., Liu, Q. R. and Levinthal. C. (1986) J. Membr. Biol. 92. 255-268 24 Papini, E., Sandona, D., Rappuoli, R. and Montecucco, C. (1988) EMBO J. 7, 3353-3359 25 Jiang, G-S., Solow, R. and Hu, V. W. (1989) J. Biol. Chem. 264, 13424-13429 26 Engelman, D. and Steitz, T. A. (1981) Cell 23, 411-422 27 Verner, K. and Schatz, G. (1988) Science 241, 1307-1313
The basic chemistry of the propagation of lipid peroxidation reactions has been known for years, but the mechanism of initiation of this process in biological membrane systems is still uncertain. Currently available assays for measuring peroxidation are reviewed - the more specific the assay used, the less peroxide is found in healthy human tissues and body fluids. Lipid peroxidation can arise as a consequence of tissue injury in many disease states and may sometimes contribute significantly to worsening the tissue injury. by museum curators interested in the oxidative degradation of valuable paintings 2. However, only since the 1950s has the relevance of lipid peroxidation to biology and medicine been extensively explored~-~E This article aims to address (1) the origin of biological lipid peroxidation studies, (2) how the mech-
© 1990,ElsevierSciencePublishersLtd,(UK) 0376-5067/90/$02.00
anisms involved are currently perceived, and (3) present thoughts on the role of lipid peroxidation in human disease. From these discussions it should become clear that methodological problems have caused severe limitations in our present understanding but the recent availability of more chemically 129
TIBS 15-APRIL 1990
Initiation
tein will clearly increase as the protein content of the membrane rises), the fatty acid composition, oxygen concentration and the presence within the membrane of chain-breaking antioxidants (A-H in Eqn 1) which interrupt the chain reaction by providing an easily-donatable hydrogen for abstraction by peroxyl radicals.
~ Removal of H • (can occur at several places in the chain)
Minor reactions
I Mol eent colarlI rearranoe Major reaction
"'-
Attack on membrane proteins;
crosslinking if two radicals
02
Attack on membrane proteins;
r Lipid peroxyl radical
reaction of two peroxyl radicals to cause singlet oxygen (102) formation /~CHO2 • +~/CHO 2 .----~ C = O
H.abstraction / from adjacent membrane lipid
+-~C - OH + 102
O~H Lipid hydroperoxide
Figure 1 An outline mechanism of lipid peroxidation. Abstraction of hydrogen from a fatty acid with three double bonds is shown. A, B and C have conjugated diene double bond structures (double bond-single bond-double bond) that absorb UV light at or around 234 nm.
sophisticated assay techniques should allow rapid progress to be made.
Mechanisms of lipid peroxidation One of the earliest descriptions of
the different stages of lipid peroxidation was given in the late 1820s by de Saussure, who used a simple mercury manometer to study the uptake of oxygen by a layer of walnut oil on water (reviewed in Ref. 3). We now know that polyunsaturated fatty acids are particularly susceptible to peroxidation and that, once initiated, the process proceeds as a free radical chain reaction. Peroxidation is initiated by the attack of any chemical species that has sufficient reactivity to abstract a hydrogen atom from a methylene carbon in the side chain (Fig. 1). The hydrogen atom is a free radical (since it has a single, unpaired electron) and its removal 130
A-H + LC~.. LO2H + A.
(1)
meet
leaves behind an unpaired electron on the carbon atom to which it was originally attached. The resulting carboncentred radical (L.) can have several fates, but the most likely one in aerobic cells is to undergo molecular rearrangement, followed by reaction with O~ to give a peroxyl radical (Fig. 1). Peroxyl radicals can combine with each other or they can attack membrane proteins, but they are also capable of abstracting hydrogen from adjacent fatty acid side chains in a membrane and so propagating the chain reaction of lipid peroxidation. Hence a single initiation event can result in conversion of hundreds of fatty acid side chains into lipid hydroperoxides (Fig. 1). The length of the propagation chain depends upon many factors, including the lipid/protein ratio in a membrane (the chance of a radical reacting with a membrane pro-
The antioxidant-derived radical (A.) might react with another LQ. molecule, it could disappear harmlessly (e.g. by dimerization to A~) or it could be converted back to A-H by reaction with another molecule. The most important ~4 (but not the only 13) chain-breaking antioxidant in human membranes is (~-tocopherol. Evidence exists that the 0¢-tocopheryl radical can be converted back to tocopherol by reduction with ascorbic acid at the surface of biological membranes 1:~-15. The occurrence of lipid peroxidation in biological membranes causes impairment of membrane functioning~'3'~, decreased fluidity, inactivation of membrane-bound receptors and enzymes and increased non-specific permeability to ions such as Ca~+. In addition, lipid hydroperoxides decompose upon exposure to iron or copper ions, simple chelates of these metal ions (e.g. with phosphate esters), haem, and some iron proteins, including haemoglobin and myoglobin (reviewed in Refs 1, 3). Products of these complex decomposition reactions include hydrocarbon gases (such as ethane and pentane), radicals that can abstract further hydrogen atoms from fatty acid side chains, and cytotoxic carbonyl compounds, of which the most noxious are the unsaturated aldehydes such as 4-hydroxy-2-trans-nonenal TM. Indeed, a major contributor to extracellular antioxidant defence in mammals is the existence in body fluids of proteins that bind copper ions (caeruloplasmin and albumin), iron ions (transferrin), haem (haemopexin) or haem proteins (haptoglobins) and stop them from accelerating lipid peroxidation and other free radical reactions ~7~8.
What reactive species initiate lipid peroxidation? High energy irradiation of aqueous solutions produces highly reactive hydroxyl radicals (.OH) that can attack all biological molecules, including
TIBS 15-APRIL 1990
membrane lipids. Attack by .OH can result in initiation of lipid peroxidation L-H + .OH , L. + H20
(2)
Such a mechanism probably accounts for initiation of peroxidation in irradiated organisms. However, most biological studies of lipid peroxidations involve transition metal ions. When Fe2. ions, Cu* ions, or simple chelates of these ions (e.g. Fe2*-ADP) are added to liposomes or to isolated biological membranes (such as microsomes, mitochondria, erythrocyte ghosts or plasma membranes) peroxidation occurs. The oxidized forms of these transition metal ions (Fe:~*, Cu2÷) can also accelerate peroxidation if a reducing agent (e.g. ascorbate) is added. Sometimes the membrane itself can provide reducing power. For example, -SH groups on membrane proteins TM can reduce Cu-'* to Cu*, and the microsomal electron transport chain can reduce Fe:~*-ADP to Fe2*-ADP if NADPH is supplied ~:'". Hydroxyl radicals can usually be detected in these various reaction mixtures. Isolated membrane fractions often reduce 02 to superoxide radical (02), which produces H202that can then react with Fe2* or Cu* to form •OH. Fig. 2 shows some of the complex reactions occurring in these allegedly 'simple' peroxidation systems. Although .OH radicals are almost always detectable in metal ion-dependent peroxidation systems, addition of •OH scavengers or of catalase (to remove H:,O2 and block .OH formation) rarely inhibits the peroxidation observed L'-'2.Thus .OH radicals are not required for the peroxidation to proceed. What then initiates the peroxidation? Figure 2 shows that reactive species additional to -OH can also be formed; these include perferryl and ferryl (a species in which iron has an oxidation number of +4). Catalase should block ferryl formation by the mechanism shown in Fig. 2, but haemassociated ferryl species may account for the ability of mixtures of haem or haem proteins and H202to accelerate peroxidation ~3.It has been argued that perferryl is a poorly reactive species that would be unable to abstract hydrogen (discussed in Ref. 3) although its participation cannot be dismissed 24. Further, it has been reproducibly observed in many membrane systems that the initial rate of peroxidation measured when Fe2÷ is added is increased if Fe3. is also present. This has led to the suggestion
Direct reduction of added ferric complexes
Membrane electron-transport systems
Fe2+
(e.g. mitochondria, microsomes, plasma membrane)
O,rec, re,u
,iono,
OF (a)
O ~ + Fe3 + ~
t. [Fe2+-O2 • . Fe3+-O~] ~
H202 Fe2++O2
Perferryl (b)
Fe2++H202 ~
[Fe(IV)=O]
D .OH+OH
+ F e 3+
Ferryl
Figure 2 Iron-dependent generation of .OH and other reactive species in biological membrane systems (based on Ref. 21). Comparable reactions may occur for copper ions although there is considerable debate about whether 'OH is actually formed. (a) 05 can reduce Fe3÷ to Fe2÷, perferryl is an intermediate. Direct reduction of metal chelates by membranebound reducing systems or by added reducing agents (e.g. ascorbate, cysteine) is also possible. (b) Fenton reaction, showing that ferryl is a likely intermediate 21 in the formation of .OH from H202plus Fe2+.
that an Fe~*-Fe:~+-O2 complex is the initiator of peroxidation 22, but considerable doubt is cast on this proposal by the observation that other metal ions (such as Pb 2.) can replace Fe:~*in stimulating Fe2*-dependent peroxidation 25. Why is there such confusion? We feel that the problem arises because the 'simple' membrane systems that have been studied to date are not simple at all (Fig. 2). Liposomes are usually prepared from commercial lipids already contaminated with hydroperoxides. Cells contain enzymes (lipoxygenases and cyclooxygenases) that produce stereospecific fatty acyl peroxides with important biological functions (i.e. many of the eicosanoids). These enzymes can be activated upon cell injury. For example, damage to the endothelial cells lining blood vessels has been suggested to activate a lipoxygenase that leads to peroxide formation in lowdensity lipoproteins and contributes to the development of the atherosclerotic lesionlL Activation of enzyme-catalysed fatty acid peroxidation, together with the fact that transition metal ions can be released from injured cells (see below) and stimulate peroxidation, means that membrane fractions isolated from disrupted cells will always contain some lipid peroxides. Initiation of peroxidation in these systems (in the sense of abstracting the first hydrogen atom)
has thus already been achieved: added metal ions may largely or entirely stimulate peroxidation by decomposing peroxides to radicals capable of abstracting hydrogen (LO., LO2') and continuing the chain reaction:
Fe
LOOH LO. alkoxyl radical
2+
.Cu
+
)
(3)
LQ. , hydrocarbon gases, peroxyl cytotoxic aldehydes radical
We feel that detailed chemical studies on the exact initiating capacity of different radical species in peroxide-free lipid systems are long overdue. Obtaining peroxide-free lipid systems is not easy, but it can be done. In order to carry out studies on the reaction of oxygen radicals with linoleic acid, Bielski et al. (pers. commun.) recrystallized the acid at low temperature under anaerobic conditions some 48 times.
Biomedical lipid peroxidation There is considerable interest in the role played by lipid peroxidation and other free radical reactions in human disease and in toxicology3-~6. Indeed, the measurement of putative 'elevated end products of lipid peroxidation' in 131
TIBS 15-APRIL 1990
Tissue injury Heat Mechanical (trauma) infections Radiation Ultrasound Toxins O2-deprivation (ischaemia)
Cell death, membrane rupture
Release of 'catalytic' transition metal ions
Activation of cyclooxygenases and lipoxygenases
Increased lipid peroxidation in surrounding tissue
/I
Significant contribution to spreading damage
\
Insignificant epiphenomenon of the tissue injury
Figure 3 Diagram showing how cell death can result in increased free radical reactions in the surrounding tissue. Sometimes this can result in a significant spreading of the injury, in other situations it may be of no biological consequence1,3.4,9-12.
human material is probably the evidence most frequently quoted in support of the involvement of free radical reactions in tissue damage by disease or toxins. Studies beginning in the 1950s provided good evidence that several halogenated hydrocarbons exert some, or all, of their toxic effects by stimulating lipid peroxidation in vivo. This is particularly true of carbon tetrachloride 5 and probably true of bromobenzeneE This early choice of halogenated hydrocarbons for study was both fortuitous (in that it gave early emphasis to the important biological role of free radical reactions) but also unfortunate, since later studies have shown that most toxins stimulating oxidative damage to cells do not appear to act by accelerating the bulk peroxidation of cell membrane lipids 7-9,17 (i.e. the sequence of reaction is not Eqn 4).
more lipid peroxidation because antioxidants are diluted out and transition metal ions that can stimulate the peroxidation process are released from disrupted cells (Fig. 3). This stimulation of lipid peroxidation as a consequence of tissue injury can sometimes make a significant contribution to worsening the injury. For example, in atherosclerosis there is good evidence that lipid peroxidation occurs within the atherosclerotic lesion and leads to foam cell generation and hence lesion growth 1L]3. In traumatic injury to the brain and spinal cord, good evidence again exists that iron ion release into the surrounding area, and consequent iron-stimulated free radical reactions, worsen the injury (reviewed in Ref. 12). It is equally likely that in some other diseases, the increased rates of free radical reactions induced as a result of tissue injury make no significant contribution to the disease pathology ]°. Each toxin -~ lipid * cell (4) proposal that free radicals in general, or peroxidation damage lipid peroxidation in particular, are important contributors to the pathology Rises in intraceilular 'free' Ca2~, with of a given disease must be carefully consequent activation of proteases and evaluated on its merits. This obviously nucleases and formation of 'membrane requires accurate methodology for blebs', oxidation of critical -SH groups measuring these processes in cells, tisand DNA damage are often more import- sues and whole organisms. Unfortunately, ant toxic events than is the bulk per- many of the methods used to date oxidation of membrane lipids 3'4'7-9'26. have been inadequate for the job. Lipid peroxidation is often (but by no Fortunately, however, better methods means always6) a late event, ac- are now available and are beginning to companying rather than causing final be used more often. Let us now look at cell death 3,7J°,26. Indeed, cell and tissue some of the problems with the predestruction (whether mediated by rad- viously used methods, and at what techicals or otherwise) can often lead to niques might be used in future work.
132
Detection and measurement of lipid peroxidation: general principles Oxidation of lipids can be measured at different stages, including (1) losses of unsaturated fatty acids, (2) measurement of primary peroxidation products, and (3) measurement of secondary carbonyls and hydrocarbon gases (Table I). Between stages 1, 2 and 3 we could detect carbon- and oxygen-centred radicals (by electron spin resonance [ESR] combined with the use of 'spin traps') and identify these radicals by their ESR spectra 38. Table I attempts to summarize the available methods for measuring lipid peroxidation in membrane systems and in body fluids. We do not have space to discuss all of these methods in detail, but Table I contains various comments and selected references. it should be noted that the chemical composition of the end products of peroxidation will depend on the fatty acid composition of the lipid substrate used and upon what metal ions (if any) are present. Thus copper and iron ions give different end-product distributions (e.g. as measured by the thiobarbituric acid [TBA] test only [see below], copper ions are good stimulators of peroxidation in low-density lipoproteins ]3 but poor stimulators in microsomes) and so the selection of only a single test to monitor peroxidation can give misleading results. Copper salts efficiently decompose peroxides, leading to low amounts of detectable peroxides but high levels of some carbonyl compounds which may react further with molecules containing amino groups to form fluorescent products (Table I). The most accurate assays of lipid peroxidation are the most chemically sophisticated ones. They also require the most sample preparation, and great care (e.g. by working under nitrogen) has to be taken to ensure that peroxidation does not occur during the handling of lipid material.
The TBA test This is probably the most widely used single assay for measuring lipid peroxidation. The lipid material is simply heated with TBA at low pH, and the formation of a pink chromogen is measured at or close to 532 nm. The chromogen is formed by reaction of one molecule of malondialdehyde (MDA) with two molecules of TBA. Several other aldehydes formed in peroxidizing lipid systems give different chromogens with TBA39. The authentic (TBA)2-MDA adduct can be separated by HPLC
TIBS15-APRIL1990 Table I. Methods used to detect and measure biological lipid peroxidation" Method
What is measured
Remarks
Analysis of fatty acids by GLC or HPLC
Loss of unsaturated fatty acids
Very useful for assessing lipid peroxidation stimulated by different metal complexes, that give different product distributions.
Oxygen electrode
Uptake of oxygen by carboncentred radicals and during peroxide decomposition reactions
Dissolved oxygen concentration is measured. Useful in vitro when spectrophotometric interference occurs or toxic chemicals interfere with enzymic techniques. Not very sensitive.
Iodine liberation
Lipid peroxides
Lipid peroxides oxidize I- to 12,for titration with thiosulphate. Useful for bulk lipids, e.g. foodstuffs. H202also oxidizes I- to 12. Method can be applied to extracts of biological samples if other oxidizing agents are absent27.
Haem degradation of peroxides often first separated by HPLC28)
Lipid peroxides
Haem moiety of proteins can decompose lipid peroxides with formation of reactive intermediates. Microperoxidase is particularly effective. Radicals produced can be reacted with isoluminol to produce light, giving a sensitivity of 10 -12 mol peroxide. Linked to a redox dye, a sensitivity of 10 9 mol hydroperoxide can be achieved.
Glutathione peroxidase GSPase)
Lipid peroxides
GSPase reacts with H202 and hydroperoxide, oxidizing GSH to GSSG. Addition of glutathione reductase and NADPH to reduce GSSG back to GSH results in consumption of NADPH which can be related to peroxide content. Sensitivity 3 nmol ml 1 peroxide. Cannot measure peroxides within membranes: they must first be cleaved out by phospholipases 29.
Cyclooxygenase
Lipid peroxides
Stimulation of cyclooxygenase activity can be used to measure trace amounts of peroxide in biological fluids. Sensitivity picomoles of peroxide3°. This assay cannot be used to identify specific peroxides present, but it is potentially interesting because it relates the presence of peroxides to one of their potential biological actions, i.e. stimulation of eicosanoid synthesis. The assay has not been widely used to date.
GLC/mass spectrometry
Lipid peroxides
Extraction, reduction (e.g. by borohydride) to alcohols, separation by GLC, identification by mass spectrometry. Several variations of these methods exist (for examples, see Refs 31, 32, 47 and 48).
Hydrocarbon gases
Pentane and ethane
GC measurement of gases formed during lipid peroxide decomposition34. Only a minor reaction pathway but can be used as a non-invasive in vivo measure of peroxidation. Results in practice have been variable: some authors have found the technique to work well and others have abandoned it. Rigorous controls are required as hydrocarbon gases are produced by bacteria and are air pollutants. Gas production is also affected by 02 concentration in vivo, and by the metabolism of pentane to pentanol. Hydrocarbon gas production depends on the presence of metal ions to decompose lipid peroxides and so may not give an adequate index of the overall peroxidation process if such ions are only available in limited amounts.
Light emission3~
Excited carbonyls, singiet oxygen
Self reaction of peroxyl radicals can produce excited-state carbonyls and singlet 02 (Fig. 1): both species emit light as they decay to the ground state. An interesting technique for use with isolated lipid systems 35. Measurement of 'low-level chemiluminescence' is a potential method for measuring generation of reactive oxygen species in whole organs, but the light appears to arise from several sources 35.
Fluorescence36
Aldehydes
Aldehydes such as malondialdehyde (MDA) can react with amino groups to form Schiff bases (at acid pH only). At neutral pH fluorescent dihydropyridines may be formed 36. Aldehydes can also polymerize to produce fluorescent products in the absence of amino groups. Formation of fluorescent products is a minor reaction pathway and has very complex chemistry, but is a highly sensitive method. It should never be assumed, without detailed characterization 3, that fluorescent products accumulating in vivo are end products of lipid peroxidation.
TBA test
TBA-reactive material (TBARS)
The test material is heated at low pH with thiobarbituric acid (TBA) and the resulting pink chromogen is measured by absorbance at ~532 nm or by fluorescence at 553 nm. The chromogen can be extracted into butan-l-oi. Most of the aldehydes that react with TBA are derived from peroxides and unsaturated fatty acids during the test procedure. Simple and non-specific assay, rigorous controls required. Discussed in text.
HPLC/antibody techniques
Cytotoxic aldehydes
Hydroxyalkenals such as 4-hydroxynonenal are products of lipid peroxidation that are cytotoxic at nanomolar concentrations 13. They can be measured by HPLC37. Several techniques have been developed which use antibodies to detect proteins modified by lipid peroxidation products, e.g. proteins modified by reaction with unsaturated aldehydes 11.
Diene conjugation
Diene-conjugated structures (see Fig. 1)
Oxidation of unsaturated fatty acids is accompanied by an increase in UV absorbance at 230-235 nm. Useful for bulk lipids. Requires extraction or separation techniques for biological use. Greater sensitivity and specificity can be gained by measuring second-derivative spectra 33. Serious problems arise when used on human body fluids (see text).
Simplified from the detailed account in Ref: 3.
133
TIBS15-APRIL1990 of the diene-conjugated peroxides (Fig. 1) from the octadeca-9,11-dienoic acid. An alternative approach that is potentially very useful is second-derivative spectroscopy33: plotting the second derivative of absorbance gives a spectrum that appears to resolve the different diene-conjugated structures present 3:1. By far the most misleading assay to use on human material, especially plasma, is the TBA test. Plasma contains many substances that react in the TBA assay, including bile pigments, amino acids and carbohydrates 41'4~. Some of these substances (e.g. bile salts) produce a different chromogen, and this interference can be overcome by separating out the authentic (TBA)~-MDA adduct (e.g. by HPLC4°) before measurement. However, this solves only part of the problem because some compounds (especially amino acids and sugars) react in the assay to form an authentic (TBA)2-MDA adduct. The lack of specificity of the TBA assay when applied to plasma is dramatically illustrated by a simple experiment performed by Lands et al. 3°. Using the cyclooxygenase assay (Table I) they measured the lipid peroxide content of some human plasma samples as about 0.5 IJM.Expressing the results of a TBA test on the same samples as 'peroxide equivalents' gave a value of 38 pM. When specific chemical assays are used, the authors and others 2~,3° find that human plasma, freshly taken from healthy subjects, has Application of lipid peroxidation assays to less than 0.1 ].tM lipid peroxide. This is human material: the problems of the past Despite the problems that can occur perhaps not surprising, since even if with assays such as the TBA test, diene peroxides do form in vivo and enter the conjugation and light emission, they circulation, they can be rapidly cleared. usually work adequately when applied For example, although lipid peroxito measurements of peroxidation in dation is now thought to be important in liposomes, microsomes or other iso- the pathology of atherosclerosis 1H3, it lated membrane fractions, provided that seems to be peroxidation in the arterial one is alert to the various artefacts that wall that matters, not peroxidation in can arise 3,2°,44. Much more serious prob- the bulk plasma H. Thus some of the lems occur when these assays are earlier suggestions that circulating applied to human body fluids or to lipid peroxides kill vascular endothelial tissue extracts. Firstly, measurement of cells and initiate atherosclerosis need diene conjugates by UV-absorbance to be reevaluated. determination on extracted lipids cannot be used as an assay upon human Lipid peroxidation in human material: samples 4s. This is because human ma- looking to the future terial contains a compound, absorbing In order to learn as much as possible UV radiation at the diene-conjugate about the real occurrence of lipid perwavelength, that does not appear to oxidation in human material, it is arise by lipid peroxidation 4s. This com- important to use techniques that give pound, octadeca-9(cis) l l (trans)-dienoic specific chemical information about acid, probably comes from the diet and what is present. Indeed, food scientists from bacterial metabolism in the have followed this principle for years 46. colon 4s. The problem could be over- Thus more and more groups are sepcome by, for example, HPLC separation arating the various peroxidation prodfrom these other chromogens before measurement, if necessary (see, for example, Ref. 40). Some of the MDA detected in the TBA test is formed during the peroxidation process itself, but most is generated by decomposition of lipid peroxides during the acid-heating stage of the test, a process that is accelerated by transition m e t a l i o n s 1.41,42in the TBA, acid and substances being tested. Thus the TBA test does not measure MDA formed in the peroxidation system; the term TBAreactive substances (TBARS) is a much better term to u s e 1,41'42. Indeed, the value of the TBA test is that the peroxidation process beginning in the reaction mixture is effectively amplified in the assay itself, so making it very sensitive. Another problem with the assay is that added metal ions, H202, antioxidants and chelating agents can influence not only peroxidation in the incubation medium, but also peroxide decomposition during the assay itselP L4~.Some groups add antioxidants (or alternatively, a large excess of iron ions) with the TBA and acid in order to attempt to standardize for these problems 2°41, but no version of the TBA test currently available is suitable for all applications. The type and concentration of acid added with the TBA also influences the rate of peroxide decomposition and hence the amount of TBA-reactive material formed 4:{.
134
ucts before measuring them. This is often done by HPLC (Table I); for example, HPLC techniques for measuring cytotoxic aldehydes are available37. However, conversion of material into volatile derivatives, separation by gas chromatography and identification by mass spectrometry is likely to give more precise chemical information when complex mixtures are being studied 31,32. We feel strongly that such analytical techniques, allowing a precise identification of what is present, should be the methods of choice for investigations of lipid peroxidation in human material. Thus derivatization and mass spectrometry have been used to characterize peroxidized fatty acids and cholesterol oxidation products in human atherosclerotic lesions 47,4~. Specificity can also be achieved by the use of antibody techniques, particularly monoclonal antibodies. Thus antibodies directed against low-density lipoprotein that has undergone peroxidation or has been treated with 4-hydroxynonenal bind to rabbit atherosclerotic lesions. In addition, lowdensity lipoproteins eluted from such lesions can bind to antibody specific for MDA-treated low-density lipoproteins (reviewed in Ref. 11). Antibody-based methods can also be applied to plasma samples. Using such specific methods, the precise role played by lipid peroxidation in cell injury and death mediated by toxins, and in human disease, should at last become clearer 49.
References 1 Gutteridge, J. M. C. (1988)in Oxygen Radicals and Tissue Injury(Halliwell, B., ed.), pp. 9-19, FASEB 2 Daniels, V. (1988) Free Radical Res. Cemmun. 5, 213-220 3 Halliwell, B. and Gutteridge, J. M. C. (1989) Free Radicals in Biology and Medicine, 2nd edn, Clarendon Press 4 Halliwell, B. (1987) FASEB J. 1,358-364 5 Slater, T. F. and Sawyer, B. C. (1971) Biochem. J. 123, 805-814 6 Comporti, M. (1987) Chem. Phys, Lipids 45, 143-169 7 Eklow-Lastbom, L., Rossi, L., Thor, H. and Orrenius, S. (1986) Free Radical Res. Commun. 2, 57-68 8 Kappus, H. (1987) Chem. Phys. Lipids 45, 105-115 90rrenius, S., McConkey, D. J., Bellomo, G. and Nicotera, P. (1989) Trends Pharmacol. Sci. 10, 281-285 10 Halliwell, B. and Gutteridge, J. M. C. (1984) Lancet i, 1396-1398 11 Steinberg, D., Parthasarathy, S., Carew, T. E., Khoo, J. C. and Witztum, J. L. (1989) N. Engl. J. Med. 320, 915-924 12 Halliwell, B. (1989) Acta Neurol. Scand. 126, 23-29 13 Esterbauer, H., Striegl, G., Puht, H. and Rotheneder, G. (1989) Free Radical Res.
TIBS 1 5 - A P R I L 1 9 9 0 Commun. 6, 67-75 14 Burton, G. W. and Ingold, K. U. (1986) Acc. Chem. Res. 19, 194-201 15 McCay, P. B. (1985) Annu. Rev. Nutr. 5, 323-340 16 Esterbauer, H., Zollner, H. and Schaur, R. J. (1988) ISI Atlas Sci. Biochem. 1,311-317 17 Halliwell, B. and Gutteridge, J. M. C. (1986) Arch. Biochem. Biophys. 246, 501-514 18 Gutteridge, J. M. C. and Smith, A. (1988) Biochem. J. 256, 861-866 19 Leblondel, G. and Allain, P. (1984) J. Inorg. Biochem. 21,241-251 20 Buege, J. A. and Aust, S. D. (1978) Methods Enzymel. 30, 302-310 21 Halliwell, B. and Gutteridge, J. M. C. (1988) ISI Atlas Sci. Biochem. 1, 48-52 22 Minotti, G. and Aust, S. D. (1987) Chem. Phys. Lipids 44, 191-208 23 Kanner, J., German, J. B. and Kinsella, J. E. (1987) CRC Crit. Rev. Food Sci. Nutr. 25, 317-364 24 Ursini, F., Maiorino, M., Hochstein, P. and Ernster, L. (1989) Free Radical Biol. Med. 6, 31-36 25 Aruoma, O. I., Halliwell, B., Laughton, M. J., Quinlan, G. J. and Gutteridge, J. M. C. (1989) Biochem. J. 258, 617-620
26 Cochrane, C. G., Schraufstatter, I. U., Hyslop, P. and Jackson, J. (1988) in Oxygen Radicals and Tissue Injury (Hatliwell, B., ed.), pp. 49-54, FASEB 27 Thomas, S. M., Jessup, W., Gebicki, J. M. and Dean, R. T. (1989) Anal. Biochem. 176, 353-359 28 Frei, B., Yamamoto, Y., Niclas, D. and Ames, B. N. (1988) Anal. Biochem. 175,120-130 29 O'Gara, C. Y., Maddipatti, K. R. and Marnett, L. J. (1989) Chem. Res. Tox. 2, 295-300 30 Marshall, P. J., Warso, M. A. and Lands, W. E. M. (1985) Anal. Biochem. 145,192-199 31 Hughes, H., Smith, C. V., Tsokos-Kuhn, J. O. and Mitchell, J. R. (1986) Anal. Biochem. 152, 107-112 32 Van Kuijk, F. J. G. M., Thomas, D. W., Stephens, R. J. and Dratz, E. A. (1986) Biochem. Biophys. Res. Commun. 139,144-149 33 Corongiu, F. P. and Milia, A. (1983) Chem.-Biol. Interact. 44, 289-297 34 Burk, R. F. and Ludden, T. M. (1989) Biochem. Pharmacol. 38, 1029-1032 35 Sies, H. (1987) Arch. Toxicol. 60, 138-143 36 Kikugawa, K. (1986) Adv. Free Radical Biol. Med. 2,389-417 37 Esterbauer, H. and Zollner, H. (1989) Free Radical Biol. Med. 7,197-203
38 Davies, M. J. (1987) Chem. Phys. Lipids 44, 149-173 39 Kosugi, H., Kate, T. and Kikugawa, K. (1987) Anal. Biochem. 165,456-464 40 Largilli~re, C. and Melancon, S. B. (1988) Anal. Biochem. 170, 123-126 41 Gutteridge, J. M. C. and Quinlan, G. J. (1983) J. Appl. Biochem. 5, 293-299 42 Gutteridge, J. M. C. (1986) Free Radical Res. Commun. 1, 173-184 43 Gutteridge, J. M. C. (1982) Int. J. Biochem. 14, 649-653 44 Nell, T., de Greet, H. and Sies, H. (1987) Arch. Biochem. Biophys. 252,284-291 45 Thompson, S. and Smith, M. T. (1985) Chem.-Biol. Interact. 55,357-366 46 Frankel, E. N. and Neff, W. E. (1983) Biochim. Biophys. Acta 754, 264-270 47 Carpenter, K. L. H., Ball, R. Y., Ardeshna, K. M., Bindman, J. P., Enright, J. H., Hartley, S. L., Nicholson, S. and Mitchinson, M. J. (1987)in Lipofuscin: State of the Art (Nagy, Z. S., ed.), pp. 245-268, Elsevier 48 Mowri, H., Chinen, K., Ohkuma, S. and Takano, T. (1986) Biochem. Int. 12,347-352 49 Steinbrecher, U. P., Lougheed, M., Kwan, W. C. and Dirks, M. (1989) J. Biol. Chem. 264, 15216-15223
LETTERS 'Rapid evolution' of the amino acid composition of proteins While preparing a chapter entitled 'Frequency of Amino Acids' for the book Proteins (Landolt-B6rnstein New Series, Vol. VII/2), we have noticed a rapid evolution in the amino acid compositions of the proteins which have been sequenced in the past decade. Table I shows the overall composition of proteins derived from databases which were available in 1978 l, 19842, and 19863, as well as the amino acid composition of the so-called open-reading-frame (ORF) proteins of the 1986 database*. Protein sequences that are derived from nucleic acid sequences have caused most of the systematic changes in composition shown in Table I. The applicability of this method does not depend on solubility or other features of the protein sequenced and thus new classes of proteins have been added to the data set. A trivial change is the increasing amount of methionine coded by the start *Baker, W. C., Hunt, L. C., George, D. G., Yeh, L. S., Chen, H. R., Blomquist, M. C., Seibel-Ross, E. I., EIzanowski, A., Hong, M. K., Ferrick, D. A., Bair, J. K., Chen, S. L. and Ledley, R. S. (1986) Protein Sequence Database, Release 11.0, December 1986 plus NEW.DAT file, National Biochemical Foundation, Georgetown University Medical Center, Washington DC, USA.
codon; N-terminal methionine belonging to a signal peptide is missing in an isolated protein. The ratio of hydrophobic and hydrophilic residues has increased, apparently because membrane-bound proteins, which cannot be sequenced as proteins by traditional methods, have been added to the database. There are also significant shifts in the relative abundances of some similar residues. For example the Arg:Lys ratio rises from 0.60 (data of 1978) to 0.86 (data of 1986), and it
is 1.17 for ORF proteins which is closer to but still far from the ratio of the number of different codons coding for Arg and Lys4.5. A significant consequence of the fact that proteins appearing in the database do not represent all proteins uniformly is that structure-prediction methods based on statistical analysis of protein, such as the widely used Chou-Fasman method for predicting secondary structures ~, or our method for predicting domain boundaries
Table I. Amino acid composition (%) of proteins a Amino acid A C D E F G H I K L M N P Q R S T V W Y
I
II
III
IV
8.3 3.1 5.5 5.7 3.8 8.9 2.4 4.1 7.0 7.5 1.6 4.0 5.5 3.8 4.2 7.3 6.1 6.5 1.2 3.4
8.11 2.28 5.13 5.97 3.83 7.57 2.38 4.98 6.25 8.76 2.25 4.29 5.10 3.94 4.94 7.12 6.02 6.46 1.40 3.29
7.75 2.14 5.16 6.06 3.97 7,35 2,36 5.07 5.97 9.08 2.27 4.23 5.28 4.02 5.11 7.10 5.90 6.53 1.39 3.74
7.28 1.77 4.07 4.87 4.63 6.18 2.34 6.14 4.85 10.93 2.99 4.49 5.76 3.42 5.67 7.70 6.09 5.79 1.58 3.45
aDerived from databases which were available in 1978 (I), in January 1984 (11),and in December 1986 (111)and from ORF proteins appearing in the 1986 database (IV) (see text).
© 1990,ElsevierSciencePublishersLtd,(UK) 0376-5067/90/$02.00
135
features of this model appear relevant to the mechanism of membrane insertion or translocation of other toxins. These features include initial interaction with the membrane via a receptor or more directly with the membrane via an electrostatic interaction, low pH conditions, a hydrophobic region accessible to the membrane and a transmembrane potential. Low pH conditions enhance initial membrane binding, expose and orientate hydrophobic regions and optimize the effect of membrane potential on translocation. Hydrophobic regions play a role in the initial insertion of the toxin into the membrane and if this region is sufficiently large, it may lead to the formation of stable pores. We would stress that the relative importance of the features we have outlined in this paper will vary from one system to another. For example, some toxins do not require a low pH medium for membrane insertion; in these cases other factors such as proteolytic cleavage, disulfide bridge reduction and the receptor may play an important role. In the near future crystal structures of a number of other toxins should become available and it will be of great interest to see how toxins
LIPID OXIDATION, leading to rancidity, has been recognized since antiquity as a problem in the storage of fats and oils. Characteristic changes associated with the oxidative deterioration of vegetable oils and animal fats include the development of unpleasant tastes and odours as well as changes in colour, viscosity, specific gravity and solubility. The mechanisms by which unsaturated lipids react with molecular oxygen to undergo 'autoxidation', or 'peroxidation' (as it is now more widely known) were established in the 1940s by Farmer and his collaborators working at the research laboratories of the British Rubber Producers Association (reviewed in Ref. 1). Peroxidation reactions have long been studied by food scientists, polymer chemists, and even J. M. C. Gutterldge is at the National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts. EN6 3QG, UK. B. Halllwell is at the Biochemistry Department, King's College London, The Strand, London WC2R 2LS, UK.
with little resemblance in structure cope with the problem of membrane transport at a molecular level.
References 1 Terwilliger, T. C., Weissman, L. and Eisenberg, D. (1982) Biophys. J. 37,353-361 2 Fussle, R., Bhakdi, S., Sziegoleit, A., TranumJensen, J., Kranz, T. and Wellensiek, H. J. (1981) J. Cell. Biol. 91, 83-94 3 Collier, R. J. (1975) Bacteriol. Rev, 39, 54-85 4 Zacks, S. I. and Sheft, M. F. (1971)in Neuropoisons, Their Pathological Actions (Simpson, L., ed.), Vol. 1, pp. 222-262, Plenum Press 5 Ramsay, G., Montgomery, D., Berger, D. and Freire, E. (1989) Biochemistry 28, 529-533 6 Morion, J., Lloubes, R., Varenne, S., Chartier, M. and Lazdunski, C. (19831 J. Mol. Biol. 170, 271-285 7 Gray, G. S. and Kehoe, M. (1984) Infect. Immun. 46, 615-618 8 Falmagne, P., Capiau, P., Lambotte, P., Zanen, J., Cabiaux, V. and Ruysschaert, J-M. (1985) Biochirn. Biophys. 4cta 827, 45-50 9 Montecucco, C., Tomasi, M. and Rappuoli, R. (1986) in Bacterial Proteins Toxins (Falmagne, P., Alouf, J. E., Fehrenbach, F. J., Jeljaszewicz, J. and Thelestam, M., eds), Zentralbl. Bakteriol. Suppl. 15, pp. 117-118, Gustav Fischer 10 Eisel, U., Jarausch, W., Goretzki, K., Henschen, A., Engels, J., Weller, U., Hudel, M., Habermann, E. and Niemann, H. (1986) EMBO J. 5, 2495-2502 11 Zalman, L. S. and Wisnieski, B. J. (1985) Infect. Immun. 50, 630-635 12 Bernheimer, A. W. and Rudy, B. (1986) Biochim. Biophys. Acta 864, 123-141
13 Bashford, C. L., Alder, G. M., Menestrina, G., Micklem, K. J., Murphy, J. J. and Pasternak, C. A. (1986) J. Biol. Chem. 261, 9300-9308 14 Ribi, H. 0., Ludwig, D. S,, Mercer, L., Schoolnik, G. K. and Komberg, R. D. (1988) Science 239, 1272-1276 15 Jiang, G-S., Solow, R. and Hu, V. W. (1989) J. Biol. Chem. 264, 13424-13429 16 Lazdunski, C., Baty, D., Geli, V., Cavard, D., Morion, J., LIoubes, R., Howard, P., Knibiehler, M., Chartier, M., Varenne, S., Frenette, M., Dasseux, J-L. and Pattus, F. (1988) Biochim. Biophys. Acta 947,445-464 17 Parker, M. W., Pattus, F., Tucker, A. D. and Tsemoglou, D. (1989) Nature 337, 93-96 18 Massotte, D., Dasseux, J-L., Sauve, P., Cyrklaff, M., Leonard, K. and Pattus, F. (1989) Biochemistry 28, 7713-7719 19 Slatin, S. L., Raymond, L. and Finkelstein, A. (1986) J. Membr. Biol. 92, 247-254 20 Williams, R. J. P., Wormald, M. R. and Cramer, W. A. (1989) Biophys. J. Abstr. 495 21 Olnes, S., Moskaug, J. 0., Stenmark, H. and Sandvig, K. (1988) Trends Biochem. Sci. 13. 265-269 22 Rees, B., Samama, J. P., Thierry, J. C., Gilibert, M., Fischer, J., Schweitz, H., Lazdunski, M. and Moras, D. (1987) Proc. Natl 4cad. Sci. USA 84, 3132-3136 23 Raymond, L., Slatin, S. L., Finkelstein, A., Liu, Q. R. and Levinthal. C. (1986) J. Membr. Biol. 92. 255-268 24 Papini, E., Sandona, D., Rappuoli, R. and Montecucco, C. (1988) EMBO J. 7, 3353-3359 25 Jiang, G-S., Solow, R. and Hu, V. W. (1989) J. Biol. Chem. 264, 13424-13429 26 Engelman, D. and Steitz, T. A. (1981) Cell 23, 411-422 27 Verner, K. and Schatz, G. (1988) Science 241, 1307-1313
The basic chemistry of the propagation of lipid peroxidation reactions has been known for years, but the mechanism of initiation of this process in biological membrane systems is still uncertain. Currently available assays for measuring peroxidation are reviewed - the more specific the assay used, the less peroxide is found in healthy human tissues and body fluids. Lipid peroxidation can arise as a consequence of tissue injury in many disease states and may sometimes contribute significantly to worsening the tissue injury. by museum curators interested in the oxidative degradation of valuable paintings 2. However, only since the 1950s has the relevance of lipid peroxidation to biology and medicine been extensively explored~-~E This article aims to address (1) the origin of biological lipid peroxidation studies, (2) how the mech-
© 1990,ElsevierSciencePublishersLtd,(UK) 0376-5067/90/$02.00
anisms involved are currently perceived, and (3) present thoughts on the role of lipid peroxidation in human disease. From these discussions it should become clear that methodological problems have caused severe limitations in our present understanding but the recent availability of more chemically 129
TIBS 15-APRIL 1990
Initiation
tein will clearly increase as the protein content of the membrane rises), the fatty acid composition, oxygen concentration and the presence within the membrane of chain-breaking antioxidants (A-H in Eqn 1) which interrupt the chain reaction by providing an easily-donatable hydrogen for abstraction by peroxyl radicals.
~ Removal of H • (can occur at several places in the chain)
Minor reactions
I Mol eent colarlI rearranoe Major reaction
"'-
Attack on membrane proteins;
crosslinking if two radicals
02
Attack on membrane proteins;
r Lipid peroxyl radical
reaction of two peroxyl radicals to cause singlet oxygen (102) formation /~CHO2 • +~/CHO 2 .----~ C = O
H.abstraction / from adjacent membrane lipid
+-~C - OH + 102
O~H Lipid hydroperoxide
Figure 1 An outline mechanism of lipid peroxidation. Abstraction of hydrogen from a fatty acid with three double bonds is shown. A, B and C have conjugated diene double bond structures (double bond-single bond-double bond) that absorb UV light at or around 234 nm.
sophisticated assay techniques should allow rapid progress to be made.
Mechanisms of lipid peroxidation One of the earliest descriptions of
the different stages of lipid peroxidation was given in the late 1820s by de Saussure, who used a simple mercury manometer to study the uptake of oxygen by a layer of walnut oil on water (reviewed in Ref. 3). We now know that polyunsaturated fatty acids are particularly susceptible to peroxidation and that, once initiated, the process proceeds as a free radical chain reaction. Peroxidation is initiated by the attack of any chemical species that has sufficient reactivity to abstract a hydrogen atom from a methylene carbon in the side chain (Fig. 1). The hydrogen atom is a free radical (since it has a single, unpaired electron) and its removal 130
A-H + LC~.. LO2H + A.
(1)
meet
leaves behind an unpaired electron on the carbon atom to which it was originally attached. The resulting carboncentred radical (L.) can have several fates, but the most likely one in aerobic cells is to undergo molecular rearrangement, followed by reaction with O~ to give a peroxyl radical (Fig. 1). Peroxyl radicals can combine with each other or they can attack membrane proteins, but they are also capable of abstracting hydrogen from adjacent fatty acid side chains in a membrane and so propagating the chain reaction of lipid peroxidation. Hence a single initiation event can result in conversion of hundreds of fatty acid side chains into lipid hydroperoxides (Fig. 1). The length of the propagation chain depends upon many factors, including the lipid/protein ratio in a membrane (the chance of a radical reacting with a membrane pro-
The antioxidant-derived radical (A.) might react with another LQ. molecule, it could disappear harmlessly (e.g. by dimerization to A~) or it could be converted back to A-H by reaction with another molecule. The most important ~4 (but not the only 13) chain-breaking antioxidant in human membranes is (~-tocopherol. Evidence exists that the 0¢-tocopheryl radical can be converted back to tocopherol by reduction with ascorbic acid at the surface of biological membranes 1:~-15. The occurrence of lipid peroxidation in biological membranes causes impairment of membrane functioning~'3'~, decreased fluidity, inactivation of membrane-bound receptors and enzymes and increased non-specific permeability to ions such as Ca~+. In addition, lipid hydroperoxides decompose upon exposure to iron or copper ions, simple chelates of these metal ions (e.g. with phosphate esters), haem, and some iron proteins, including haemoglobin and myoglobin (reviewed in Refs 1, 3). Products of these complex decomposition reactions include hydrocarbon gases (such as ethane and pentane), radicals that can abstract further hydrogen atoms from fatty acid side chains, and cytotoxic carbonyl compounds, of which the most noxious are the unsaturated aldehydes such as 4-hydroxy-2-trans-nonenal TM. Indeed, a major contributor to extracellular antioxidant defence in mammals is the existence in body fluids of proteins that bind copper ions (caeruloplasmin and albumin), iron ions (transferrin), haem (haemopexin) or haem proteins (haptoglobins) and stop them from accelerating lipid peroxidation and other free radical reactions ~7~8.
What reactive species initiate lipid peroxidation? High energy irradiation of aqueous solutions produces highly reactive hydroxyl radicals (.OH) that can attack all biological molecules, including
TIBS 15-APRIL 1990
membrane lipids. Attack by .OH can result in initiation of lipid peroxidation L-H + .OH , L. + H20
(2)
Such a mechanism probably accounts for initiation of peroxidation in irradiated organisms. However, most biological studies of lipid peroxidations involve transition metal ions. When Fe2. ions, Cu* ions, or simple chelates of these ions (e.g. Fe2*-ADP) are added to liposomes or to isolated biological membranes (such as microsomes, mitochondria, erythrocyte ghosts or plasma membranes) peroxidation occurs. The oxidized forms of these transition metal ions (Fe:~*, Cu2÷) can also accelerate peroxidation if a reducing agent (e.g. ascorbate) is added. Sometimes the membrane itself can provide reducing power. For example, -SH groups on membrane proteins TM can reduce Cu-'* to Cu*, and the microsomal electron transport chain can reduce Fe:~*-ADP to Fe2*-ADP if NADPH is supplied ~:'". Hydroxyl radicals can usually be detected in these various reaction mixtures. Isolated membrane fractions often reduce 02 to superoxide radical (02), which produces H202that can then react with Fe2* or Cu* to form •OH. Fig. 2 shows some of the complex reactions occurring in these allegedly 'simple' peroxidation systems. Although .OH radicals are almost always detectable in metal ion-dependent peroxidation systems, addition of •OH scavengers or of catalase (to remove H:,O2 and block .OH formation) rarely inhibits the peroxidation observed L'-'2.Thus .OH radicals are not required for the peroxidation to proceed. What then initiates the peroxidation? Figure 2 shows that reactive species additional to -OH can also be formed; these include perferryl and ferryl (a species in which iron has an oxidation number of +4). Catalase should block ferryl formation by the mechanism shown in Fig. 2, but haemassociated ferryl species may account for the ability of mixtures of haem or haem proteins and H202to accelerate peroxidation ~3.It has been argued that perferryl is a poorly reactive species that would be unable to abstract hydrogen (discussed in Ref. 3) although its participation cannot be dismissed 24. Further, it has been reproducibly observed in many membrane systems that the initial rate of peroxidation measured when Fe2÷ is added is increased if Fe3. is also present. This has led to the suggestion
Direct reduction of added ferric complexes
Membrane electron-transport systems
Fe2+
(e.g. mitochondria, microsomes, plasma membrane)
O,rec, re,u
,iono,
OF (a)
O ~ + Fe3 + ~
t. [Fe2+-O2 • . Fe3+-O~] ~
H202 Fe2++O2
Perferryl (b)
Fe2++H202 ~
[Fe(IV)=O]
D .OH+OH
+ F e 3+
Ferryl
Figure 2 Iron-dependent generation of .OH and other reactive species in biological membrane systems (based on Ref. 21). Comparable reactions may occur for copper ions although there is considerable debate about whether 'OH is actually formed. (a) 05 can reduce Fe3÷ to Fe2÷, perferryl is an intermediate. Direct reduction of metal chelates by membranebound reducing systems or by added reducing agents (e.g. ascorbate, cysteine) is also possible. (b) Fenton reaction, showing that ferryl is a likely intermediate 21 in the formation of .OH from H202plus Fe2+.
that an Fe~*-Fe:~+-O2 complex is the initiator of peroxidation 22, but considerable doubt is cast on this proposal by the observation that other metal ions (such as Pb 2.) can replace Fe:~*in stimulating Fe2*-dependent peroxidation 25. Why is there such confusion? We feel that the problem arises because the 'simple' membrane systems that have been studied to date are not simple at all (Fig. 2). Liposomes are usually prepared from commercial lipids already contaminated with hydroperoxides. Cells contain enzymes (lipoxygenases and cyclooxygenases) that produce stereospecific fatty acyl peroxides with important biological functions (i.e. many of the eicosanoids). These enzymes can be activated upon cell injury. For example, damage to the endothelial cells lining blood vessels has been suggested to activate a lipoxygenase that leads to peroxide formation in lowdensity lipoproteins and contributes to the development of the atherosclerotic lesionlL Activation of enzyme-catalysed fatty acid peroxidation, together with the fact that transition metal ions can be released from injured cells (see below) and stimulate peroxidation, means that membrane fractions isolated from disrupted cells will always contain some lipid peroxides. Initiation of peroxidation in these systems (in the sense of abstracting the first hydrogen atom)
has thus already been achieved: added metal ions may largely or entirely stimulate peroxidation by decomposing peroxides to radicals capable of abstracting hydrogen (LO., LO2') and continuing the chain reaction:
Fe
LOOH LO. alkoxyl radical
2+
.Cu
+
)
(3)
LQ. , hydrocarbon gases, peroxyl cytotoxic aldehydes radical
We feel that detailed chemical studies on the exact initiating capacity of different radical species in peroxide-free lipid systems are long overdue. Obtaining peroxide-free lipid systems is not easy, but it can be done. In order to carry out studies on the reaction of oxygen radicals with linoleic acid, Bielski et al. (pers. commun.) recrystallized the acid at low temperature under anaerobic conditions some 48 times.
Biomedical lipid peroxidation There is considerable interest in the role played by lipid peroxidation and other free radical reactions in human disease and in toxicology3-~6. Indeed, the measurement of putative 'elevated end products of lipid peroxidation' in 131
TIBS 15-APRIL 1990
Tissue injury Heat Mechanical (trauma) infections Radiation Ultrasound Toxins O2-deprivation (ischaemia)
Cell death, membrane rupture
Release of 'catalytic' transition metal ions
Activation of cyclooxygenases and lipoxygenases
Increased lipid peroxidation in surrounding tissue
/I
Significant contribution to spreading damage
\
Insignificant epiphenomenon of the tissue injury
Figure 3 Diagram showing how cell death can result in increased free radical reactions in the surrounding tissue. Sometimes this can result in a significant spreading of the injury, in other situations it may be of no biological consequence1,3.4,9-12.
human material is probably the evidence most frequently quoted in support of the involvement of free radical reactions in tissue damage by disease or toxins. Studies beginning in the 1950s provided good evidence that several halogenated hydrocarbons exert some, or all, of their toxic effects by stimulating lipid peroxidation in vivo. This is particularly true of carbon tetrachloride 5 and probably true of bromobenzeneE This early choice of halogenated hydrocarbons for study was both fortuitous (in that it gave early emphasis to the important biological role of free radical reactions) but also unfortunate, since later studies have shown that most toxins stimulating oxidative damage to cells do not appear to act by accelerating the bulk peroxidation of cell membrane lipids 7-9,17 (i.e. the sequence of reaction is not Eqn 4).
more lipid peroxidation because antioxidants are diluted out and transition metal ions that can stimulate the peroxidation process are released from disrupted cells (Fig. 3). This stimulation of lipid peroxidation as a consequence of tissue injury can sometimes make a significant contribution to worsening the injury. For example, in atherosclerosis there is good evidence that lipid peroxidation occurs within the atherosclerotic lesion and leads to foam cell generation and hence lesion growth 1L]3. In traumatic injury to the brain and spinal cord, good evidence again exists that iron ion release into the surrounding area, and consequent iron-stimulated free radical reactions, worsen the injury (reviewed in Ref. 12). It is equally likely that in some other diseases, the increased rates of free radical reactions induced as a result of tissue injury make no significant contribution to the disease pathology ]°. Each toxin -~ lipid * cell (4) proposal that free radicals in general, or peroxidation damage lipid peroxidation in particular, are important contributors to the pathology Rises in intraceilular 'free' Ca2~, with of a given disease must be carefully consequent activation of proteases and evaluated on its merits. This obviously nucleases and formation of 'membrane requires accurate methodology for blebs', oxidation of critical -SH groups measuring these processes in cells, tisand DNA damage are often more import- sues and whole organisms. Unfortunately, ant toxic events than is the bulk per- many of the methods used to date oxidation of membrane lipids 3'4'7-9'26. have been inadequate for the job. Lipid peroxidation is often (but by no Fortunately, however, better methods means always6) a late event, ac- are now available and are beginning to companying rather than causing final be used more often. Let us now look at cell death 3,7J°,26. Indeed, cell and tissue some of the problems with the predestruction (whether mediated by rad- viously used methods, and at what techicals or otherwise) can often lead to niques might be used in future work.
132
Detection and measurement of lipid peroxidation: general principles Oxidation of lipids can be measured at different stages, including (1) losses of unsaturated fatty acids, (2) measurement of primary peroxidation products, and (3) measurement of secondary carbonyls and hydrocarbon gases (Table I). Between stages 1, 2 and 3 we could detect carbon- and oxygen-centred radicals (by electron spin resonance [ESR] combined with the use of 'spin traps') and identify these radicals by their ESR spectra 38. Table I attempts to summarize the available methods for measuring lipid peroxidation in membrane systems and in body fluids. We do not have space to discuss all of these methods in detail, but Table I contains various comments and selected references. it should be noted that the chemical composition of the end products of peroxidation will depend on the fatty acid composition of the lipid substrate used and upon what metal ions (if any) are present. Thus copper and iron ions give different end-product distributions (e.g. as measured by the thiobarbituric acid [TBA] test only [see below], copper ions are good stimulators of peroxidation in low-density lipoproteins ]3 but poor stimulators in microsomes) and so the selection of only a single test to monitor peroxidation can give misleading results. Copper salts efficiently decompose peroxides, leading to low amounts of detectable peroxides but high levels of some carbonyl compounds which may react further with molecules containing amino groups to form fluorescent products (Table I). The most accurate assays of lipid peroxidation are the most chemically sophisticated ones. They also require the most sample preparation, and great care (e.g. by working under nitrogen) has to be taken to ensure that peroxidation does not occur during the handling of lipid material.
The TBA test This is probably the most widely used single assay for measuring lipid peroxidation. The lipid material is simply heated with TBA at low pH, and the formation of a pink chromogen is measured at or close to 532 nm. The chromogen is formed by reaction of one molecule of malondialdehyde (MDA) with two molecules of TBA. Several other aldehydes formed in peroxidizing lipid systems give different chromogens with TBA39. The authentic (TBA)2-MDA adduct can be separated by HPLC
TIBS15-APRIL1990 Table I. Methods used to detect and measure biological lipid peroxidation" Method
What is measured
Remarks
Analysis of fatty acids by GLC or HPLC
Loss of unsaturated fatty acids
Very useful for assessing lipid peroxidation stimulated by different metal complexes, that give different product distributions.
Oxygen electrode
Uptake of oxygen by carboncentred radicals and during peroxide decomposition reactions
Dissolved oxygen concentration is measured. Useful in vitro when spectrophotometric interference occurs or toxic chemicals interfere with enzymic techniques. Not very sensitive.
Iodine liberation
Lipid peroxides
Lipid peroxides oxidize I- to 12,for titration with thiosulphate. Useful for bulk lipids, e.g. foodstuffs. H202also oxidizes I- to 12. Method can be applied to extracts of biological samples if other oxidizing agents are absent27.
Haem degradation of peroxides often first separated by HPLC28)
Lipid peroxides
Haem moiety of proteins can decompose lipid peroxides with formation of reactive intermediates. Microperoxidase is particularly effective. Radicals produced can be reacted with isoluminol to produce light, giving a sensitivity of 10 -12 mol peroxide. Linked to a redox dye, a sensitivity of 10 9 mol hydroperoxide can be achieved.
Glutathione peroxidase GSPase)
Lipid peroxides
GSPase reacts with H202 and hydroperoxide, oxidizing GSH to GSSG. Addition of glutathione reductase and NADPH to reduce GSSG back to GSH results in consumption of NADPH which can be related to peroxide content. Sensitivity 3 nmol ml 1 peroxide. Cannot measure peroxides within membranes: they must first be cleaved out by phospholipases 29.
Cyclooxygenase
Lipid peroxides
Stimulation of cyclooxygenase activity can be used to measure trace amounts of peroxide in biological fluids. Sensitivity picomoles of peroxide3°. This assay cannot be used to identify specific peroxides present, but it is potentially interesting because it relates the presence of peroxides to one of their potential biological actions, i.e. stimulation of eicosanoid synthesis. The assay has not been widely used to date.
GLC/mass spectrometry
Lipid peroxides
Extraction, reduction (e.g. by borohydride) to alcohols, separation by GLC, identification by mass spectrometry. Several variations of these methods exist (for examples, see Refs 31, 32, 47 and 48).
Hydrocarbon gases
Pentane and ethane
GC measurement of gases formed during lipid peroxide decomposition34. Only a minor reaction pathway but can be used as a non-invasive in vivo measure of peroxidation. Results in practice have been variable: some authors have found the technique to work well and others have abandoned it. Rigorous controls are required as hydrocarbon gases are produced by bacteria and are air pollutants. Gas production is also affected by 02 concentration in vivo, and by the metabolism of pentane to pentanol. Hydrocarbon gas production depends on the presence of metal ions to decompose lipid peroxides and so may not give an adequate index of the overall peroxidation process if such ions are only available in limited amounts.
Light emission3~
Excited carbonyls, singiet oxygen
Self reaction of peroxyl radicals can produce excited-state carbonyls and singlet 02 (Fig. 1): both species emit light as they decay to the ground state. An interesting technique for use with isolated lipid systems 35. Measurement of 'low-level chemiluminescence' is a potential method for measuring generation of reactive oxygen species in whole organs, but the light appears to arise from several sources 35.
Fluorescence36
Aldehydes
Aldehydes such as malondialdehyde (MDA) can react with amino groups to form Schiff bases (at acid pH only). At neutral pH fluorescent dihydropyridines may be formed 36. Aldehydes can also polymerize to produce fluorescent products in the absence of amino groups. Formation of fluorescent products is a minor reaction pathway and has very complex chemistry, but is a highly sensitive method. It should never be assumed, without detailed characterization 3, that fluorescent products accumulating in vivo are end products of lipid peroxidation.
TBA test
TBA-reactive material (TBARS)
The test material is heated at low pH with thiobarbituric acid (TBA) and the resulting pink chromogen is measured by absorbance at ~532 nm or by fluorescence at 553 nm. The chromogen can be extracted into butan-l-oi. Most of the aldehydes that react with TBA are derived from peroxides and unsaturated fatty acids during the test procedure. Simple and non-specific assay, rigorous controls required. Discussed in text.
HPLC/antibody techniques
Cytotoxic aldehydes
Hydroxyalkenals such as 4-hydroxynonenal are products of lipid peroxidation that are cytotoxic at nanomolar concentrations 13. They can be measured by HPLC37. Several techniques have been developed which use antibodies to detect proteins modified by lipid peroxidation products, e.g. proteins modified by reaction with unsaturated aldehydes 11.
Diene conjugation
Diene-conjugated structures (see Fig. 1)
Oxidation of unsaturated fatty acids is accompanied by an increase in UV absorbance at 230-235 nm. Useful for bulk lipids. Requires extraction or separation techniques for biological use. Greater sensitivity and specificity can be gained by measuring second-derivative spectra 33. Serious problems arise when used on human body fluids (see text).
Simplified from the detailed account in Ref: 3.
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TIBS15-APRIL1990 of the diene-conjugated peroxides (Fig. 1) from the octadeca-9,11-dienoic acid. An alternative approach that is potentially very useful is second-derivative spectroscopy33: plotting the second derivative of absorbance gives a spectrum that appears to resolve the different diene-conjugated structures present 3:1. By far the most misleading assay to use on human material, especially plasma, is the TBA test. Plasma contains many substances that react in the TBA assay, including bile pigments, amino acids and carbohydrates 41'4~. Some of these substances (e.g. bile salts) produce a different chromogen, and this interference can be overcome by separating out the authentic (TBA)~-MDA adduct (e.g. by HPLC4°) before measurement. However, this solves only part of the problem because some compounds (especially amino acids and sugars) react in the assay to form an authentic (TBA)2-MDA adduct. The lack of specificity of the TBA assay when applied to plasma is dramatically illustrated by a simple experiment performed by Lands et al. 3°. Using the cyclooxygenase assay (Table I) they measured the lipid peroxide content of some human plasma samples as about 0.5 IJM.Expressing the results of a TBA test on the same samples as 'peroxide equivalents' gave a value of 38 pM. When specific chemical assays are used, the authors and others 2~,3° find that human plasma, freshly taken from healthy subjects, has Application of lipid peroxidation assays to less than 0.1 ].tM lipid peroxide. This is human material: the problems of the past Despite the problems that can occur perhaps not surprising, since even if with assays such as the TBA test, diene peroxides do form in vivo and enter the conjugation and light emission, they circulation, they can be rapidly cleared. usually work adequately when applied For example, although lipid peroxito measurements of peroxidation in dation is now thought to be important in liposomes, microsomes or other iso- the pathology of atherosclerosis 1H3, it lated membrane fractions, provided that seems to be peroxidation in the arterial one is alert to the various artefacts that wall that matters, not peroxidation in can arise 3,2°,44. Much more serious prob- the bulk plasma H. Thus some of the lems occur when these assays are earlier suggestions that circulating applied to human body fluids or to lipid peroxides kill vascular endothelial tissue extracts. Firstly, measurement of cells and initiate atherosclerosis need diene conjugates by UV-absorbance to be reevaluated. determination on extracted lipids cannot be used as an assay upon human Lipid peroxidation in human material: samples 4s. This is because human ma- looking to the future terial contains a compound, absorbing In order to learn as much as possible UV radiation at the diene-conjugate about the real occurrence of lipid perwavelength, that does not appear to oxidation in human material, it is arise by lipid peroxidation 4s. This com- important to use techniques that give pound, octadeca-9(cis) l l (trans)-dienoic specific chemical information about acid, probably comes from the diet and what is present. Indeed, food scientists from bacterial metabolism in the have followed this principle for years 46. colon 4s. The problem could be over- Thus more and more groups are sepcome by, for example, HPLC separation arating the various peroxidation prodfrom these other chromogens before measurement, if necessary (see, for example, Ref. 40). Some of the MDA detected in the TBA test is formed during the peroxidation process itself, but most is generated by decomposition of lipid peroxides during the acid-heating stage of the test, a process that is accelerated by transition m e t a l i o n s 1.41,42in the TBA, acid and substances being tested. Thus the TBA test does not measure MDA formed in the peroxidation system; the term TBAreactive substances (TBARS) is a much better term to u s e 1,41'42. Indeed, the value of the TBA test is that the peroxidation process beginning in the reaction mixture is effectively amplified in the assay itself, so making it very sensitive. Another problem with the assay is that added metal ions, H202, antioxidants and chelating agents can influence not only peroxidation in the incubation medium, but also peroxide decomposition during the assay itselP L4~.Some groups add antioxidants (or alternatively, a large excess of iron ions) with the TBA and acid in order to attempt to standardize for these problems 2°41, but no version of the TBA test currently available is suitable for all applications. The type and concentration of acid added with the TBA also influences the rate of peroxide decomposition and hence the amount of TBA-reactive material formed 4:{.
134
ucts before measuring them. This is often done by HPLC (Table I); for example, HPLC techniques for measuring cytotoxic aldehydes are available37. However, conversion of material into volatile derivatives, separation by gas chromatography and identification by mass spectrometry is likely to give more precise chemical information when complex mixtures are being studied 31,32. We feel strongly that such analytical techniques, allowing a precise identification of what is present, should be the methods of choice for investigations of lipid peroxidation in human material. Thus derivatization and mass spectrometry have been used to characterize peroxidized fatty acids and cholesterol oxidation products in human atherosclerotic lesions 47,4~. Specificity can also be achieved by the use of antibody techniques, particularly monoclonal antibodies. Thus antibodies directed against low-density lipoprotein that has undergone peroxidation or has been treated with 4-hydroxynonenal bind to rabbit atherosclerotic lesions. In addition, lowdensity lipoproteins eluted from such lesions can bind to antibody specific for MDA-treated low-density lipoproteins (reviewed in Ref. 11). Antibody-based methods can also be applied to plasma samples. Using such specific methods, the precise role played by lipid peroxidation in cell injury and death mediated by toxins, and in human disease, should at last become clearer 49.
References 1 Gutteridge, J. M. C. (1988)in Oxygen Radicals and Tissue Injury(Halliwell, B., ed.), pp. 9-19, FASEB 2 Daniels, V. (1988) Free Radical Res. Cemmun. 5, 213-220 3 Halliwell, B. and Gutteridge, J. M. C. (1989) Free Radicals in Biology and Medicine, 2nd edn, Clarendon Press 4 Halliwell, B. (1987) FASEB J. 1,358-364 5 Slater, T. F. and Sawyer, B. C. (1971) Biochem. J. 123, 805-814 6 Comporti, M. (1987) Chem. Phys, Lipids 45, 143-169 7 Eklow-Lastbom, L., Rossi, L., Thor, H. and Orrenius, S. (1986) Free Radical Res. Commun. 2, 57-68 8 Kappus, H. (1987) Chem. Phys. Lipids 45, 105-115 90rrenius, S., McConkey, D. J., Bellomo, G. and Nicotera, P. (1989) Trends Pharmacol. Sci. 10, 281-285 10 Halliwell, B. and Gutteridge, J. M. C. (1984) Lancet i, 1396-1398 11 Steinberg, D., Parthasarathy, S., Carew, T. E., Khoo, J. C. and Witztum, J. L. (1989) N. Engl. J. Med. 320, 915-924 12 Halliwell, B. (1989) Acta Neurol. Scand. 126, 23-29 13 Esterbauer, H., Striegl, G., Puht, H. and Rotheneder, G. (1989) Free Radical Res.
TIBS 1 5 - A P R I L 1 9 9 0 Commun. 6, 67-75 14 Burton, G. W. and Ingold, K. U. (1986) Acc. Chem. Res. 19, 194-201 15 McCay, P. B. (1985) Annu. Rev. Nutr. 5, 323-340 16 Esterbauer, H., Zollner, H. and Schaur, R. J. (1988) ISI Atlas Sci. Biochem. 1,311-317 17 Halliwell, B. and Gutteridge, J. M. C. (1986) Arch. Biochem. Biophys. 246, 501-514 18 Gutteridge, J. M. C. and Smith, A. (1988) Biochem. J. 256, 861-866 19 Leblondel, G. and Allain, P. (1984) J. Inorg. Biochem. 21,241-251 20 Buege, J. A. and Aust, S. D. (1978) Methods Enzymel. 30, 302-310 21 Halliwell, B. and Gutteridge, J. M. C. (1988) ISI Atlas Sci. Biochem. 1, 48-52 22 Minotti, G. and Aust, S. D. (1987) Chem. Phys. Lipids 44, 191-208 23 Kanner, J., German, J. B. and Kinsella, J. E. (1987) CRC Crit. Rev. Food Sci. Nutr. 25, 317-364 24 Ursini, F., Maiorino, M., Hochstein, P. and Ernster, L. (1989) Free Radical Biol. Med. 6, 31-36 25 Aruoma, O. I., Halliwell, B., Laughton, M. J., Quinlan, G. J. and Gutteridge, J. M. C. (1989) Biochem. J. 258, 617-620
26 Cochrane, C. G., Schraufstatter, I. U., Hyslop, P. and Jackson, J. (1988) in Oxygen Radicals and Tissue Injury (Hatliwell, B., ed.), pp. 49-54, FASEB 27 Thomas, S. M., Jessup, W., Gebicki, J. M. and Dean, R. T. (1989) Anal. Biochem. 176, 353-359 28 Frei, B., Yamamoto, Y., Niclas, D. and Ames, B. N. (1988) Anal. Biochem. 175,120-130 29 O'Gara, C. Y., Maddipatti, K. R. and Marnett, L. J. (1989) Chem. Res. Tox. 2, 295-300 30 Marshall, P. J., Warso, M. A. and Lands, W. E. M. (1985) Anal. Biochem. 145,192-199 31 Hughes, H., Smith, C. V., Tsokos-Kuhn, J. O. and Mitchell, J. R. (1986) Anal. Biochem. 152, 107-112 32 Van Kuijk, F. J. G. M., Thomas, D. W., Stephens, R. J. and Dratz, E. A. (1986) Biochem. Biophys. Res. Commun. 139,144-149 33 Corongiu, F. P. and Milia, A. (1983) Chem.-Biol. Interact. 44, 289-297 34 Burk, R. F. and Ludden, T. M. (1989) Biochem. Pharmacol. 38, 1029-1032 35 Sies, H. (1987) Arch. Toxicol. 60, 138-143 36 Kikugawa, K. (1986) Adv. Free Radical Biol. Med. 2,389-417 37 Esterbauer, H. and Zollner, H. (1989) Free Radical Biol. Med. 7,197-203
38 Davies, M. J. (1987) Chem. Phys. Lipids 44, 149-173 39 Kosugi, H., Kate, T. and Kikugawa, K. (1987) Anal. Biochem. 165,456-464 40 Largilli~re, C. and Melancon, S. B. (1988) Anal. Biochem. 170, 123-126 41 Gutteridge, J. M. C. and Quinlan, G. J. (1983) J. Appl. Biochem. 5, 293-299 42 Gutteridge, J. M. C. (1986) Free Radical Res. Commun. 1, 173-184 43 Gutteridge, J. M. C. (1982) Int. J. Biochem. 14, 649-653 44 Nell, T., de Greet, H. and Sies, H. (1987) Arch. Biochem. Biophys. 252,284-291 45 Thompson, S. and Smith, M. T. (1985) Chem.-Biol. Interact. 55,357-366 46 Frankel, E. N. and Neff, W. E. (1983) Biochim. Biophys. Acta 754, 264-270 47 Carpenter, K. L. H., Ball, R. Y., Ardeshna, K. M., Bindman, J. P., Enright, J. H., Hartley, S. L., Nicholson, S. and Mitchinson, M. J. (1987)in Lipofuscin: State of the Art (Nagy, Z. S., ed.), pp. 245-268, Elsevier 48 Mowri, H., Chinen, K., Ohkuma, S. and Takano, T. (1986) Biochem. Int. 12,347-352 49 Steinbrecher, U. P., Lougheed, M., Kwan, W. C. and Dirks, M. (1989) J. Biol. Chem. 264, 15216-15223
LETTERS 'Rapid evolution' of the amino acid composition of proteins While preparing a chapter entitled 'Frequency of Amino Acids' for the book Proteins (Landolt-B6rnstein New Series, Vol. VII/2), we have noticed a rapid evolution in the amino acid compositions of the proteins which have been sequenced in the past decade. Table I shows the overall composition of proteins derived from databases which were available in 1978 l, 19842, and 19863, as well as the amino acid composition of the so-called open-reading-frame (ORF) proteins of the 1986 database*. Protein sequences that are derived from nucleic acid sequences have caused most of the systematic changes in composition shown in Table I. The applicability of this method does not depend on solubility or other features of the protein sequenced and thus new classes of proteins have been added to the data set. A trivial change is the increasing amount of methionine coded by the start *Baker, W. C., Hunt, L. C., George, D. G., Yeh, L. S., Chen, H. R., Blomquist, M. C., Seibel-Ross, E. I., EIzanowski, A., Hong, M. K., Ferrick, D. A., Bair, J. K., Chen, S. L. and Ledley, R. S. (1986) Protein Sequence Database, Release 11.0, December 1986 plus NEW.DAT file, National Biochemical Foundation, Georgetown University Medical Center, Washington DC, USA.
codon; N-terminal methionine belonging to a signal peptide is missing in an isolated protein. The ratio of hydrophobic and hydrophilic residues has increased, apparently because membrane-bound proteins, which cannot be sequenced as proteins by traditional methods, have been added to the database. There are also significant shifts in the relative abundances of some similar residues. For example the Arg:Lys ratio rises from 0.60 (data of 1978) to 0.86 (data of 1986), and it
is 1.17 for ORF proteins which is closer to but still far from the ratio of the number of different codons coding for Arg and Lys4.5. A significant consequence of the fact that proteins appearing in the database do not represent all proteins uniformly is that structure-prediction methods based on statistical analysis of protein, such as the widely used Chou-Fasman method for predicting secondary structures ~, or our method for predicting domain boundaries
Table I. Amino acid composition (%) of proteins a Amino acid A C D E F G H I K L M N P Q R S T V W Y
I
II
III
IV
8.3 3.1 5.5 5.7 3.8 8.9 2.4 4.1 7.0 7.5 1.6 4.0 5.5 3.8 4.2 7.3 6.1 6.5 1.2 3.4
8.11 2.28 5.13 5.97 3.83 7.57 2.38 4.98 6.25 8.76 2.25 4.29 5.10 3.94 4.94 7.12 6.02 6.46 1.40 3.29
7.75 2.14 5.16 6.06 3.97 7,35 2,36 5.07 5.97 9.08 2.27 4.23 5.28 4.02 5.11 7.10 5.90 6.53 1.39 3.74
7.28 1.77 4.07 4.87 4.63 6.18 2.34 6.14 4.85 10.93 2.99 4.49 5.76 3.42 5.67 7.70 6.09 5.79 1.58 3.45
aDerived from databases which were available in 1978 (I), in January 1984 (11),and in December 1986 (111)and from ORF proteins appearing in the 1986 database (IV) (see text).
© 1990,ElsevierSciencePublishersLtd,(UK) 0376-5067/90/$02.00
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