Oxidised low density lipoproteins induce iron release from activated myoglobin

Volume 326, number 1,2,3, 177-182 FEBS 12685 © 1993 Federation of European BiochemicalSocieties00145793193l$6.00

July 1993

Oxidised low density lipoproteins induce iron release from activated myoglobin Catherine Rice-Evans a, E m m a Green a, George Paganga a, Christopher C o o p e r b and John Wrigglesworth c aFree Radical Research Group, Division of Biochemistry, UMDS-Guy's Hospital, St. Thomas Street, London, SE1 9RT, UK, bDepartment of Paediatrics, University College London, School of Medicine, The Rayne Institute, University Street, London, WC1E 6JJ, UK a n d CMetals in Biology and Medicine Centre, Division of Life Sciences, Kings College London, Campden Hill Road, London, W8 7AH, UK

Received 13 May 1993 Recent reports have detected the presence of iron in human atherosclerotic lesions [Biochem. J. 286 (1992) 901-905]. This study provides evidence for a biochemical mechanism whereby iron is released from myoglobin by low density lipoprotein (LDL) which has become oxidised by the ferryl myoglobin species. The haem destabilisation and iron release are inhibited by monohydroxamate compounds and desferrioxamine through their ability to inhibit the propagation of LDL oxidation. Thus, iron may derive from the myoglobin released from ruptured cells in the oxidising environment of the atherosclerotic lesion. Oxidised LDL; Monohydroxamate; Desferrioxamine; Iron; Myoglobin; Low density lipoprotein

1. INTRODUCTION The mechanisms underlying the oxidation of low density lipoproteins (LDL) in the artery wall [2] are yet to be elucidated. Evidence has been provided that a range of cell types capable of generating superoxide radicals induce the oxidation of LDL in culture, particularly macrophages/monocytes [3], smooth muscle cells [4] and endothelial cells [5], but only in iron-containing media [6]. Superoxide radical and hydrogen peroxide, its dismutation product, are not reactive per se with polyunsaturated fatty acids, but their reactivity may be amplified by interaction either with available transition metal ions, such as iron or copper, generating the hydroxyl radical [7], or with specific peroxidases, such as myoglobin [8 13] and haemoglobin [14,15], producing the ferryl forms of the haem proteins. In this study we have investigated the comparative mechanisms by which metmyoglobin and its activated counterpart, ferryl myoglobin, enhance LDL oxidation and how the oxidised LDL influences the reactivity and stability of the haem protein. Analysis of the propensity for the novel monohydroxamate compound, N-methylN-hexanoyl hydroxamate (NMHH) [16] to inhibit the oxidative modification of LDL compared with the trihydroxamatate, desferrioxamine (DFO), have afforded interpretation of their comparative modes of action in terms of the inhibition of the initiating species Correspondence address:C. Rice-Evans, Free Radical Research Group, Division of Biochemistry, UMDS Guy's Hospital, St. Thomas Street, London, SE1 9RT, UK. Fax: (44) (71) 955 4983.

Published by Elsevier Science Publishers B. E

or chain-breaking scavenging of propagating lipid peroxyl radicals. The results show that, on oxidation mediated by ferryl myoglobin, the resulting oxidised LDL attacks the haem protein, destabilising the haem ring, inducing haem destruction and iron release. N M H H and DFO inhibit the release of iron by suppressing LDL oxidation through the reduction of the initiating species, as well as by acting as inhibitors of the propagation of lipid peroxidation. During metmyoglobin-induced propagation of LDL oxidation under comparative conditions of interaction, the LDL oxidation is less extensive and iron is not made available. 2. MATERIALS AND METHODS Desferrioxamine mesylate (desferal; DFO) was from CIBA-Geigy; NMHH was synthesized using standard literature methods [17]. Hydrogen peroxide was of Aristar grade, and all other chemicals were of Analar grade and supplied by Merck (Darmstadt, Germany) or Sigma (Poole, UK). Myoglobin (ferric from, horse heart, type III) was prepared as described previously [18] and purified by oxidation with potassium ferricyanide and subsequent separation on a Sephadex G15 column. Visible spectroscopy was performed on a Beckman DU-65 spectrophotometer fitted with Quant 1 software. Incubations of metmyoglobin (final concentration 20/.tM) were carried out in 10 mM phosphate-buffered saline, pH 7.4, and spectra were run at timed intervals for up to 1.5 h. The concentration of the different myoglobin species were estimated by applying the extinction coefficients for ferryl, met- and oxymyoglobin [13]. Fresh human blood was obtained from human volunteers (with informed consent). LDL was prepared from the fresh plasma in a Kontron 2070 ultracentrifuge fitted with a fixed-angle Kontron rotor, according to the method of Chung et al. [19], as described previously [15]. The concentration of protein was determined by a modified procedure of [20]. LDL (final concentration 0.25 mg/ml protein) was reacted with metmyoglobin (20 ,uM) in phosphate-buffered saline at

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Fig. 1. Altered electrophoretic mobility of LDL (0.25 mg/ml LDL protein) induced by myoglobin (20 HM + 25/~M H202) after 1.5 h incubation, and its inhibition by hydroxamates. Lanes 1,8, native LDL; lanes 2,3, LDL + metmyoglobin or ferryl myoglobin, respectively; lanes 4,6, LDL + metmmyoglobin + DFO (10¢tM) or NMHH (10 ,uM); lanes 5,7, LDL + ferryl myoglobin + DFO (10 ¢tM) or NMHH (10 ¢tM).

pH 7.4 in the absence, as well as in the presence, of hydrogen peroxide (myoglobin : H202 = 1 : 1.25 mol ratio) to initiate ferryl myoglobin radical formation. Oxidative modification of the LDL was assessed by measuring the increased electrophoretic mobility on agarose gels (Beckman paragon lipo electrophoresis system) stained with Sudan black B stain. The extent of lipid peroxidation was assayed using a modified thiobarbituric acid assay [21] with the absorbance of the chromophore measured at 532 nm, corrected for background absorbance at 580 nm due to possible contributions from haem compounds, and incorporating appropriate controls [22]. Observations of the effects of LDL on the oxidation state of myoglobin as a function of time were measured as difference spectra with prior subtraction of the LDL spectrum. The concentration of chelatable iron was determined by low temperature (30 K) EPR spectroscopy using a Bruker ESP300 spectrometer fitted with a TEl03 rectangular cavity and an Oxford instruments liquid helium flow cryostat ESR900. Spectra were baseline-corrected by subtraction of a cavity spectrum or water/buffer under identical conditions. To allow accurate comparative quantitation, the chelatable iron was always converted to ferrioxamine prior to freezing. To accomplish this, DFO was either present already in the experiment or added 1 min prior to freezing the sample in the EPR tube. To determine the absolute concentration of DFO chelatable iron in the sample, the size of the g = 4.3 EPR signal from ferrioxamine was compared to a standard curve, constructed by adding varying amounts of ferrous sulphate to an aerobic solution of DFO (note: desferrioxamine cannot chelate iron out of undamaged haem proteins, such as myoglobin). 3. R E S U L T S To investigate the p o t e n t i a l for release o f iron f r o m the h a e m p r o t e i n on i n t e r a c t i o n with L D L the effects o f ferryl m y o g l o b i n as an initiating oxidising species, a n d the p r o p a g a t i o n effects o f m e t m y o g l o b i n , were studied. 178

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A f t e r 90 min i n t e r a c t i o n b o t h m e t m y o g l o b i n and, to a greater extent, ferryl m y o g l o b i n cause an increase in the relative e l e c t r o p h o r e t i c m o b i l i t y o f the L D L , i n d i c a t i n g o x i d a t i o n a n d change in the surface charge o f the L D L p r o t e i n (Fig. 1). The level o f t h i o b a r b i t u r i c acid-reactive c o m p o u n d s , as a m e a s u r e o f b r e a k d o w n p r o d u c t s o f lipid p e r o x i d a t i o n , m e d i a t e d b y m e t m y o g l o b i n a n d ferryl m y o g l o b i n , reached 6.9 + 0.5 n m o l / m g L D L p r o t e i n ( c o r r e s p o n d i n g to A532-A58 o 0.69/mg L D L protein) a n d 40 __ 1.0 n m o l / m g L D L protein, respectively, at 1.5 h (n = 8). C h a n g e s in the o x i d a t i o n state as the h a e m p r o t e i n is a c t i v a t e d by h y d r o g e n p e r o x i d e in the presence a n d absence o f L D L are shown in Fig. 2. The difference spectra were a n a l y s e d for the presence o f ferryl m y o globin a p p l y i n g the W h i t b u r n algorithms. A c t i v a t i o n o f m e t m y o g l o b i n in the absence o f the L D L s u b s t r a t e gene r a t e d ferryl m y o g l o b i n to the extent o f 60% o f the total h a e m , with very little change after 90 m i n i n t e r a c t i o n (Fig. 2, panels a), as p r e v i o u s l y r e p o r t e d [23]. In the presence o f L D L the m a x i m a l ferryl f o r m a t i o n is att a i n e d m o r e r a p i d l y a n d the a c t u a l level is e n h a n c e d initially, 72% b y 6.5 min interaction, sustained u p to 14.5 min, b u t declines progressively as the r e a c t i o n with L D L continues, b e c o m i n g progressively r e d u c e d to m e t m y o g l o b i n with only 39% ferryl m y o g l o b i n r e m a i n ing after 90 m i n interaction. I n t e r a c t i o n between L D L a n d m e t m y o g l o b i n in the absence o f h y d r o g e n p e r o x i d e s h o w e d no d e t e c t a b l e changes in the optical s p e c t r u m o f the h a e m protein. The o x i d a t i v e m o d i f i c a t i o n o f the L D L is shown clearly b o t h in terms o f lipid p e r o x i d a t i o n a n d altered surface charge, a n d is evidence for m e t m y o g l o b i n - m e d i a t e d p r o p a g a t i o n o f oxidation. P r e t r e a t m e n t o f m y o g l o b i n with m o n o h y d r o x a m a t e and trihydroxamate hydrogen-donating compounds p r i o r to a c t i v a t i o n with h y d r o g e n p e r o x i d e (in the absence o f L D L ) s u b s t a n t i a l l y inhibited the d e v e l o p m e n t o f the ferryl m y o g l o b i n (Fig. 2). In the presence o f L D L , p r e t r e a t m e n t with N M H H ( 1 0 0 / I M ) was m o r e effective at r e d u c i n g the ferryl m y o g l o b i n b a c k to the m e t state t h a n D F O when at c o n c e n t r a t i o n s in excess o f the m y o g l o b i n b u t n o t at lower c o n c e n t r a t i o n s ( 1 0 / I M ) . A t hydroxamate concentrations of l0/IM, both DFO and N M H H inhibited the o x i d a t i o n o f L D L , as shown b y the s u p p r e s s i o n o f the altered e l e c t r o p h o r e t i c m o b i l i t y (Fig. 1), the m o n o h y d r o x a m a t e being m o r e effective t h a n the t r i h y d r o x a m a t e . A t excess c o n c e n t r a t i o n s o f b o t h drugs ( 1 0 0 / I M ) the c h a n g e in surface charge is t o t a l l y inhibited. I n h i b i t i o n o f p e r o x i d a t i o n o f the p o l y u n s a t u r a t e d fatty acyl chains o f the L D L by the drugs when present at the time o f a c t i v a t i o n to ferryl m y o g l o b i n in the presence o f the L D L is shown in Table I. A t l 0 / I M levels, the m o n o h y d r o x a m a t e was a l m o s t as effective as 100 ¢tM c o n c e n t r a t i o n s , b u t 10 ¢lM D F O only inhibited to the extent o f 60%. M e t m y o g l o b i n - i n d u c e d p r o p a g a -

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B

A a

e" , m

,,Q 0

D

I

I

I

20

40

60

I

1

20

40

L

I

bt

o>,

E L_

IJ.

C

~ J 0

~p~.~ ...................~......... ~ ~ 60

~r

80

Time (min) Fig. 2. Changes in the oxidation state as metmyoglobin is activated to ferryl myoglobin in the absence (A) and presence (B) of LDL and the effects of hydrogen-donating compounds. Panels a, no drugs; panels b, + DFO 10 p M (O), 100 ,uM (V); panels c, + NMHH 10 HM (O), 100/IM (V). Reaction concentrations: [metmyoglobin] 20/IM, [hydrogen peroxide] 25 ,uM, [LDL] 0.25 mg/ml, [hydroxamates] 10/~M, 100 HM.

tion of oxidation was again inhibited extensively by 100 /.tM monohydroxamate, but, in this case, 100/IM and 10 HM D F O were only effective to 55% and 30% inhibition, respectively. 10/IM N M H H is twice as effective as D F O as a chain-breaking antioxidant of L D L oxidation. The stability of the haem ring of the myoglobin on interaction with L D L was assessed by studying the increase in non-haem iron by EPR spectroscopy and by calculating the decrease in the amount of haem remaining by visible spectroscopy applying the Whitburn algorithms, and from the intensity of the absorption maximum in the Soret region applying the extinction coefficient. The visible spectroscopic results reveal a decline in the total haem content of 25% after interaction of ferryl myoglobin with L D L with time of exposure up to 90 min, whereas the haem ring remains intact when metmyoglobin was applied under the same conditions

and when metmyoglobin is activated by hydrogen peroxide in the absence of L D L (data not shown). Confirmation of the iron release from ferryl myoglobin consequent to its interaction with L D L is also provided from experiments applying low temperature EPR spectroscopy (Fig. 3) from the g-values at 6 and 4.3, respectively [24]. After the 90 min interaction between ferryl myoglobin and LDL, 25% of the original total haem was detected as non-haem iron; thus after this duration of interaction, the loss of haem is stoichiometric with the detection of non-haem iron. In the case of metmyoglobin as pro-oxidant, where the L D L oxidation was much less extensive, no non-haem iron was detected after 90 min of exposure to L D L (data not shown). This former finding supports the contention that, on oxidation by ferryl myoglobin, the oxidised L D L attacks the haem protein, destabilising the haem ring and releasing the iron. The pre-treatment of L D L with the monohy179

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~rg =4-3 a

___L '1300

1 . . . . . .

~.400

J_ . . . . . .

1,500

___

J__

1600

~1 . . . . 1 1700 tBO0

l _

1900

MAGNETIC FIELD (Gauss)

Fig. 3. Low-temperature EPR spectrum showing iron release from myoglobin in the presence of LDL and ferryl myoglobin. Samples were frozen at the times indicated. DFO was added to chelate the released iron for identification. (Trace a) MetMb + H202 + DFO + LDL, time = 5 s; (trace b) MetmMb + H202 + LDL; DFO was added at the end of incubation at time = 90 min; (trace c) 50 ¢tM ferrioxamine (at one-tenth the gain of (a) and (b), thus depicting 5 ~M non-haem iron). Reaction concentrations: [MetMb] 20/IM, [H202] 25/gM, [DFO] 100/~M, [LDL] 0.25 mg/ml. EPR conditions: Sweep time 3G/s; microwavepower 20 mW; microwavefrequency 9.37 GHz; modulation amplitude 10 G; time constant 330 ms; signal is average of 2 scans; temperature 30 K. Spectra (a) and (b) are displayed at 10-timesthe gain of (c). droxamate compound prior to exposure to ferryl myoglobin prevents the oxidation of LDL. Thus the haem ring is protected from destabilisation, and the release of iron is prevented. 4. D I S C U S S I O N Our previous studies suggested that ruptured myocytes under oxidative stress generate ferryl myoglobin radicals which damage the lipids of membranes [10]. In addition, haem proteins from ruptured erythrocytes have been shown to oxidise L D L in vitro via activation to the ferryl state [14], and to modify its recognition properties affording uptake by macrophages. This can be prevented by enhancing the antioxidant status of the L D L [251. The results reported here show that oxidised L D L , oxidatively modified by interaction of the L D L with ferryl myoglobin, can interact with the myoglobin destabilising the haem ring and inducing iron release. In the presence of hydroxamate compounds, the haem ring is protected and iron release does not occur due to their hydrogen-donating antioxidant properties. It has been reported by other workers that at levels of hydrogen peroxide in excesss of the haem protein, with prolonged exposure, destabilisation of the haem ring occurs and iron may be released [11]. This work demonstrates that under conditions of interaction between myoglobin and hydrogen peroxide (in the absence of L D L ) in which destabilisation of the haem ring of the ferryl myoglobin 180

does not occur, the additional presence of oxidised L D L augments the peroxide level and the haem ring is disrupted. There are two potential mechanisms by which myoglobin may promote the peroxidation of LDL. Firstly, ferryl myoglobin can initiate peroxidation of the polyunsaturated fatty acid side chains by hydrogen abstraction. Secondly, in the absence of hydrogen peroxide, metmyoglobin amplifies the propagation of the L D L peroxidation by catalysing the decomposition of trace amounts of preformed lipid hydroperoxides within the LDL, according to the mechanisms for haem-mediated decomposition of lipid hydroperoxides (Eqn. 1-3) [26]. L O O H + H X - F e 3+ ~ LO" + HX-[Fe ~v = 0] 2 "4-H + (1) L O O H + H X - F e 3+ ~ LOO" + H X - F e 2+ + H +

(2)

L O O H + H X - F e 2+ --) LO" + H X - F e 3+ + O H -

(3)

The alkoxyl and peroxyl radicals and the ferryl species formed as the haem iron species recycle, are capable of re-initiating further L D L peroxidation, although only a small proportion of the metmyoglobin cycling through the ferryl state is spectroscopically detectable by this mechanism. The ability of D F O and N M H H to function as hydrogen donors [9,10,23,27-30], independently of their iron-chelating properties, and to protect the haem protein from destabilisation by reducing the initiating spe-

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cies, m a y be an i m p o r t a n t c o n t r i b u t o r to the m e c h a n i s m o f the h y d r o x a m a t e - m e d i a t e d i n h i b i t i o n o f ferryl m y o globin-induced oxidation of LDL. The hydroxamates m a y also inhibit the p r o p a g a t i o n process b y acting as c h a i n - b r e a k i n g a n t i o x i d a n t s a n d i n t e r c e p t i n g the p r o p a g a t i o n phase o f L D L o x i d a t i o n by H d o n a t i o n to a l k o x y l or p e r o x y l species. T h e results suggest that, when in excess, the m o n o h y d r o x a m a t e is a m o r e efficient i n h i b i t o r o f ferryl m y o g l o b i n f o r m a t i o n a n d hence its effectiveness in inhibiting lipid p e r o x i d a t i o n , b e c a u s e o f its scavenging o f the initiator. D F O has a lower rate c o n s t a n t t h a n N M H H for i n t e r a c t i o n with the ferryl m y o g l o b i n species [23] a n d thus is n o t so effective in directly r e d u c i n g the latter; however, at h y d r o x a m a t e c o n c e n t r a t i o n s well b e l o w t h a t o f m y o g l o b i n , D F O suppresses the ferryl m y o g l o b i n species m o r e effectively t h a n it reduces lipid p e r o x i d a t i o n , suggesting t h a t D F O is n o t acting as a c h a i n - b r e a k i n g a n t i o x i d a n t at these low c o n c e n t r a t i o n s . In fact, N M H H is m o r e effective in inhibiting m e t m y o g l o b i n - m e d i a t e d L D L o x i d a t i o n t h a n D F O a n d is thus a m o r e efficacious c h a i n - b r e a k i n g ant i o x i d a n t . I m p o r t a n t l y , since the h y d r o x a m a t e s possess the d u a l activities o f a n t i o x i d a n t a n d i r o n - c h e l a t i n g p r o p e r t i e s , they also have the p o t e n t i a l to inhibit the f o r m a t i o n o f h y d r o x y l radicals, which m a y be g e n e r a t e d on release o f i r o n f r o m the h a e m p r o t e i n by oxidised L D L . It is possible, in vivo, t h a t h a e m proteins, l e a k i n g f r o m r u p t u r e d cells, m a y be c a p a b l e o f e n h a n c i n g the o x i d a t i o n o f L D L which has p e n e t r a t e d the e n d o t h e lium o f the c o r o n a r y vessels. This m a y o c c u r b y h a e m protein-mediated decomposition of LDL hydroperoxides which have a l r e a d y been oxidised by, for e x a m p l e , c o n t a c t with n e i g h b o u r i n g cells or the e n z y m a t i c activity o f lipoxygenases. As the level o f L D L o x i d a t i o n increases on p r o l o n g e d i n t e r a c t i o n with the h a e m p r o -

Table I The percentage inhibition of myoglobin-mediated LDL oxidation by desferrioxamine (DFO) and N-methyl-N-hexanoyl hydroxamate (NMHH) % decrease in A532-Asso/mg LDL protein

n

Ferryl myoglobin 100,uM DFO 100/2M NMHH 10/2M DFO 10pM NMHH

94 + 95 + 60+ 90 +

3 2 5 6

8 8 4 4

Metmyoglobin 100pM DFO 100,uM NMHH 10pM DFO 10/2M NMHH

55 + 12 89 + 5 30-+ 5 58 -+ 6

6 8 4 4

Hydroxamates were added to LDL (0.25 mg/ml) prior to addition of metmyoglobin (20 pM) _+hydrogen peroxide (25 pM) and incubated for 1.5 h at 37°C. Concentrations stated are final concentrations.

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tein, i r o n release m a y occur. P r e v i o u s studies [31] have r e p o r t e d the d e s t r u c t i o n o f h a e m a n d the release o f i r o n i n t e r a c t i o n o f L D L with h a e m i n , an oxidative den a t u r a t i o n p r o d u c t o f h a e m o g l o b i n , b u t only in the presence o f excessive h y d r o g e n p e r o x i d e levels (10 or 20-fold m o l a r excesses). A t these c o n c e n t r a t i o n s the presence o f oxidised L D L is n o t necessary to drive the i r o n release. R e c e n t analyses o f the c o n t e n t s o f h u m a n a t h e r o s c l e r o t i c lesions [1] have suggested t h a t this is a p o t e n t i a l l y p r o - o x i d a n t e n v i r o n m e n t with the possible a v a i l a b i l i t y o f i r o n a n d copper. O u r findings suggest a p o t e n t i a l m e c h a n i s m w h e r e b y i r o n m a y be derived f r o m m y o g l o b i n released f r o m r u p t u r e d cells in a d v a n c e d lesions.

Acknowledgements: We thank the British Technology Group and British Heart Foundation for funding this study, and King's College, London, for providing the EPR facilities. REFERENCES [1] Smith, C., Mitchinson, M.J., Aruoma, O.I. and Halliwell, B. (1992) Biochem. J. 286, 901-905. [2] Steinberg, D., Parthasarathy, S., Carew, T.E., Khoo, J.C. and Witztum, J.L. (1989) N. Engl. J. Med. 320, 915 924. [3] Cathcart, M.K., Morel, D.W. and Chisolm, G.M. (1985) J. Leukoc. Biol. 38, 341-350. [4] Morel, D.W., DiCorleto, P.E. and Chisolm, G.M. (1984) Arteriosclerosis 4, 357 364. [5] Henriksen, T., Mahoney, E.M. and Steinberg, D. (1981) Proc. Natl. Acad. Sci. USA 78, 6499-6503. [6] Leake, D. and Rankin, S. (1990) Biochem. J. 270, 741 748. [7] Halliwell, B. and Gutteridge, J.M.C. (1990) Methods Enzymol. 186, 1 85. [8] George, P. and Irvine, D.H. (1952) Biochem. J. 52, 51l 517. [9] Harel and Kanner, J. (1987) Free Rad. Res. Commun. 3, 309317. [10] Turner, J.J.O., Rice-Evans, C., Davies, M.J. and Newman, E.S.R. (1991) Biochem. J. 277, 833-837. [1 l] Puppo, A. and Halliwell, B. (1988) Free Rad. Res. Commun. 4, 415422. [12] Galaris, D., Sevanian, A., Cadenas, E. and Hochstein, P. (1990) Arch. Biochem. Biophys. 281, 163 169. [13] Whitburn, K.D., Shieh, J.J., Sellers, R.M., Hoffman, M.Z. and Taub, I.A. (1982) J. Biol. Chem. 257, 1860 1869. [14] Giulivi, C. and Davies, K.J.A. (1991) J. Biol. Chem. 265, 19453 19460. [15] Paganga, G., Rice-Evans, C., Rule, R. and Leake, D. (1992) FEBS Lett. 303, 154-158. [16] Rice-Evans, C. and Davies, M.J. (1990) United Kingdom Patent Application No 9024820.4. [17] Ulrich, H. and Sayigh, A.R. (1963) J. Chem. Soc. 1098-1101. [18] Dee, G., Rice-Evans, C., Obeyeskera, S., Meraji, S., Jacobs, M. and Bruckdorfer, K.R. (1991) FEBS Lett. 294, 3842. [19] Chung, B.H., Wilkinson, T., Geer, J.C. and Segrest, J.P. (1980) J. Lipid Res. 221,284-317. [20] Markwell, M.A., Haas, S.M., Biever, L.L. and Tubert, N.E. (1978) Anal. Biochem. 87, 106-110. [21] Walls, R., Kumar, K.S. and Hochstein, P. (1976) Arch. Biochem. Biophys. 172, 463468. [22] Gutteridge, J.M.C. (1986) Free Rad. Res. Commun. 1, 173-184. [23] Green, E.S.R., Evans, H., Rice-Evans, P., Davies, M.J., Salah, N. and Rice-Evans, C. (1993) Biochem. Pharmacol. 45,357 366. [24] Palmer, G. (1985) Biochem. Soc. Trans. 13, 548 560. [25] Labeque, R. and Marnett, L. (1988) Biochemistry 27, 7960-7970. 181

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[26] Andrews, B., Paganga, G. and Rice-Evans, C. (1993) Biochem. Soc. Trans. 21 (in press). [27] Morehouse, K.M., Flitter, W.D. and Mason, R.P. (1987) FEBS Lett. 222, 24(~550. [28] Darley-Usmar, V.M., Hersey, A., Garland, L.G., Leonard, E and Wilson, M.T. (1989) in: Free Radicals, Disease States and Antiradical Interventions (Rice-Evans, C. ed.) pp. 183 200, Richelieu Press, London.

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[29] Rice-Evans, C., Okunade, G. and Khan, R. (1989) Free Rad. Res. Commun. 7, 45-54. [30] Hartley, A., Davies, M. and Rice-Evans, C. (1990) FEBS Lett. 264, 145-148. [31] Balla, G., Jacob, H.S., Eaton, J.W., Belcher, J.D. and Vercellotti, G.M. (1991) Arteriosclerosis Thrombosis 11, 1700-1711.