Enzymatic production of hydroperoxides of unsaturated fatty acids by injury of mammalian cells

Chemistry and Physics of

ELSEVIER

Chemistry and Physics of Lipids 79 (1996) 113-121

LIPIDS

Enzymatic production of hydroperoxides of unsaturated fatty acids by injury of mammalian cells Michael

Herold*,

Gerhard

Spiteller*

Institut Jher Organische Chemie I, Universitaet Bayreuth, Universitaetsstrasse 30, 95440 Bayreuth, Germany

Received 31 July 1995; revision received 28 November 1995; accepted 29 November 1995

Abstract

Hydroperoxides of unsaturated fatty acids (LOOHs) are generated by homogenisation of liver tissue, but not if the liver is boiled before homogenisation. This observation indicates that the LOOHs are produced in an enzymatic reaction. This assumption is corroborated by an analysis of the reduction products of LOOHs by gas chromatography/mass spectrome.try (GC/MS). A main part of LOOHs is derived from linoleic acid and not from arachidonic acid. Massive cell damage occurs by myocardial infarction or other severe injuries; these events were found to be connected with generation of LOOHs. We suspect - - considering the above outlined experiment - - that the LOOH production is also mainly caused in these cases by activation of enzymes and not - - as postulated - - by an autocatalytic process. Increased amounts of LOOHs are found in many chronic diseases, e.g. in rheuma, atherosclerosis or psoriasis, obviously caused by a gradual damage of cells. Thus, the common root of an increased LOOH level might be cell injury. Keywords: Cell injury; Lipoxygenases; Hydroperoxy fatty acids; Linoleic acid; Arachidonic acid

1. Introduction

Injury of plants causes, as first reported by Galliard [1], fast degradation of phospholipids and galactosides, paralleled by a decrease in the amount of linoleic acid [2]. Galliard demonstrated that plant cell injury is connected with an activation of hydrolases and lipoxygenases [1] which convert linoleic acid (the main unsaturated fatty acid in m a n y plant membranes) and other polyunsaturated fatty acids (PUFAS) to the correspond-

ing L O O H s [1]. The decrease in linoleic acid can easily be recognised if the acid fractions of boiled and unboiled plant samples are compared after homogenisation and appropriate derivatisation by G C / M S [2-4], since boiling deactivates the enzymes. Bergers et al. [5] demonstrated by a comparison of boiled and unboiled homogenised skin samples that only in the latter were membrane phospholipids attacked, which was considered as p r o o f that phospholipase A2 was involved in the libera-

0009-3084/96/$15.00 ~-3 1996 Elsevier Science Ireland Ltd. All rights reserved SSD1 0009-3084(95)02518-N

M. Heroid, G. Spiteller /Chemistry and Physics qfLipids 79 (1996) 113 121

114 100.

100-

80.

801

60.

60~

40.

~"

20. 0.

401 20-

5

6

7

8

9

10 11 12 13 14 15

~

Position of OH

0

5

6

7

8

9

10

11 12

13 14

Position of OH

(a) (b) Fig. 1. Typical pattern of isomers of monohydroxy methyleicosanoates after reduction and derivatisation of the autocatalytically obtained oxidation products of arachidonic acid (a) and after an enzymatic lipid peroxidation (b).

tion of free acids in the homogenisation process. In addition, injury of mammalian cells is reported to cause not only the activation of cyclooxygenase - - responsible for production of prostaglandins [6,7], thromboxans [8] and prostacyclins [9,10] - but also of lipoxygenases which transform PUFAS to leukotriens [11]. Thus, a similar response of mammalian cells to injury - - by activation of esterases and lipoxygenases - - as reported for plant cells seems possible [12]. We report here on an investigation which strengthens this assumption. In addition, the observation that tissue injury induces the generation of LOOHs is of practical importance since enhanced levels of hydroperoxides of unsaturated acids were detected in a great number of diseased states listed in a recent review [13], e.g. in rheuma [14-16], atherosclerosis [1722], psoriasis [23], burn injury [24-29] or a myocardial infarction [30-33]. Although some of these diseased states, e.g. a myocardial infarction or severe burn injury, are obviously connected with massive tissue damage, it was postulated that the increased amounts of LOOHs observed in these diseases [24,30] are the result of an autocatalytical process, induced by radicals, e.g O 2 - , OH'. Especially the latter was suspected to react with double allylically activated CH2 groups to produce finally in a chain reaction LOOHs [34,35]. Therefore, a distinction between enzymatically and autocatalytically induced processes is required, which can be done by a method based on experiments of Yamagata [36]: Yamagata showed

that oxidation of arachidonic acid in air under autocatalytic conditions produces a distinct pattern of hydroxy fatty acids (LOHs) obtained after hydrogenation of the LOOHs: Due to the fact that arachidonic acid contains three equally double allylically activated CH2 groups in position 7, 10 and 13, six isomeric hydroperoxy acids with a functional group in position 5, 8, 9, 11, 12 or 15 are expected to be produced by an autocatalytic reaction in about equal amounts. The investigation of Yamagata revealed that the isomers with inner hydroperoxyl groups are degraded faster than outer ones [36]. Thus a typical pattern of derived hydroxy acids with equal high amounts of the 5- and 15-hydroxy eicosanoic acids and much lower ones of their 8, 9, 11 and 12 isomers was obtained (Fig. la). We were able to confirm these deductions by oxidation of arachidonic acid by "OH radicals (A. Mlakar and G. Spiteller, to be published). Similarly 9- and 13-hydroxy stearic acid are found in equal amounts after reduction of the hydroperoxides of linoleic acid - - obtained after its autocatalytic oxidation (A. Mlakar and G. Spiteller, to be published). In contrast enzymes react by regio and stereo specific hydrogen-abstraction resulting in the production of only distinct isomers (Fig. lb). In this paper we show that the pattern of hydroxy acids derived from arachidonic acid hydroperoxides obtained after homogenisation of liver does not indicate initiation by an autocatalytic process but rather shows the pattern of an enzymatically induced reaction.

M. Heroid, G. Spiteller / Chemistry and Physics of Lipids 79 (1996) 113-121

2. Materials and methods

A porcine liver was removed immediately after slaughtering and divided in two parts.

2.1. Tissue incubation One part (100 g) was homogenised in 200 ml water and stirred at room temperature. Aliquots containing 25 g liver were withdrawn in time intervals (0, 6 and 24 h). In a control experiment another 100 g part of the same liver was boiled for 30 min and homogenised. Then 25 mg arachidonic and 50 mg linoleic acid were added. The homogenate was stirred at room temperature and samples were withdrawn after 0 and 6 h. All samples of boiled and unboiled homogenates were processed in exactly the same way: Aliquots of wJithdrawn samples were extracted instantly in a one-phase-system containing 75 g of the homogenate, 87.5 ml chloroform and 185 ml methanol. Subsequent addition of each 87.5 ml water and chloroform caused separation into two layers according to Bligh and Dyer [37]. After centrifugation at 1000 × g for 10 min the chloroform layer was dried under reduced pressure. 2.2. Reduction The lipids obtained from the chloroform layer were dissolved in a solution of 20 ml chloroform, 40 ml methanol and 16 ml water. Then, 100 mg SnCI2"2H20 were added and the pH was adjusted to 2-3 by addition of 20% hydrochloric acid. After stirring for 1 h at room temperature, separation of the phases was achieved as described above by addition of 20 ml each of water and chloroform. After evaporation of the solvent, the lipids were stored at - 2 0 ° C under argon. 2.3. Liquid chromatography The eluent for Sep-Pak cartridges and for high performance liquid chromatography (HPLC) was a mixture, of 85% methanol in water containing 0.1% acetic acid (v/v). Then, 10 mg of each raw lipid fraction were dissolved in 1 ml eluent. This mixture was transferred to a Sep-Pak Plus C-18 cartridge to separate free fatty acids from choles-

115

terol and ester lipids. The cartridge was extracted with 15 ml eluent. The eluate containing the free fatty acids was brought to dryness. The residue was dissolved in 100/zl eluent and injected onto a 250 x 8 mm Spherisorb ODS II HPLC-column [38]. HPLC was performed at a flow rate of 4 ml/min. Ultraviolet (UV) absorption was monitored at 235 nm (A235) (maximum of the conjugated diene system) [39] and 212 nm. The fraction eluting between 4-8 min, showing high A235, was collected [38]. The oxygenated fatty acids collected from HPLC were hydrogenated in ethyl acetate using a PtO2-catalyst (H2 pressure 0.3 Mpa, 1 h) to achieve saturation of the double bonds. The product mixture was filtered through a 0.2 ~tm PP disposable syringe filter. After evaporation of the solvent, 2% ethereal diazomethane (w/v) was added. The solution was kept at room temperature for 15 min. Then the dried methylesters were dissolved in 25 ~tl N-methyl-N-(trimethylsilyl)trifluoroacetamid and kept at room temperature for 12 h under argon to obtain the saturated trimethylsilyloxy-methylesters. Aliquots were injected onto the GC column (DB-1, carrier gas H2, 3°/rain from 80 to 300°C). Saturated monohydroxy fatty acids were identified by their typical mass spectra (EI, 70 eV) due to c~-cleavage products. Quantification was achieved by mass spectrometric single ion monitoring of the peak resulting by ~-cleavage at the > CH-OTMS group which contained both the OTMS and the methylcarboxylate residue. Another sample of each raw lipid was processed and examined by GC/MS exactly in the same way but without catalytic hydrogenation. This allowed to distinguish if the liver samples already contained saturated hydroxy fatty acids.

3. Results

A porcine liver was removed immediately after slaughtering and divided into two parts. The one part was boiled in order to destroy the enzymes, a method which had already been proofed in our laboratory useful in efficiently destroying plant

116

M. Heroid, G. Spiteller / Chemistry and Physics q/Lipids 79 (1996) 113 121

enzymes [3,4]. Then the sample was homogenised. The second part of the liver was homogenised without boiling. In comparison to the homogenate of raw liver, the boiled liver sample showed no lipase activity and contained therefore nearly no fatty acids. If formation of LOOHs would be an autoxidative process requiring free fatty acids and oxygen, the production of LOOHs would have been excluded in this sample due to the lack of free fatty acids. Therefore, arachidonic acid and linoleic acid were added to the homogenate of boiled liver. Aliquots were withdrawn immediately after homogenisation from boiled and unboiled liver samples, and then by continuous stirring at room temperature in the air after 6 and 24 h. From these aliquots the free acids were extracted. The direct measurement of the characteristic absorption of the diene system at 235 nm does not only indicate LOOHs but also derived LOHs. Since most of produced LOOHs are reduced enzymatically very fast to LOHs in a biological system [40], the diene measurement comprises mainly LOHs. Considering these facts, the content of conjugated dienes was used in this investigation as a measure for lipid peroxidation instead of the more common determination of one of the decomposition products of LOOHs, malondialdehyde (MDA), which is reacted with thiobarbituric acid to give a coloured product [41]. Besides the well-known unspecifity of this method [42], we found that the amount of produced MDA is dependent on the number of double bonds: LOOHs derived from linoleic acid generate only about one third of MDA compared with LOOHs derived from arachidonic acid (A. Dudda and G. Spiteller, to be published). At room temperature (that is under physiological conditions) MDA is produced only in much lower amounts. The amount of conjugated dienes was low in the boiled sample immediately after homogenisation. It had not increased after stirring for 6 h in air (Fig. 2), in spite of addition of free acids. This demonstrates that an autocatalytic oxidation is negligible.

A 2 3,,.,5 , [mAUl

100 -

5000

I

I

I

I

5

10

15

20

Ip rain

Fig. 2. Diene absorption of the boiled liver sample extract 6 h after homogenisation, obtained by HPLC separation. Within 6 h in the unboiled sample, the amount of dienes rose to values l0 times higher than in the boiled sample (Fig. 3). Unfortunately HPLC chromatography on a reversed phase column does not allow entire separation of different LOHs (Figs. 2 and 3) and consequently the detection of the isomer pattern was not possible for further confirmation of the enzymatic character of diene formation. Yamagata [36], following a procedure outlined by Hamberg [43], had determined the pattern of different isomeric hydroxy eicosanoic acids by reduction of the LOOHs with NaBH4, hydrogenation of double bonds, methylation of the acid function and trimethylsilylation. This procedure allowed at least partly gas chromatographic sepaA235

[mAUl

300 -

250

-

200 -

150 -

100 -

I 0

I 5

I 10

I 15

I 20

,P m,n

Fig. 3. Diene absorption of the unboiled liver sample extract 6 h after homogenisation, obtained by HPLC separation.

ll7

M. Heroid, G. Spitel/er / Chemistry and Physics of Lipids" 79 (1996) 113 121 % lOO-

%

I~ c18] @~ c2o

801 6oi 4oi 2oi ol

100 80 60 40 20

0

(a)

6

[hi

O,

0

0

24

[hi

(b) Fig. 4. Comparison .)f C-18 and C-20 monohydroxy acids obtained after reduction and derivatisation of LOOHs in boiled liver homogenates immediately and 6 h after homogenisation (a), and in unboiled liver homogenates immediately, 6 and 24 h after homogenisation (b). ration of the isomers [36]. Quantification of the different hydroxy acids was achieved by measuring the ion currents of selected characteristic ions of each compound. If this procedure is applied we do not consider the eventual occurrence of saturated hydroxy fatty acids in the original compound mixture (these are not detected by measurement of diene absorption). The:refore, we checked if such saturated hydroxy aciids had been present in the original acid fraction, and indeed we detected that the biological samples contain traces of 9-, 10-, 12and 13-hydroxy stearic acid. Saturated C-20 monohydroxy acids could not be detected. Thus the method is at least applicable for the quantification of LOOHs derived from arachidonic acid. Further difficulties were encountered in the reduction step. Besides NaBH 4, SnCl2 or triphenylphosphine are also recommended for reduction of LOOHs [44]. We tried all three methods. Reduction with triphenylphosphine is problematic since separation of the obtained hydroxy acids from accompanying triphenylphosphine proved difficult. If unsaturated fatty acids are treated with NaBH4 we observed the generation of hydroxy zLcids in tiny amounts. A further investigation revealed that NaBH4 is added to double bonds of unsaturated acids in the form of a complex. Thes.e products are transformed to hydroxy acids by the following reduction step. Although this reaction requires a free carboxylic group, and can be circumvented by preparation of esters (S. Thiemt and G. Spiteller, to be published), we decided to use SnC12 for reduction.

Monoepoxides of unsaturated fatty acids are found in injured tissue [45]. We showed in a model experiment that they are partly converted by catalytic hydrogenation in methanolic solution as proposed in the literature [40] to monohydroxy acids. Since we detected in another model experiment that hydrogenation of epoxides, using ethyl acetate as solvent, does not produce monohydroxy acids we used this solvent for hydrogenation. Thus we became able to recognise the contribution of the different hydroxy acids to the diene absorption. Already immediately after homogenisation (about 5 min had passed from the begin of homogenisation until sample removal) the amount of hydroperoxy fatty acids (measured as saturated derivatives) is increased in the unboiled sample (Fig. 4b) compared to the boiled one (Fig. 4a) by a factor of three for C-18 acids and by a factor of two for C-20 acids. In the unboiled sample the amount of C-18 acids increases with time, that of C-20 acids remained nearly constant; 6 h after homogenisation the C-18 hydroxy acids reached a level three times higher than that of C-20 acids. This may reflect the increased tendency of conjugated diens derived from arachidonic acid to undergo a second oxygenation reaction, followed by further degradation reactions compared to C-18 acids (A. Mlakar and G. Spiteller, to be published). After 6 h the amount of hydroxy acids decreased gradually (Fig. 4b). This continuous de-

M. Heroid, G. Spiteller / Chemistry and Physics of Lipids 79 (1996) 113 121

118 100-

100-

80-

80-

60-

60-

40-

=~ 40-

20 -

20-

4

(a)

S

6' 7

8

9

10 11 12 13 14 15 16

Position of - O H

--- --~ 4

(b)

5

6

7

8

9

~--

I~

10 11 12 13 14 15 16

Position of - O H

Fig. 5. Pattern of C-20 hydroxy eicosanoic acids derived from the boiled (a) and unboiled (b) liver sample 6 h after homogenisation.

crease is obviously due to the usual metabolic degradation of fatty acids. As pointed out above the hydroxy acid pattern after an autocatalytic lipid peroxidation of arachidonic acid, reduction, hydrogenation and derivatisation should indicate nearly equal amounts of the C-5 and C-15 hydroxy eicosanoic acid derivatives (Fig. la). The pattern of the C-20 hydroxy acids in the boiled sample withdrawn 6 h after homogenisation (Fig. 5a) shows the prevalence of 5-hydroxy eicosanoic acid. 5-Hydroxy eicosanoic acid is derived from 5-hydroperoxy eicosa-6t r a n s - 8 - c i s - l l - c i s - 1 4 - c i s - t e t r a e n o i c acid, a wellknown product of the lipoxygenase-induced oxidation of arachidonic acid [46]. Therefore, one must conclude that the precursor of the main C-20 hydroxy acid was produced enzymatically by action of lipoxygenase on arachidonic acid. Obviously the 5-hydroperoxide of arachidonic acid is either produced in a steady process in the intact liver so that a certain level of 5-LOOH is always present or LOOHs were generated from arachidonic acid after injury of cells in heating denaturation if the enzymes were not inactivated immediately. The fact that the amount of C-20 monohydroxy fatty acids had not increased in the boiled sample during 6 h of stirring in air in spite of addition of arachidonic acid (Fig. 4a) and the prevalence of the 5-hydroxy eicosanoic acid (Fig. 5a) prove that a non-enzymatic oxidation had not occurred to a considerable extent. The sample withdrawn from the unboiled liver 6 h after homogenisation, which showed a dramatic increase in LOOHs (Fig. 4), also contained

after processing as main hydroxy derivative, the 5-hydroxy eicosanoic acid (Fig. 5b), again pointing to the enzymatic origin of the products. This sample showed the presence of additional hydroxy acids (Fig. 5b). We attribute their presence to an unspecific process: The additional presence of 6-, 8-, 9-, 11-, 12-, 14- and 15-hydroxy isomers is in perfect agreement with earlier investigations which demonstrated that injury of liver activates a P450 epoxidase, which is able to attack all four double bonds of arachidonic acid. If the epoxides are enzymatically metabolised, corresponding hydroxy acids are obtained. Thus the 5- and 6-, the 8- and 9-, the 11- and 12-, and the 14- and 15-hydroxy acid derivatives of eicosanoic acid are produced in about equal amounts (additional amounts of the 5-isomer are produced by lipoxygenase) as indicated in the diagram (Fig. 5b). The distribution picture of the C-18 hydroxy acids is practically identical in samples taken from boiled and unboiled liver samples as well as immediately after homogenisation and 6 h later (Fig. 6). The main C-18 hydroxy acid observed is 9-hydroxy stearic acid, the hydrogenation product of 9-hydroxyperoxy octadeca- l O-trans-12-cis-dienoic acid. The latter acid is produced by action of the same cyclooxygenase which produces also 11-hydroperoxy eicosa-6-trans-8-cis - l 1-cis- 14-cis -tetraenoic acid, an intermediate in prostaglandin synthesis [47,48]. Beside this acid as second main hydroxy acid, 13-hydroxy octadeca-9-cis-11-transdienoic acid, is found. This acid is derived from the 13-hydroperoxide of linoleic acid. Unexpect-

M. Heroid, G. Spiteller / Chemistry and Physics of Lipids 79 (1996) 113-/21 100 80

100-

i

80-

I

60 !

200~

119

4

(a)

5

6

7

~

8

9

60-

20. 0~ ~ ' ~ - - ' - ~ - - ~ - - - ~ 4 5 6 7 8 9 10 11 12 13 14 15 16

10 11 12 13 14 15 16

Position of - O H

(b)

Position of - O H

Fig. 6. Pattern of hydroxy stearic acids obtained after reduction and derivatisation of LOOHs from boiled (a) and unboiled (b) liver samples 6 h after homogenisation.

edly 12- and 10-hydroxy stearic acid were also produced to a considerable extend. At present we are unable to explain the generation of these high amounts of 10 and 12 isomers, probably they were not produced in an autocatalytic process - in this case, the 10 and the 12 isomers are found only in traces (A. Mlakar and G. Spiteller, to be published).

4. Discussion

The above described experiments indicate that injury of mammalian cells (nearly similar results are obtained by homogenisation of heart tissue except that the oxidation is slower) activates lipoxygenases and epoxidases. This behaviour parallels the answer of plants after mechanical injury or cell damage by fungi [49-51]. As already mentioned increased amounts of LOOHs were found in many diseased states. A review of the literature reveals that especially high amounts of LOOH were detected in diseases caused by spontaneous events, e.g. in fulminant hepatitis [52], heart infarction [30-33] and severe burn injury [24-29]. In all these diseases massive cell injury is observed. As a consequence hydrolases and lipoxygenases are also expected to be activated, which should produce LOOHs. Most of them should be converted to corresponding LOHs and in fact these products were detected after a myocardial infarction (A. Dudda and G. Spiteller, to be published).

Unfortunately cell damage also causes the liberation of iron ions. These are able to react with LOOHs in a non-enzymatic reaction by production of LO" radicals. Some of these are decomposed to aldehydes [53], but others are able to abstract a hydrogen atom - - in a non-enzymatic reaction - - from the activated CH2 groups adjacent to the conjugated double bond system of LOOHs or LOHs [54]. Thus, the produced mesomeric radicals can add a second oxygen molecule, resulting finally in production of e-hydroxyhydroperoxy acids. These are decomposed to ~-hydroxyaldehydes (Scheme 1) which were shown to induce an oxidative burst in stimulated macrophages (H. Heinle, N. Gugeler, R. Felde and G. Spiteller, to be published). Thus, production of damaging products must be regarded as a three step process: after cell injury phospholipases produce free fatty acids. These are oxidised by lipoxygenases to LOOHs, which are finally decomposed in a third non-enzymatic step with the aid of similarly liberated iron ions to ~-hydroxyaldehydes and other products. Bearing this in mind we are able to explain the high increase of lipid peroxidation products immediately after reperfusion after a myocardial infarction [55]: during the ischemic period injured cells activate hydrolases. These produce free fatty acids. Injury also activates lipoxygenases, but they cannot act, since in the damaged tissue no fresh blood is available and consequently 02 is lacking. If reperfusion is started, oxygen becomes available and the lipoxygenases can react with unsaturated fatty acids. Thus, unusual large amounts of LOOHs are produced which induce damage [56].

120

M. Heroid, G. Spiteller / Chemistry and Physics of Lipids 79 (1996) 113 121

--

--

(CH2)7--COOH

OOH ~[[enzymoticaUyl ~CHz--(CH2)6~COOH I °nzy~ot,o~,y [

CH--(CH2)e--COOH "H-O=I H/ OH-LOH In°n-anzym°t|c°liY~ ~ C H - - ( C H 2I )

oH

J

~ ( C H = ) I - - C O O H + O=, + LOH [ - ( L C H - H e)~,

OH

6-COOH

OOH

~ ( C H z ) s

~COOH

o.

=

: . ~ ~ ~ ' - ~

(CH2)a~COOH

+ Oz, + LOH 1 - (L(3H- HI) OH ~

~

-

~

(CH2)a~ COOH

[ OH°OH

CHO

/"~~CHO Scheme 1. Formation of hydroxyaldehydes.

We speculate that similar processes may occur after a shock, in fulminant hepatitis and, of course, after severe injuries, e.g. after burning. Caution must also be paid to these processes in long lasting surgery, e.g. organ transplantations. A moderate increase of LOOHs is also reported in psoriasis [23], rheuma [14-16], atherosclerosis [17-22] and other chronic diseases [13,57]. In the case of psoriasis, mainly enzymatic lipid peroxidation was proofed to occur by the detection of chiral LOHs [23]. Injury was also recognised to be able to induce atherosclerosis [58]. This assumption was later rejected due to the observation that fatty streaks, first signs of an atherogenesis, were observed beneath an intact epidermis; however, at that time obviously one had not considered that enzymes might be liberated within a cell just by pressure, e.g. increased blood pressure, which deforms a

cell. Therefore, it might well be that atherosclerosis is also caused by previous cell injury or deformation.

Acknowledgements We thank 'Deutschen Forschungsgemeinschaft' and 'Fond der Chemischen Industrie' for financial support. We are obliged Mr. D. Laatsch for maintenance of the gas chromatograph and Mr. M. Glaessner for running the mass spectra. We are also grateful to Mr. W. Kern for purification of solvents.

References

[1] T. Galliard (1970) Phytochemistry9(8), 1725-1734.

M. Heroid, G. Spiteller / Chemistry and Physics of Lipids 79 (1996) 113-121 [2] A. Graveland (1973) Lipids 8(11), 599 605. [3] H. Spreitzer, J. Schmidt and G. Spiteller (1989) Fett Wiss. Technol. 91(3), 108-113. [4] H. Spreitzer, J. Schmidt, U. Kratzel, G. Spiteller (1989) Monatsh. Chem. 120(2), 169-176. [5] M. Bergers, A.R. Verhagen, M. Jongerius and P.D. Mier (1986) Biochim Biophys. Acta 876, 327-332. [6] S. Bergstr6m and J. Sjvall (1957) Acta Chem. Scand. 11, 1086. [7] S. Bergstr6m, R. Ryhage, B. Samuelsson and J. Sjvall (1962) Acta Chem. Scand. 16, 501-502. [8] M. Hamberg, J. Svensson and B. Samuelsson (1975) Proc. Natl. Acad. Sci. USA 72(8), 2994-2998. [9] S. Moncada, R. Gryglewski, S. Bunting and J.R. Vane (1976) Nature (London) 263(5579) 663 665. [10] S. Moncada, R. Korbut, S. Bunting and J.R. Vane (1978) Nature (London) 273(5665) 767-768. [11] L.S. Wolfe (1982) J. Neurochem. 38(I) 1-14. [12] G. Spiteller (19'93) J. Lipid Mediators 7(3), 199 221. [13] B. Halliwell and M. Grootveld (1987) FEBS Lett. 213(1), 9-14. [14] C.R. Wade, P.G. Jackson, J. Highton and A.M. Van Rij (1987) Clin. Chim. Acta 164(3), 245-250. [15] D. Rowley, J.M.C. Gutteridge, D. Blake and M. Farr (1984) Clin. Sci. 66(6), 691-695. [16] D.R. Blake, N.D. Hall, P.A. Bacon, P.A. Dieppe, B. Halliwell and J.M.C. Gutteridge (1981) Lancet 2(8256), 1142 1144. [17] J. Glavind, S. Hartman, J. Clemmesen, K.E. Jessen and H. Dam (1952) Acta Pathol. Microbiol. Scand. 30, 1-6. [18] R. Dargel (1992) Exp. Toxicol. Pathol. 44(4), 169-181. [19] H. Esterbauer, J. Gebicki, H. Puhl and G. Juergens (1992) Free Radical Biol. Med. 13(4), 341-390. [20] D. Harman (1969) J. Am. Geriat. Soc. 17(8), 721-735. [21] R.L. Jackson, G. Ku and C.E. Thomas (1993) Med. Res. Rev. 13(2), 161--182. [22] U.P. Steinbrecher, H. Zhang and M. Lougheed (1990) Free Radical Biol. Med. 9(2), 155-168. [23] A.N. Baer, P.B. Costello and F.A. Green (1990) J. Lipid Res. 31(1), 125-130. [24] I. Nishigaki, M. Hagihara, M. Hiramatsu, Y. Izawa and K. Yagi (1980) Biochem. Med. 24(2), 185-189. [25] M. Hiramatsu, Y. Izawa, M. Hagihara, I. Nishigaki and K. Yagi (1984) Burns 11, 111-113. [26] G.O. Till, J.R. Hatherill and P.A. Ward (1986) in: G.P. Novelli and U.F. Karger (Eds.), Oxygen Free Radicals Shock, Int. Workshop 1985, Basel, Switzerland, pp. 3441. [27] J.O. Wooliscroft, J.K. Prasad, P. Thomson, G.O. Till and R, Fox (1990) Burns 16(2), 92-96. [28] K, Yagi (1987) Chem. Phys. Lipids 45(2-4), 337-351. [29] Y.K. Youn, C. Lalonde and R. Demling (1992) Free Radical Biol. Med. 12(5), 409 415. [30] S.S. Kumari and V.P. Menon (1987) Indian J. Exp. Biol. 25(6), 419 423.

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[31] F.Z. Meerson, V.E. Kagan, Y.P. Kozlov and L.M. Belkina (1982) Basic Res. Cardiol. 77(5), 465--485. [32] P.S. Rao, N.V. Cohen and H.S. Mueller (1983) in: G. Cohen and R.A. Greenwald (Eds.), Oxygen Radicals Their Scavenger Systems, Proc. 3rd Int. Conf. Superoxide/Superoxide Dismutase 1982, Vol. 2, Elsevier, New York, pp. 357-360. [33] A.K. Kogan, A.N. Kudrin and N.I. Losev (1992) Cor Vasa 34(3), 273-282. [34] B. Halliwell (1993) Haemostasis 23 (Suppl. 1), 118-126. [35] B. Halliwell, J.M.C. Gutteridge and C.E. Cross (1992) J. Lab. Clin. Med. 119(6), 598 620. [36] S. Yamagata, H. Murakami, J. Terao and S. Matsushita (1983) Agric. Biol. Chem. (12), 2791-2799. [37] E.G. Bligh and W.J. Dyer (1959) Can. J. Biochem. Physiol. 37, 911 917. [38] H. Kuehn, J. Belkner and R. Wiesner (1990) Eur. J. Biochem. 191(1), 221-227. [39] H.D. Berkeley and T. Galliard (1976) Phytochemistry 15(10), 1475-1479. [40] W.D. Lehmann, M. Stephan and G. Fuerstenberger (1992) Anal. Biochem. 204(1), 158-170. [41] K. Yagi (1976) Biochem. Med. 15(2), 212 216. [42] D.R. Janero (1990) Free Radical Biol. Med. 9 (6), 515540. [43] M. Hamberg and B Samuelsson (1965) Biochem. Biophys. Res. Commun. 21, 531-536. [44] B. Frei, Y. Yamamoto, D. Niclas and B.N. Ames (1988) Anal. Biochem. 175(1), 120-130. [45] E.H. Oliw, F.P. Guengerich and J.A. Oates (1982) J. Biol. Chem. 257(7), 3771-3781. [46] P. Borgeat, M. Hamberg and B. Samuelsson (1976) J. Biol. Chem. 251(24), 7816-7820. [47] A.N. Baer, P.B. Costello and F.A. Green (1991) Biochim. Biophys. Acta 1085(1), 45 52. [48] M. Hamberg and B. Samuelsson (1980) Biochim. Biophys. Acta 617, 545-547. [49] H. Grisebach and J. Ebel (1978) Angew. Chem. 90(9), 668-681. [50] T. Kato, Y. Yamaguchi, T. Hirano, T. Yokoyama, T. Uyehara, T. Namai, S. Yamanaka and N. Harada (1984) Chem. Lett. (3), 409-412. [51] R.M. Bostock, H. Yamamoto, D. Choi, K.E. Ricker and B.L. Ward (1992) Plant Physiol. 100(3), 1448-1456. [52] T. Suematsu, T. Kamada, H. Abe, S. Kikuchi and K. Yagi (1977) Clin. Chim. Acta 79(1), 267-270. [53] H.W. Gardner (1989) Free Radical Biol. Med. 7(1), 6586. [54] A. Loidl-Stahlhofen, K. Hannemann and G. Spiteller (1994) Biochim. Biophys. Acta 1213, 140-148. [55] T. Grune, W.G. Siems, K. Schoenheit and I.E. Blasig (1993) Int. J. Tissue React. 15(4), 145-150. [56] C. Hoelzel and G. Spiteller (1995) Naturwissenschaften 82, 452-460. [57] B. Halliwell and J.M.C. Gutteridge (1992) FEBS Lett. 307(1), 108 112. [58] R. Ross (1981) Arteriosclerosis (Dallas) 1(5), 293-311.