Comparative antioxidant potential of anaesthetics and perioperative drugs in vitro

Clinica Chimica Acta 301 (2000) 41–53 www.elsevier.com / locate / clinchim

Comparative antioxidant potential of anaesthetics and perioperative drugs in vitro a, a b c David Mantle *, Fadel Eddeb , Kiri Areni , Christopher Snowden , A. David Mendelow b a

Department of Neurochemistry, Regional Neurosciences Centre, Newcastle General Hospital, Newcastle-upon-Tyne NE4 6 BE, UK b Department of Neurosurgery, Regional Neurosciences Centre, Newcastle General Hospital, Newcastle-upon-Tyne NE4 6 BE, UK 3 Freeman Hospital Anaesthesia Research Group, Freeman Hospital, Newcastle-upon-Tyne NE7 7 DN, UK Received 25 February 2000; received in revised form 4 May 2000; accepted 12 May 2000

Abstract We have investigated the comparative antioxidant capacity of a range of anaesthetics (inhaled and intravenous) and perioperative neurosurgical drugs (at clinically relevant concentrations) using different radical species and assay methods in vitro. The highest levels of antioxidant activity against the ABTS ?1 radical were obtained with propofol (100 mmol / LTE) and dopamine (1080 mmol / LTE), respectively. However, only dopamine (12 mmol / l) showed antioxidant activity in protecting proteins in normal brain tissue from oxidative damage (assessed via SDS–PAGE analysis) induced by OH ? or O 2? generated radiolytically in vitro. Neither dopamine nor propofol 2 showed antioxidant activity against O 22? generated chemically via reaction between xanthine and xanthine oxidase in vitro. From these data, together with data on the relative antioxidant properties of anaesthetics / drugs obtained by other research groups which we have reviewed, we conclude that the apparent antioxidant activity of a given compound may depend entirely on the free radical species and / or the method of generation or assay employed. Finally, we suggest that on the basis of data obtained showing protection of brain proteins from oxidative damage induced by OH ? , or O 2? 2 in vitro, further investigation into the in vivo antioxidant therapeutic potential of dopamine (or its analogues) on neurosurgical patients may be warranted.  2000 Elsevier Science B.V. All rights reserved. Keywords: Anaesthetics; Perioperative drugs; Free radicals; Antioxidants

*Corresponding author. 0009-8981 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 00 )00315-6

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1. Introduction Free radicals are highly reactive transient chemical species formed in all cells as unwanted by-products of normal aerobic metabolism. Cells are protected from free radical induced damage by a variety of radical scavenging antioxidant proteins, enzymes and chemical compounds. Cellular damage arising from an imbalance between free radical generating and scavenging systems (‘oxidative stress’) has been implicated in the pathogenesis of a wide range of human disorders, including ischemia–reperfusion injury characteristic of neurological disorders such as stroke [1]. Recent research has suggested that anaesthetics such as propofol may function as antioxidants, thereby protecting brain tissue from free radical induced damage to the lipid components of cell membranes [2]. The objectives of the present investigation were: (i) to compare the levels of antioxidant activity of a range of anaesthetics and perioperative neurosurgical drugs (at clinically relevant concentrations) via different assay methods / free radical species, since there is evidence that the latter may influence apparent antioxidant activity [3]; (ii) to determine the effect of compounds with high antioxidant activity identified in (i) above on oxidative damage to brain proteins (arguably of equal importance to lipid peroxidation in free radical induced cell death) induced via Co 60 d irradiation in vitro.

2. Materials Compounds investigated for antioxidant activity in vitro were obtained from the following sources; volatile anaesthetics: halothane (Rhone Poulenc Rorer, Eastbourne, UK), ethane and isofluorane (Abbot Labs, Maidenhead, UK); sedatives / inducing agents: propofol (DIPRIVAN, 10 mg / ml, ICI, Wilmslow, UK) midazolam HCl (HYPNOVEL, 2 mg / ml, Roche, Welwyn Garden City, UK), sodium thiopentone (INTRAVOL Na 2.5%, May & Baker, Dagenham, UK); antiepileptics: phenobarbitone Na (30 mg / ml, Martindale Pharmaceutical, Romford, UK), clonazepam (RIVOTRIL, 1 mg / ml, Roche), phenytoin Na (50 mg / ml, South Devon Healthcare, UK) sodium valproate (EPILIM, 100 mg / ml, Sanofi Winthrop, Guildford, UK); steroids: methyl prednisolone (MEDRONE, 62 mg / ml, Upjohn, Crawley, UK), sodium hydrocortisone (50 mg / ml, Upjohn), dexamethasone (5 mg / ml, Organum Labs, Cambridge, UK); sympathomimetics: dopamine HCl (INTROPIN, 40 mg / ml, DuPont, Stevenage, UK), dobutamine (DOBUTEX, 12 mg / ml, Eli Lilly, Basingstoke, UK), noradrenaline (LEVOPHED, 2 mg / ml, Sanofi Winthrop), isoprenaline HCl (SAVENTRINE, 1 mg / ml, Pharmax Ltd, Bexley, UK); miscellaneous agents: nimodipine (NIMOTOP, 0.2 mg / ml, Bayer Diagnostics, Slough, UK), chlorpromazine

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(LARGACTIL, 25 mg / ml, Rhone Poulenc Rorer), ephidrine HCl (30 mg / ml, Martindale Pharmaceutical), sodium heparin (HEPLOK, 10 u / ml, Leo Labs, Aylesbury, UK), mannitol (20%, Baxter Healthcare, Thetford, UK), intralipid (20%, Pharmacia, Milton Keynes, UK). All other reagents were obtained from Sigma, Poole, UK and were of analytical grade where available.

3. Methods In the various experimental methods to assess antioxidant activity described below, volatile anaesthetic agents were delivered into spectrometer cells or reaction vials closed with self-sealing stoppers, using a calibrated microsyringe.

3.1. Determination of in vitro antioxidant activity against ABTS ?1 radical The antioxidant activity of anaesthetics or neurosurgical drugs (at clinically relevant concentrations, as shown in Table 1) was initially assessed via scavenging of the ABTS ?1 (2,29 azinobis-3-ethyl-benzthiazoline-6-sulphonic acid) radical generated by a metmyoglobin / hydrogen peroxide system as described previously [4]. The test sample (15 ml) was added to a 1 cm pathlength spectrometer cuvette (1 ml capacity) containing 20 mmol / l phosphate buffer pH 7.4, 2.5 mmol / l metmyoglobin and the reaction initiated by addition of 75 mmol / l hydrogen peroxide, and the absorbance change at 734 nm monitored at 308C (sealed cuvette). A quantitative relation exists between the absorbance at 734 nm after 6 min and the antioxidant status of the test sample, determined relative to Trolox (water soluble vitamin E analogue) antioxidant standards (0–2.5 mmol / l) and expressed in terms of mmol / l Trolox equivalent (mmol / LTE).

3.2. Determination of in vitro antioxidant activity against O ?2 2 radical ( generated chemically) The antioxidant activity of anaesthetics or neurosurgical drugs (concentrations as in Table 1 above) against the O ?2 2 radical was determined via the method of McCord and Fridovich [5], using the following assay medium: 100 mmol / l phosphate buffer pH 7.5, 0.1 mmol / l ETDA, 1 3 10 25 M ferricytochrome c, 5 3 10 25 mol / l xanthine and xanthine oxidase (0.004 units / assay); the rate of absorbance change at 550 nm (a quantitative measure of O 2? generation) was 2 0.25 absorbance units / min, and sufficient test sample was added to the above to

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Table 1 Comparison of antioxidant capacity of anaesthetics and neurosurgical drugs against ABTS ?1 in vitro a Compound [(mmol / l)]

Antioxidant activity (mmol / LTE)

Dopamine HCl (261) Propofol (56) Dobutamine (40) Noradrenalin (6) Isoprenaline HCl Thiopentone Na (94) Halothane Midazolam HCl (6) Prednisolone (173) Hydrocortisone Na (138) Dexamethasone (12) Phenobarbitone Na (129) Valproate Na (694) Clonazepam (3) Phenytoin Na (198) Nimodipine (0.5) Mannitol (1100) Chlorpromazine (78) Ephedrine HCl (182) Isofluorane Ethrane

10806162 100618 80616 62616 1664.2 1564.5 1.360.2 Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil

a The assay of antioxidant activity against ABTS ?1 was as described under Methods; data shown as mean6S.D. from three separate experiments, each compound was tested (15 ml / assay) at the appropriate clinically relevant concentration, the molar value of which is shown in parenthesis, together with the corresponding antioxidant activity in terms of mmol / l Trolox equivalent.

attempt to reduce the latter rate by 50% (equivalent to 1 unit of superoxide dismutase activity by definition).

3.3. Determination of in vitro antioxidant activity against OH ? or O 2? 2 ( generated radiolytically) Human brain tissue (cerebral cortex) was obtained at autopsy (within 15 h of death) from cases with no history of neurodegenerative disorders. Tissue samples were extracted via homogenization 1:10 (w / v) in 50 mmol / l phosphate buffer pH 7.5 using an Ultra-Turrax homogenizer (2 3 10 s at 5000 rev / min), and the homogenates centrifuged (5000 g, 15 min). After removal of the supernatants (comprising soluble, principally enzymatic proteins), the pellets were washed (via rehomogenization / centrifugation) three times in extraction buffer, and reconstituted in the original volume of the latter buffer. Supernatant or reconstituted pellet (comprising principally structural cytoskeletal proteins)

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fractions were gassed to saturation with N 2 O (for subsequent generation of OH ? radicals) or with O 2 (following addition of 20 mmol / l sodium formate, for subsequent generation of O 2? 2 radicals) immediately prior to irradiation (up to 40 h) using a Co 60 d irradiation source. The quantification of free radical dosage (equivalent to 100 kRads / h) was determined via standard dosimetric techniques [6]. The theoretical and methodological aspects of generation of OH ? and O 2? 2 via irradiation have been described in detail previously [7]. Free radical induced oxidative damage to supernatant or pellet proteins in the absence or presence of putative antioxidant test compounds (at concentrations shown in Table 2) was assessed via SDS polyacrylamide gel electrophoresis (SDS–PAGE) as described previously [8], using a 5% stacking gel and 5–20% linear acrylamide gradient in the separating gel. After staining, gels were scanned using an Alpha Multi Image Cabinet (with Alpha 3.3 software analysis) to determine quantitative oxidative damage (as % control band intensity lost) to individual proteins. Table 2 Comparative antioxidant capacity of dopamine and propofol in protecting brain soluble proteins from OH ? induced damage in vitro a Protein

Protein band degradation (% control remaining)

molecular mass (kDa)

No scavanger

Dopamine (2 mg / ml)

Propofol (2 mg / ml)

209 160 136 89 77 60 56 51 48 46 43 40 37 35 27 22 20 16 13

1362.9 4265.8 5468.6 1062.5 260.8 2665.3 2765.9 4368.9 5366.4 6165.5 6169.2 6366.6 7168.5 2565.1 50610 68612 5468.3 93611 7368.6

10068.8 84610 9567.9 95611 80611 8667.7 7368.5 94613 9469.1 99610 10065.2 9668.1 100612 10064.5 9469.0 10064.3 10066.6 100613 81611

1564.1 50611 58613 1263.3 1062.3 3065.5 3162.9 4066.0 6068.1 6565.8 6067.2 5564.8 6569.1 2765.2 5569.0 6665.9 5367.7 85615 75613

procedures for the generation of OH ? radiolytically, and quantification of oxidative damage to brain tissue proteins via SDS–PAGE and densitometric analysis were as described under Methods; data for protein band degradation (as % control) shown as mean6S.D. from three separate experiments, the final concentration of dopamine or propofol in this experiment is approx. 12 mmol / l. a

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Alternatively, antioxidant activity against OH ? or O 2? 2 generated radiolytically in vitro was assessed via protection against oxidative damage to a target enzyme, human brain alanyl aminopeptidase, with loss of activity quantitated via fluorimetric assay. Briefly, alanyl aminopeptidase (a key enzyme in intracellular protein metabolism / neuropeptide processing) was chromatographically purified to homogeneity from normal human cortical brain tissue as described previously [9]. Enzyme solutions (approx. 20 mg / ml in 50 mmol / l phosphate buffer pH 7.5) were exposed to OH ? or O 2? 2 radicals generated radiolytically (5–10 min) as outlined above, in the absence or presence of the antioxidant test compounds (at concentrations shown in Table 3). Samples processed as above but not irradiated served as appropriate controls. The activity of the alanyl aminopeptidase samples was then determined via fluorimetric assay [9] as follows: enzyme samples (50 ml) were incubated (10–30 min) at 378C in a medium (total volume 0.3 ml) containing 50 mmol / l Tris / acetate buffer pH 7.5 at 378C, 1 mmol / l 2-mercaptoethanol, 5 mmol / l CaCl 2 and 0.125 mmol / l alanyl-7-amido-4methyl-coumarin (AMC) as substrate. Ethanol (0.6 ml) was added to terminate the reaction, and the fluorescence of the liberated AMC measured ( lex 380 nm, lem 440 nm) by reference to an appropriate fluorescence standard. Assay blanks were run in which enzyme was added to medium immediately before ethanol addition. Enzyme activities were determined following exposure to various OH ? or O 2? 2 dosages in the presence or absence of antioxidant test compound, and the relative free radical scavenging capacity determined by comparison of the % enzyme activity remaining after irradiation in the absence and presence of each antioxidant.

4. Results and discussion The results of the initial screening of anaesthetics and neurosurgical drugs (at clinically relevant concentrations) for antioxidant activity against the ABTS ?1 radical are shown in Table 1. Of the three inhalational anaesthetics, only halothane showed measurable activity (1.3 mmol / LTE) against ABTS ?1 ; the intravenous sedative / inducing agents propofol and thiopentone gave values of 100 and 15 mmol / LTE, respectively. Of the neurosurgical drugs investigated, the highest level of antioxidant activity was obtained for dopamine (1080 mmol / LTE), with many compounds tested showing no measurable antioxidant activity against ABTS ?1 . The assessment of antioxidant activity determined against ABTS ?1 in human plasma / serum for routine use in clinical biochemistry has been proposed (a procedure based on the above for automated analysis in general hospital pathology laboratories is currently marketed by Randox Ltd, Crumlin, N. Ireland) [10], a specific example of which is the monitoring of antioxidant activity / free radical generation during aortic aneurysm repair

Irradiation dosage (kRADS)

0 3 8 16 24

Alanyl aminopeptidase activity (% control) OH ?

O 2? 2

No scavenger

Glutathione (0.1 mmol/l)

Propofol (1 mg/ml)

Intralipid (10%)

No scavenger

Glutathione (0.1 mmol/l)

Propofol (1 mg/ml)

Intralipid (10%)

100 1762.5 5.561.0 0 0

100 5068.3 4369.1 3567.5 2566.3

100 55611.1 4566.8 2064.6 1062.4

100 5969.4 4868.6 2364.0 0

100 2864.2 1563.5 561.2 0

100 100 100 9067.5 8069.9

100 3563.8 2565.3 1563.9 0

100 3367.3 2564.0 1564.2 0

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Table 3 Effect of putative antioxidants on oxidative damage to alanyl aminopeptidase activity induced radiolytically in vitro. The generation of OH ? or O 22? radiolytically, and quantification of alanyl aminopeptidase activity in the absence or presence of antioxidants were as described under Methods; data shown as mean6S.D. from three separate experiments

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surgery [11]. However, the use of this assay method to determine antioxidant levels in other experimental situations is less well defined. There has been a recent report describing the antioxidant potential of i.v. fluids determined against ABTS ?1 , N-acetyl cysteine showed the highest level of antioxidant activity (502 mmol / LTE), with gelofusine, haemacel and albumin having activities , 1 mmol / LTE. Some i.v. fluids, including mannitol and heparin showed no measurable antioxidant activity against ABTS ?1 [12], in agreement with data from the present investigation. This finding is of note since mannitol is considered clinically to be a potent antioxidant, on the basis of its activity against OH ? radicals reported previously [13]. This apparent discrepancy may result from compounds showing different antioxidant activity against different radical species and / or assay procedures, as discussed below. There has been a further recent report to determine the antioxidant potential of various perioperative drugs in vitro, based on scavenging of free radical species generated via thermal decomposition of 2,-29-azobis (2-amidinopropane) dihydrochloride (AAPH), using changes in fluorescence of b-phycoerthyrin (as a reporter target protein) to quantify oxidative damage [14]. Three categories of compounds were identified; those with no measurable antioxidant activity (heparin, pentazocine, bupivacaine, atropine, suxamethonium, prostaglandin E); those with intermediate antioxidant activity (including nicardipine, verapamil, ephidrine, lidocaine, mepivacaine, prednisolone, hydrocortisone, dexamethasone); and those with a high degree of antioxidant activity (including dopamine, epinephrine, dobutamine, isoprotenerol). On the basis of the above, it is possible to identify compounds with high levels of antioxidant activity (e.g. dopamine, dobutamine) or no antioxidant activity (e.g. heparin) using different assay methods based on ABTS ?1 or AAPH derived (i.e. ‘non-physiological’) radical species. However, even with these broadly similar assay methods, there is some disparity in that prednisolone, hydrocortisone and dexamethasone show some antioxidant activity against AAPH derived radicals [14], but not against ABTS ?1 (Table 1, present investigation). In addition, it is not always possible to predict the potential antioxidant activity based on a knowledge of the chemical structure and / or primary mode of action. Thus, steroids such as prednisolone, which have a similar structure to that of the 21 amino steroid based drugs (‘lazeroids’), which have been shown to be effective in preventing free radical induced lipid damage following CNS trauma [15], did not show antioxidant activity against ABTS ?1 (Table 1). None of the compounds investigated in the present study (including those with high activity against ABTS ?1 showed measurable activity against the O 2? 2 radical generated chemically via reaction between xanthine / xanthine oxidase. It has been reported that several compounds, including thiopentone, can inhibit the release of O 2? 2 from polymorphonuclear leukocytes in vitro, although the precise

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mechanism involved (i.e. direct radical scavenging or, e.g. inhibition of O 2? 2 producing enzymes) was not determined [16]. A separate investigation showed that the local anaesthetics lidocaine and meprivacaine reduced the rate of free radical production by neutrophils in vitro, but failed to reduce O 2? 2 produced via reaction between xanthine / xanthine oxidase (i.e. cell free system) [17]. However, pretreatment with isoprotenerol or dexamethosone reduced O 2? 2 production in cultured microglial cells, induced via exposure to phorbol myristate acetate [18]. On the basis of the above data, together with that obtained in the current investigation, we conclude that the apparent antioxidant activity of a given compound may depend on the radical species or assay method utilized. In order to further investigate this hypothesis, the two compounds representing the highest levels of antioxidant activity of the anaesthetics (propofol) and neurosurgical drugs (dopamine), respectively, against ABTS ?1 , were studied with regard to their capacity for scavenging OH ? or O 2? radicals generated 2 radiolytically in vitro. The results of a typical experiment to determine the effect of oxidative damage to proteins in normal brain tissue induced by OH ? , or O 2? 2 are shown in Fig. 1; SDS–PAGE analysis showed substantial oxidative degradation of brain supernatant protein bands with increasing exposure to OH ? (500 kRads, lane 3; 2000 kRads, lane 5), compared to corresponding nonirradiated control tissue extracts (lanes 1 and 4). The overall SDS–PAGE protein fractionation pattern showed a generalised progressive loss in band staining intensities in a dose dependent manner, with essentially complete destruction of most protein species at a dosage of 4000 kRads. It was also evident (from densitometric analysis) that different protein species showed different relative susceptibility to oxidative damage by OH ? ; with higher molecular mass proteins ( . 70 kDa) in general terms showing greater susceptibility to oxidative degradation (Table 2). There was no evidence for the formation of protein fragments or aggregates after exposure of brain extracts to OH ? or O 22? as reported previously following exposure of model proteins (lysozyme, haemoglobin) to free radical species generated radiolytically [19,20]. Presumably the disappearance of protein bands induced by free radicals in the present study corresponds with the formation of either very large aggregates or very small peptides / free amino acids, which are not resolved via SDS–PAGE analysis. Broadly, similar results were obtained following exposure of brain supernatant ? 2? proteins to O 2? 2 or cytoskeletal proteins to OH or O 2 in vitro (data not shown). The addition of dopamine to brain supernatants prior to irradiation resulted in substantial protection of proteins from oxidative damage by OH ? (Fig. 1, lanes 4 and 6), as summarised in Table 2. The addition of dopamine to non-irradiated brain supernatant samples did not result in any alteration to the SDS–PAGE fractionation pattern (Fig. 1, lane 2). Similar results were obtained showing a substantial degree of protection by dopamine in preventing oxidative damage to

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Fig. 1. SDS–PAGE analysis of oxidative damage to brain soluble proteins via OH ? in absence and presence of dopamine. Procedures for the generation of OH ? radiolytically and analysis of oxidative damage to brain soluble proteins via SDS–PAGE were as described under Methods. The interpretation of fractionation pattenrs in lanes 1–6 is given in the discussion.

? 2? supernatant proteins by O 2? 2 , or cytoskeletal proteins to OH or O 2 in vitro (data not shown). However, when the above experimental procedures were repeated using propofol, there was no evidence for the protection of brain supernatant or cytoskeletal proteins from oxidative damage by OH ? O 22? in vitro (Table 2), in contrast to the protective effect obtained with dopamine. To confirm the apparent absence of antioxidant activity of propofol in protecting brain proteins from generalised antioxidant damage in vitro, a further experiment was carried out to investigate the effect of propofol in protecting alanyl aminopeptidase from damage induced by OH ? or O 22? (since changes in enzyme activity are arguably the most sensitive index of free radical induced oxidative damage to proteins). The data presented in Table 3 show the loss of activity of purified alanyl aminopeptidase (using relatively low irradiation dosages compared to the above experiments for whole tissue extracts), following exposure to OH ? or O 22? in

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vitro; this loss of activity was partially prevented following addition of the free radical scavenger glutathione, which was used as a ‘positive control’ in this experiment [21]. Although there was some degree of apparent antioxidant protection conferred on alanyl aminopeptidase by propofol, this was subsequently attributed to the presence of the intralipid carrier, rather than the anaesthetic agent itself (Table 3). There was therefore no evidence that propofol can act as an antioxidant in protecting brain proteins from oxidative damage by OH ? or O 2? in vitro, even under experimental conditions designed for optimum 2 sensitivity of detection of damage / protection as above. In this regard, the use of alanyl aminopeptidase in an isolated, purified form as a biomolecular target in vitro would be predicted to render this enzyme more susceptible to free radical damage than would be the case in vivo; i.e. in the presence of other intracellular proteins, which would effectively act as competitive biomolecular free radical targets. In addition, the free radical dosages to which alanyl aminopeptidase was exposed in vitro were predicted to be substantially greater than those to which the enzyme would be likely to be exposed in vivo. It is therefore of note that even under such extreme experimental conditions, there was no evidence for any significant antioxidant activity for propofol (in contrast to that observed with glutathione) in the above experimental system. It has been reported that isofluorane reduces the degradation of microtubule associated protein 2 during forebrain ischaemia in the rat [22], and the free radical scavenging properties of dopamine in protecting fatty acids from AAPH induced oxidation in vitro [23], or following traumatic brain injury in cats [24] has been recognised. However, there have been no previous investigations into the effects of anaesthetics or neurosurgical drugs in protecting brain proteins against OH ? or O 22? generated radiolytically in vitro. In summary, we have identified a number of anaesthetics with antioxidant activity against the ABTS ?1 radical (although it was not possible to predict antioxidant activity from a knowledge of chemical structure or primary action). The highest levels of antioxidant activity against ABTS ?1 were obtained with dopamine and propofol; however only dopamine showed antioxidant activity in protecting proteins in normal brain tissue from oxidative damage induced by OH ? or O 2? generated 2 radiolytically in vitro. Neither dopamine nor propofol showed antioxidant activity against O 2? generated chemically via reaction between xanthine and 2 xanthine oxidase in vitro. From these data, together with corresponding data reviewed from other research groups, we conclude that the apparent antioxidant activity of a given anaesthetic agent or neurosurgical drug may depend entirely on the free radical species and / or method of generation or assay employed. Finally we conclude on the basis of data obtained demonstrating the protection of brain proteins from oxidative damage induced by physiologically relevant radical species in vitro, further investigations into the in vivo therapeutic potential of dopamine in neurosurgical patients may be warranted.

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Acknowledgements We acknowledge Newcastle Hospitals Trust Research Committee for financial support.

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