The generation of byproducts of lipid peroxidation following carotid endarterectomy

Eur J Vasc Endovasc Surg 12, 455--458(1996)

The Generation of Byproducts of Lipid Peroxidation following Carotid Endarterectomy C. V. Soong .1, I. S. Young 2, J. M. Hood 1, B. J. Rowlands 3, E. R. Trimble 2 and A, A. B, Barros D'Sa 1 1Vascular Surgery Unit of the Royal Victoria Hospital, Belfast and Departments of 2ClinicaI Biochemistry and 3Surgery of the Queen's University of Belfast, Grosvenor Road, Belfast BT12 6BJ, U.K. The aim of this study was to determine whether free radical-induced lipid peroxidation occurs following transient carotid clamping. Jugular vein plasma levels of malondialdehyde (MDA) and diene conjugates (DC) were estimated in 24 patients undergoing carotid endarterectomy, at the beginning of the operation (To), just prior to clamping the carotid artery before the shunt was removed for closure of the arteriotomy (Ts), and at 30 (T30), 60 (T60), 120 (T120), 180 (T180) and 300 (T300) seconds after the clamps were released. Carotid clamp times were recorded. Significant elevations in the concentrations of both MDA and DC were observed at T60 after clamp release (MDA = 559 + 64 pmol/ml, DC = 428 + 32 units/ml), in comparison to concentrations at To (MDA = 408 +_34 pmol/ml, p < 0.01; DC = 374 +-28 units/ml, p < 0.05), returning to baseline at T300. There was a significant correlation between the percentage rise in MDA concentration and the duration of clamp-induced ischaemia (r = 0.45, p = 0.03). The significance of this burst of MDA and DC is unclear especially as the one patient who sustained a postoperative neurological deficit displayed no rise in the concentration of either. If this rise is related to free radical generation following ischaemia-reperfusion injury it may play an important role in influencing the clinical outcome in the patients. Key Words: Lipid peroxidation; Malondialdehyde; Carotid endarterectomy.

Introduction The production of oxygen-derived free radicals has been observed in many pathological states, increasing the severity of tissue damage, particularly in situations when ischaemic tissues are reperfused, such as after coronary and lower limb arterial bypass surgery. 1'2 Free radicals are cytotoxic species, normally generated in small amounts by the mitochondria and endoplasmic reticulum of cells via the electron transport chain. 3 They are also produced by polymorphonuclear leucocytes as part of the body's defences against invading micro-organisms. 4 Under physiological conditions, free radicals are neutralised by the presence of endogenous anti-oxidants and free radical scavengers, but when these protective mechanisms are overwhelmed injury to the surrounding tissues occurs as a consequence of processes such as lipid peroxida-

tion. 5 Lipid peroxidation is a process in which polyunsaturated fatty acids incorporated in lipid membranes of cells are damaged releasing byproducts such as malondialdehyde (MDA) and diene conjugates (DC). As a result, the integrity of the cell membrane is altered, rendering it more permeable and the cell becomes prone to oedema. This study investigates free radical-induced lipid peroxidation following transient hypoperfusion of the cerebrum during carotid clamping in patients undergoing carotid endarterectomy.

Method

Twenty-one patients undergoing 24 carotid endarterectomies were recruited into the study after obtaining informed written consent. Three patients underwent staged bilateral operations. All operations were performed by two consultant vascular surgeons. After *Pleaseaddress all correspondenceto: C. V. Soong,VascularSurgery Unit, Royal Victoria Hospital Grosvenor Road, Belfast BT126BJ, routine endotracheal anaesthesia and neck exposure, a cannula was inserted into the internal jugular vein via U.K. 1078-5884/96/040455+ 04 $12.00/0 © 1996W. B. Saunders Company Ltd.

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the common facial vein. Intravenous heparin (5000 units) was given to all patients prior to cross clamping the carotid artery. A Brenner shunt was inserted in all cases. The carotid system was cross-clamped twice, once during shunt placement and the second following shunt removal prior to completing closure of the arteriotomy. Stump pressure, being a measure of the pressure within the internal carotid artery distal to the site of endarterectomy, was recorded routinely via the side arm of the shunt. Blood samples for measurements of MDA and DC were collected in EDTA bottles at the commencement of surgery (TO), during shunting (Ts) and at 30 (T30), 60 (T60), 120 (T120), 180 (T180) and 300 (T300) seconds after release of the carotid clamps following closure of the arteriotomy. The plasma from these samples was isolated within 4h of collection by centrifugation at 2000 rpm and then stored at -70°C until assayed. It has previously been shown that the plasma concentrations of these lipid peroxidation products are stable for at least 4 h at room temperature. 6 Plasma MDA concentrations were measured using a high performance liquid chromatography technique with fluorimetric detection as described by Young and Trimble 6 while DC measurements were made using second derivative spectroscopy. 7 Eight patients undergoing abdominal aortic surger~ who had central lines inserted for monitoring purposes, were used as controls. To assess the effect of jugular vein cannulation and anaesthesia on MDA concentrations, samples of blood were collected at previously stated time points during the operation beginning after the placement of an aortic clamp and ending prior to its removal. All results are expressed as the mean _+ S.E.M. Statistical analyses of the data were performed using the Wilcoxon-Signed Rank test, analysis of variance (ANOVA) and Spearman's Rank correlation w h e r e appropriate with significance taken at the 5% level. This study was approved by the Research Ethical Committee of the Queen's University of Belfast.

(4%) suffered a mild neurological deficit postoperatively. Carotid clamp time during closure of the arteriotomy (177_+9s) was significantly longer than that at shunt placement (83 _+5s). There were significant elevations in the MDA and DC (Fig. 1) concentrations at T60 (MDA --= 559 + 64 pmol/ml, DC = 428 + 32 units/ml), compared to TO (MDA = 408 -+ 34 pmol/ml, p < 0.01; DC = 374 + 28 units/ml, p<0.05). Carotid clamp times showed a correlation with the percentage rise in the MDA concentrations (Fig. 2) but not with the percentage rise in concentrations of DC (r = 0.06, p = 0.73), even though the changes in these two byproducts of lipid peroxidation correlated significantly (r = 0.59, p = 0.003). No correlation was detected between the percentage fall in the distal systolic stump pressure and the rise in either MDA (r = 0.04, p = 0.98) or DC (r = 0.26, p = 0.20). In contrast to the carotid endarterectomy patients, no rise in MDA concentrations was detected in the control group of patients (Fig. 3). There was however no evidence of a rise in MDA or DC in the one patient who suffered postoperative neurological deficit (Fig. 4).

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Of the 21 patients studied, eight were females and the rest males. The age ranged between 45 and 74 years with a mean of 65 _+7 years. Fifteen operations were for transient ischaemic attacks, three for recurrent amaurosis fugax, three for a combination of the two symptom complexes; in three cases the carotid lesion, a high grade stenosis, was asymptomatic. One patient Eur J Vasc Endovasc Surg Vol 12, November 1996

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Fig. 1. (a) Mean (_+S.E.M.) malondialdehyde concentrations in patients undergoing carotid endarterectomy (**p < 0.01; ANOVA). Co) Mean (+S.E.M.) diene conjugate concentrations in carotid endarterectomy patients (*p < 0.05; ANOVA).

Lipid Peroxidation following Carotid Endarterectomy

Discussion

Oxygen-derived free radicals have been implicated in the pathogenesis of many disease states, including reperfusion injury of ischaemic tissues. The re-establishment of blood flow either to an ischaemic myocardium or to the lower limb has been shown to generate oxygen-derived free radicals which can cause further damage to tissues already injuredJ '2 It has been suggested that the production of these free radicals causes brain oedema following transient cerebral hypoperfusion in an animal m o d e l s In support of this proposition evidence exists to show that the damage to the cerebrum can be reduced by pretreating animals with anti-oxidants in anticipation

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Fig. 2. Correlation b e t w e e n the percentage rise in MDA and carotid clamp time in individual patients, r = 0.45, p = 0.03, Spearman's Rank Correlation.

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of reperfusion injury.8-~° Panetta et al. ~1 demonstrated that reperfusion of the brain in patients undergoing carotid surgery produced changes in both blood pressure and somatosensory evoked potentials, even after short periods of carotid clamping. Many theories have been postulated to explain the generation of these free radicals one of which is the xanthine dehydrogenase/xanthine oxidase pathway. ~2 By the inactivation of xanthine oxidase using tungsten and the administration of dimethylthiourea (a hydrogen peroxide scavenger) Patt et al. ° were able to reduce brain oedema in gerbils subjected to temporary unilateral carotid artery occlusion. Others however, propose that the release of transition metal ions together with the increase in tissue lactate concentration leads to the formation of hydroxyl radicals or closely related iron-containing species. 1~ This hypothesis is supported by workers who were able to minimise reperfusion injury using deferoxamine.9 Regardless of the mechanism for free radical production, brain tissue is rich in lipid and is particularly susceptible to peroxidation damage. We have demonstrated that following even short periods of varying degrees of cerebral hypoperfusion there is a rise in MDA and DC with a weak correlation existing between the duration of diminished flow. The origin of this increased concentration of MDA is difficult to determine. While accepting the fact that the brain is prone to free radical damage, it should be remembered that the vascular endothelium itself is rich in xanthine oxidase and being susceptible to ischaemia-reperfusion injury may well be the source of the MDA rise. 24 This peroxidative damage to the lining of the vessels, can produce endothelial swelling which in turn may further reduce cerebral blood flow thereby compounding the insult to the brain. In

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Fig. 3. Mean ( _+ S.E.M.) malondialdehyde concentration in the control group of patients undergoing abdominal aortic surgery. (p = NS, ANOVA). F = first sample which was taken at the start of cross clamping the aorta. O = second sample which was about 30 min after the first. The other samples were taken at 30, 60, 120, 180 and 300 s after the second to make it as similar as possible to the carotid group of patients. All samples were taken at the time of aortic crossclamping.

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Fig. 4. The concentrations of MDA (C) p m o l / m l ) and DC (@ units/ ml) in the patient w h o sustained postoperative neurological deficit.

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addition, xanthine oxidase driven superoxide production may lead to neutralisation of endothelium derived relaxing factor (nitric oxide) giving rise to vasoconstriction and narrowing of vessels. 15 It must be borne in mind, however, that one plausible source for the MDA is the atherosclerotic plague, which at the time of carotid artery manipulation during dissection and when squeezed intermittently, during clamping and unclamping, may lead to its release. 16 Unfortunately, this possibility is not easy to exclude. Therefore, the production of MDA and DC as a result of free radical induced lipid peroxidation is difficult to prove in this situation. Even if the rise in MDA concentration at 60 s after clamp release is not an artefact its significance remains moot as the incidence of neurological deficit is relatively low despite the fact that MDA itself is cytotoxicY"is In addition, the one patient who suffered a postoperative neurological complication did not show a rise in either MDA nor DC; it could be argued that such a rise might have been missed or that it did not occur because the patient did not sustain a reperfusion injury as blood flow was not re-established. The lack of elevation of byproducts of lipid peroxidation during the period of shunting might be attributed to a very short clamp time during shunt placement in comparison to the final stages of arteriotomy closure. Secondly, as the only sample taken at the time of shunting (Ts) was at a point long after clamp removal following insertion of the shunt, a short-lived rise in MDA or DC might well have passed. To establish whether a direct correlation exists between the degree of lipid peroxidation and the actual development of postoperative neurological deficit in patients undergoing carotid endarterectomy will require a much larger study, particularly as such complications occur infrequently. The physical inaccessibility of the cerebrum makes it impossible to ascertain exactly how much these damaging species contribute to the cerebral injury observed after global hypoperfusion related to cardiac arrest and other hypotensive states.

Acknowledgements This study was supported by a Fellowship of the Royal Victoria Hospital, Belfast with additional support from the British Heart Foundation, Department of Health and Social Services in Northern Ireland, and the Chest, Heart and Stroke Association. We are most grateful for the generosity of these establishments and also to all the

Eur J Vasc Endovasc Surg Vol 12, November 1996

staff in the Vascular Surgery Unit and Theatre for their contributions.

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