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Lipid peroxidation as a cause of lower limb swelling following femoro-popliteal bypass grafting
Eur J VascSurg 7, 540-545(1993)
Lipid Peroxidation as a Cause of Lower Limb Swelling Following Femoro-popliteal Bypass Grafting C. V. Soong 1, I.S. Young 2, P. H. B. Blair 1, J. M. Hood ~, B. J. Rowlands 3, E. R. Trimble 2 and A. A. B. Barros D'Sa I ~Vascular Surgery Unit, 2Department of Clinical Biochemistry and 3Department of Surgery, Queen's University of Belfast Royal Victoria Hospital, Belfast, Northern Ireland, U.K. We examined the role of free radical induced lipid peroxidation in lower limb swelling in patients following femoro-popliteal bypass grafting. In 20 patients undergoing this operation blood samples were taken from the femoral vein via a cannula before the femoral artery clamp was applied, just prior to and immediately after clamp release and at 10 min intervals thereafter for I h for measurements of malondialdehyde (MDA) and vitamin E. The concentration of M D A was significantly elevated at 40 min after reperfusion (mean + S.E.M., 573 + 83 pmol/ml) compared to just before clamp release (359 ± 41 pmol/ml; p < 0.01). This was associated with a corresponding fall in the concentration of vitamin E at the time of peak MDA rise (5.68 +_ 0.28 to 5.29 + 0.28 p~mol/mM cholesterol, p ~ 0.05) suggesting its utilisation as an antioxidant. The degree of oedema was related to the changes in MDA and vitamin E. Thus, in the 15 patients with greater than 10% increase in lower limb volume the rise in the concentration of M D A was 364 +_ 44 to 693 + 76pmol/mI (p = 0.0001) while that in the five, whose swelling was less than 10%, was 344 + 40 to 559 + 243 pmol/ml (p = 0.25). A significant fall in vitamin E was found only in the group with greater than 10% lower limb oedema (5.90 + 0.33 to 5.40 + 0.34~mol/mM cholesterol, p < 0.01), in comparison to those with less than 10% swelling (5.01 + 0.35 to 5.04 + 0.50~mol/m~ cholesterol). 'lhese results suggest that Jree radzcal reduced peroxidation occurs following femoro-popliteal bypass grafting and that there is more free radical damage in those who subsequently develop significant lower limb oedema. Oxygen-derived free radicals may play a significant role in causing lower limb oedema in this situation. Key Words: Malondialdehyde; Vitamin E; Femoro-popliteal bypass grafting.
Introduction Lower limb swelling is a common problem following femoro-popliteal bypass grafting, occurring in up to 100% in some series. 1-4 The oedema can be significant with an average increase of 30% of initial limb volume. 3 This lower limb oedema can delay re-mobilisation and compromise healing not only in patients with associated ischaemic ulcers but also in those who have undergone distal amputations, and the requirement to use a compression stocking in the elderly may demand additional supervision. All these factors contribute towards prolonged hospital stay. Rarely, the oedema may be severe enough to cause compartmental syndrome, further compromising blood flow to an already ischaemic limb by impairing the collateral circulation, s Many theories have been proposed for the causation of lower limb swelling but the exact aetiology 0950-821X/93/070540+06$08.00/0© 1993Grune & Stratton Ltd.
remains controversial. These hypotheses include an increase in capillary filtration and disruption of the lymphatics at the time of dissection. 1-4"6"7 It is agreed, however, that deep venous thrombosis is not a frequent contributor. Recently, it has been suggested that oxygen-derived free radicals m a y play a role in swelling of the lower limb in this situation, s It has been shown that free radicals are generated following reperfusion of ischaemic muscle and may cause cellular damage giving rise to changes in the transmembrane potential difference, and increases in skeletal muscle vascular permeability. 9"10 Oxygen-derived free radicals are cytotoxic species which can cause damage to cells via processes such as lipid peroxidation, the end products of which include malondialdehyde (MDA). This chain reaction is limited in vivo by free radical scavengers such as vitamin E, the major lipid phas e antioxidant, 11 which is incorporated into the lipid membrane of cells. Free
Lipid Peroxidation Following Femoro-popliteal Bypass
radical damage to cellular membranes leads to an increase in cell permeability which in turn can give rise to oedema. Unfortunately, direct measurement of oxygenderived free radicals using' electron spin resonance is difficult and impractical in biological systems and many indirect methods are therefore used. These techniques involve either the measurements of byproducts of free radical induced tissue damage, like MDA, or the assessment of the consumption of endogenous antioxidants. Although lower limb swelling is likely to be multifactorial in origin, oxygen-derived free radicals generated during reperfusion may have a major role to play. Therefore the aim of this study is to investigate whether free radical-induced lipid peroxidation occurs following femoro-popliteal bypass grafting and if the degree of lipid peroxidation can be correlated to the severity of lower limb oedema.
541
ings were taken after the patient had been recumbent for 30 min. The volume of the lower leg was calculated using the formula of a truncated cone; V = h x 3.142 x (C2 + c2 + Cc)/3. Where: V = volume; C = bulkiest portion of calf; c = narrowest portion of ankle; and h = distance between C and c. This technique of assessing volume change has been shown to be as accurate as the water displacement technique. 3'12 Swelling was taken as the increase in volume in the operated leg minus the change in the contralateral leg. This takes into account the volume change that may result from central causes such as, fluid overload and heart failure. This swelling is expressed as a percentage of the initial limb volume and an increase of 10% or more was taken as significant.
Malondialdehyde and vitamin E measurements
Materials and Methods Patient selection Patients undergoing femoro-popliteal bypass grafting for relief of intermittent claudication were recruited into the study after informed written consent was obtained. Patients were excluded if they had: (1) preexisting gross lower limb oedema; (2) previous lower limb arterial surgery or (3) undergone previous above or below knee amputation on the contralateral leg. After routine endotracheal anaesthesia, conventional longitudinal groin and medial popliteal incisions were employed. A Gortex graft was used in all the patients except one, in whom a reversed vein graft was used. The proximal anastomosis was to the common femoral artery in all patients while the distal anastomosis was to the popliteal artery above knee level in all cases but one. Six patients undergoing surgery for varicose veins were recruited as controls.
Lower limb measurements Daily circumference measurements were made of the lower leg at two points, namely at the bulkiest and narrowest parts, just before operation and continued until discharge from hospital or until an elastic compression stocking was applied. The two points chosen were marked carefully using a permanent ink marker to aid subsequent measurements. These read-
Blood samples were taken from the femoral vein via a cannula before the application of the femoral artery clamp, just prior to and immediately after clamp release and at 10 min intervals thereafter for I h. The blood samples were collected in EDTA bottles and the plasma isolated by centrifugation at 2000r/min for 10 min within 4 h of collection. The plasma was then stored at -70°C until analysed. The samples were analysed for MDA using high performance liquid chromatography with fluorimetric detection as described by Young and Trimble. 13 Vitamin E concentrations were measured at five key time points: at the time of peak MDA rise, 10 min on either side of this maximum MDA rise and just prior to and immediately after clamp release. Vitamin E measurements were made using a high performance liquid chromatograhy (HPLC) technique with concentrations corrected for plasma concentrations of cholesterol. 14"15 Creatine kinase was measured on a Beckman CX5 centrifugal analyser. In the six control patients femoral venous blood was taken in the same manner as that in femoropopliteal bypass patients. Blood was taken at the start of the operation, towards the end of the operation, about 5min after this and then at 10min intervals thereafter for l h . These samples were assayed for MDA.
Statistics Measurements of MDA and creatine kinase were Eur J Vasc Surg Vol 7, September 1993
C.V. Soong et al.
542
a s s e s s e d u s i n g analysis of variance (ANOVA). To c o m p a r e paired variables, such as c o m p a r i s o n bet w e e n the p e a k a n d baseline concentrations of M D A a n d c h a n g e s in v i t a m i n E concentrations, the Wilcoxon Signed Rank test w a s used. All results are e x p r e s s e d as the m e a n + S.E.M. This s t u d y w a s a p p r o v e d b y the Research Ethical C o m m i t t e e of the Q u e e n ' s U n i v e r s i t y of Belfast.
700 600 ~
*p < 0-001
,T,
500
E 400
< 300 X 200
n = 20
100
Results T w e n t y patients w e r e recruited into the study. Of these, 12 w e r e males a n d the a v e r a g e age w a s 60 + 5 years. The m e a n a n k l e - b r a c h i a l systolic p r e s s u r e index w a s 0.60 + 0.03. The increase in l o w e r limb volume post-operatively ranged between 0-30% and eight patients required t r e a t m e n t w i t h a c o m p r e s s i o n stocking. In 15 patients l o w e r limb swelling w a s greater t h a n 10%. T h e r e w a s n o significant difference in the age, sex distribution, p r e - o p e r a t i v e a n k l e brachial systolic p r e s s u r e index a n d intra-operative f e m o r a l artery c l a m p time b e t w e e n those w h o s e swelling w a s greater t h a n 10% c o m p a r e d to those w h o s e swelling w a s less t h a n 10% (Table 1).
I
I
v
PC
BR
800
(a)
I
T0
I
I
I
I
I
I
10
20 30 40 50 60 Time (min) Fig. 1. Malondialdehyde concentrations (mean _+ S.E.M.) in patients undergoing femoro-popliteal bypass grafting. PC, preclamp; BR, before clamp release. Arrow indicates time of clamp release.
*
n = 20
*p < 0.001 60o
~ 400 <~ 200
T a b l e 1. Characteristics of patients w i t h and w i t h o u t significant l o w e r l i m b s w e l l i n g ( m e a n + S.E.M.)
Significant swelling (>10%)
No significant swelling (<1o°/,,)
Age (years)
64.7 +_ 1.3
64.3 + 3.3
Sex (re:f)
9:6
3:2
~
5.8
Ankle-brachial index
0.62 ± 0.03
0.61 ± 0.03
~
5.6
Clamp time (min)
48 + 2.5
54 + 2.0
~
5.4
6.2
(b)
n = 20 *p < 0.05
6
5.2 The p r e - c l a m p concentration of M D A w a s o b s e r v e d to be 407 + 40 p m o l / m l . Following c l a m p release the concentration of M D A rose gradually, p e a k i n g b e t w e e n 30 a n d 5 0 m i n in the majority of patients. This elevation in M D A r e a c h e d a significant level at 40 m i n of r e p e r f u s i o n (573 + 83 pmol/ml) in c o m p a r i s o n to the concentrations at m a x i m u m ischaemia, i.e. before c l a m p release (359 + 41 p m o l / ml; p < 0.01, A N O V A ) (Fig. 1). This w a s associated w i t h a c o r r e s p o n d i n g significant fall in v i t a m i n E concentrations at the time of p e a k M D A rise (5.68 + 0.28 to 5.29 + 0.28 p~mol/mM cholesterol; p < 0.05 Wilcoxon Signed Rank) (Fig. 2). Eur J Vasc Surg Vol 7, September 1993
~
5
+ 10 min 10 min Peak Time Fig. 2. Correlation between mean concentrations (+_ S.E.M,) of (a) MDA and (b) vitamin E at the five time points. BR, time before clamp release. Peak, time of peak MDA rise in individual patients. BR
The p e a k M D A rise, w a s significant in those w h o s u b s e q u e n t l y d e v e l o p e d a greater t h a n 10% swelling (364 + 44 to 693 + 76 pmol/ml; p = 0.0001) while t h o s e w i t h less t h a n 10% swelling did not h a v e a statistically significant increase (344 + 40 to 559 + 243 p m o l / ml; p = 0.25) (Fig. 3). The fall in v i t a m i n E w a s f o u n d
Lipid Peroxidation Following Femoro-popliteal Bypass 1000
6O
n = 15
800
543
n=5
*
T
*p < 0.001
60O 45
< 400
n
200
4o~85
I I I Significant swelling No significant swelling Fig. 3. MDA concentrations (_+ S.mM.) before clamp release (@) and at peak rise (O) in patients with and without significant leg swelling. 0
n=15
30
I PC
I BR
I 0
_1 10
t I I I t 20 30 40 50 60 Time (min) Fig. 6. Concentrations (mean + S.E.M.) of creatine kinase before, during and after ischaemia. PC, preclamp; BR, before clamp release.
n=5
6.2 o
~6
*p < 0.01 5.7
N.S.
Discussion
T h e a e t i o l o g y of l o w e r limb o e d e m a f o l l o w i n g femoro-popliteal bypass grafting remains unresolved although many theories have been proposed. Some h a v e s u g g e s t e d t h a t the s w e l l i n g is a result of > 4.7 i Significant swelling No significant swelling n c r e a s e d capillary filtration, 6' 7 w h i l e o t h e r s felt t h a t Fig. 4. Changes in vitamin E concentrations (mean + S.E.M.) in d i s r u p t i o n of the l y m p h a t i c s d u r i n g d i s s e c t i o n of the patients with and without significant lower limb swelling ([] = g r o i n a n d p o p l i t e a l fossa is responsible.l"2 A l t h o u g h before clamp release, [] = peak MDA time). s o m e i n v e s t i g a t o r s w e r e able to r e d u c e the i n c i d e n c e a n d s e v e r i t y of the o e d e m a b y p e r f o r m i n g l y m p h a t i c 800 s p a r i n g incisions, t h e y w e r e u n a b l e to p r e v e n t this s w e l l i n g c o m p l e t e l y . 2"4 M o r e r e c e n t l y it w a s p r o 600 p o s e d that o x y g e n - d e r i v e d free radicals g e n e r a t e d d u r i n g r e p e r f u s i o n of the i s c h a e m i c limb m a y h a v e a © role to play. 8 Free radicals h a v e b e e n s h o w n to c a u s e 400 v a r i o u s d e g r e e s of d a m a g e to cells s u b j e c t e d to < i s c h a e m i a - r e p e r f u s i o n injury; this d a m a g e varies p = 0.88 (ANOVA) 2o0 f r o m a c h a n g e in cellular t r a n s m e m b r a n e p o t e n t i a l to cell necrosis. 9' 10,16,17 T h e p r i m a r y s o u r c e of this free radical g e n e r a t i o n 0 I i l I I I I I I is n o t e n t i r e l y clear b u t it h a s b e e n s u g g e s t e d b y s o m e 80 GO 0 10 20 30 40 50 60 Time (min) t h a t it m a y be p r o d u c e d via the x a n t h i n e o x i d a s e system.lO,16,18,19 X a n t h i n e o x i d a s e occurs n a t u r a l l y , Fig. 5. Mean MDA concentrations (+ S.E.M.) in control patients undergoing surgery for varicose veins. SO, start of operation; EO, m a i n l y as t h e d e h y d r o g e n a s e in v a r i o u s tissues, s u c h end of operation. as t h e intestine, brain, liver, heart, skeletal m u s c l e a n d e n d o t h e l i a l cells. T h e d e h y d r o g e n a s e f o r m catao n l y in t h o s e w i t h g r e a t e r t h a n 10% s w e l l i n g (5.90 + l y s e s the reaction: 0.33 to 5.40 + 0.34 txmol/mM cholesterol; p < 0.01) in c o m p a r i s o n to t h o s e w h o s e s w e l l i n g w a s less t h a n x a n t h i n e + H 2 0 + NAD+---~uric acid + N A D H + H + 10% (5.01 ± 0.35 to 5.04 ± 0.50 ~xmol/mM cholesterol) D u r i n g i s c h a e m i a it is c o n v e r t e d to t h e o x i d a s e f o r m (Fig. 4). A rise in M D A c o n c e n t r a t i o n w a s n o t b y a p r o t e a s e a n d utilises o x y g e n to g e n e r a t e s u p e r o b s e r v e d in the c o n t r o l g r o u p (Fig. 5). W e did n o t oxide: d e m o n s t r a t e a n y e l e v a t i o n in the creatine k i n a s e conc e n t r a t i o n w i t h i n o u r s a m p l i n g t i m e in the b y p a s s h y p o x a n t h i n e + H 2 0 + 202---~uric acid + 2 0 2 " + g r o u p of p a t i e n t s (Fig. 6). 2H + 5.2
Eur J Vasc Surg Vol 7, September 1993
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C.V. Soong et aL
Neglen et al. demonstrated an abundance of plasma hypoxanthine, a breakdown product of adenosine monophosphate, during ischaemia in the limb. 18 Therefore, with the accumulation of hypoxanthine and xanthine oxidase during ischaemia, favourable conditions are created for the generation of oxygenderived free radicals when reperfusion occurs. 19 A reduction in the degree of cell damage brought about either by the administration of catalase, superoxide dismutase or allopurinol, or by controlling the delivery of oxygen during reperfusion of ischaemic tissues is further corroborative evidence of the role played by free radicals in ischaemia-reperfusion injury. 16"17 As an indicator of free radical-induced lipid peroxidation we have serially measured MDA by an HPLC technique with fluorimetric detection. 13 The use of this technique minimises interference by creatinine, urea, glucose and bilirubin which otherwise reduces the specificity of MDA measurements. Elevation of MDA together with the fall in vitamin E, the major lipid-phase chain breaking anti-oxidant, following reperfusion in patients undergoing femoropopliteal bypass grafting in this study provide strong evidence of increased free radical production. As expected, the lowest vitamin E concentrations were observed at the time of maximum lipid peroxidation. In the presence of increased free radical production, vitamin E levels are expected to fall as tocopherol is converted to the tocopheryl radical. The subsequent rise in vitamin E levels may be attributed to recycling of the stable tocopheryl radical by aqueous phase anti-oxidants. The late increase in MDA concentration, occurring at 40 min following reperfusion, may account for the failure by others to detect its occurrence at an earlier time. 8 The reason for this late generation of MDA is not clear but it may be due to the pattern of flow in the vessels following reperfusion of an ischaemic limb, whereby a low reflow state occurs after an ischaemic insult, the duration of which depends on the length of the ischaemia. 2° This variable period of low flow may cause a delay in reperfusion injury in an ischaemic limb subjected to transient ischaemia. Although Persson et al. 8 did not show a rise in thiobarbituric acid reactive material in reperfused ischaemic muscle, they demonstrated that the concentration of this substance was increased with iron stimulation, suggesting a greater susceptibility to free radical induced lipid peroxidation following reperfusion. Further, subcutaneous tissue is rich in polyunsaturated fatty acids and is likely to be particularly vulnerable to lipid peroxidation, a factor which may also have contributed to the observed rise in MDA in our study. Eur J Vasc Surg Vol 7, September 1993
Even though there was no change in MDA levels in the control varicose vein group of patients, baseline MDA concentrations were higher. The control group was selected to assess the non-specific effects of surgery, anaesthesia and cannulation of the femoral vein on serial MDA levels. Variables which might affect baseline MDA levels, such as smoking status and triglyceride concentrations, were not assessed, and therefore we do not believe that any pathophysiological significance should be ascribed to differences in baseline levels between the two groups. Although the rise in MDA might be deemed a consequence of cell damage during ischaemia, the rise reflecting a "wash out" phenomenon following reperfusion, we felt that this was not so for the following reasons. Firstly, ischaemia was not complete and the period of relative hypoperfusion was short; secondly, the time lapse of 40 rain from ischaemia to the peak rise in MDA must be regarded as rather prolonged; thirdly, this phenomenon would not explain the observed fall in tocopherol levels; and finally, we did not detect any elevation in the level of creatine kinase, an accepted indicator of muscle cell death. Those patients who developed lower limb swelling greater than 10% had a significantly higher peak elevation in MDA concentration. As the fall in vitamin E concentration was only evident in these patients it might be inferred that the degree of lipid peroxidation is more severe in those who subsequently developed significant lower limb oedema, thus supporting the view that oxygen-derived free radicals play a significant role in causing lower limb oedema following femoro-popliteal bypass grafting. Consequently, it might be possible to reduce lower limb oedema in this situation by prior treatment of patients with appropriate antioxidants, and we are currently investigating this prophylactic option.
Acknowledgement This study has been supported by a Royal Victoria Hospital Belfast Research Fellowship with additional support from the British Heart Foundation, Chest, Heart and Stroke Association (NI) and Department of Health and Social Services in Northern Ireland. We are grateful to these bodies and all the staff in the Vascular Surgery Unit and Theatre for their contribution.
References 1 EICKHOFF Jtt, ENGELL HC. Local regulation of blood flow and the occurrence of edema after reconstruction of the lower limbs. Ann Surg 1982; 195: 474-478.
Lipid Peroxidation Following Femoro-popliteal Bypass
2 PORTER TM, LINDELL TD, LAKIN PC. Leg edema following femoropopliteal autogenous vein bypass. Arch Surg 1972; 105: 883-888. 3 PERSSONNH, TAKOLANDERR, BERGQVISTD. Lower limb oedema after arterial reconstructive surgery. Acta Chir Scand 1989; 155: 259-266. 4 ABu-RAHMAN AF, WOODRUFF BA, LUCENTEFC. Edema after femoropopliteal bypass surgery: lymphatic and venous theories of causation. J Vasc Surg 1990; 11: 461-467. 5 PATMANRD. Compartmental syndromes in peripheral vascular surgery. Clin Orthop 1975; 113: 103-110. 6 STRANDENE. Transcapillary fluid filtration in patients with leg oedema following arterial reconstruction for lower limb atherosclerosis. VASA 1983; 12: 219-224. 7 HUSNI EA. The edema of arterial reconstruction. Circulation Suppl 1967; 35-35: I169-I173. 8 PERSSONNH, BERGQVISTD, FEx G, MARKLUNDSL, NILSSON B, TAKOLANDERR. Lipid peroxidation and activity of anti-oxidant enzymes in muscle of the lower leg before and after arterial reconstruction. Eur J Vasc Surg 1989; 3: 399-403. 9 PERRYMO, FANTIN! n. Ischaemia: profile of an enemy. J Vasc Surg 1987; 6: 231-234. 10 KORTHIUSRJ, GRANGERDN, TOWNSLEYMI, TAYLORAE. The role of oxygen-derived free radicals in ischaemia-induced increases in canine skeletal muscle vascular permeability. Circ Res 1985; 57: 599-609. 11 HALLIWELLB, GUTTERIDGEJMC. Lipid peroxidation: a radical chain reaction. In: Free Radicals in Biology and Medicine. New York: Oxford Press 1988: 188-276.
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12 STRANDEN E. A comparison between surface measurements and
13 14 15
16 17 18 19 20
water displacement volumetry for the quantification of leg oedema. J Oslo City Hosp 1981; 31: 153-155. YOUNG IS, TRIMBLE ER. Measurement of malondialdehyde in plasma by high performance liquid chromatography with fluorimetric detection. Ann Clin Biochern 1991; 28: 504-508. CATIGNANIGL, B~ER~JG. Simultaneous determination of retinol and alpha-tocopherol in serum or plasma by liquid chromatography. Clin Chem 1983; 29: 708-712. THURNHAMDI, DAVIESJA, CRUMPBJ, SITUNAYAKERD, DAWSM. The use of different lipids to express serum tocopherol: lipid ratios for the measurement of vitamin E status. Ann Clin Biochem 1986; 23: 514-520. OREDSSONS, PLATEG, QVARTORDTP. Allopurinol--a free radical scavenger--reduces reperfusion injury in skeletal muscle. Eur ] Vasc Surg 1991; 5: 47-52. WALKERPM, LINDSAYTF, LABBER, MICKLEDA, ROMASCHINAD. Salvage of skeletal muscle with free radical scavengers. J Vasc Surg 1987; 5: 68-75, NEGLEN P, JABS CM, EKLOF B. Plasma metabolic disturbances and reperfusion injury following partial limb ischaemia in Man. Eur ] Vasc Surg 1989; 3: 165-172. McCoRD JM. Oxygen-derived free radicals in postischaemic tissue injury. N Engl J Med 1985; 312: 159-163. HARDYSC, HOMER-VANNIASINKAMS, GOUGHMJ. The triphasic pattern of skeletal muscle blood flow in reperfusion injury: an experimental model with implications for surgery on the acutely ischaemic lower limb. Eur J Vasc Surg 1990; 4: 587-590.
Accepted 25 March 1993
Eur J Vasc Surg Vol 7, September 1993
Lipid Peroxidation as a Cause of Lower Limb Swelling Following Femoro-popliteal Bypass Grafting C. V. Soong 1, I.S. Young 2, P. H. B. Blair 1, J. M. Hood ~, B. J. Rowlands 3, E. R. Trimble 2 and A. A. B. Barros D'Sa I ~Vascular Surgery Unit, 2Department of Clinical Biochemistry and 3Department of Surgery, Queen's University of Belfast Royal Victoria Hospital, Belfast, Northern Ireland, U.K. We examined the role of free radical induced lipid peroxidation in lower limb swelling in patients following femoro-popliteal bypass grafting. In 20 patients undergoing this operation blood samples were taken from the femoral vein via a cannula before the femoral artery clamp was applied, just prior to and immediately after clamp release and at 10 min intervals thereafter for I h for measurements of malondialdehyde (MDA) and vitamin E. The concentration of M D A was significantly elevated at 40 min after reperfusion (mean + S.E.M., 573 + 83 pmol/ml) compared to just before clamp release (359 ± 41 pmol/ml; p < 0.01). This was associated with a corresponding fall in the concentration of vitamin E at the time of peak MDA rise (5.68 +_ 0.28 to 5.29 + 0.28 p~mol/mM cholesterol, p ~ 0.05) suggesting its utilisation as an antioxidant. The degree of oedema was related to the changes in MDA and vitamin E. Thus, in the 15 patients with greater than 10% increase in lower limb volume the rise in the concentration of M D A was 364 +_ 44 to 693 + 76pmol/mI (p = 0.0001) while that in the five, whose swelling was less than 10%, was 344 + 40 to 559 + 243 pmol/ml (p = 0.25). A significant fall in vitamin E was found only in the group with greater than 10% lower limb oedema (5.90 + 0.33 to 5.40 + 0.34~mol/mM cholesterol, p < 0.01), in comparison to those with less than 10% swelling (5.01 + 0.35 to 5.04 + 0.50~mol/m~ cholesterol). 'lhese results suggest that Jree radzcal reduced peroxidation occurs following femoro-popliteal bypass grafting and that there is more free radical damage in those who subsequently develop significant lower limb oedema. Oxygen-derived free radicals may play a significant role in causing lower limb oedema in this situation. Key Words: Malondialdehyde; Vitamin E; Femoro-popliteal bypass grafting.
Introduction Lower limb swelling is a common problem following femoro-popliteal bypass grafting, occurring in up to 100% in some series. 1-4 The oedema can be significant with an average increase of 30% of initial limb volume. 3 This lower limb oedema can delay re-mobilisation and compromise healing not only in patients with associated ischaemic ulcers but also in those who have undergone distal amputations, and the requirement to use a compression stocking in the elderly may demand additional supervision. All these factors contribute towards prolonged hospital stay. Rarely, the oedema may be severe enough to cause compartmental syndrome, further compromising blood flow to an already ischaemic limb by impairing the collateral circulation, s Many theories have been proposed for the causation of lower limb swelling but the exact aetiology 0950-821X/93/070540+06$08.00/0© 1993Grune & Stratton Ltd.
remains controversial. These hypotheses include an increase in capillary filtration and disruption of the lymphatics at the time of dissection. 1-4"6"7 It is agreed, however, that deep venous thrombosis is not a frequent contributor. Recently, it has been suggested that oxygen-derived free radicals m a y play a role in swelling of the lower limb in this situation, s It has been shown that free radicals are generated following reperfusion of ischaemic muscle and may cause cellular damage giving rise to changes in the transmembrane potential difference, and increases in skeletal muscle vascular permeability. 9"10 Oxygen-derived free radicals are cytotoxic species which can cause damage to cells via processes such as lipid peroxidation, the end products of which include malondialdehyde (MDA). This chain reaction is limited in vivo by free radical scavengers such as vitamin E, the major lipid phas e antioxidant, 11 which is incorporated into the lipid membrane of cells. Free
Lipid Peroxidation Following Femoro-popliteal Bypass
radical damage to cellular membranes leads to an increase in cell permeability which in turn can give rise to oedema. Unfortunately, direct measurement of oxygenderived free radicals using' electron spin resonance is difficult and impractical in biological systems and many indirect methods are therefore used. These techniques involve either the measurements of byproducts of free radical induced tissue damage, like MDA, or the assessment of the consumption of endogenous antioxidants. Although lower limb swelling is likely to be multifactorial in origin, oxygen-derived free radicals generated during reperfusion may have a major role to play. Therefore the aim of this study is to investigate whether free radical-induced lipid peroxidation occurs following femoro-popliteal bypass grafting and if the degree of lipid peroxidation can be correlated to the severity of lower limb oedema.
541
ings were taken after the patient had been recumbent for 30 min. The volume of the lower leg was calculated using the formula of a truncated cone; V = h x 3.142 x (C2 + c2 + Cc)/3. Where: V = volume; C = bulkiest portion of calf; c = narrowest portion of ankle; and h = distance between C and c. This technique of assessing volume change has been shown to be as accurate as the water displacement technique. 3'12 Swelling was taken as the increase in volume in the operated leg minus the change in the contralateral leg. This takes into account the volume change that may result from central causes such as, fluid overload and heart failure. This swelling is expressed as a percentage of the initial limb volume and an increase of 10% or more was taken as significant.
Malondialdehyde and vitamin E measurements
Materials and Methods Patient selection Patients undergoing femoro-popliteal bypass grafting for relief of intermittent claudication were recruited into the study after informed written consent was obtained. Patients were excluded if they had: (1) preexisting gross lower limb oedema; (2) previous lower limb arterial surgery or (3) undergone previous above or below knee amputation on the contralateral leg. After routine endotracheal anaesthesia, conventional longitudinal groin and medial popliteal incisions were employed. A Gortex graft was used in all the patients except one, in whom a reversed vein graft was used. The proximal anastomosis was to the common femoral artery in all patients while the distal anastomosis was to the popliteal artery above knee level in all cases but one. Six patients undergoing surgery for varicose veins were recruited as controls.
Lower limb measurements Daily circumference measurements were made of the lower leg at two points, namely at the bulkiest and narrowest parts, just before operation and continued until discharge from hospital or until an elastic compression stocking was applied. The two points chosen were marked carefully using a permanent ink marker to aid subsequent measurements. These read-
Blood samples were taken from the femoral vein via a cannula before the application of the femoral artery clamp, just prior to and immediately after clamp release and at 10 min intervals thereafter for I h. The blood samples were collected in EDTA bottles and the plasma isolated by centrifugation at 2000r/min for 10 min within 4 h of collection. The plasma was then stored at -70°C until analysed. The samples were analysed for MDA using high performance liquid chromatography with fluorimetric detection as described by Young and Trimble. 13 Vitamin E concentrations were measured at five key time points: at the time of peak MDA rise, 10 min on either side of this maximum MDA rise and just prior to and immediately after clamp release. Vitamin E measurements were made using a high performance liquid chromatograhy (HPLC) technique with concentrations corrected for plasma concentrations of cholesterol. 14"15 Creatine kinase was measured on a Beckman CX5 centrifugal analyser. In the six control patients femoral venous blood was taken in the same manner as that in femoropopliteal bypass patients. Blood was taken at the start of the operation, towards the end of the operation, about 5min after this and then at 10min intervals thereafter for l h . These samples were assayed for MDA.
Statistics Measurements of MDA and creatine kinase were Eur J Vasc Surg Vol 7, September 1993
C.V. Soong et al.
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a s s e s s e d u s i n g analysis of variance (ANOVA). To c o m p a r e paired variables, such as c o m p a r i s o n bet w e e n the p e a k a n d baseline concentrations of M D A a n d c h a n g e s in v i t a m i n E concentrations, the Wilcoxon Signed Rank test w a s used. All results are e x p r e s s e d as the m e a n + S.E.M. This s t u d y w a s a p p r o v e d b y the Research Ethical C o m m i t t e e of the Q u e e n ' s U n i v e r s i t y of Belfast.
700 600 ~
*p < 0-001
,T,
500
E 400
< 300 X 200
n = 20
100
Results T w e n t y patients w e r e recruited into the study. Of these, 12 w e r e males a n d the a v e r a g e age w a s 60 + 5 years. The m e a n a n k l e - b r a c h i a l systolic p r e s s u r e index w a s 0.60 + 0.03. The increase in l o w e r limb volume post-operatively ranged between 0-30% and eight patients required t r e a t m e n t w i t h a c o m p r e s s i o n stocking. In 15 patients l o w e r limb swelling w a s greater t h a n 10%. T h e r e w a s n o significant difference in the age, sex distribution, p r e - o p e r a t i v e a n k l e brachial systolic p r e s s u r e index a n d intra-operative f e m o r a l artery c l a m p time b e t w e e n those w h o s e swelling w a s greater t h a n 10% c o m p a r e d to those w h o s e swelling w a s less t h a n 10% (Table 1).
I
I
v
PC
BR
800
(a)
I
T0
I
I
I
I
I
I
10
20 30 40 50 60 Time (min) Fig. 1. Malondialdehyde concentrations (mean _+ S.E.M.) in patients undergoing femoro-popliteal bypass grafting. PC, preclamp; BR, before clamp release. Arrow indicates time of clamp release.
*
n = 20
*p < 0.001 60o
~ 400 <~ 200
T a b l e 1. Characteristics of patients w i t h and w i t h o u t significant l o w e r l i m b s w e l l i n g ( m e a n + S.E.M.)
Significant swelling (>10%)
No significant swelling (<1o°/,,)
Age (years)
64.7 +_ 1.3
64.3 + 3.3
Sex (re:f)
9:6
3:2
~
5.8
Ankle-brachial index
0.62 ± 0.03
0.61 ± 0.03
~
5.6
Clamp time (min)
48 + 2.5
54 + 2.0
~
5.4
6.2
(b)
n = 20 *p < 0.05
6
5.2 The p r e - c l a m p concentration of M D A w a s o b s e r v e d to be 407 + 40 p m o l / m l . Following c l a m p release the concentration of M D A rose gradually, p e a k i n g b e t w e e n 30 a n d 5 0 m i n in the majority of patients. This elevation in M D A r e a c h e d a significant level at 40 m i n of r e p e r f u s i o n (573 + 83 pmol/ml) in c o m p a r i s o n to the concentrations at m a x i m u m ischaemia, i.e. before c l a m p release (359 + 41 p m o l / ml; p < 0.01, A N O V A ) (Fig. 1). This w a s associated w i t h a c o r r e s p o n d i n g significant fall in v i t a m i n E concentrations at the time of p e a k M D A rise (5.68 + 0.28 to 5.29 + 0.28 p~mol/mM cholesterol; p < 0.05 Wilcoxon Signed Rank) (Fig. 2). Eur J Vasc Surg Vol 7, September 1993
~
5
+ 10 min 10 min Peak Time Fig. 2. Correlation between mean concentrations (+_ S.E.M,) of (a) MDA and (b) vitamin E at the five time points. BR, time before clamp release. Peak, time of peak MDA rise in individual patients. BR
The p e a k M D A rise, w a s significant in those w h o s u b s e q u e n t l y d e v e l o p e d a greater t h a n 10% swelling (364 + 44 to 693 + 76 pmol/ml; p = 0.0001) while t h o s e w i t h less t h a n 10% swelling did not h a v e a statistically significant increase (344 + 40 to 559 + 243 p m o l / ml; p = 0.25) (Fig. 3). The fall in v i t a m i n E w a s f o u n d
Lipid Peroxidation Following Femoro-popliteal Bypass 1000
6O
n = 15
800
543
n=5
*
T
*p < 0.001
60O 45
< 400
n
200
4o~85
I I I Significant swelling No significant swelling Fig. 3. MDA concentrations (_+ S.mM.) before clamp release (@) and at peak rise (O) in patients with and without significant leg swelling. 0
n=15
30
I PC
I BR
I 0
_1 10
t I I I t 20 30 40 50 60 Time (min) Fig. 6. Concentrations (mean + S.E.M.) of creatine kinase before, during and after ischaemia. PC, preclamp; BR, before clamp release.
n=5
6.2 o
~6
*p < 0.01 5.7
N.S.
Discussion
T h e a e t i o l o g y of l o w e r limb o e d e m a f o l l o w i n g femoro-popliteal bypass grafting remains unresolved although many theories have been proposed. Some h a v e s u g g e s t e d t h a t the s w e l l i n g is a result of > 4.7 i Significant swelling No significant swelling n c r e a s e d capillary filtration, 6' 7 w h i l e o t h e r s felt t h a t Fig. 4. Changes in vitamin E concentrations (mean + S.E.M.) in d i s r u p t i o n of the l y m p h a t i c s d u r i n g d i s s e c t i o n of the patients with and without significant lower limb swelling ([] = g r o i n a n d p o p l i t e a l fossa is responsible.l"2 A l t h o u g h before clamp release, [] = peak MDA time). s o m e i n v e s t i g a t o r s w e r e able to r e d u c e the i n c i d e n c e a n d s e v e r i t y of the o e d e m a b y p e r f o r m i n g l y m p h a t i c 800 s p a r i n g incisions, t h e y w e r e u n a b l e to p r e v e n t this s w e l l i n g c o m p l e t e l y . 2"4 M o r e r e c e n t l y it w a s p r o 600 p o s e d that o x y g e n - d e r i v e d free radicals g e n e r a t e d d u r i n g r e p e r f u s i o n of the i s c h a e m i c limb m a y h a v e a © role to play. 8 Free radicals h a v e b e e n s h o w n to c a u s e 400 v a r i o u s d e g r e e s of d a m a g e to cells s u b j e c t e d to < i s c h a e m i a - r e p e r f u s i o n injury; this d a m a g e varies p = 0.88 (ANOVA) 2o0 f r o m a c h a n g e in cellular t r a n s m e m b r a n e p o t e n t i a l to cell necrosis. 9' 10,16,17 T h e p r i m a r y s o u r c e of this free radical g e n e r a t i o n 0 I i l I I I I I I is n o t e n t i r e l y clear b u t it h a s b e e n s u g g e s t e d b y s o m e 80 GO 0 10 20 30 40 50 60 Time (min) t h a t it m a y be p r o d u c e d via the x a n t h i n e o x i d a s e system.lO,16,18,19 X a n t h i n e o x i d a s e occurs n a t u r a l l y , Fig. 5. Mean MDA concentrations (+ S.E.M.) in control patients undergoing surgery for varicose veins. SO, start of operation; EO, m a i n l y as t h e d e h y d r o g e n a s e in v a r i o u s tissues, s u c h end of operation. as t h e intestine, brain, liver, heart, skeletal m u s c l e a n d e n d o t h e l i a l cells. T h e d e h y d r o g e n a s e f o r m catao n l y in t h o s e w i t h g r e a t e r t h a n 10% s w e l l i n g (5.90 + l y s e s the reaction: 0.33 to 5.40 + 0.34 txmol/mM cholesterol; p < 0.01) in c o m p a r i s o n to t h o s e w h o s e s w e l l i n g w a s less t h a n x a n t h i n e + H 2 0 + NAD+---~uric acid + N A D H + H + 10% (5.01 ± 0.35 to 5.04 ± 0.50 ~xmol/mM cholesterol) D u r i n g i s c h a e m i a it is c o n v e r t e d to t h e o x i d a s e f o r m (Fig. 4). A rise in M D A c o n c e n t r a t i o n w a s n o t b y a p r o t e a s e a n d utilises o x y g e n to g e n e r a t e s u p e r o b s e r v e d in the c o n t r o l g r o u p (Fig. 5). W e did n o t oxide: d e m o n s t r a t e a n y e l e v a t i o n in the creatine k i n a s e conc e n t r a t i o n w i t h i n o u r s a m p l i n g t i m e in the b y p a s s h y p o x a n t h i n e + H 2 0 + 202---~uric acid + 2 0 2 " + g r o u p of p a t i e n t s (Fig. 6). 2H + 5.2
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C.V. Soong et aL
Neglen et al. demonstrated an abundance of plasma hypoxanthine, a breakdown product of adenosine monophosphate, during ischaemia in the limb. 18 Therefore, with the accumulation of hypoxanthine and xanthine oxidase during ischaemia, favourable conditions are created for the generation of oxygenderived free radicals when reperfusion occurs. 19 A reduction in the degree of cell damage brought about either by the administration of catalase, superoxide dismutase or allopurinol, or by controlling the delivery of oxygen during reperfusion of ischaemic tissues is further corroborative evidence of the role played by free radicals in ischaemia-reperfusion injury. 16"17 As an indicator of free radical-induced lipid peroxidation we have serially measured MDA by an HPLC technique with fluorimetric detection. 13 The use of this technique minimises interference by creatinine, urea, glucose and bilirubin which otherwise reduces the specificity of MDA measurements. Elevation of MDA together with the fall in vitamin E, the major lipid-phase chain breaking anti-oxidant, following reperfusion in patients undergoing femoropopliteal bypass grafting in this study provide strong evidence of increased free radical production. As expected, the lowest vitamin E concentrations were observed at the time of maximum lipid peroxidation. In the presence of increased free radical production, vitamin E levels are expected to fall as tocopherol is converted to the tocopheryl radical. The subsequent rise in vitamin E levels may be attributed to recycling of the stable tocopheryl radical by aqueous phase anti-oxidants. The late increase in MDA concentration, occurring at 40 min following reperfusion, may account for the failure by others to detect its occurrence at an earlier time. 8 The reason for this late generation of MDA is not clear but it may be due to the pattern of flow in the vessels following reperfusion of an ischaemic limb, whereby a low reflow state occurs after an ischaemic insult, the duration of which depends on the length of the ischaemia. 2° This variable period of low flow may cause a delay in reperfusion injury in an ischaemic limb subjected to transient ischaemia. Although Persson et al. 8 did not show a rise in thiobarbituric acid reactive material in reperfused ischaemic muscle, they demonstrated that the concentration of this substance was increased with iron stimulation, suggesting a greater susceptibility to free radical induced lipid peroxidation following reperfusion. Further, subcutaneous tissue is rich in polyunsaturated fatty acids and is likely to be particularly vulnerable to lipid peroxidation, a factor which may also have contributed to the observed rise in MDA in our study. Eur J Vasc Surg Vol 7, September 1993
Even though there was no change in MDA levels in the control varicose vein group of patients, baseline MDA concentrations were higher. The control group was selected to assess the non-specific effects of surgery, anaesthesia and cannulation of the femoral vein on serial MDA levels. Variables which might affect baseline MDA levels, such as smoking status and triglyceride concentrations, were not assessed, and therefore we do not believe that any pathophysiological significance should be ascribed to differences in baseline levels between the two groups. Although the rise in MDA might be deemed a consequence of cell damage during ischaemia, the rise reflecting a "wash out" phenomenon following reperfusion, we felt that this was not so for the following reasons. Firstly, ischaemia was not complete and the period of relative hypoperfusion was short; secondly, the time lapse of 40 rain from ischaemia to the peak rise in MDA must be regarded as rather prolonged; thirdly, this phenomenon would not explain the observed fall in tocopherol levels; and finally, we did not detect any elevation in the level of creatine kinase, an accepted indicator of muscle cell death. Those patients who developed lower limb swelling greater than 10% had a significantly higher peak elevation in MDA concentration. As the fall in vitamin E concentration was only evident in these patients it might be inferred that the degree of lipid peroxidation is more severe in those who subsequently developed significant lower limb oedema, thus supporting the view that oxygen-derived free radicals play a significant role in causing lower limb oedema following femoro-popliteal bypass grafting. Consequently, it might be possible to reduce lower limb oedema in this situation by prior treatment of patients with appropriate antioxidants, and we are currently investigating this prophylactic option.
Acknowledgement This study has been supported by a Royal Victoria Hospital Belfast Research Fellowship with additional support from the British Heart Foundation, Chest, Heart and Stroke Association (NI) and Department of Health and Social Services in Northern Ireland. We are grateful to these bodies and all the staff in the Vascular Surgery Unit and Theatre for their contribution.
References 1 EICKHOFF Jtt, ENGELL HC. Local regulation of blood flow and the occurrence of edema after reconstruction of the lower limbs. Ann Surg 1982; 195: 474-478.
Lipid Peroxidation Following Femoro-popliteal Bypass
2 PORTER TM, LINDELL TD, LAKIN PC. Leg edema following femoropopliteal autogenous vein bypass. Arch Surg 1972; 105: 883-888. 3 PERSSONNH, TAKOLANDERR, BERGQVISTD. Lower limb oedema after arterial reconstructive surgery. Acta Chir Scand 1989; 155: 259-266. 4 ABu-RAHMAN AF, WOODRUFF BA, LUCENTEFC. Edema after femoropopliteal bypass surgery: lymphatic and venous theories of causation. J Vasc Surg 1990; 11: 461-467. 5 PATMANRD. Compartmental syndromes in peripheral vascular surgery. Clin Orthop 1975; 113: 103-110. 6 STRANDENE. Transcapillary fluid filtration in patients with leg oedema following arterial reconstruction for lower limb atherosclerosis. VASA 1983; 12: 219-224. 7 HUSNI EA. The edema of arterial reconstruction. Circulation Suppl 1967; 35-35: I169-I173. 8 PERSSONNH, BERGQVISTD, FEx G, MARKLUNDSL, NILSSON B, TAKOLANDERR. Lipid peroxidation and activity of anti-oxidant enzymes in muscle of the lower leg before and after arterial reconstruction. Eur J Vasc Surg 1989; 3: 399-403. 9 PERRYMO, FANTIN! n. Ischaemia: profile of an enemy. J Vasc Surg 1987; 6: 231-234. 10 KORTHIUSRJ, GRANGERDN, TOWNSLEYMI, TAYLORAE. The role of oxygen-derived free radicals in ischaemia-induced increases in canine skeletal muscle vascular permeability. Circ Res 1985; 57: 599-609. 11 HALLIWELLB, GUTTERIDGEJMC. Lipid peroxidation: a radical chain reaction. In: Free Radicals in Biology and Medicine. New York: Oxford Press 1988: 188-276.
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12 STRANDEN E. A comparison between surface measurements and
13 14 15
16 17 18 19 20
water displacement volumetry for the quantification of leg oedema. J Oslo City Hosp 1981; 31: 153-155. YOUNG IS, TRIMBLE ER. Measurement of malondialdehyde in plasma by high performance liquid chromatography with fluorimetric detection. Ann Clin Biochern 1991; 28: 504-508. CATIGNANIGL, B~ER~JG. Simultaneous determination of retinol and alpha-tocopherol in serum or plasma by liquid chromatography. Clin Chem 1983; 29: 708-712. THURNHAMDI, DAVIESJA, CRUMPBJ, SITUNAYAKERD, DAWSM. The use of different lipids to express serum tocopherol: lipid ratios for the measurement of vitamin E status. Ann Clin Biochem 1986; 23: 514-520. OREDSSONS, PLATEG, QVARTORDTP. Allopurinol--a free radical scavenger--reduces reperfusion injury in skeletal muscle. Eur ] Vasc Surg 1991; 5: 47-52. WALKERPM, LINDSAYTF, LABBER, MICKLEDA, ROMASCHINAD. Salvage of skeletal muscle with free radical scavengers. J Vasc Surg 1987; 5: 68-75, NEGLEN P, JABS CM, EKLOF B. Plasma metabolic disturbances and reperfusion injury following partial limb ischaemia in Man. Eur ] Vasc Surg 1989; 3: 165-172. McCoRD JM. Oxygen-derived free radicals in postischaemic tissue injury. N Engl J Med 1985; 312: 159-163. HARDYSC, HOMER-VANNIASINKAMS, GOUGHMJ. The triphasic pattern of skeletal muscle blood flow in reperfusion injury: an experimental model with implications for surgery on the acutely ischaemic lower limb. Eur J Vasc Surg 1990; 4: 587-590.
Accepted 25 March 1993
Eur J Vasc Surg Vol 7, September 1993