Influence of low dose allopurinol on ischaemia — Reperfusion injury during abdominal aortic surgery

Eur J VascEndovasc Surg (1995)9, 162-169

Influence of Low Dose AIIopurinol on Ischaemia - Reperfusion Injury During Abdominal Aortic Surgery H a r m J. S m e e t s , 1 Jan C a m p s , 2 A . W . M a r c van Milligen de Wit, 1 Job Kievit, ~ J. H a j o van Bockel, ~ Jo H e r m a n s 4 and H o w a r d M. B e r g e r 3

Departments of 1Surgery, 2Diagnostic Radiology and Nuclear Medicine and 3paediatrics, University Hospital Leiden and 4Department of Medical Statistics, State University Leiden, Leiden, The Netherlands. Objectives: To ascertain whether surgery causes ischaemia-reperfusion (I-R) related injury, if this injury is augmented by preoperative shock, and reduced with low dose atlopurinol. Design: Randomised blind placebo controlled trial. Setting: Surgical laboratory. Material and methods: 22 pigs were randomly allocated to four groups; OP = operation~placebo, OA = operation/ allopurinol, SOP = shock + operation~placebo, SOA = shock + operation/allopurinol. An aortic tube prosthesis was inserted in all. In groups SOP and SOA preoperative shock was induced by exsanguination. Allopurinol was adminstered in group OA on the preoperative day and peroperatively, in group SOA during shock and peroperatively. Chief outcome measures: Perioperative blood concentrations of thiobarbituric acid reactive species (TBARS), ascorbic acid (AA), albumin, 99mTc-albumin and creatine phosphokinase (CPK) as indicators of oxidative membrane damage, antioxidant activity, microvascular permeability changes and muscular cell damage respectively. Main results: In the OP and OA groups TBARS gradually increased, while ~ , 99mTc-albumin and CPK remained unchanged and albumin decreased. No effect of allopurinol was observed in these groups. In the SOP group TBARS and AA were not significantly different from groups OP and OA. Yet, albumin, 9 9 m Tc-albumin and CPK decreased significantly more in the SOP group. Compared with the SOP group, allopurinol treatment (SOA) produced lower TBARS and higher AA levels, and reduced the effect of shock on albumin, 99mTc-albumin and CPK concentrations. Conclusion: Aortic surgery causes no I-R related damage. Pre-operative shock produces I-R related damage, which is reduced by allopurinol. Key Words: Aortic surgery; Allopurinol; Ischaemia-reperfusion; Thiobarbituric acid reactive species; Ascorbic acid; Albumin; Microvascular permeability.

Introduction

Surgery for ruptured abdominal aortic aneurysm is associated with a high incidence of postoperative morbidity and mortality.l'2 These complications also occur after elective aortic surgery, but less frequently so. The pre- and peroperative hypovolaemic shock is possibly the main cause for this difference in morbidity and mortality. Hypovolaemic shock followed by resuscitation can b e regarded as a "whole-body" ischaemia-reperfusion (I-R) insult. In addition to this, temporary cross-clamping of the aorta during the operation causes a second I-R episode. Please address all correspondence to: H.J. Smeets, Department of Surgery, BronovoHospital, P.O. Box 96900,2509JH 's-Gravenhage, The Netherlands.

Following I-R oxygen derived free radicals (OFR) cause endothelial and tissue damage by lipid peroxidation of cell membranes. 3'4 In response to this, polymorphonuclear neutrophil granulocytes (PMN) are attracted and activated, adhere to the endothelium and subsequently infiltrate into tissues causing additional damage by OFR formation and release of proteolytic enzymes. 4--6 The xanthine-oxidase inhibitor allopurinol may reduce endothelial and tissue injury after I-R, because it blocks the production of OFR. Several authors have reported a beneficial effect of allopurinol in intestinal, cardiac, lower torso and whole body I-R models. 7-12 However, in these experiments animals were either pretreated with allopurinol for many days before the ischaemia, or extremely high doses were used (50 to 200 m g / k g / d a y ) . In aortic surgery for ruptured aneurysm the relevance of the

1078-5884/95/020162+ 08 $08'00/0 © 1995W. B. Saunders Company Ltd.

AIIopurinol in Aortic Surgery

aforementioned studies is limited, because pretreatment is impossible and because the advised maximum dose in humans is approximately 10 m g / k g / d a y . 13 In the present study we tested the hypothesis (1) that aortic surgery causes I-R related injur3~ (2) that preoperative shock causes additional I-R related injur~ and (3) that this injury can be reduced with a clinically applicable dose of allopurinol. The study was approved by the Leiden University Committee on Animal Experiments.

Material and Methods

Animal model Twenty-two female piglets, with an average bodyweight of 33 kg, were subjected to aortic surgery. On the day prior to the operation a carotid artery catheter and a jugular vein catheter were inserted for blood sampling and circulatory control. Each pig was then randomly allocated to one of four groups; group OP = operatioi~/placebo (n = 6), group OA = operation/allopurinol (n = 5), group SOP = preoperative shock + operation/placebo (n = 6), group SOA= preoperative shock + operation/allopurinol (n = 5). Randomisation for placebo or allopurinol was blind to the investigators. After an overnight fast anaesthesia was induced with azaperon 70 mgi.m, and metomidate 200 mgi.v. Before endotracheal intubation 1.75 mg atropine was given. Anaesthesia was continued with isoflurane 0.5-1% and a mixture of 0 2 / N 2 0 . Additional azaperon or metomidate was administered peroperatively if necessary to keep the animals sedated. The abdominal aorta was exposed using a transperitoneal approach. After systemic heparin (25 mg) the aorta was cross-clamped proximally and distally and an 8 cm Dacron tube bypass (Microvel TM Double Velour graft, Meadox Medicals, Oakland, U.S.A.) with a diameter of 10mm was anas[omosed with the infrarenal aorta by two end-side anastomoses. After restoration of the blood flow, the section of the aorta between the two anastomoses was ligated. During the operation 1500ml of Ringers lactate was infused routinely. If necessary additional Ringers was given to maintain mean arterial blood pressure above 40 mm Hg and central venous pressure above i m m H g . Peroperative blood loss was monitored. Procainebenzylpenicillin and gentamycin were administered prophylactically and warfarin was given to prevent prosthetic thrombosis. Postoperatively pigs received a 1000 ml Ringers lactate infusion at a rate of

163

50ml/hr, until the first postoperative day. They were then regularly fed and had access to water. The occurrence of complications was monitored until the fifth postoperative day. Animals in groups SOP and SOA were subjected to preoperative shock. After sedation a catheter was inserted in the right jugular vein. After systemic heparinisation shock was induced by exsanguination to a mean arterial pressure of 20 mm Hg, at a rate of 30ml/min. The blood was collected in heparinised transfusion bags. The mean arterial pressure of 20 mm Hg was maintained, either by further exsanguination or b y retransfusion of the collected blood, until 20% of the maximally exsanguinated blood was retransfused, according to the model described by Crowell and others. 11' 12 The residual blood was then retransfused at a rate of 50 ml/min. Surgery started 15 minutes after complete retransfusion. Pigs without preoperative shock were given saline (group OP) or 10mg/kgi.v. allopurinol (group OA) on the evening before surgery. In pigs with preoperative shock saline (group SOP) or 10 mg/kgi.v, allopurinol (group SOA) was administered during shock, just prior to retransfusion. All animals received booster doses of allopurinol (2 x 5 m g / k g ) or saline, one before cross-clamping of the aorta and one just prior to recirculation. Allopurinol (Apurin TM) was purchased from Multipharma, Weesp, The Netherlands.

Measurements From carotid artery blood we determined the concentrations of thiobarbituric acid reactive species (TBARS) as indicator of endothelial and tissue cell membrane lipid peroxidation, total ascorbic acid as indicator of plasma antioxidant activit~ granulocytes as indicator of granulocyte endothelial adherence and tissue infiltration, albumin and technetium labelled human serum albumin (99mTc-HSA) as parameters of microvascular damage causing increased permeabilit~ and creatine phosphokinase (CPK) as indicator of muscular cell damage. Blood samples were obtained preoperatively (t = PRE), at aortic cross-clamping (t = CLAMP), 5 minutes after clamp removal (t = CLOFF), skin closure (t = PO), and I and 4 hours postoperatively (t = PO + 1/t = PO + 4). For determination of TBARS and ascorbic acid, blood was placed on ice immediately after sampling and subsequently centrifuged. Next, plasma was stored at -80°C for pooled measurement of TBARS concentrations by spectrofluorometry, 14 and of ascorbic acid concentrations by HPLC. is Albumin and CPK serum concentrations were measured with the SMA-C II autoanalyser Eur J VascEndovasc Surg Vol 9, February 1995

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H.J. Smeets et aL

Table 1. Population characteristics

Number

Operation/ placebo

Operation/ allopurinol

Shock + operation/ placebo

Shock + operation/ allopurinol

6

5

6

5

Operating time (rain)

124 _+ 4

110 + 10

128 + 8

118 + 13

Aorta-clamping (rain)

41 + 4

43 _+ 2

42 _+ 2

44 + 4

Exsanguination max. (ml)

-

-

1075 + 74

1235 _+ 68

Shock duration (min)

-

-

140 + 15

148 + 7

Operative blood loss (ml)

138 _+ 26

139 + 24

242 _+ 62"t

233 + 42"t

Perioperative fluids (ml)

1667 + 167

1800 + 200

3283 + 497"t

3300 +_ 734"t

54 + 7

47 + 5

53 + 4

49 _+ 6

6 _+ 2

4 +2

5 +2

4 + 1

Mean arterial pressure (mmHg)1 Central venous pressure (mmHg)lI

Mean _+ S.E.M.1 at the end of the operation. * significant difference between vs. combined operation groups (Mann-Whitney), t significant effect of preoperative shock (two-way ANOVA), all at p < 0.05.

(Technicon, Tarrytown, U.S.A.). On the p r e o p e r a t i v e d a y 350 MBq 99mTc-HSA was injected in the jugular vein. Samples were obtained f r o m the carotid artery at t = 20, 30, 40, 50, 60, 90, 120, 180, 240, 300 a n d 360 minutes after injection. After centrifugation, radioactivity in p l a s m a w a s d e t e r m i n e d in a universal g a m m a - c o u n t e r (LKB Wallac, Finland). The radioactivity of each s a m p l e w a s corrected for the radioactivity at t = 20 min, p r e s u m i n g an equilibration period of 20 minutes. The p r o c e d u r e w a s r e p e a t e d on the d a y of surgery, w h e n 99mTc-HSA w a s injected at the start of the operation. To identify the influence of aortic surgery, shock a n d allopurinol, the corrected radioactivity of each sample obtained on the d a y of s u r g e r y w a s expressed as percentage of the corrected radioactivity of the c o r r e s p o n d i n g s a m p l e obtained on the p r e o p e r a t i v e day.

Results

Twenty-one of the 22 animals could be completely evaluated. One animal in g r o u p SOA died 3 h o u r s after surgery due to anastomotic leakage. Only peroperative data f r o m this animal were u s e d for evaluation. N o other postoperative complications occurred. N o differences b e t w e e n the four groups w e r e o b s e r v e d as for the duration of operation a n d aortic cross-clamping, postoperative b l o o d pressure, pulse rate a n d central v e n o u s pressure (Table 1). In the animals with p r e o p e r a t i v e shock the m a x i m u m a m o u n t of b l o o d w i t h d r a w n a n d the duration of shock were comparable. Peroperative b l o o d loss w a s greater a n d m o r e fluid h a d to be supplied in the animals with p r e o p e r a t i v e shock c o m p a r e d with those w i t h o u t p r e o p e r a t i v e shock.

Thiobarbituric acid reactive species Statistics Statistical analysis w a s p e r f o r m e d with the Wilcoxon test for c o m p a r i n g changes within a g r o u p a n d with the M a n n - W h i t n e y test for c o m p a r i n g groups. Additionally a t w o - w a y analysis of variance (ANOVA) w a s u s e d to test the effect of allopurinol (vs. placebo), of p r e o p e r a t i v e shock (vs. s u r g e r y only), a n d to test the possible interaction of d r u g (allopurinol) a n d condition (preoperative shock) at each time point in this 2 × 2 designed experiment. Eur J Vasc Endovasc Surg Vol 9, February 1995

Figure 1 s h o w s the t e n d e n c y of the p l a s m a concentrations of the lipid peroxidation p r o d u c t s (TBARS) to g r a d u a l l y increase w i t h time. This increase w a s only significant if groups OP a n d OA were combined, if g r o u p s SOP a n d SOA w e r e combined, or if all g r o u p s were considered together (all groups: m e a n _+ S.E.M. at t = P R E 1.95 + 0.37 vs. at t = P O 3.89 + 0.42, p---0.0004). Although TBARS s e e m e d to increase m o r e in g r o u p SOP c o m p a r e d with the other groups, this w a s not significant. At t = CLOFF TBARS concentration in the SOP g r o u p w a s significantly higher

AIIopurinol in Aortic Surgery

compared with the group SOA. In animals without shock no effect of allopurinol was present. We thus observed an interaction between drug (allopurinol) and condition (shock). Although this interaction was significant only at t = CLOFF, the trend was similar at t = CLAMP and t = PO (Fig. 1).

Ascorbic acid Figure 2 illustrates that ascorbic acid concentrations remained relatively unchanged during the experiment, although there seemed to be an increase in the SOA group (not significant). However, at t = CLOFF the ascorbic acid concentration in the SOA group was significantly higher than in the SOP group. At this point we also observed a significant over-all effect of allopurinol.

Granulocytes The granulocyte count in peripheral blood decreased in all groups, although this decrease was significant only in groups OP, OA and SOP (Table 2). In group SOP the decrease was more severe than in the other groups, as illustrated by a significant difference at t -- PO + 4, and the similar trend observed during the whole experiment (Table 2). Allopurinol was effective in preventing this effect of preoperative shock, but

7-

165

had no effect in groups without shock. The two-way ANOVA did not identify a significant interaction between allopurinol and preoperative shock.

Albumin The serum albumin concentration decreased to the same extent in groups OP and OA. Compared with these groups albumin concentrations were significantly lower in the placebo treated shock group (Table 3). This effect of preoperative shock was present during the whole experiment, and was attenuated by allopurinol (group SOA). The interaction between allopurinol and preoperative shock failed to reach significance (2-way ANOVA). 99mTc_HS A

The per- and postoperative plasma radioactivity of 99mTc-HSA was expressed as percentage of the plasma radioactivity obtained after injection of the isotope on the preoperative day. In groups OP and OA this percentage averaged 100% at all sample times (Table 4). Compared to these animals the percentages in group SOP were significantly lower. This effect lasted from t = 120 rain until the end of the experiment. In group SOA this effect of preoperative shock was reduced. The interaction between allopurinol and preoperative shock was not significant (two-way ANOVA). Test samples obtained peroperatively showed that 98.2% of 99rnTc was bound to albumin.

TBARS (iJmol/I)

-~-OP --El-OA -=~-SOP "EP'SOA

6 5 4 J

z

jJ

J f

Ascorbic acid (pmol/t) 0.6-~-OP HE]-OA "-~"SOP "-E~SOA

0.5-

0.4- I 0.3-

J

0.2 -

---. ~

#

1

0.1 0

# ##

~

PRE

CLAMP

CLOFF

PO

Time

0

~

PRE

Fig. 1. Thiobarbituric acid reactive species. Mean + S.E.M. concentration in plasma (~mol/1). * significant difference vs. PRE value, all groups combined (Wilcoxon, p<0.01), # significant difference between SOP a n d SOA groups (Mann-Whitney, p < 0.02). • * significant interaction between allopurinol a n d pre-operative shock (two-way ANOVA, p<0.05). O P = o p e r a t i o n / p l a c e b o , OA = operation/allopurinol, SOP = shock + operation/placebo, SOA = shock + operation/allopurinol.

CLAMP

E

CLOFF

PO

Time

Fig. 2. Ascorbic acid. M e a n + S.E.M. concentration in plasma (~mol/1). # significant difference between SOP a n d SOA groups (two-way ANOVA, p<0.02). ## significant allopurinol effect (two-way ANOVA, p = 0.01). OP = operation/placebo, OA = operation/allopurinol, SOP = shock + operation/placebo, SOA = shock + operation/allopurinol.

Eur J Vasc Endovasc Surg Vol 9, February 1995

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H,J. Smeets et aL

Table 2. Granulocyte count Operation/ placebo

Operation/ allopurinol

Shock + o p e r a t i o n / placebo

Shock + o p e r a t i o n / allopurinol

10.1 _+ 1.7

10.0 + 1.6

11.1 + 1.0

10.9 + 2.6

CLAMP

7.2 _+ 1.6

7.3 _+ 2.1

3.8 + 0.4*

7.9 + 1.9

CLOFF

7.0 _+ 2.2

6.7 _+ 1.8"

2.8 + 1.0"

6.7 + 1.8

PO

5.1 _+ 1.5"

4.3 + 1.2"

2.7 + 0.8*

5.0 + 0.6

PO+I

3.1 + 1.5"

1.9 + 0.3*

1.5 + 0.3

3.9 _+ 1.5

PO+4

5.0 _+ 1.4"

5.0 _+ 1.6"

1.3 _+ 0.3*t§

6.1 + 3.2§

Time PRE

M e a n concentration (109/1) + S.E.M. * significant difference vs. PRE value (Wilcoxon, p < 0.05), t significant difference vs. c o m b i n e d operation g r o u p s (Mann-Whitne3~ p < 0.05), § significant difference b e t w e e n shock + operation g r o u p s (Mann-Whitne)~ ~ < 0.01).

Table 3. Albumin and creatine phosphokinase Operation/ placebo

Operation/ allopurinol

Shock + o p e r a t i o n / placebo

Shock + o p e r a t i o n / allopurinol

PRE

28.8 -+ 1.1

28.2 -+ 0.9

27.5 +- 0.7

27.2 -+ 1.6

CLAMP

25.7 -+ 0.6*

25.6 -+ 1.1

21.8 + 1.0*f

24.6 -+ 1.3

CLOFF

24.8 _+ 0.6*

24.2 _+ 1.2

19.5 -+ 1.5"t

21.0 _+ 1.8"

PO

24.0 _+ 0.6*

23.2 -+ 1.1"

16.3 -+ 1.5*f

19.4 _+ 2.2*

PO+ 1

23.5 -+ 0.6*

23.4 _+ 0.9*

18.7 _+ 1.2*t

20.4 + 2.6

PO+4

23.3 + 0.7*

23.0 _+ 0.9*

19.3 _+ 1.5*t

20.8 _+ 1.4

PRE

1.8 _+ 0.6

1.6 _+ 0.6

1.3 + 0.2

1.0 _+ 0.2

CLAMP

1.8 _+ 0.5

1.4 + 0.4

1.0 + 0.2

0.9 + 0.1

CLOFF

1.6 + 0.4

1.1 + 0.4

0.9 + 0.2*

0.8 + 0.2

PO

1.6 _+ 0.4

1.4 + 0.3

0.8 + 0.2*

0.7 + 0.1

PO+ 1

1.6 + 0.4

1.6 + 0.4

1.3 + 0.3

1.0 _+ 0.1

PO+4

2.1 + 0.5

1.9 + 0.4

4.8 + 1.3"

3.0 + 0.5

Time A l b u m i n (g/l)

CPK (x 103 U / l )

M e a n s e r u m concentrations _+ S.E.M. * significant difference vs. PRE value (Wilcoxon, p < 0.05), t significant difference vs. c o m b i n e d operation g r o u p s (Mann-Whitne)9 p < 0.05).

Creatine

In groups remained

Phosphokinase

OP and OA serum essentially unchanged

CPK concentration ( T a b l e 3). I n t h e

Eur J Vasc Endovasc Surg Vol 9, February 1995

groups

with preoperative

tendency

to decrease

increase

postoperatively.

were

significant

shock

CPK levels showed

a little peroperativel)~ However,

only in group

SOP.

these

and

a to

changes

AIIopurinol in Aortic Surgery

Discussion

Allopurinol has been proven to protect against I-R related injury in a number of experimental animal models. 7-12 It might therefore be a promising drug in reducing morbidity and mortality in humans during vascular surgery and resuscitation following shock. However, in previous experimental models animals were pretreated with allopurinol during one or more days before the I-R injury,7 or extremely high doses (50-200mg/kg) were used. s-22 Therefore the clinical relevance of these studies is limited, especially regarding haemorrhagic shock and emergency vascular surgery. In the present study we tried to simulate the clinical situation more closel)4 by applying the maximum recommended allopurinol dose for humans ( 1 0 m g / k g ) 9 This dose was administered during ischaemia in the animals with preoperative shock, and on the evening prior to surgery in case of aortic reconstruction without preoperative shock. A peroperative booster dose was given to compensate for allopurinol lost with haemorrhage. The shock model we used proved to be reproducible and to account for individual variance in compensation mechanisms. The surgical procedure yielded highly reproducible aortic cross-clamping time, operating time, haemodynamic values and blood loss. Thus the model proved satisfactory to study if aortic cross-clamping and reconstruction causes I-R related injury, if preoperative shock causes

167

additional I-R related injury and if this injury can be reduced with allopurinol. Aortic reconstruction (operation only) caused an increase of plasma TBARS concentration, suggesting membrane lipid peroxidation. Although TBARS measurement is not entirely specific for measuring membrane lipid peroxidation products, it correlates well with other methods for determining membrane lipid peroxidation, such as diene conjugation and direct malondialdehyde measurement with high pressure liquid chromatography. 26 Aortic reconstruction also caused a decrease in granulocyte count. This was interpreted as granulocyte endothelial adhesion and tissue infiltration, as has been reported to occur following oxidative membrane damage. 6' 27, 28 Paterson et aI. also interpreted the decrease of peripheral leucocyte count following abdominal aortic reconstruction as leucosequestration in lungs, 29 while Klausner et al. proved such leucosequestration in lungs following temporary lower torso ischaemia. 28 Additional support for membrane damage was the postoperative reduction of serum albumin concentration, which was interpreted as albumin redistribution due to increased microvascular permeability following endothelial membrane damage, as proposed by others. 2°-22 In contrast with these findings were the unchanged levels of ascorbic acid and of 99mTc-HSA following aortic reconstruction, which suggested no antioxidant activity and changes of membrane permeability respectively. However, serum concentrations

Table 4. 99mTc-HSA Operation/ placebo

Operation/ allopurinol

Shock + operation/ placebo

Shock + operation/ allopurinol

20

100 + 0

100 + 0

100 + 0

100 _+ 0

30

101 + 3

98 -+ 4

95 + 3

105 _+ 5

40

103 + 1

97 _+ 3

98 + 3

93 -+ 4

50

105 + 2

97 + 5

94 + 4

97 + 3

60

103 + 2

97 + 5

94 + 4

99 + 3

90

97 -+ 3

94 _+ 6

92 + 6

90 _+ 3

120

95 + 5

94 + 5

77 + 7*

93 + 4

180

107 _+ 7

99 -+ 4

78 + 3*

90 _+ 6

240

102 + 6

98 + 5

84 _+ 4*

95 + 5

300

107 + 5

95 + 4

83 + 5*

94 -+ 7

360

104 + 7

97 + 5

82 _+ 5*

93 + 4

T i m e (min)

P l a s m a r a d i o a c t i v i t y e x p r e s s e d as p e r c e n t a g e of r a d i o a c t i v i t y i n c o r r e s p o n d i n g s a m p l e o n p r e - o p e r a t i v e day. R a d i o a c t i v i t y at t = 20 m i n w a s the s t a n d a r d o n b o t h d a y s . M e a n + S.E.M. * s i g n i f i c a n t d i f f e r e n c e v s . c o m b i n e d o p e r a t i o n g r o u p s ( M a n n - W h i t n e y , p < 0.05).

E u r J Vasc E n d o v a s c S u r g Vo]. 9, F e b r u a r y 1995

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H.J. Smeets et al.

of these parameters have to be interpreted with care. A subtle reduction of plasma ascorbic acid concentration may have been obscured by the release of ascorbic acid from blood cells, which contain high quantities of ascorbic acid. 23 It cannot be excluded that some damage of these cells occurred during the experiment. Small changes of membrane permeability may be missed with the use of 99mTc-albumin, because this tracer has a rapid initial disappearance rate from plasma after injection. 24 We conclude that infrarenal aortic cross-clamping and reconstruction causes only limited oxidative membrane damage. No convincing effect of aUopurinol compared with placebo was observed in pigs subjected to aortic reconstruction without preoperative shock. The absence of such an effect suggests that temporary aortic cross-clamping causes insufficient ischaemia in the lower torso to produce OFR related damage following reperfusion. This was confirmed by strong backbleeding during the operation and the absence of postoperative muscular cell injury, as indicated by unchanged CPK levels. We must therefore presume that the oxidative membrane damage due to aortic cross-clamping and reconstruction is caused by OFR generated by different pathways than I-R, e.g. by neutrophil granulocytes activated by tissue trauma. Addition of preoperative shock did not cause a significant increase of TBARS or a decrease of ascorbic acid levels, although TBARS tended to be higher in group SOP at t = CLAMP. Shock-induced increase of TBARS and decrease of ascorbic acid levels may have been obscured by the release of intracellular ascorbic acid, due to mechanical damage of blood cells during exsanguination and retransfusion. Yet, other parameters deteriorated due to the presence of preoperative shock (Group SOP). In this group granulocyte counts, albumin concentrations and 99rnTc-HSA plasma radioactivity were significantly lower and levels of CPK significantly higher compared with groups in which the animals were not subjected to pre-operative shock. Some of these differences were already present during the operation and changed little afterwards, which is an argument in favour of a predominant role of preoperative shock. This is supported by the increased need of fluid supply to maintain adequate blood pressure peroperatively. These data suggest that preoperative shock and resuscitation increased endothelial cell membrane damage, causing redistribution of albumin, 99mTc-HSA and fluid. Although preoperative shock was severe, no postoperative complications occurred, probably due to the relatively short duration of shock. An important finding was that administration of allopurinol attenuated the influence of preoperative shock (Group SOA), suggesting that the shockEur J Vasc Endovasc Surg Vol 9, February 1995

induced augmentation of membrane damage, granulocyte adherence and muscle cell damage is related to I-R. This is confirmed by others reporting a favourable effect of allopurinol in hypovolaemic shock per se. 11" 12

Our study shows that a low dose of allopurinol is sufficient to reduce I-R related damage. This is supported by Megison et al. who found that pretreatment with 5 m g / k g / d a y was as effective as 50mg/ k g / d a y in attenuating intestinal I-R injury, 7 and by Johnson et aI. who recently reported the favourable effect of a single dose of 5 m g / k g allopurinol in coronary bypass surgery.2s We doubled the latter dose to obtain adequate serum levels. The maximum re'commended dose in humans is 800mg/day. 13 The present study also shows that it is not obligatory to administer allopurinol prior to the ischaemia. Some have argued that pretreatment with allopurinol is necessary because oxypurinol, the metabolite of allopurinol, is a more effective xanthine oxidase inhibitor.9, 26 Yet, others have shown significant effectivity of allopurinol administered during or just prior to the ischaemia. 12"27'~'8 Also, the fact that allopurinol and oxypurinol have been reported to be equally effective, suggests that pretreatment is not necessary,a7' 28 These findings are important for the future use of allopurinol in surgical patients admitted with shock, or in patients undergoing emergency vascular surgery. In conclusion, the findings of this study suggest that temporary aortic cross-clamping combined with aortic reconstruction causes limited oxidative membrane damage, which is not related to I-R. Preoperative shock augments the membrane damage. This augmentation is related to I-R and is reduced with allopurinol in a dosage safe for human use, administered during shock. These findings warrant a prospective study in human aortic surgery.

Acknowledgements We thank Toos van Kesteren, Adri6nne Brouwers, Frans Dulfer, the staff of the Laboratory of Surgery of the Leiden University Hospital and Ernst Houdkamp for their technical assistance. This study was supported by a grant from the foundation Stichting Fonds ter Bevordering van de Gezondheidszorg, The Netherlands.

References 1 HOLLIERLH, TAYLORLM, OCHSNERJ. Recommended indications for operative treatment of abdominal aortic aneurysms. J Vasc Surg 1992; 15: 1046-1056. 2 JENKINSAM, RUCKLEYCV, NOLAN B. Ruptured abdominal aortic aneurysm. Br J Surg 1986; 73: 395-398.

AIIopurinol in Aortic Surgery

3 McCoRD JM. Oxygen-derived free radicals in postischemic tissue

4 5

6 7 8 9 10 11 12 13 14 15

16

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Accepted 3 May 1994

Eur J Vasc Endovasc Surg Vol 9, February 1995