Chem.-Biol~ Interactions, 76 {1990) 141 - 161 Elsevier Scientific Publishers Ireland Ltd.

141

Review Article

OXYGEN R A D I C A L S IN I N T E S T I N A L I S C H E M I A A N D REPERFUSION

M.H. SCHOENBERG and H.G. BEGER*

Dept. of General Surgery, University of Ulm, Ulm fF.R.G.} {Received April 30th, 1990) {Revision received June 25th, 1990) {Accepted June 29th, 1990)

SUMMARY

Intestinal ischemia, however, caused, is still a serious and growing clinical problem with an unacceptable mortality rate of over 60%. This high mortality rate is mainly due to the fact that the patients are not admitted to the hospital or not treated early enough. Even if the patients are operated on within 24 h, their mortality rate is still over 500/0, and those surviving the initial treatment suffer from postischemic complications. These damages have been accounted until now to tissue ischemia. It has been proven experimentally that also reperfusion or revascularization after time-limited ischemia add to the tissue damages observed, due to the formation of 02radicals. Thereby the prequisites for the production of these radicals {the conversion of xanthine dehydrogenase to xanthine oxidase and the increase of hypoxanthine concentrations in the tissue and plasma) are generated during tissue ischemia. These radicals damage directly or initiate several vicious circles leading to mucosal lesions, impaired intestinal function and an enhanced absorption of bacteria and endotoxin. Various substances (SOD, catalase, DMSO, allopurinol, deferoxamine etc.) detoxify oxygen radicals or inhibit the pathomechanisms leading to the enhanced radical generation. Hopefully, the combination of early revascularization with these already available scavengers will improve the high mortality and morbidity of patients suffering from intestinal ischemia.

Key words: Intestinal ischemia -- Reperfusion -- Oxygen radicals

*Address for correspondence: Prof. H.G. Beger, Dept: of General Surgery, University Ulm, 7900 Ulm, F.R.G. 0009-2797/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

142 I. INTESTINAL ISCHEMIA - CLINICALRELEVANCEAND PROGNOSIS Intestinal ischemia used to be rare, its frequency, however, has increased in the last decade [1]. There are basically two types of intestinal ischemia. First, intestinal ischemia may derive from an acute occlusion of the mesenteric art4ries or veins. The etiology of acute mesenteric vessel occlusion is in most cases an embolic occlusion of the mesenteric arteries. The source of emboli are most often atrial fibrillation and/or rheumatic heart disease [2]. An increasing number of patients suffer from an acute thrombosis of mesentery vessels [3]. Venous thrombosis occurs seldom and is often secondary to sepsis, the use of birth control pills, thrombocytosis and neoplasia [4]. The second type of intestinal ischemia is called "non-occlusive intestinal ischemia" and is defined as intestinal ischemia in the presence of patent mesenteric arteries and veins [5]. The cause of non-occlusive intestinal ischemia is unknown. It is believed that a decrease in cardiac output, due to cardiogenic failure as well as hemorrhagic hypotension, leads to a reduced intestinal perfusion pressure and the intestinal mucosa becomes ischemia [6]. The prognosis of acute intestinal ischemia, regardless of its etiology, is still poor. The overall mortality rate ranges from 50 to 70% and has improved only little over the last three decades despite the possibilities of updated intensive care medicine. The survival of the patient depends firstly on the cause of intestinal ischemia and the extent of the infarction and secondly on the time elapsing from onset of the first symptoms to proper treatment [7]. In cases of embolic occlusion of the mesenteric arteries, rapid surgical treatment has the greatest impact on the long-term survival of the patient. If the patient is operated within 12 h after the onset of the symptoms, embolectomy alone leads to a survival rate of over 80%. Within 24 h embolectomy has to be combined with resection of the ischemic bowel in order to achieve similar low mortality rates of less than 20% [8]. Only 30% of all patients, however, are operated within this time interval. If the patient is treated after 24 h, the mortality rate increased to 65%, regardless which operation procedure was chosen [3]. Patients suffering from thrombosis of the mesenteric arteries have the worst prognosis. Only 5--17% of all patients survive this type of bowel infarction [3,9]. This high mortality rate is mainly due to a delayed diagnosis, since this group of patients has a more insidious onset of symptoms than patients suffering from embolic occlusions [3]. Patients with a mesenteric venous thrombosis have the lowest mortality rate ranging from 36.4 to 50% [4,10]. Khoudadi et al. [11] reported a 100% survival of this patient group undergoing resection, postoperative heparinization and a mandatory second-look operation. The outcome of the non-occlusive intestinal ischemia is also invariably fatal. There are two publications reporting survival rates of 0% and 29%, respectively [4,6]. Two main reasons are responsible for this high mortality rate of acute

143 intestinal ischemia. Firstly, most patients are old (70-80 years) and suffer from various other diseases such as atrial fibrillation, congestive heart failure and/or generalized occlusive arteriosclerotic disease [2,3,12]. Often acute mesenteric ischemia is only a symptom or the consequence of these diseases. Moreover, in cases of non-occlusive intestinal ischemia, the patients are or were hypotensive, due to cardiac insufficiency or hemorrhage. Secondly, it is believed that cardiodepressant substances are formed in the ischemic gut and are washed-out mto the systemic blood circulation after successful treatment and revascularisation of the intestine [13]. These cardiodepressant factors are supposed to act negatively inotropic and impair the cardiac function. This could initiate a vicious circle leading to irreversible cardiac failure and shock. The hypothesis of cardiodepressant substances released from the ischemic gut is only based on experimental observations. In spite of almost two decades of extensive research neither origin nor structure of the so called "myocardial depressant factor" has been revealed [14,15]. Moreover, cardiodepressant substances have never been observed in patients, although most patients who die after treatment suffer mostly from irreversible cardiac insufficiency and multiorgan failure [8]. II. INTESTINALDAMAGESBY ISCHEMIA Independently from its cause (i.e. acute mesenteric infarction, thrombosis or non-occlusive intestinal ischemia) reduction or stop of the nutritive blood flow to the intestine, if not compensated for by collateral circulation, leads after a certain period of time to an irreversible ischemic damage of the gut. Also, short and small decreases of intestinal blood flow may initiate ischemic tissue lesions. Thereby the overall intestinal circulation is sufficiently maintained, the tops of the villi, however, are already severely hypoxic. The susceptability of the villous tips to limited reduction of the blood flow is due to an extravascular short-circuiting of oxygen at the base of the villi [16]. Consequently the ischemic damage of the intestine starts at the villous layer of the mucosa [5,17]. Damages at the mucosal layer allow an enhanced uptake of proteolytic enzymes, bacteria and endotoxins from the intestinal lumen into the blood circulation [18] and adversely affect the respiratory and cardiac function of the patients. Thereby, together with the release of cardiodepressant substances, a vicious circle is created, in which the impaired heart function leads to a progressive deterioration of the intestinal blood circulation. This is turn results in a further reduction of flow also to the other layers of the intestine followed by total bowel infarction (see Fig. 1). In line with this concept, Haglund et al. [14] could show experimentally that the extent of mucosal lesions after 2 h of ischemia, correlated well with the decrease in mean arterial pressure and the impairment of the cardiac function seen after reperfusion (Fig. 2). Sinee morphologically identical alterations of the mucosal layer have been observed in patients suffering from intestinal ischemia [19], the described pathomechanisms seem to be also of clinical relevance [20].

144

Hypotensive states: Haemorrhage Heart infarction ?

1 Arterial blood I pressure reduction Decrease of villous blood flow velocity

l I Short-circuiting of 1 oxygen in the villi I Villous hypoxia '1 and damage J

_l Releaseof cardiotoxic -I material

I Intestinal gangrene ~ ' ~ " Fig. 1. Cause and pathophysiological effects of intestinal blood flow reduction {from Haglund et al. [13]).

BLOOD PRESSURE FALL, mm Hg

80-

t

604020O-

0-1 n=10

2-3 n=10

4-5 n= 9

GRADE

OF

MUCOSAL LESIONS

Fig. 2. Correlation between the grade of mucosal damage and the fall in systemic arterial blood pressure following regional intestinal shock (from Haglund et al. [14]).

145 III.INTESTINAL D A M A G E S

DURING REPERFUSION

A growing number of patients, suffering from intestinal ischemia, are treated within 12 h after onset of the first symptoms. In this group of patients (about 30%), the intestine can be revascularized successfully without resection. Retrospective analysis of patients treated accordingly reveal that cardiopulmonary as well as intestinal problems arise also after revascularisation of the ischemic gut [7]. The revascularized intestine often may require several weeks to recover [7]. This phase is usually characterized by diarrhea and malabsorbation. Frequently mucosal ulcerations and intraluminal bleeding are observed which seldom are life threatening, but require blood transfusions [3,7]. In a later phase these mucosal lesions result in intestinal strictures and mechanical obstruction of the bowel, which in some cases have to be treated by surgery [7]. In the last 8 years, it could be shown in various organs, including the small intestine, that after temporary ischemia, tissue edema and morphological damages as well as an impaired organ function do not only develop during the hypoxic period but aggravate after reoxygenation. Granger et al. could show that temporary intestinal ischemia and reperfusion lead to an increased interstitial edema, resulting in net fluid movement into the bowel lumen [21]. The reason for this enhanced tissue edema which they observed after a short period of intestinal ischemia (reducing the local inflow pressure to 30 mmHg by stenosis of the superior mesenteric artery for 1 h) and reperfusion, was an increased vascular permeability of the intestine. If the duration of intestinal ischemia is longer than one hour, the mucosa of the small intestine is severely damaged, leading to hemorrhagic ulcerations of the intestinal mucosa. It was shown by Schoenberg et al., that after 2 h of intestinal ischemia, these characteristic mucosal lesions develop mainly after reperfusion [22]. Moreover, the absorptive function of the intestine, as measured by net transmucosal water flux, is significantly reduced during intestinal ischemia of 3 h. After reperfusion and parallel to the aggravation of the mucosal lesions, the intestine does not resume its ability to absorb water, leading to a net water loss into the lumen of the gut [23]. This water shift is due to a fluid loss from the interstitial space over the damaged mucosal layer into the intestinal lumen [24]. It was believed that tissue hypoxia played the key role in the pathogenesis of mucosal lesions and the impaired intestinal function seen after reperfusion. This view was stronly supported by the observation, that either introduction of gaseous oxygen into the small bowel lumen or perfusion of the gut lumen with oxygenated saline reduced or even prevented the development of lesions and increased the survival rate of the experimental animals [25,26]. Other endogenous substances such as histamine, prostaglandins, lysosomal enzymes, and endotoxin have been implicated in the pathogenesis of the observed increase in permeability and mucosal damages after ischemia and reperfusion [27--30]. In fact, these substances are released or washed

146 out from the ischemic intestine and enhance the permeability of the intestinal vasculature, when infused intraarterially into the normal gut [28,3032]. Pretreatment neither with indomethacin or antihistamine nor instillation of albumin or activated charcoal into the intestinal lumen attenuated, however, the mucosal damages [25,26,33]. IV. INVOLVEMENTOF OXYGEN RADICALS IN THE POSTHYPOTENSIVE ALTERATIONS OF THE INTESTINE In contrast to the result mentioned above, treatment with superoxide dismutase (SOD), a superoxide radical scavenging enzyme, normally present intracellularly, decreased the enhanced vascular permeability normally observed after 1 h stenosis of the superior mesenteric artery [33]. Furthermore, it was shown in a non-occlusive intestinal ischemia model, that treatment with SOD (15 000 units/kg body wt. after ligation of the renal artery), given during ischemia and shortly before reperfusion, significantly prevented the further aggravation of mucosal lesions normally observed after reperfusion [22]. Similarly, SOD prevented net water loss into the bowel lumen, due to an improved water absorption capacity after reperfusion [23]. Since SOD is regarded to be a highly specific enzyme, scavenging O~ [34], the aforementioned observation implicates the participation of oxygen radicals in the reperfusion injury of the gut. Apparently these oxygen radicals are produced in the tissue during the early reoxygenation or reperfusion phase of the ischemic gut and add significantly to the lesions observed. This notion was supported recently by Nilsson and coworkers. They reduced in cats the intestinal blood flow to 10°/o of the normal blood flow (less than 5 ml min -1 • 100 g-i) and could show, by using a modified electron spin resonance (ESR) technique, that free radicals are generated in the first minutes of reperfusion [35]. All results mentioned above, however, were obtained from experiments in which the intestine of cats was subjected to a moderate or severe level of partial ischemia. In these experiments the superior mesenteric artery was stenosized but not completely occluded over a time period of 1 - 3 h, respectively. At reperfusion the ischemic injury exacerbated substantially, this could be prevented by SOD and other scavengers [23,33]. If the mesenteric vessels were completely occluded, however, the mucosal permeability of the gut in dogs was significantly increased and the villi appeared severely damaged already after 1 h of intestinal ischemia [36,37]. Similarly, Haglind et al. observed after 1 h strangulation of the rats' intestine severe damages of the villous layer [38]. Amano et al. could show that after 4 h of strangulation the mucosa became necrotic, after 8--12 h a transmural infarction and subsequent bowel wall disintegration developed. In these experimental settings of severe intestinal ischemia induced by complete occlusion of the mesentery vessels no obvious aggravation of the tissue damage was observed after reperfusion. Moreover, SOD or other free radical scavengers had no effect on the increase in permeability or tissue damage [39]. Apparently complete vascular occlusion leads to a rapid development of •

147 intestinal injury without any significant reperfusion component. These results support the conventional concept of progressive tissue damage due to hypoxia and the lack of other nutrients. On the other hand, Nilsson et al. could show in the same model as mentioned above that reduction of intestinal blood flow only to about 25% of the resting level (8--15 ml • min -1 • 100 g-~) for 2 h followed by 30 min of reperfusion also induced intestinal damages. In these experiments, however, no radicals could be detected [35]. There exists apparently a "severity window" in which the formation of free radicals add significantly to the damages observed. Intestinal damage may occur without an enhanced generation of free radicals but radicals were never observed in the absence of intestinal lesions. Since damages due to ischemia and reperfusion are probably caused by several pathophysiological pathways, the contribution of the different components to the lesions vary according to the severity and time duration of the pathological condition. Damages of the mucosa layer seen after partial ischemia and reperfusion have probably little clinical relevance regarding late stages of mesenterial infarction. These lesion, however, might be of importance in non-occlusive intestinal ischemia and in early forms of bowel infarction with sustained collateral circulation [40]. A. Generation of oxygen radicals There are various pathomechanisms which induce an enhanced production of oxygen radicals and their derivatives. After hypoxia and reperfusion of the gut the hypoxanthine-xanthine oxidase system seems to be an important and probably the initial source of free radical production [41]. Purine metabolism during intestinal ischemia~ The production of ATP stops during hypoxia, but the use of ATP continues. Consequently, energyrich phosphates are degraded from ATP to AMP further to adenosine. This latter substance rapidly diffuses extracellularly, where it is further degraded over inosine to hypoxanthine [42]. Hypoxanthine is not further degraded to uric acid and accumulates in the ischemic tissue. In an experimental model of non-occlusive intestinal ischemia in cats it was shown, that 2 h of ischemia {intestinal arterial pressure decrease to 2 5 - 3 0 mmHg) reduce the ATP concentrations to about 40% of the preischemic value. The decrease in ATP is associated with an increase of AMP (7.6-fold) and hypoxanthine (10-fold) in the intestinal tissue [41]. Moreover, the relationship between the duration of ischemia and the decrease of ATP in the intestinal tissue has been determined for the rat. Using 31p nuclear magnetic resonance spectroscopy, Blum et al. could show a complete depletion of ATP within 20 min of complete ischemia. Further periods of ischemia did not lead to a further decline in ATP concentrations [43]. This observation is consistent with reports that only 30 min of ischemia are needed to induce irreversible damages to the rat intestine [44]. Xanthine oxidase: activity, localisation and conversion. In most species the intestine is the richest source of xanthine dehydrogenase and/or oxidase in the body [45,46]. In order t~ localize the enzyme activity in the tissue, the

148 intestine of several animal species has been examined by histochemical and immunohistochemical methods. Pickett et al. observed the highest enzyme concentration in the villous layer of small intestine [47]. Thereby the xanthine oxidase activity exhibits an increasing gradient from the villus base to the tip. This would explain the observation that the villous tips are more susceptible to the postischemic damage than the base [23]. Using immunohistochemical methods Jarascb et al. saw only in the capillary endothelial cells of the lamina propria and lamina muscularis mucosae significant concentrations of xanthine oxidase [48]. The reason for these differences is still unclear and might be due to the different methods used. In the normal healthy cell, predominantly xanthine dehydrogenase (Dform) is found. Xanthine dehydrogenase catalyses the metabolism of hypoxanthine to uric acid. Thereby NAD ÷ is the electronacceptor [49]. During tissue ischemia~ however, Roy et al. could show that in various organs including the small intestine xantbine debydrogenase (XDH) is converted into xanthine oxidase (XO). In the intestine, the rate of conversion from XDH to XO was extremely rapid. Within only 1 rain of intestinal ischemia XDH was completely converted to XO, whereas other organs examined (i.e., heart, kidney, spleen, liver) bad to be iscbemic for over 1 hour [50]. Parks et al., however, could not reproduce the aforementioned results. They observed in a similar study a slower conversion rate, that is after 2 b and 3 h of intestinal ischemia only 46% and 61% of the enzyme was present in its oxidaseform [51]. Nevertheless, already after a short period of intestinal ischemia a substantial part of xanthine dehydrogenase is converted to xanthine oxidase (see Fig. 3). The pathomechanisms governing the D-to-O conversion during tissue ischemia, however, are still poorly understood and mainly based on circumstantial evidence. The two most likely candidates initiating the conversion are limited proteolysis and oxidation by sulbydryl groups [50]. It was shown

Ischernia

ATP ADP AMP 4 IMP Adenosine Xanthine dehydrogenase Inosine 1' ~1, proteinase ~activatEi Ca~+intracellular t

!

I Hypoxanthine

Xanthine oxidase

Fig. 3. The development of the purine metabolism and the conversion of xanthine dehydrogenase into its oxidase form during tissue ischemia.

149 "in vitro" but not "in vivo" that both limited proteolysis and sulfhydryl oxidation are capable of converting the enzyme from D-form into the O-form. Roy and McCord suggested, that the "in vivo" enzyme conversion during intestinal ischemia is induced by proteolysis, which in turn is triggered by the influx of calcium [50]. It is well established that during hypoxia of the cell, the energy consuming calcium pump does not function, consequently leading to an increase of calcium concentration in the cells [52]. This influx of calcium is supposed to activate the calmodulin-regulated intracellular proteases, which in turn trigger the D-to-0 conversion. The aforementioned hypothesis of Roy and McCord was based on the observation that inhibition of the intracellular proteases with a serine protease inhibitor (STI -- soybean trypsin inhibitor; dosage: pretreatment with 15 mg/kg body weight intraperitoneally) prevented the conversion of XDH into XO. Concomitantly, this treatment reduced the enhanced vascular permeability of the intestine after short-term ischemia [50]. STI, however, is a protein and therefore unlikely to enter the cytosol where apparently the conversion takes place. Therefore this simple hypothesis is not very convincing. Roy and McCord proposed the following explanation: Serine protease inhibitors (such as STI) can block cell receptors, which normally trigger directly or indirectly biochemical mechanisms intracellularly. Possibly these protease inhibitors could therefore prevent indirectly the initiation of metabolic events, that would lead to the activation of intracellular proteases [50]. It was shown, however, that also other protease inhibitors such as aprotinin protect the intestine against ischemic and postischemic tissue injury. Bounous et al. suggested that the pancreatic~proteases in the intestinal lumen induce the mucosal lesions seen after ischemia. They could demonstrate that the instillation of aprotinin into the intestinal lumen attenuated the mucosal lesions significantly [18,53]. Also i.v. application improved the tissue damages normally observed after time-limited strangulation of the gut [54]. Moreoever, it was suggested that proteases inhibitors prevent the damaging effects of numerous proteases released by activated PMN-leukocytes which accumulated after intestinal ischemia and reperfusion [55]. Furthermore, Hallet et al. could show in an "in vitro" study, that aprotinin could inhibit the production of oxygen radicals by stimulated PMN-leukocytes [56]. Apparently the beneficial effects of protease inhibitors can be explained in many ways and the true mechanisms of action remain unclear. In order to evaluate the significance of the calcium influx for the D~to-O conversion in the intestine, Roy and McCord pretreated rats with Stelazine, a calcium-calmodulin inhibitor, and could show that the conversion still occurred during ischemia, the rate of conversion, however, was significantly reduced [50]. In contrast to the STI-treatment, Stelazine was not able to attenuate the postischemic permeability changes in the intestine [50,57]. This could be due to the fact that the calcium-calmodulin inhibitor merely reduces, but does not completely inhibit the conversion of the enzyme.

150

Reperfusion

ATP ADP AMP IMP

Adenosine

Xanthine

Inosine

dehydrogenase O~ H,O, OH"

t $

!

Xanthine

Hypoxanthine

oxidase .,,,~#

#gll I1~-

uric acid

,iiI~

O2 Fig. 4. Generation of oxygen free radicals after reoxygenation.

Apparently more studies are needed to fully understand and substantiate the hypothesis of the calcium triggered and proteolysis induced D-to-O conversion in intestinal ischemia. After reperfusion, that is after reoxygenation, hypoxanthine which has accumulated during the ischemic period in the intestinal tissue is degraded to uric acid [41]. As described above, this reaction is catalysed mainly by xanthine oxidase. Thereby the electrons are not transferred to NAD ÷ but to molecular oxygen, both single or in pairs, thus reducing oxygen to superoxide radical and to hydrogen peroxide (see Fig. 4.) [58]. These reactions lead consequently to an enhanced generation of cytotoxic oxygen radicals which overwhelm the protective intracellular mechanisms. Inhibition of xanthine oxidase. The assumption that XO is a major and possibly the initial source of free radical production in the ischemic and reperfused intestine is based largely on the observation, that competitive inhibition by allopurinol is as effective as SOD in preventing the increase of vascular permeability and the aggravation of mucosa damages seen after reperfusion [23,41,59--61]. Moreover, inactivation of the enzyme by folic acid and pterin aldehyde also reduces the increase in vascular permeability seen after reperfusion of the intestine although the specificity of both substances is rather low [62,63]. Another approach assessing the role of XO after ischemia and reperfusion is to feed rats a molybdenum-deficient, tungsten supplemented diet. This treatment leads by an incorporation of tungsten rather than molybdenum into XO, to a reduction of the mucosal XO activity by 75% [64]. This diet attenuates the increase in microvascular permeability seen after ischemia and reperfusion. The competitive inhibition of XO by allopurinol, however, does not only decrease the generation of oxygen radicals and their derivatives but also

151 preserves the nucleotide pool. The preservation of energy rich phosphates is frequently invoked as an alternate explanation for the effects of this treatment [65]. Allopurinol does attenuate the reduction of tissue-ATP normally seen during ischemia, however, the maintenance of energy rich phosphates by continuous infusion of inosine has no protective effect against reperfusion injury in cat intestine [41]. Moreover, Morris et al. could show that the administration of allopurinol shortly before reperfusion was equally effective in preventing the reperfusion damage as compared to the same treatment starting before ischemia. It seems therefore most unlikely that purine salvage plays an important role preventing the development of postischemic lesions [66]. Furthermore, it has been suggested that allopurinol is a reasonably potent OH L radical scavenger [67,68] and the results of Morris et al. do not exclude this possibility for explanation. In "in vitro" studies it was shown that the allopurinol~xypurinol concentration must exceed 500 ~M to effectively scavenge oxygen radicals. The regimen of allopurinol treatment, used in the experiments described above, however, leads to an allopurinol and/or oxypurinol concentration of about 20 ~M in the extracellular compartment and of 4.15 _+ 0.98 nmol/mg protein in the intestinal tissue [41,69]. These concentrations, which effectively inhibit XO, are probably too low to scavenge significantly hydroxyl radicals. It is therefore reasonable to assume that the protection afforded by allopurinol is due to the competitive inhibition of XO which prevents an enhanced production of oxygen radicals. Other sources of free radical generation after tissue ischemia and reperfusion seem also possible (i.e. damage to mitochondria and subsequently disruption of the respiratory chain, catecholamine oxidation}, however, have not been evaluated yet.

B. Chemistry and cytotoxicity of oxygen radicals Although the results obtained after SOD treatment strongly support the notion, that oxygen free radicals participate in the post-ischemic damages of the gut, they do not indicate whether 02 per se or its derivatives such as H202 or the hydroxyl radical {OH'} are responsible for the enhanced permeability, aggravation of the tissue lesions and impairment of the intestinal function after ischemia and reperfusion. The univalent reduction of 02 leads to the formation of superoxide anion radical 02. The superoxide radical is rather unstable and spontaneously dismutates to produce H202 and 02. Both oxygen species are either a weak or slow oxidant and are apparently not the predominant oxygen metabolite inducing the cell and tissue damage observed [70]. Parks et al. could show that not only the treatment with SOD but also with catalase, an enzyme reducing H20 e to oxygen and water attenuated the tissue permeability and mueosal damages, normally observed after reperfusion [59,71]. Moreover, similar results were obtained with dimethyl sulfoxide (DMSO), and OH" scavenger [61,71]. The protection provided by SOD, catalase and DMSO strongly suggests that the oxygen radical inducing tissue damage is the l~ghly cyto-

152 toxic hydroxyl radical, which is generated by 02 and HeO2, according to the following reaction: , 20H + 2OH" + 202

20~ + 2H202

This so called Haber-Weiss reaction, however, is far too slow to be of physiological significance [72,73]. It therefore takes place only in the presence of certain transition metals, metal chelates, or hemoproteins to yield the potent OH'. This step has been termed "Fenton reaction". In biological systems, the transition metal is most likely iron, in the form of iron-containing compounds, such as transferrin, lactoferrin, ferritin, hemoglobin and ADP-Fe 3÷. Thereby 02 reduces the ferric to ferrous iron, which in turn reduces H202 into OH" (see Fig. 5) [24]. In line with this hypothesis, Hernandez et al. could show that pretreatment with deferoxamine, an iron chelator, and apotransferrin, an iron-binding protein, attenuated the increase in vascular permeability normally observed after reperfusion of the ischemic intestine. Iron-loaded deferoxamine and transferrin, however, did not protect against the postischemic damages of the intestine [74]. These observations support the hypothesis of a hydroxyl radical formation by the iron-catalysed Haber-Weiss reaction after reperfusion and argue against an unspecific protective action of these ironbinding compounds. It cannot exclude, however, that the beneficial effects of iron chelator treatment as observed is due to an inhibition of highly reactive ferryl- or perferryl radicals which are generated by the reaction of Fe 2÷ with oxygen, superoxide radical or hydrogen peroxide [75].

Scavengers l 02

Men* o

I soDl'°ntan'~ f

i

,

O

=

~

/wOH. C

RH

\

; o,

"~H=O= RO0" ,1Catalase1RH H,O

ROOH+ R.

Fig. 5. Schematic illustration of the metal-catalysedHaber-Weiss reaction, generating OH. The hydroxyl radical reacts preferentially with lipids, forming other radicals (Rt (from R. Del Maestro: Acta Physiol.Scand. (Suppl.), 492 (1980) 153--168).

153

02 -radicals I lipidperoxidation

chemotactic factor

l

activation of PG-metabolism

accumulation of PMN-leukocytes

membrane desintegration

1

phagocytosis ('respiratory burst')

cel!-death

\

tissue damage 4,

leukocyte sticking plugging of capillaries

1 0 2

-radicals

ischemia

Fig. 6. The direct and indirect effects of oxygen free radicals in the tissue.

C. Effects of free radicals (Fig. 6) The hydroxyl radical is the most reactive and short lived oxy-radical [70,76]. It reacts with all biological substances such as proteins, polysaccharides and nucleic acids. Most readily, however, polyunsaturated fatty acids are attacked especially by the OH-radical. This reaction results in the peroxidation of the lipids (see Fig. 5). [77--79]. Polyunsaturated fatty acids are present in high concentrations in the cellular membrane and most susceptible against free radical attack. As mentioned above, reaction of radicals, especially of OH', with these membrane constituents lead to lipid peroxidation within the membranes followed by desintegration of the cells and ultimately to cell death [78]. Damages to this extent occurring in the endothelial layer of the capillary and venuoles result in an increased permeability with extravasation of plasma and even erythrocytes [80]. In an experimental model of non~)cclusive intestinal ischemia in the cat, it was in fact shown, that shortly after reperfusion the tissue levels of conjugated dienes, an indirect measure for lipid peroxidation was significantly increased. Parallel to the increase in lipid peroxidation products, the mucosal layer became edematous and later on a pronounced epithelial lifting as well as hemorrhagic ulcerations were observed [81]. Besides their direct damaging effects on the tissue, free radicals seem to trigger the accumulation of granulocytes in the tissue involved. Microcirculation studies reveal that PMN-leukocytes adhere to the capillary wall ("leuko-

154 cyte sticking") and in some instances even plug the entire vessel which adds to microcirculatory derangements observed in the reperfusion phase [82]. Besides adhering to the endothelial cells or even plugging the capillary, activated PMN-leukocytes secrete various enzymes (i.e., myeloperoxidase, elastase, neutral and acid proteases), prostaglandin metabolites and oxygen radicals as byproducts of phagocytosis [83]. These various enzymes and metabolites are also able to induce the mucosal lesions observed. Concomitantly recent studies examined the changes of the PMN°leukocyte flux in the cat mucosa during intestinal ischemia and after reperfusion measuring the activity of myeloperoxidase in the tissue. Grisham et al. could show that after reperfusion of the ischemic gut the myeloperoxidase activity in the mucosa increased 18-fold as compared to the control levels. In an attempt to determine the relation between enhanced oxygen radical generation and PMN-leukocyte accumulation, the authors pretreated the cats with SOD and allopurinol [55] and more recently with catalase, deferoxamine and dimethylthiourea, a hydroxyl radical scavenger [84,85]. The pretreatment attenuated the increase of myeloperoxidase in the mucosa suggesting that postischemic accumulation of PMN-leukocytes was almost completely prevented. The pathomechanism governing the interaction between oxygen radicals and granulocyte accumulation is yet unknown. In vitro, Petrone et al. could demonstrate, that superoxide radicals generate or activate in human plasma a "chemotactic factor". If this pretreated plasma was injected intracutaneously, it induced a pronounced leukocyte infiltration around the site of injection [86]. The structure of this chemotactic factor, however, has not been analysed yet. Moreover, these "in vitro" results could not be reproduced "in vivo". Exposing feline plasma to superoxide by various methods, Zimmerman et al. could not demonstrate an enhanced chemotactic activity of the feline extracellular fluid thereafter [85]. The important question arises whether the accumulation of PMN-leukocytes is caused by or induce the postischemic lesions of the intestine. In order to answer this question, Hernandez et al. treated cats either with an antineutrophil serum or with a monoclonal antibody specific for the /~-chain of the CD 18 complex that prevents neutrophil adherence and extravasation. The study could demonstrate that both PMN-leukocyte depletion and prevention of its adherence attenuated significantly the postischemic increase in microvascular permeability normally observed [87]. These findings and the observation that various scavengers prevent the influx of PMN-leukocytes into the tissue, support the hypothesis that a major part of the reperfusion injury is mediated by leukocytes which accumulate in the mucosa in response to the initial generation of oxygen radicals by xanthine oxidase. It remains, however, unclear, if this injury process can be attributed to oxygen radicals, secreted during the "respiratory burst" alone, or to other nonoxidative toxins liberated from the granulocytes after activation. It was shown in vitro that due to the generation of lipid peroxides oxygen

155 free radicals indirectly stimulate the arachidonic acid metabolism and lead to increased concentrations of prostaglandins, thromboxane and leucotrienes which by themselves contribute to the permeability changes and micro- and macrocirculatory derangements [82,88]. In various experimental models concerning intestinal ischemia the development of prostaglandins and thromboxanes was measured. It could be shown that during intestinal ischemia the prostaglandin and thromboxane levels were nearly unchanged [27,89]. After reperfusion of the intestine, however, prostaglandin metabolites (PGI2, PGF2a, 13, 14 dihydro 15-keto PGF2a} increased significantly, indicating a stimulation of the cyclooxygenase cascade [89]. These elevated prostaglandin concentrations possibly contribute to cardiodepression commonly observed after reperfusion. The enhanced PG-metabolism, however, seems to be independent from the generation of lipid peroxides by oxygen radicals. SOD and catalase treatment before reperfusion prevented the increase in conjugated dienes in the intestinal tissue, but bad no influence on the development of the prostaglandin metabolites [90,91].

D. Treatment of oxygen radical tissue damage Since oxygen radicals are highly reactive oxygen species, an indirect approach of evidence had to be used in the past in order to prove the involvement of this 02-radicals in the postischemic mucosal lesions. Therefore the endogenous antioxidant enzymes, such as superoxide dismutase (SOD} and catalase, which are normally present in high concentrations in the cells, were infused intravenously. These enzymes are highly specific substances, detoxifying oxygen free radicals, their beneficial effects after treatment served as an indirect proof for the postulated involvement of free radicals in various pathophysiological situations [34,91,92]. Concomitantly, it also points out a new option for treatment. In order to effectively scavenge 02 or H202 in patients, however, SOD or catalase have to reach a concentration of 10 ~g/ml and therefore should be applied continuously, i.e, as an infusion to the patient, since the circulating half-lives of SOD and catalase are only 6 - 9 rain [93-95]. The chronic administration of these enzymes, however, possibly induces an antibody production against the enzymes injected. It is not clearly understood how SOD and catalase applied intravenously can protect the tissues from oxygen radical induced damages. It was generally assumed that both enzymes are membrane impermeable and therefore cannot reach and scavenge oxygen radicals, produced intracellularly. Recently, however, Dini and Rotilio could show in cultured hepatocytes that SOD, conjugated to colloidal gold, was internalized into the cells possibly by receptor-mediated endocytosis [96]. Moreover several possibilities for the superoxide radical to cross the membrane have been proposed, i.e. diffusion of O~ through anion channels of the membrane [97] or lipid soluble redoxactive factors crossing the cell membrane and autooxidize extracellularly, forming again 02 [98]. Nevertheless, the mechanisms involved are not clear. In order to overcome the problems of continuous intravenous substitution

156 of the enzymes SOD and catalase, either the enzymes were attached to inert macromolecules such as Ficoll, dextran or polyethylene glycol (PEG), or entrapped by liposomes. Conjugation of the antioxidant enzymes to various macromolecules increases the circulating half-lives up to 30--40 h [93]. Furthermore, PEG promotes the cellular fixation and possibly the intracellular penetration of the enzymes coupled to this molecule [94]. Liposomes containing SOD have a half-life in the circulation of 2--5 h and in the tissue over 12 h. These liposomes are taken up by the cells mainly through endocytosis. In fact it was shown in cultured endothelial cells, that treatment with liposomes entrapped SOD increased the activity of the antioxidant enzyme in the cells and its resistance to 02 toxicity [99]. Nevertheless, both modifications of antioxidant enzymes have not been used in an intestinal ischemia model yet. Recently, Marklund et al. isolated extracellular SOD from the lung tissue, where it accounts for about 5% of the whole body activity. This enzyme is a 135 000-Da homotetramer containing 4 Cu and possibly 4 Zn atoms. The physiological role of extracellular SOD is not completely understood, however, it might have great potentials in scavenger therapy [100]. Another possibility to prevent the development of postischemic damages is to block various pathophysiological pathways leading to the generation of oxygen radicals. Most promising is the competitive inhibition of xanthine oxidase by allopurinol. As mentioned above, it prevents effectively the formation of oxygen radicals [23,41]. It is commercially available also as an i.v. injection and is well known as treatment for gout. Another commercially available drug is deferoxamine. Deferoxamine chelates iron and is approved for human use in treatment of acute and chronic iron toxicity. It was shown experimentally that this synthetic iron chelator also inhibits the iron-catalysed Harber-Weiss reaction, thus the formation of OH [74]. Besides the specific antioxidant enzymes and drugs inhibiting the formation of oxygen radicals, there is a fairly large group of substances which detoxify oxygen free radicals and their derivatives in various ways. These substances, such as mannitol, DMSO, vitamin C and E are used in clinical practice for various other indications [101]. Their beneficial effects in treating the postischemic lesions were in some instances already known {i.e. mannitol, DMSO), the mode of actions in this pathophysiological situation was, however, interpreted differently. The specificity of some scavengers has, however, been questioned. For instance, DMSO is not only an effective OH-scavenger but also interferes with PMN-leukocytes migration and the arachidonic acid metabolism [102]. REFERENCES 1 S.J. Boley,R.F. Fenstein, R. Sammartano, L.J. Brandt and S. Sprayregen, New concepts in the managementof emboli of the superior mesenteric artery, Surg. Gynecol.Obstet., 153 (1981) 561--569. 2 R. Andersson, H. P~irrson, B. Isaksson and L. Norgren, Acute intestinal ischemia, Acta. Chir. Scand., 150 (1984) 217-221. 3 L.W. Ottinger, The surgical management of acute occlusion of the superior mesenteric artery, Ann. Surg. 188 (6) (1978)721--731.

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