Eur J Appl Physiol (1986) 55:410--412

European Journal of

Applied Physiology and Occupational Physiology �9 Springer-Verlag 1986

Increase in rat intestinal permeability to endotoxin during hyperthermia

Y. Shapiro 1, M. Alkan 1, Y. Epstein 2, F. Newman 2, and A. Magazanik 2

1 Ben-Gurion University Faculty of Health Sciences and Soroka Medical Center, Beer-Sheva, and 2 Heller Institute of Medical Research, Chaim Sheba Medical Center, 52621 Tel-Hashomer, Israel

Summary. Victims of heat stroke exhibit several clinical features which are also encountered in en- dotoxaemia. In order to investigate these similari- ties hyperthermic rats were used to explore the possibility that high body temperature results in increased permeability of intestinal wall to endo- toxin. 1251 endotoxin was introduced into intesti- nal segments taken from non-heat exposed rats. The segments were then incubated at 37~ or 45 ~ C. Intestinal segments from heat stressed rats were similarly prepared and incubated at 37~ Leakage of endotoxin from segments taken from heat stressed rats was three times greater than from those from non-heat stressed rats, as were the segments from non-heat stressed rats which were incubated at 45 ~ C. These results indicate that the intestinal membrane is damaged by heat and that an increase in outward leakage of micro- bial endotoxins from the gut then occurs. This might contribute to the pathophysiological pic- ture of heatstroke.

Key words: Endotoxin -- Hyperthermia

Introduction

Victims of heatstroke exhibit several clinical and pathological features which are also encounterr in endotoxaemia, these being massive increase in capillary permeability, oedema, gastrointestinal bleeding, general circulatory failure, blood clot- ting disturbances, altered acid base balance, kid- ney failure, shock and a high death rate (Rosen- thal et al. 1971 ; Shibolet et al. 1976; Ollodart et al. 1967; Dietzman et al. 1969; Cuevas et al. 1972;

Offprint requests to: Y. Epstein at the above address

Wolff 1973; Wardle 1974; Magazanik et al. 1980).

Bacterial endotoxin is present in very large amounts in the intestine, but normally only min- ute quantities penetrate into the circulation (Car- idis et al. 1972; Hamer-Hodges et al. 1974; Prytz et al. 1976). It has been suggested that endotoxins are involved in the development of the patholog- ical picture of heatstroke (Shibolet et al. 1976; As- sia et al. 1985).

The appearance of measurable amounts of enteric endotoxin in the peripheral blood could be the cause or the result of increased permeabil- ity of the intestinal epithelium (Walker et al. 1978) with or without a decrease in hepatic detoxifica- tion (Aballi et al. 1978). Increased concentration of endotoxin in the intestinal lymphatics could bypass the liver and directly elevate the levels in the systemic circulation. Hepatic failure directly causes systemic endotoxaemia (Wilkinson et al. 1974) which results in further damage to this or- gan (Nolan 1975). This has been shown to contri- bute to mortality from large burns (Cuevas et al. 1974). The origin of endotoxin in all these cases seems to be from the intestinal flora (Gans et al. 1974).

The present study investigates the possibility that high temperature results in increased permea- bility of the intestinal wall to endotoxin, enabling its passage into the circulation. This may contri- bute further to the laypothesis that endotoxins play a role in the pathological picture of heat- stroke.

Methods

Endotoxin (DIFCO E. coli 055:85) was separated from low molecular weight contaminants by a Sephadex G-200 column.

Y. Shapiro et al.: Increase in rat intestinal permeat~ility to endgtoxin during hyperthermia 411

The void volume, containing the endotoxin was labeled with 1251-NaI (Amersham) according to K_imura (1976) and rechro- matographed on a Sephadex G-50 column within one day of use. A specific activity of 64 mCi �9 r ag - 1 of endotoxin was ob- tained.

Charles River Albino male rats (mean wight 250 g) were divided into three experimental groups: 1. Non-heat exposed rats, intestinal segments incubated at

37~ (9 rats) 2. Non-heat exposed rats, intestinal segments incubated at

45~ (15 rats) 3. Heat exposed rats (exposed to 45~ and 25% relative hu-

midity until body temperature reached 45 ~ C). Intestinal segments of these rats were incubated at 37~ (14 rats). Rats were anaesthetized and then sacrified. A 6 cm seg-

ment of the terminal portion of the ileum was immediately re- moved, washed and flushed with Ringer's solution at 37~ Both ends of the intestinal segments were ligated with silk su- tures. 0.5 ml of the radiolabelled endotoxin solution was intro- duced into the segment lumen: this volume did not inflate the intestine. The segments were incubated in 25 ml of Tyrode's solution (pH 7.4) at the appropriate temperature with air flow through the test solution. Aliquots of 0.2 ml were removed from the solution for radioactivity assessment at different time intervals.

Plasma was obtained under anaerobic conditions from rats of groups 1 and 3, and endotoxin presence was deter- mined by the limulus lysate (LAL) test. The LAL test was chosen because of its specificity and sensitivity in the presence of plasma endotoxin levels above 0.0001 lig- ml-1 (Randolph et al. 1971).

Results were expressed as the increment of counts in the medium over the total counts in the intestinal segment. Statis- tical analysis was performed by analysis of variance (ANOVA).

Resu l t s

Leakage of radioactive material from the intesti- nal loops was approximately linear: more was

25-

- E 2 0 . "o

'-~ 15-

10- tM

5 ~

0 o is zb 4s c~ TIME (rain)

Fig. 1. Endotoxin leakage from isolated rat intestine. 125I-en- dotoxin in incubation medium expressed as percent of initial radioactivity in intestinal lumen ( + s t a n d a r d deviation) over time. Group 1: Control rats, incubation temperature 37 ~ ( �9 Group 2: Control rats incubation temperature 45 ~ (A). Hy- perthermic rats, incubation temperature 37 ~ (@)

found in the medium of specimens exposed to heat than in controls (P<0.001). There was no significant difference between segments obtained from hyperthermic rats and those heated in vitro only (Fig. 1). The limulus lysate test was positive in all hyperthermic rat plasma and negative in all control rats.

D i s c u s s i o n

Increased serum endotoxin levels might account for the common clinical signs and symptoms found in heat stroke and gram negative bacterial infections. Low levels of endotoxin can be found in conditions other than these infections, but high levels have occured in patients in whom endo- toxin load is excessive or in whom the liver fails to remove this substance from the circulation (Waiter et al. 1978). The present study indicates that elevated body temperature in the rat results in a measurable increase in serum endotoxin lev- els. Leakage of radioactive material from isolated bowel segments increased when animals were ex- posed to conditions which caused an increase of body temperature to 45 ~ as well as in speci- mens from control animals which had been heated in vitro to the same temperature. This leads to the assumption that the bowel ceases to be an effective barrier against intestinal endotoxin at these temperatures, irrespective of liver func- tion, intestinal circulation or other pathological changes observed in heat stroke (Shibolet et al. 1976). Identification of endotoxin in a biological assay in the plasma of hyperthermic rats supports the assumption that the leaked radioactive mate- rial is indeed endotoxin. This might suggest that endotoxaemia is one of the contributory factors to the complex syndrome of heat-stroke. This hypo- thesis has been suggested by others (Graber et al. 1971). Furthermore, DuBose et al. (1983) linked such findings to mortality of rats with heatstroke. The combination of temperature induced damage to intestinal cells, circulatory failure and impair- ment of liver function (Shibolet et al. 1976) leads to an increase in serum endotoxin, which contri- butes to the clinical and pathological picture of heatstroke.

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Accepted January 28, 1986