ETHANOL INGESTION AND INCORPORATION OF 32p INTO PHOSPHOLIPIDS OF PANCREAS IN THE RAT

Transcription

1 GASTROENTEROLOGY Copyright 1969 by The Williams & Wilkins Co. Vol. 56, No.2 Printed in U.S.A. ETHANOL INGESTION AND INCORPORATION OF 32p INTO PHOSPHOLIPIDS OF PANCREAS IN THE RAT H. ORREGo-MA'ITE, M.D., E. NAVIA, CHEM.D., A. FEREs, M.D., AND L. COSTAMAILLERE, M.D. Gastroenterology Unit, J. J. Aguirre Hospital, University of Chile, Santiago, Chile The incorporation of 32p into pancreas phospholipids was studied in the following groups of rats: (a) normal; (b) chronic ethanol ingestion (4.8 g per kg of body weight for 75 days); (c) acute ethanol ingestion (4.8 g per kg of body weight in two doses); (d) chronic glucose administration by intra gastric tube in amounts isocaloric with the amount of ethanol given in group b; (e) chronic saline administration by intragastric tube (1.5 ml daily for 75 days); (f) vagotomized; and (g) sham-operated. The effect of stimulation with tl-methylcholine was investigated (1.25 mg administered subcutaneously) in normal control rats, in rats under chronic ethanol ingestion, and in vagotomized rats. Rats under acute or chronic ethanol ingestion have significantly lower incorporation of 32p into pancreas phospholipids than controls and rats in which ethanol had been replaced by isocaloric amounts of glucose. This difference is abolished in the group with chronic ethanol ingestion by the administration of tl-methylcholine. The same dose of tl-methylcholine has no effect on 32p incorporation in normal rats. Withdrawal of ethanol for 7 days in the group with acute ethanol ingestion returns the labeling of pancreas phospholipids to normal levels; on the other hand, in the group with chronic ethanol ingestion, only after 27 days of ethanol withdrawal was there a significant increase in 32p incorporation, but without reaching normal values. Vagotomy reduces the incorporation of 32p into pancreas phospholipids in the rat. This effect is not shown in sham-operated animals. After tl-methylcholine administration, there is a significant increase in olp incorporation, but the values are still significantly below normal levels. The possibility of ethanol interfering with a cholinergic mechanism, necessary for normal 32p incorporation into pancreas phospholipids, is discussed. It is known that acute and chronic pancreatitis are frequently associated with alcoholism,l but there are no studies on the biochemical effects of ethanol on the pan- Received January 31, Accepted July 24, Address requests for reprints to: Dr. Hector Orrego-Matte, Centro de Gastroenterologia, Hospital J. J. Aguirre, Universidad de Chile, Santos Dumont 999, Santiago, Chile. This research was supported by the Faculty of Medicine, University of Chile, Grant (67-39) The authors are grateful to Dr. Jorge Espinoza for all of his help and criticism. 280 creas. The object of this paper was to study the effects of chronic and acute ethanol ingestion on the incorporation of 32p into pancreatic phospholipids. We chose to study this biochemical parameter in view of the important role that phospholipids play in pancreatic secretion. 2 4 Material and Methods Female rats weighing approximately 150 g were used in all of the experiments. The rats came from a laboratory stock which originated from a Sprague-Dawley strain. The rats were distributed by random allocation in the different experimental groups.

2 February 1969 PHOSPHOLIPIDS OF PANCREAS IN RAT 281 In all of the rats tested, 100 p,c of sodium orthophosphate-32p (N. V. Philips-Duphar, Holland) were injected intravenously after a 12-hr fast. Thirty minutes later the rats were killed with ether. This interval was chosen on the basis of experiments which showed that 32p incorporation into pancreatic phospholipids reached plateau values at this time in normal rats. The pancreas was removed along with the spleen and part of the upper small intestine, washed repeatedly in saline to remove the blood and small clots, and carefully dissected. The separation of pancreatic from adipose tissue was easily accomplished because the former does not float in saline. The pancreatic tissue was blotted gently with filter paper and weighed. Four milliliters of saline were added and the cells were destroyed in a high intensity ultrasonic probe (Bronwill Biosonik, Model BP ll). The homogenous fluid obtained was treated with cold trichloroacetic acid with a final concentration of 10%. The precipitate was centrifuged at 2000 g for 20 min and extracted twice by refluxing for 20 min with 8 ml of an ethanol-ether mixture (3: 1) in the centrifuge tube. This mixture extracts total phospholipids from blood and tissues,5 and Penman 6 has shown that it extracts over 95% of choline- 14 C labeled phospholipids. The mixture was then centrifuged at 750 g for 15 min. The supernatant was collected, and the ethanol-ether extracts were evaporated to dryness at 70 C with an air stream. The contents of the tubes were redissolved in 4 ml of petroleum ether and extracted with a solution of Na2HP04 to remove inorganic phosphorus. 7 Aliquots, 0.2 ml, of the petroleum ether supernatant were plated in aluminum planchets, evaporated under infrared light, and counted in a gas-flow counter (Nuclear-Chicago) with a micromil window and using Q gas. Samples were plated in triplicate and each planchet counted three times. Adequate corrections were made for the isotopic decay. From each extract two 1-ml samples were taken to determine the total phosphorus with the method of GomorL 8 The results were expressed as counts per minute per milligram of phosphorus, using the mean plus or minus the standard error. The data were analyzed statistically by paired t test for the difference of the means of paired data. When not paired, the test significance was analyzed as the difference between two group means. In comparing the different groups with the control group, P values of less than 0.05 were considered significant. Histological sections of the liver in the alcohol-treated rats were studied by light microscopy stained with hematoxylin and eosin. No specific stain or chemical measurements for fat were done. Chronic Experiments The study was based on a pair-feeding technique for the animals subjected to chronic experiments. Female rats weighing 130 to 150 g were paired into groups according to body weight and maintained in separate cages at an ambient temperature of 20 to 28 C. The animals were given a stock diet. It provided: proteins, 19.3%; carbohydrates, 57.5%; fat, 3.5%; 312 kcal per 100 g; and an adequate amount of minerals and vitamins. During this 75-day period the average daily intake of ground stock ration remained close to 15 g in both the experimental and pair-fed control groups. Chronic ethanol ingestion. Ethanol was given to 8 rats by. forced feeding through an intragastric polyethylene tube for 75 days. The daily dose was 1.5 ml of 40% ethanol solution per 100 g of body weight (4.8 g per kg). This group was studied paired with two control groups: one which received no ethanol, and another group of 8 rats which received by forced feeding for 75 days 1.5 ml per 100 g of body weight of a glucose solution providing the same amount of calories as the ethanol. At the end of the period of ethanol and glucose administration, the experimental animals and the controls were fasted for 12 hr. 32p was injected and uptake in total pancreatic phospholipids was measured as described. Chronic ethanol ingestion and stimuwtion with p-methylcholine. Eight rats, also paired with 8 control animals, received together with the 32p injection a standard dose of 1.25 mg of p-methylcholine (Urekolin, Silesia). 32p incorporation into pancreatic phospholipids was measured as described. This dose was chosen because it produced obvious signs of parasympathetic stimulation: sialorrhea, lacrimation, and diarrhea. Chronic ethanol ingestion and different periods of abstinence. Nineteen rats subjected to chronic ethanol ingestion as described above, paired with normal controls, were divided in three subgroups. The first, comprising 6 controls and 6 rats with chronic ethanol ingestion, was studied 7 days after withdrawal of ethanol; the second group, 6 rats and their respective controls, were studied 20 days after withdrawal; and the remaining 7 rats with their controls were studied 27 days after ethanol withdrawal. Rats given isotonic saline by forced feeding.

3 282 ORREGO-MATTE ET AL. Vol. 56, No.2 The effect of forced feeding per se on the incorporation of 32p into pancreatic phospholipids was studied in 8 rats paired with 8 control animals. They received 1.5 ml of isotonic saline per 100 g of body weight for 75 days through the same type of polyethylene tube used in the groups given ethanol. Acute Experiments Acute ethanol ingestion. The effect of a single dose of ethanol on 32p incorporation into pancreatic phospholipids was studied. Eleven rats received 1.5 ml of 40% ethanol per 100 g of body weight by forced feeding. On the following day, the same dose was given again, and 1 hr later 32p was injected and measured in pancreatic phospholipids as described. Six rats were studied 7 days after the single dose. This group was compared with 7 rats receiving a solution of glucose, at exactly the same time and providing the same amount of calories and volume as the solution of ethanol. Vagotomized rats. In 21 rats the vagi were severed immediately below the diaphragm. In order to eliminate vagal fibers that might exist in the esophageal wall, the esophagus was sectioned and ligated at both ends. The rats were kept under total fasting for 18 hr and 32p incorporation into pancreatic phospholipids was measured as described. At the end of the fasting period, these rats had lost about 20 g of weight; otherwise they were in good general condition. This group was compared with 7 rats that were laparotomized under ether anesthesia and in which the stomach and esophagus were handled in the same way and for the same time as the vagotomized rats, without sectioning the esophagus. This group of sham-operated rats was submitted to the same period of total fasting of 18 hr as the vagotomized group. The average weight loss of the sham-operated rats was also about 20 g. In the other 6 vagotomized rats, a standard dose of 1.25 mg of,8-methylcholine was injected subcutaneously along with the 32P. Results Initially (first 2 weeks) the rats showed obvious signs of alcoholic intoxication after each administration of ethanol: ataxia, lethargy, and other behavior abnormalities, lasting for at least 1 hr. There was no mortality. The animals were in good general condition. The food intake in the group of rats subjected to continuous ethanol ingestion was the same as that in the paired control rats. If the rate of growth of the 16 rats with chronic ethanol ingestion (8 with chronic ethanol ingestion and 8 that were stimulated with /1-methylcholine 30 min before being killed) is compared with the respective paired controls not given glucose, a statistically higher rate of growth was observed in the rats with ethanol (table 1). However, the group of rats in which the extra amount of calories provided by ethanol was replaced by glucose grew at the same rate as the experimental group given ethanol. There was no difference in the weight of the pancreas between the groups with chronic ingestion of ethanol, glucose, and the controls. Chronic ethanol administration did not change the total phospholipid phosphorous of the pancreas. Histological sections of the liver in the alcoholic group observed by light microscopy showed no alterations, and specifically there was no increase in visible fat. Effects of chronic ethanol ingestion on TABLE 1. Body weight and weight and phospholipid phosphorous of the pancreas in controls and in rats after chronic ethanol ingestion" A, Controls B, Chronic ethanol ingestion C, Chronic glucose ingestion Weight of rats (g) ± 5.10 (16) ± 3.68 (16) ± 4.46 (8) Weight of pancreas (mg) ± (16) ± (16) ± (8) Phospholipid phosphorous (J'g/mI) ± 8.97 (8) ± (8) "Values are means ± SE. Number of rats are given in parentheses. P < 0.05 of A compared with B (rat weight) ; P > 0.50 of B compared with C (rat weight); P > 0.05 of B compared with C (pancreas weight) ; and P > 0.70 of A compared with C (phospholipid phosphorous).

4 February 1969 PHOSPHOLIPIDS OF PANCREAS IN RAT P incorporation into pancreatic phospholipids with and without {3-methylcholine stimulation. The rats with chronic ethanol ingestion had a much lower 32p incorporation into the phospholipids of the pancreas than the control group; the difference was highly significant (P < 0.01) (table 2). This difference in incorporation was abolished if the rats ingesting ethanol were injected with {3- methylcholine at the moment of the 32p administration (table 3). {3-Methylcholine did not stimulate uptake in controls, as can be seen by comparing control values in tables 2 and 3. The group of rats in which ethanol was replaced isocalorically by glucose had a lower 32p incorporation than the one in control animals, but the difference was at the limit of significance ( P > 0.05). On the other hand, the difference with the group with chronic ethanol ingestion was highly significant (P < 0.01). Isotonic saline, given by forced feeding for 75 days, had no effect on 32p incorporation into pancreas phospholipids. Withdrawal of ethanol for 7 and 20 days in rats after chronic ethanol ingestion did not increase significantly the 32p incorporation, but 27 days after withdrawal of ethanol there was a significant increase in incorporation over the rats studied immediately after 75 days of ethanol ingestion, but the incorporation of 32p was still sig:nificantly below the control values (P < 0.05) (table 4). Acute ethanol ingestion. The rats with acute ethanol intake had an incorporation of 32p into pancreatic phospholipids as low as in the animals ingesting ethanol chronically (P > 0.70). Isocaloric replacement of ethanol by glucose had no effect on 32p incorporation in comparison with normal rats. Mter 7 days of ethanol withdrawal, the counts were again at the same level as in the animals with acute glucose administration (P > 0.30) (table 5). Effects of vagotomy on 32p incorporation into pancreas phospholipids. After vagotomy, the incorporation of 32p into pancreas phospholipids was significantly lower than in the group of animals submitted to a sham operation. When vagotomized rats had their pancreases stimulated with {3-methylcholine, the incorporation of 32p increased significantly (P < 0.001) but was still significantly below the values obtained in previous controls (P > 0.001) (table 6). Discussion Our data show that in the rat chronic ingestion of ethanol in doses of 4.8 g of ethanol per kilogram of body weight produces a decrease in 32p incorporation into phospholipids of the pancreas. However, the total phospholipid phosphorus of the pancreas did not change. This effect lasted for more than 27 days after ethanol withdrawal. The progressive increase in 32p TABLE 3. Effect of stimulation with (:J-methylcholine on 32p incorporation into phospholipids of pancreas of normal rats and of rats with chronic ethanol ingestiona No. of A No. of rats Control rats -- counts/min/mg of phosphorus B Chronic ethanol ingestion counts/min/mg of phosphorus 8 47,088 ± 8, ,722 ± 6,455 a Values are means ± SE. TABLE 2. Effect of chronic ethanol ingestion on 32I; incorporation into phospholipids of pancreasa A, Controls B, Chronic ethanol ingestion I C, Chronic glucose ingestion I D, Chronic saline ingestion 42,935 ± 3,840 (8) 47,924 ± 4,593 (8) 14,206 ± 1,578 (8) (P < O.Ol)b counts/min/mg of phosphorus 29,493 ± 1,903 (8) (P > 0.05)b 52,666 ± 8,066 (8) a Values are means ± SE. Number of rats are given in parentheses. P < 0.01 of B compared with C. b Compared with controls.

5 284 ORREGO-MATTE ET AL. Vol. 56, No. 2 TABLE 4. Effects of different periods of abstinence after chronic ethanol ingestion (75 days) on 32p incorporation into phospholipids of pancreasa Control Animals with chronic ethanol pb Days of abstinence after 75 days ingestion ethanol ingestion counts/min/mg of phosphorus counts/min/mg of phosphorus 42,935 ± 3,840 (8) 14,206 ± 1,578 (8) 0 44,926 ± 4,532 (6) 18,516 ± 4,403 (6) > ,927 ± 2,980 (6) 18,092 ± 3,232 (6) > ,417 ± 5,325 (7) 25,556 ± 4,509 (7) < a Values are means ± SE. Number of rats are given in parentheses. b Compared to 75 days of ethanol ingestion but no period of abstinence. TABLE 5. Effect of acute ethanol ingestion on 32p incorporation into phospholipids of pancreasa A, Controls with acute glucose ingestion.. B, Acute ethanol ingestion. C, Acute ethanol ingestion and 7 days abstinence..... No. of rats Counts per min per mg of phosphorus 7 46,899 ± 9, ,818 ± 3, ,474 ± 2,536 a Values are means ± SE. Difference between A and B = P < 0.001; difference between A and C = P > TABLE 6. Effects of vagotomy on 32p incorporation into phospholipids of pancreas, with and without {3-methylcholine (1.25 mg subcutaneously)a No. Without No. With of Ii-methyl- of Ii-methylrats choline rats choline counts/min/mg of phosphorus counts/min/mg of phosphorus A, Sham-oper- 7 48,615 ated ± 12,830 B, Vagoto- 6 6, ,440 mized ± 1,544 ± 2,536 a Values are means ± SE. Difference between A and B = P > 0.001; difference between vagotomized rats and vagotomized rats plus {3-methylcholine = P > incorporation as time after withdrawal elapsed suggests, but does not prove, that the alteration can be reversible. In vagotomized rats, as in the animals with ethanol ingestion, there was a decrease in 32p incorporation in phospholipids. After IJ-methylcholine stimulation vagotomized rats also increased the incorporation of 32p into pancreatic phospholipids; this increase, however, did not reach the values obtained in normal rats. Vagotomy seems to be the important factor interfering with pancreatic metabolism as suggested by the control experiments for surgical stress and fasting for 18 hr. Contrary to the findings of other authors using tissue slices,3 we did not detect an increase in 32p incorporation into pancreas phospholipids in normal rats stimulated with IJ-methylcholine. This could be due to several reasons. (1) The methods were different; we did not attempt to separate the different species of phospholipids and we studied whole pancreas without separation of cell fractions. (2) Hokin and Hokin3 found that the highest increases in incorporation occur in in vitro studies in the pigeon; in vivo, the same authors found a much smaller increase in incorporation of 32p into pancreas phospholipids in mice after stimulation with carbamylcholine. 9 (3) There is also the possibility of a species difference. Our results could be tentatively explained postulating that the normal pancreas of the rat is under a basal cholinergic stimulation, probably through the vagus nerve. This could explain the lack of increase of 32p incorporation into phospholipids after IJ-methylcholine stimulation in normal rats. Ethanol might interfere with this mechanism, and this could explain the normalization of 32PincOrpO_ ration into pancreatic phospholipids after administration of IJ-methylcholine. It is of interest in this respect that Kalant et

6 February 1969 PHOSPHOLIPIDS OF PANCREAS IN RAT 285 ai. IO have shown that ethanol in a concentration compatible with moderate to severe intoxication in vivo in the rat decreases the amount of acetylcholine released from unstimulated cerebral cortex. These authors conclude that die effect of ethanol in vivo may be due in part to an inhibition of acetylcholine release at central cholinergic synapses. Further work with other parameters and with separation of the different phosphojipids and fractioning of pancreatic cells will be needed to establish the real significance of these findings. REFERENCES 1. Sarles, H., J. C. Sarles, R. Camatte, R. Muratore, M. Cain:, C. Guien, J. Pastor, and F. LeRoy Observations on 205 confirmed cases of acute pancreatitis, recurring pancreatitis and chronic pancreatitis. Gut 6: Hokin, M. R., and L. E. Hokin Enzyme secretion and the incorporation of p 32 into phospholipids of pancreas slices. J. Biol. Chern. 203: Hokin, L. E., and M. R. Hokin Studies of pancreatic tissue in vitro. Gastroenterology 36: Hokin, L. E., and M. R. Hokin The ribonucleic acid content of pancreas and parotid glands during enzyme synthesis and secretion in vitro. Biochirn. Biophys. Acta 13: Oser, B. L. led.] Hawk's physiological chemistry, Ed. 14, p Blakiston Division. McGraw-Hill Book Company, Inc., New York. 6. Penman, S Stimulation of the incorporation of choline in poliovirus infected cells. Virology 25: Fries, B. A., H. Schachner, and 1. L. Chaikoff The in vitro formation of phospholipid by brain and nerve with radioactive phosphorus as indicator. J. BioI. Chern. 144: Gomori, G A modification of the colorimetric phosphorus determination for use with the photoelectric colorimeter. J. Lab. Clin. Med. 27: Hokin, M. R., and L. E. Hokin Effects of acetylcholine on phospholipids in the pancreas. J. Biol. Chern. 209: Kalant, H., Y. Israel, and M. A. Mahon The effect of ethanol on acetylcholine synthesis, release, and degradation in brain. Canad. J. Physiol. Pharrnacol. 45: