INTESTINAL LYMPH FORMATION AND FAT ABSORPTION: STIMULATION BY ACUTE ETHANOL ADMINISTRATION AND INHIBITION BY CHRONIC ETHANOL FEEDING

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1 GASTROENTEROLOGY 68: , 1975 Copyright 1975 by The Williams & Wilkins Co. Vol. 68, No.3 Printed in U.S.A. INTESTINAL LYMPH FORMATION AND FAT ABSORPTION: STIMULATION BY ACUTE ETHANOL ADMINISTRATION AND INHIBITION BY CHRONIC ETHANOL FEEDING ENRIQUE BARAONA, M.D., AND CHARLES S. LIEBER, M.D. Laboratory of the Section of Liver Disease and Nutrition, Veterans Administration Hospital, Bronx, New York, and Department of Medicine, Mount Sinai School of Medicine of the City University of New York, New York, New York The acute administration of ethanol, either in lipid emulsions administered intraduodenally or in liquid diets given by gastric tube, increased the flow of intestinal lymph and the output of proteins and dietary lipids into the lymph, mainly in the 1st hr after administration, During this time, the intraduodenal administration of ethanol (0.75 g per kg of body weight), without exogenous lipids, increased the flow of lymph without changing the lymph lipid output. Stimulation of the lymph flow with neostigmine or by increasing the fluid load also enhanced the output of lymph proteins and the transport of exogenous lipids from the intestinal lumen into the lymph. To study the chronic effects of ethanol, rats were pair-fed liquid diets containing either ethanol (36% of calories) or isocaloric carbohydrate for 3 to 4 weeks. Thereafter, the lymph changes were measured after administration of equal lipid loads with and without ethanol. The administration of an acute ethanol dose to rats chronically fed alcohol moderately increased the lymph flow, but did not change the output of dietary lipids. Furthermore, rats chronically fed alcohol responded to a dietary challenge devoid of ethanol with increases in both lymph flow and dietary lipid output which were not as great as those of pair-fed controls. Thus, acute ethanol administration has a marked stimulatory effect both on the formation of intestinal lymph and on the transport of dietary fat. By contrast, chronic ethanol feeding inhibits these acute effects of ethanol, and, in addition, appears to have moderate inhibitory effect on lipid absorption. Steatorrhea is common in alcoholics with 1 or without 2 liver cirrhosis, but the respective roles of ethanol and associated Received June 18, Accepted September 18, Address requests for reprints to: Dr. E. Baraona, Section of Liver Disease and Nutrition, Veterans Administration Hospital, 130 West Kingsbridge Road, Bronx, New York This investigation was supported by United Stated Public Health Service Grants AM 12511, AA 00224, the Veterans Administration, and Research Scientist Development Award AA The expert assistance of Miss Frances Finkelman is gratefully acknowledged. 495 malnutrition in its pathogenesis are unknown. Acute and chronic administration of ethanol inhibit the intestinal absorption of various water-soluble nutrients. 3-7 Ethanol also affects intestinal lipid metabolism, 8-10 but there is little information concerning direct effects of ethanol on the absorption of dietary fat. This study was undertaken to investigate the effects of both acute and chronic ethanol administration on the transport of dietary lipids into the lymph which is the major route of absorption of dietary fat. 11 Acute and chronic effects of ethanol are usually compared in experimental models

2 496 BARAONA AND LIEBER Vol. 68. No.3 which differ in many respects, including the dose of ethanol and the previous dietary treatment. The model proposed here is designed to minimize these differences in order to distinguish the effects depending on the actual presence of ethanol (acute effects) from those resulting from prolonged ethanol administration and which are manifest even in the absence of ethanol (chronic effects). Methods To study the effects of chronic ethanol feeding, male rat littermates (CD rats purchased from Charles River Breeding Laboratories, Wilmington, Mass.), weighing 130 to 150 g, were pair-fed previously described liquid dietsl2 for 3 to 4 weeks. These diets contain 18% of the total calories as protein, 35% as fat, 11% as carbohydrate, and 36% either as ethanol or additional carbohydrate (control diet). Alcohol-fed rats consumed 12 to 15 g of ethanol per kg of body weight per day. Under pentobarbital anesthesia, the superior mesenteric lymph duct was cannulated 13 and the accessory intestinal lymphatics were ligated. The animals were then restrained in Bollman cages 14 while the pairfeeding continued for 2 additional days by administration of the diets, at 6-hr intervals, through a nasogastric polyethylene tube put in place at the time of lymph diversion and fixed to the scalpl. The acute effects of ethanol were assessed by comparing the response of the same animal to the randomized administration of equal amounts of lipids with either ethanol or isocaloric carbohydrate. These acute effects were measured in lymph-fistula rats previously fed Purina chow (11.3% of calories as fat) ad libitum, in rats fed alcohol-containing diets for 3 to 4 weeks, and in rats pair-fed diets containing isocaloric carbohydrate instead of ethanol. Six hours after the last administration of food through the nasogastric tube, each rat received at random an intragastric dose (60 ml per kg of body weight) of either control or alcohol-containing diet, labeled with [carboxyl-hc Jtripalmitin 02.2 mc per mmole; 1 f..lc per ml of diet) (New England Nuclear Corp., Boston, Mass.). Six hours after the administration of the first diet. an equal dose of the alternate diet was given intragastrically. These doses supplied 2.4 g of mixed dietary oils" per kg of body weight, and, in the case of the alcohol-containing diet, the dose was equivalent to 3 g of ethanol per kg of body weight. To exclude the possibility that the observed effects were due either to changes in gastric emptying or to alterations in lipid digestion, another series of animals (12 pair-fed and 7 chow-fed rats) was tested after intraduodenal administration of a fatty acid, monoglyceride, and bile salt mixture. In these rats, a polyethylene cannula was inserted in the first portion of the duodenum (without occlusion of the lumen) at the time of lymph diversion. For 48 hr after surgery, the rats were restrained and deprived of dietary lipids. The pair-feeding was maintained by inclusion of ethanol or isocaloric glucose in the drinking saline solution. With this procedure the daily ethanol intake amounted to 7 to 9 g of ethanol per kg of body weight. Then, at 5-hr intervals, the rats were given intraduodenally 30 ml per kg of an emulsion containing 6 mm [1-"C]palmitic acid (0.166 mc per mmole), 3 mm glyceryl monoleate, 24 mm sodium taurocholate, and either 0.54 M ethanol or isocaloric glucose, dissolved in Krebs-Ringer phosphate buffer (devoid of calcium and magnesium) at ph The dose of ethanol given with the lipid emulsion was reduced to 0.75 g per kg of body weight in order to provide a local ethanol concentration of 2.5 g per loo ml. The administration of ethanol in this concentration into the first portion of the duodenum is likely to result in intraluminal concentrations similar to those achieved in the upper small intestine after intragastric administration of ethanol (3 g per kg of body weight) in diet. 17 In three of these experiments, the output of lipids was also determined in the chylomicron and in the very low density lipoproteins fractions of the lymph; these fractions were obtained by ultracentrifugation as previously described. 18 To dissociate the effects of ethanol on the lymph flow from the changes in lymph lipid output, 5 additional chow-fed rats were tested after intraduodenal infusion of either ethanol (0.75 g per kg) or isocaloric glucose without the administration of exogenous lipids. To study whether the stimulation of lymph flow affects the output of aietary lipids into the lymph, the flow was increased by two procedures other than ethanol administration. In chow-fed rats, neostigmine (0.05 mg per kg of body weight) was administered intraduodenally together with the lipid emulsion and its effects were compared in the same animal with those of a similar lipid load without the drug. In 8 chow-fed rats, 180 f..lmoles of palmitate, 90 f..lmoles of monoleate, and 720 f..lmoles of taurocholate were diluted in a volume of saline amounting to either 7.5 ml or 45 ml per kg of

3 March 1975 ETHANOL AND FAT ABSORPTION 497 I...-. ETHANOL-CONTAINING DIET CONTROL DIET RATS FED ETHANOL PAIR-FED CONTROLS ~ 20 CHRONICALLY body weight. The different loads of fluid were given at random to the same animal and their effects on lymph were compared. The lymph was collected hourly before and after the administration of the test substances. Total lipids were extracted from the lymph and their contents and radioactivity were measured as previously described. 18 Lymph proteins were measured according to the method of Lowry et al. 19 Ethanol was measured by the method of Bonnichsen 20 in samples of blood obtained from the tail. Statistics. Student's t-test was applied to the mean of the individual differences between values obtained in each alcohol-fed rat and its pair-fed control, or values obtained during the control period compared to those observed in the same rat when the test substance was administered (paired comparisons). 21 All values are expressed as their means ± SEM. Results Effects of acute ethanol administration on lymph flow and lipid output. The administration of liquid diets by gastric tube to lymph-fistula rats increased the flow of intestinal lymph, and, despite dilution of lymph lipids, significantly increased the total output of lipids into the lymph. This response to the intake of diet was maximal in the 1st hr and lasted for 3 hr. In rats not previously fed alcohol, the lymph flow and lipid output were significantly greater in the 1st hr after the administration of diet containing ethanol than after control diet (fig. 1). This was associated with increased recovery of dietary tripalmitin label in the lymph lipids. The order of administration of alcohol and control diets made no difference. During the 1st hr, blood ethanol concentrations increased from an undetectable level to 104 ± 5 mg per 100 ml. Similar effects were observed when either ethanol or isocaloric glucose were included in lipid emulsions containing fatty acids, monoglycerides, and bile salts and were administered into the duodenal lumen (fig. 2, and table 1). When the fatty acids of the emulsion were marked with [14Clpalmitate, the lymph lipids became more labeled after ethanol than after isocaloric glucose administration (table 1). Blood ethanol concentration increased up to 64.0 ± 5 mg per 100 ml and r:. :0;;,12 ~ ~ ~ /1;..... ~ 4 / _- _ ~ I "''''... - i I,---, ~ 500 ~ : ; i 300 ~ 100 ~. 1 ~ i o I 2 3 HOURS ~ o 2 3 FIG. 1. Time course of intestinal lymph changes after intragastric administration of single doses of diets containing either ethanol (3 g per kg) (solid lines) or isocaloric carbohydrate (broken lines) to rats pretreated for 3 to 4 weeks with either alcoholcontaining or control diets. Dietary lipids were labeled with [carboxyl-he ltripalmitin.... ETHANOL ISOCALORIC GLUCOSE WITH EXOGENOUS LIPIDS o 2 3 HOURS WITHOUT EXOGENOUS LIPIDS ~ l ~ " ~ o 2 3 FIG. 2. Intestinal lymph changes after intraduodenal administration of either ethanol (0.75 g per kg) (solid lines) or isocaloric glucose (broken lines), with (left) and without (right) emulsions containing palmi tate, monoleate, and taurocholate. 59 ± 7 mg per 100 ml at 30 and 60 min after administration, respectively. The increased output and labeling of lymph lipids occurred mainly in chylomicrons, whereas no significant changes were observed in the output of lipids in lymph very low density

4 498 BARAONA AND LIEBER Vol. 68, No. 3 TABLE 1. Effects of either neostigmine or ethanol on lymph changes in the 1st hr after the intraduodenal administration of lipids a No. of experiments ntestinal lymph Lymph lipid Lymph lipid Lymph protein flow output labeling output ml/kg/hr mg/kg/hr dpm x JO-'/kg/hr mg/kg/hr Controls ± ± ± ± 10.7 Neostigmine ± 1.5" ± 20.0" ± 1.5" ± 13.4" (0.05 mg/kg) Ethanol ± 0.8" ± 14.4" +9.0 ± 0.6" ± 43.5" (0.75 gikg) a Mean ± SEM of lymph changes (compared to the values found in the hour prior to the tests) in chow-fed rats given intraduodenally an emulsion containing 6 mm ["C]palmitate, 3 mm monoleate, 24 mm taurocholate, and either ethanol (2.5 g per 100 ml) or isocaloric glucose (both in the controls and neostigmine-treated rats). " P < 0.01, compared to the effects of equal fluid (30 ml per kg) and lipid (270 ILmoles per kg) loads to the same animal (paired comparisons). lipoproteins. The administration of an equal dose of ethanol without concomitant administration of exogenous lipids produced a marked increase in the flow of intestinal lymph_ The magnitude of this increase was similar to that produced by the simultaneous administration of ethanol and lipids. However, despite this rise in lymph flow, the output of endogenous lipids into the lymph was similar to that observed after administration of isocaloric glucose (fig. 2). The recoveries of fat in the lymph (compared with the lymph lipid outputs observed when no exogenous fat was given) were 28.0% of the administered fat in the 1st hr, 28.0% in the 2nd, and 15.3% in the 3rd hr after the administration of lipid emulsions with ethanol. After ethanol, 81% of the exogenous lipids were recovered in the 1st hr, 10.2% in the 2nd, and none in the 3rd. Acute ethanol administration also increased the output of proteins into the mesenteric lymph, mainly in the 1st hr (table 1); 90% of this increase was due to proteins other than those contained in chylomicrons or very low density lipoproteins. Effects of chronic ethanol administration on lymph.responses to ethanol or lipids. In rats fed alcohol for 3 to 4 weeks, the intragastric administration of a single dose of ethanol-containing diet slightly increased lymph flow, but the output of dietary lipids into the lymph was similar to that observed after a control diet (fig. 1). Compared with their pair-fed controls, the rats fed -ethanol chronically had smaller lymph responses not only to the intragastric administration of ethanol-containing diets but also to that of diets without ethanol. Within the 1st hr after administration of diets devoid of alcohol, the rats fed alcohol chronically showed a smaller lymph flow (4.7 ± 0.7 ml per kg of body weight) and lesser output of labeled lipids (1.8 ± 0.5 dpm x 10 6 per kg of body weight) than their pair-fed controls (11.2 ± 0.9 ml and 9.3 ± 1.8 dpm x 10 6, respectively) (P < 0.01). Total output of lipids also appeared decreased, but this difference did not reach a level of statistical significance. After the 1st hr, there were no significant differences between rats fed alcohol chronically and their pair-fed controls. Previous chronic ethanol feeding also resulted in a lack of acute effects of ethanol when the latter was administered intraduodenally together with lipid emulsions. In the 1st hr, the flow of lymph increased by 4.3 ± 1.6 ml per kg of body weight after ethanol administration and 2.4 ± 0.8 ml after isocaloric glucose. The output oflipidsincreased 52.7 ± 14.2 mg per kg of body weight after ethanol and 47.9 ± 13.6 mg after glucose. None of these differences was statistically significant. By contrast, in the rats pair-fed isocaloric carbohydrate instead of ethanol for 3 to 4 weeks, the increases in lymph flow and lipid output (per kg of body weight) were significantly greater (P < 0.01 and P < 0.02, respectively) after ethanol (15.6 ± 3.2 ml and 89.1 ± 20.1 mg) than after isocaloric glucose (5.4 ± 1.1 ml and 41.2 ± 11.0 mg). These acute ethanol effects were similar to those observed in rats fed chow diet ad libitum (table 1).

5 March 1975 ETHANOL AND FAT ABSORPTION 499 Effects of increased lymph flow on lymph lipid output. The intraduodenal administration of neostigmine (0.05 mg per kg of body weight) together with lipid emulsions produced an increase in the flow of lymph of comparable magnitude to that produced by ethanol. The output of both lymph proteins and dietary fat was also significantly increased. The maximal lymph changes were observed in the 1st hr after the administration of the drug and these are reported in table 1. A 6-fold increase in the volume of the duodenal infusion solution (without changes in lipid load) also increased significangly the lymph flow (P < 0.01) and the output of proteins (P < 0.01) and lipids (P < 0.05) (table 2). These increases were less than those after ethanol or neostigmine, and generally were maximal in the 1st hr after administration (table 2), except in three experiments in which even greater increases occurred in the 2nd hr. Discussion The administration of ethanol-containing diets to rats not previously fed alcohol increased both the flow of intestinal lymph and the output of lipids into the lymph. The increase in lymph lipids was due at least in part, to enhanced transport of dietary fat, as indicated by the increased recovery in the lymph of labeled lipids administered into the gastrointestinal lumen. The increased lipid output occurred predominantly in chylomicrons. These effects were not due to changes in the rate of gastric emptying since they could be reproduced by intra duodenal administration of ethanol in a dose (0.75 g per kg) which, within 1 hr, produced intraluminal and blood ethanol concentrations comparable to those observed after intragastric administration of the diet. Also, these effects are not likely to be due to alterations in lipolysis or emulsification since similar changes were observed after administration of emulsions containing fatty acids, monoglycerides, and bile salts. The most striking effect of acute ethanol administration was the marked increase in the flow of intestinal lymph. This was not a consequence of the associated increase in fat transport because the administration of ethanol without exogenous lipids similarly increased the flow without affecting the output of lymph lipids. Ethanol (0.63 g per kg of body weight per hr) has been reported to increase the output of endogenous lipids, but this occurred after intraduodenal infusions of an ethanol solution at a concentration of 10 g per 100 ml for 8 hr.10 The reported effect could be due to the prolonged exposure of the small intestine to unusually high ethanol concentrations Furthermore, the flow increased more than the lipid output and the lipid concentration of the lymph actually decreased. However, the converse relationship had to be considered, namely that the stimulatory effect of ethanol on the transport of dietary fat could in some way be linked to the increased flow of lymph. Interference with lymphatic circulation is a recognized cause of lipid malabsorption, but the possibility that increases in flow could favor the absorption of fat has not received much attention. Therefore, we assessed whether other procedures which increase the flow of lymph are also associated with enhanced fat transport. Neostigmine and other cholinergic drugs, as well as the ingestion of saline solutions,25 have been shown to increase the flow of lymph. We also found TABLE 2. Effects of fluid loading on lymph changes in the 1st hr after the intra duodenal administration of lipids a Volume of No. of Intestinal lymph Lymph lipid Lymph lipid Lymph protein infusion solution experiments flow output labeling output ml/kg ml/kg/hr mg/kg/hr dpm x 10-'/kg/hr mg/kg/hr ± ± ± ± ± l= ± ± 10.9 P < 0.01 P < 0.05 P < 0.02 P < O.or a Mean ± SEM of lymph changes (compared to the values found in the hour prior to the tests) in chow-fed rats given intraduodenally an emulsion containing 180 ",moles of [He jpalmitate, 90 ",moles of monoleate, and 720 ",moles of taurocholate per kg of body weight.

6 500 BARAONA AND LIEBER VoI.68,No.3 increases in the intestinal lymph flow either after intraduodenal injection of neostigmine (0.05 mg per kg of body weight) or by augmenting 6-fold the volume of saline in which the duodenal load of lipids was administered. Under both conditions, the increased flow was associated with enhanced output of lymph lipids and increased recovery in the lymph of labeled fatty acids administered intraduodenally. The mechanism of these effects is unknown. The possibility that they could be due to changes in intestinal motility is unlikely. Although neostigmine and distention by fluids can produce hypermotility, ethanol has been reported to produce only a transient increase in the duodenal motility,26 a decreased jejunal segmenting activity (type I waves), and augmented ileal propulsive activity (type III waves. 27 Furthermore, changes in intestinal motility do not produce consistent effects on lymph flow and fat transport Shepherd and Simmonds 30 also observed increases in the output of fat into the thoracic duct lymph when the flow was stimulated by intraduodenal fluid loading. These findings were interpreted as "washing out" of fat through the intestinal wall by the increased passage of water from the intestinal lumen into the lymph. However, the lymphagogue effects observed in this study were associated with increas'ed output of proteins in the lymph far beyond that accounted for by the increase in lymph lipoproteins. The passage of proteins from plasma into the lymph is known to occur and to depend on filtering pressures and capillary permeability. 31 Fat,32, 33 ethanol,34 cholinergic drugs,35 and even saline 36 produce profound hemodynamic changes in the splanchnic circulation, which could be responsible for the increased passage of protein into the lymph. The significance of this outpouring of plasma proteins into the intestinal lymph during fat absorption 37, 38 remains obscure. Although the small intestine can synthesize lipid and protein components oflymph lipoproteins,39 significant fat absorption into the lymph persists during inhibition of protein synthesis This suggests that preformed plasma apolipoproteins may play a role in the formation of lyrrph chylomicrons. Recent evidence in isolated small intestine 43 also indicates that a major protein fraction of lymph lipoproteins derives from plasma proteins. It is unknown to what extent fat absorption can be facilitated by the increased availability of plasma apolipoproteins. The parallelism between the lymphaguoge effect of ethanol and the enhanced fat transport does not exclude the possibility that ethanol could also act on other steps of fat absorption. There are conflicting reports on the acute effects of ethanol on triglyceride synthesis by intestinal slices: 1 hr after intragastric administration of ethanol (3 g per kg) this process was inhibited,44 whereas it was stimulated 16 hr after the administration of a higher dose (7.5 g per kg).9 After prolonged ethanol administration, the intestinal triglyceride synthesis is enhanced 9, 44; however, the output of lipids into the lymph is not increased: the acute administration of ethanol and lipids to rats previously fed diets containing alcohol for several weeks, resulted in moderate stimulation of the lymph flow, but did not produce a greater output of lipids than the administration of isocaloric carbohydrate (fig. 1). Chronic ethanol feeding not only prevented the acute effects of ethanol, but also inhibited the intestinal absorption of lipids administered without ethanol. When rats pair-fed with diets containing either ethanol or isocaloric carbohydrate were given control diet acutely, the alcohol pretreated rats had lesser lymph flows and lipid outputs than the pair-fed controls. The cause of this decreased response (fig. 1) is not known. One possibility could be a reduction in the absorptive surface, since a decreased number of jejunal villus cells has been reported in rats fed alcohol chronically.17 However, the possibility that prior chronic feeding with ethanol could have resulted in a delay of the gastric emptying of control.diet has not been ruled out. Thus, acute administration of ethanol exerts a marked stimulatory effect both on the flow of lymph and on the transport of dietary fat into the lymph. These findings suggest that alcohol drinking may indeed

7 March 1975 ETHANOL AND FA T ABSORPTION 501 favor fat absorption temporarily, but this effect would tend to be abolished by prolonged ingestion of alcohol. Chronic ethanol ingestion (with maintenance of adequate nutrition) not only prevented the acute effects of ethanol, but also resulted in a moderate decrease of fat absorption during periods of ethanol withdrawal in comparison to rats fed the control diet. REFERENCES 1. Baraona E, Orrego H, Fernandez 0, et al: Absorptive function of the small intestine in liver cirrhosis. Am J Dig Dis 7: , Roggin GM, Iber FL, Kater RMH, et al: Malabsorption in the chronic alcoholic. Johns Hopkins Med J 125: , Small MD, Gershoff SN, Broitman SA, et al: Effects of alcohol and dietary deprivation on absorption of xylose from the rat small intestine. Am J Dig Dis 5: , Chang T, Lewis J, Glazko AJ: Effects of ethanol and other alcohols on the transport of amino acids and glucose by everted sacs of rat s mall intestine. Biochim Biophys Acta 135: , Israel Y, Salazar I, Rosenmann E : Inhibitory effects of alcohol on intestinal amino acid transport in vivo and in vitro. J Nutr 96: , Lindenbaum J, Lieber CS: Alcohol-induced malabsorption of vitamin B12 in man. Nature 224 : , Lindenbaum J, Shea N, Saha JR, et al: Alcoholinduced impairment of carbohydrate (CHO) absorption (abstr). Gastroenterology 64:762, Middleton, WRJ, Carter EA, Drummey GD, et al: Effects of oral ethanol administration on intestinal cholesterogenesis in the rat. Gastroenterology 60: , Carter EA, Drummey GD, Isselbacher, KJ: Ethanol stimulates triglyceride synthesis by the intestine. Science 174: , Mistilis SP, Ockner RK: Effects of ethanol on endogenous lipid and lipoprotein metabolism in small intestine. J Lab Clin Med 80:34-46, Bloom B, Chaikoff IL, Reinhardt WO, et al: The quantitative significance of the lymphatic pathway in transport of absorbed fatty acids. J Bioi Chem 184:1-8, DeCarli LM, Lieber CS: Fatty liver in the rat after prolonged intake of ethanol with nutritionally adequate new liquid diet. J Nutr 91: , Bollman JL, Cain JC, Grindlay JH: Techniques for the collection of lymph from the liver, small intestine, or thoracic duct of the rat. J Lab Clin Med 33: , Bollman JL: A cage which limits the activity of rats. J Lab Clin Med 33: 1348, Epstein AN: Water intake without the act of drinking. Science 131: , Thompson GR, Ockner RK, lsselbacher KJ: Effect of mixed micellar lipid on the absorption of cholesterol and vitamin D, into lymph. J Clin Invest 48:87-95, Baraona E, Pirola RC, Lieb.er CS: Small intestinal damage and changes in cell population produced by ethanol ingestion In the.rat. Gastroenterology 66: , Baraona E, Pirola RC, Lieber CS: Pathogenesis of postprandial hyperlipemia in rats fed ethanolcontaining diets. J Clin Invest 52: , Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Bioi Chem 193: , Bonnichsen R: Ethanol determination with alcohol dehydrogenase. In Methods of Enzymatic Analysis. Edited by HU Bergmeryer. New York, Academic Press, 1963, p Snedecor GW, Cochran WG: Statistical Methods. Sixth edition. Ames, Iowa, Iowa State University Press, Halsted CH, Robles EA, Mezey E: Distribution of ethanol in the human gastrointestinal tract. Am J Clin Nutr 26: , Kolmen SN, Daily LJ, Traber DL: Antonomic involvement in the lymphatic delivery of fibrinogen. Am J Physiol 209: , Lee JS: Motility, lymphatic contractility. and distention pressure in intestinal absorption. Am J Physiol 208: , Simmonds WJ: The effects of fluid, electrolyte and food intake on thoracic duct lymph flow in unanesthetized rats. Aust J Exp Bioi Med Sci 32: , Pirola RC, David AE: Effect of intravenous alcohol on motility of the duodenum and of the sphincter of Oddi. Aust Ann Med 19:1-6, Robles EA, Mezey E. Halsted CH, et al: Effects of ethanol on motility of the small intestine. Johns Hopkins Med J 135:17-24, Simmonds WJ: The relationship between intestinal motility and the flow and rate of fat output in thoracic duct lymph in unanaesthetized rats. Q J Exp Physiol 42: , Bennett S, Shepherd P, Simmonds WJ: The effects of alterations in intestinal motility induced by morphine and atropine on fat absorption in the rat. Aust J Exp Bioi Med Sci 40: , Shepherd P, Simmonds WJ: Some conditions affecting the maintenance of a steady lymphatic absorption of fat. Aust J Exp Bioi Med Sci 37:1-10, Yoffey JM, Court ice FC: Lymphatics. In Lymph and Lymphoid Tissue. Cambridge, Mass, Harvard University Press, 1956

8 502 BARAONA AND LIEBER Vol. 68, No Kilmore MA, McCormick JT, Flentge RL, et al: Gastrointestinal blood flow as influenced by changes in dietary fat level. Fed Proc 26:472, Fara JW, Rubinstein EH, Sonnenschein RR: Visceral and behavioral responses to intraduodenal fat. Science 166: 110-Ill, Stein SW, Lieber CS, Leevy CM et al: The effect of ethanol upon systemic and hepatic blood flow in man. Am J Clin Nutr 13:68-74, Price WE, Shehadeh Z, Thompson GH et al: Effects of acetylcholine on intestinal blood flow and motility. Am J Physiol 216: , Varro V, Csernay L, Szarvas F, et al: Effect of glucose and glycine solution on the circulation of the isolated jejunal loop in the dog. Am J Dig Dis 12:60-64, Borgstrom B, Laurell GB: Studies on lymph and lymph-proteins during absorption of fat and saline by rats. Acta Physiol Scand 29: , Simmonds WJ: Some observations on the increase in thoracic duct lymph flow during intestinal absorption of fat in unanaesthetized rats. Aust J Exp Bioi Med Sci 33: , Kessler JI, Stein J, Dannacker D, et al: Biosynthesis of low density lipoprotein by cell-free preparations of rat intestinal mucosa. J Bioi Chern 245: , Redgrave TG: Inhibition of protein synthesis and absorption of lipid into thoracic duct lymph of rats. Proc Soc Exp Bioi Med 130: , Redgrave TG, Zilversmit DB: Does puromycin block release of chylomicrons from the intestine? Am J Physiol 217: , Allen CH, Gibbs PS, Jersild RA: Intestinal fat transport during inhibition of protein synthesis. An electron microscopic study. Exp Mol Pat hoi 14:90-102, Windmueller HG, Herbert PN, Levy RI: Biosynthesis of lymph and plasma lipoprotein apoproteins by isolated perfused rat liver and intestine. J Lipid Res 14: , Baraona E, Pirola RC, Lieber CS: Pathogenesis of alcoholic hyperlipemia (abstr). J Clin Invest 51:8a, 1972