Nutr. Metabol. 19: 192 200(1975)
A Model System for Direct in vivo Measurement of Absorbed Nutrients in Portal Venous Blood A pplicability to A lcohol Studies'
William N. Hovland, George W. Thomas and Edward S. Kline2 Department of Biochemistry, Virginia Commonwealth University, Medical College of Virginia, Health Sciences Center, Richmond, Va.
Key Words. Alcohol • Intestinal transport • Alcohol and venous blood nutrients Abstract. In order to investigate the in vivo effect of alcohol on intestinal absorption and on other factors which influence the rate of appearance of nutrients into the portal venous system, an experimental system has been developed in which separate cannulas have been implanted in the duodenum and portal vein of the rat. Following recovery from surgical procedures, unanesthetized animals may be administered nutrients directly into the duodenum, with and without alcohol, and the rate of appearance of nutrients in the portal system may be monitored by rapid sampling of the portal blood. Potential applicability of this experimental animal is demonstrated by the kinetics obtained with an amino acid, ¿-phenylalanine, both in the presence and the absence of alcohol.
Introduction Ethanol-induced alterations in the rate of appearance of nutrients in the portal venous system (via malabsorption, effects on blood flow, etc.) may con tribute to the hepatotoxicity associated with the ingestion of alcoholic bever ages. The effect of alcohol on intestinal absorption has been investigated under a variety of experimental conditions, both in experimental animals (1, 3, 8, 14), and people (5, 9, 11, 13). In several of these studies, evidence for an alcoholinduced malabsorption of amino acids or other nutrients was presented (3, 8, 9, 1 This study was supported in part by United States Public Health Service grant MH 16809 and a grant from the Licensed Beverage Industries, Inc. 2 We appreciate the helpful assistance of Miss K. C. Fehskens and Mrs. D. McMath for the preparation of this manuscript.
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Received: June 25, 1975: accepted: September 26, 1975.
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14). The animal studies employed either in vitro experiments (3, 8, 14) or in vivo experiments in which transport was assayed indirectly (1, 8), under condi tions which did not delineate between effects of alcohol on absorption and other processes, e.g., stomach emptying. All of the studies with people were in vivo and, by necessity, involved indirect measurements of intestinal absorption. Two of these (5 ,9 ) involved aspiration of intestinal contents after administration of the nutrient directly into the small intestine and, thus, interpretative difficulties were minimized. Thus far, no interrelationships amongst alcohol ingestion, defects in nutrient supply to the liver, and hepatotoxicity have been elucidated. The goal of the research reported herein was to develop an experimental system with which one might delineate the physiologic effects of ethanol on rates of appearance of nutrients in the portal venous system and with which such anomalies could be related to alcohol-induced hepatotoxicity. To meet this goal, it was considered important that the investigation should be performed in the live, unanesthetized animal-employing methodology that would permit rapid, direct monitoring of intestinal absorption and related processes in animals administered alcohol either acutely or chronically. This report describes a procedure which satisfies these criteria. With this procedure, rapid kinetic analysis can be performed. The appli cability of the procedure has been demonstrated by measuring the rate of ap pearance of ¿-phenylalanine label in the portal blood, in the presence and absence of acutely administered ethanol - with both materials administered directly into the duodenum. The procedure is also suitable for experiments involving chronic ethanol administration by mouth.
Methods and Results
Surgical Procedures (Illustrated in Figure 1) Starting 5 mm below the xyphoid, a 3-cm midline incision was made through the skin and extended through the musculature into the peritoneal cavity. The area around the incision was draped with gauze saturated with plasma-lyte 148, pH 7.4 (Travenol Labora tories). The cecum and sufficient small intestine were exposed and oriented with the superior mesenteric vein visible and lying in a straight line toward the portal vein. All exposed tissues were kept moist with plasma-lyte saturated gauze. The ileocecal vein was freed of covering facia and surrounding fat. A 3 -0 nylon suture was placed under this vein, slightly distal to where it enters the superior mesenteric vein and left untied. A second 3 - 0 suture was also placed under the vein and tied 1 cm distal to the first suture. Tire cannula to be implanted (intramedic polyethylene tubing, PE-10, i.d. 0.011 and o.d. 0.024 inches, Clay
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Animals All experiments employed male Holtzman rats weighing between 300 and 500 g. The animals had free access to Purina Rat Chow and water and were maintained on a 12-hour photoperiod, i.e., 12 h light and 12 h dark. Animals were housed in constant temperature and humidity quarters.
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Adams) was prepared in advance by placing the tubing slightly above an electrically heated wire in order to produce a small bubble in the tubing. An 18-cm length of tubing was used and the bubble was made about 8 cm from one end. The short end of the cannula was cut to a length that would allow the beveled tip of the cannula to pass the entrance of the splenic vein into the portal vein with the bubble just beyond the proximal suture after insertion of the cannula into the superior mesenteric vein. To insert the cannula, a small incision was made halfway through the ileocecal vein between the two sutures, while applying tension to the proximal suture in order to prevent back bleeding through the vein. The cannula, filled with plasma-lyte, was inserted through the hole into the vein and pushed through the vein until the beveled tip was in the portal vein with the bubble situated beyond the proximal suture. The proximal suture was tied and a few milliliters of plasma-lyte infused through the cannula into the portal vein, leaving the cannula filled with this solution. Cecum and small intestine were then placed back into the abdominal cavity. The duodenum was then prepared for insertion of the intestinal cannula by making a small puncture through the abmesenteric side of the duodenum opposite to the entry of the bile duct. A previously prepared cannula containing a bubble similar to the portal vein cannula was used. The bubble was 5 mm from the end of the tube and the end was not beveled. The 5-mm length of the cannula was inserted through the hole in the duodenum into the lumen of that organ with the bubble remaining external to the hole. A 3 -0 suture was anchored in the gut musculature and tied around the noninserted portion of the cannula so as to hold the bubble tightly against the external wall of the duodenum. This cannula was flushed and left filled with distilled water. The gut was then replaced into the abdominal cavity. In this study it was desired to have the nonimplanted ends of each cannula external to the animal. To accomplish this, the rat was placed on his stomach and a 2-cm incision was made through the skin in the mid-lower back. This skin was then separated from the subcutaneous tissue. With a needle, a hole was punctured through the abdominal muscula ture into the abdominal cavity via the aforementioned incision on the back. The portal vein cannula was filled with sodium heparin (1,000 U.S.P. U/ml, Upjohn Co.), sealed by touching the nonimplanted end to a hot hemostat and brought through the needle to bring it external to the animal. The needle was withdrawn and reintroduced similarly into the abdominal cavity. The intestinal cannula, filled with distilled water, was sealed and withdrawn through the needle to bring it external to the animal. After removal of the needle, both external cannulas were coiled into a subcutaneous pouch on the back of the rat and the skin closed with wound clips. The abdominal musculature and skin were sutured separately and the animals were housed singly, without restraint, and allowed to recover from surgical proce dures.
Fig. I. In vivo implantation of cannulas in (a) duodenum and (b) portal vein of the rat, a schematic drawing.
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Measurement o f Appearance Rate o f Nutrients in Portal Venous Blood (General Procedure) Animals were used for experiments about 1 week after surgery (after weight loss had ceased or after weight regain commenced). The evening before an experiment, food was removed from the animal. On the morning of an experiment (performed 8 -1 0 a.m.) the animal was restrained without anesthesia, the two cannulas exposed and the sealed ends cut off. The portal vein cannula was flushed with plasma-lyte. Gentle suction was applied to the syringe to bring blood into the cannula. Slight pressure on the abdomen helped to stimulate
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Appearance Rate o f L-Phenylalanine Label in Portal Venous Blood, in Presence and Absence o f Ethanol The experimental animal described previously was developed to investigate potential effects of ethanol on the rate of appearance of nutrients in portal venous blood. In this initial report, experiments with ¿-phenylalanine are described. Figure 2 shows the level of label in portal blood as a function of time after administra tion of the amino acid, with and without alcohol, into the duodenum. Without alcohol, one observes a rapid increase in appearance of label, with a peak occurring at about 6 min, followed by an initially rapid decline and subsequently slow decline in blood labeling. In the presence of alcohol, one observes a marked alteration in these kinetics. In addition to a significant and appreciable dimunition in the maximum amount of label appearing in the blood, there is also a decreased rate of labeling and no obvious appearance of the early peak.
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blood flow. Lack of free blood flow occasionally occurred, and these animals were not used. Once bleeding was established, the portal vein cannula was flushed and refilled with plasmalyte. The animals were administered 0.3 ml of sodium heparin (500 U/ml in plasma-lyte) via the portal vein cannula and the cannula rinsed and left filled with plasma-lyte. The intestinal cannula was flushed with 0.1 ml of distilled water. After allowing heparin to act, bleeding was induced through the portal vein cannula and after discarding the first few drops of blood, successive 5-iul aliquots were collected (baseline samples). As with all blood samples, these were added to scintillation vials containing 0.3 ml NCS tissue solubilizer (AmershamSearle) for digestion. The portal vein cannula was flushed again and left filled with plasmalyte. In rapid succession, the following were added to the intestinal cannula: 1 ml of an alcohol solution to an experimental animal or 1 ml of distilled water to a control animal, 1 ml of radiolabeled amino acid with or without added carrier amino acid, 0.2 ml distilled water to flush the cannula. Time of isotope addition represented zero time for data presen tations. Following these additions, flow of blood from the portal vein cannula was induced and the first few drops discarded. Successive 5-/j 1 samples were collected from the portal vein cannula. This blood was treated similarly to the baseline samples. In our current procedure, an attempt has been made to sample all of the blood flowing from the portal vein cannula. Sampling time was when blood started to accumulate on the tip of the cannula and samples were collected directly from the tip of the cannula. Although desirable, it has not always been possible to collect all the blood flowing from the portal vein cannula. If animals stopped bleeding freely during an experiment, plasma-lyte was flushed through the portal vein cannula, bleeding reinitiated and sampling continued. Rarely, additional heparin was administered during an experiment to help maintain blood flow. After termination of an experiment, the portal vein cannula was filled with heparin and the intestinal cannula filled with distilled water. The two cannulas were sealed and, under light ether anesthesia, were reimplanted subcutaneously on the back of the animal. In initial experiments, a slight ly different experimental procedure was employed. In some cases, animals have been used for more than one experiment - with no less than 1 week interval between experiments. Such rats have been employed both as control and experimental animals. The digesting blood samples were usually incubated at 60 °C for about 1 h in order to affect complete digestion; after which a few drops of benzoyl peroxide solution (1 g benzo yl peroxide in 5-ml toluene) were added to samples and the bottles incubated at 60 °C for 1 h. This treatment decolorized the samples. The scintillation fluor solution (75 mg POPOP, 6 g PPO, 1 liter toluene) was added to the samples and they were counted in a Nuclear-Chicago, Mark 1, Liquid Scintillation Spec trometer. The external standard was used for quench correction and baseline activities were subtracted from subsequent samples.
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800
700
600
500 E ü 400
r
300
LW
200
100
0
L —
_____ 1________ 1________ 1________ 1________ l 5 10 15 20 25 T IM E , min
The eventual level of label observed is similar both with and without alcohol and may be approaching a steady-state level. Dilution of blood by plasma-lyte in the cannula would not affect the kinetic patterns, since a volume of blood greater than that contained in the cannula is discarded just prior to sampling. Table 1 shows these data with the corresponding standard errors. Between replicate experiments, large variations may occur with respect to the amount of radioactivity observed in portal blood. However, the composite curve ob tained without alcohol is clearly different from the composite curves obtained with alcohol (p < 0.001) and the differences in peak levels of label are unambiguous.
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Fig. 2. Influence of ethanol on uptake of ¿-phenylalanine label by portal venous blood - composite of several separate/« vivo experiments, o = '"¿'-¿-phenylalanine minus ethanol, average of four experiments; * = '"C-/.-phenylalanine plus 276 mg ethanol, average of two experiments; o = l4CV,-phenylalanine plus 395 mg ethanol, average of two experiments. For each experiment, freeze-dried ¿-3-phenylalanine-l4C(U) of 10 mCi/mmol specific activity was used. The labeled amino acid was checked for chemical and radiochemical purity by paper chromotography. An aqueous solution containing lOpCi of the labeled amino acid and 20 mg of added carrier ¿-phenylalanine was injected into the duodenal cannula as described in the text. Each time point represents DPM per 5 pi of blood. Differences among the experimental curves were evaluated with the use of the paired t test (least squares method). The curve derived from the experiments without alcohol was considered to be different from each of the curves derived from experiments in which alcohol was present (p < 0.001). The two curves derived from experiments in which alcohol was present were not considered to be different from one another (p > 0.05).
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Table /. Influence of ethanol on uptake of ¿-phenylalanine label by portal venous blood' Sampling time min
Radioactivity in portal blood (mean DPM ± SEM) ------------------------------------------------------------------------------'"C-phe ,4C-phe plus 14C-phe plus minus ethanol 276 mg ethanol 395 mg ethanol
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0
43 ± 17 58 ± 23 145 + 49 208 ± 104 293 ± 120 284 ± 90 396 ± 101 456 ± 119 568 ± 124 725 ± 185 773 + 211 733 ± 237 758 ± 229 698 ± 167 671 ± 156 605 ± 149 503 ± 160 428 ± 177 385 ± 164 340 ± 144 210 ± 105 258 ± 94 238 ± 82 240 ± 66 254 ± 74 236 ± 66 229 ± 138 221 ± 78 223 ± 70 219 ± 63 229 t 74 170 ± 66 170 ± 72 171 ± 74 163 + 63 166 ± 72 156 + 61
1 These data are from the same experiments as used for figure 2.
33 ± 18 38 + 3 53 ± 18 80 ± 15 68 ± 33 90 + 30 98 ± 8 180 ± 50 235 ± 65 235 ± 15 230 ± 20 175 ± 15 195 ± 55 200 ± 65 240 ± 60 180 ± 10 208 ± 58 200 ± 40 225 ± 25 233 ± 73 230 ± 80 220 ± 10 280 ± 110 225 ± 75 185 t 95 215 ± 25 170 ± 5 115 ± 5 140 + 5 208 + 78 255 ± 65 255 t 75 230 ± 60 265 ± 65 275 ± 35 273 ± 48 300 ± 10
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29 + 27 28 ± 23 25 ± 20 40 ± 30 113 ± 98 165 ± 125 162 ± 107 135 ± 100 155 i 120 160 ± 115 178 ± 128 160 ± 90 185 ± 90 230 ± 90 248 ± 83 240 ± 70 220 ± 60 205 i 55 215 ± 95 230 ± 100 230 ± 100 210 ± 100 233 ± 103 260 ± 100 200 t 60 230 ± 25 185 ± 50 183 ± 58 193 ± 48 240 ± 120 395 ± 295 405 ± 275 295 ± 140 263 ± 153 265 ± 155 225 ± 135 208 ± 118
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In initial studies, the rats had a cannula in the portal vein only and materials were administered by stomach intubation rather than by addition directly into the duodenum. This resulted in bizarre portal blood labeling kinetics which were quite complicated and difficult to interpret, very likely because of complications due to stomach emptying. Since alcohol alters the rate of stomach emptying (1) we chose to deliver materials directly into the intestine even though this is not the usual route by which people ingest spirits. Administered via the duodenum, relatively uncomplicated kinetics are observed. As seen in figure 2, a rather sharp peak of label may be observed about 6 min after administration of ¿-phenyl alanine in the absence of alcohol. Unpublished data with ¿-leucine are consistent with an even more rapid peak labeling in the absence of ethanol. Thus, it is important to obtain rapid and frequent samples of blood in order to observe significant events and obtain meaningful kinetic data. The design of this experi mental system allows for rapid administration of materials and rapid, frequent sampling of blood. However, proper development of both the surgical and ex perimental techniques, do require practice and patience. Several techniques have been developed for implantation of cannulas in the intestine or portal vein of animals other than rats (2, 10, 12). Cannulation of these organs in the rat poses a more difficult problem, particularly, with the small and fragile portal vein. At the time our studies were initiated, the only suitable procedure known to us for rats employed portal cannulation via the splenic vein, a method which involved ligation of all blood vessels connected to the spleen and splenectomy (7). For this investigation, it was not desirable to remove organs. Therefore, we chose a different means of portal cannulation, one that might allow the animal to maintain a more normal physiologic state. Re cently, a procedure for direct implantation of a cannula in the portal vein has been described (4). In this procedure, the tube is glued into the portal vein of the rat. It would seem that this method causes less trauma than other methods. These investigators also implanted a cannula in the duodenum and measured appearance of label in portal blood after administration of the dipeptide, glycylglycine. Their kinetics were very similar to that seen in figure 2 for phenylala nine in the absence of ethanol. In the experiments described herein, a pronounced effect of ethanol on appearance of ¿-phenylalanine label in portal blood was observed. The experi mental conditions employed, however, were chosen to test the validity of the experimental animal; not to investigate whether or not ethanol produced a spe cific effect on absorption or a physiologically significant anomaly. In these experiments, the observed results may be related to the high alcohol concentra tions employed (although total amounts of ethanol used were small), osmotic effects, blood flow rate effects, etc. However, even effects such as these may
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Discussion
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relate to the mode of action of alcohol in people since alterations of portal blood nutrient profiles need not result only from alterations in specific absorp tion mechanisms. A variety of factors, in addition to absorptive ones, may influence the concentration profiles in portal blood — both in the presence and absence of alcohol. Regarding alcohol concentrations that can be observed in the intestine, it is interesting that hitherto unsuspectedly high concentrations can be found in experimental human subjects (6).
References
Dr. E.S. Kline, Department of Biochemistry, Virginia Commonwealth University, Medical College of Virginia, Health Sciences Center, Richmond, VA 23298 (USA)
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1 Barboriak, J.J. and Meade, R.C.: Impairment of gastro-intestinal processing of fat and protein by ethanol in rats. J. Nutr. 98: 373 -378 (1969). 2 Ban, W.H. and Riegelman. S.: Intestinal drug absorption and metabolism. I. Compari son of methods and models to study physiological factors of in vitro and in vivo intestinal absorption. J. pharm. Sei. 59: 154-163 (1970). 3 Chang, T.: Lewis, J., and Glazko, A.J.: Effect of ethanol and other alcohols on the transport of amino acids and glucose by everted sacs of rat small intestine. Biochim. biophys. Acta 135: 1000-1007 (1967). 4 Gallo-Tones, H.E. and Ludorf, J.: Techniques for the in vivo catheterization of the portal vein in the rat. Proc. Soc. exp. Biol. Med. 145: 249-254 (1974). 5 Halsted, C.H.; Robles. E.A., and Mezey, E.: Decreased jejunal uptake of labeled folic acid (3H-PGA) in alcoholic patients. Roles of alcohol and nutrition. New Engl. J. Med. 285: 701 706 (1971). 6 Halsted, C.H.: Robles, E.A., and Mezey, E.: Distribution of ethanol in the human gastrointestinal tract. Am. J. clin. Nutr. 26: 831-834 (1973). 7 Hyun, S.A.: Vahouny, G. V, and Treadwell, C.R.: Portal absorption of fatty acids in lymph- and portal vein-cannulated rats. Biochim. biophys. Acta ¡37: 296-305 (1967). 8 Israel, Y.; Salazar, /., and Rosentnan, E.: Inhibitory effects of alcohol on intestinal amino acid transport in vivo and in vitro. J. Nutr. 96: 499-504 (1968). 9 Israel, Y.; Valenzulla, J.E.; Salazar. I., and Ugarte, G.: Alcohol and amino acid trans port in the human small intestine. J. Nutr. 98: 222 -224 (1969). 10 Lydtin, II.: Pieper, M.: Kusus. T. Schnelle, K. und Zoliner, N.: Die Implantation von Verweilkathetern in die Vena portae, die Vena cava, den rechten Vorhof und in den Magen des Miniaturschweines. Z. ges. exp. Med. 149: 279-282 (1969). 11 Mezey, E.: Jow, E.; Slavin, R.E., and Tabon, F.: Pancreatic functions and intestinal absorption in chronic alcoholism. Gastroenterology 59: 657-664 (1970). 12 Mouzas, G.L and Smith, G. A method for withdrawing blood from the portal vein in dogs using silastic tubing. Int. surg. Dig. 51: 495-498 (1969). 13 Roggin, G.M.: Iber, F.L.: Kater, R.M.H., and Tabon, F : Malabsorption in the chronic alcoholic. Johns Hopkins med. J. 125: 321 -330 (1969). 14 Spencer, R.P.; Brody, K.R., and Lutters, B.M.: Some effects of ethanol on the gastro intestinal tract. Am. J. dig. Dis. 9: 599 -604 (1964).
A Model System for Direct in vivo Measurement of Absorbed Nutrients in Portal Venous Blood A pplicability to A lcohol Studies'
William N. Hovland, George W. Thomas and Edward S. Kline2 Department of Biochemistry, Virginia Commonwealth University, Medical College of Virginia, Health Sciences Center, Richmond, Va.
Key Words. Alcohol • Intestinal transport • Alcohol and venous blood nutrients Abstract. In order to investigate the in vivo effect of alcohol on intestinal absorption and on other factors which influence the rate of appearance of nutrients into the portal venous system, an experimental system has been developed in which separate cannulas have been implanted in the duodenum and portal vein of the rat. Following recovery from surgical procedures, unanesthetized animals may be administered nutrients directly into the duodenum, with and without alcohol, and the rate of appearance of nutrients in the portal system may be monitored by rapid sampling of the portal blood. Potential applicability of this experimental animal is demonstrated by the kinetics obtained with an amino acid, ¿-phenylalanine, both in the presence and the absence of alcohol.
Introduction Ethanol-induced alterations in the rate of appearance of nutrients in the portal venous system (via malabsorption, effects on blood flow, etc.) may con tribute to the hepatotoxicity associated with the ingestion of alcoholic bever ages. The effect of alcohol on intestinal absorption has been investigated under a variety of experimental conditions, both in experimental animals (1, 3, 8, 14), and people (5, 9, 11, 13). In several of these studies, evidence for an alcoholinduced malabsorption of amino acids or other nutrients was presented (3, 8, 9, 1 This study was supported in part by United States Public Health Service grant MH 16809 and a grant from the Licensed Beverage Industries, Inc. 2 We appreciate the helpful assistance of Miss K. C. Fehskens and Mrs. D. McMath for the preparation of this manuscript.
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Received: June 25, 1975: accepted: September 26, 1975.
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14). The animal studies employed either in vitro experiments (3, 8, 14) or in vivo experiments in which transport was assayed indirectly (1, 8), under condi tions which did not delineate between effects of alcohol on absorption and other processes, e.g., stomach emptying. All of the studies with people were in vivo and, by necessity, involved indirect measurements of intestinal absorption. Two of these (5 ,9 ) involved aspiration of intestinal contents after administration of the nutrient directly into the small intestine and, thus, interpretative difficulties were minimized. Thus far, no interrelationships amongst alcohol ingestion, defects in nutrient supply to the liver, and hepatotoxicity have been elucidated. The goal of the research reported herein was to develop an experimental system with which one might delineate the physiologic effects of ethanol on rates of appearance of nutrients in the portal venous system and with which such anomalies could be related to alcohol-induced hepatotoxicity. To meet this goal, it was considered important that the investigation should be performed in the live, unanesthetized animal-employing methodology that would permit rapid, direct monitoring of intestinal absorption and related processes in animals administered alcohol either acutely or chronically. This report describes a procedure which satisfies these criteria. With this procedure, rapid kinetic analysis can be performed. The appli cability of the procedure has been demonstrated by measuring the rate of ap pearance of ¿-phenylalanine label in the portal blood, in the presence and absence of acutely administered ethanol - with both materials administered directly into the duodenum. The procedure is also suitable for experiments involving chronic ethanol administration by mouth.
Methods and Results
Surgical Procedures (Illustrated in Figure 1) Starting 5 mm below the xyphoid, a 3-cm midline incision was made through the skin and extended through the musculature into the peritoneal cavity. The area around the incision was draped with gauze saturated with plasma-lyte 148, pH 7.4 (Travenol Labora tories). The cecum and sufficient small intestine were exposed and oriented with the superior mesenteric vein visible and lying in a straight line toward the portal vein. All exposed tissues were kept moist with plasma-lyte saturated gauze. The ileocecal vein was freed of covering facia and surrounding fat. A 3 -0 nylon suture was placed under this vein, slightly distal to where it enters the superior mesenteric vein and left untied. A second 3 - 0 suture was also placed under the vein and tied 1 cm distal to the first suture. Tire cannula to be implanted (intramedic polyethylene tubing, PE-10, i.d. 0.011 and o.d. 0.024 inches, Clay
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Animals All experiments employed male Holtzman rats weighing between 300 and 500 g. The animals had free access to Purina Rat Chow and water and were maintained on a 12-hour photoperiod, i.e., 12 h light and 12 h dark. Animals were housed in constant temperature and humidity quarters.
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Adams) was prepared in advance by placing the tubing slightly above an electrically heated wire in order to produce a small bubble in the tubing. An 18-cm length of tubing was used and the bubble was made about 8 cm from one end. The short end of the cannula was cut to a length that would allow the beveled tip of the cannula to pass the entrance of the splenic vein into the portal vein with the bubble just beyond the proximal suture after insertion of the cannula into the superior mesenteric vein. To insert the cannula, a small incision was made halfway through the ileocecal vein between the two sutures, while applying tension to the proximal suture in order to prevent back bleeding through the vein. The cannula, filled with plasma-lyte, was inserted through the hole into the vein and pushed through the vein until the beveled tip was in the portal vein with the bubble situated beyond the proximal suture. The proximal suture was tied and a few milliliters of plasma-lyte infused through the cannula into the portal vein, leaving the cannula filled with this solution. Cecum and small intestine were then placed back into the abdominal cavity. The duodenum was then prepared for insertion of the intestinal cannula by making a small puncture through the abmesenteric side of the duodenum opposite to the entry of the bile duct. A previously prepared cannula containing a bubble similar to the portal vein cannula was used. The bubble was 5 mm from the end of the tube and the end was not beveled. The 5-mm length of the cannula was inserted through the hole in the duodenum into the lumen of that organ with the bubble remaining external to the hole. A 3 -0 suture was anchored in the gut musculature and tied around the noninserted portion of the cannula so as to hold the bubble tightly against the external wall of the duodenum. This cannula was flushed and left filled with distilled water. The gut was then replaced into the abdominal cavity. In this study it was desired to have the nonimplanted ends of each cannula external to the animal. To accomplish this, the rat was placed on his stomach and a 2-cm incision was made through the skin in the mid-lower back. This skin was then separated from the subcutaneous tissue. With a needle, a hole was punctured through the abdominal muscula ture into the abdominal cavity via the aforementioned incision on the back. The portal vein cannula was filled with sodium heparin (1,000 U.S.P. U/ml, Upjohn Co.), sealed by touching the nonimplanted end to a hot hemostat and brought through the needle to bring it external to the animal. The needle was withdrawn and reintroduced similarly into the abdominal cavity. The intestinal cannula, filled with distilled water, was sealed and withdrawn through the needle to bring it external to the animal. After removal of the needle, both external cannulas were coiled into a subcutaneous pouch on the back of the rat and the skin closed with wound clips. The abdominal musculature and skin were sutured separately and the animals were housed singly, without restraint, and allowed to recover from surgical proce dures.
Fig. I. In vivo implantation of cannulas in (a) duodenum and (b) portal vein of the rat, a schematic drawing.
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Measurement o f Appearance Rate o f Nutrients in Portal Venous Blood (General Procedure) Animals were used for experiments about 1 week after surgery (after weight loss had ceased or after weight regain commenced). The evening before an experiment, food was removed from the animal. On the morning of an experiment (performed 8 -1 0 a.m.) the animal was restrained without anesthesia, the two cannulas exposed and the sealed ends cut off. The portal vein cannula was flushed with plasma-lyte. Gentle suction was applied to the syringe to bring blood into the cannula. Slight pressure on the abdomen helped to stimulate
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Appearance Rate o f L-Phenylalanine Label in Portal Venous Blood, in Presence and Absence o f Ethanol The experimental animal described previously was developed to investigate potential effects of ethanol on the rate of appearance of nutrients in portal venous blood. In this initial report, experiments with ¿-phenylalanine are described. Figure 2 shows the level of label in portal blood as a function of time after administra tion of the amino acid, with and without alcohol, into the duodenum. Without alcohol, one observes a rapid increase in appearance of label, with a peak occurring at about 6 min, followed by an initially rapid decline and subsequently slow decline in blood labeling. In the presence of alcohol, one observes a marked alteration in these kinetics. In addition to a significant and appreciable dimunition in the maximum amount of label appearing in the blood, there is also a decreased rate of labeling and no obvious appearance of the early peak.
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blood flow. Lack of free blood flow occasionally occurred, and these animals were not used. Once bleeding was established, the portal vein cannula was flushed and refilled with plasmalyte. The animals were administered 0.3 ml of sodium heparin (500 U/ml in plasma-lyte) via the portal vein cannula and the cannula rinsed and left filled with plasma-lyte. The intestinal cannula was flushed with 0.1 ml of distilled water. After allowing heparin to act, bleeding was induced through the portal vein cannula and after discarding the first few drops of blood, successive 5-iul aliquots were collected (baseline samples). As with all blood samples, these were added to scintillation vials containing 0.3 ml NCS tissue solubilizer (AmershamSearle) for digestion. The portal vein cannula was flushed again and left filled with plasmalyte. In rapid succession, the following were added to the intestinal cannula: 1 ml of an alcohol solution to an experimental animal or 1 ml of distilled water to a control animal, 1 ml of radiolabeled amino acid with or without added carrier amino acid, 0.2 ml distilled water to flush the cannula. Time of isotope addition represented zero time for data presen tations. Following these additions, flow of blood from the portal vein cannula was induced and the first few drops discarded. Successive 5-/j 1 samples were collected from the portal vein cannula. This blood was treated similarly to the baseline samples. In our current procedure, an attempt has been made to sample all of the blood flowing from the portal vein cannula. Sampling time was when blood started to accumulate on the tip of the cannula and samples were collected directly from the tip of the cannula. Although desirable, it has not always been possible to collect all the blood flowing from the portal vein cannula. If animals stopped bleeding freely during an experiment, plasma-lyte was flushed through the portal vein cannula, bleeding reinitiated and sampling continued. Rarely, additional heparin was administered during an experiment to help maintain blood flow. After termination of an experiment, the portal vein cannula was filled with heparin and the intestinal cannula filled with distilled water. The two cannulas were sealed and, under light ether anesthesia, were reimplanted subcutaneously on the back of the animal. In initial experiments, a slight ly different experimental procedure was employed. In some cases, animals have been used for more than one experiment - with no less than 1 week interval between experiments. Such rats have been employed both as control and experimental animals. The digesting blood samples were usually incubated at 60 °C for about 1 h in order to affect complete digestion; after which a few drops of benzoyl peroxide solution (1 g benzo yl peroxide in 5-ml toluene) were added to samples and the bottles incubated at 60 °C for 1 h. This treatment decolorized the samples. The scintillation fluor solution (75 mg POPOP, 6 g PPO, 1 liter toluene) was added to the samples and they were counted in a Nuclear-Chicago, Mark 1, Liquid Scintillation Spec trometer. The external standard was used for quench correction and baseline activities were subtracted from subsequent samples.
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800
700
600
500 E ü 400
r
300
LW
200
100
0
L —
_____ 1________ 1________ 1________ 1________ l 5 10 15 20 25 T IM E , min
The eventual level of label observed is similar both with and without alcohol and may be approaching a steady-state level. Dilution of blood by plasma-lyte in the cannula would not affect the kinetic patterns, since a volume of blood greater than that contained in the cannula is discarded just prior to sampling. Table 1 shows these data with the corresponding standard errors. Between replicate experiments, large variations may occur with respect to the amount of radioactivity observed in portal blood. However, the composite curve ob tained without alcohol is clearly different from the composite curves obtained with alcohol (p < 0.001) and the differences in peak levels of label are unambiguous.
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Fig. 2. Influence of ethanol on uptake of ¿-phenylalanine label by portal venous blood - composite of several separate/« vivo experiments, o = '"¿'-¿-phenylalanine minus ethanol, average of four experiments; * = '"C-/.-phenylalanine plus 276 mg ethanol, average of two experiments; o = l4CV,-phenylalanine plus 395 mg ethanol, average of two experiments. For each experiment, freeze-dried ¿-3-phenylalanine-l4C(U) of 10 mCi/mmol specific activity was used. The labeled amino acid was checked for chemical and radiochemical purity by paper chromotography. An aqueous solution containing lOpCi of the labeled amino acid and 20 mg of added carrier ¿-phenylalanine was injected into the duodenal cannula as described in the text. Each time point represents DPM per 5 pi of blood. Differences among the experimental curves were evaluated with the use of the paired t test (least squares method). The curve derived from the experiments without alcohol was considered to be different from each of the curves derived from experiments in which alcohol was present (p < 0.001). The two curves derived from experiments in which alcohol was present were not considered to be different from one another (p > 0.05).
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Table /. Influence of ethanol on uptake of ¿-phenylalanine label by portal venous blood' Sampling time min
Radioactivity in portal blood (mean DPM ± SEM) ------------------------------------------------------------------------------'"C-phe ,4C-phe plus 14C-phe plus minus ethanol 276 mg ethanol 395 mg ethanol
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0
43 ± 17 58 ± 23 145 + 49 208 ± 104 293 ± 120 284 ± 90 396 ± 101 456 ± 119 568 ± 124 725 ± 185 773 + 211 733 ± 237 758 ± 229 698 ± 167 671 ± 156 605 ± 149 503 ± 160 428 ± 177 385 ± 164 340 ± 144 210 ± 105 258 ± 94 238 ± 82 240 ± 66 254 ± 74 236 ± 66 229 ± 138 221 ± 78 223 ± 70 219 ± 63 229 t 74 170 ± 66 170 ± 72 171 ± 74 163 + 63 166 ± 72 156 + 61
1 These data are from the same experiments as used for figure 2.
33 ± 18 38 + 3 53 ± 18 80 ± 15 68 ± 33 90 + 30 98 ± 8 180 ± 50 235 ± 65 235 ± 15 230 ± 20 175 ± 15 195 ± 55 200 ± 65 240 ± 60 180 ± 10 208 ± 58 200 ± 40 225 ± 25 233 ± 73 230 ± 80 220 ± 10 280 ± 110 225 ± 75 185 t 95 215 ± 25 170 ± 5 115 ± 5 140 + 5 208 + 78 255 ± 65 255 t 75 230 ± 60 265 ± 65 275 ± 35 273 ± 48 300 ± 10
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29 + 27 28 ± 23 25 ± 20 40 ± 30 113 ± 98 165 ± 125 162 ± 107 135 ± 100 155 i 120 160 ± 115 178 ± 128 160 ± 90 185 ± 90 230 ± 90 248 ± 83 240 ± 70 220 ± 60 205 i 55 215 ± 95 230 ± 100 230 ± 100 210 ± 100 233 ± 103 260 ± 100 200 t 60 230 ± 25 185 ± 50 183 ± 58 193 ± 48 240 ± 120 395 ± 295 405 ± 275 295 ± 140 263 ± 153 265 ± 155 225 ± 135 208 ± 118
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In initial studies, the rats had a cannula in the portal vein only and materials were administered by stomach intubation rather than by addition directly into the duodenum. This resulted in bizarre portal blood labeling kinetics which were quite complicated and difficult to interpret, very likely because of complications due to stomach emptying. Since alcohol alters the rate of stomach emptying (1) we chose to deliver materials directly into the intestine even though this is not the usual route by which people ingest spirits. Administered via the duodenum, relatively uncomplicated kinetics are observed. As seen in figure 2, a rather sharp peak of label may be observed about 6 min after administration of ¿-phenyl alanine in the absence of alcohol. Unpublished data with ¿-leucine are consistent with an even more rapid peak labeling in the absence of ethanol. Thus, it is important to obtain rapid and frequent samples of blood in order to observe significant events and obtain meaningful kinetic data. The design of this experi mental system allows for rapid administration of materials and rapid, frequent sampling of blood. However, proper development of both the surgical and ex perimental techniques, do require practice and patience. Several techniques have been developed for implantation of cannulas in the intestine or portal vein of animals other than rats (2, 10, 12). Cannulation of these organs in the rat poses a more difficult problem, particularly, with the small and fragile portal vein. At the time our studies were initiated, the only suitable procedure known to us for rats employed portal cannulation via the splenic vein, a method which involved ligation of all blood vessels connected to the spleen and splenectomy (7). For this investigation, it was not desirable to remove organs. Therefore, we chose a different means of portal cannulation, one that might allow the animal to maintain a more normal physiologic state. Re cently, a procedure for direct implantation of a cannula in the portal vein has been described (4). In this procedure, the tube is glued into the portal vein of the rat. It would seem that this method causes less trauma than other methods. These investigators also implanted a cannula in the duodenum and measured appearance of label in portal blood after administration of the dipeptide, glycylglycine. Their kinetics were very similar to that seen in figure 2 for phenylala nine in the absence of ethanol. In the experiments described herein, a pronounced effect of ethanol on appearance of ¿-phenylalanine label in portal blood was observed. The experi mental conditions employed, however, were chosen to test the validity of the experimental animal; not to investigate whether or not ethanol produced a spe cific effect on absorption or a physiologically significant anomaly. In these experiments, the observed results may be related to the high alcohol concentra tions employed (although total amounts of ethanol used were small), osmotic effects, blood flow rate effects, etc. However, even effects such as these may
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Discussion
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relate to the mode of action of alcohol in people since alterations of portal blood nutrient profiles need not result only from alterations in specific absorp tion mechanisms. A variety of factors, in addition to absorptive ones, may influence the concentration profiles in portal blood — both in the presence and absence of alcohol. Regarding alcohol concentrations that can be observed in the intestine, it is interesting that hitherto unsuspectedly high concentrations can be found in experimental human subjects (6).
References
Dr. E.S. Kline, Department of Biochemistry, Virginia Commonwealth University, Medical College of Virginia, Health Sciences Center, Richmond, VA 23298 (USA)
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1 Barboriak, J.J. and Meade, R.C.: Impairment of gastro-intestinal processing of fat and protein by ethanol in rats. J. Nutr. 98: 373 -378 (1969). 2 Ban, W.H. and Riegelman. S.: Intestinal drug absorption and metabolism. I. Compari son of methods and models to study physiological factors of in vitro and in vivo intestinal absorption. J. pharm. Sei. 59: 154-163 (1970). 3 Chang, T.: Lewis, J., and Glazko, A.J.: Effect of ethanol and other alcohols on the transport of amino acids and glucose by everted sacs of rat small intestine. Biochim. biophys. Acta 135: 1000-1007 (1967). 4 Gallo-Tones, H.E. and Ludorf, J.: Techniques for the in vivo catheterization of the portal vein in the rat. Proc. Soc. exp. Biol. Med. 145: 249-254 (1974). 5 Halsted, C.H.; Robles. E.A., and Mezey, E.: Decreased jejunal uptake of labeled folic acid (3H-PGA) in alcoholic patients. Roles of alcohol and nutrition. New Engl. J. Med. 285: 701 706 (1971). 6 Halsted, C.H.: Robles, E.A., and Mezey, E.: Distribution of ethanol in the human gastrointestinal tract. Am. J. clin. Nutr. 26: 831-834 (1973). 7 Hyun, S.A.: Vahouny, G. V, and Treadwell, C.R.: Portal absorption of fatty acids in lymph- and portal vein-cannulated rats. Biochim. biophys. Acta ¡37: 296-305 (1967). 8 Israel, Y.; Salazar, /., and Rosentnan, E.: Inhibitory effects of alcohol on intestinal amino acid transport in vivo and in vitro. J. Nutr. 96: 499-504 (1968). 9 Israel, Y.; Valenzulla, J.E.; Salazar. I., and Ugarte, G.: Alcohol and amino acid trans port in the human small intestine. J. Nutr. 98: 222 -224 (1969). 10 Lydtin, II.: Pieper, M.: Kusus. T. Schnelle, K. und Zoliner, N.: Die Implantation von Verweilkathetern in die Vena portae, die Vena cava, den rechten Vorhof und in den Magen des Miniaturschweines. Z. ges. exp. Med. 149: 279-282 (1969). 11 Mezey, E.: Jow, E.; Slavin, R.E., and Tabon, F.: Pancreatic functions and intestinal absorption in chronic alcoholism. Gastroenterology 59: 657-664 (1970). 12 Mouzas, G.L and Smith, G. A method for withdrawing blood from the portal vein in dogs using silastic tubing. Int. surg. Dig. 51: 495-498 (1969). 13 Roggin, G.M.: Iber, F.L.: Kater, R.M.H., and Tabon, F : Malabsorption in the chronic alcoholic. Johns Hopkins med. J. 125: 321 -330 (1969). 14 Spencer, R.P.; Brody, K.R., and Lutters, B.M.: Some effects of ethanol on the gastro intestinal tract. Am. J. dig. Dis. 9: 599 -604 (1964).
