Effect of ethanol on glycylsarcosine absorption

Life Sciences, Vol. 14, pp. Printed in the U.S.A.

1403-1407

Pergamon Pres

EFFECT OF ETHANOL ON GLYCYLSARCOSINE ABSORPTION J.J. Hajjar, K. Krasner, B. Van Linda, and R. Scheig Research Service, Veterans Administration Medical Center, Newington, CT; Department of Medicine, University of Connecticut, Farmington, CT 06032 (Received in final form July 24, 1981) Summary Glycylsarcosine (GlySar) absorption by the rat intestine is not altered by acute ethanol administration (luminal perfusion of a 0.7 M ethanol solution) or by chronic consumption of a 15% ethanol solution in drinking water. Both total absorption, per entire rat small intestine, and specific absorption per mg dry weight of mucosa, were unaffected by ethanol. During the absorption of GlySar, glycine, produced by hydrolysis of the peptide in the cytosol of the intestinal cells, appears in the intestinal lumen. During acute ethanol administration the luminal appearance of glycine is decreased probably due to a reduction in intracellular hydrolysis of the dipeptide. Protein malnutrition, a frequent complication of alcoholism, is believed to be due to a decrease in intake (i), digestion or absorption of dietary proteins (2). Ethanol is known to inhibit amino acid absorption (3-7) but amino acid constituents of dietary proteins are also absorbed as oligopeptides (8,9) In the absence of any information on the effect of ethanol on peptide transport, the present studies were undertaken using the in vivo rat intestine as an experimental model. We studied the acute and chronic effects of ethanol on glycylsarcosine (GlySar) absorption. GlySar was chosen since it is resistant to hydrolysis by brush border peptidases (i0) and slowly hydrolyzed in the cytosol of the intestinal cells (ii). Methods Animals: An inbred strain of male Sprague-Dawley rats was used. The rat were housed individually after weaning in screen-bottom stainless steel cages in a temperature controlled room (25-27oc) that was maintained on 12:12 hour light-dark schedule. They were fed Purina Lab Chow (Ralston Purina Co., St. Louis, MO) and water ad lib for seven weeks at the end of which their weights ranged between 290-380 g. They were then divided into three groups, A (8 rats), B (6 rats) and C (8 rats). Groups A and B were selected to have a simi lar mean initial weight of about 300 g, while the heavier rats were selected as group C with a mean initial weight of 364 g. The rats in group C were given a ground chow diet ad lib in rimmed feeding dishes which prevented spillage and losses during feeding. They were given 15% (V/V) ethanol as the sole water source. Groups A and B were fed tap water ad lib and a diet of ground rat chow, isocaloric to that consumed by the alcohol-fed rats. Calculation of the exact amount of rat chow administered to the pair fed rats was done as described by Wang et al (12). The controlled diets were administered for seven weeks during which the average nutrient intake of the three groups was about 75-80 Kcal/rat/day. Intake of the alcohol group was 68% from rat chow and 32% from ethanol (6-7 g/Kg/day). 0024-3205/81/141403-05502.00/0

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Ethanol and Dipeptide Absorption

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Perfusion studies: The rats were anesthetized by intraperitoneal Napentobarbital (50 mg/Kg). The abdomen was opened by a mid-line incision and the intestine was cannulated with an inlet cannula placed 2 cm caudad to the ligament of Treitz and an oulet cannula 2 cm orad to the ileocecal valve. The rats were perfused while lying on their backs on a heating pad that maintained their rectal temperature at 37oc. The test solution contained 5 m M GlySar (Sigma Chemical Co., St. Louis, MO) and 156 mM NaCI and was perfused at a constant rate of 0.5 ml/min using a peristaltic pump (Manostat, New York, NY). Labelled polyethylene glycol (14C-PEG, New England Nuclear, Boston, MA) was added to the perfusate (i0 ~Ci/L) as a nonabsorbable marker. In experiments on group B, ethanol was added to the perfusates in a 0.7 M concentration without altering the sodium concentration. Effluent solutions were collected after 30 and 90 min of perfusion. The sample collected after 30 min was considered a wash sample during the establishment of steady-state and was discarded. The other subsequent 60 min sample was placed in a tube that contained 3.5 mg/ml sulfosalicylic acid to prevent intraluminal peptidase activity. Immediately after perfusion, the intestine was resected and its length was determined after stretching it with a constant weight on a flat surface. It was then slit open along its longitudinal axis. Its mucosal surface was scraped with a microscope glass slide and the dry weight of the mucosal scrapings was determined after drying in an oven at 90oc for 24 hours. In group B, the concentration of ethanol in the perfusates was checked at the end of the experiments and it decreased by about 15-20% of the original concentration. Analysis and Calculations: The test solution and effluent samples were analyzed for GlySar and glycine concentrations using an amino acid analyzer (Beckman Instruments, Fullerton, CA). 14C-PEG was determined by liquid scintillation (Nuclear Chicago, Isocap 300, Des Plaines, IL). GlySar absorption was calculated by the following formula. A = (Ci - {CfxR}) V where A is total absorption by the perfused intestine in ~mol/h, Ci is initial, Cf is final GlySar concentrations in ~mol/ml. R is the ratio of initial over final 14C-PEG concentrations and V is the perfusion rate in ml/h. Specific absorption is calculated by dividing A by mg dry weight of mucosa or by i00 g rat body weight. The luminal appearance rate of glycine was calculated as follows: G =

((CfxR}-

Ci) V

where G is glycine appearance in ~mol/ml; Cf and Ci are final and initial glycine concentrations in Dmol/ml. R and V are the same as above. The data are presented as mean values ± S.E. for groups of 6-8 rats. Statistical analysis for significance was done using Student's t test (12). Results The initial and final body weights of the three groups of rats are shown in Table I. The ethanol-fed rats do not gain as much weight as any of the pair fed groups. The average weight gain of all the rats was variable during the first two weeks, but during the last five weeks of ethanol treatment the gain of the ethanol fed rats was significantly less (p < .05) than controls being 3.89 ± 0.25 g/day for the ethanol fed and 4.76 ± 0.29 for controls. At the end of seven weeks of the controlled diets, the mean values of the body weights of the three groups studied were about the same. The dry weight of the mucosa of the ethanol fed rats seemed slightly increased in comparison with the pair fed controls, the increase however was not statistically significant. Table II shows GlySar absorption for the three groups of rats. Both total and specific absorption rates were equal in the three groups. Absorption per i00 g rat body weight, an index of the contribution of the dipeptide absorp-

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tion to homeostasis nol administration.

Ethanol

and body mass,

and Dipeptide

was not affected

TABLE Rat groups

Absorption

by acute

or chronic

etha-

I

studied Rat weight Initial

(g) Final

Dry wt. m u c o s a

(g)

Pair

fed, A

300 ± 5

544 ± 8

1.47 ± 0.09

Pair

fed, B

308 ± 6

543 ÷ 8

1.55 ± 0.ii

364 ± 7

558 ± i0

1.70 ± 0.09

Ethanol

1405

fed,

C

Values are means ± S.E. pair fed Groups A and B were given the same diet, Group B was used to test the effect of acute ethanol a d m i n i s t r a t i o n while A served as control

GlySar

absorption

Group Control

TABLE II (~mol/h) Total per Entire Specific per mg Small Intestine Dry Wt. Mucosa

Per 100g Body Wt.

206 ± 4

0.14 ± 0.02

37.8 ± 2.5

202 ± 6

0.13 ± 0.02

37.2 ± 2.6

204 ± 3

0,12 ± 0.02

36.5 ± 1.6

(A) Acute

Ethanol

(B) Chronic

Ethanol

(c) Values are means ± S.E. G l y c y l s a r c o s i n e concentrations in the perfusion solutions was 5 mM. Ethanol in acute experiments was added in a 0.7 M concentration. TABLE III Luminal appearance of free glycine (~mol/h) Total per Entire Specific per mg Small Intestine Dry W!. Mucosa Control

13.15 ± 1.17

Per 100g Body Wt.

8.79 + 1.83

2.64 ± 0.53

3.98 ± 1.13"

1.25 ± 0.i0"

5.98 ± 1.15

2.09 ± 0.59

(A) Acute

Ethanol

6.91 ± 0.80*

(B) Chronic

Ethanol

12.33 ± 1.53

(c) Values

are means

± S.E.

* P<.05

as compared

to control.

Total glycine secretion into the intestinal lumen was significantly decreased (p
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Ethanol

and Dipeptide

Absorption

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Discussion When ethanol is given to rats and their total nutrient intake is duplicated in pair fed controls, the ethanol fed rats usually gain weight at a slower rate than controls (12,14-16). In this study we evaluated the effect ol ethanol on GlySar absorption without the interference of malnutrition, an inherent c o m p l i c a t i o n of alcohol ingestion. The deficit in the growth rate cf the ethanol fed rats was anticipated in advance and the rats that were selected to receive ethanol were chosen to have a heavier mean initial weight (364g) than the controls (300g). After seven weeks of controlled dieting the body weights of the ethanol fed and pair fed animals became equal. This was selected as an appropriate time to do the absorptive studies while the body weight variable was controlled. The intestines of the rats at the time of study were also about equal in length (139 ± 3 cm for A, 140 ± 3 for B and 139 ± for C) and in mucosal dry weight (Table I). The atrophic changes of the intestinal m u c o s a that are described to occur with chronic ethanol administration (17) seemed to be absent in this study since the mucosal dry weights of the ethanol fed and the control rats were about equal. This may be due to regenerative hypertrophic changes of the intestinal villi of the ethanol fed rats. Ananna et al (18) described such changes to occur after an initial phase of villous atrophy within the first 15 days of chronic ethanol administration in rats. Although ethanol is reported to inhibit amino acid transport across the rat intestine (3-7) it does not have an effect on GlySar absorption both when acutely or chronically administered. In the acute experiments we tested ethanol in a 0.7 M solution (about 4% V/V), a c o n c e n t r a t i o n which is in the range of the intra-jejunal ethanol concentrations achieved in humans after moderate ingestion of alcohol (5,12-20). Chronic ethanol ingestion in group C (6-7 g/Kg/day) was r e l a t i v e l y modest but was comparable to the amounts that were found to produce h i s t o l o g i c a l changes of the intestinal m u c o s a by other investigators (17,18). The lack of inhibition of GlySar absorption by ethanol is another situation that d e m o n s t r a t e s the lack of responsiveness of the peptide transport m e c h a n i s m to stimuli. In several studies where intestinal amino acid a b s o r p t i o n rates were shown to be altered, dipeptide transport in contrast, was found unaffected. This difference was seen in diabetes (21) untreated celiac sprue (22) and after jejunoiliac bypass operations (23). The insensitivity of the peptide transport system to ethanol could be a factor that may explain, at least in part, the lack of change in fecal nitrogen excretion after ethanol ingestion in rats (24). During in vivo perfusion of peptides, constituent amino acids produced by the hydrolysis of either brush border or cytosol enzymes, appear in the intestinal lumen. GlySar is hydrolyzed in the cytosol (ii) and glycine appearance in the intestinal lumen represents an indirect estimate of the intracellular hydrolysis of the dipeptide. The results in Table III suggest that acute exposure of the intestine to ethanol inhibits GlySar hydrolysis. Thls effect, however, does not persist with prolonged ethanol ingestion. Ethanol inhibition of GlySar hydrolysis is similar to the inhibition by alcohol of other intestinal enzyme activities such as lactase and thymidine kinase (17). This ethanol effect is different, however, from that observed with dietary restriction which causes an increase in the activities of intestinal peptidases in the cytosol (25). In the present experiments pair feeding and dietary restrictions to all rat groups may have increased GlySar hydrolysis to values that exceed those present in rats that are fed a normal unrestricted diet.

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Ethanol and Dipeptide Absorption

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Finally, the transport and hydrolysis of GlySar are differently affected by acute ethanol administration. This suggests that transport and hydrolysis of the dipeptide are not immediately related and brush border hydrolysis is not a major component of GlySar disappearance from the intestinal lumen. Acknowledgements Supported by the Medical Research Service of the Veterans Administration. We gratefully acknowledge the assistance of Mrs. T. K. Tomicic in carrying out these experiments, and of Dr. E. Khairallah in the use of the amino acid analyzer. We also thank Michelle F. Toucey and Eleanor LaBier for secretarial assistance. References i.

2. 3. 4.

5. 6.

7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19 20 21 22 23 24

25.

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