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Chronic Alcoholism and Canine Exocrine Pancreas Secretion
72:238-243, 1977 Copyright © 1977 by The Williams & Wilkins Co.
Vol 72, No.2
GASTROENTEROLOGY
Printed in U.8A.
CHRONIC ALCOHOLISM AND CANINE EXOCRINE PANCREAS SECRETION
A long term follow-up study H.
SARLES,
O.
G. P ALASCIANO
TISCORNIA, AND
Unite de Recherches de PathoLogie Digestive, Institute NationaLe de la Sante et La Recherche Medicale U 31, Marseille, France
Sequential changes in pancreatic secretion were evaluated during a 2- to 3-year follow-up study in 8 dogs provided with Thomas gastric and duodenal fistulas. Four animals were given intragastric ethanol (2 g kg- I) daily for 3 years; 4 others served as controls. In alcohol-fed animals: (1) Flow rate and bicarbonate output in response to 1 clinical unit kg-I hr- I of secretin were increased at the end of 2 years but not after 1 year of alcohol feeding. (2) At the end of 2 years, the dose-response curve of pancreatic secretion to cholecystokinin was unchanged but the maximal bicarbonate and water secretion in response to high doses of secretin were increased. Modifications (1) and (2) are explained by reduplication of pancreatic ducts secondary to the development of chronic pancreatitis, verified by biopsy. (3) The previous report by our group of an increased cholecystokinin release in the course of chronic alcohol consumption in dogs seems to be attributable to an artefact. This work shows that the pancreatic response to intraduodenal oleic acid is not significantly modifed by 3 years of alcohol feeding. When men, rats, and dogs l - 5 are not accustomed to the regular consumption of ethanol, an intravenous infusion of ethanol decreases pancreatic exocrine secretion. But when dogs are submitted to daily intragastric administration of ethanol, intravenous ethanol increases pancreatic exocrine secretion. 6 , 7 These two opposite actions affect protein more than water and bicarbonate secretion, and both are mediated by cholinergic nerves. 8 (D. Celener, P. Lechene, O. M. Tiscornia, et al., manuscript submitted for publication). These nerve-mediated changes are associated with modifications of gastrointestinal hormone release. In alcoholfed dogs, a meal induces a greater gastrin release than in controls9 but secretin release after intraduodenal injection of HCl is decreased. 11 On the other hand, protein output in response to an intraduodenal infusion of oleic acid (expressed as a percentage of pre infusion levels) is increased, which has been interpreted as being caused by an increased release of cholecystokinin (CCK).IO The aim of the present study was to analyze after 2 to 3 years of intragastric ethanol feeding: (1) the sequential changes of pancreatic secretion induced either by secretin infusion alone or by the association of secretin and CCK; (2) the changes in the sensitivity and in the maximal secretory response to secretin and to CCK; (3) the sequential modifications of pancreatic secretion in response to intraduodenal infusion of oleic acid. These
studies were carried out in comparison with a control group of dogs matched by weight with alcohol-fed dogs. Methods Animal Preparations Eight mongrel dogs (12 to 30 kg) were provided with a duodenal Thomas cannula 12 opposite the main pancreatic duct; the accessory pancreatic duct was ligated and divided. A Thomas cannula was also inserted in the most dependent part of stomach. At least 4 weeks were allowed for recovery. Dogs were deprived of food, but not water, (or 18 hr before tests. During tests, the gastric cannula was kept open to prevent endogenous release of duodenal hormones by gastric contents. Before each test a glass cannula, connected to a piece of polyethylene tubing, was inserted into the main pancreatic duct papilla. Pancreatic juice was continuously collected into ice-chilled graduated centrifuge tubes and divided into 20-min samples. Dogs were allotted to two groups of 4 animals. Group A: after completion of standard tests as described below, these dogs were made "alcoholic" by being given 2 g kg- I of ethanol daily for 36 months. Ethanol was diluted to 50% (v/v) with normal saline and was given through the gastric cannula. In the fasting state (morning), a protein-supplemented diet was then given, and in the afternoon, dogs were coaxed to eat an extra food ration. Group B: a second group of dogs was used as control and received the same diet without ethanol. Each control dog was weight-matched to 1 experimental (alcoholic) dog. All dogs maintained excellent nutritional status. Three kinds of protocols were applied to both groups.
Protocols Protocoll . Pancreatic response to secretin alone or secretin and cholecystokinin-pancreozymin (CCJ(-PZ) was tested repeatedly before and after (1.5, 3, 12, and 24 months) the
Received September 18, 1975. Accepted July 26, 1976. Address requests for reprints to: Professeur H. Sarles, Unite de Recherches de Pathologie Digestive, INSERM U 31, 46 chemin de la Gaye, 13009 Marseille, France. 238
February 1977
CHRONIC ALCOHOLISM AND PANCREATIC SECRETION
beginning of intragastric ethanol administration in group A dogs and at an interval of 24 months in group B dogs. Both hormones were purchased from the Gastrointestinal Hormone Research Laboratory, Karolinska Institutet, Stockholm, Sweden. Hormones were diluted with normal saline and were infused into a leg vein at a constant rate of 100 ml per hr. The doses administered were: secretin alone, 1 clinical unit kg- 1 hr- 1 ; and secretin, 1 clinical unit kg-I hr- I + CCK-PZ, 3 CrickHarper-Raper (CHR) units kg-I hr-l. The infusions lasted 100 min. The mean value during the last 40 min was considered as the plateau. Protocol 2. Dose-response to secretin and to CCK-PZ were performed in 3 alcoholic dogs, 24 months after beginning ethanol feeding, and in 3 paired control dogs after the same period. Dose-response curves were constructed by intravenous infusion of increasing doses, each dose being doubled every 60 min. Dose-response to secretin was obtained with 5 doses: 0.5, 1, 2, 4, and 8 clinical units kg- 1 hr- I. A dose-response curve to CCK-PZ was also obtained with 5 doses: 1.5, 3, 6, 12 and 24 CHR units kg- 1 hr- I, superimposed on a 1 clinical unit kg-I hr- I secretin background. To construct dose-response curves, mean values of the last 40 min at each dose were used. Protocol 3. Endogenous release of duodenal hormones (CCK-PZ) was repeatedly studied by infusing oleic acid through the duodenal cannula at 4, 24, and 36 months after starting ethanol feeding; again, control dogs were studied twice at an interval of 24 months. Oleic acid solution (20 ml) was infused over a 10-min period, when pancreatic secretion had reached its plateau under secretin (1 clinical unit kg- 1 hr- 1 ) stimulation. Pancreatic response was expressed as secretion after oleic acid infusion and as a ratio: (peak secretion after oleic acid infusion -;. secretin plateau value) x 100. TABLE
239
At the end of 2 years, 1 control dog was given 2 g kg- 1 of ethanol daily as previously described and oleic acid experiments were repeated after 12 months of alcohol feeding.
Chemical Determinations The volume of every pancreatic juice sample was read to the nearest 0.1 ml. Samples were then taken to determine bicarbonate, protein, and calcium concentrations. '2 Outputs were calculated as concentration times volume. HC0 3 concentration was determined by the VanSlyke volumetric method; protein concentration was determined by absorption at 280 nm (E:~~· = =:; 20) ; calcium was determined by atomic absorption spectrophotometry. Statistical Analysis Experimental data were statistically evaluated with Student's t-test, for paired or nonpaired values '3 according to which protocol had been followed.
Results Sequential Changes in Pancreatic Response to Exogenous Hormones Response to secretin alone (1 clinical unit kg- 1 hr- 1) (table 1). In group A, alcoholic dogs, response to secretin was as follows. When compared to the pre-ethanol value in the same dogs, pancreatic secretion during ethanol administration was not modified during the 1st year of alcohol feeding but later on showed a dissociation between volume and HC03 secretion on the one hand and protein secretion on the other: 24 months of
1. Secretin-induced pancreatic secretion of 4 dogs before and during alcohol-fed stage and of 4 controls Alcohol-fed dogs
Parameter
Nonalcoholic period"
Flow rate (ml/20 min) RC03 - (mEq/liter) RC0 3 - output (mEq/20 min) Protein (mg/mD Protein output (mg/20 min)
15.9 144 2.3 2.4 43.7
± ± ± ± ±
2.8 2.6 0.4 0.4 13.1
Control dogs
Alcohol-fed stage 3 moO
11.6 158 1.9. 2.3 26.5
± ± ± ± ±
0.5 3.4 0.1 0.3 3.3
12 moO
14.9 142 2.14 1.2 18.6
± ± ± ± ±
2.0 5.8 0.4 0.3 d 4.5
24 moO
23 144 3.3 0.1 24.3
± ± ± ± ±
1.6 c
1.6 0 ..2 0.2d 6.0
Stage 0 mo·
15.4 131.5 2.0 1.4 24.4
± ± ± ± ±
2.1 2.8 0.3 0.1 4.9
Stage 24 mo·
20.9 148 3.1 1.6 40.0
± ± ± ± ±
1.2 3.0 0.2 0.2 4.7
Means ± SEM of 8, 5, 4, and 24 experiments, respectively, on 4 alcohol-fed dogs. Means ± SEM of 13 and 17 tests, respectively, on control dogs. Statistical analysis by the t-test applied to a pairs comparison showed no significant changes. c Significant difference (P = 0.025) from nonalcoholic period. d Significant difference (P = 0.002) from nonalcoholic period. a b
TABLE
2. Secretin + cholecystokinin (CCK)-induced pancreatic secretion of 4 dogs before and during alcohol-fed stage and of 4 controls Alcohol-fed dogs Parameter
Flow rate (ml/20 min) RC03 - (mEq/liter) RCOa- output (mEq/20 min) Protein (mg/mD Protein output (mg/20 min)
Nonalcoholic period"
Control dogs
Alcohol-fed stage 6 wk"
3 moO
12 moO
24 moO
Stage 0 mo·
Stage 24 mo·
24.8 ± 3.1
28.4 ± 1.0
28.8 ± 5.5
24.1 ± 6.6
26.1 ± 4.0
27.1 ± 2.1
32.4 ± 2.7
152 ± 3.5 3.77 ± 0.5
150 ± 3.5 4.32 ± 0.2
160 ± 2.6c 4.0 ± 0.9
151 ± 1.8 3.8 ± 1.0
141 ± 2.9 3.8 ± 0.5
152.5 ± 1.1 4.4 ± 0.3
140.5 ± 5.5 4.5 ± 0.4
2.6 ± 0.6 59.0 ± 4.1
2.9 ± 0.3 77.7 ± 6.1
2.3 ± 0.5 70.9 ± 29.1
1.9 ± 0.5 46.9 ± 18.1
1.8 ± 0.3 50.2 ± 11.4
2.6 ± 0.3 71.5 ± 9.8
2.1 ± 0.2 69.5 ± 10.5
Means ± SEM of 13,4, 4, 4, and 30 experiments, respectively, on 4 alcohol-fed dogs. Means ± SEM of 13 and 7 tests on 3 control dogs. Statistical analysis by the t-test applied to a paired comparison showed no significant changes. c Significant difference with the nonalcoholic period at 0.05 level. a
b
240
SARLES ET AL.
ethanol administration increased both volume and HC03 output by about 40% as compared to pre-ethanol values; bicarbonate output increase was statistically significant, but volume increase was not. Protein concentration and output decreased by 60 and 40%, respectively, as against pre-ethanol values; protein output decrease was statistically significant. In contrast to ethanol-fed dogs, group B, control dogs, did not show any statistically significant changes after 24 months. Response to secretin (1 clinical unit kg- 1 hr- 1 ) + CCK-PZ (3 CHR units kg- 1 hr- 1 ) (table 2). Mter 1.5 to 24 months of alcohol feeding, pancreatic response to a combined secretin + CCK-PZ stimulation was not significantly different from that before ethanol administration in group A dogs, nor from that after 24 months in group B dogs. TABLE
Dose-Response Studies in Alcoholic and Control Dogs Dose response to secretin (table 3, fig. 1). Mter 24 months of ethanol administration, observed maximal responses of volume, HC0 3 concentration, and output were significantly higher in the 3 alcoholic dogs than in their matched controls. On the contrary, responses to sub maximal doses of volume, protein, bicarbonate, and calcium concentrations and outputs did not show any difference between groups. Calcium values were almost always lower in alcoholic dogs than in controls. Dose response to CCK-PZ (table 4, fig. 2). After 24 months of ethanol feeding, observed maximal responses in volume and HC0 3 - output were similar in both groups, but HC0 3 - concentration after 12 CHR U kg- 1 hr-l was significantly higher in alcoholic dogs, which is in keeping with the fact that with these doses
3. Exocrine pancreatic secretion response of control (C) and alcohol-fed (AF) dogs after different doses of a continuous intravenous injection of secretin a Intravenous secretin (U/kg/hr) Parameter Group 0.5
Flow rate (JLl/kg/min) HC03 - (mEq/liter) HC03 - output (JLEq/kg/min) Protein concentration {mg/mll Protein output (JLg/kg/min) Ca++ (mg/liter) Ca++ output (JLg/kg/min) a b C
C AF C AF C AF C AF C AF C AF C AF
9.1 11.1 95.7 100.7 0.9 1.2 6.7 7.6 68.5 89.3 2.8 3.1 25.2 32.1
± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.0
1.5 3.8 7.8 14.4 0.2 0.6 2.5 1.3 35.4 34.1 0.3 0.6 4.7 10.7
25.5 31.4 128.2 127.7 3.4 4.3 2.5 3.7 52.9 100.0 1.9 1.3 55.0 37.3
1.5
HC03 - (mEq/liter) HC0 3 - output (JLEq/kg/min) Protein concentration {mg/mll Protein output (JLg/kg/min) Cl- (mEq/liter) Cl- output (JLEq/kg/min)
C
d
2.0
5.2 6.6 7.2 10.5 0.8 1.1 0.5 0.7 11.8 19.5 0.6 0.1 26.2 7.0
45.9 48.3 140.8 142.3 6.5 6.9 1.4 1.7 67.1 72.6 1.4 0.8 69.7 38.9
± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.0
5.1 6.1 1.7 5.8 0.7 1.0 0.3 0.5 19.0 16.0 0.4 0.1 23.4 8.1
59.1 75.0 123.5 137.5 7.3 10.3 0.7 0.8 45.2 63.1 0.9 0.9 55.2 63.6
± ± ± ± ± ± ± ± ± ± ± ± ± ±
8.0
4.3 3.2b
1.7
2.8 e
0.5
0.6 e
0.0 0.2 7.8 16.1 0.1 0.2 10.5 17.4
70.7 80.7 107.7 134.3 7.6 10.8 0.9 0.7 60.8 57.8 0.9 0.5 69.5 37.3
± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.9 1.3 1.2
4.6 e
0.6
0.5 b
0.1 0.2 5.7 18.9 0.2 0.2 21.8 14.8
4. Exocrine pancreatic secretion response of control (C) and alcohol-fed (AF) dogs after different doses of cholecystokinin (CCK) superimposed on a continuous intravenous secretin perfusion a CCK (U/kg/hr) + secretin 1.0 U/kg/h- 1 Parameter Group
Flow rate (JLl/kg/min)
a
± ± ± ± ± ± ± ± ± ± ± ± ± ±
Values are means ± SEM of six experiments on 3 control and 3 alcohol-fed dogs. Significantly different from control (P = 0.02). Significantly different from control (P = 0.005).
TABLE
b
Vol. 72, No.2
C AF C AF C AF C AF C AF C AF C AF
33.5 33.6 124.7 126.5 4.5 4.2 3.2 4.4 100.6 149.7 30.5 24.7 0.8 0.8
± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.0
7.0 1.5 9.7 1.5 1.1 0.1 0.6 0.7 25.6 30.0 7.9 2.7 0.1 0.1
37.9 38.1 126.2 126.0 4.8 4.9 3.4 3.1 133.4 119.7 29.1 22.2 1.0 0.8
± ± ± ± ±
± ± ± ± ± ± ± ± ±
6.0
6.9 4.6 4.9 10.3 0.9 1.0 0.6 0.6 36.3 28.1 3.6 3.8 0.2 0.1
Values are means ± SEM of six experiments on 3 control and 3 alcohol-fed dogs. Significantly different from control (P = 0.005). Significantly different from control (P = 0.05). Significantly different from control (P = 0.01).
60.1 58.3 132.2 138.5 7.9 8.1 3.4 4.0 201.5 229.3 26.5 16.1 1.6 0.9
± ± ± ± ± ± ± ± ± ± ± ± ± ±
12.0
4.5 6.6 2.8 2.7 0.6 1.0 0.2 0.5 11.8 37.5 3.5 2.6 e
0.2 O.le
71.4 69.1 116.3 139.2 8.3 9.5 4.8 4.1 334.9 286.6 39.0 20.7 2.8 1.5
± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.0 6.4 4.9
3.6 b
0.7 0.7 0.5 0.6 31.8 51.2 2.7
3.6 b
0.3 0.3 d
24
90.6 93.0 140.7 135.0 12.6 12.5 5.5 3.8 498.5 359.2
± ± ± ± ± ± ± ± ± ±
6.9 5.4 9.9 3.5 0.2 0.4 0.8 1.0 57.0 100.4
Control· 4th month AF' 24th month AFD 36th month AF' Control· 4th month AF' 24th month AFD 36th month AF' Control· 4th month AF' 24th month AFD 36th month AF' Control· 4th month AF' 24th month AFD 36th month AF' Control· 4th month AF' 24th month AFD 36th month AF'
Dogs
17.9 19.0 15.9 15.8 144.2 161 136.2 139.6 2.6 3.0 2.2 2.3 1.7 0.8 1.3 2.3 30.6 15.2 29.9 33.0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.3 1.5 2.2 2.8 2.4 1.1 3.9 3.5 0.2 0.4 0.3 0.5 0.1 0.2 0.1 0.5 3.7 5.0 7.8 6.0
Plateau
25.5 31.8 30.4 27.6 145.2 161 145.3 151.8 3.7 5.1 4.4 4.2 4.3 3.4 3.7 4.7 112.4 108.6 119.8 130.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.4e 0.7 e 0.6 d 16.9 d 17.0e 23.1J 18.0 d
3.3'
0.8 e 0.6 d
0.7
0.3 d
3.6 4.6 6.1 3.3
2.3' 2.3
SEM
5.6 e 3.2e
Mean ±
0-20
+42 +67 +91 +75 +0.6 0.0 +7 +9 +42 +69 +100 +83 +158 +308 +183 +100 +267 +614 +301 +297
change a
%
25.9 31.6 27.9 35.0 139.2 160 146 157.8 3.6 5.1 4.1 5.5 4.5 3.5 4.5 4.93 116.3 115.6 116.4 167.3 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
21.3 J 1.0 h
12.0J
0.5
2.0e 0.2d
4.2 4.8 4.8
4.6 e 2.4 h
2.2
1.8 d
SEM
0.7 e O.4 h 2.0h O.4 e O.4 d O.4 h 12.7 d
Mean ±
20-40
+45 +66 +77 +122 -3 -0.6 +7 +13 +38 +68 +88 +139 +169 +327 +245 +110 +279 +661 +290 +407
change a
%
26.6 31.2 27.7 34.7 136.6 156 141 157.8 3.7 4.9 3.9 5.5 4.4 3.5 4.3 5.0 115.2 112.4 118.0 165.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.9 4.0 5.3 7.7 2.10.3 0.6 0.7'
4.8 e
2.41 d 2.8
SEM
24.5 J 14.0h
15.0J
0.5 h O.4 h 0.5 e 0.6 e 0.5 h 14.9 d
Mean ±
40-60
+49 +64 +74 +120 -5 -3 +4 +13 +41 +63 +78 +137 +161 +320 +234 +112 +276 +639 +295 +403
change a
% ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.2
3.0 e
SEM
60-80
25.3 J 12.0h
1S.0
0.6 h 15.4 d
0.9J 0.8 J
0.5 d 0.5 h 0.4h
3.4 8.4 8.3 0.9' 0.4' 0.4
4.5 d 3.1h
Mean ± 24.9 27.8 28.8 30.3 131.9 153 141.3 152.6 3.3 4.4 3.9 4.6 4.1 4.2 4.1 4.8 101.6 121.2 98.4 130.9
Secretin (1.0 CU/k~ '/hr ') after intraduodenal oleIc acid (min)
+39 +44 +81 +92 -9 -5 +4 +9 +27 +46 +81 +100 +145 +410 +215 +101 +232 +697 +229 +297
change a
%
23.8 25.4 26.6 28.0 128.7 157 141.3 151.0 3.0 4.0 3.7 4.3 4.2 3.7 4.0 5.2 101.9 105.0 100.7 132.3
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
28.5 J 17.0h
25.0
0.7 e 18.2d
0.7 J 0.8 J
o.e 0.6 e O.4 h
4.2 6.8 9.0 2.3 0.4 0.5
3.6 h 3.W
SEM
80-100
3.5' 3.4
Mean ±
5. Exocrine pancreatic secretion response elicited by intraduodenal oleic acid infusion superimposed on secretin perfusion in control and alcohol-fed (AF) dogs
a Percentage difference compared with plateau level. • As the results of control dogs at the beginning and at the 24th month of experiment were not statistically different, they were pooled together; mean of 12 tests on 4 control dogs. , Significantly different from plateau (P = 0.05). d Significantly different from plateau (P = 0.005). e Significantly different from plateau (P = 0.01). , Mean of eight tests on 4 dogs. D Mean of six tests on 3 dogs. h Significantly different from plateau (P = 0.001). , Mean of eight tests on 2 dogs. J Significantly different from plateau (P = 0.02).
Protein output (mg/20 min)
Protein (mg/m!)
HC03 - output (mEq/20 min)
HC03 - (mEq/ liter)
Flow rate (m1/20 min)
Parameter
TABtE
+33 +34 +67 +77 -11 -2 +4 +8 +15 +32 +69 +83 +149 +351 +211 +120 +233 +591 +237 +310
change a
%
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242
SARLES ET AL .
IlEq / kg/min
T J '-e~
11
Alcohol- Fed Dogs
9
7
j
I
"".-'
.... I-
.
•• .-' '-'1-
Control Dogs
5
3
Ii
1
2
0,5 SECRETIN
4
8
(Clinical Units)
FIG . 1. Bicarbonate output in control and alcohol-fed dogs after different doses of continuous intravenous infusion of secretin. Significant differences at 0.05 and 0.02 levels are indicated by *, ** respectively. 1l9/kg/min Non- Alcoholic Dogs
500
400 Alcoholic Dogs
300
200
100
•
I
Vol . 72, No . 2
of hormone, chloride concentration and output were lower. Therefore, the sum of chloride and bicarbonate is constant. Protein secretion was not significantly different between the two groups, although lower values were obtained in alcoholic dogs in response to high CCK-PZ doses.
Intraduodenal Oleic Acid Infusion Tests (Table 5) In alcohol-fed dogs, there was a relative increase in protein output and concentration after oleic acid, when expressed as: (protein secretion (output and concentration) in response to oleic acid -;- protein secretion before oleic acid) x 100, although this difference diminished from the 4th to the 36th month. But protein secretion in response to secretin before oleic acid infusion was significantly lower at the 4th month than in controls, and the peak secretory response to oleic acid was not significantly different in controls and after 4, 24, and even 36 months of alcohol consumption. In the control dog that was subsequently fed ethanol for 12 months, peak pancreatic secretion in 20-min samples after oleic acid was as shown in table 6. Discussion The pancreatic secretory response to sub maximal doses of secretin and of secretin + CCK-PZ was not modified either during the 24-month follow-up of control dogs or during the first 12 months of alcohol feeding. After 24 months of alcohol feeding, bicarbonate output was significantly increased in comparison with output before alcohol feeding. Increased response to secretin could be attributable either to an increased sensitivity of ductal cells to secretin or to an increased number of ductal cells. This question can be answered by a pathological study realized in the same animals, 14 and indeed ductal reduplication was evident in the pancreatic biopsies after 2 years of alcohol feeding. A similarly increased secretion of water and bicarbonate response to secretin has been observed in human chronic pancreatitis. 15 The fact that in dogs this change appears only at the end of 2 years of alcohol consumption, when protein plugs and microstones are found as early as the 3rd month,6 shows that modifications of water and bicarbonate secretion attributable to ductal hyperplasia are not the cause of chronic pancreatitis but are relatively late phenomena during the course of chronic alcohol consumption and its resulting pancreatic lesions. On the contrary, protein secretory response to secretin diminished during the course of alcohol feeding. TABLE
6. Peak pancreatic secretion after oleic acid in control dog subsequently fed tehanol" Parameter
1.5
3
6
12
24
PANCREOZYMIN (C .H.R. Units)
FIG . 2. Protein output in control and alcohol-fed dogs after different doses of cholecystokinin (CCK) superimposed on a continuous intravenous infusion of secretin (1.0 clinical unit kg- 1 hr- l).
Volume (m1!20 min) Bicarbonate output (mEq/20 min) Protein output (mg/20 min)
Before alcohol feed- Alcohol-fed ing
24.7 83.7 3.2
23.6 91.4
3.0
These results, which are the mean of three experiments, are not significantly different. a
February 1977
CHRONIC ALCOHOLISM AND PANCREATIC SECRETION
243
Although significant, this decrease is probably fortui- release, CCK release is not greatly modified by chronic tous, as the response to secretin before oleic acid infu- alcohol consumption. sion tends to increase during the same time in the REFERENCES same dogs (table 5). This illustrates the irregularity of 1. Mott CB, Sarles H, Tiscornia OM, et al: Inhibitory action of the protein response to secretin in contrast to the staalcohol on human exocrine pancreatic secretion. Am J Dig Dis bility of protein response to secretin + CCK-PZ. In17:902-910, 1972 deed, in none of the controls or alcohol-fed animals was 2. Tiscornia OM, Gullo L, Sarles H: The inhibition of canine exocrine pancreatic secretion by intravenous ethanol. Digestion the protein secretion after CCK-PZ significantly modi9:231-240, 1973 fied. In previous work6 it has been found that in another 3. Tiscornia OM, Palasciano G, Dzieniszewski J, et al: Simultaneous changes in pancreatic and gastric secretion induced by acute series of dogs protein secretory response to CCK was intravenous ethanol infusion. Effects of atropine and reserpine. increased at the end of 6 weeks and 3 months of alcohol Am J Gastroenterol 63:389-395, 1975 feeding, and indeed, in the present series of alcohol-fed 4. Tiscornia OM, Hage G, Palasciano G, et al: The effects of pentodogs, protein output was increased after the same pelinium and vagotomy on the inhibition of canine exocrine panriod of alcohol consumption, although not significreatic secretion by intravenous ethanol. Biomedicine 18:159cantly; this lack of significance is probably attributa163, 1973 ble to a smaller number of experiments. It is therefore 5. Cavarsan A, Teixeira A, Sarles H, et al: Action of intragastric ethanol on the pancreatic secretion of conscious rats. Digestion probable that in the first months of alcohol consump13:145-152, 1975 tion, at least before the onset of pancreatic lesions, pancreatic secretion of protein in response to submaxi- 6. Sarles H, Tiscornia OM, Palasciano G, et al: Effects of chronic intragastric ethanol administration on canine exocrine panmal doses of CCK is increased, which is in keeping creatic secretion. Scand J Gastroenterol 8:85-96, 1973 with the assumption of an increased cholinergic tone. 16 7. Tiscornia OM, Palasciano G, Sarles H: Effects of chronic ethanol The dose-response curve to CCK is not significantly administration on canine exocrine pancreatic secretion. Further modified by alcohol consumption. The bicarbonate and studies. Digestion 11:172-182, 1974 volume response to submaximal doses of secretin is 8. Tiscornia OM, Palasciano G, Sarles H: Atropine and exocrine also similar in both groups of dogs. The increase of flow pancreatic secretion in alcohol-fed dogs. Am J Gastroenterol rate, bicarbonate output, and concentration in re63:33-36, 1975 9. Treffot MJ, Tiscornia OM, Palasciano G, et al: Chronic alcoholsponse to submaximal doses of secretin and of bicarism and endogenous gastrin. Am J Gastroenterol 63:29-36, 1975 bonate output in response to CCK confirms the in10. Palasciano G, Tiscornia OM, Hage G, et al: Chronic alcoholism creased volume of secreting ductal cells. and endogenous cholecystokinin-pancreozymin. Biomedicine In previous work, we concluded that CCK release 21:94-97, 1974 was probably increased in chronic alcoholic dogs com11. Thomas JE: An improved cannula for gastric and intestinal pared to controlslO and that this difference was supfistulas. Proc Soc Exp BioI Med 46:260-261, 1941 17 pressed by atropin infusion. Percentage change of 12. Henry RJ: Clinical Chemistry. Principles and Technics. New protein output from plateau level under secretin perfuYork, Harper & Row, 1966, p 402-409 sion had been taken as a measure of CCK release after 13. Bradford Hill A: Principles of Medical Statistics. New York, intraduodenal oleic acid infusion. Indeed, percentage Oxford University Press, 1961, p 149-150 increase of protein secretion was greater after 4 14. Sarles H, Sahel J, Lebreuil G, et al: Alcoholic experimental pancreatitis in dog. Pathological study. BioI Gastroenterol months of alcohol consumption than in controls. But (Paris) 8:363, 1975 this could be an artefact caused by the unexplained low protein secretion induced by secretin infusion after 4 15. Dreiling DA, Greenstein A, Bordalo 0: The hypersecretory states of the pancreas. Am J Gastroenterol 59:505-511, 1973 months: the peak secretory response to oleic acid after 16. Vaysse N, Pascal JP, Roux P, et al: Role of cholinergic mecha4, 24, and 36 months is not different from control valnism in the response to secretin of isolated canine pancreas. ues. The fact that 1 control dog submitted to alcohol Gastroenterology 69:1269-1277, 1975 feeding for 12 months responded to intraduodenal oleic 17. Voirol M, Bretholz A, Laugier R, et al: Atropine-induced inhibiacid infusion after alcohol feeding as before confirms tion of the enhanced CCK release observed in alcoholic dogs. Digestion (in press) that, contrary to the pattern of gastrin and secretin
Vol 72, No.2
GASTROENTEROLOGY
Printed in U.8A.
CHRONIC ALCOHOLISM AND CANINE EXOCRINE PANCREAS SECRETION
A long term follow-up study H.
SARLES,
O.
G. P ALASCIANO
TISCORNIA, AND
Unite de Recherches de PathoLogie Digestive, Institute NationaLe de la Sante et La Recherche Medicale U 31, Marseille, France
Sequential changes in pancreatic secretion were evaluated during a 2- to 3-year follow-up study in 8 dogs provided with Thomas gastric and duodenal fistulas. Four animals were given intragastric ethanol (2 g kg- I) daily for 3 years; 4 others served as controls. In alcohol-fed animals: (1) Flow rate and bicarbonate output in response to 1 clinical unit kg-I hr- I of secretin were increased at the end of 2 years but not after 1 year of alcohol feeding. (2) At the end of 2 years, the dose-response curve of pancreatic secretion to cholecystokinin was unchanged but the maximal bicarbonate and water secretion in response to high doses of secretin were increased. Modifications (1) and (2) are explained by reduplication of pancreatic ducts secondary to the development of chronic pancreatitis, verified by biopsy. (3) The previous report by our group of an increased cholecystokinin release in the course of chronic alcohol consumption in dogs seems to be attributable to an artefact. This work shows that the pancreatic response to intraduodenal oleic acid is not significantly modifed by 3 years of alcohol feeding. When men, rats, and dogs l - 5 are not accustomed to the regular consumption of ethanol, an intravenous infusion of ethanol decreases pancreatic exocrine secretion. But when dogs are submitted to daily intragastric administration of ethanol, intravenous ethanol increases pancreatic exocrine secretion. 6 , 7 These two opposite actions affect protein more than water and bicarbonate secretion, and both are mediated by cholinergic nerves. 8 (D. Celener, P. Lechene, O. M. Tiscornia, et al., manuscript submitted for publication). These nerve-mediated changes are associated with modifications of gastrointestinal hormone release. In alcoholfed dogs, a meal induces a greater gastrin release than in controls9 but secretin release after intraduodenal injection of HCl is decreased. 11 On the other hand, protein output in response to an intraduodenal infusion of oleic acid (expressed as a percentage of pre infusion levels) is increased, which has been interpreted as being caused by an increased release of cholecystokinin (CCK).IO The aim of the present study was to analyze after 2 to 3 years of intragastric ethanol feeding: (1) the sequential changes of pancreatic secretion induced either by secretin infusion alone or by the association of secretin and CCK; (2) the changes in the sensitivity and in the maximal secretory response to secretin and to CCK; (3) the sequential modifications of pancreatic secretion in response to intraduodenal infusion of oleic acid. These
studies were carried out in comparison with a control group of dogs matched by weight with alcohol-fed dogs. Methods Animal Preparations Eight mongrel dogs (12 to 30 kg) were provided with a duodenal Thomas cannula 12 opposite the main pancreatic duct; the accessory pancreatic duct was ligated and divided. A Thomas cannula was also inserted in the most dependent part of stomach. At least 4 weeks were allowed for recovery. Dogs were deprived of food, but not water, (or 18 hr before tests. During tests, the gastric cannula was kept open to prevent endogenous release of duodenal hormones by gastric contents. Before each test a glass cannula, connected to a piece of polyethylene tubing, was inserted into the main pancreatic duct papilla. Pancreatic juice was continuously collected into ice-chilled graduated centrifuge tubes and divided into 20-min samples. Dogs were allotted to two groups of 4 animals. Group A: after completion of standard tests as described below, these dogs were made "alcoholic" by being given 2 g kg- I of ethanol daily for 36 months. Ethanol was diluted to 50% (v/v) with normal saline and was given through the gastric cannula. In the fasting state (morning), a protein-supplemented diet was then given, and in the afternoon, dogs were coaxed to eat an extra food ration. Group B: a second group of dogs was used as control and received the same diet without ethanol. Each control dog was weight-matched to 1 experimental (alcoholic) dog. All dogs maintained excellent nutritional status. Three kinds of protocols were applied to both groups.
Protocols Protocoll . Pancreatic response to secretin alone or secretin and cholecystokinin-pancreozymin (CCJ(-PZ) was tested repeatedly before and after (1.5, 3, 12, and 24 months) the
Received September 18, 1975. Accepted July 26, 1976. Address requests for reprints to: Professeur H. Sarles, Unite de Recherches de Pathologie Digestive, INSERM U 31, 46 chemin de la Gaye, 13009 Marseille, France. 238
February 1977
CHRONIC ALCOHOLISM AND PANCREATIC SECRETION
beginning of intragastric ethanol administration in group A dogs and at an interval of 24 months in group B dogs. Both hormones were purchased from the Gastrointestinal Hormone Research Laboratory, Karolinska Institutet, Stockholm, Sweden. Hormones were diluted with normal saline and were infused into a leg vein at a constant rate of 100 ml per hr. The doses administered were: secretin alone, 1 clinical unit kg- 1 hr- 1 ; and secretin, 1 clinical unit kg-I hr- I + CCK-PZ, 3 CrickHarper-Raper (CHR) units kg-I hr-l. The infusions lasted 100 min. The mean value during the last 40 min was considered as the plateau. Protocol 2. Dose-response to secretin and to CCK-PZ were performed in 3 alcoholic dogs, 24 months after beginning ethanol feeding, and in 3 paired control dogs after the same period. Dose-response curves were constructed by intravenous infusion of increasing doses, each dose being doubled every 60 min. Dose-response to secretin was obtained with 5 doses: 0.5, 1, 2, 4, and 8 clinical units kg- 1 hr- I. A dose-response curve to CCK-PZ was also obtained with 5 doses: 1.5, 3, 6, 12 and 24 CHR units kg- 1 hr- I, superimposed on a 1 clinical unit kg-I hr- I secretin background. To construct dose-response curves, mean values of the last 40 min at each dose were used. Protocol 3. Endogenous release of duodenal hormones (CCK-PZ) was repeatedly studied by infusing oleic acid through the duodenal cannula at 4, 24, and 36 months after starting ethanol feeding; again, control dogs were studied twice at an interval of 24 months. Oleic acid solution (20 ml) was infused over a 10-min period, when pancreatic secretion had reached its plateau under secretin (1 clinical unit kg- 1 hr- 1 ) stimulation. Pancreatic response was expressed as secretion after oleic acid infusion and as a ratio: (peak secretion after oleic acid infusion -;. secretin plateau value) x 100. TABLE
239
At the end of 2 years, 1 control dog was given 2 g kg- 1 of ethanol daily as previously described and oleic acid experiments were repeated after 12 months of alcohol feeding.
Chemical Determinations The volume of every pancreatic juice sample was read to the nearest 0.1 ml. Samples were then taken to determine bicarbonate, protein, and calcium concentrations. '2 Outputs were calculated as concentration times volume. HC0 3 concentration was determined by the VanSlyke volumetric method; protein concentration was determined by absorption at 280 nm (E:~~· = =:; 20) ; calcium was determined by atomic absorption spectrophotometry. Statistical Analysis Experimental data were statistically evaluated with Student's t-test, for paired or nonpaired values '3 according to which protocol had been followed.
Results Sequential Changes in Pancreatic Response to Exogenous Hormones Response to secretin alone (1 clinical unit kg- 1 hr- 1) (table 1). In group A, alcoholic dogs, response to secretin was as follows. When compared to the pre-ethanol value in the same dogs, pancreatic secretion during ethanol administration was not modified during the 1st year of alcohol feeding but later on showed a dissociation between volume and HC03 secretion on the one hand and protein secretion on the other: 24 months of
1. Secretin-induced pancreatic secretion of 4 dogs before and during alcohol-fed stage and of 4 controls Alcohol-fed dogs
Parameter
Nonalcoholic period"
Flow rate (ml/20 min) RC03 - (mEq/liter) RC0 3 - output (mEq/20 min) Protein (mg/mD Protein output (mg/20 min)
15.9 144 2.3 2.4 43.7
± ± ± ± ±
2.8 2.6 0.4 0.4 13.1
Control dogs
Alcohol-fed stage 3 moO
11.6 158 1.9. 2.3 26.5
± ± ± ± ±
0.5 3.4 0.1 0.3 3.3
12 moO
14.9 142 2.14 1.2 18.6
± ± ± ± ±
2.0 5.8 0.4 0.3 d 4.5
24 moO
23 144 3.3 0.1 24.3
± ± ± ± ±
1.6 c
1.6 0 ..2 0.2d 6.0
Stage 0 mo·
15.4 131.5 2.0 1.4 24.4
± ± ± ± ±
2.1 2.8 0.3 0.1 4.9
Stage 24 mo·
20.9 148 3.1 1.6 40.0
± ± ± ± ±
1.2 3.0 0.2 0.2 4.7
Means ± SEM of 8, 5, 4, and 24 experiments, respectively, on 4 alcohol-fed dogs. Means ± SEM of 13 and 17 tests, respectively, on control dogs. Statistical analysis by the t-test applied to a pairs comparison showed no significant changes. c Significant difference (P = 0.025) from nonalcoholic period. d Significant difference (P = 0.002) from nonalcoholic period. a b
TABLE
2. Secretin + cholecystokinin (CCK)-induced pancreatic secretion of 4 dogs before and during alcohol-fed stage and of 4 controls Alcohol-fed dogs Parameter
Flow rate (ml/20 min) RC03 - (mEq/liter) RCOa- output (mEq/20 min) Protein (mg/mD Protein output (mg/20 min)
Nonalcoholic period"
Control dogs
Alcohol-fed stage 6 wk"
3 moO
12 moO
24 moO
Stage 0 mo·
Stage 24 mo·
24.8 ± 3.1
28.4 ± 1.0
28.8 ± 5.5
24.1 ± 6.6
26.1 ± 4.0
27.1 ± 2.1
32.4 ± 2.7
152 ± 3.5 3.77 ± 0.5
150 ± 3.5 4.32 ± 0.2
160 ± 2.6c 4.0 ± 0.9
151 ± 1.8 3.8 ± 1.0
141 ± 2.9 3.8 ± 0.5
152.5 ± 1.1 4.4 ± 0.3
140.5 ± 5.5 4.5 ± 0.4
2.6 ± 0.6 59.0 ± 4.1
2.9 ± 0.3 77.7 ± 6.1
2.3 ± 0.5 70.9 ± 29.1
1.9 ± 0.5 46.9 ± 18.1
1.8 ± 0.3 50.2 ± 11.4
2.6 ± 0.3 71.5 ± 9.8
2.1 ± 0.2 69.5 ± 10.5
Means ± SEM of 13,4, 4, 4, and 30 experiments, respectively, on 4 alcohol-fed dogs. Means ± SEM of 13 and 7 tests on 3 control dogs. Statistical analysis by the t-test applied to a paired comparison showed no significant changes. c Significant difference with the nonalcoholic period at 0.05 level. a
b
240
SARLES ET AL.
ethanol administration increased both volume and HC03 output by about 40% as compared to pre-ethanol values; bicarbonate output increase was statistically significant, but volume increase was not. Protein concentration and output decreased by 60 and 40%, respectively, as against pre-ethanol values; protein output decrease was statistically significant. In contrast to ethanol-fed dogs, group B, control dogs, did not show any statistically significant changes after 24 months. Response to secretin (1 clinical unit kg- 1 hr- 1 ) + CCK-PZ (3 CHR units kg- 1 hr- 1 ) (table 2). Mter 1.5 to 24 months of alcohol feeding, pancreatic response to a combined secretin + CCK-PZ stimulation was not significantly different from that before ethanol administration in group A dogs, nor from that after 24 months in group B dogs. TABLE
Dose-Response Studies in Alcoholic and Control Dogs Dose response to secretin (table 3, fig. 1). Mter 24 months of ethanol administration, observed maximal responses of volume, HC0 3 concentration, and output were significantly higher in the 3 alcoholic dogs than in their matched controls. On the contrary, responses to sub maximal doses of volume, protein, bicarbonate, and calcium concentrations and outputs did not show any difference between groups. Calcium values were almost always lower in alcoholic dogs than in controls. Dose response to CCK-PZ (table 4, fig. 2). After 24 months of ethanol feeding, observed maximal responses in volume and HC0 3 - output were similar in both groups, but HC0 3 - concentration after 12 CHR U kg- 1 hr-l was significantly higher in alcoholic dogs, which is in keeping with the fact that with these doses
3. Exocrine pancreatic secretion response of control (C) and alcohol-fed (AF) dogs after different doses of a continuous intravenous injection of secretin a Intravenous secretin (U/kg/hr) Parameter Group 0.5
Flow rate (JLl/kg/min) HC03 - (mEq/liter) HC03 - output (JLEq/kg/min) Protein concentration {mg/mll Protein output (JLg/kg/min) Ca++ (mg/liter) Ca++ output (JLg/kg/min) a b C
C AF C AF C AF C AF C AF C AF C AF
9.1 11.1 95.7 100.7 0.9 1.2 6.7 7.6 68.5 89.3 2.8 3.1 25.2 32.1
± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.0
1.5 3.8 7.8 14.4 0.2 0.6 2.5 1.3 35.4 34.1 0.3 0.6 4.7 10.7
25.5 31.4 128.2 127.7 3.4 4.3 2.5 3.7 52.9 100.0 1.9 1.3 55.0 37.3
1.5
HC03 - (mEq/liter) HC0 3 - output (JLEq/kg/min) Protein concentration {mg/mll Protein output (JLg/kg/min) Cl- (mEq/liter) Cl- output (JLEq/kg/min)
C
d
2.0
5.2 6.6 7.2 10.5 0.8 1.1 0.5 0.7 11.8 19.5 0.6 0.1 26.2 7.0
45.9 48.3 140.8 142.3 6.5 6.9 1.4 1.7 67.1 72.6 1.4 0.8 69.7 38.9
± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.0
5.1 6.1 1.7 5.8 0.7 1.0 0.3 0.5 19.0 16.0 0.4 0.1 23.4 8.1
59.1 75.0 123.5 137.5 7.3 10.3 0.7 0.8 45.2 63.1 0.9 0.9 55.2 63.6
± ± ± ± ± ± ± ± ± ± ± ± ± ±
8.0
4.3 3.2b
1.7
2.8 e
0.5
0.6 e
0.0 0.2 7.8 16.1 0.1 0.2 10.5 17.4
70.7 80.7 107.7 134.3 7.6 10.8 0.9 0.7 60.8 57.8 0.9 0.5 69.5 37.3
± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.9 1.3 1.2
4.6 e
0.6
0.5 b
0.1 0.2 5.7 18.9 0.2 0.2 21.8 14.8
4. Exocrine pancreatic secretion response of control (C) and alcohol-fed (AF) dogs after different doses of cholecystokinin (CCK) superimposed on a continuous intravenous secretin perfusion a CCK (U/kg/hr) + secretin 1.0 U/kg/h- 1 Parameter Group
Flow rate (JLl/kg/min)
a
± ± ± ± ± ± ± ± ± ± ± ± ± ±
Values are means ± SEM of six experiments on 3 control and 3 alcohol-fed dogs. Significantly different from control (P = 0.02). Significantly different from control (P = 0.005).
TABLE
b
Vol. 72, No.2
C AF C AF C AF C AF C AF C AF C AF
33.5 33.6 124.7 126.5 4.5 4.2 3.2 4.4 100.6 149.7 30.5 24.7 0.8 0.8
± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.0
7.0 1.5 9.7 1.5 1.1 0.1 0.6 0.7 25.6 30.0 7.9 2.7 0.1 0.1
37.9 38.1 126.2 126.0 4.8 4.9 3.4 3.1 133.4 119.7 29.1 22.2 1.0 0.8
± ± ± ± ±
± ± ± ± ± ± ± ± ±
6.0
6.9 4.6 4.9 10.3 0.9 1.0 0.6 0.6 36.3 28.1 3.6 3.8 0.2 0.1
Values are means ± SEM of six experiments on 3 control and 3 alcohol-fed dogs. Significantly different from control (P = 0.005). Significantly different from control (P = 0.05). Significantly different from control (P = 0.01).
60.1 58.3 132.2 138.5 7.9 8.1 3.4 4.0 201.5 229.3 26.5 16.1 1.6 0.9
± ± ± ± ± ± ± ± ± ± ± ± ± ±
12.0
4.5 6.6 2.8 2.7 0.6 1.0 0.2 0.5 11.8 37.5 3.5 2.6 e
0.2 O.le
71.4 69.1 116.3 139.2 8.3 9.5 4.8 4.1 334.9 286.6 39.0 20.7 2.8 1.5
± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.0 6.4 4.9
3.6 b
0.7 0.7 0.5 0.6 31.8 51.2 2.7
3.6 b
0.3 0.3 d
24
90.6 93.0 140.7 135.0 12.6 12.5 5.5 3.8 498.5 359.2
± ± ± ± ± ± ± ± ± ±
6.9 5.4 9.9 3.5 0.2 0.4 0.8 1.0 57.0 100.4
Control· 4th month AF' 24th month AFD 36th month AF' Control· 4th month AF' 24th month AFD 36th month AF' Control· 4th month AF' 24th month AFD 36th month AF' Control· 4th month AF' 24th month AFD 36th month AF' Control· 4th month AF' 24th month AFD 36th month AF'
Dogs
17.9 19.0 15.9 15.8 144.2 161 136.2 139.6 2.6 3.0 2.2 2.3 1.7 0.8 1.3 2.3 30.6 15.2 29.9 33.0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.3 1.5 2.2 2.8 2.4 1.1 3.9 3.5 0.2 0.4 0.3 0.5 0.1 0.2 0.1 0.5 3.7 5.0 7.8 6.0
Plateau
25.5 31.8 30.4 27.6 145.2 161 145.3 151.8 3.7 5.1 4.4 4.2 4.3 3.4 3.7 4.7 112.4 108.6 119.8 130.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.4e 0.7 e 0.6 d 16.9 d 17.0e 23.1J 18.0 d
3.3'
0.8 e 0.6 d
0.7
0.3 d
3.6 4.6 6.1 3.3
2.3' 2.3
SEM
5.6 e 3.2e
Mean ±
0-20
+42 +67 +91 +75 +0.6 0.0 +7 +9 +42 +69 +100 +83 +158 +308 +183 +100 +267 +614 +301 +297
change a
%
25.9 31.6 27.9 35.0 139.2 160 146 157.8 3.6 5.1 4.1 5.5 4.5 3.5 4.5 4.93 116.3 115.6 116.4 167.3 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
21.3 J 1.0 h
12.0J
0.5
2.0e 0.2d
4.2 4.8 4.8
4.6 e 2.4 h
2.2
1.8 d
SEM
0.7 e O.4 h 2.0h O.4 e O.4 d O.4 h 12.7 d
Mean ±
20-40
+45 +66 +77 +122 -3 -0.6 +7 +13 +38 +68 +88 +139 +169 +327 +245 +110 +279 +661 +290 +407
change a
%
26.6 31.2 27.7 34.7 136.6 156 141 157.8 3.7 4.9 3.9 5.5 4.4 3.5 4.3 5.0 115.2 112.4 118.0 165.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.9 4.0 5.3 7.7 2.10.3 0.6 0.7'
4.8 e
2.41 d 2.8
SEM
24.5 J 14.0h
15.0J
0.5 h O.4 h 0.5 e 0.6 e 0.5 h 14.9 d
Mean ±
40-60
+49 +64 +74 +120 -5 -3 +4 +13 +41 +63 +78 +137 +161 +320 +234 +112 +276 +639 +295 +403
change a
% ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.2
3.0 e
SEM
60-80
25.3 J 12.0h
1S.0
0.6 h 15.4 d
0.9J 0.8 J
0.5 d 0.5 h 0.4h
3.4 8.4 8.3 0.9' 0.4' 0.4
4.5 d 3.1h
Mean ± 24.9 27.8 28.8 30.3 131.9 153 141.3 152.6 3.3 4.4 3.9 4.6 4.1 4.2 4.1 4.8 101.6 121.2 98.4 130.9
Secretin (1.0 CU/k~ '/hr ') after intraduodenal oleIc acid (min)
+39 +44 +81 +92 -9 -5 +4 +9 +27 +46 +81 +100 +145 +410 +215 +101 +232 +697 +229 +297
change a
%
23.8 25.4 26.6 28.0 128.7 157 141.3 151.0 3.0 4.0 3.7 4.3 4.2 3.7 4.0 5.2 101.9 105.0 100.7 132.3
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
28.5 J 17.0h
25.0
0.7 e 18.2d
0.7 J 0.8 J
o.e 0.6 e O.4 h
4.2 6.8 9.0 2.3 0.4 0.5
3.6 h 3.W
SEM
80-100
3.5' 3.4
Mean ±
5. Exocrine pancreatic secretion response elicited by intraduodenal oleic acid infusion superimposed on secretin perfusion in control and alcohol-fed (AF) dogs
a Percentage difference compared with plateau level. • As the results of control dogs at the beginning and at the 24th month of experiment were not statistically different, they were pooled together; mean of 12 tests on 4 control dogs. , Significantly different from plateau (P = 0.05). d Significantly different from plateau (P = 0.005). e Significantly different from plateau (P = 0.01). , Mean of eight tests on 4 dogs. D Mean of six tests on 3 dogs. h Significantly different from plateau (P = 0.001). , Mean of eight tests on 2 dogs. J Significantly different from plateau (P = 0.02).
Protein output (mg/20 min)
Protein (mg/m!)
HC03 - output (mEq/20 min)
HC03 - (mEq/ liter)
Flow rate (m1/20 min)
Parameter
TABtE
+33 +34 +67 +77 -11 -2 +4 +8 +15 +32 +69 +83 +149 +351 +211 +120 +233 +591 +237 +310
change a
%
~
>-' """
t-:l
~
~
~
~
C1
::j
~ ~
~
~
>
~
~
~
C1
~
~ C1
~
o
::ti
'0 '-l '-l
'-
<:!
§
0-
242
SARLES ET AL .
IlEq / kg/min
T J '-e~
11
Alcohol- Fed Dogs
9
7
j
I
"".-'
.... I-
.
•• .-' '-'1-
Control Dogs
5
3
Ii
1
2
0,5 SECRETIN
4
8
(Clinical Units)
FIG . 1. Bicarbonate output in control and alcohol-fed dogs after different doses of continuous intravenous infusion of secretin. Significant differences at 0.05 and 0.02 levels are indicated by *, ** respectively. 1l9/kg/min Non- Alcoholic Dogs
500
400 Alcoholic Dogs
300
200
100
•
I
Vol . 72, No . 2
of hormone, chloride concentration and output were lower. Therefore, the sum of chloride and bicarbonate is constant. Protein secretion was not significantly different between the two groups, although lower values were obtained in alcoholic dogs in response to high CCK-PZ doses.
Intraduodenal Oleic Acid Infusion Tests (Table 5) In alcohol-fed dogs, there was a relative increase in protein output and concentration after oleic acid, when expressed as: (protein secretion (output and concentration) in response to oleic acid -;- protein secretion before oleic acid) x 100, although this difference diminished from the 4th to the 36th month. But protein secretion in response to secretin before oleic acid infusion was significantly lower at the 4th month than in controls, and the peak secretory response to oleic acid was not significantly different in controls and after 4, 24, and even 36 months of alcohol consumption. In the control dog that was subsequently fed ethanol for 12 months, peak pancreatic secretion in 20-min samples after oleic acid was as shown in table 6. Discussion The pancreatic secretory response to sub maximal doses of secretin and of secretin + CCK-PZ was not modified either during the 24-month follow-up of control dogs or during the first 12 months of alcohol feeding. After 24 months of alcohol feeding, bicarbonate output was significantly increased in comparison with output before alcohol feeding. Increased response to secretin could be attributable either to an increased sensitivity of ductal cells to secretin or to an increased number of ductal cells. This question can be answered by a pathological study realized in the same animals, 14 and indeed ductal reduplication was evident in the pancreatic biopsies after 2 years of alcohol feeding. A similarly increased secretion of water and bicarbonate response to secretin has been observed in human chronic pancreatitis. 15 The fact that in dogs this change appears only at the end of 2 years of alcohol consumption, when protein plugs and microstones are found as early as the 3rd month,6 shows that modifications of water and bicarbonate secretion attributable to ductal hyperplasia are not the cause of chronic pancreatitis but are relatively late phenomena during the course of chronic alcohol consumption and its resulting pancreatic lesions. On the contrary, protein secretory response to secretin diminished during the course of alcohol feeding. TABLE
6. Peak pancreatic secretion after oleic acid in control dog subsequently fed tehanol" Parameter
1.5
3
6
12
24
PANCREOZYMIN (C .H.R. Units)
FIG . 2. Protein output in control and alcohol-fed dogs after different doses of cholecystokinin (CCK) superimposed on a continuous intravenous infusion of secretin (1.0 clinical unit kg- 1 hr- l).
Volume (m1!20 min) Bicarbonate output (mEq/20 min) Protein output (mg/20 min)
Before alcohol feed- Alcohol-fed ing
24.7 83.7 3.2
23.6 91.4
3.0
These results, which are the mean of three experiments, are not significantly different. a
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CHRONIC ALCOHOLISM AND PANCREATIC SECRETION
243
Although significant, this decrease is probably fortui- release, CCK release is not greatly modified by chronic tous, as the response to secretin before oleic acid infu- alcohol consumption. sion tends to increase during the same time in the REFERENCES same dogs (table 5). This illustrates the irregularity of 1. Mott CB, Sarles H, Tiscornia OM, et al: Inhibitory action of the protein response to secretin in contrast to the staalcohol on human exocrine pancreatic secretion. Am J Dig Dis bility of protein response to secretin + CCK-PZ. In17:902-910, 1972 deed, in none of the controls or alcohol-fed animals was 2. Tiscornia OM, Gullo L, Sarles H: The inhibition of canine exocrine pancreatic secretion by intravenous ethanol. Digestion the protein secretion after CCK-PZ significantly modi9:231-240, 1973 fied. In previous work6 it has been found that in another 3. Tiscornia OM, Palasciano G, Dzieniszewski J, et al: Simultaneous changes in pancreatic and gastric secretion induced by acute series of dogs protein secretory response to CCK was intravenous ethanol infusion. Effects of atropine and reserpine. increased at the end of 6 weeks and 3 months of alcohol Am J Gastroenterol 63:389-395, 1975 feeding, and indeed, in the present series of alcohol-fed 4. Tiscornia OM, Hage G, Palasciano G, et al: The effects of pentodogs, protein output was increased after the same pelinium and vagotomy on the inhibition of canine exocrine panriod of alcohol consumption, although not significreatic secretion by intravenous ethanol. Biomedicine 18:159cantly; this lack of significance is probably attributa163, 1973 ble to a smaller number of experiments. It is therefore 5. Cavarsan A, Teixeira A, Sarles H, et al: Action of intragastric ethanol on the pancreatic secretion of conscious rats. Digestion probable that in the first months of alcohol consump13:145-152, 1975 tion, at least before the onset of pancreatic lesions, pancreatic secretion of protein in response to submaxi- 6. Sarles H, Tiscornia OM, Palasciano G, et al: Effects of chronic intragastric ethanol administration on canine exocrine panmal doses of CCK is increased, which is in keeping creatic secretion. Scand J Gastroenterol 8:85-96, 1973 with the assumption of an increased cholinergic tone. 16 7. Tiscornia OM, Palasciano G, Sarles H: Effects of chronic ethanol The dose-response curve to CCK is not significantly administration on canine exocrine pancreatic secretion. Further modified by alcohol consumption. The bicarbonate and studies. Digestion 11:172-182, 1974 volume response to submaximal doses of secretin is 8. Tiscornia OM, Palasciano G, Sarles H: Atropine and exocrine also similar in both groups of dogs. The increase of flow pancreatic secretion in alcohol-fed dogs. Am J Gastroenterol rate, bicarbonate output, and concentration in re63:33-36, 1975 9. Treffot MJ, Tiscornia OM, Palasciano G, et al: Chronic alcoholsponse to submaximal doses of secretin and of bicarism and endogenous gastrin. Am J Gastroenterol 63:29-36, 1975 bonate output in response to CCK confirms the in10. Palasciano G, Tiscornia OM, Hage G, et al: Chronic alcoholism creased volume of secreting ductal cells. and endogenous cholecystokinin-pancreozymin. Biomedicine In previous work, we concluded that CCK release 21:94-97, 1974 was probably increased in chronic alcoholic dogs com11. Thomas JE: An improved cannula for gastric and intestinal pared to controlslO and that this difference was supfistulas. Proc Soc Exp BioI Med 46:260-261, 1941 17 pressed by atropin infusion. Percentage change of 12. Henry RJ: Clinical Chemistry. Principles and Technics. New protein output from plateau level under secretin perfuYork, Harper & Row, 1966, p 402-409 sion had been taken as a measure of CCK release after 13. Bradford Hill A: Principles of Medical Statistics. New York, intraduodenal oleic acid infusion. Indeed, percentage Oxford University Press, 1961, p 149-150 increase of protein secretion was greater after 4 14. Sarles H, Sahel J, Lebreuil G, et al: Alcoholic experimental pancreatitis in dog. Pathological study. BioI Gastroenterol months of alcohol consumption than in controls. But (Paris) 8:363, 1975 this could be an artefact caused by the unexplained low protein secretion induced by secretin infusion after 4 15. Dreiling DA, Greenstein A, Bordalo 0: The hypersecretory states of the pancreas. Am J Gastroenterol 59:505-511, 1973 months: the peak secretory response to oleic acid after 16. Vaysse N, Pascal JP, Roux P, et al: Role of cholinergic mecha4, 24, and 36 months is not different from control valnism in the response to secretin of isolated canine pancreas. ues. The fact that 1 control dog submitted to alcohol Gastroenterology 69:1269-1277, 1975 feeding for 12 months responded to intraduodenal oleic 17. Voirol M, Bretholz A, Laugier R, et al: Atropine-induced inhibiacid infusion after alcohol feeding as before confirms tion of the enhanced CCK release observed in alcoholic dogs. Digestion (in press) that, contrary to the pattern of gastrin and secretin