141
Atherosclerosis, 24 (1976) 141-148 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CHOLESTEROL THE PIG
METABOLISM
FOLLOWING
PORTACAVAL
SHUNT IN
H. PETER CHASE and TOM MORRIS Department ofpediatrics, (U.S.A.) and Department (New Zealand)
University of Colorado Medical Center, Denver, Cola. 80220 of Surgery, University of Auckland School of Medicine, Auckland
(Received 12th August, 1975) (Revised, received 9th November, 1975) (Accepted 9th November, 1975)
Summary Portacaval shunting resulted in lower serum cholesterol and low density lipoprotein levels in comparison with values for littermate control pigs. Triglyceride levels were lower in the shunted animals only while receiving a low fat diet. The reductions in serum cholesterol and triglyceride levels of shunted pigs while receiving a standard pig diet were shown to be related to reduced hepatic synthesis of cholesterol and triglycerides. Key words: Cholesterol - Lipoprotein
-Pigs
- Portacaval shunt - Triglyceride
Introduction Portacaval shunting has been shown to result in a lowering of blood cholesterol levels and disappearance of skin xanthomas in some patients with homozygous Type II hypercholesterolemia [1,2] . In at least the initial patient regression of atherosclerosis was also suspected [3] . The reason for the lowering of serum cholesterol levels is unknown but could be related to reduced cholesterol synthesis and or increased degradation. In a preliminary report of the effects of portacaval shunting in dogs [4] , serum cholesterol levels were found to This work was financially supported by the Auckland Medical Research Foundation 01AT0376Alllll. NIH Retardation Studies, from the National Institutes of Health, Please send correspondence and reprint requests to H.P.C. in Denver, Colorado.
and Grant HS 0315Bethesda, Md.
142
decrease; however, studies of the rate of hepatic cholesterol synthesis were uninterpretable due to fasting and anesthesia. In the present report, the effects of portacaval shunting on cholesterol and triglyceride metabolism were investigated in young littermate pigs. Methods A litter of ten 5-week-old in-bred New Zealand white pigs was obtained from the Hellaby Research Farm, Auckland, N.Z. They were divided into 2 groups of 5 to have animals of similar sex and weight in each group. Three overnight fasting blood samples were drawn on all animals over a 2-week period (‘l-day intervals) while they were fed 1200 g/day of a 23% protein, 6% fat, 50% carbohydrate standard pig weaning diet, SMR-1, obtained from N.Z. Stock Foods Company, Auckland, N.Z. The third sample was drawn on the day of operation, just following anaesthesia induced with 0.5% halothane, gradually increased to 2.5%, in a mixture of nitrous oxide : oxygen (60 : 40). Test animals underwent end-to-side portacaval shunt whereas control animals had sham operations. Both groups received fluids and bicarbonate during surgery [5] . The control and test pigs were then fed a high fat diet [6] consisting of 800 g of the standard weaning diet, 400 g of butterfat (ghee) and 12 g of cholesterol per day for 4 weeks, followed by 1200 g/day of the standard pig weaning diet for another 4 weeks (Fig. 1). The animals were individually fed and except for 2 days when one control animal had diarrhea, consumed all food offered. Weekly fasting blood samples were drawn from a leg vein without anaesthesia, except at the fourth week when they were drawn during light anaesthesia induced by 2.5% thiopentone sodium with nitrous oxide : oxygen (60 : 40) for an initial open liver biopsy. Intermittent intravenous pentobarbitone was used
40.
30.
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.
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0’
.
? Suigery
’
234+5678
.
.
Bidpsy
Fig. 1. Body weiyhfs. Results represent the mean +l hemi-standard deviation for control sham-operated ahd their littermates with the portacaval shunt (- - - - - -). The horizontal axis represents the pigs ( -_) time in weeks following surgery. with the initial liver biopsy at 4 weeks and the final biopsy at 8 weeks. Weights were significantly different (I ’ i 0.01 or less) at the 5th week and thereafter.
143
as needed during the operation. Serum cholesterol levels were determined by the method of Franey and Amador [7], triglycerides using the BoehringerMannheim Kit, and lipoproteins by the method of Hatch et al. [8] using agarose gel and fat red 7-B stain. Lipoprotein results are reported in integrator units because the precise lipid composition of pig lipoproteins is unknown and would be required for expression of results in mg/lOO ml [8]. Weights and blood samples were not taken during the sixth postoperative week. Animals were sacrificed at 8 weeks by exsanguination without anaesthesia and a final liver specimen taken to measure the rate of cholesterol synthesis. The animals were fed approximately 2 h prior to each of the 2 liver biopsies. The rate of hepatic synthesis of cholesterol and other lipids was measured in tissue slices [9] using 1 PCi [2-14C] acetate and 2 m&f cold acetate, which was shown in preliminary experiments to result in a maximal rate of cholesterol synthesis in porcine liver. After stopping the reactions with 0.5 ml 10 M H,S04, lipids were extracted by the method of Folch [lo]. An initial aliquot of the lipid extract was removed and counted in a liquid scintillation counter to determine [‘“Cl acetate incorporation into total lipids. The remainder of the lipid extract and appropriate standards were applied to silica gel thin layer plates and chromatographed in ether, benzene, ethanol and acetic acid (80 : 100 : 4 : 0.4) [ll]. The plates were placed in an iodine chamber fcr 15 min, the individual bands marked, and the iodine allowed to subliminate away. Desired areas, including a blank in order to subtract background counts, were scraped from the plates and transferred to scintillation vials for countin?,, with recovery of total lipid counts ranging from 93 to 101% of the value for the initial aliquot. Statistical analyses were done using the Student t-test. Results Body weights Mean body weights were similar (P> 0.05) for control (13.2 f 2.6 kg) and test (12.7 f 1.3 kg) animals prior to surgery (Fig. 1). They remained similar through the 4 weeks on high-fat diet, at which time the controls weighed 21.5 f 3.5 kg and the shunted animals 21.7 rf. 1.2 kg. However, one week after reverting to the standard pig diet and thereafter, weights of the control animals were statistically greater (P < 0.01). One of the shunted animals died of sepsis secondary to a wound abscess during its third week on the high-fat diet. The shunt was patent at post-mortem, as it was in the other animals following sacrifice. Serum cholesterol Mean serum cholesterol levels were similar in the control and test groups prior to operation (Fig. 2), but were significantly lower in the shunted animals at 2 (P< 0.05) and 3 (P< 0.02) weeks after operation, when all animals were receiving the high-fat diet. While receiving the high-fat diet, the test animals continued to have levels similar to the preshunt values, whereas levels for the sham-operated animals increased on the high-fat diet. Cholesterol levels were also significantly lower in the shunted pigs at 5 (P < 0.02), 7 (P < 0.01) and 8 (P < 0.001) weeks, when all animals were receiving the standard pig diet. At
144
180. E 160.
I
1,1,1,1~,1//1, Pliigh-fat diet Oweeks 0 1 /,,A,,
20. 50-
Wzeksq
1
234t5678
Surgery Fig. 2. Serum cholesterol sham-operated pigs (-----cantly different (P < 0.05 week.
Biopsy
+ Surgery
‘I--___
I
,“‘Y
23:5678 Biopsy
leuels. Results represent the mean *l hem&standard deviation for the control ) and their littermates with the portacaval shunt (- - - - - -). Results were signifior less) from the 3rd week following surgery and thereafter. except at the 4th
Fig. 3. Serum triglyceride leuels. Results represent the mean +l hem&standard deviation for the control sham operated pigs (----) and their littermates with the portacaval shunt (- - - - - -). Results were significantly different (P < 0.01 or less) from the 5th week, when all animals were changed back to standard pig diet.
this time levels were lower for the shunted animals in comparison tive values when they also received the standard diet.
to preopera-
Serum triglyceride Mean serum triglyceride levels were similar in the control and test groups prior to operation (Fig. 3). There was considerable variation within each group following surgery, and although the mean levels were consistently lower in the shunted group, significant differences were found only at 5 (P < O.Ol), 7 (P < 0.001) and 8 (P < 0.001) weeks, when all pigs were receiving the standard pig diet.
Serum lipoproteins Pig low density lipoprotein (LDL) bands had a similar mobility to human LDL. The weekly mean LDL levels were statistically lower in the shunted compared with control animals at 2, 3, 4, 7 and 8 weeks (Table 1). In comparison to their own preoperative levels, control animals had a statistically higher LDL level 3 weeks after starting the high fat diet, whereas shunted animals had statistically lower levels at 2, 4, 5, 7 and 8 weeks following surgery (Table 1). Very low density lipoprotein bands were less marked than in the human and
1
shunts
(5) 132 *29 >0.05
>0.05
166 +55
156 ?26
146 ?31
1.
Atherosclerosis, 24 (1976) 141-148 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CHOLESTEROL THE PIG
METABOLISM
FOLLOWING
PORTACAVAL
SHUNT IN
H. PETER CHASE and TOM MORRIS Department ofpediatrics, (U.S.A.) and Department (New Zealand)
University of Colorado Medical Center, Denver, Cola. 80220 of Surgery, University of Auckland School of Medicine, Auckland
(Received 12th August, 1975) (Revised, received 9th November, 1975) (Accepted 9th November, 1975)
Summary Portacaval shunting resulted in lower serum cholesterol and low density lipoprotein levels in comparison with values for littermate control pigs. Triglyceride levels were lower in the shunted animals only while receiving a low fat diet. The reductions in serum cholesterol and triglyceride levels of shunted pigs while receiving a standard pig diet were shown to be related to reduced hepatic synthesis of cholesterol and triglycerides. Key words: Cholesterol - Lipoprotein
-Pigs
- Portacaval shunt - Triglyceride
Introduction Portacaval shunting has been shown to result in a lowering of blood cholesterol levels and disappearance of skin xanthomas in some patients with homozygous Type II hypercholesterolemia [1,2] . In at least the initial patient regression of atherosclerosis was also suspected [3] . The reason for the lowering of serum cholesterol levels is unknown but could be related to reduced cholesterol synthesis and or increased degradation. In a preliminary report of the effects of portacaval shunting in dogs [4] , serum cholesterol levels were found to This work was financially supported by the Auckland Medical Research Foundation 01AT0376Alllll. NIH Retardation Studies, from the National Institutes of Health, Please send correspondence and reprint requests to H.P.C. in Denver, Colorado.
and Grant HS 0315Bethesda, Md.
142
decrease; however, studies of the rate of hepatic cholesterol synthesis were uninterpretable due to fasting and anesthesia. In the present report, the effects of portacaval shunting on cholesterol and triglyceride metabolism were investigated in young littermate pigs. Methods A litter of ten 5-week-old in-bred New Zealand white pigs was obtained from the Hellaby Research Farm, Auckland, N.Z. They were divided into 2 groups of 5 to have animals of similar sex and weight in each group. Three overnight fasting blood samples were drawn on all animals over a 2-week period (‘l-day intervals) while they were fed 1200 g/day of a 23% protein, 6% fat, 50% carbohydrate standard pig weaning diet, SMR-1, obtained from N.Z. Stock Foods Company, Auckland, N.Z. The third sample was drawn on the day of operation, just following anaesthesia induced with 0.5% halothane, gradually increased to 2.5%, in a mixture of nitrous oxide : oxygen (60 : 40). Test animals underwent end-to-side portacaval shunt whereas control animals had sham operations. Both groups received fluids and bicarbonate during surgery [5] . The control and test pigs were then fed a high fat diet [6] consisting of 800 g of the standard weaning diet, 400 g of butterfat (ghee) and 12 g of cholesterol per day for 4 weeks, followed by 1200 g/day of the standard pig weaning diet for another 4 weeks (Fig. 1). The animals were individually fed and except for 2 days when one control animal had diarrhea, consumed all food offered. Weekly fasting blood samples were drawn from a leg vein without anaesthesia, except at the fourth week when they were drawn during light anaesthesia induced by 2.5% thiopentone sodium with nitrous oxide : oxygen (60 : 40) for an initial open liver biopsy. Intermittent intravenous pentobarbitone was used
40.
30.
$0. E .-0)
.
r” 10.
0’
.
? Suigery
’
234+5678
.
.
Bidpsy
Fig. 1. Body weiyhfs. Results represent the mean +l hemi-standard deviation for control sham-operated ahd their littermates with the portacaval shunt (- - - - - -). The horizontal axis represents the pigs ( -_) time in weeks following surgery. with the initial liver biopsy at 4 weeks and the final biopsy at 8 weeks. Weights were significantly different (I ’ i 0.01 or less) at the 5th week and thereafter.
143
as needed during the operation. Serum cholesterol levels were determined by the method of Franey and Amador [7], triglycerides using the BoehringerMannheim Kit, and lipoproteins by the method of Hatch et al. [8] using agarose gel and fat red 7-B stain. Lipoprotein results are reported in integrator units because the precise lipid composition of pig lipoproteins is unknown and would be required for expression of results in mg/lOO ml [8]. Weights and blood samples were not taken during the sixth postoperative week. Animals were sacrificed at 8 weeks by exsanguination without anaesthesia and a final liver specimen taken to measure the rate of cholesterol synthesis. The animals were fed approximately 2 h prior to each of the 2 liver biopsies. The rate of hepatic synthesis of cholesterol and other lipids was measured in tissue slices [9] using 1 PCi [2-14C] acetate and 2 m&f cold acetate, which was shown in preliminary experiments to result in a maximal rate of cholesterol synthesis in porcine liver. After stopping the reactions with 0.5 ml 10 M H,S04, lipids were extracted by the method of Folch [lo]. An initial aliquot of the lipid extract was removed and counted in a liquid scintillation counter to determine [‘“Cl acetate incorporation into total lipids. The remainder of the lipid extract and appropriate standards were applied to silica gel thin layer plates and chromatographed in ether, benzene, ethanol and acetic acid (80 : 100 : 4 : 0.4) [ll]. The plates were placed in an iodine chamber fcr 15 min, the individual bands marked, and the iodine allowed to subliminate away. Desired areas, including a blank in order to subtract background counts, were scraped from the plates and transferred to scintillation vials for countin?,, with recovery of total lipid counts ranging from 93 to 101% of the value for the initial aliquot. Statistical analyses were done using the Student t-test. Results Body weights Mean body weights were similar (P> 0.05) for control (13.2 f 2.6 kg) and test (12.7 f 1.3 kg) animals prior to surgery (Fig. 1). They remained similar through the 4 weeks on high-fat diet, at which time the controls weighed 21.5 f 3.5 kg and the shunted animals 21.7 rf. 1.2 kg. However, one week after reverting to the standard pig diet and thereafter, weights of the control animals were statistically greater (P < 0.01). One of the shunted animals died of sepsis secondary to a wound abscess during its third week on the high-fat diet. The shunt was patent at post-mortem, as it was in the other animals following sacrifice. Serum cholesterol Mean serum cholesterol levels were similar in the control and test groups prior to operation (Fig. 2), but were significantly lower in the shunted animals at 2 (P< 0.05) and 3 (P< 0.02) weeks after operation, when all animals were receiving the high-fat diet. While receiving the high-fat diet, the test animals continued to have levels similar to the preshunt values, whereas levels for the sham-operated animals increased on the high-fat diet. Cholesterol levels were also significantly lower in the shunted pigs at 5 (P < 0.02), 7 (P < 0.01) and 8 (P < 0.001) weeks, when all animals were receiving the standard pig diet. At
144
180. E 160.
I
1,1,1,1~,1//1, Pliigh-fat diet Oweeks 0 1 /,,A,,
20. 50-
Wzeksq
1
234t5678
Surgery Fig. 2. Serum cholesterol sham-operated pigs (-----cantly different (P < 0.05 week.
Biopsy
+ Surgery
‘I--___
I
,“‘Y
23:5678 Biopsy
leuels. Results represent the mean *l hem&standard deviation for the control ) and their littermates with the portacaval shunt (- - - - - -). Results were signifior less) from the 3rd week following surgery and thereafter. except at the 4th
Fig. 3. Serum triglyceride leuels. Results represent the mean +l hem&standard deviation for the control sham operated pigs (----) and their littermates with the portacaval shunt (- - - - - -). Results were significantly different (P < 0.01 or less) from the 5th week, when all animals were changed back to standard pig diet.
this time levels were lower for the shunted animals in comparison tive values when they also received the standard diet.
to preopera-
Serum triglyceride Mean serum triglyceride levels were similar in the control and test groups prior to operation (Fig. 3). There was considerable variation within each group following surgery, and although the mean levels were consistently lower in the shunted group, significant differences were found only at 5 (P < O.Ol), 7 (P < 0.001) and 8 (P < 0.001) weeks, when all pigs were receiving the standard pig diet.
Serum lipoproteins Pig low density lipoprotein (LDL) bands had a similar mobility to human LDL. The weekly mean LDL levels were statistically lower in the shunted compared with control animals at 2, 3, 4, 7 and 8 weeks (Table 1). In comparison to their own preoperative levels, control animals had a statistically higher LDL level 3 weeks after starting the high fat diet, whereas shunted animals had statistically lower levels at 2, 4, 5, 7 and 8 weeks following surgery (Table 1). Very low density lipoprotein bands were less marked than in the human and
1
shunts
(5) 132 *29 >0.05
>0.05
166 +55
156 ?26
146 ?31
1.
