Exp. Geront. Vo I. 14, pp. 201-204. ~, Pergamon Press Ltd. 1979. Pri,qted in Great Britain.
0531-5565/79/0801-0201 $02.00[0
INCREASE IN CHOLESTEROL INTESTINAL ABSORPTION WITH AGING IN THE RAT* D. HOLLANDER a n d D. MORGAN Division t~l Gastroenterology, Wayne State University and tlarper Grace ttospitals, Detroit, MI 48201, U.S.A.
(Received 24 July 1978) INTRODUCTION ARTERIOSCt,EROTIC c a r d i o v a s c u l a r disease, a d i s e a s e a s s o c i a t e d w i t h a g i n g , is c a u s e d by e x c e s s i v e a m o u n t s o f c h o l e s t e r o l d e p o s i t s in t h e l i n i n g o f b l o o d vessels. T h e s m a l l i n t e s t i n e is t h e site o f a b s o r p t i o n o f c h o l e s t e r o l f r o m d i e t a r y s o u r c e s ( B o r g s t r o m , 1960). E n d o g e n o u s l y s y n t h e s i z e d c h o l e s t e r o l w h i c h is e x c r e t e d w i t h bile i n t o t h e l u m e n o f t h e d u o d e n u m is a l s o a b s o r b e d b y t h e s m a l l i n t e s t i n e ( T r e a d w e l l et al., 1959). S i n c e a r t e r i o s c l e r o t i c c a r d i o v a s c u l a r d i s e a s e is a d i s e a s e o f a g i n g a n d since t h e s m a l l b o w e l is t h e site o f c h o l e s t e r o l a b s o r p t i o n , we d e c i d e d t o i n v e s t i g a t e t h e r e l a t i o n s h i p b e t w e e n a g i n g a n d i n t e s t i n a l a b s o r p tion of cholesterol. Our studies showed that cholesterol absorption increases linearly with a g i n g , e v e n a t t h e e x t r e m e e n d o f t h e a n i m a l ' s life e x p e c t a n c y . MATERIALS AND METHODS Animal.~ Intestinal absorption of cholesterol was studied in male Sprague-Dawley rats varying in ages from I to 42 months. The rats were allowed free access to regular chow and water and were kept under identical conditions before experimentation. Three to six rats were studied in each age group.
Surgical preparation Each rat was anesthetized with ether, its abdomen was opened, and an inflow cannula was inserted into the lumen of the small bowel 3 cm distal to the entry of the common bile duct into the duodenum. The distal end of the small bowel was cannulated with a glass cannula just prior to the junction of the ileum and cecum. The rat was placed in a restraint cage which allowed mobility but prevented dislodgement of the catheters (Hollander et al., 1977). The animal's normal body temperature was maintained with a forced-air heating device which was activated by a thermostatic temperature controller (Hollander and Muralidhara, 1977). Experimental perfusions The infusate solution was composed of 10 mM sodium taurocholate in a Krebs phosphate buffer at pH 7.4. Inulin, a 14C-labeled nonabsorbable sugar, was added to the perfusate to detect changes in cholesterol concentration secondary to water transport and to correct the final data for this artifact (Miller and Schedl, 1970). SH-la, 2u (n) cholesterol with specific activity of 43 Ci/mmol was used as a tracer compound and was added to the nonradioactive cholesterol in the infusate to a final concentration of 50 taM. A micellar solution was prepared by ultrasound irradiation with a sonicator. The solution was infused into the proximal small bowel at a constant rate of 1 ml/min over a 2 h period and the outflow was collected in 6 separate 20 rain aliquots. Determination of absorption rate The absorption of cholesterol during each 20 rain period was calculated by subtracting the total amount of cholesterol collected out of the outflow cannula from the amount known to have been infused into the proximal small intestine during the same 20 min period. 8H-cholesterol was separated from ~aC-inulin by total combustion of triplicate aliquots of the intestinal perfusate with a tissue oxidizer (Hollander et al., 1976). Radioactivity was than assayed with a beta liquid scintillation counter to an accuracy of ± 1 ° o, The corrected net absorption rate of cholesterol in each 20 min period was calculated and all 6 values for absorption from each animal were pooled with the values obtained from other animals of the same age group, *Mail requests for reprints to: Daniel Hollander, M.D., Department of Medicine, 5901 East 7th St.. Long Beach, CA 90801, U.S.A. 201
202
D. HOLLANDERAND 1). MORGAN
for calculation of the mean i S.E. rate of absorption of cholesterol. Cholesterol absorption by each age group was also expressed in terms of percentage absorption of the amount of cholesterol infused per h. Determination o f intestinal size The animals were killed with an overdose of ether at the completion of perfusion and the perfused intestinal segments were removed. Well-standardized methods (Hollander and Muralidhara, 1977) of stretching and desiccating the bowel were used for measuring the intestinal weight and length in the dry state, so that the segments from the various age groups could be compared.
RESULTS When the rate of cholesterol absorption in each age group was plotted against the age of the animals, a linear relationship was found between the two parameters (Fig. 1). Absorption of cholesterol continued to increase until the extreme end of the animal's life expectancy (42 months) (Hollander, 1976). When cholesterol absorption was expressed as a percentage of the amount infused, 14~o was absorbed at the age of 1 month, as compared to 38 o/ /o at the age of 42 months (Table 1). The body weight and the intestinal length and weight of each group of animals showed a rapid rise during their first 12 months of life (Table 1). However, during the latter 30 months of life the animals showed no further significant increase in these three parameters (Table 1).
I0001
CHOLESTEROL ABSORPTION
900:E
(_)
o
800-
700i.i.i
/ = 4 4 5 ~ Ilx
~. 6oo500-~
I IO
2'0 MONTHS
p
0531-5565/79/0801-0201 $02.00[0
INCREASE IN CHOLESTEROL INTESTINAL ABSORPTION WITH AGING IN THE RAT* D. HOLLANDER a n d D. MORGAN Division t~l Gastroenterology, Wayne State University and tlarper Grace ttospitals, Detroit, MI 48201, U.S.A.
(Received 24 July 1978) INTRODUCTION ARTERIOSCt,EROTIC c a r d i o v a s c u l a r disease, a d i s e a s e a s s o c i a t e d w i t h a g i n g , is c a u s e d by e x c e s s i v e a m o u n t s o f c h o l e s t e r o l d e p o s i t s in t h e l i n i n g o f b l o o d vessels. T h e s m a l l i n t e s t i n e is t h e site o f a b s o r p t i o n o f c h o l e s t e r o l f r o m d i e t a r y s o u r c e s ( B o r g s t r o m , 1960). E n d o g e n o u s l y s y n t h e s i z e d c h o l e s t e r o l w h i c h is e x c r e t e d w i t h bile i n t o t h e l u m e n o f t h e d u o d e n u m is a l s o a b s o r b e d b y t h e s m a l l i n t e s t i n e ( T r e a d w e l l et al., 1959). S i n c e a r t e r i o s c l e r o t i c c a r d i o v a s c u l a r d i s e a s e is a d i s e a s e o f a g i n g a n d since t h e s m a l l b o w e l is t h e site o f c h o l e s t e r o l a b s o r p t i o n , we d e c i d e d t o i n v e s t i g a t e t h e r e l a t i o n s h i p b e t w e e n a g i n g a n d i n t e s t i n a l a b s o r p tion of cholesterol. Our studies showed that cholesterol absorption increases linearly with a g i n g , e v e n a t t h e e x t r e m e e n d o f t h e a n i m a l ' s life e x p e c t a n c y . MATERIALS AND METHODS Animal.~ Intestinal absorption of cholesterol was studied in male Sprague-Dawley rats varying in ages from I to 42 months. The rats were allowed free access to regular chow and water and were kept under identical conditions before experimentation. Three to six rats were studied in each age group.
Surgical preparation Each rat was anesthetized with ether, its abdomen was opened, and an inflow cannula was inserted into the lumen of the small bowel 3 cm distal to the entry of the common bile duct into the duodenum. The distal end of the small bowel was cannulated with a glass cannula just prior to the junction of the ileum and cecum. The rat was placed in a restraint cage which allowed mobility but prevented dislodgement of the catheters (Hollander et al., 1977). The animal's normal body temperature was maintained with a forced-air heating device which was activated by a thermostatic temperature controller (Hollander and Muralidhara, 1977). Experimental perfusions The infusate solution was composed of 10 mM sodium taurocholate in a Krebs phosphate buffer at pH 7.4. Inulin, a 14C-labeled nonabsorbable sugar, was added to the perfusate to detect changes in cholesterol concentration secondary to water transport and to correct the final data for this artifact (Miller and Schedl, 1970). SH-la, 2u (n) cholesterol with specific activity of 43 Ci/mmol was used as a tracer compound and was added to the nonradioactive cholesterol in the infusate to a final concentration of 50 taM. A micellar solution was prepared by ultrasound irradiation with a sonicator. The solution was infused into the proximal small bowel at a constant rate of 1 ml/min over a 2 h period and the outflow was collected in 6 separate 20 rain aliquots. Determination of absorption rate The absorption of cholesterol during each 20 rain period was calculated by subtracting the total amount of cholesterol collected out of the outflow cannula from the amount known to have been infused into the proximal small intestine during the same 20 min period. 8H-cholesterol was separated from ~aC-inulin by total combustion of triplicate aliquots of the intestinal perfusate with a tissue oxidizer (Hollander et al., 1976). Radioactivity was than assayed with a beta liquid scintillation counter to an accuracy of ± 1 ° o, The corrected net absorption rate of cholesterol in each 20 min period was calculated and all 6 values for absorption from each animal were pooled with the values obtained from other animals of the same age group, *Mail requests for reprints to: Daniel Hollander, M.D., Department of Medicine, 5901 East 7th St.. Long Beach, CA 90801, U.S.A. 201
202
D. HOLLANDERAND 1). MORGAN
for calculation of the mean i S.E. rate of absorption of cholesterol. Cholesterol absorption by each age group was also expressed in terms of percentage absorption of the amount of cholesterol infused per h. Determination o f intestinal size The animals were killed with an overdose of ether at the completion of perfusion and the perfused intestinal segments were removed. Well-standardized methods (Hollander and Muralidhara, 1977) of stretching and desiccating the bowel were used for measuring the intestinal weight and length in the dry state, so that the segments from the various age groups could be compared.
RESULTS When the rate of cholesterol absorption in each age group was plotted against the age of the animals, a linear relationship was found between the two parameters (Fig. 1). Absorption of cholesterol continued to increase until the extreme end of the animal's life expectancy (42 months) (Hollander, 1976). When cholesterol absorption was expressed as a percentage of the amount infused, 14~o was absorbed at the age of 1 month, as compared to 38 o/ /o at the age of 42 months (Table 1). The body weight and the intestinal length and weight of each group of animals showed a rapid rise during their first 12 months of life (Table 1). However, during the latter 30 months of life the animals showed no further significant increase in these three parameters (Table 1).
I0001
CHOLESTEROL ABSORPTION
900:E
(_)
o
800-
700i.i.i
/ = 4 4 5 ~ Ilx
~. 6oo500-~
I IO
2'0 MONTHS
p
