716
Specialia
s a m e es. Meanwhile, it is k n o w n t h a t t h e distribution and t h e n u m b e r of receptors v a r y n o t o n l y w i t h t h e degree of differentiation of m y o t u b e s 2,, b u t also w i t h its s t a t e of i n n e r v a t i o n 2~, t h e s a m e holds t r u e for cholinesterase ~s. R e c e n t l y is has been shown 10 t h a t t h e n u m b e r of acetylcholine receptors of n e u r o m u s c u l a r junction-free muscle strips of r a t soleus (red) was a l m o s t 2 t i m e s greater t h a n t h a t of t h e e x t e n s o r d i g i t o r u m longus (white). T h e difference between t h e A C h E a c t i v i t y in t h e 2 cultures suggest t h a t this is related to the different s t r u c t u r a l properties of excitable m e m b r a n e s of pectoralis and add u c t o r muscle d i f f e r e n t i a t e d in vitro. These differences in t h e A C h E a c t i v i t y , as in I C D H , show t h a t m y o b l a s t s o b t a i n e d f r o m 2 different h o m o g e n e o u s muscles differentiate, in vitro, u n d e r t h e s a m e conditions of d e v e l o p m e n t , into muscle fibres w i t h different functional and m e t a b o l i c properties. This result b e t w e e n t h e 2 cultures m a y be related to t h e existence of m y o g e n i c cells w i t h different potentialities. I n a h i s t o c h e m i c a l s t u d y of in v i t r o differentiated muscle fibres f r o m b r e a s t and leg muscles m y o b l a s t s of 10-dayold chick embryos, Askanas et al. 1~ found no difference in phosphorylase (glycolytic metabolism) staining b e t w e e n t h e 2 cultures, this result being in a g r e e m e n t w i t h our o b s e r v a t i o n for aldolase, a n o t k e r e n z y m e of t h e glycolytic p a t h w a y . As we observed significant differences in I C D H activities (citric acid cycle), these a u t h o r s also observed a difference in succinate d e h y d r o g e n a s e (oxidative metabolism) : staining for this e n z y m e was m o r e p r o n o u n c e d
EXPERIENTIA 33/6
in c u l t u r e d leg muscles t h a n c u l t u r e d breast muscle, and this difference could be related to a greater a m o u n t of m i t o c h o n d r i a as observed in electron micrographs of c u l t u r e d leg muscles. H o w e v e r , these leg muscle cultures were established from ill defined p o p u l a t i o n s of m y o g e n i c cells coming f r o m heterogeneous muscles samples, t h e m a j o r i t y of t h e m being of m i x e d fibre p o p u l a t i o n s : I n these circumstances it is difficult to relate the observations to t h e t y p e of m y o g e n i c cell. W e feel t h a t w i t h our results, p a r t i c u l a r l y in t h e case of a d d u c t o r muscle cultures, we can w i t h c e r t a i n t y relate t h e differences to t h e t y p e of m y o b l a s t f r o m which t h e y originated. This is evidence, therefore, t h a t m y 0 b l a s t s f r o m p o t e n tially different skeletal muscle differentiate in different w a y s in tissue culture, e v e n in t h e absence of t h e n e r v e supply. This c e r t a i n l y seems to be true as far as e n z y m e activities are concerned. F r o m a general p o i n t of view, this seems to be the expression of those characteristics which are m y o g e n i c a l l y determined, o t h e r characteristics m a y well be d e t e r m i n e d b y exogenous factors including t h e n e r v e Supply.
25 E.A. Barnard, J. Wieekowski and T. H. Chin, Nature 234, 207 (1971). 26 A. J. Sytkowski, Z. Vogel and M. W. Nirenberg, Proe. nat. Aead. Set. USA 70, 270 (1973). 27 G.D. Fischbach and S. A. Cohen, Devl Biol. 3J, 147 (1973). 28 A.L. Harvey and W. F. Dryden; Differentiation 2, 237 (1974).
Reduction of gallstone formation by ascorbic acid in hamsters E. Ginter and LI Miku~
Research Institute/or Human Nutrition, ham. d. aprila 7, 88030 Bratislava (Czechoslovakia), 13 August ?976 Summary. The a d d i t i o n of 0.5 % of ascorbic acid to t h e lithogenic diet of golden h a m s t e r s whose b o d y pool was labelled w i t h 26-~4C-cholesterol, lowered t h e f o r m a t i o n of gallstones, t h e cholesterol c o n c e n t r a t i o n and half-life in blood p l a s m a a n d in the liver, and accelerated cholesterol t r a n s f o r m a t i o n to bile acids. Bile acids and lecithin are the m a j o r solubilizing agents for biliary cholesterol, t h e p r e d o m i n a n t c o m p o n e n t of at least 90% of gallstones. An initial phase in gallstone f o r m a t i o n is a disorder of liver cell metabolism, the prod u c t i o n of bile t h a t is s u p e r s a t u r a t e d w i t h cholesterol 1. Persons w i t h cholesterol gallstones h a v e a decrease in bile acid pool size 2. The increase in biliary cholesterol secretion and t h e r e d u c t i o n of bile acid pool size could lead to a decrease in t h e p r o p o r t i o n of bile salts to cholesterol which could result in the p r e c i p i t a t i o n of cholesterol and aggregation of cholesterol crystals into gallstones. A similar m e t a b o l i c s i t u a t i o n occurs in guineapigs during a chronic l a t e n t v i t a m i n C deficiency: t h e r a t e of cholesterol t r a n s f o r m a t i o n to bile acids in t h e liver is decreased s, 4 and t h e size of t h e bile acid po01 is also reduced 5. Marginal v i t a m i n C deficiency interferes w i t h t h e biosynthesis of bile acids a t t h e stage of m i c r o s o m a l 7 e - h y d r o x y l a t i o n of t h e cholesterol nucleus s. B j S r k h e m and Kallner 7 h a v e suggested t h a t ascorbate affects the synthesis or b r e a k d o w n of the cholesterol 7 ~ - h y d r o x y l a t ing system, in p a r t i c u l a r the c y t o c h r o m e P-450 c o m p o n e n t . I n scorbutic guinea-pigs, f r e q u e n t occurence of gallstones was observed 8-10. These facts h a v e p r o m p t e d us to consider t h e p o t e n t i a l role of a chronic l a t e n t v i t a m i n C deficiency in t h e pathogenesis of cholelithiasis and t h e possibility of p r e v e n t i n g t h e f o r m a t i o n of gallstones b y
a p e r m a n e n t supply of v i t a m i n C 11. The aim of t h e exp e r i m e n t r e p o r t e d here has been to v e r i f y this hypothesis. Materials and methods. 200 male golden h a m s t e r s aged a p p r o x i m a t e l y 6 weeks (body weight a b o u t 70 g) were p u t on a semi-purified fat-free, high-glucose d i e t 12 k n o w n to produce cholesterol gallstones. 100 animals were fed this diet w i t h o u t a n y v i t a m i n C addition, t h e others had t h e same diet plus 5 g of ascorbic acid per kg of diet. The W. Admirand and D. Small, J. clin. Invest. 47, 1043 (1968). Z. Vlahcevic, C. Bell, I. Buhac,J. Farrar and L. Swell, Gastroenterology 59, 165 (1970). E. Ginter, J. Cerven, R. Nemee and L. Mikuw Am. J. elin. Nutr. 3 2g, 1238 (1971). 4 E. Ginter, Science 179, 702 (1973). 5 D. Hornig and H. Weiser, Experientia 32, 687 (1976). 6 E. Ginter, Ann. N. Y. Acad. Sci. 258, 410 (1975). 7 I. Bj6rkhem and A. Kallner, 7- Lipid Res. 17, 360 (1976). 8 I. Pavel, N. Chisiu and D. Sdrobiei, Nutritio Dicta Jl, 60 (1969). 9 N. M. Di Filippo and H. J. Blumenthal, 7. Am. osteop. Ass. 72, 284 (1972). H. Bellmann, E. Rauchfuss, B. Wohlgemut, S. Schubert, K. F. 10 Fuchs, F. Geissler, R. Haupt, G. Conradi, W. Sch6nlebe, E. Daniel and O. Giinther, Z. inn. Ned. 29, 997 (1974). 11 E. Ginter, Lancet 2, 1198 (1971). 12 H. Dam, Proc. 6th Int. Congress Nutr., p. 6. Livingstone Ltd, Edinburg-London 1964. 1 2
15. 6. 1977
Specialia
w e i g h t curves and m o r t a l i t y in b o t h t h e groups were similar (7% w i t h i n 8 weeks). A f t e r 8 weeks, 50 prospering animals w i t h a m i n i m u m b o d y w e i g h t r a n g e (90-110 g), were picked o u t f r o m each group. B o d y w e i g h t was t h e o n l y selection criterium. The animals were all given an i.p. injection of 26A4C-cholesterol (Radiochemical Center, A m e r s h a m ; specific a c t i v i t y 55.8 mCi/mM) emulsified w i t h t w e e n 20 in saline, in a dose of 3.36 [zCi per animal. The n u t r i t i o n a l regimen in b o t h t h e groups r e m a i n e d unaltered. A t w e e k l y i n t e r v a l s from w e e k 1 to 8 following a d m i n i s t r a t i o n of labeled cholesterol, t h e 24-h Output of 1'CO,, v i t a m i n C c o n c e n t r a t i o n in the liver, t h e conc e n t r a t i o n and specific a c t i v i t y of cholesterol in blood p l a s m a and liver were d e t e r m i n e d b y procedures similar to those used in our previous s t u d y *. 6 animals were autopsied in each w e e k (8 in w e e k 8) of b o t h t h e groups. The presence of gallstones was registered: t h e occurence of small stones, a p p r o x i m a t e l y 0.2 m m in diameter, was classified as incipient cholelithiasis, while t h a t of a larger n u m b e r of m i n u t e stones or of one large aggregate a b o v e 1 m m in d i a m e t e r was classified as a d v a n c e d cholelithiasis. The decline of the log specific a c t i v i t y of plasma and liver cholesterol in t i m e f r o m t h e second w e e k after labelled cholesterol a d m i n i s t r a t i o n p r o v e d to be linear (correlation coefficient of log specific a c t i v i t y - t i m e was 0.985 in b o t h t h e groups). These d a t a served for a calculation of t h e half-life of plasma and liver cholesterol and f u r t h e r par a m e t e r s of cholesterol t u r n o v e r in t e r m s of 0ne-pool m o d e l ~s. The r a t e of cholesterol t r a n s f o r m a t i o n to bile acids was d e t e r m i n e d as the ratio of disintegrations in tile 14CO2 to the cholesterol specific a c t i v i t y similarly as in our preceding s t u d y 4. The results were statistically e v a l u a t e d b y m e a n s of standardized p r o g r a m m e s (Stud e n t ' s t-test, linear regression, )c2-test) on a c o m p u t e r . The effect of vitamin C on the incidence of gallstones and on cholesterol metabolism in golden hamsters fed fat-free high-glucose diet Parameter
Ascorbic acid in the diet Zero 0.5%
Body weight (g) Animals free of gallstones (%) Animals with incipient eholelithiasis (%) Animals with advanced eholelithiasis (%) Ascorbie acid in the liver (rag/100 g wet tissue) Total cholesterol in plasma (mg/100 ml) Total cholesterol in the liver (mg/100 g wet tissue) Cholesterol transformation to bile acids (mg/24 h/100 g b.wt) Body pool of miscible cholesterol (rag per animal) Half-life of plasma cholesterol (days) Half-life of liver cholesterol (days) Cholesterol turnover rate (mg per 24 h per animal) Fractional turnover rate (% 9 f pool renewed in 24 h)
92 -4- 1 32 (16)
93 4- 1 56 (28)*
26 (13)
20 (10)
42 (21)
24 (12)*
14.7 -t- 0.5
26.8 4- 0.6**
210 4- 8
181 ! 4 (49)**
751 i 49
582 4- 26**
2.16 -4- 0.12 (32) 2.53 • 0.11 (30)* 162
128
21.5
15.7"*
21.5
16.3"*
5.23
5.67
3.2
4.4
Figures represent mean values :j= SEM. The number of animals observed for each determination was 50, unless indicated otherwise in parenthesis. *p ~ 0.05-0.02; **p < 0.01-0,001.
717
Results and discussion. The b o d y weight from t h e t i m e of t h e a d m i n i s t r a t i o n of labelled cholesterol until d e a t h r e m a i n e d u n c h a n g e d and was a l m o s t identical in t h e 2 groups. N o t a single one of t h e 100 animals perished. z/s of t h e h a m s t e r s k e p t on v i t a m i n C-free diet had gallstones. The a d d i t i o n of ascorbic acid to t h e diet signific a n t l y depressed b o t h t h e occurence and the degree of e x p e r i m e n t a l cholelithiasis (table). E v e n t h o u g h h a m s t e r s possess t h e c a p a c i t y to s y n t h e t i z e ascorbate in t h e liver, a l o n g - t e r m a d m i n i s t r a t i o n of an u n b a l a n c e d diet dep r i v e d of v i t a m i n C p r o d u c e d in t h e m a r e l a t i v e v i t a m i n C deficiency: t h e i r ascorbate level in the liver was subs t a n t i a l l y lower t h a n in h a m s t e r s fed o n cereals and vegetables 14. The a d d i t i o n of 0.5% ascorbic acid to tile diet doubled t h e v i t a m i n C c o n c e n t r a t i o n in t h e liver. U n d e r t h e effect of ascorbic acid, the c o n c e n t r a t i o n and halflife of cholesterol decreased in the p l a s m a and liver, and s i m u l t a n e o u s l y t h e rate of cholesterol t r a n s f o r m a t i o n to bile acids slightly increased. The effect of v i t a m i n C on cholesterol m e t a b o l i s m in h a m s t e r s is t h u s v e r y similar to t h a t seen in guinea-pigs ~, ~. D a t a from a kinetic analysis of cholesterol t u r n o v e r in t e r m s of one-pool m o d e l o v e r e s t i m a t e t h e true size of miscible pools 1~. H o w e v e r , t h e decrease in t h e size of miscible pool in animals receiving v i t a m i n C is in good correlation w i t h tile decline in the levels of plasma and liver cholesterol. The metabolic situation developing ill h a m s t e r s p u t on a fat-free, ascorbate-free, high-glucose diet, is p r o b a b l y as follows: t h e i n t a k e of e n o r m o u s q u a n t i t i e s of glucose enhances t h e endogenous synthesis of cholesterol ~6. As a consequence of a deficiency of essential f a t t y acids and v i t a m i n C, these animals are incapable of c o m p e n s a t i n g for tile e n h a n c e d cholesterol biosynthesis b y increasing its c a t a b o l i s m to bile acids ~, 17. H y p e r c h o l e s t e r o l e m i a sets in these animals, w i t h an a c c u m u l a t i o n of cholesterol in t h e liver, a prolongation of t h e half-life of p l a s m a and liver cholesterol, an increase in the ratio cholesterol/bile salts in t h e bilelL and u l t i m a t e l y leads to t h e f o r m a t i o n of gallstones. Similarly in guinea-pigs l a t e n t ascorbate deficiency increases tile p r o p e n s i t y for gallstone f o r m a t i o n (unpublished results). On t h e o t h e r hand, a d d i t i o n of v i t a m i n C to t h e diet enhances the c o n c e n t r a t i o n of ascorbate in hepatic cells so t h a t the liver becomes at least p a r t i a l l y capable of c o m p e n s a t i n g for the e n h a n c e d synthesis of endogenous cholesterol b y its increased transf o r m a t i o n to bile acids. As a result of this, t h e cholesterol a c c u m u l a t i o n in t h e plasma and liver is smaller and the increase in the ratio cholesterol/bile salts in t h e gallbladder bile is less striking. The overall o u t c o m e is a lowered gallstone f o r m a t i o n u n d e r t h e effect of v i t a m i n C. The results o b t a i n e d indicate t h a t s a t u r a t i o n of the liver w i t h v i t a m i n C significantly depresses the occurrence of gallstones in t h e hamster. The k e y question t h a t remains open is w h e t h e r these results m a y be e x t r a p o l a t e d to the h u m a n organism. Large doses of v i t a m i n C did n o t significantly alter the chemical composition of the bile in h e a l t h y subjects 18, b u t in these e x p e r i m e n t s ascorbic acid was u n f o r t u n a t e l y a d m i n i s t e r e d o n l y o v e r a v e r y short period (1-2 weeks).
13 A. V. Chobanian, B. A. Burrows and W. Hollander, J. elin. Invest. 41, 1738 (1962). 14 E. Ginter, O. Cerna, R. Ondreieka, V. Roeh and V. Balaz, Food Chem. l, 23 (1976). 15 S.M. Grundy and E. H. Ahrens Jr, J. Lipid Res. 70, 91 (1969). 16 H. Muroya, R. Suzue and Y. Hikasa, Arehs Bioehem. Biophys. 12d, 12 (1968). 17 R.F. MeGovern andF. W. Quackenbush, Lipids 8, 466 (1973). 18 L. Pedersen, Seand. J. Gastroent. 10, 311 (1975).
Specialia
s a m e es. Meanwhile, it is k n o w n t h a t t h e distribution and t h e n u m b e r of receptors v a r y n o t o n l y w i t h t h e degree of differentiation of m y o t u b e s 2,, b u t also w i t h its s t a t e of i n n e r v a t i o n 2~, t h e s a m e holds t r u e for cholinesterase ~s. R e c e n t l y is has been shown 10 t h a t t h e n u m b e r of acetylcholine receptors of n e u r o m u s c u l a r junction-free muscle strips of r a t soleus (red) was a l m o s t 2 t i m e s greater t h a n t h a t of t h e e x t e n s o r d i g i t o r u m longus (white). T h e difference between t h e A C h E a c t i v i t y in t h e 2 cultures suggest t h a t this is related to the different s t r u c t u r a l properties of excitable m e m b r a n e s of pectoralis and add u c t o r muscle d i f f e r e n t i a t e d in vitro. These differences in t h e A C h E a c t i v i t y , as in I C D H , show t h a t m y o b l a s t s o b t a i n e d f r o m 2 different h o m o g e n e o u s muscles differentiate, in vitro, u n d e r t h e s a m e conditions of d e v e l o p m e n t , into muscle fibres w i t h different functional and m e t a b o l i c properties. This result b e t w e e n t h e 2 cultures m a y be related to t h e existence of m y o g e n i c cells w i t h different potentialities. I n a h i s t o c h e m i c a l s t u d y of in v i t r o differentiated muscle fibres f r o m b r e a s t and leg muscles m y o b l a s t s of 10-dayold chick embryos, Askanas et al. 1~ found no difference in phosphorylase (glycolytic metabolism) staining b e t w e e n t h e 2 cultures, this result being in a g r e e m e n t w i t h our o b s e r v a t i o n for aldolase, a n o t k e r e n z y m e of t h e glycolytic p a t h w a y . As we observed significant differences in I C D H activities (citric acid cycle), these a u t h o r s also observed a difference in succinate d e h y d r o g e n a s e (oxidative metabolism) : staining for this e n z y m e was m o r e p r o n o u n c e d
EXPERIENTIA 33/6
in c u l t u r e d leg muscles t h a n c u l t u r e d breast muscle, and this difference could be related to a greater a m o u n t of m i t o c h o n d r i a as observed in electron micrographs of c u l t u r e d leg muscles. H o w e v e r , these leg muscle cultures were established from ill defined p o p u l a t i o n s of m y o g e n i c cells coming f r o m heterogeneous muscles samples, t h e m a j o r i t y of t h e m being of m i x e d fibre p o p u l a t i o n s : I n these circumstances it is difficult to relate the observations to t h e t y p e of m y o g e n i c cell. W e feel t h a t w i t h our results, p a r t i c u l a r l y in t h e case of a d d u c t o r muscle cultures, we can w i t h c e r t a i n t y relate t h e differences to t h e t y p e of m y o b l a s t f r o m which t h e y originated. This is evidence, therefore, t h a t m y 0 b l a s t s f r o m p o t e n tially different skeletal muscle differentiate in different w a y s in tissue culture, e v e n in t h e absence of t h e n e r v e supply. This c e r t a i n l y seems to be true as far as e n z y m e activities are concerned. F r o m a general p o i n t of view, this seems to be the expression of those characteristics which are m y o g e n i c a l l y determined, o t h e r characteristics m a y well be d e t e r m i n e d b y exogenous factors including t h e n e r v e Supply.
25 E.A. Barnard, J. Wieekowski and T. H. Chin, Nature 234, 207 (1971). 26 A. J. Sytkowski, Z. Vogel and M. W. Nirenberg, Proe. nat. Aead. Set. USA 70, 270 (1973). 27 G.D. Fischbach and S. A. Cohen, Devl Biol. 3J, 147 (1973). 28 A.L. Harvey and W. F. Dryden; Differentiation 2, 237 (1974).
Reduction of gallstone formation by ascorbic acid in hamsters E. Ginter and LI Miku~
Research Institute/or Human Nutrition, ham. d. aprila 7, 88030 Bratislava (Czechoslovakia), 13 August ?976 Summary. The a d d i t i o n of 0.5 % of ascorbic acid to t h e lithogenic diet of golden h a m s t e r s whose b o d y pool was labelled w i t h 26-~4C-cholesterol, lowered t h e f o r m a t i o n of gallstones, t h e cholesterol c o n c e n t r a t i o n and half-life in blood p l a s m a a n d in the liver, and accelerated cholesterol t r a n s f o r m a t i o n to bile acids. Bile acids and lecithin are the m a j o r solubilizing agents for biliary cholesterol, t h e p r e d o m i n a n t c o m p o n e n t of at least 90% of gallstones. An initial phase in gallstone f o r m a t i o n is a disorder of liver cell metabolism, the prod u c t i o n of bile t h a t is s u p e r s a t u r a t e d w i t h cholesterol 1. Persons w i t h cholesterol gallstones h a v e a decrease in bile acid pool size 2. The increase in biliary cholesterol secretion and t h e r e d u c t i o n of bile acid pool size could lead to a decrease in t h e p r o p o r t i o n of bile salts to cholesterol which could result in the p r e c i p i t a t i o n of cholesterol and aggregation of cholesterol crystals into gallstones. A similar m e t a b o l i c s i t u a t i o n occurs in guineapigs during a chronic l a t e n t v i t a m i n C deficiency: t h e r a t e of cholesterol t r a n s f o r m a t i o n to bile acids in t h e liver is decreased s, 4 and t h e size of t h e bile acid po01 is also reduced 5. Marginal v i t a m i n C deficiency interferes w i t h t h e biosynthesis of bile acids a t t h e stage of m i c r o s o m a l 7 e - h y d r o x y l a t i o n of t h e cholesterol nucleus s. B j S r k h e m and Kallner 7 h a v e suggested t h a t ascorbate affects the synthesis or b r e a k d o w n of the cholesterol 7 ~ - h y d r o x y l a t ing system, in p a r t i c u l a r the c y t o c h r o m e P-450 c o m p o n e n t . I n scorbutic guinea-pigs, f r e q u e n t occurence of gallstones was observed 8-10. These facts h a v e p r o m p t e d us to consider t h e p o t e n t i a l role of a chronic l a t e n t v i t a m i n C deficiency in t h e pathogenesis of cholelithiasis and t h e possibility of p r e v e n t i n g t h e f o r m a t i o n of gallstones b y
a p e r m a n e n t supply of v i t a m i n C 11. The aim of t h e exp e r i m e n t r e p o r t e d here has been to v e r i f y this hypothesis. Materials and methods. 200 male golden h a m s t e r s aged a p p r o x i m a t e l y 6 weeks (body weight a b o u t 70 g) were p u t on a semi-purified fat-free, high-glucose d i e t 12 k n o w n to produce cholesterol gallstones. 100 animals were fed this diet w i t h o u t a n y v i t a m i n C addition, t h e others had t h e same diet plus 5 g of ascorbic acid per kg of diet. The W. Admirand and D. Small, J. clin. Invest. 47, 1043 (1968). Z. Vlahcevic, C. Bell, I. Buhac,J. Farrar and L. Swell, Gastroenterology 59, 165 (1970). E. Ginter, J. Cerven, R. Nemee and L. Mikuw Am. J. elin. Nutr. 3 2g, 1238 (1971). 4 E. Ginter, Science 179, 702 (1973). 5 D. Hornig and H. Weiser, Experientia 32, 687 (1976). 6 E. Ginter, Ann. N. Y. Acad. Sci. 258, 410 (1975). 7 I. Bj6rkhem and A. Kallner, 7- Lipid Res. 17, 360 (1976). 8 I. Pavel, N. Chisiu and D. Sdrobiei, Nutritio Dicta Jl, 60 (1969). 9 N. M. Di Filippo and H. J. Blumenthal, 7. Am. osteop. Ass. 72, 284 (1972). H. Bellmann, E. Rauchfuss, B. Wohlgemut, S. Schubert, K. F. 10 Fuchs, F. Geissler, R. Haupt, G. Conradi, W. Sch6nlebe, E. Daniel and O. Giinther, Z. inn. Ned. 29, 997 (1974). 11 E. Ginter, Lancet 2, 1198 (1971). 12 H. Dam, Proc. 6th Int. Congress Nutr., p. 6. Livingstone Ltd, Edinburg-London 1964. 1 2
15. 6. 1977
Specialia
w e i g h t curves and m o r t a l i t y in b o t h t h e groups were similar (7% w i t h i n 8 weeks). A f t e r 8 weeks, 50 prospering animals w i t h a m i n i m u m b o d y w e i g h t r a n g e (90-110 g), were picked o u t f r o m each group. B o d y w e i g h t was t h e o n l y selection criterium. The animals were all given an i.p. injection of 26A4C-cholesterol (Radiochemical Center, A m e r s h a m ; specific a c t i v i t y 55.8 mCi/mM) emulsified w i t h t w e e n 20 in saline, in a dose of 3.36 [zCi per animal. The n u t r i t i o n a l regimen in b o t h t h e groups r e m a i n e d unaltered. A t w e e k l y i n t e r v a l s from w e e k 1 to 8 following a d m i n i s t r a t i o n of labeled cholesterol, t h e 24-h Output of 1'CO,, v i t a m i n C c o n c e n t r a t i o n in the liver, t h e conc e n t r a t i o n and specific a c t i v i t y of cholesterol in blood p l a s m a and liver were d e t e r m i n e d b y procedures similar to those used in our previous s t u d y *. 6 animals were autopsied in each w e e k (8 in w e e k 8) of b o t h t h e groups. The presence of gallstones was registered: t h e occurence of small stones, a p p r o x i m a t e l y 0.2 m m in diameter, was classified as incipient cholelithiasis, while t h a t of a larger n u m b e r of m i n u t e stones or of one large aggregate a b o v e 1 m m in d i a m e t e r was classified as a d v a n c e d cholelithiasis. The decline of the log specific a c t i v i t y of plasma and liver cholesterol in t i m e f r o m t h e second w e e k after labelled cholesterol a d m i n i s t r a t i o n p r o v e d to be linear (correlation coefficient of log specific a c t i v i t y - t i m e was 0.985 in b o t h t h e groups). These d a t a served for a calculation of t h e half-life of plasma and liver cholesterol and f u r t h e r par a m e t e r s of cholesterol t u r n o v e r in t e r m s of 0ne-pool m o d e l ~s. The r a t e of cholesterol t r a n s f o r m a t i o n to bile acids was d e t e r m i n e d as the ratio of disintegrations in tile 14CO2 to the cholesterol specific a c t i v i t y similarly as in our preceding s t u d y 4. The results were statistically e v a l u a t e d b y m e a n s of standardized p r o g r a m m e s (Stud e n t ' s t-test, linear regression, )c2-test) on a c o m p u t e r . The effect of vitamin C on the incidence of gallstones and on cholesterol metabolism in golden hamsters fed fat-free high-glucose diet Parameter
Ascorbic acid in the diet Zero 0.5%
Body weight (g) Animals free of gallstones (%) Animals with incipient eholelithiasis (%) Animals with advanced eholelithiasis (%) Ascorbie acid in the liver (rag/100 g wet tissue) Total cholesterol in plasma (mg/100 ml) Total cholesterol in the liver (mg/100 g wet tissue) Cholesterol transformation to bile acids (mg/24 h/100 g b.wt) Body pool of miscible cholesterol (rag per animal) Half-life of plasma cholesterol (days) Half-life of liver cholesterol (days) Cholesterol turnover rate (mg per 24 h per animal) Fractional turnover rate (% 9 f pool renewed in 24 h)
92 -4- 1 32 (16)
93 4- 1 56 (28)*
26 (13)
20 (10)
42 (21)
24 (12)*
14.7 -t- 0.5
26.8 4- 0.6**
210 4- 8
181 ! 4 (49)**
751 i 49
582 4- 26**
2.16 -4- 0.12 (32) 2.53 • 0.11 (30)* 162
128
21.5
15.7"*
21.5
16.3"*
5.23
5.67
3.2
4.4
Figures represent mean values :j= SEM. The number of animals observed for each determination was 50, unless indicated otherwise in parenthesis. *p ~ 0.05-0.02; **p < 0.01-0,001.
717
Results and discussion. The b o d y weight from t h e t i m e of t h e a d m i n i s t r a t i o n of labelled cholesterol until d e a t h r e m a i n e d u n c h a n g e d and was a l m o s t identical in t h e 2 groups. N o t a single one of t h e 100 animals perished. z/s of t h e h a m s t e r s k e p t on v i t a m i n C-free diet had gallstones. The a d d i t i o n of ascorbic acid to t h e diet signific a n t l y depressed b o t h t h e occurence and the degree of e x p e r i m e n t a l cholelithiasis (table). E v e n t h o u g h h a m s t e r s possess t h e c a p a c i t y to s y n t h e t i z e ascorbate in t h e liver, a l o n g - t e r m a d m i n i s t r a t i o n of an u n b a l a n c e d diet dep r i v e d of v i t a m i n C p r o d u c e d in t h e m a r e l a t i v e v i t a m i n C deficiency: t h e i r ascorbate level in the liver was subs t a n t i a l l y lower t h a n in h a m s t e r s fed o n cereals and vegetables 14. The a d d i t i o n of 0.5% ascorbic acid to tile diet doubled t h e v i t a m i n C c o n c e n t r a t i o n in t h e liver. U n d e r t h e effect of ascorbic acid, the c o n c e n t r a t i o n and halflife of cholesterol decreased in the p l a s m a and liver, and s i m u l t a n e o u s l y t h e rate of cholesterol t r a n s f o r m a t i o n to bile acids slightly increased. The effect of v i t a m i n C on cholesterol m e t a b o l i s m in h a m s t e r s is t h u s v e r y similar to t h a t seen in guinea-pigs ~, ~. D a t a from a kinetic analysis of cholesterol t u r n o v e r in t e r m s of one-pool m o d e l o v e r e s t i m a t e t h e true size of miscible pools 1~. H o w e v e r , t h e decrease in t h e size of miscible pool in animals receiving v i t a m i n C is in good correlation w i t h tile decline in the levels of plasma and liver cholesterol. The metabolic situation developing ill h a m s t e r s p u t on a fat-free, ascorbate-free, high-glucose diet, is p r o b a b l y as follows: t h e i n t a k e of e n o r m o u s q u a n t i t i e s of glucose enhances t h e endogenous synthesis of cholesterol ~6. As a consequence of a deficiency of essential f a t t y acids and v i t a m i n C, these animals are incapable of c o m p e n s a t i n g for tile e n h a n c e d cholesterol biosynthesis b y increasing its c a t a b o l i s m to bile acids ~, 17. H y p e r c h o l e s t e r o l e m i a sets in these animals, w i t h an a c c u m u l a t i o n of cholesterol in t h e liver, a prolongation of t h e half-life of p l a s m a and liver cholesterol, an increase in the ratio cholesterol/bile salts in t h e bilelL and u l t i m a t e l y leads to t h e f o r m a t i o n of gallstones. Similarly in guinea-pigs l a t e n t ascorbate deficiency increases tile p r o p e n s i t y for gallstone f o r m a t i o n (unpublished results). On t h e o t h e r hand, a d d i t i o n of v i t a m i n C to t h e diet enhances the c o n c e n t r a t i o n of ascorbate in hepatic cells so t h a t the liver becomes at least p a r t i a l l y capable of c o m p e n s a t i n g for the e n h a n c e d synthesis of endogenous cholesterol b y its increased transf o r m a t i o n to bile acids. As a result of this, t h e cholesterol a c c u m u l a t i o n in t h e plasma and liver is smaller and the increase in the ratio cholesterol/bile salts in t h e gallbladder bile is less striking. The overall o u t c o m e is a lowered gallstone f o r m a t i o n u n d e r t h e effect of v i t a m i n C. The results o b t a i n e d indicate t h a t s a t u r a t i o n of the liver w i t h v i t a m i n C significantly depresses the occurrence of gallstones in t h e hamster. The k e y question t h a t remains open is w h e t h e r these results m a y be e x t r a p o l a t e d to the h u m a n organism. Large doses of v i t a m i n C did n o t significantly alter the chemical composition of the bile in h e a l t h y subjects 18, b u t in these e x p e r i m e n t s ascorbic acid was u n f o r t u n a t e l y a d m i n i s t e r e d o n l y o v e r a v e r y short period (1-2 weeks).
13 A. V. Chobanian, B. A. Burrows and W. Hollander, J. elin. Invest. 41, 1738 (1962). 14 E. Ginter, O. Cerna, R. Ondreieka, V. Roeh and V. Balaz, Food Chem. l, 23 (1976). 15 S.M. Grundy and E. H. Ahrens Jr, J. Lipid Res. 70, 91 (1969). 16 H. Muroya, R. Suzue and Y. Hikasa, Arehs Bioehem. Biophys. 12d, 12 (1968). 17 R.F. MeGovern andF. W. Quackenbush, Lipids 8, 466 (1973). 18 L. Pedersen, Seand. J. Gastroent. 10, 311 (1975).
