15. 8. 1977
1055
Specialia
T h e a m i n o acid c o m p o s i t i o n of r a t bile i U. R. F61sch e a n d K. G. W o r m s l e y 3
Department o/ Therapeutics, University o/ Dundee, Dundee DD2 1UB (Scotland), 25 January 1977 Summary. The p a t t e r n of a m i n o acids in t h e bile of r a t s differs f r o m t h e p a t t e r n in t h e s e r u m of t h e s e animals, since bile c o n t a i n s s i g n i f i c a n t l y g r e a t e r a m o u n t s of acidic a n d s u l p h u r - c o n t a i n i n g a m i n o acids a n d glycine t h a n serum, while t h e s e r u m c o n t a i n e d m o r e basic a m i n o acids t h a n bile, i n d i c a t i n g t h a t secretion of a m i n o acids i n t o bile m a y involve specific t r a n s p o r t processes. As p a r t of a s t u d y of t h e i n c o r p o r a t i o n of a m i n o acids i n t o p a n c r e a t i c e n z y m e s a n d t h e s e c r e t i o n of t h e s e e n z y m e s 4, we s t u d i e d t h e a p p e a r a n c e of a m i n o acids in t h e bile of r a t s . Our f i n d i n g s i n d i c a t e t h a t t h e secretion of a m i n o acids i n t o bile is selective a n d specific. Materials and methods. Male W i s t a r rats, w e i g h i n g 300 g (=L 30 g) were s t u d i e d . 2 e x p e r i m e n t a l schedules were employed. 1. 9 r a t s were f a s t e d for 24 h a n d t h e n a n a e s t h e t i z e d w i t h u r e t h a n e (0.6 ml/100 g b . w t of a solution c o n t a i n i n g 250 g/l) b y i.p. injection. D u r i n g e a c h s t u d y , t h e b o d y t e m p e r a t u r e of t h e r a t s was m a i n t a i n e d a t 37 ~ The bile d u c t was c a t h e t e r i z e d n e a r t h e liver a n d t h e d u c t distal to t h e c a n n u l a was ligated. A c a t h e t e r was placed in t h e j u g u l a r v e i n a n d 0.15 moles/1 s o d i u m chloride solution w a s i n f u s e d a t 0.9 ml/h. Bile was collected in 30-min b a t c h e s in p l a s t i c t u b e s a n d t h e n pooled. Blood was obt a i n e d b y aortic p u n c t u r e a t t h e end of e a c h s t u d y . 2. I n o r d e r to s t u d y t h e effect of f a s t i n g a n d feeding on t h e biliary e x c r e t i o n of a m i n o acids, a f u r t h e r 12 r a t s were d i v i d e d i n t o 2 s u b g r o u p s , h a l f of w h i c h were f a s t e d for 24 h a n d s t u d i e d as above, while t h e r e m a i n i n g 6 r a t s were fed u n t i l t h e t i m e of s t u d y a n d t h e n h a d bile collected, as above. S a m p l e s (100 ixl) of bile or s e r u m were t r e a t e d w i t h trifluoroacetic acid (5 vi) a t 10 ~ A f t e r c e n t r i f u g a t i o n , t h e s u p e r n a t a n t a n d 4 w a s h i n g s of t h e p r e c i p i t a t e (each w a s h i n g 50 txl of triple-distilled water) were c o m b i n e d a n d e v a p o r a t e d in a v a c u u m desiccator. T h e a m i n o - a c i d c o m p o s i t i o n of t h e r e s u l t i n g residues w a s d e t e r m i n e d w i t h a L o C a r t e A m i n o - a c i d A n a l y s e r (single 22 c m c o l u m n ;
b u f f e r c h a n g e s - p H 3.25 for 65 min, 4.25 for 60 min, 6.65 for 45 rain; b u f f e r t e m p e r a t u r e - - 5 2 ~ for first 45 rain, t h e n 60~ t h e r e a f t e r ) a n d A u t o l a b C o m p u t i n g I n t e g r a t o r S y s t e m AA. Results. Bile c o n t a i n e d s i g n i f i c a n t l y g r e a t e r a m o u n t s of t h e acidic a m i n o acids a s p a r t i c a n d g l u t a m i c (table) (p < 0.01) while t h e a m o u n t s of t h e s e a m i n o acids in s e r u m were small. On t h e o t h e r h a n d , s e r u m c o n t a i n e d m o r e of t h e basic a m i n o acids lysine a n d o r n i t h i n e t h a n did bile (p ~ 0.001). W h i l e bile c o n t a i n e d a p p r e c i a b l e a m o u n t s of t h e s u l p h u r - c o n t a i n i n g a m i n o acids c y s t i n e a n d m e t h i o n i n e , c y s t i n e was a b s e n t f r o m s e r u m a n d only t r a c e s of m e t h i o n i n e could b e d e t e c t e d (table) (p < 0.001). F u r t h e r m o r e , t h e r e were s i g n i f i c a n t l y g r e a t e r a m o u n t s of glycine in bile c o m p a r e d w i t h s e r u m (p < 0.001). The levels of histidine, valine, t y r o s i n e , isoleucine, leucine a n d p h e n y l a l a n i n e in bile a n d s e r u m did n o t differ signifi-
1
2 3 4
Acknowledgments. We thank Mr M. Earlam of Pharmaceuticals Division, Imperial Chemical Industries Limited for the aminoacid analyses, and Dr J. s. Morley for helpful discussion. URF gratefully acknowledges a grant (FO 73/2) from the Deutsche Forsehungsgemeinschaft, Bonn-Bad Godesberg, Fed. Rep. of Germany. KGW is recipient of a grant from the Scottish Hospital Research Endowments Trust. Present address: DW of Gastroenterology and Metabolism, Department of Medicine, University of G6ttingen, D-34 GSttingen, Fed. Rep. of Germany. Address communications to: K. G. Wormsley, Ninewells Hospital, Dundee, Scotland. DD2 1UB. U.R. F61sch and K. G. Wormsley, Br. J. Nutr. 36, 281 (1976).
Amino acid composition of serum and bile Group I Fasted Serum Median Histidine 0.050 Lysine 0.237 Arginine 0.027 Ornithine 0.134 Aspartie acid 0 Threonine +Gln +Asn + Serine 0 . 5 4 8 Glutanfie acid 0.147 Glycine 0.235 Alanine 0.264 Half cystine 0 Valine 0.131 Methionine 0.012 Isoleucine 0.078 Leucine 0.164 Tyrosine 0.055 Phenylalanine 0.082 All values in mmoles]l.
Range
Bile Median
Range
Group II Serum Fasted Median
0.025-0.148 0.158-0.325 0 -0.078 0.096-0.273 0 -0.041 0.426-0.606 0.063-0.206 0.190-0.309 0.181-0.428 0 -0.024 0.098~).162 0 -0.020 0.058-0.107 0.115-0.219 0.038-0.090 0.058-0.122
0.076 0.089 0 0.066 0.770 0.659 1.356 1.074 0.233 0.472 0.187 0.086 0.145 0.362 0.091 0.132
0.023-0.100 0.052-0.181 0 -0.017 0.042-0.078 0.371-1.118 0.128-0.902 0.788-2.536 0.611-2.649 0.175-0.376 0.373-0.929 0.106-0.247 0.051-0.408 0.108-0.247 0.124-0.666 0.077-0.118 0.085-0.219
0.056 0.381 . . 0.261 0.016 0.890 0.197 0.378 0.573 0 0.299 0.027 0.134 0.285 0.133 0.134
Fed Median 0.057 0.328 . 0.282 0 0.637 0.143 0.408 0.590 0 0.154 0.031 0.062 0.206 0.152 0.162
Bile Fasted Median
Fed Median
0.062 0.103
0.045 0.070
0.067 >2.500 0.357 2.049 1.008 0.143 0.540 0.231 0.382 0.327 0.446 0.150 0.210
0.049 >2.500 0.236 1.742 0.937 0.152 0.130 0.179 0.383 0.153 0.347 0.091 0.132
.
Specialia
1056
c a n t l y . A n u n i d e n t i f i e d peak~ t r a v e l l i n g b e t w e e n leucine a n d t y r o s i n e in bile, was n o t f o u n d in a n y s e r u m sample. Discussion. W h i l e t h e r e h a v e b e e n m a n y s t u d i e s of t h e p r o t e i n c o n t e n t of bile 5, v e r y little i n f o r m a t i o n is available a b o u t t h e a m i n o acid c o m p o s i t i o n of bile. Miller s a n a l y s e d c a d a v e r i c bile a n d f o u n d free lysine, t y r o s i n e a n d glycine w h i c h h e c o n s i d e r e d r e p r e s e n t e d t h e e x c r e t i o n of w a s t e p r o d u c t s . Dassi a n d Gianni 7 e x a m i n e d h u m a n bile o b t a i n e d d u r i n g l a p a r o t o m y a n d d e t e c t e d w a t e r soluble s u b s t a n c e s r e a c t i n g w i t h n i n h y d r i n , w h i c h t h e y c o n s i d e r e d c o m p a t i b l e w i t h t h e p r e s e n c e of free a m i n o acids or p o l y p e p t i d e s . The p r e s e n t s t u d y is t h e r e f o r e t h e f i r s t to q u a n t i f y t h e a m i n o acids in bile a n d to c o m p a r e t h e p a t t e r n of a m i n o acids in bile w i t h t h o s e f o u n d in serum. The h i g h c o n c e n t r a t i o n s of acidic a n d s u l p h u r - c o n t a i n i n g a m i n o acids in bile a n d t h e r e l a t i v e lack of basic a m i n o acids h a s n o t b e e n e x p l a i n e d . I n p a r t , t h e c o n c e n t r a t i o n s in bile m a y reflect t h e c o n c e n t r a t i o n s of t h e a m i n o acids inside t h e h e p a t i c cells, since it h a s b e e n s h o w n t h a t t h e r e are s i g n i f i c a n t l y g r e a t e r a m o u n t s of s o m e a m i n o acids ( g l u t a m a t e , a s p a r t a t e a n d glycine) in t h e liver cells t h a n in s e r u m 8. H o w e v e r , o t h e r a m i n o acids are also f o u n d to be c o n c e n t r a t e d in t h e h e p a t o c y t e s (e.g. alanine) b u t are n o t f o u n d c o n c e n t r a t e d in bile, while a m i n o acids w h i c h are n o t c o n c e n t r a t e d b y t h e liver cells (e.g. cystine) m a y be f o u n d in g r e a t e r a m o u n t s in bile t h a n in s e r u m . L y s i n e h a s b e e n s h o w n to be specifically t r a n s p o r t e d b y t h e m u c o s a of t h e gall b l a d d e r , b u t so are m e t h i o n i n e a n d glycin.e 9, so t h a t t h e r e l a t i v e lack of lysine in t h e bile is p r o b a b l y n o t a t t r i b u t a b l e t o selective t r a n s p o r t of t h e lysine o u t of t h e l u m e n of t h e b i l i a r y t r a c t . Unlike p r o t e i n s , w h i c h a p p e a r in bile b y processes inv o l v i n g b u l k t r a n s f e r a n d molecular sieving of t h e p l a s m a p r o t e i n s 10,11, it a p p e a r s t h a t t h e t r a n s f e r of a m i n o acids i n t o t h e bile i n v o l v e s specific t r a n s p o r t processes. I t has p r e v i o u s l y b e e n s h o w n t h a t organic a n i o n s TM a n d cat-
EXPERIENTIA 33/8
ionsla c a n be s e c r e t e d into bile b y a c t i v e t r a n s p o r t p r o cesses b u t m u l t i p l e e x c r e t o r y processes are p r o b a b l y i n v o l v e d 14 a n d t h e role of t h e s e processes in t h e t r a n s p o r t of a m i n o acids h a s n o t b e e n s t u d i e d . I n t h e small i n t e s t i n e , t h e t r a n s p o r t of t h e n e u t r a l , acidic a n d basic a m i n o acids is h i g h l y specific 15 so t h a t t h e r e m a y be similar p r o c e s s e s i n v o l v e d in t h e h e p a t i c s e c r e t i o n of t h e s e a m i n o acids, in v i e w of t h e s i g n i f i c a n t l y d i f f e r e n t h a n d l i n g of t h e s e 3 g r o u p s of a m i n o acids b y t h e liver cells. T h e f u n c t i o n of t h e biliary a m i n o acids h a s n o t b e e n defined, b u t a m i n o acids are k n o w n t o s t i m u l a t e p a n creatic exocrine s e c r e t i o n 16 and, p e r h a p s in c o n j u n c t i o n w i t h bile salts (which also s t i m u l a t e p a n c r e a t i c secret i o n 1~) m a y h a v e some f u n c t i o n a l role in t h e e a r l y s t a g e s of t h e digestive r e s p o n s e to t h e e n t r y of food into t h e alimentary tract.
5 6 7 8 9 10 11 12 13 14 15 16 17
C. Dive, Les prot6ines de la bile. Editions Arsica SA, Bruxelles 1971. E.F.W. Mtiller, Hoppe-Seylers Z. physiol. Chem. 242, 201 (1936). P, Dassi and A. M. Gianni, Panminerva med. 3, 461 (1961). H. Tarver, in: The Liver, vol. 1, p. 450. Ed. C. Rouiller. Academic Press, New York and London 1963. V. Mirkoviteh, F. V. Sepulveda, H. Menge and J. W. L. Robinson, Pfltigers Arch. 355, 319 (1975). C. Dive and J. F. Heremans, Eur. J. clin. Invest. d, 235 (1974). C. Dive, R. A. Nadalini, J.-P. Vaerman and J. F. Heremans, Eur. J. clin. Invest. 4, 241 (1974). I. Sperber, in: The Biliary System, p. 457. Ed. W. Taylor. Blaekwell, Oxford 1965. L.S. Sehanker, in: The Biliary System, p. 469. Ed. W. Taylor. Blaekwell, Oxford 1965. V. Hoenig and R. Preisig, Biomedicine 18, 23 (1973). P . F . Curran, Archs int. Med. 129, 258 (1972). S, J. Konturek, T. Radecki, P. Thor and A. Dembinski, Proc. Soc. exp. Biol. Med. 143, 305 (1973). M. M. Forell, M. Otte, H. J. Kohl, P. Lehnert and H. P. Stahlheber, Scand. J. Gastroent. 6, 261 (1971).
Nerve e n d i n g s isolated f r o m chick e m b r y o n i c optic t e c t u m . 2. D e v e l o p m e n t a l aspects of s y n a p t o s o m a l m e m b r a n e F. Gremo, G. C, P a n z i c a a n d C. P a n z i c a Viglietti
Istituto di Istologia e Embriologia generale, Facolta di Medicina e Chirurgia, Universith di Torino, corso M . D'Azeglio 52, 1-10726 Torino (Italy), 31 August 1976 S u m m a r y . F r a c t i o n s e n r i c h e d in n e r v e e n d i n g s ( s y n a p t o s o m e s ) h a v e b e e n isolated f r o m chick e m b r y o n i c o p t i c t e c t u m d u r i n g d e v e l o p m e n t . A f t e r o s m o t i c shock, t h e s e f r a c t i o n s a p p e a r e d t o be enriched in m e m b r a n e s w h i c h d u r i n g d e v e l o p m e n t a c q u i r e t y p i c a l f e a t u r e s of m a t u r e s y n a p t o s o m a l m e m b r a n e s . T h e d e v e l o p m e n t of i m p r o v e d f r a c t i o n a t i o n t e c h n i q u e s h a s p e r m i t t e d b i o c h e m i c a l a n d m o r p h o l o g i c a l s t u d i e s to be carried o u t i n t o t h e n a t u r e of i n d i v i d u a l c o m p o n e n t s of n e r v e e n d i n g s ( s y n a p t o s o m e s ) . T h e m o s t a t t e n t i o n h a s b e e n focused on s y n a p t o s o m a l m e m b r a n e s , b o t h in analysis of gross c o m p o s i t i o n a n d i d e n t i f i c a t i o n of specific c o m p o n e n t s 1 b o t h in u l t r a s t r u c t u r a l o r g a n i z a t i o n of s y n a p t i c densities 2, in correla-
16th Homogenate Crude mitochondrial fraction Synaptosomes Membranes
a)
b)
100 19.86 4.34 1.86
48.35 9.60 2.10 0.90
tion with its critical role played in synaptic transmission. It is also generally accepted that synaptosomal membrane is to a certain extent involved in neuronal receptor recognition and specificity of cell adhesion during the development of neuronal circuits 3. Consequently, isolation of embryonic synaptic membrane can be regarded as an essential step for biochemical and morphological studies on properties of maturing synaptic complexes.
18th
a)
b)
2nd ,)
b)
100 17.32 6.90 5.68
91.45 15.84 6.31 5.20
100 20.72 5.21 7.15
66.00 13,68 3.44 4.72
~) Percentage of protein content of each fraction based on the homogenate being 100% ; b) total protein content of the fractions (mg).
1055
Specialia
T h e a m i n o acid c o m p o s i t i o n of r a t bile i U. R. F61sch e a n d K. G. W o r m s l e y 3
Department o/ Therapeutics, University o/ Dundee, Dundee DD2 1UB (Scotland), 25 January 1977 Summary. The p a t t e r n of a m i n o acids in t h e bile of r a t s differs f r o m t h e p a t t e r n in t h e s e r u m of t h e s e animals, since bile c o n t a i n s s i g n i f i c a n t l y g r e a t e r a m o u n t s of acidic a n d s u l p h u r - c o n t a i n i n g a m i n o acids a n d glycine t h a n serum, while t h e s e r u m c o n t a i n e d m o r e basic a m i n o acids t h a n bile, i n d i c a t i n g t h a t secretion of a m i n o acids i n t o bile m a y involve specific t r a n s p o r t processes. As p a r t of a s t u d y of t h e i n c o r p o r a t i o n of a m i n o acids i n t o p a n c r e a t i c e n z y m e s a n d t h e s e c r e t i o n of t h e s e e n z y m e s 4, we s t u d i e d t h e a p p e a r a n c e of a m i n o acids in t h e bile of r a t s . Our f i n d i n g s i n d i c a t e t h a t t h e secretion of a m i n o acids i n t o bile is selective a n d specific. Materials and methods. Male W i s t a r rats, w e i g h i n g 300 g (=L 30 g) were s t u d i e d . 2 e x p e r i m e n t a l schedules were employed. 1. 9 r a t s were f a s t e d for 24 h a n d t h e n a n a e s t h e t i z e d w i t h u r e t h a n e (0.6 ml/100 g b . w t of a solution c o n t a i n i n g 250 g/l) b y i.p. injection. D u r i n g e a c h s t u d y , t h e b o d y t e m p e r a t u r e of t h e r a t s was m a i n t a i n e d a t 37 ~ The bile d u c t was c a t h e t e r i z e d n e a r t h e liver a n d t h e d u c t distal to t h e c a n n u l a was ligated. A c a t h e t e r was placed in t h e j u g u l a r v e i n a n d 0.15 moles/1 s o d i u m chloride solution w a s i n f u s e d a t 0.9 ml/h. Bile was collected in 30-min b a t c h e s in p l a s t i c t u b e s a n d t h e n pooled. Blood was obt a i n e d b y aortic p u n c t u r e a t t h e end of e a c h s t u d y . 2. I n o r d e r to s t u d y t h e effect of f a s t i n g a n d feeding on t h e biliary e x c r e t i o n of a m i n o acids, a f u r t h e r 12 r a t s were d i v i d e d i n t o 2 s u b g r o u p s , h a l f of w h i c h were f a s t e d for 24 h a n d s t u d i e d as above, while t h e r e m a i n i n g 6 r a t s were fed u n t i l t h e t i m e of s t u d y a n d t h e n h a d bile collected, as above. S a m p l e s (100 ixl) of bile or s e r u m were t r e a t e d w i t h trifluoroacetic acid (5 vi) a t 10 ~ A f t e r c e n t r i f u g a t i o n , t h e s u p e r n a t a n t a n d 4 w a s h i n g s of t h e p r e c i p i t a t e (each w a s h i n g 50 txl of triple-distilled water) were c o m b i n e d a n d e v a p o r a t e d in a v a c u u m desiccator. T h e a m i n o - a c i d c o m p o s i t i o n of t h e r e s u l t i n g residues w a s d e t e r m i n e d w i t h a L o C a r t e A m i n o - a c i d A n a l y s e r (single 22 c m c o l u m n ;
b u f f e r c h a n g e s - p H 3.25 for 65 min, 4.25 for 60 min, 6.65 for 45 rain; b u f f e r t e m p e r a t u r e - - 5 2 ~ for first 45 rain, t h e n 60~ t h e r e a f t e r ) a n d A u t o l a b C o m p u t i n g I n t e g r a t o r S y s t e m AA. Results. Bile c o n t a i n e d s i g n i f i c a n t l y g r e a t e r a m o u n t s of t h e acidic a m i n o acids a s p a r t i c a n d g l u t a m i c (table) (p < 0.01) while t h e a m o u n t s of t h e s e a m i n o acids in s e r u m were small. On t h e o t h e r h a n d , s e r u m c o n t a i n e d m o r e of t h e basic a m i n o acids lysine a n d o r n i t h i n e t h a n did bile (p ~ 0.001). W h i l e bile c o n t a i n e d a p p r e c i a b l e a m o u n t s of t h e s u l p h u r - c o n t a i n i n g a m i n o acids c y s t i n e a n d m e t h i o n i n e , c y s t i n e was a b s e n t f r o m s e r u m a n d only t r a c e s of m e t h i o n i n e could b e d e t e c t e d (table) (p < 0.001). F u r t h e r m o r e , t h e r e were s i g n i f i c a n t l y g r e a t e r a m o u n t s of glycine in bile c o m p a r e d w i t h s e r u m (p < 0.001). The levels of histidine, valine, t y r o s i n e , isoleucine, leucine a n d p h e n y l a l a n i n e in bile a n d s e r u m did n o t differ signifi-
1
2 3 4
Acknowledgments. We thank Mr M. Earlam of Pharmaceuticals Division, Imperial Chemical Industries Limited for the aminoacid analyses, and Dr J. s. Morley for helpful discussion. URF gratefully acknowledges a grant (FO 73/2) from the Deutsche Forsehungsgemeinschaft, Bonn-Bad Godesberg, Fed. Rep. of Germany. KGW is recipient of a grant from the Scottish Hospital Research Endowments Trust. Present address: DW of Gastroenterology and Metabolism, Department of Medicine, University of G6ttingen, D-34 GSttingen, Fed. Rep. of Germany. Address communications to: K. G. Wormsley, Ninewells Hospital, Dundee, Scotland. DD2 1UB. U.R. F61sch and K. G. Wormsley, Br. J. Nutr. 36, 281 (1976).
Amino acid composition of serum and bile Group I Fasted Serum Median Histidine 0.050 Lysine 0.237 Arginine 0.027 Ornithine 0.134 Aspartie acid 0 Threonine +Gln +Asn + Serine 0 . 5 4 8 Glutanfie acid 0.147 Glycine 0.235 Alanine 0.264 Half cystine 0 Valine 0.131 Methionine 0.012 Isoleucine 0.078 Leucine 0.164 Tyrosine 0.055 Phenylalanine 0.082 All values in mmoles]l.
Range
Bile Median
Range
Group II Serum Fasted Median
0.025-0.148 0.158-0.325 0 -0.078 0.096-0.273 0 -0.041 0.426-0.606 0.063-0.206 0.190-0.309 0.181-0.428 0 -0.024 0.098~).162 0 -0.020 0.058-0.107 0.115-0.219 0.038-0.090 0.058-0.122
0.076 0.089 0 0.066 0.770 0.659 1.356 1.074 0.233 0.472 0.187 0.086 0.145 0.362 0.091 0.132
0.023-0.100 0.052-0.181 0 -0.017 0.042-0.078 0.371-1.118 0.128-0.902 0.788-2.536 0.611-2.649 0.175-0.376 0.373-0.929 0.106-0.247 0.051-0.408 0.108-0.247 0.124-0.666 0.077-0.118 0.085-0.219
0.056 0.381 . . 0.261 0.016 0.890 0.197 0.378 0.573 0 0.299 0.027 0.134 0.285 0.133 0.134
Fed Median 0.057 0.328 . 0.282 0 0.637 0.143 0.408 0.590 0 0.154 0.031 0.062 0.206 0.152 0.162
Bile Fasted Median
Fed Median
0.062 0.103
0.045 0.070
0.067 >2.500 0.357 2.049 1.008 0.143 0.540 0.231 0.382 0.327 0.446 0.150 0.210
0.049 >2.500 0.236 1.742 0.937 0.152 0.130 0.179 0.383 0.153 0.347 0.091 0.132
.
Specialia
1056
c a n t l y . A n u n i d e n t i f i e d peak~ t r a v e l l i n g b e t w e e n leucine a n d t y r o s i n e in bile, was n o t f o u n d in a n y s e r u m sample. Discussion. W h i l e t h e r e h a v e b e e n m a n y s t u d i e s of t h e p r o t e i n c o n t e n t of bile 5, v e r y little i n f o r m a t i o n is available a b o u t t h e a m i n o acid c o m p o s i t i o n of bile. Miller s a n a l y s e d c a d a v e r i c bile a n d f o u n d free lysine, t y r o s i n e a n d glycine w h i c h h e c o n s i d e r e d r e p r e s e n t e d t h e e x c r e t i o n of w a s t e p r o d u c t s . Dassi a n d Gianni 7 e x a m i n e d h u m a n bile o b t a i n e d d u r i n g l a p a r o t o m y a n d d e t e c t e d w a t e r soluble s u b s t a n c e s r e a c t i n g w i t h n i n h y d r i n , w h i c h t h e y c o n s i d e r e d c o m p a t i b l e w i t h t h e p r e s e n c e of free a m i n o acids or p o l y p e p t i d e s . The p r e s e n t s t u d y is t h e r e f o r e t h e f i r s t to q u a n t i f y t h e a m i n o acids in bile a n d to c o m p a r e t h e p a t t e r n of a m i n o acids in bile w i t h t h o s e f o u n d in serum. The h i g h c o n c e n t r a t i o n s of acidic a n d s u l p h u r - c o n t a i n i n g a m i n o acids in bile a n d t h e r e l a t i v e lack of basic a m i n o acids h a s n o t b e e n e x p l a i n e d . I n p a r t , t h e c o n c e n t r a t i o n s in bile m a y reflect t h e c o n c e n t r a t i o n s of t h e a m i n o acids inside t h e h e p a t i c cells, since it h a s b e e n s h o w n t h a t t h e r e are s i g n i f i c a n t l y g r e a t e r a m o u n t s of s o m e a m i n o acids ( g l u t a m a t e , a s p a r t a t e a n d glycine) in t h e liver cells t h a n in s e r u m 8. H o w e v e r , o t h e r a m i n o acids are also f o u n d to be c o n c e n t r a t e d in t h e h e p a t o c y t e s (e.g. alanine) b u t are n o t f o u n d c o n c e n t r a t e d in bile, while a m i n o acids w h i c h are n o t c o n c e n t r a t e d b y t h e liver cells (e.g. cystine) m a y be f o u n d in g r e a t e r a m o u n t s in bile t h a n in s e r u m . L y s i n e h a s b e e n s h o w n to be specifically t r a n s p o r t e d b y t h e m u c o s a of t h e gall b l a d d e r , b u t so are m e t h i o n i n e a n d glycin.e 9, so t h a t t h e r e l a t i v e lack of lysine in t h e bile is p r o b a b l y n o t a t t r i b u t a b l e t o selective t r a n s p o r t of t h e lysine o u t of t h e l u m e n of t h e b i l i a r y t r a c t . Unlike p r o t e i n s , w h i c h a p p e a r in bile b y processes inv o l v i n g b u l k t r a n s f e r a n d molecular sieving of t h e p l a s m a p r o t e i n s 10,11, it a p p e a r s t h a t t h e t r a n s f e r of a m i n o acids i n t o t h e bile i n v o l v e s specific t r a n s p o r t processes. I t has p r e v i o u s l y b e e n s h o w n t h a t organic a n i o n s TM a n d cat-
EXPERIENTIA 33/8
ionsla c a n be s e c r e t e d into bile b y a c t i v e t r a n s p o r t p r o cesses b u t m u l t i p l e e x c r e t o r y processes are p r o b a b l y i n v o l v e d 14 a n d t h e role of t h e s e processes in t h e t r a n s p o r t of a m i n o acids h a s n o t b e e n s t u d i e d . I n t h e small i n t e s t i n e , t h e t r a n s p o r t of t h e n e u t r a l , acidic a n d basic a m i n o acids is h i g h l y specific 15 so t h a t t h e r e m a y be similar p r o c e s s e s i n v o l v e d in t h e h e p a t i c s e c r e t i o n of t h e s e a m i n o acids, in v i e w of t h e s i g n i f i c a n t l y d i f f e r e n t h a n d l i n g of t h e s e 3 g r o u p s of a m i n o acids b y t h e liver cells. T h e f u n c t i o n of t h e biliary a m i n o acids h a s n o t b e e n defined, b u t a m i n o acids are k n o w n t o s t i m u l a t e p a n creatic exocrine s e c r e t i o n 16 and, p e r h a p s in c o n j u n c t i o n w i t h bile salts (which also s t i m u l a t e p a n c r e a t i c secret i o n 1~) m a y h a v e some f u n c t i o n a l role in t h e e a r l y s t a g e s of t h e digestive r e s p o n s e to t h e e n t r y of food into t h e alimentary tract.
5 6 7 8 9 10 11 12 13 14 15 16 17
C. Dive, Les prot6ines de la bile. Editions Arsica SA, Bruxelles 1971. E.F.W. Mtiller, Hoppe-Seylers Z. physiol. Chem. 242, 201 (1936). P, Dassi and A. M. Gianni, Panminerva med. 3, 461 (1961). H. Tarver, in: The Liver, vol. 1, p. 450. Ed. C. Rouiller. Academic Press, New York and London 1963. V. Mirkoviteh, F. V. Sepulveda, H. Menge and J. W. L. Robinson, Pfltigers Arch. 355, 319 (1975). C. Dive and J. F. Heremans, Eur. J. clin. Invest. d, 235 (1974). C. Dive, R. A. Nadalini, J.-P. Vaerman and J. F. Heremans, Eur. J. clin. Invest. 4, 241 (1974). I. Sperber, in: The Biliary System, p. 457. Ed. W. Taylor. Blaekwell, Oxford 1965. L.S. Sehanker, in: The Biliary System, p. 469. Ed. W. Taylor. Blaekwell, Oxford 1965. V. Hoenig and R. Preisig, Biomedicine 18, 23 (1973). P . F . Curran, Archs int. Med. 129, 258 (1972). S, J. Konturek, T. Radecki, P. Thor and A. Dembinski, Proc. Soc. exp. Biol. Med. 143, 305 (1973). M. M. Forell, M. Otte, H. J. Kohl, P. Lehnert and H. P. Stahlheber, Scand. J. Gastroent. 6, 261 (1971).
Nerve e n d i n g s isolated f r o m chick e m b r y o n i c optic t e c t u m . 2. D e v e l o p m e n t a l aspects of s y n a p t o s o m a l m e m b r a n e F. Gremo, G. C, P a n z i c a a n d C. P a n z i c a Viglietti
Istituto di Istologia e Embriologia generale, Facolta di Medicina e Chirurgia, Universith di Torino, corso M . D'Azeglio 52, 1-10726 Torino (Italy), 31 August 1976 S u m m a r y . F r a c t i o n s e n r i c h e d in n e r v e e n d i n g s ( s y n a p t o s o m e s ) h a v e b e e n isolated f r o m chick e m b r y o n i c o p t i c t e c t u m d u r i n g d e v e l o p m e n t . A f t e r o s m o t i c shock, t h e s e f r a c t i o n s a p p e a r e d t o be enriched in m e m b r a n e s w h i c h d u r i n g d e v e l o p m e n t a c q u i r e t y p i c a l f e a t u r e s of m a t u r e s y n a p t o s o m a l m e m b r a n e s . T h e d e v e l o p m e n t of i m p r o v e d f r a c t i o n a t i o n t e c h n i q u e s h a s p e r m i t t e d b i o c h e m i c a l a n d m o r p h o l o g i c a l s t u d i e s to be carried o u t i n t o t h e n a t u r e of i n d i v i d u a l c o m p o n e n t s of n e r v e e n d i n g s ( s y n a p t o s o m e s ) . T h e m o s t a t t e n t i o n h a s b e e n focused on s y n a p t o s o m a l m e m b r a n e s , b o t h in analysis of gross c o m p o s i t i o n a n d i d e n t i f i c a t i o n of specific c o m p o n e n t s 1 b o t h in u l t r a s t r u c t u r a l o r g a n i z a t i o n of s y n a p t i c densities 2, in correla-
16th Homogenate Crude mitochondrial fraction Synaptosomes Membranes
a)
b)
100 19.86 4.34 1.86
48.35 9.60 2.10 0.90
tion with its critical role played in synaptic transmission. It is also generally accepted that synaptosomal membrane is to a certain extent involved in neuronal receptor recognition and specificity of cell adhesion during the development of neuronal circuits 3. Consequently, isolation of embryonic synaptic membrane can be regarded as an essential step for biochemical and morphological studies on properties of maturing synaptic complexes.
18th
a)
b)
2nd ,)
b)
100 17.32 6.90 5.68
91.45 15.84 6.31 5.20
100 20.72 5.21 7.15
66.00 13,68 3.44 4.72
~) Percentage of protein content of each fraction based on the homogenate being 100% ; b) total protein content of the fractions (mg).
