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Absorption of nicotinic acid and nicotinamide across human buccal mucosa in vivo
Life Sciences, Vol. 27, pp. P r i n t e d in the U.S.A.
ABSORPTION
D.F.
1649-1651
Pergamon
OF N I C O T I N I C A C I D AND N I C O T I N A M I D E H U M A N B U C C A L M U C O S A in vlvo
Evered,
F. S a d o o g h - A b a s l a n
D e p a r t m e n t of Baochemastry, o f London), Chelsea, (Received
in final
and P.D.
ACROSS
Patel
Chelsea College London SW3 6LX
(Unlverslty
form A u g u s t
1980)
22,
Press
Summary B u c c a l a b s o r p t i o n rates of n ~ c o t l n ~ c acld and n ~ c o t i n a m z d e were llnear w l t h r e s p e c t to Inltlal c o n c e n t r a t l o n b e t w e e n 2 and iO mlllimolar. A b s o r p t l o n was s o d i ~ n - d e p e n d e n t as in the small zntestlne a n d s u g g e s t e d c a r r l e r - m e d l a t e d transport. T r a n s p o r t of n l c o t ~ n l c acad and n l c o t i n a m l d e has b e e n s t u d l e d uslng e v e r t e d sacs of rat small i n t e s t l n e in vltro. The results w e r e compatible w l t h c a r r l e r - m e d l a t e d f a c ~ l l t a t e d d l f f u s l o n at low concentratlons m a s k e d by p a s s i v e d l f f u s i o n at h a g h e r c o n c e n t r a t l o n s (i). S~nce a b s o r p t l o n of c e r t a l n n u t r i e n t s from the small intestane and across human b u c c a l m u c o s a ls b r o a d l y sam~lar we h a v e i n v e s t l g a t e d a b s o r p t l o n of n l c o t l n l c acld and n l c o t l n a m ~ d e u s l n g the latter slte of the g a s t r o i n t e s t l n a l tract. Methods Buccal a b s o r p t a o n w a s s t u d l e d by a p u b l i s h e d m e t h o d (2) i n v o l v l n g c a r c u l a t l o n of a p r e - i n c u b a t e d b u f f e r e d s o l u t a o n of the test c o m p o u n d in the m o u t h for fave minutes. The solution, of k n o w n inltlal concentratlon, was e 3 e c t e d and f o l l o w e d by a f l v e - s e c o n d w a s h w l t h fresh b u f f e r solutaon. The p o o l e d sample and w a s h l n g s w e r e d l l u t e d and a s s a y e d for n a c o t l n l c acid, n l c o t l n a m a d e or ~ s o n l c o t l n l c a c z d using a s p e c t r o p h o t o m e t r a c m e t h o d (3). C o m p a r a s o n of the amount remaining, found by analysis, w a t h the lnitaal c o n c e n t r a t a o n of the test c o m p o u n d gave a m e a s u r e of the a b s o r p t l o n from the m o u t h an ~moles/5 mln. Results Buccal a b s o r p t l o n of these two compounds was llnear w l t h znltl~l conc e n t r a t l o n b e t w e e n 2 and iO m i l l i ~ o l a r (Fig. i). O m i s s i o n of Na ions in the test buffer, r e p l a c e d by K ions, d e c r e a s e d v z t a m i ~ a b s o r p t i o n across the mucosa. Thus for 4 m M n l c o t i n i c a c i d w l t h Na ions p r e s e n t a b s o r p t i o n was 7.2 ± 0.92 ~ m o l e s / 5 m i n (4 experiments) a n d w l t h o u t Na + • ons, 4.1 ± 0.81 (n = 4), s t a t 1 ~ t l c a l s l g n i f i c a n c e P < 0.025. For 4 m M n l c o t l n a m l ~ e a b s o r p t i o n w i t h Na ions p r e s e n t was 4.2 ± 0 . 5 0 (n = 4) and w i t h o u t Na ions 3.0 ± 0 . 3 0 (n = 4), P < 0.05. Thls e f f e c t w a s c o n f i r m e d by the a d d i t a o n of e t h a c r y n a c acad. N i c o t i n l c acid (4 mM) a b s o r p t i o n in the absence of e t h a c r y n a c a c a d was 4.8 ± 0 . 5 0 (n = 4) but in the p r e s e n c e of e t h a c r y n i c a c i d (i mM) at w a s 3.0 ± 0 . 3 0 (n = 4), P < 0.025. Similarly n a c o t l n a m i d e a b s o r p t a o n alone was 4.4 ± 0.52 (n = 4) but In the p r e s e n c e of e t h a c r y n i c acid a b s o r p t a o n was 2.2 ± 0 . 6 0 (n = 4), P < 0.025. 0024-3205/80/441649-03502.00/0 Copyright (c) 1980 P e r g a m o n Press Ltd.
1650
Absorption
of N i c o t i n a t e
and N i c o t l n a m l d e
Vol.
27, No.
18,
Buccal m u c o s a was also p e r m e a b l e to an ~somer of n ~ c o t ~ n l c acid, n a m e l y i s o n l c o t l n ~ c acid. For 4 mM n ~ c o t ~ n l c acid as control, a b s o r p t l o n was 5.4 ± 0.35 (n = 4). For I s o n l c o t l n z c acid the a b s o r p t l o n was m u c h lower: 3.3 ± 0.42 (n = 4), P < 0.005.
E ~
12
12
lO
¢ I0 E
i.
.m
~
4
2
2
4
6
8
lO
2
Initial c o n c e n t r a t i o n of n i c o t i n i c acid [mM]
Fig.
la
4
~
a
*0
Initial c o n c e n t r a t i o n of m c o t l n a m l d e [ m M ]
Fig.
lb
E f f e c t of inltzal c o n c e n t r a t l o n on the a b s o r p t i o n and (ib) n l c o t l n a m l d e across h u m a n b u c c a l mucosa. mean of 4 to 7 e x p e r i m e n t s and the bars r e p r e s e n t
of
(la) n l c o t l n l c ac±d Each p o l n t is the the SEM.
The l~nes w e r e drawn f o l l o w ~ n g llnear r e g r e s s i o n analys~s. Correlatlon c o e f f ~ c l e n t s w e r e n l c o t l n ~ c acld, 0.99 and n l c o t ~ n a m l d e , 0.996. Dlscusslon
For drugs p a s s l n g across b u c c a l mucosa, p e r m e a b l l l t y is a f f e c t e d by l l p l d s o l u b l l l t y and by the p e r c e n t a g e of u n ~ o n l z e d m o l e c u l e s p r e s e n t (4). At pH 6, the pH at w h i c h our b u c c a l e x p e r l m e n t s w e r e performed, n l c o t l n l c acld is 6% u n l o n ~ z e d b u t n l c o t ~ n a m l d e is a l m o s t fully l o n l z e d (99.99%). F u r t h e r m o r e , b o t h compounds are s o l u b l e ~n w a t e r and p o o r l y soluble in llpld. Hence, in spite of a c o n s i d e r a b l e d l f f e r e n c e In the p e r c e n t a g e l o n l z e d of n l c o t l n l c acid and n l c o t l n a m l d e at pH 6 thelr a b s o r p t l o n rates across the b u c c a l m u c o s a are very s l m ~ l a r (Fig. i). Th~s b e h a v l o u r w o u l d imply t r a n s p o r t of the u n l o n l z e d m o l e c u l e s . The a b s o r p t l o n rates of n l c o t l n l c acld and n l c o t l n a m l d e dld not reach a m a x l m u m even at i0 mM c o n c e n t r a t l o n (Fig. i). Th~s s u g g e s t s e ~ t h e r p a s s l v e d ~ f f u s l o n or c a r r l e r - m e d z a t e d t r a n s p o r t w l t h a v e r y h l g h d l s s o c l a t l o n constant, K tT r a n s p o r t o c c u r r e d w l t h the c o n c e n t r a t i o n g r a d l e n t "downh~ll" ~n our e x p e r l m e n t s since the ~nltlal test c o n c e n t r a t l o n s always e x c e e d e d the b l o o d level. The assay p r o c e d u r e was n o t s e n s l t l v e enough to m e a s u r e a c c u r a t e l y t r a n s p o r t of n l c o t l n l c acld b e l o w the b l o o d level of about 56 ~ m o l a r (5). The n l c o t l n l c a c l d c o n t e n t of r e s t l n g s a l l v a is about 0.25 ~ m o l a r (6). T h e r e f o r e ~ n v e s t l g a t l o n s of t r a n s p o r t a g a i n s t the c o n c e n t r a t l o n g r a d i e n t c o u l d n o t be c o n d u c t e d w ~ t h the p r e s e n t experlm e n t a l system. The use of I s o t o p l c a l l y - l a b e l l e d compounds w l t h h u m a n b u c c a l m u c o s a in v l v o Is c o n t r a l n d l c a t e d on the grounds of safety and ethlcal c o n s l d e r a t l o n s .
1980
Vol.
27, No.
18, 1980
A b s o r p t i o n of N 1 c o t l n a t e and N 1 c o t i n a m l d e
P a s s ± v e d l f f u s l o n s e e m s u n l l k e l y s l n c e a b s o r p t l o n of b o t h n ± c o t l n l c a c l d a n d n ± c o t l n a m l d e a c r o s s b u c c a l m u c o s a w a s d e p e n d e n t o n the p r e s e n c e o f s o d l u m ions. T h l s f l n d l n g ±s s u b s t a n t l a t e d b y the a b l l ± t y of e t h a c r y n l c a c l d to i n h l b l t the t r a n s p o r t of n l c o t l n l c a c l d a n d n l c o t l n a m ± d e a c r o s s b u c c a l mucosa. E t h a c r y n l c a c i d is k n o w n to i n h l b l t e x t r u s i o n of Na + f r o m cells (7). Isonlcotlnlc acld and nlcotln±c acid have identlcal molecular welghts and v e r y s l m l l a r p K a values. 4.84 a n d 4.77 r e s p e c t ± v e l y (8) y e t the f o r m e r has a l o w e r rate o f a b s o r p t i o n . Thls r e s u l t is a l s o c o m p a t l b l e w i t h e x c l u d l n g p a s s ± v e d l f f u s l o n as the m o d e of t r a n s p o r t for n ± c o t l n l c ac±d. A c c o r d l n g to our p r e v l o u s o b s e r v a t l o n s (1) a c a r r l e r - m e d l a t e d t r a n s p o r t system, n a m e l y f a c l l l t a t e d d l f f u s l o n , is r e s p o n s l b l e for the a b s o r p t l o n of n l c o t l n l c a c i d a n d n l c o t ± n a m l d e f r o m m a m m a l l a n s m a l l intestlne. It seems l l k e l y t h a t t h e s e c o m p o u n d s are also a b s o r b e d a c r o s s b u c c a l m u c o s a by a c a r r l e r - m e d l a t e d p r o c e s s . T h u s n l c o t l n l C a c l d a n d n l c o t l n a m l d e 3 o l n the llst of n u t r l e n t s w h l c h are a b s o r b e d f r o m b u c c a l m u c o s a ±n a r a t h e r s l m l l a r w a y to a b s o r p t l o n f r o m the s m a l l intestlne. F o o d s u g a r s (9) and a n o t h e r v l t a m l n , a s c o r b l c a c l d (2) are t y p l c a l examples. Acknowledgment We t h a n k Dr. P.J. data.
B u t t e r w o r t h for the l l n e a r r e g r e s s i o n a n a l y s l s of o u r
References i. F. S A D O O G H - A B A S I A N and D.F. EVERED, Bloch±m. b l o p h y s . A c t a 598 3 8 5 - 3 9 1 (1980). 2. F. S A D O O G H - A B A S I A N a n d D.F. EVERED, Br. J. Nutr. 42 1 5 - 2 0 (1979). 3. L.A. C A R L S O N , C l l n l c a chlm. A c t a 13 3 4 9 - 3 5 1 (1966). 4. A.H. B E C K E T T a n d R.D. H O S S I E , Handb. exp. P h a r m a c o l . 28 24-46 (1971). 5. C.W. DENKO, W.E. G R U N D Y a n d J.W. P O R T E R , Archs. Blochem. 13 4 8 1 - 4 8 4
(1947). 6. J.E. EASTOE, B l o c h e m l s t ' s Handbook; C. LONG, ed. 9 0 7 - 9 1 0 Spon, L o n d o n (1961). 7. G. W H I T T E M B U R Y , J. gen. Phys±ol. 51 3 0 3 s - 3 1 4 s (1968). 8. R.W. G R E E N a n d H.K. TONG, J. Am. chem. Soc. 78 4 8 9 6 - 4 9 0 0 (1956). 9. A.S. M A N N I N G a n d D.F. EVERED, Cl±n. Scl. 51 1 2 7 - 1 3 2 (1976).
1651
ABSORPTION
D.F.
1649-1651
Pergamon
OF N I C O T I N I C A C I D AND N I C O T I N A M I D E H U M A N B U C C A L M U C O S A in vlvo
Evered,
F. S a d o o g h - A b a s l a n
D e p a r t m e n t of Baochemastry, o f London), Chelsea, (Received
in final
and P.D.
ACROSS
Patel
Chelsea College London SW3 6LX
(Unlverslty
form A u g u s t
1980)
22,
Press
Summary B u c c a l a b s o r p t i o n rates of n ~ c o t l n ~ c acld and n ~ c o t i n a m z d e were llnear w l t h r e s p e c t to Inltlal c o n c e n t r a t l o n b e t w e e n 2 and iO mlllimolar. A b s o r p t l o n was s o d i ~ n - d e p e n d e n t as in the small zntestlne a n d s u g g e s t e d c a r r l e r - m e d l a t e d transport. T r a n s p o r t of n l c o t ~ n l c acad and n l c o t i n a m l d e has b e e n s t u d l e d uslng e v e r t e d sacs of rat small i n t e s t l n e in vltro. The results w e r e compatible w l t h c a r r l e r - m e d l a t e d f a c ~ l l t a t e d d l f f u s l o n at low concentratlons m a s k e d by p a s s i v e d l f f u s i o n at h a g h e r c o n c e n t r a t l o n s (i). S~nce a b s o r p t l o n of c e r t a l n n u t r i e n t s from the small intestane and across human b u c c a l m u c o s a ls b r o a d l y sam~lar we h a v e i n v e s t l g a t e d a b s o r p t l o n of n l c o t l n l c acld and n l c o t l n a m ~ d e u s l n g the latter slte of the g a s t r o i n t e s t l n a l tract. Methods Buccal a b s o r p t a o n w a s s t u d l e d by a p u b l i s h e d m e t h o d (2) i n v o l v l n g c a r c u l a t l o n of a p r e - i n c u b a t e d b u f f e r e d s o l u t a o n of the test c o m p o u n d in the m o u t h for fave minutes. The solution, of k n o w n inltlal concentratlon, was e 3 e c t e d and f o l l o w e d by a f l v e - s e c o n d w a s h w l t h fresh b u f f e r solutaon. The p o o l e d sample and w a s h l n g s w e r e d l l u t e d and a s s a y e d for n a c o t l n l c acid, n l c o t l n a m a d e or ~ s o n l c o t l n l c a c z d using a s p e c t r o p h o t o m e t r a c m e t h o d (3). C o m p a r a s o n of the amount remaining, found by analysis, w a t h the lnitaal c o n c e n t r a t a o n of the test c o m p o u n d gave a m e a s u r e of the a b s o r p t l o n from the m o u t h an ~moles/5 mln. Results Buccal a b s o r p t l o n of these two compounds was llnear w l t h znltl~l conc e n t r a t l o n b e t w e e n 2 and iO m i l l i ~ o l a r (Fig. i). O m i s s i o n of Na ions in the test buffer, r e p l a c e d by K ions, d e c r e a s e d v z t a m i ~ a b s o r p t i o n across the mucosa. Thus for 4 m M n l c o t i n i c a c i d w l t h Na ions p r e s e n t a b s o r p t i o n was 7.2 ± 0.92 ~ m o l e s / 5 m i n (4 experiments) a n d w l t h o u t Na + • ons, 4.1 ± 0.81 (n = 4), s t a t 1 ~ t l c a l s l g n i f i c a n c e P < 0.025. For 4 m M n l c o t l n a m l ~ e a b s o r p t i o n w i t h Na ions p r e s e n t was 4.2 ± 0 . 5 0 (n = 4) and w i t h o u t Na ions 3.0 ± 0 . 3 0 (n = 4), P < 0.05. Thls e f f e c t w a s c o n f i r m e d by the a d d i t a o n of e t h a c r y n a c acad. N i c o t i n l c acid (4 mM) a b s o r p t i o n in the absence of e t h a c r y n a c a c a d was 4.8 ± 0 . 5 0 (n = 4) but in the p r e s e n c e of e t h a c r y n i c a c i d (i mM) at w a s 3.0 ± 0 . 3 0 (n = 4), P < 0.025. Similarly n a c o t l n a m i d e a b s o r p t a o n alone was 4.4 ± 0.52 (n = 4) but In the p r e s e n c e of e t h a c r y n i c acid a b s o r p t a o n was 2.2 ± 0 . 6 0 (n = 4), P < 0.025. 0024-3205/80/441649-03502.00/0 Copyright (c) 1980 P e r g a m o n Press Ltd.
1650
Absorption
of N i c o t i n a t e
and N i c o t l n a m l d e
Vol.
27, No.
18,
Buccal m u c o s a was also p e r m e a b l e to an ~somer of n ~ c o t ~ n l c acid, n a m e l y i s o n l c o t l n ~ c acid. For 4 mM n ~ c o t ~ n l c acid as control, a b s o r p t l o n was 5.4 ± 0.35 (n = 4). For I s o n l c o t l n z c acid the a b s o r p t l o n was m u c h lower: 3.3 ± 0.42 (n = 4), P < 0.005.
E ~
12
12
lO
¢ I0 E
i.
.m
~
4
2
2
4
6
8
lO
2
Initial c o n c e n t r a t i o n of n i c o t i n i c acid [mM]
Fig.
la
4
~
a
*0
Initial c o n c e n t r a t i o n of m c o t l n a m l d e [ m M ]
Fig.
lb
E f f e c t of inltzal c o n c e n t r a t l o n on the a b s o r p t i o n and (ib) n l c o t l n a m l d e across h u m a n b u c c a l mucosa. mean of 4 to 7 e x p e r i m e n t s and the bars r e p r e s e n t
of
(la) n l c o t l n l c ac±d Each p o l n t is the the SEM.
The l~nes w e r e drawn f o l l o w ~ n g llnear r e g r e s s i o n analys~s. Correlatlon c o e f f ~ c l e n t s w e r e n l c o t l n ~ c acld, 0.99 and n l c o t ~ n a m l d e , 0.996. Dlscusslon
For drugs p a s s l n g across b u c c a l mucosa, p e r m e a b l l l t y is a f f e c t e d by l l p l d s o l u b l l l t y and by the p e r c e n t a g e of u n ~ o n l z e d m o l e c u l e s p r e s e n t (4). At pH 6, the pH at w h i c h our b u c c a l e x p e r l m e n t s w e r e performed, n l c o t l n l c acld is 6% u n l o n ~ z e d b u t n l c o t ~ n a m l d e is a l m o s t fully l o n l z e d (99.99%). F u r t h e r m o r e , b o t h compounds are s o l u b l e ~n w a t e r and p o o r l y soluble in llpld. Hence, in spite of a c o n s i d e r a b l e d l f f e r e n c e In the p e r c e n t a g e l o n l z e d of n l c o t l n l c acid and n l c o t l n a m l d e at pH 6 thelr a b s o r p t l o n rates across the b u c c a l m u c o s a are very s l m ~ l a r (Fig. i). Th~s b e h a v l o u r w o u l d imply t r a n s p o r t of the u n l o n l z e d m o l e c u l e s . The a b s o r p t l o n rates of n l c o t l n l c acld and n l c o t l n a m l d e dld not reach a m a x l m u m even at i0 mM c o n c e n t r a t l o n (Fig. i). Th~s s u g g e s t s e ~ t h e r p a s s l v e d ~ f f u s l o n or c a r r l e r - m e d z a t e d t r a n s p o r t w l t h a v e r y h l g h d l s s o c l a t l o n constant, K tT r a n s p o r t o c c u r r e d w l t h the c o n c e n t r a t i o n g r a d l e n t "downh~ll" ~n our e x p e r l m e n t s since the ~nltlal test c o n c e n t r a t l o n s always e x c e e d e d the b l o o d level. The assay p r o c e d u r e was n o t s e n s l t l v e enough to m e a s u r e a c c u r a t e l y t r a n s p o r t of n l c o t l n l c acld b e l o w the b l o o d level of about 56 ~ m o l a r (5). The n l c o t l n l c a c l d c o n t e n t of r e s t l n g s a l l v a is about 0.25 ~ m o l a r (6). T h e r e f o r e ~ n v e s t l g a t l o n s of t r a n s p o r t a g a i n s t the c o n c e n t r a t l o n g r a d i e n t c o u l d n o t be c o n d u c t e d w ~ t h the p r e s e n t experlm e n t a l system. The use of I s o t o p l c a l l y - l a b e l l e d compounds w l t h h u m a n b u c c a l m u c o s a in v l v o Is c o n t r a l n d l c a t e d on the grounds of safety and ethlcal c o n s l d e r a t l o n s .
1980
Vol.
27, No.
18, 1980
A b s o r p t i o n of N 1 c o t l n a t e and N 1 c o t i n a m l d e
P a s s ± v e d l f f u s l o n s e e m s u n l l k e l y s l n c e a b s o r p t l o n of b o t h n ± c o t l n l c a c l d a n d n ± c o t l n a m l d e a c r o s s b u c c a l m u c o s a w a s d e p e n d e n t o n the p r e s e n c e o f s o d l u m ions. T h l s f l n d l n g ±s s u b s t a n t l a t e d b y the a b l l ± t y of e t h a c r y n l c a c l d to i n h l b l t the t r a n s p o r t of n l c o t l n l c a c l d a n d n l c o t l n a m ± d e a c r o s s b u c c a l mucosa. E t h a c r y n l c a c i d is k n o w n to i n h l b l t e x t r u s i o n of Na + f r o m cells (7). Isonlcotlnlc acld and nlcotln±c acid have identlcal molecular welghts and v e r y s l m l l a r p K a values. 4.84 a n d 4.77 r e s p e c t ± v e l y (8) y e t the f o r m e r has a l o w e r rate o f a b s o r p t i o n . Thls r e s u l t is a l s o c o m p a t l b l e w i t h e x c l u d l n g p a s s ± v e d l f f u s l o n as the m o d e of t r a n s p o r t for n ± c o t l n l c ac±d. A c c o r d l n g to our p r e v l o u s o b s e r v a t l o n s (1) a c a r r l e r - m e d l a t e d t r a n s p o r t system, n a m e l y f a c l l l t a t e d d l f f u s l o n , is r e s p o n s l b l e for the a b s o r p t l o n of n l c o t l n l c a c i d a n d n l c o t ± n a m l d e f r o m m a m m a l l a n s m a l l intestlne. It seems l l k e l y t h a t t h e s e c o m p o u n d s are also a b s o r b e d a c r o s s b u c c a l m u c o s a by a c a r r l e r - m e d l a t e d p r o c e s s . T h u s n l c o t l n l C a c l d a n d n l c o t l n a m l d e 3 o l n the llst of n u t r l e n t s w h l c h are a b s o r b e d f r o m b u c c a l m u c o s a ±n a r a t h e r s l m l l a r w a y to a b s o r p t l o n f r o m the s m a l l intestlne. F o o d s u g a r s (9) and a n o t h e r v l t a m l n , a s c o r b l c a c l d (2) are t y p l c a l examples. Acknowledgment We t h a n k Dr. P.J. data.
B u t t e r w o r t h for the l l n e a r r e g r e s s i o n a n a l y s l s of o u r
References i. F. S A D O O G H - A B A S I A N and D.F. EVERED, Bloch±m. b l o p h y s . A c t a 598 3 8 5 - 3 9 1 (1980). 2. F. S A D O O G H - A B A S I A N a n d D.F. EVERED, Br. J. Nutr. 42 1 5 - 2 0 (1979). 3. L.A. C A R L S O N , C l l n l c a chlm. A c t a 13 3 4 9 - 3 5 1 (1966). 4. A.H. B E C K E T T a n d R.D. H O S S I E , Handb. exp. P h a r m a c o l . 28 24-46 (1971). 5. C.W. DENKO, W.E. G R U N D Y a n d J.W. P O R T E R , Archs. Blochem. 13 4 8 1 - 4 8 4
(1947). 6. J.E. EASTOE, B l o c h e m l s t ' s Handbook; C. LONG, ed. 9 0 7 - 9 1 0 Spon, L o n d o n (1961). 7. G. W H I T T E M B U R Y , J. gen. Phys±ol. 51 3 0 3 s - 3 1 4 s (1968). 8. R.W. G R E E N a n d H.K. TONG, J. Am. chem. Soc. 78 4 8 9 6 - 4 9 0 0 (1956). 9. A.S. M A N N I N G a n d D.F. EVERED, Cl±n. Scl. 51 1 2 7 - 1 3 2 (1976).
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