830

Specialia

Material und Methoden. Zur Isolierung wurde Phallolysin aus kalt-w/isserigen E x t r a k t e n lyophilisierter Pilze m i t 40 % A m m o n i u m s u l f a t gef/illt nnd durch Ionenaust a u s c h c h r o m a t o g r a p h i e an D E A E - C e l l u l o s e (0,03 M Tris-HCl-Puffer p H 8,0; 2 ~ und wiederholte Gelchrom a t o g r a p h i e an Biogel P-30 (Off M N a - P h o s p h a t p u f f e r p H 5,5, NaC1 ad 0,3 M ; 2~ gereinigt. Zur Reinheitspriifung diente Celluloseacetat-Elektrophorese (pH 8,5), S t a n d a r d - D i s k - E l e k t r o p h o r e s e (pH 4,0; p H 8,5) und N a t r i u m d o d e c y l s u l f a t ( S D S ) - D i s k - E l e k t r o p h o r e s e ; gef/~rbt wurde m i t Coomassie Brilliant Blue. Das a p p a r e n t e Molekulargewicht e r m i t t e l t e n wir dnrch gelchromatographischen Vergleich m i t S t a n d a r d p r o t e i n e n an S e p h a d e x G-75, den Isoelektrischen P u n k t d u r c h Elektrofokussier u n g in Ampholine. P h o s p h a t w u r d e n a c h AMES~ bestimmt. Ergebnisse und Diskussion. Die h~tmolytische A k t i v i t / i t liess sich aus C e l l u l o s e a c e t a t - E l e k t r o p h e r o g r a m m e n und P o l y a c r y l a m i d g e l e n eluieren und entsprach einer m i t Coomassie Blue f~rbbaren Bande. Bei der SDS-DiskElektrophorese erwies sich das isolierte Phallolysin als zu e t w a 50 % rein. Bei der E l e k t r o p h o r e s e v e r h i e l t es sich basisch, der Isoelektrische P u n k t lag bei 8,25. Es war nicht dialysabel. Ftir das n a t i v e HS,molysin wurde ein Molekulargewicht von 30.000 ermittelt. Phallolysin w u r d e durch 0 , 1 % N a t r i u m d o d e c y l s u l f a t (60 min/37~ vollst~ndig und irreversibel inaktiviert. D u t c h 8 M H a r n s t o f f wurde es langsam i n a k t i v i e r t (nach 24 h bei 20 ~ R e s t a k t i v i t / i t < 1-15 % ) D a b e i v e r s c h w a n d m i t der h g m o l y t i s c h e n A k t i v i t / i t auch die Toxizitgt. Der i n a k t i v i e r t e Anteil liess sich nicht reaktivieren durch Dialyse, Verdiinnung oder gelchromatographische Abt r e n n u n g des Harnstoffs. Es war t h e r m o l a b i l und verlor bei 65 ~ innerhalb yon 5 rain 90 % seiner Aktivit~t. Es war s~turelabil und wurde bei p H 2,0 bei 37 ~ innerhalb yon 10 min i n a k t i v i e r t ; in der K/~lte war es gegen S~ure best~ndiger. Gegen Alkali w a r es stabiler. Obwohl die physikalisch-chemischen E i g e n s c h a f t e n v o n Phallolysin auf ein P r o t e i n hinwiesen, war die hXmolytische A k t i v i t ~ t resistent gegen Pepsin, Trypsin, Chymotrypsin, Subtilisin und Pronase (48h/37 ~ Dabei h e m m t e es die A k t i v i t g t dieser Proteasen gegen andere S u b s t r a t e nicht; auch/~nderte u n t e r der E n z y m b e h a n d l u n g die der

LDso (mg/kg~) mit Vertrauensgrenzen Pilztrockenpulver (in Agar suspendiert) ~thanolischer Extrakt (50% Nthanol, 25~ Wfissriger Ex~rakt (2 • 24 h, 2~

26,0

(22,2-30,5)

112,0 (102,0-123,0) 2O,2

(15,6-26,1)

~Ausgedriickt als Pilztrockenpulver.

EXPERIENTIA29/7

h~molytischen A k t i v i t g t entsprechende B a n d e ihr diskelektrophoretisches Verhalten nicht. Die h~molytische A k t i v i t / i t war anch resistent gegen B r o m e l i n und Proteinase K (48 h/37 ~ A u c h d u t c h a-Amylase oder P a n k r e a tin (24 h/37 ~ wurde Phallolysin nicht inaktiviert. Pro Molekfil eines e t w a 50 % reinen Phallolysin (MolGew. 30.000) w u r d e n nur 3-4 Molekiile P h o s p h a t nachgewiesen, die R e s t e nicht a b g e t r e n n t e r Puffersalze darstellen konnten. Phallolysin hXmolysierte gewaschene E r y t h r o c y t e n v o n Mensch, Maus, Kaninchen, Meerschweinchen, R a t t e , H u n d und Schwein direkt; gegen menschliche E r y t h r o c y t e n war es 20 mal a k t i v e r als Digitonin; Rinder- und H a m m e l e r y t h r o c y t e n w a r e n w e i t g e h e n d resistent. Es w u r d e durch Cholesterin nicht g e h e m m t , zeigte keine Phospholipase A-Aktivit~tt und auch keine Phospholipase C - A k t i v i t g t 3 Es wirkte c y t o t o x i s c h auf H e L a - Z e l l e n und M~useAscites-Tumorzellen in v i t r o 4. Es war i.p. hoch toxisch. Die m i t verschieden welt gereinigtem Phallolysin b e s t i m m t e T o x i z i t ~ t war nach A b t r e n n u n g der toxischen Cyclopeptide der h/imolytischen A k t i v i t S t korreliert. U n a b h ~ n g i g v o m R e i n h e i t s g r a d b e t r u g die LDs0 fiir M~use (i.p.) ca 1000 h~molytische E i n h e i t e n / k g . (Eine H/imolytische E i n h e i t h/imolysiert 1 ml 1 % i g e Suspension gewaschener K a n i n c h e n e r y t h r o c y t e n bei 37 ~ innerhalb yon 2 h total.) ; je nach AktivitS, t des Materials waren dies 0,3-0,6 m g isoliertes, iyophilisiertes Phallolysin. In griinen Knollenblgtterpilzen v o n verschiedenen F u n d often und J a h r e u wurde Phallolysin k o n s t a n t und in hoher Aktivit/~t nachgewiesen 5. Infolge seiner T h e r m o und S/iurelabilit/it dfirfte dieses Toxin jedoch an der K n o l l e n b l ~ t t e r p i l z v e r g i f t u n g des Menschen nicht beteiligt sein. E i n e eingehende Darstellung erfolgt an anderer Stelle.

Summary. Phallolysin, a toxic hemolysin of high molecular weight, was isolated from aqueous e x t r a c t s of Amanita phalloides. I t s properties are discussed. ~c~UTH

SEEGER 6

u n t e r M i t a r b e i t yon H. SCHARRER7 und MELITTA H A U P T

Institut /fir Pharmakologie und Toxikologie der Universitiit, Koellikerstrasse 2, D-8700 Wi~rzburg (Germany), 10. Februar t973.

t Tm WIELA~D, Fortschr. Chem. org. NatStoffe 25, 214 (1967). 2 B.N. AMES,in Methods in Enzymology (Eds. S. N. COLOWlCKand N. O. KAPLA~; Academic Press, New York), vol. 8, p. 115 (1966). 3 R. SEZGZR, G. B~RKnARDTand G. NEEB, Naunyn-Schmiedebergs Arch. Pharmak. 277, R 72 (1973). 4 D. LEHMANN,Zahnmed. Diss. Wtirzburg, in Vorbereitung. 5 R. SEEG~R, I-I. KRA~JSund R. WI~DMANN,Archs Toxic. 30, 215 (1973). s Mit Unterstiitzung tier Deutschen Forschungsgemeinschaft. 7 Ein Tell der Ergebnisse entstammt der Dissertation yon H. SCHARRER.

Changes of the fl-Receptor Binding Sites of the Rabbit Ileum under the Influence of High Temperature Several authors observed t h a t an elevation of t e m p e rature was accompanied b y a decrease of the a f f i n i t y for s y m p a t h o m i m e t i c drugs to t h e fl-receptors on the isolated guinea-pig a t r i u m 1-5 and on the r a b b i t ileum ".

On r a b b i t ileum, this p r o n o u n c e d t h a n on of a f f i n i t y caused b y c o m p l e t e l y reversible

decrease of affinity was m u c h more guinea-pig atrium. The d i m i n u t i o n high t e m p e r a t u r e was found to be b y the m e t a b o l i c inhibitor iodoaee-

15.7.1973

tic acid on a t r i u m as well as on i l e u m L F u r t h e r m o r e , e x p e r i m e n t s on a t r i a s h o w e d t h e a f f i n i t y for f l - s y m p a t h o m i m e t i c s to b e n e a r l y i d e n t i c a l e i t h e r a t 25~ u n d e r c o n t r o l c o n d i t i o n s or a t 25~ a f t e r recooling f r o m 42~ ~. The c o n t r a c t i l e a m p l i t u d e a n d t h e m a x i m a l increase of t h e a m p l i t u d e i n d u c e d b y s y m p a t h o m i m e t i c s were n o t a l t e r e d b y t h i s procedure. T h e s e o b s e r v a t i o n s on a t r i a are in a c c o r d a n c e w i t h t h e idea t h a t in t h i s o r g a n t h e increase of m e t a b o l i c r a t e is t h e m a i n f a c t o r r e s p o n s i b l e for t h e d i m i n u t i o n of t h e a f f i n i t y for f i - s y m p a t h o m i m e t i c drugs. W e were, therefore, i n t e r e s t e d t o see w h e t h e r in t h e ileum a d d i t i o r m l f a c t o r s for t h e o b s e r v e d d i m i n u t i o n of f l - s y m p a t h o m i m e t i c a f f i n i t y h a v e t o be t a k e n i n t o c o n s i d e r a t i o n . M a t e r i a l and methods. Pieces f r o m r a b b i t i l e u m were k e p t in K r e b s - H e n s e l e i t s o l u t i o n c o n t a i n i n g 6.3 • 10 -7 M p h e n t o l a m i n e in o r d e r t o b l o c k t h e c~-receptors. T h e s p o n t a n e o u s m o t i l i t y was r e g i s t e r e d b y a s t r a i n gauge. D o s e - r e s p o n s e curves for t h e f l - s y m p a t h o m i m e t i c d r u g s isoprenaline a n d T h 1165a 6 were d e t e r m i n e d b y a d d i n g single doses. The e q u i l i b r a t i o n t i m e for e v e r y n e w t e m p e r a t u r e s t e p was 60 rain. A f t e r c o m p l e t i o n of t h e d o s e - r e s p o n s e c u r v e s a t 25~ t h e o r g a n b a t h was h e a t e d up t o 42~ a n d m a i n t a i n e d for 120 m i n a t t h i s t e m p e r a t u r e w i t h o u t f u r t h e r experim e n t a l p r o c e d u r e . The s y s t e m was t h e n recooled t o 25~

~

831

Specialia

maximal response 100 I PN

-~ 100

50

Th 1165a

.,~

,.~

,,s>-

//y//

1~]-9 10-8

10-7

16-6

10-5

10-4

again a n d a n o t h e r d o s e - r e s p o n s e c u r v e was d e t e r m i n e d a t 25~ Also a d o s e - r e s p o n s e c u r v e 5 h a f t e r recooling t o 25~ was p e r f o r m e d for i s o p r e n a l i n e . I n a parallel set of e x p e r i m e n t s , d o s e - r e s p o n s e curves for t h e s y m p a t h o m i m e t i c s a t 42~ were d e t e r m i n e d . The intrinsic a c t i v i t y (~) a n d t h e pD2-values were e s t i m a t e d a c c o r d i n g t o ARtENS s a n d VAN R o s s u M ~, resp. F o r f u r t h e r details of t h e m e t h o d s see p r i o r p u b l i c a t i o n s 8, 6, v. Results. On t h e isolated r a b b i t i l e u m an increase of t h e b a t h t e m p e r a t u r e f r o m 25 ~ t o 42~ r e s u l t e d in a s h i f t of t h e d o s e - r e s p o n s e curves for i s o p r e n a l i n e (IPN) a n d T h 1165a t o t h e r i g h t (Figure). T h e r e s u l t i n g d i m i n u t i o n of a f f i n i t y is e x p r e s s e d b y m e a n s of t h e pD2-values. F o r I P N t h e y declined s i g n i f i c a n t l y f r o m 7.70 a t 25~ to 6.05 a t 42~ a n d e v e n m o r e p r o n o u n c e d for T h 1165a f r o m 6.80 t o 4.06 (Table). I n o r d e r t o p r o v e w h e t h e r t h e d i m i n u t i o n of a f f i n i t y d e s c r i b e d was r e v e r s i b l e or not, recooling e x p e r i m e n t s were p e r f o r m e d . A f t e r 120 m i n u t e s a t 42~ recooling to 25~ i n c r e a s e d t h e affinities of t h e t w o s y m p a t h o m i m e t i c s i n v e s t i g a t e d , b u t n o t t o t h e i r initial values a t 25~ recooling f r o m 42~ increased t h e pD~-value for I P N f r o m 6.05 to 6.82 a n d for T h 1165a f r o m 4.06 to 6.03. W h e n t h e d o s e - r e s p o n s e c u r v e for I P N was r e p e a t e d 5 h a f t e r recooling a t 25~ no f u r t h e r r e s h i f t o c c u r r e d as is i n d i c a t e d b y t h e pD2-value of 7.03 • 0.07 (n = 7). These findings imply t h a t the temperature-induced d i m i n u t i o n of a f f i n i t y consists of a n irreversible a n d a r e v e r s i b l e p a r t . The irreversible decrease of affinity, w h i c h is d e f i n e d b y t h e difference of t h e d o s e - r e s p o n s e curves a t 25 ~ a n d a t 25~ a f t e r recooling, is of t h e s a m e o r d e r of m a g n i t u d e for b o t h a m i n e s as can be d e r i v e d f r o m t h e d o s e - r e s p o n s e curves for I P N a n d T h 1165a (Figure). T h e d i s t a n c e s b e t w e e n t h e d o s e - r e s p o n s e curves a t 42 ~ a n d a t 25~ a f t e r recooling, d e t e r m i n i n g t h e reversible p a r t , show, on t h e c o n t r a r y , a clear c u t difference, i.e. t h e r e v e r s i b l e p a r t for T h 1165a a m o u n t s to a b o u t 2 decades, t h a t for I P N less t h a n one. Since we o b s e r v e d t h a t t h e d i m i n u t i o n of a f f i n i t y e v o k e d b y h i g h t e m p e r a t u r e is p a r t l y due t o a n irrever-

10-3ff

Dose-response curves for isoprenaline (IPN) and Th 1165a on rabbit ileum at 25 ~ before (@--@) and after heating up the organ bath to 42~ (A--A), and at 42~ ( 9 9 Number of experiments are given in the Table.

1 S. GARB, M. PENNA,J. appl. Physiol. 9, 431 (1956). 2 j. A. OPPnR~ANN,C. F. RVAN and C. O. HAAVlK,Life Set. 70, 613 (1977). 3 D. REINHARDT, J . WAGNER and H. J. SCHO~tANN, NaunynSchmiedeberg's Arch. Pharmac. 275, 95 (1972). 4 U. TRENDELENBURG, J. Pharmae. exp. Ther. 167,222 (1968). 5 W. W6PPEL, Dissertation, Wfirzburg 1972. 6 j. WAGNER, D. REINHARDT and H. J. SCH'0MANN, Arch. int. PharmacodYn. 197, 290 (1972). 7 H. J. SCH0~ANN, J. WAGNER and D. REINHARDT, NaunynSehmiedeberg's Arch. Pharmac. 275, 105 (1972). s ]~. j. ARIENS,Molecular Pharmacology (Academic Press, New York and London 1964), vol. 1. 9 j. M. VAN Rossu~, Arch. int. Pharmacodyn. 743, 299 (1963).

pD2-values and intrinsic activity (c~) of isoprenaline (IPN) and Th 1165 a at 25 ~ after reeooling from 42 ~ and at 42 ~ 25 ~

25 ~ after recooling from 42 ~

42 ~

IPN

pD~-values Intrinsic activity (~)

7.70 J= 0.08 (7) 1

6.82 • 0.10 b (7) 0.71

6.05 4- 0.11 b (5) 0.55

Th 1165a

pD2-values Intrinsic activity (~)

6.80 4- 0.11 (5) 1

6.03 -4- 0.20 9 (5) 0.94

4.06 2~ 0.11 b (5 0.90

pD2-values: means • S.E.; number of experiments in brackets, a p < 0.05; b p < 0.001 compared with 25 ~

832

Specialia

sible d i m i n u t i o n , it was of special i n t e r e s t t o e x a m i n e t h e i n t r i n s i c a c t i v i t i e s of t h e t w o s y m p a t h o m i m e t i c s u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s . A t 25~ both amines relaxed the preparation completely, that means the i n t r i n s i c a c t i v i t y a m o u n t e d t o 1.0. T r o u g h o u t t h e e x p e r i m e n t a l p r o c e d u r e , no c h a n g e of t h e i n t r i n s i c a c t i v i t y for T h 1165a occured (Table). T h e i n t r i n s i c a c t i v i t y for I P N , o n t h e o t h e r h a n d , d e c r e a s e d t o 0.55 a t 42~ a n d was o n l y p a r t l y r e s t o r e d t o 0.71 a f t e r recooIing. Discussion. O u r r e s u l t s s h o w e d t h a t t h e s h i f t t o t h e r i g h t of t h e dose-response c u r v e s for I P N a n d T h 1165a c a u s e d b y a n e l e v a t i o n of t e m p e r a t u r e f r o m 25 ~ t o 42~ could n o t b e c o m p l e t e l y reversed, i.e. a n i r r e v e r s i b l e s h i f t of t h e dose-response c u r v e s for t h e f i - s y m p a t h o m i m e t i c s r e m a i n e d a f t e r recooling to 25~ T h i s could b e due to a d e n a t u r a t i o n of t h e c o n t r a c t i l e a p p a r a t u s or t o a n a c t u a l c o n f o r m a t i o n a l c h a n g e of t h e fl-receptors o n r a b b i t ileum. T h e d e n a t u r a t i o n c a n b e e x c l u d e d since p a p a v e r i n e s h o w e d n e i t h e r a d i m i n u t i o n of a f f i n i t y n o r a n y a l t e r a t i o n of t h e i n t r i n s i c a c t i v i t y a t h i g h t e m p e r a t u r e o n r a b b i t i l e u m ( u n p u b l i s h e d results). W e w o u l d like to suggest, therefore, t h a t fl-receptor sites u n d e r g o irrev e r s i b l e c o n f o r m a t i o n a l changes. T h a t a c c o u n t s n o t o n l y for a d r e n e r g i c fl-receptors b u t also for r e c e p t o r s m e d i a t i n g t h e r e s p o n s e s for a n g i o t e n s i n , v a s o p r e s s i n 1~ a n d serotoninl0,1~ since o n r a t f u n d u s strip, recooling f r o m 47 ~ to 37.5~ s h o w e d all i r r e v e r s i b l e s h i f t of t h e i r doser e s p o n s e c u r v e s t o t h e r i g h t a n d also a decrease of t h e i n t r i n s i c activities. O n t h e o t h e r h a n d , h e a t i n g is w i t h o u t a n y i n f l u e n c e o n t h e dose-response c u r v e s for acetylcholine, KC1 a n d b r a d y k i n i n ~~ T h e s e results are in f a v o u r of t h e e x i s t e n c e of h e a t - l a b i l e a n d - s t a b l e r e c e p t o r s on s m o o t h muscle of t h e d i g e s t i v e t r a c t . A c c o r d i n g to o u r results, t h e i r r e v e r s i b l e p a r t of t h e d i m i n u t i o n of t h e s y m p a t h o m i m e t i c a f f i n i t y is n e a r l y e q u a l for b o t h amines. T h i s implies t h a t t h e p o p u l a t i o n of t h e /?-receptors w h i c h u n d e r g o a n a l t e r a t i o n of t h e i r a c t i v e sites b y o u r p r o c e d u r e is of t h e s a m e o r d e r of magnitude.

EXPERIENTIA 29/7

O n t h e o t h e r h a n d , t h e e x t e n t of t h e r e v e r s i b l e d i m i n u t i o n of a f f i n i t y s h o w e d g r e a t differences for b o t h amines. T h i s p a r t is g r e a t e r for T h 1165a t h a n for isoprenaline. T h a t was n o t s u r p r i s i n g since t h e decrease of a f f i n i t y for T h 1165a 12 e v o k e d b y h i g h t e m p e r a t u r e s was m u c h m o r e p r o n o u n c e d t h a n for I P N . T h e s e results are in a c c o r d a n c e w i t h t h e o b s e r v a t i o n t h a t p r e i n c u b a t i o n w i t h t h e m e t a b o l i c i n h i b i t o r , iodoacetic acid, i n c r e a s e d t h e a f f i n i t y of T h 1165a t o a h i g h e r degree t h a n for i s o p r e n a l i n e ~. From the present study the conclusion can be drawn t h a t in r a b b i t i l e u m t h e decrease of a f f i n i t y i n d u c e d b y a l t e r a t i o n of t e m p e r a t u r e is n o t o n l y d e p e n d e n t u p o n a n increase of t h e m e t a b o l i c rate, as for i n s t a n c e in t h e g u i n e a - p i g a t r i u m s , b u t in a d d i t i o n b y a n i r r e v e r s i b l e i m p a i r m e n t of t h e fi-receptor sites. T h e a l t e r a t i o n of t h e i n t r i n s i c a c t i v i t y o b s e r v e d for i s o p r e n a l i n e c a n n o t b e e x p l a i n e d so far a n d n e e d s f u r t h e r elucidation by experiments.

Zusammen~assung. A m K a n i n c h e n - I l e u m b e s t e h t die durch Temperaturerh6hung bedingte Rechtsverschiebung der Dosis-Wirkungs-Kurven yon Isoprenalin und T h 1165a aus e i n e m r e v e r s i b l e n u n d e i n e m i r r e v e r s i b l e n Anteil. D. REINHARDT, J. WAGNER a n d H. J. SCHUMANNlS Pharmakologisches lnstitut der Universitiit Essen (Gesamthochschule) Hu/elandstr. 55, D-43 Essen (Germany), 37 January 1973. 13 j. H.. FLEISCH and S. EHRENPREIS,J. Pharmac. exp. Ther. 162, 21 (1968). 11 A. VACCARI,Pharmacology 5, 321 (1971). 12 Our thanks are due to Dr. A. ENG}gLHARDT, C. H. Boehringer Ingelheim, for generous gift of the drugs. 18 The authors wish to thank Mrs. GABRIELEG~AF and Miss BETTINA SCHEEPERS for skilful technical assistance. This work was supported by the Deutsche Forschungsgemeinsehaft.

A s c o r b i c Acid: Effect of H i g h D o s e s on Brain and Heart C a t e c h o l a m i n e Levels in G u i n e a - P i g s and Rats T h e g r o w i n g i n t e r e s t in m e g a v i t a m i n t h e r a p y for m e n t a l illness 1-~ h a s p r o m p t e d us to s t u d y t h e effect of r e l a t i v e l y h i g h doses of a s c o r b i c acid o n c a t e c h o l a m i n e levels in t h e h e a r t a n d b r a i n of t h e guinea-pig, a species w h i c h like m a n , c a n n o t s y n t h e s i z e its o w n ascorbic acid. THOA et al. ~ h a v e s h o w n t h a t h e a r t n o r e p i n e p h r i n e is d e c r e a s e d in t h e scorbutic g u i n e a - p i g while m o r e r e c e n t l y LOKOSHKO a n d LESNYKn 6 h a v e r e p o r t e d t h a t in guineapigs w i t h h y p o v i t a m i n o s i s C, h e a r t e p i n e p h r i n e is no d i f f e r e n t f r o m n o r m a l h e a l t h y animals. I n t h e p r e s e n t c o m m u n i c a t i o n we r e p o r t t h e effect of a r e l a t i v e l y h i g h d i e t a r y i n t a k e of v i t a m i n C o n h e a r t a n d b r a i n levels of c a t e c h o l a m i n e s in t h e guinea-pig. B y w a y of c o m p a r i s o n , we also r e p o r t t h e effect of a h i g h d i e t a r y i n t a k e of ascorbic acid in t h e rat, a species w h i c h is c a p a b l e of s y n t h e s i z i n g its o w n ascorbic acid. Materials and methods. A s c o r b i c acid-free p o w d e r e d R e i d - B r i g g s diet, p u r c h a s e d f r o m G e n e r a l Biochemicals, C h a g r i n Falls, Ohio, was s u p p l e m e n t e d w i t h v i t a m i n C sufficient t o s u p p l y g u i n e a - p i g s w i t h 150 or 1000 m g / k g / d a y of a s c o r b i c acid. F o r t h e r a t studies, p o w d e r e d P u r i n a R a t C h o w w a s s u p p l e m e n t e d w i t h a s c o r b i c acid so as t o s u p p l y t h e a n i m a l s w i t h 1000 m g / k g / d a y . T h e d i e t s were p r e p a r e d b y m i x i n g i n a r o t a r y m i x e r for 3 h ; fresh d i e t was p r e p a r e d e v e r y 4 days. T h e a n i m a l s were

allowed food a n d w a t e r a d l i b i t u m . I n t h e s e studies guinea-pigs a n d r a t s c o n s u m e d 20-25 a n d 18-20 g of food p e r a n i m a l p e r day, respectively. Male, S p r a g u e - D a w l e y r a t s (200-250 g) or male, H a r t l e y s t r a i n guinea-pigs (300-400 g) were used in our studies. W h e n t h e guinea-pigs were r e c e i v e d f r o m t h e supplier, t h e y were a c c l i m a t e d t o o u r a n i m a l r o o m for 10 d a y s o n a s c o r b u t a g e n i c diet w h i c h w a s s u p p l e m e n t e d w i t h ascorbic acid so as t o s u p p l y t h e a n i m a l s w i t h 150 m g / k g / d a y of t h e v i t a m i n . T h i s a m o u n t of ascorbic acid is a p p r o x i m a t e l y e q u i v a l e n t t o t h e a m o u n t of v i t a m i n C w h i c h guinea-pigs i n g e s t w h e n t h e y are fed normal guinea-pig lab chow supplemented with a daily r a t i o n of lettuce. R a t s were a c c l i m a t e d o n s t a n d a r d P u r i n a l a b chow. T h e a n i m a l s were t h e n p l a c e d o n t h e

1 L. PAULING, Science 160, 265 (1968). 2 W. L. DALTON, J. Indiana State reed. Ass. 55, 1151 (1962). s M. L. RUCCITELLI,J. Am. geriat. Soc. 20, 34 (1972). 4 L. PAULING, Vitamin C and the Common Cold (W. H. Freeman Co., San Francisco, California 1970). N. t3. THOA, R. J. WURTMANand J. AXELROD, Proc. Soc. exp. Biol. Med. 121,267 (1965). 60. A. LOKOS~tKO and L. D. LESNYKH, Ukr. Biokhem. Zh. 43, 539

(1971).