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A study of some pharmacological properties of certain alpha-glyceryl ethers. Klier, Gail Dianne Bellward 1963

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A STUDY OP SOME PHARMACOLOGICAL PROPERTIES OP CERTAIN ALPHA-GLYCERYL ETHERS  by GAIL DIANNE BELLWARD KLIER  A t h e s i s submitted i n p a r t i a l f u l l f i l m e n t of the requirements f o r the degree of MASTER OP SCIENCE IN PHARMACY i n the D i v i s i o n of Pharmacology of the F a c u l t y of Pharmacy  We accept t h i s t h e s i s as conforming to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA October,  1963  In presenting  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  the requirements f o r an advanced degree at the U n i v e r s i t y  of  B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and .study. mission for extensive purposes may  I f u r t h e r agree that per-  copying of t h i s t h e s i s f o r s c h o l a r l y  be granted by the Head of my Department or  h i s representatives,,  I t i s understood that copying, or  by publi-  c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n .  Department of The U n i v e r s i t y of B r i t i s h Columbia,. Vancouver 8, Canada. Date  i  A STUDY OP SOME PHARMACOLOGICAL  PROPERTIES OP  CERTAIN ALPHA-GLYCERYL ETHERS by  GAIL DIANNE BELLWARD KLIER  ABSTRACT The a l p h a - g l y c e r y l ether3 (AGE) are w i d e l y d i s t r i b u t e d i n n a t u r e , o c c u r r i n g i n many marine organisms, and humans.  l a n d animals, p l a n t s ,  A s e r i e s o f p r e l i m i n a r y experiments  was begun t o  determine whether these compounds possessed p o s s i b l e pharma c o l o g i c a l a c t i o n s a g a i n s t inflammation.  Anti-inflammatory t e s t s  u t i l i z e d both the granuloma pouch and c o t t o n p e l l e t g r a n u l a t i o n methods.  P o s s i b l e g l u c o c o r t i c o i d a c t i v i t y was checked by  measuring  l i v e r g l y c o g e n d e p o s i t i o n . The e f f e c t s o f AGE on  growth and weight  o f the animals was a l s o noted.  N e i t h e r s e l a c h y l d i h e m i s u c c i n a t e sodium ( s e l a c h y l DHSS) nor s e l a c h y l a l c o h o l decreased the exudate f o r m a t i o n i n t h e granuloma pouch experiments, a l t h o u g h e x c e l l e n t r e s u l t s were obtained^with hydrocortisone.  The h i g h dose o f the AGE was the e q u i v a l e n t o f  t e n m i l l i g r a m s ; low doses were t h r e e hundred and s i x hundred micrograms.  Routes o f a d m i n i s t r a t i o n used were o r a l ,  p e r i t o n e a l , and  intra-  subcutaneous.  In the c o t t o n p e l l e t granuloma t e s t , some i n d i c a t i o n o f a n t i inflammatory a c t i v i t y was o b t a i n e d , i n accordance w i t h p r e v i o u s r e s u l t s i n t h i s and other l a b o r a t o r i e s .  S e l a c h y l DHSS decreased  g r a n u l a t i o n t i s s u e f o r m a t i o n by o n l y 11.7$ i n growing  rats,  ii  which i s not s i g n i f i c a n t ; however, i n mature r a t s , there was a decrease o f 29.$%.  The dosage used i n t h i s s e r i e s was 30  m i l l i g r a m s per k i l o g r a m d a i l y by the subcutaneous r o u t e , A d e f i n i t e and comparatively glycogen  large increase i n l i v e r  d e p o s i t i o n was observed i n r a t s allowed t o eat f r e e l y ,  when g i v e n s e l a c h y l DHSS subcutaneously. were decreased  These glycogen  values  r a d i c a l l y by a p a i r e d f e e d i n g study i n which the  t r e a t e d r a t s were allowed  t o eat o n l y the same amount o f food as  t h e i r p a i r e d c o n t r o l animal.  Thus f o o d i n t a k e appears t o be one  o f the f a c t o r s r e s p o n s i b l e f o r the i n c r e a s e d glycogen  storage.  The A G E appear to a f f e c t growth only by the I n t r a p e r i t o n e a l route of administration. r e t a r d e d by 29.6$.  I n t h i s case, the growth o f r a t s was  I t was a l s o noted t h a t these animals a t e  somewhat l e s s f o o d t h a n the c o n t r o l s , although appear t o be an important  t h i s d i d not  enough d i f f e r e n c e t o account f o r the  l a c k o f weight g a i n . Throughout these experiments one o f the most  notable  o b s e r v a t i o n s was a l a c k o f u n i f o r m i t y o f the r e s u l t s .  Factors  which appeared t o cause a v a r i a b i l i t y i n the outcome o f t e s t s i n c l u d e d the age o f the animals, housing  l e n g t h o f time o f p r e o p e r a t i v e  i n new q u a r t e r s , r o u t e of a d m i n i s t r a t i o n , and dosage.  A n o n - l i n e a r r e l a t i o n s h i p between dose and e f f e c t appears probable. Signature  o f Examiners  iii  TABLE OP CONTENTS Page ABSTRACT. . . . . . . .  i  LIST OP TABLES  v  LIST OP FIGURES  vi  INTRODUCTION Occurrence and S e p a r a t i o n o f Alpha G l y c e r y l E t h e r s . .  1 I4.  Proof of S t r u c t u r e Synthesis  h  •  Chemical and P h y s i c a l P r o p e r t i e s . Physiological Properties. . .  l\. • .  6  Fate.  6  Central Effects  8  Hematological  Effects  .  . . . . .  8  Growth S t u d i e s  12  Toxicity.  13  Anti-inflammatory  Properties. . . . . . . . . . .  11+  EXPERIMENTAL Methods  •  15  Ant I-inflammatory  l£  Granuloma Pouch Technique Cotton P e l l e t Method  . . . . . 0  16 18  L i v e r Glycogen Determination.  19  Growth Studies  23  Results Anti-inflammation  •  2I4. 2i+  iv  Page 2i\.  Granuloma Pouch C o t t o n P e l l e t Granuloma L i v e r Gycogen Determinations Growth S t u d i e s  ••••-••••..26 . . . . . . . . . . . .  27 28  DISCUSSION  33  SUMMARY  37  BIBLIOGRAPHY  38  V  LIST OP TABLES Table I II  III IV V VI  VII VIII IX  Page General C h a r a c t e r i s t i c s of some A l p h a - g l y c e r y l Ethers  $  E f f e c t of S e l a c h y l Dihemlsuccinate Sodium Compared with H y d r o c o r t i s o n e A c e t a t e using the Subcutaneous Route  2\  E f f e c t of S e l a c h y l A l c o h o l Compared w i t h Hydrocortisone  25  E f f e c t o f S e l a c h y l Dihemlsuccinate Sodium Compared with S e l a c h y l A l c o h o l  26  Comparison of G r a n u l a t i o n T i s s u e Formation i n Young and Mature Rats  27  Comparison of L i v e r Glycogen i n Animals T r e a t e d w i t h S e l a c h y l Dihemisuccinate Sodium and Hydrocortisone  27  E f f e c t s o f the A l p h a - g l y c e r y l Ethers Rats during Granuloma Pouch Studies  30  E f f e c t s of A l p h a - g l y c e r y l Ethers during Cotton P e l l e t S t u d i e s . .  on Growth o f . .  on Weight o f Rats"  Long Term Study of the E f f e c t s o f S e l a c h y l Dihemlsuccinate Sodium i n D r i n k i n g Water o f Normal Rats. .  30  31  LIST OP FIGURES Figure  Page  1.  Standard Curve f o r O p t i c a l D e n s i t y of Glucose.  2.  E f f e c t of S e l a c h y l Dihemisuccinate Sodium oh L i v e r Glycogen D e p o s i t i o n of Rats  3.  E f f e c t of S e l a c h y l Dihemisuccinate Sodium on Growth and Food Consumption o f Rats  . .  22 29  . «  32  vii  ACKNOWLEDGEMEFT  The author i s indebted  t o Western Chemical  L i m i t e d f o r s u p p l i e s o f the a l p h a - g l y c e r y l  Industries  ethers.  Thanks i s a l s o extended to Dr. J.E. H a l l i d a y f o r h i s h e l p f u l guidance and c r i t i c i s m .  INTRODUCTION  Occurrence and S e p a r a t i o n The  Ethers  t h r e e most important alpha g l y c e r y l ethers  i n nature are b a t y l , chimyl,  and s e l a c h y l a l c o h o l s .  they were i n v e s t i g a t i n g the chemical (1).  fish oils  (AGE) found Their  demonstrated by Tsujimoto and Toyaraa i n 1922  presence was f i r s t while  of Alpha-Glyceryl  structure of c e r t a i n  At t h i s time, the AGE were i s o l a t e d from the  unsaponifiable  f r a c t i o n o f the l i v e r o i l s of v a r i o u s s p e c i e s o f  Elasmobranchii.  (2).  Large amounts are a l s o found i n chimaeroids  AGE have been i s o l a t e d from t h e f o l l o w i n g sources: the b l a c k shark (3);  the gorgonia,  Plexaur.a  liver  fflexuosa,  o i l of  (I4.); the  s t a r f i s h , A s t e r i a s rubens (§.), when " A s t r o l " was found t o be i d e n t i c a l w i t h b a t y l a l c o h o l ; u n s a p o n i f i a b l e f r a c t i o n o f whale and h e r r i n g o i l , (6); rollestoni Bell., unsaponifiable  unsaponifiable  substance o f A s t e r i a  and Cucumaria chronh.jelmi  f r a c t i o n of l i v e r  Theel.  (7); (8);  o i l o f C e n t r i n a c e n t r i n a L,  A d r i a t i c Elasmobranchia l i v e r o i l s , C e n t r i n a s a l v i a n i .  (9);  s t e r o l - f r e e f r a c t i o n o f the u n s a p o n i f i a b l e f r a c t i o n o f Liolophura  .japonica ( c h i t o n s ) , ( l ) j A s t e r i a s amurenis,  cow shark l i v e r , Hexanchus g r i s e u s , (12); of C o s c i n a s t e r i a s a c u t e s p i n a Asterias  ffopbesi.  sea anemones, (16); oil,  (17);  (llj.);  u n s a p o n i f i a b l e matter  and Comanthus j a p o n i c a ,  fatty oils  o f Stickopus  (19);  (13);  .laponicus.  (15);  Tachypleus t r i d e n t a t u s f a t and M y t i l u s e d u l l s  l i v e r o i l of Squalus s u c k l e y i ( s h a r k ) , (18);  l e u t e o 3 t o m a ,  (11);  and f a t t y o i l s o f Pentacta  doliolum  Tonna  (20).  2 In t h e i r  review,  t h e AGE i n f i s h remaining  Bodman a n d M a i s i n (21) c i t e d  o i l s may be p r e s e n t a s f a t t y  two h y d r o x y l s b e i n g  oils  acid esters, the  w i t h a subnormal degree o f u n s a t u r a t i o n .  vegetable  o i l c o n t a i n i n g a n y q u a n t i t y o f AGE was t u n g o i l ,  However, l a t e r  vegetable  oils,  Intestinal  s t u d i e s showed t h e p r e s e n c e  as i n d i c a t e d  In higher animals,  yolk  t h a t the only  t h i s p a r t i c u l a r l i n k a g e m i g h t be c h a r a c t e r i s t i c  fats.  AGE a r e f o u n d  f a t and l i v e r  (21}.), s p l e e n o f p i g s (25),  (29).  Reduction  isolated tissue  i n t h e dog.  much a s o t h e r  (30), b a t y l  down a n i n o s i t o l Nakagawa  phosphatide  and McKibbin  (31)  m i c r o m o l e s AGE p e r gram o f d r y l i p i d - f r e e R a b b i t b o n e marrow c o n t a i n e d t e n t i m e s a s  tissues.  H a l l g r e n a n d L a r s son highest  (27).  o f b r a i n colaraine  I n a s t u d y o n human b r a i n  the c e p h a l i n f r a c t i o n .  .2 - 1.35  aortas  o f chimyl and b a t y l a l c o h o l s i n  a l c o h o l was f o u n d a f t e r b r e a k i n g i s o l a t e d from  h e n (21), egg  from r e d u c e d t o t a l - p i g - h e a r t - p h o s p h a t i d e s ,  gave a m i x t u r e  Debuch's s t u d y  (21).  i n t h e body c a v i t y f a t ,  o i l o f the r a t and domestic  a n d c e p h a l i n (28).  plasmalogen  o f animal  o f AGE I n o t h e r  human e r y t h r o c y t e s (26), a n d human a r t e r i o s c l e r o t i c A n AGE was i s o l a t e d  so  i n Bodman a n d M a i s i n ' s r e v i e w  (23), bone marrow o f c a t t l e  lecithin,  acid.  o f AGE i s m a i n l y i n  K a r n o v s k y e t . a l . (22) i n 191+6 c o n c l u d e d  that  that  combined w i t h a h i g h e r f a t t y  I t was a l s o p o i n t e d o u t t h a t t h e o c c u r r e n c e fish  evidence  (2lj.) s t u d i e d AGE i n man a n d cows.  c o n c e n t r a t i o n was f o u n d  o f l i p i d s ) a n d human m i l k  i n human r e d bone marrow  (0.1$ o f l i p i d s ) .  e x t r a c t i o n o f r e d b l o o d c e l l s was p o o r ,  The  (0.2$  The method o f  and t h e r e f o r e a low  3  c o n c e n t r a t i o n was r e p o r t e d .  However even the h i g h e s t con-  c e n t r a t i o n shown i n the human c o u l d not compare w i t h the 10 - 30$ AGE i n l i p i d s o f Elasmobranch l i v e r o i l .  S e v e r a l other g l y c e r y l  ethers b e s i d e s s e l a c h y l , b a t y l , and chimyl a l c o h o l s were a l s o reported.  These unknown a l c o h o l s formed 30$ o f the AGE i n human  bone marrow.  T h i s group concluded t h a t the AGE are found i n  m i l k , f e t a l t i s s u e s , or r a p i d l y r e p r o d u c i n g t i s s u e s such as bone marrow. Bodman and M a i s i n (21) concluded t h a t AGE i n l a n d animals are present i n those t i s s u e s c o n t a i n i n g c e l l s  o f mesenchymal  o r i g i n , such as bone marrow, s p l e e n , and i n t e s t i n e , from which t i s s u e c e l l s a r e c o n t i n u a l l y shed i n t o a body c a v i t y .  Since the  I n t e s t i n e i s concerned w i t h metabolism of h i g h e r a l c o h o l s and r e l a t e d compounds, they h y p o t h e s i z e d that the gut mucosa might be concerned w i t h AGE s y n t h e s i s . In r e c e n t y e a r s , the most w i d e l y used methods o f s e p a r a t i o n of g l y c e r y l e t h e r s a r e v a r i o u s forms of chromatography. (32) o x i d i z e d t h e AGE t o aldehydes b e f o r e u t i l i z i n g  Eramerie  ascending  paper chromatography, and a f u s h i n - s u l f u r o u s a c i d reagent f o r color production.  Blomstrand  and G u r t l e r (33) found t h a t  complete s e p a r a t i o n of AGE was o b t a i n e d by g a s - l i q u i d chromatography a f t e r a c y l a t i o n of the two hydroxy groups.  Hallgren  and L a r s s o n (3^,35) e x t r a c t e d AGE w i t h d i e t h y l oxide from an a l k a l i n e h y d r o l y z a t e o f f i s h l i v e r o i l s , and separated these by column chromatography on alumina.  The ethers were c o n v e r t e d t o  dimethoxy d e r i v a t i v e s , rechromatographed on alumina, and then were r u n through the g a s - l i q u i d chromatograph.  Malins  (36)  u t i l i z e d both t h i n - l a y e r s i l i c i c chromatography w h i l e studying  0CH(CH20H)2..  1933  Adam  Structure  b a t y l a l c o h o l was  (37)  and  considered  synthetic alpha  were i d e n t i c a l , s i m i l a r l y c h i m y l a l c o h o l  a l p h a hexadecyl g l y c e r y l ether were i d e n t i c a l . (38)  confirmed t h i s r e s u l t by  compounds.  t o be  In t h i s way,  C^QH-^-  other workers u t i l i z e d s u r f a c e  on water t o show t h a t b a t y l a l c o h o l and g l y c e r y l ether  gas  dogfish l i v e r o i l .  P r o o f of P r e v i o u s to  a c i d chromatography and  octadecyl and  Davies e t . a l .  the o x i d a t i o n o f the  s e l a c h y l a l c o h o l was  films  various  found t o be  alpha  o l e y l g l y c e r y l ether. The  n a t u r a l b a t y l and  to the d - s e r i e s . to b a t y l , i t was  chimyl a l c o h o l s were known t o belong  When s e l a c h y l a l c o h o l was also assigned  t o the  shown to be  d-series  reducable  (39).  Synthesis V a r i o u s methods have been u t i l i z e d f o r s y n t h e s i z i n g AGE  and  t h e i r analogues, i n c l u d i n g those of Kornblum and Holmes (I4.0), Gupta and Kummerow (I4.I)> Kumamura (l\Z), B a y l i s e t . a l . (1^3)» Bevan and M a l k i n  &  nd  (I4J4.). Chemical and  Physical  Properties  General c h a r a c t e r i s t i c s of these compounds may  be  seen i n  Table I. The  double bond i n s e l a c h y l a l c o h o l f l u o r e s c e s at 3 6 5  microns under an u l t r a - v i o l e t lamp.  milli-  S e l a c h y l a l c o h o l occurs i n  TABLE I General C h a r a c t e r i s t i c s o f some A l p h a - g l y c e r y l  Compound batyl  alcohol  Ethers  Chemical Name  Components  Melting Point  alpha-octadecylg l y c e r y l ether  stearyl a l c o h o l and glycerol  70 - 71  Formula c  CH 0(CH ) 70H3 2  2  1  GHOH i CH 0H 2  chimyl  alcohol  alpha-hexadecylg l y c e r y l ether  cetyl alcohol and g l y c e r o l  60.5  (  eH 0.(GH .)i£CH3 2  2  CHOH I  CH 0H 2  selachyl alcohol  alpha, 9 - o c t a decehyl g l y c e r y l ether  oleyl alcohol (cis-9-octadecahol) and glycerol  8.  0  CH O(CH ) CH 2  2  2  CHOH I  CH 0H 2  =  8  'CH(CH 7 CH 7  3  cis  and t r a n s isomers,  The  molecule  the lower readily also  the c i s being  o f t h e c i s Isomer i s much l e s s  melting point.  interchanged,  occur  the form found  In v i t r o ,  extended, b u t I t has  t h e e i s - t r a n s forms a r e  a n d t h e r e i s some e v i d e n c e  optical rotation.  F o r example, b a t y l  c e n t r a t i o n s has a n e g a t i v e t o z e r o a t 10$  may  t h r e e AGE h a v e a n  alcohol i n high  r o t a t i o n , which g r a d u a l l y  concentration.  condiminishes  A p o s i t i v e r o t a t i o n i s seen on  dilutions.  C a u t i o n must be u s e d when d e t e r m i n i n g these  that this  i n vivo.  B o t h n a t u r a l and s y n t h e t i c forms o f t h e s e  further  i n nature.  compounds s i n c e t h e y  form e u t e c t i c  the melting points o f  mixtures.  This fact  account  f o r the v a r i a t i o n s i n melting p o i n t s r e p o r t e d i n the  earlier  literature.  According oleophobic  t o Bigelow e t . a l .  films.  (1+5),  batyl  a l c o h o l forms  The AGE a r e a l s o s u r f a c t a n t s , a l t h o u g h t h e  e m u l s i f y i n g power o f b a t y l a l c o h o l i s somewhat monostearin  (1+6).  may  less  I t has been p o i n t e d out t h a t  than  alpha-  t h e AGE a r e a n  (1+7).  i n t e r m e d i a t e l i n k b e t w e e n g l y c e r i d e s ( t r u e f a t s ) a n d waxes Physiological Properties Fate The  a b s o r p t i o n and metabolism o f chirayl a l c o h o l has been  extensively rats  (1+8)  absorbed recovered  s t u d i e d by Blomstrand  i t was f o u n d after  that t h i s  oral intubation.  and co-workers  (1+8,1+9,50).  compound was n i n e t y - f i v e The r e m a i n i n g  from the u n s a p o n i f i a b l e f r a c t i o n  In  percent  five  percent  of fecal  lipids.  was  7  Approximately  h a l f of the absorbed chimyl a l c o h o l was  present i n  the lymph l i p i d s .  H a l f a g a i n o f t h i s p o r t i o n was  the other h a l f was  f i f t y percent f r e e a l c o h o l and the r e s t  esterified.  Thus chimyl a l c o h o l appeared  (1+9)  appeared  acid; was  to be w e l l absorbed  e x t e n s i v e l y m e t a b o l i z e d during t h i s a b s o r p t i o n . active investigations  palmitic  but  Further r a d i o -  t o show that the g l y c e r y l  ether can be absorbed unchanged, but i s m e t a b o l i z e d t o a l a r g e extent i n the I n t e s t i n a l mucosa c e l l s .  T h i s w a 3 i n d i c a t e d by  the  f a c t t h a t i n the lymph l i p i d s more than h a l f of the r a d i o a c t i v i t y present was  i n the form o f p a l m i t i c a c i d due t o a s p l i t t i n g  the ether bond i n the i n t e s t i n a l mucosa.  These workers  of  concluded  t h a t a dynamic s i t u a t i o n i s present i n the i n t e s t i n e , where r e s y n t h e s i s of g l y c e r i d e e s t e r bonds occurs s i m u l t a n e o u s l y w i t h hydrolysis. Blomstrand  and Ahrens (5>0)  f u r t h e r examined the f a t e o f  chimyl a l c o h o l by u t i l i z i n g two human p a t i e n t s , one w i t h c h y l u r i a and the other w i t h a b i l a t e r a l c h y l o t h o r a x . was  almost  A g a i n the compound  completely absorbed, w i t h the ether l i n k a g e o f a  p o r t i o n of the amount a d m i n i s t e r e d r u p t u r e d i n the  intestinal  mucosa, and the p a l m i t y l a l c o h o l moiety r e a d i l y o x i d i z e d t o palmitic acid.  H a l f the r a d i o a c t i v i t y i n the lymph was  a l c o h o l ; the r e s t was p a l m i t i c a c i d .  Cleavage  chimyl  of the ether  l i n k a g e suggested "a h i t h e r t o u n d e f i n e d enzymatic  mechanism."  T h i s paper i n d i c a t e d the p o s s i b i l i t y t h a t a l l the compound i s t r a n s p o r t e d v i a the lymphatic pathway.  absorbed  8  Central E f f e c t s During the course of Berger's systematic  ($1,52), b a t y l a l c o h o l  the p h a r m a c o l o g i c a l p r o p e r t i e s o f AGE studied.  I t was  reported  i n v e s t i g a t i o n of  that nine millimole:s p e r  was  kilogram  subcutaneously i n j e c t e d i n mice and r a t s d i d not r e a c h e i t h e r the mean p a r a l y z i n g dose or the mean l e t h a l dose.  However, many other  substances of the type R-0-CH2-CHOH-CH2OH d i d produce muscular r e l a x a t i o n and p a r a l y s i s due  to c e n t r a l nervous system  e s p e c i a l l y a f f e c t i n g the s p i n a l cord.  In the a l i p h a t i c  group, s t r a i g h t c h a i n a l k y l s gave the b e s t amyl ether being  the most p o t e n t .  destroyed  the p a r a l y z i n g  radical  e f f e c t , with the  Increasing  s u b s t i t u t i o n on the carbon atom or h y d r o x y l  depression,  chain  n-  length,  groups decreased or  activity.  Hematological E f f e c t s In 191+9. Sandler e f f e c t s of yellow and humans.  (I4.7) p u b l i s h e d  the  bone marrow e x t r a c t s and b a t y l a l c o h o l on r a t s  Although other groups had  bone marrow e x t r a c t s b e f o r e  this,  c o n t a i n i n g b a t y l a l c o h o l had now  s t u d i e d the e f f e c t s o f  t h e i r work was  haphazard and r a t h e r i n c o n c l u s i v e .  various  a paper d e s c r i b i n g  The  generally  f r a c t i o n of the  extracts  been w e l l e s t a b l i s h e d , as were  syntheses of the compounds.  Sandler t r e a t e d benzene-  d e p l e t e d r a t s subcutaneously w i t h bone marrow c o n c e n t r a t e s ,  and  showed t h a t such measures i n c r e a s e d the r e s i s t a n c e of the r a t s as shown by b e t t e r g e n e r a l  p h y s i c a l c o n d i t i o n and  m o r t a l i t y , i n c r e a s e d the number o f c i r c u l a t i n g decreased the  s l i g h t l y lower erythrocytes,  subcutaneous abscesses caused by the benzene,  and  9  decreased  the inflammation  the t a i l was  cut f o r b l o o d sampling.  used, a g a i n there was The  i n the t a i l of c e r t a i n groups i n which When normal animals were  an i n c r e a s e In the e r y t h r o c y t e  count.  h e a l t h y human s u b j e c t s showed an i n c r e a s e i n t h e i r  r e t i c u l o c y t e value of s i x times the s t a n d a r d , d e v i a t i o n , which l a s t e d e i g h t to n i n e days a f t e r t e r m i n a t i o n of treatment. Neither hemoglobin nor increased.  The  e r y t h r o c y t e values were s i g n i f i c a n t l y  compound appeared t o have a cumulative  type  of  effect. Sandler's  paper aroused a great d e a l of i n t e r e s t i n the  f i e l d of g e n e r a l d e p r e s s i o n of the bone marrow. by de Gaetani  and B a i o t t i  (53)  confirmed  t h a t b a t y l a l c o h o l post-treatment prolonged  l i f e , and pretreatment  A l a t e r paper  Sandler's work, s t a t i n g  to benzene-intoxicated a c t e d as a p r o t e c t i o n .  rats It  was  a l s o found t o i n c r e a s e the osmotic r e s i s t a n c e of the r e d b l o o d cells,  r e s t o r e the a c t i v i t y o f the bone marrow, and  favor  reticulocytes!s. A s e r i e s o f papers on the hemopoietic p r o p e r t i e s o f b a t y l a l c o h o l has been p u b l i s h e d by Linman and h i s co-workers (51+.55, 56,57).  They found that t h i s AGE  stimulated erythropoiesis i n  normal r a t s by a c c e l e r a t e d e r y t h r o b l a s t i c c e l l u l a r without  an i n c r e a s e i n hemoglobin formation.  thrombopoiesis,  and  Reticulocytosis,  some g r a n u l o p o i e s i s were a l s o  B a t y l a l c o h o l and the thermostable,  division  present.  e t h e r - s o l u b l e plasma  e r y t h r o p o i e t i c f a c t o r share many common p h y s i c a l and p r o p e r t i e s , such as the f o l l o w i n g : e r y t h r o c y t e p r e c u r s o r s without  chemical  both s t i m u l a t e d i v i s i o n of  d e c r e a s i n g hemoglobin p r o d u c t i o n ,  10 b o t h are r e s i s t a n t to heat at 100°C f o r p r o l o n g e d p e r i o d s , are s i m i l a r i n m o l e c u l a r s i z e and  are l i p i d - l i k e  both  compounds.  T h e r e f o r e t h i s group o f workers concluded t h a t b a t y l a l c o h o l the e t h e r - 3 o l u b l e  f a c t o r are probably the  and  same or c l o s e l y r e l a t e d  compounds. Later  s t u d i e s added supporting  leukopoietic; neither response i s o b t a i n e d  evidence.  Both compounds are  i n c r e a s e the h e m a t o c r i t ; an i n vivo.  If t h i s conclusion  b a t y l a l c o h o l or some other AGE  c o u l d be  one  identical i s correct,  o f the main  c o n t r o l l i n g f a c t o r s f o r the primary e r y t h r o c y t o g e n i c Since  s e l a c h y l a l c o h o l i s the most abundant AGE  i t s e f f e c t on the bone marrow was results.  i n nature,  compared w i t h the above  No hemopoietic s t i m u l a t o r y a c t i v i t y i n normal r a t s  found, a l t h o u g h b o t h the used  stimulus.  s y n t h e t i c and  n a t u r a l compounds were  (57). B a t y l a l c o h o l has been t e s t e d as a p r o t e c t i v e or  agent i n many c o n d i t i o n s marrow.  E d l u n d (58)  l e t h a l i t y was  s i g n i f i c a n t l y lowered.  thirty-day  During t h i s experiment i t  on human p a t i e n t s w i t h r a d i a t i o n leukopenia was by B r o h u l t  cases g i v e n a l k o x y g l y c e r o l immediate i n c r e a s e  bone  gave subcutaneous i n j e c t i o n s to mice  shown that an overdosage i n c r e a s e d the l e t h a l i t y .  simultaneously  therapeutic  c h a r a c t e r i z e d by a h y p o p l a s t i c  i r r a d i a t e d w i t h x-rays w i t h the r e s u l t t h a t the  was  was  and Holmberg (26).  group concluded t h a t the AGE l e u k o p e n i a of r a d i a t i o n .  c e l l count.  study  published  Of the  e s t e r s o r a l l y , twenty-five  i n white b l o o d  A  thirty-six showed an  Therefore t h i s  were promising as a therapy  against  11  B a t y l a l c o h o l was  a l s o s t u d i e d i n eases of b r a c k e n  poisoning  i n c a t t l e (59 )> s i n c e t h i s c o n d i t i o n i s c h a r a c t e r i z e d by a severe l e u k o p e n i a and bone marrow. c o n t r o l and was  thrombocytopenia caused by a d e p r e s s i o n  of  the  A n t i b i o t i c s were a d m i n i s t e r e d at the same time t o t r e a t e d animals.  An i n c r e a s e d l e u k o c y t i c response  o b t a i n e d , w i t h the r e s u l t t h a t the animals  survived.  A s i m i l a r type of a p l a s t i c anemia i s caused by trichloroethylene-extracted calves  (60)  and  soybean o i l meal.  then b a t y l a l c o h o l was  b e f o r e h e m a t o l o g i c a l symptoms had e f f e c t was  found.  type o f anemia may  f e d to  i n j e c t e d by v a r i o u s  developed.  However, v a r i o u s  T h i s was  No  routes  protective  d i f f e r e n c e s i n technique  e x p l a i n the c o n f l i c t i n g r e s u l t s .  c o n d i t i o n there i s a decrease i n hemoglobin.  and  In t h i s  S i n c e i t has  a l r e a d y been shown t h a t b a t y l a l c o h o l does not a f f e c t hemoglobin l e v e l s , t h i s c o u l d be p a r t o f the reason f o r a l a c k of response. A l s o the t o x i c agent i n the meal may  a f f e c t some b i o c h e m i c a l  mechanism i r r e v e r s i b l y b e f o r e i n i t i a t i o n o f the therapy was P o s i t i v e r e s u l t s were shown by  subcutaneous or o r a l b a t y l  a l c o h o l when i t p a r t i a l l y prevented the l e u c o p e n i a mustard N-oxide i n r a t s (61).  Further  supporting  of  nitrogen  evidence  shown i n r a b b i t s w i t h n i t r o m i n - c a u s e d anemia and p a t i e n t s organic  solvent  begun.  was with  i n t o x i c a t i o n (62), although the b a t y l a l c o h o l  produced no hemopoietic s t i m u l a t i n g a c t i o n on h e a l t h y r a t s or rabbits. Ghys (63)  challenged  of b a t y l alcohol.  the l e u c o p o i e t i c - p r o m o t i n g  properties  F a i r l y l a r g e doses of a f r a c t i o n o f the  liver  o i l o f Somniosus microcephalus were g i v e n o r a l l y t o p a t i e n t s  with  12  x-ray l e u c o p e n i a .  Only f o u r out o f f i f t e e n cases showed any  leukopoietic effect.  However, some o f these anomalies may be* due  to u s i n g doses other than the maximum e f f e c t i v e dose.  Brohult  (6i|.) has shown that t h e white b l o o d c e l l count rose to a maximum as the dose i n c r e a s e d , b u t f e l l o f f with i n c r e a s i n g amounts o f o i l g i v e n above t h i s In an attempt  dose.  t o e v a l u a t e the Perm t e s t f o r cancer, R i l e y  e t . a l . (65,66) i d e n t i f i e d b a t y l and chimyl a l c o h o l s from t h e u n s a p o n i f i a b l e l i v e r f r a c t i o n of cancerous  individuals.  These  compounds were two o f the major c o n s t i t u e n t s r e s p o n s i b l e f o r the serological flocculation reaction.  The f a c t o r I n the serum which  combines with these a l c o h o l s t o produce i d e n t i f i e d as a C - r e a c t i v e p r o t e i n .  the f l o c c u l a t i o n was  T h i s compound was thought t o  be p r e s e n t i n the serum o f p a t i e n t s w i t h cancer, but not i n normal serum.  T h i s was d i s p r o v e n when i t was shown that the p r o t e i n  appears  i n inflammatory  s t a t e s and not i n those  specifically  a s s o c i a t e d w i t h cancer. Growth S t u d i e s R e c e n t l y s e v e r a l papers have been p u b l i s h e d d e s c r i b i n g t h e g l y c e r y l e t h e r s as growth-promoting substances.  Working w i t h  dwarf and A l a s k a peas, Stowe (67) observed that although e x c i s e d s e c t i o n s o f the p l a n t s f a i l e d t o o b t a i n h a l f t h e i r normal s i z e , p a r t o f t h i s m i s s i n g growth c o u l d be r e s t o r e d by s e v e r a l types of fatty acid esters.  The methyl  one o f these substances. promotion  e s t e r o f s e l a c h y l a l c o h o l was  In order t o e x h i b i t any growth-  i n a l l experiments  sugar and a u x i n were a l s o n e c e s s a r y .  G i b b e r e l l i c a c i d enhanced t h e i r a c t i o n f u r t h e r .  This indicated  13  t h a t the growth s t i m u l a t i o n was  due  t o an enhancement of  and g i b b e r e l l i n a c t i o n by the e s t e r s .  auxin  These e f f e c t s were maximal  when s m a l l q u a n t i t i e s o f e s t e r s were used, f o r example, 10 raibromoles;  £0  to  t h e r e f o r e , the a c t i o n appeared to be hormonal r a t h e r  than n u t r i t i o n a l . Brohult l a c t i s and  (68) examined the matter f u r t h e r , u s i n g L a c t o b a c i l l u s A s t i m u l a t i o n o f growth was  selachyl alcohol.  observed even at low  concentrations  o f the AGE.  Brohult  clearly also  observed t h a t when i r r a d i a t e d r a t s were p r e t r e a t e d w i t h s e l a c h y l a l c o h o l , they showed a greater controls.  However, t h i s may  Increase i n weight than the  not be connected e x c l u s i v e l y w i t h  growth e f f e c t s since many other of  f a c t o r s e n t e r i n t o the problem  irradiation. In t h e i r study on the presence of g l y c e r y l ethers  other AGE  i n man  and  animals (2l+), H a l l g r e n and L a r s s o n concluded that s i n c e  are found i n milk,  f e t a l t i s s u e , or r a p i d l y  the  reproducing  t i s s u e such as bone marrow, these compounds "might s t i m u l a t e  the  growth o f the newborn." AGE  have a l s o been found t o promote stage one  wound h e a l i n g has  (21)  but  of i r r a d i a t i o n  do not a c c e l e r a t e normal h e a l i n g which  a l r e a d y begun.  Toxicity Extremely l i t t l e work has been done on the t o x i c i t y o f the AGE.  As  s t a t e d i n Sandler's a r t i c l e  (1+7),  an i n v e s t i g a t i o n i n t o  the t o x i c i t y of cod l i v e r o i l showed that prolonged caused degenerative changes i n the and kidneys of a l b i n o mice.  The  suprarenal  gland,  administration heart,  liver,  t o x i c agents were b a t y l a l c o h o l  lit-  and  octadecyl  alcohol.  I t has a l s o been observed t h a t t h e f r e e a l c o h o l s  a r e much  more t o x i c t h a n t h e i r e s t e r s which a r e t h e n a t u r a l l y - o c c u r r i n g form.  However no evidence of t o x i c i t y i n t h e human s t u d i e s has  been shown, a l t h o u g h some nausea was present (1|7). Anti-inflammatory  Properties  There has been some i n d i c a t i o n i n the l i t e r a t u r e t h a t the AGE might possess an a n t i - i n f l a m m a t o r y a c t i o n . study o f t h i s p o s s i b i l i t y small  doses.  effectiveness  A  preliminary  (2) gave p o s i t i v e r e s u l t s w i t h v e r y  There have a l s o been u n p u b l i s h e d r e p o r t s  o f the  o f AGE i n some cases o f a r t h r i t i s ( 2 ) .  I t has long been hoped t h a t an a n t i - i n f l a m m a t o r y compound c o u l d be found which would not possess t h e exaggerated hormonal m a n i f e s t a t i o n s of t h e c o r t i c o s t e r o i d s o r the t o x i c s i d e - e f f e c t s o f phenylbutazone, t h e s a l i c y l a t e s , and t h e i r d e r i v a t i v e s .  Since  the t o x i c dose o f t h e AGE appears t o be very h i g h i n comparison to the p h a r m a c o l o g i c a l l y e f f e c t i v e dose, i t was f e l t  that  further  i n v e s t i g a t i o n i n t o the a n t i - i n f l a m m a t o r y a c t i o n o f t h i s group o f compounds was warranted.  Their  i n t e r e s t i n g e f f e c t on growth a l s o  appeared t o have been underemphasized.  15  EXPERIMENTAL  Methods Ant i - i n f lammat ory A g r e a t many types o f experiments have been developed f o r the purpose o f t e s t i n g a n t i - i n f l a m m a t o r y t e s t s a r e the f o l l o w i n g :  activity.  Some o f these  tuberculin skin sensitivity,  induced s w e l l i n g o f f o o t or trunk area, l i v e r g l y c o g e n d e p o s i t i o n and a d r e n a l  irritant-  histamine-sensitization,  cortex a l t e r a t i o n s , a l l o f  which a r e d e s c r i b e d b y D'Arcy e t . a l . (69), " c o l d s t r e s s " (*7Q), "swimming" s t r e s s (71)* i m m o b i l i z a t i o n  (72),  f o r e i g n body  granulomas (73) > n d granuloma pouch f o r m a t i o n a  (7I4. and 75) •  A r e c e n t review by Wilhelm (76) c r i t i c i z e s the methods which utilize  the s w e l l i n g o f t h e f e e t or trunk o f s m a l l animals  because the a n e s t h e s i a  used depresses the p e r m e a b i l i t y  of the p e r i p h e r a l b l o o d v e s s e l s .  The intravenous  response  i n j e c t i o n of  dyes which accumulate i n t r e a t e d s i t e s , p l u s the " s e n s i t i v i t y " t e s t s , were a l s o s a i d t o be a poor means o f t e s t i n g a n t i inflammatory compounds since they a r e so s u b j e c t i v e .  Many other  f a c t o r s i n t e r f e r e w i t h the p r e v i o u s l y l i s t e d t e s t s , such as temperature (77)* &ge and s p e c i e s o f a n i m a l . Previous  studies i n this lab u t i l i z i n g  the " c o t t o n  method have i n d i c a t e d t h a t s e l a c h y l dihemlsuccinate  pellet"  sodium i s  the most c o n s i s t e n t l y e f f e c t i v e drug o f the compounds t e s t e d . per r a t ( 7 8 ) .  The  dosage u t i l i z e d was f i v e t o t e n m i l l i g r a m s  The  g r a n u l a t i o n response which i s used as a measure o f the degree  16  of inflammation i n t h i s t e s t , i n c r e a s e s not o n l y w i t h time but with mechanical i n j u r y  (79).  The l a t t e r e f f e c t tends t o cause  . v a r i a b i l i t y i n r e s u l t s , thus i t was  d e c i d e d to t r y methods which,  e n t a i l e d l e s s trauma due t o o p e r a t i v e procedures.  The  tests  d e c i d e d upon were a m o d i f i e d c o t t o n p e l l e t method, and the granuloma pouch assay (75)* where i t i s p o s s i b l e t o o b t a i n q u a n t i t a t i v e measurements f o r purposes  of  comparison,  (a) Granuloma pouch technique A m o d i f i c a t i o n of the method d e s c r i b e d by Robert and Nezamis (75)  was used.  Female W i s t a r r a t s , with weights v a r y i n g from  150 to 200 grams, were d i v i d e d i n t o groups o f f i v e t o t e n animals each, and matched f o r weight. animals to a d j u s t to t h e i r new  In order t o a l l o w the  surroundings, they were l e f t i n  t h e i r cages f o r f o r t y - e i g h t hours b e f o r e the s t a r t of the experiment.  A f i v e percent stock s o l u t i o n o f c r o t o n o i l i n  c o t t o n s e e d o i l was  s t o r e d i n the r e f r i g e r a t o r .  a two percent s o l u t i o n f o r the i n j e c t i o n s . the experiment  was  s t e r i l i z e d by immersion  chloride solution.  I t was  diluted to  Apparatus used d u r i n g i n benzalkonium  A l l i n j e c t i o n s were g i v e n i n a room other  t h a n the one used t o shave and house the animals, and the o p e r a t i n g a r e a was  washed w i t h benzalkonium  solution,  also.  A f t e r a n e s t h e t i z i n g the animals w i t h p e n t o b a r b i t a l , t h e i r backs were c l o s e l y shaved w i t h an e l e c t r i c w i t h the a n t i s e p t i c .  c l i p p e r and swabbed  Twenty-five c . c . o f a i r was  i n j e c t e d under  the d o r s a l s k i n j u s t s l i g h t l y caudad to the center o f the animal's back.  A t h i r t y c.c. s y r i n g e and l / I f . i n c h , 26 gauge  needle were used.  In order to form the pouch p r o p e r l y , i t was  17  g e n t l y p r e s s e d forward while the needle was needle was  still  inserted.  The  t h e n removed w h i l e p r e s s i n g the s k i n together at the  s i t e of I n j e c t i o n . A f t e r one group of animals was (2%)  was  mla o f c r o t o n o i l  i n j e c t e d subcutaneously d i r e c t l y Into t h e top of the  pouch, u s i n g a I / I 4 . i n c h , 27 in  done, 0.5  f o r a few minutes  gauge needle.  The needle was  to a l l o w the i r r i t a n t  o i l t o d r a i n out.  T h i s procedure guarded a g a i n s t l o c a l n e c r o s i s . a d m i n i s t e r e d at t h i s time and d a i l y The a i r was the pouch.  left  The AGE  was  thereafter.  withdrawn f o r t y - e i g h t hours a f t e r f o r m a t i o n o f  T h i s was  done through a 2l\. gauge needle a t t a c h e d by  rubber t u b i n g , through a water t r a p , to the a s p i r a t o r f i t t i n g a water o u t l e t .  No a n e s t h e t i c was  c a u t i o n s were s t i l l  maintained.  on  necessary, but a s e p t i c p r e -  S i n c e the l a y e r s of the pouch  sometimes adhere together a f t e r the withdrawal  of the a i r , the  pouches were g e n t l y manipulated the next day t o break up  any  adhesions. On the f o u r t h or f i f t h day a f t e r the i n i t i a l pouch f o r m a t i o n (day z e r o ) , the animals were s a c r i f i c e d u s i n g e t h e r . the exudate was  c o l l e c t e d u s i n g a l a r g e s y r i n g e and  i n j e c t e d d i r e c t l y i n t o the pouch, and the remainder through a cut In the  Part of needle collected  skin.  The e f f e c t s on exudate f o r m a t i o n produced by  selachyl  a l c o h o l and s e l a c h y l d i h e m i s u c c i n a t e were compared w i t h that o f hydrocortisone acetate. 2 percent tween 20  The h y d r o c o r t i s o n e was  s o l u t i o n i n one  s o l u t i o n s i n t h i s group was  experiment;  2% tween 20,  suspended  in a  the v e h i c l e f o r a l l  therefore.  In another  18 experiment, which was  the h y d r o c o r t i s o n e was  suspended i n cottonseed o i l ,  the v e h i c l e f o r t h i s group.  V a r i o u s dosage l e v e l s  and  r o u t e s of a d m i n i s t r a t i o n o f the compounds were used.  Throughout  a l l experiments,  AGE,  i t was  found that i n j e c t i o n s o f the  e s p e c i a l l y s e l a c h y l dihemlsuccinate sodium, caused  local  irritation. (b) Cotton p e l l e t method T h i s technique, o r i g i n a l l y d e s c r i b e d by Meier was  e t . a l . (73)  m o d i f i e d to e l i m i n a t e some o f the v a r i a b i l i t y .  A  cylindrical  compressed c o t t o n sponge o f the type used by d e n t i s t s was i n t o c i r c u l a r p e l l e t s weighing approximately J4.O mgm p e l l e t produced i n t h i s manner was surface area. pentobarb,  sliced  each.  o f a uniform weight  The  and  Male a l b i n o M i s t a r r a t s were a n e s t h e t i z e d w i t h  and one weighed p e l l e t c a r e f u l l y implanted  cutaneously i n the l o o s e d o r s a l neck t i s s u e .  sub-  This eliminated  much of the trauma to surrounding t i s s u e s by i m p l a n t a t i o n of f o u r p e l l e t s i n the usual method. At t e r m i n a t i o n of the experiment, and extraneous p e l l e t was  the i n c i s i o n was  t i s s u e cut away u n t i l the granuloma  a b l e t o be p r e s s e d out o f i t s c a v i t y .  l e n g t h of such an experiment the drugs g i v e n d a i l y .  was  reopened  surrounded The  usual  seven days, w i t h i n j e c t i o n s o f  The p e l l e t s were p l a c e d on t a r e d  p l a n c h e t s and oven d r i e d at 80°C f o r twenty-four hours b e f o r e reweighing. D a i l y subcutaneous i n j e c t i o n s of 10 mg per k i l o g r a m o f h y d r o c o r t i s o n e and 30 mg P©r k i l o g r a m o f s e l a c h y l sodium were compared i n young r a t s weighing  100  dihemlsuccinate  to 125  grams, and  19 mature r a t s of 220  t o 300  grams s i n c e i n p r e l i m i n a r y  experiments  more c o n s i s t e n t r e s u l t s were o b t a i n e d i n o l d e r r a t s . used was  normal  The  vehicle  saline.  L i v e r - g l y c o g e n Determination Although i t i s p o s s i b l e f o r a compound to possess  anti-  inflammatory a c t i v i t y without p o s s e s s i n g g l u c o c o r t i c o i d (80), i t was  activity  f e l t t h a t the r e s u l t s of a g l y c o g e n d e p o s i t i o n t e s t  would be o f i n t e r e s t f o r the f o l l o w i n g reasons: - p r e v i o u s experiments (2) have shown the AGE  in this,  (78) and other l a b o r a t o r i e s  to have some a n t i - i n f l a m m a t o r y a c t i v i t y ,  although the r e s u l t s were i r r e g u l a r ; - an i n c r e a s e i n the r e s i s t a n c e to s t r e s s has been a t t r i b u t e d to  the AGE,  (2);  - v a r i o u s workers have p u b l i s h e d r e s u l t s i n d i c a t i n g some hormonal or hormone-stimulating response (5i{.,55>,67); - o b s e r v a t i o n s on the growth of p l a n t s and animals the AGE  implicate  i n t h i s p h y s i o l o g i c a l process (67,68,2l|.).  The l o g i c a l c o r r e l a t i o n of t h i s data appeared t o i n d i c a t e t h a t the a d r e n a l - p i t u i t a r y a x i s c o u l d perhaps be a f f e c t e d by the AGE.  In order to determine  i f these compounds d i d have a  g l u c o c o r t i c o i d e f f e c t , the d e p o s i t i o n of glycogen i n t h e l i v e r of r a t s was  investigated.  The P f l u g e r method as m o d i f i e d by Colowick and Kaplan used w i t h minor changes (81). tubes each c o n t a i n i n g 3*5 were t a r e d .  Male Wis t a r  A number of 15 ml Pyrex  was  centrifuge  mis o f 30$ potassium hydroxide  solution  r a t s were k i l l e d by ether a n e s t h e s i a and  approximately one gram of l i v e r from the l e f t lobe was  quickly  20  e x c i s e d , weighed, and immersed i n the potassium h y d r o x i d e . tubes were reweighed exact weight bath.  on an a n a l y t i c a l balance t o determine  the  of t i s s u e used, and t h e n immersed i n a b o i l i n g water  The t i s s u e was  d i g e s t e d f o r approximately t h i r t y  or u n t i l a r e l a t i v e l y c l e a r s o l u t i o n i s o b t a i n e d . time the tubes were shaken f r e q u e n t l y t o a s s i s t  sodium s u l f a t e was  added.  volumes of 95$  The glycogen was  ethanol, s t i r r e d ,  washed w i t h 60$ e t h a n o l .  minutes,  During  this  i n the d i g e s t i o n .  The tubes were removed from the bath, and 0.5  t o 1.2  The  mis of s a t u r a t e d  precipitated  1.1  and the s t i r r i n g r o d  The tubes were heated j u s t to b o i l i n g  i n the hot water bath, c o o l e d , and c e n t r i f u g e d at 3000 r.p.m. The  supernatant l i q u i d was  allowed t o d r a i n .  decanted, and the glycogen p r e c i p i t a t e  In order t o expel any a l c o h o l which may  be  l e f t , the tubes were heated a g a i n f o r a s h o r t time. The glycogen was r e d i s s o l v e d i n 2.0 r e p r e c i p i t a t e d with 2.5  mis o f 95$  mis o f d i s t i l l e d  ethanol, s t i r r e d ,  water,  heated,  c e n t r i f u g e d , decanted and d r a i n e d as b e f o r e . These more p u r i f i e d samples were d i s s o l v e d i n d i s t i l l e d water and t r a n s f e r r e d q u a n t i t a t i v e l y t o 50 ml v o l u m e t r i c f l a s k s . A f t e r d i l u t i n g t o volume w i t h water and mixing thoroughly, a second d i l u t i o n was and 1.25  mis was  experiment. with  made.  U s u a l l y an a l i q u o t of between  necessary depending  T h i s a l i q u o t was  0.75  on t h e c o n d i t i o n s of t h e  d i l u t e d v o l u m e t r i c a l l y t o 50  mis  water. F i v e mis of t h i s second d i l u t i o n were t r a n s f e r r e d t o c l e a n  f i f t e e n ml tubes f o r the "unknowns."  The  standard was  prepared  by u s i n g 5 mis of a f r e s h g l u c o s e s o l u t i o n c o n t a i n i n g 100  t  hexose f o r 5 mis.  The blank was 5 mis of d i s t i l l e d water. A l l  the tubes were submerged i n a c o l d water bath, and 10 mis of anthrone reagent (0.2$ anthrone i n 95$ s u l f u r i c a c i d , prepared d a i l y ) was added t o each tube from a f a s t - f l o w i n g p i p e t t e .  The  tubes were s w i r l e d t o a s s i s t i n t h e mixing o f the components, and covered w i t h g l a s s marbles. minutes  After heating f o r exactly ten  i n the b o i l i n g water bath, the s o l u t i o n s were c o o l e d i n  the c o l d bath.  The o p t i c a l d e n s i t y was r e a d immediately on a  S p e c t r o n i c 20 at 620 ny/.. A standard curve ( F i g u r e 1) was u t i l i z e d t o determine the amount of g l u c o s e present i n the v a r i o u s s o l u t i o n s , and the g l y c o g e n content of the t i s s u e , c a l c u l a t e d from t h i s .  A factor of  0 . 9 was used f o r c o n v e r t i n g glucose to glycogen (82). L i v e r glycogen determinations were done both on the r a t s used f o r the c o t t o n - p e l l e t t e s t s , , and on otherwise normal  rats.  Using normal r a t s , a s e r i e s o f experiments was r u n t o determine the dosage of s e l a c h y l sodium s u c c i n a t e , and the l a t e n t necessary to e l i c i t subcutaneous  a response.  period,  I t was found that t h r e e  i n j e c t i o n s , g i v e n over a p e r i o d o f twelve hours,  were e f f e c t i v e when the r a t s were k i l l e d twenty-four hours the f i r s t 9:30  a.m.,  injection. 3*30 p.m.,  For example, the i n j e c t i o n s were g i v e n at and 9 : 3 0 p.m.,  w i t h the experiment  t e r m i n a t e d a t 9 : 3 0 the f o l l o w i n g morning.  Effective  u t i l i z e d i n t h i s study were 3 0 , tf.0, 50 and 60 mgm rat.  after  doses  per 100 gram  The groups o f animals were matched f o r weight, and were  a l l o w e d t o eat and d r i n k ad l i b .  0  0 ^  Ho  IS  Ho  2~h  CONCEHTBATION( mg./lOO ml. )  Figure 1. A typical standard curve for the optical density of glucose.  3h  3T5  4.'o  23 A. s e c o n d  type o f s t u d y u t i l i z e d  E a c h cage was partition.  divided  Each p a i r  the  i n two,  the  w i t h one  paired feeding  r a t on  o f r a t s were e x a c t l y  experiment.  the  of  three  d a y s , a f t e r w h i c h i n j e c t i o n s were b e g u n .  controls.  only  Thus t h e i r  the  food  eating  each s i d e of  onset  a n i m a l s were g i v e n  Their  was  the  same w e i g h t a t  h a b i t s were o b s e r v e d  amount o f f o o d intake  method.  no  The  e a t e n by  longer  an  the  for  treated their  paired  uncontrolled  factor.  Growth  Studies  During  the  c o u r s e of the  i n c o n s i s t e n t e f f e c t s of the of was  food  e a t e n , and  tabulated.  to t r y to  Also,  determine  (a) S e l a c h y l  the  anti-inflammatory  AGE  the  e f f e c t s of  dihemisuccinate  P o o d and  selachyl  the  c o n s u m p t i o n was recorded  t e s t s were done on the  AGE  every f i v e  days.  tap  i n excess  5 t o £0  water.  the weight  Length of t h i s  studies,  growth. water:  so  into  that  c o n t r o l group r e c e i v e d  i n their  amount  normal r a t s  grams e a c h , were d i v i d e d  The  measured d a i l y ;  on  sodium I n d r i n k i n g  t r e a t e d groups r e c e i v e d  sodium s u c c i n a t e  The  animals used i n these  w a t e r were g i v e n  amounts were a l w a y s p r e s e n t . water, w h i l e  the  other  Male Wis t a r r a t s , I4.O t o 72 groups o f s i x .  on g r o w t h were n o t e d .  growth of several  experiments,  parts  Pood and  o f the  tap  per  million  water  animals  s t u d y was  ample  was  thirty-one  days. (b) A 100  Selachyl  dihemisuccinate  short-terra experiment  grams o f t h e  drug  s o d i u m by  various  o f s i x d a y s was  ( v e h i c l e was  w a t e r ) and  routes:  done u s i n g five  rats  5 mg per  per  21*  group.  The  r o u t e s o f a d m i n i s t r a t i o n were o r a l  cutaneous and  intraperitoneal  intraperitoneal  saline  injections.  The  i n t u b a t i o n , sub-  .  controls received  injections. Results  Anti-inf1aramation (a) G r a n u l o m a p o u c h It  c a n be  seen  from  Table  I I t h a t a somewhat g r e a t e r v o l u m e  o f exudate formed i n the pouches of r a t s DHSS t h a n i n c o n t r o l r a t s .  Hydrocortisone  e x u d a t e f o r m a t i o n as m i g h t be potent  treated with  expected  completely  selachyl inhibited  i n v i e w o f i t s known  anti-inflammatory properties. TABLE I I Effect  o f S e l a c h y l DHSS Compared w i t h H y d r o c o r t i s o n e A c e t a t e u s i n g the Subcutaneous Route  Group  Number o f Rats  Dose (rag/rat/day)  Mis o f Exudate  % of Control 100  Controls  i\.  -  6.13  S e l a c h y l DHSS  5  5-  7.92  $  5  0.0  129.2  Hydrocortisone acetate (vehicle  - 2%  t w e e n 20  Thus i t a p p e a r e d effect  for this  t r e a t e d animals  solution;  t h a t t h i s AGE  p r e p a r a t i o n and was  d u r a t i o n of experiment  also  showed no  dose.  The  anti-inflammatory  weight o f t h e  comparable t o t h a t o f the  a l t h o u g h h y d r o c o r i t s o n e group l o s t  - 5> d a y s ) .  AGE-  controls,  weight.  #' S e l a c h y l d i h e r a i s u c c i n a t e s o d i u m w i l l h e r e a f t e r be r e f e r r e d s e l a c h y l DHSS.  to  as  25  Since  i t had been shown that very s m a l l doses o f some  administered (2), 600  o r a l l y reduce f l u i d formation  AGE  i n the granuloma pouch  performed i n which r a t s were g i v e n 300  an experiment was  micrograms o r a l l y of s e l a c h y l a l c o h o l each day.  The  and  results  are shown i n Table I I I . TABLE I I I E f f e c t of S e l a c h y l A l c o h o l Compared w i t h Hydrocortisone - O r a l I n t u b a t i o n Group  Number o f Rats  Control  Dose (mcg/rat/day)  Mis. o f Exudate  % of Control  5  -  7.2  5  300  8.1  112.5  5  600  7.9  109.7  5  500  lf.,9  100  Selachyl alcohol Selachyl alcohol Hydrocortisone acetate  ( v e h i c l e - cottonseed  oil;  d u r a t i o n of t e s t - 5  No decrease i n exudate due I t was,  68.0  days.)  t o s e l a c h y l a l c o h o l was  observed.  i n f a c t , i n c r e a s e d over the c o n t r o l l e v e l , c o n t r a r y  previous  reports.  As f a r as i t was  p o s s i b l e to a s c e r t a i n ,  o n l y d i f f e r e n c e between t h i s method and t h a t which was to give a decrease i n exudate was drugs were a d m i n i s t e r e d  the  reported  t h a t i n t h i s experiment  the  by o r a l i n t u b a t i o n , whereas i n the  p r e v i o u s l y r e p o r t e d t e s t the compounds were f e d to the o r a l l y by  to  animals  dropper.  A t h i r d study was p e r i t o n e a l route,  performed, t h i s time u t i l i z i n g the  to compare the above two AGE.  The  intra-  r e s u l t s are  26  t a b u l a t e d below.  Equivalent  doses o f the two compounds, a s  c a l c u l a t e d from molecular weights, were used. TABLE IV E f f e c t o f S e l a c h y l DHSS Compared w i t h S e l a c h y l Alcohol - Intraperitoneal Injections Group  Number o f Rats  Dose (mg/rat/day)  Mis. of Exudate  -  7.08  9  Controls  $ of Control 100  Selachyl 10  DHSS  13.8  6.5*+  92.1+  Selachyl alcohol 8 10.0 6.25 88.3 ( v e h i c l e f o r c o n t r o l and a l c o h o l - peanut o i l ; f o r s u c c i n a t e water; d u r a t i o n o f experiment - 5 days.) Again, t h e decrease i n the amount o f exudate was i n s i g n i f i c a n t , s i n c e a v a r i a t i o n o f a t l e a s t + 10$ n o r m a l l y occurs i n c o n t r o l groups o f such methods, (b) Cotton p e l l e t  granuloma  The e f f e c t of the age o f the animals o f t h i s method was investigated.  S i m i l a r experiments were c a r r i e d out on young  growing r a t s weighing 100 t o 125 grams, and mature r a t s weighing 220 t o 300 grams.  I n each case f i v e r a t s were used per group and  i n j e c t i o n s were made once d a i l y by t h e subcutaneous  route.  C o n t r o l r a t s r e c e i v e d s a l i n e only, and the d u r a t i o n o f the experiment was seven days.  The r e s u l t s a r e g i v e n I n T a b l e V.  I t was noted that i n the mature animals a more pronounced anti-Inflammatory e f f e c t was produced i n b o t h t r e a t e d groups. T h i s c o u l d perhaps be due to the f a c t that these r a t s a r e not  27  growing although the young r a t s a r e . TABLE V Comparison o f G r a n u l a t i o n T i s s u e Formation i n Young and Mature Rats  Group  Dose (mg/kg)  Control Hydrocortisone acetate  Young Animals mg. o f gran% of ulation tissue control  Mature Animals mg. o f gran"% o f ulation tissue control  55. l ( l | . l - - ° 7 . 8 ) 100  100  -  i4.1.6(30.2-61|.5)  10  25.2(22.8-30.7 )  60.6  27.0(22.9-31.1)  30  36.8(2lj..5-65.7)  88.3  38.8(26.0-50.2) 70.5  l|.9.1  Selachyl  DHSS  L i v e r Glycogen Determinations The r e s u l t s o f the experiments done on t h e animals w i t h c o t t o n granulomas a r e shown i n Table VI.  The c o n d i t i o n s o f t h i s  experiment a r e the same as f o r the experiments : r e f e r r e d t o i n T a b l e V. TABLE V I Comparison o f L i v e r Glycogen i n Animals T r e a t e d w i t h S e l a c h y l DHSS and H y d r o c o r t i s o n e Acetate L i v e r glycogen Young AnimalsT Mature Animals mg/Gm o f l i v e r % o f mg/Gm o f l i v e r % o f . \ control control  Group  2i^.5(ll|.5-31.3)  Control  100  25.1(11.25-36.8) 100  Hydrocortisone  acetate Selachyl  DHSS  20.0(15.9-25.95)  81,6  31.7(27.5-36.1)  129.2  3 7 . 7 ( 3 5 . M - 1 . 6 r 150.8 28.1(20.3-1}.2.7)  112.0  (# Mature group r e c e i v e d a d d i t i o n a l dose three hours b e f o r e s a c r i f i c i n g - see e x p l a n a t i o n f o l l o w i n g ) .  28  There appeared to be a compensatory g l y c o g e n o l y s i s i n the young r a t s t r e a t e d w i t h h y d r o c o r t i s o n e s i n c e the h y d r o c o r t i s o n e , which i s e f f e c t i v e f o r o n l y a few hours, had been a d m i n i s t e r e d twelve hours b e f o r e the death of the young animals. glycogen r e a d i n g was  Thus a low  o b t a i n e d f o r the h y d r o c o r t i s o n e group.  To  p r o v i d e a f a i r e r b a s i s f o r comparisons, the mature animals were g i v e n i n j e c t i o n s of the drugs t h r e e hours b e f o r e t e r m i n a t i o n of the experiment. c o n t r o l was The  In t h i s case, an i n c r e a s e of 50.8$ over  the  obtained.  i n c r e a s e i n glycogen content  i n the s e l a c h y l DHSS  group made f u r t h e r s t u d i e s on t h i s aspect appear n e c e s s a r y . F i g u r e 2 summarizes the r e s u l t s o b t a i n e d from f o u r l i v e r glycogen experiments on r a t s w i t h no substances  present.  mg/lOO Gms.  The  inflammatory-stimulating  l o g doses r e p r e s e n t 30, i+0, 50,  F i v e groups o f animals  Two  groups gave low v a l u e s .  gave a value of only 13$  over  60  showed a l a r g e i n c r e a s e over  c o n t r o l v a l u e s , i n the same range t h a t was studies.  and  shown i n p r e l i m i n a r y  The p a i r e d f e e d i n g group  controls.  Growth S t u d i e s The  o b s e r v a t i o n s on growth during the granuloma pouch and  cotton p e l l e t  s t u d i e s are summarized i n Tables V I I and V I I I .  29  R  O normal feeding  -i  CO  ^  paired feeding  o  \  /  /  O \  /  /  9, -  N  /  /  /  • o /  /  I  o  6  I  I  I  I  o  i-H  X) 1  1.45  1.55  1 1.65  1 1.75  f  1.85  LOG DOSE  Figure 2 . Effect of subcutaneous selachyl DHSS on l i v e r glycogen deposition of rats. Connected dots represent single experiments. Controls=100$.  30 TABLE V I I E f f e c t s o f the AGE on Growth o f Rats During Granuloma Pouch S t u d i e s Compound  S e l a c h y l DHSS " Selachyl alcohol  Route o f Administration  Dose  5 rag/rat 13.8 mg/rat  Weight Gain (fo C o n t r o l )  subcutaneous  n e g l i g i b l e (+)  intraperitoneal  63$  300 meg/rat  oral  n e g l i g i b l e (+)  600 meg/rat  oral  negligible  10 mg/rat  intraperitoneal  (-)  n e g l i g i b l e (+)  The o n l y route o f a d m i n i s t r a t i o n by which s e l a c h y l DHSS a f f e c t e d weight g a i n was t h e i n t r a p e r i t o n e a l r o u t e . a l c o h o l , by t h i s r o u t e , d i d not a p p r e c i a b l y a f f e c t  Selachyl growth.  TABLE V I I I E f f e c t s o f AGE on Weight o f Rats During Cotton P e l l e t S t u d i e s Group  Controls  Dose  Pood Consumption (Gm. of f o o d eaten/ rat/day) Young ' Mature llf.O  XI1—I4. (2.5  Hydrocortisone acetate  10 mg per Kg. 12.8  12.2  Selachyl DHSS  30 mg p e r Kg. 16.5  18*k  (subcutaneous i n j e c t i o n s )  Weight' G a i n (Gm/rat/day) Young  3.8  t o i(..5)  Mature (~3.k  0.03  to 1.1)  04 -7.7 (-2.1 to 2.1) (-11.0 t o -6.1) (2.5  k-7 t o 6.3)  0.7  (-0.7 t o l.ij.)  31  The young r a t a g i v e n s e l a c h y l DHSS g a i n e d 23.7$ more weight than t h e i r c o n t r o l s , w h i l e the mature r a t s m a i n t a i n e d t h e i r weight, Growth s t u d i e s where no inflammation was present a r e summarized  i n T a b l e IX and F i g u r e 3»  The f i g u r e shows the  average weight g a i n and f o o d consumption as a percent o f the control.  A l s o i n c l u d e d a r e l i n e s showing the extremes i n each  group. TABLE IX Long Term Study o f the E f f e c t s o f S e l a c h y l DHSS i n D r i n k i n g Water o f Normal Rats Group  Dose Water ( p a r t s per Consumption million) (mis/rat/day)  Control Selachyl DHSS  5  50  Food Consumption (Gm/rat/day)  Weight G a i n ($ o f c o n t r o l )  20.96  13.7  21.90  13.1+  91+.1  20.20  13.1+  88.9  100  ( d u r a t i o n o f study - 31 days.) No s i g n i f i c a n t changes were brought about by these doses orally. F i g u r e 3 a g a i n shows that the i n t r a p e r i t o n e a l route i s the o n l y one a f f e c t i n g weight g a i n , and i t had the g r e a t e s t on f o o d consumption.  effect  o  o  C\J .  o u +»  oo  g o o  8-  8-  o o  § o • o  o•  VD  VO  O  control  oral  s.c  0  i.p.  o_  control  ROUTE OP ADMINISTRATION  oral  s.c.  Figure3. E f f e c t o f s e l a c h y l dihemisuccinate sodium on growth and food consumption o f rats„ Various routes o f a d m i n i s t r a t i o n . Dose- 5 0 . mg. per k i l o g r a m .  33 DISCUSSION  S e l a c h y l DHSS and s e l a c h y l a l c o h o l showed no s i g n i f i c a n t anti-inflammatory  e f f e c t w i t h the granuloma pouch method a t the  dosage l e v e l s and routes  of a d m i n i s t r a t i o n used.  These doses had  g i v e n p o s i t i v e r e s u l t s i n other s t u d i e s ( 2 , 7 8 ) , and i n c l u d e d both v e r y s m a l l and r e l a t i v e l y l a r g e doses. anti-inflammatory  Some evidence o f  a c t i v i t y was r e v e a l e d by t h e c o t t o n p e l l e t  granuloma t e s t .  Although s e l a c h y l DHSS decreased  t i s s u e formation  i n young r a t s by only l l « 7 $ j which i s not  c o n s i d e r e d t o be s i g n i f i c a n t , i t decreased f o r m a t i o n i n mature r a t s by 2 9 * 5 $ . dose o f 30 mg/Kg subcutaneously.  the g r a n u l a t i o n  t h e granuloma  T h i s was accomplished a t a Hydrocortisone  a c e t a t e , on the  other hand, gave r e s u l t s ranging from complete d e p r e s s i o n o f exudate i n the f i r s t granuloma pouch assay (Table II) t o depressions (Table V)  o f 39 »h  5 0 . 9 $ i n the c o t t o n p e l l e t  granulations.  These r e s u l t s were e l i c i t e d u s u a l l y by much lower  dosages than those  o f the AGE.  Subcutaneous s e l a c h y l DHSS caused an i n c r e a s e i n l i v e r glycogen  i n a l l except one o f the experiments.  (Figure 2 )  In t h i s p a r t i c u l a r case the 30 and 1+0 mg/lOO Gm. doses gave g l y c o g e n v a l u e s near the c o n t r o l l e v e l . h i g h values i n the f o l l o w i n g experiment.  The same dosages gave This large  discrepancy  may probably be accounted f o r i n the f o l l o w i n g d i s c u s s i o n , on t h e method used. In t h e p a i r e d f e e d i n g experiment there was an i n c r e a s e o f o n l y 1 3 $ over c o n t r o l v a l u e s .  Therefore,  increased  food  3%  consumption or u t i l i z a t i o n appears  t o be a t l e a s t one o f the  f a c t o r s i n the i n c r e a s e d storage o f glycogen i n the l i v e r . However, the s m a l l number o f animals, l i m i t e d dosage range, and s i n g l e r o u t e o f a d m i n i s t r a t i o n used i n these experiments,  plus  the i n h e r e n t i n a c c u r a c y o f the method, make i t , i m p o s s i b l e t o draw any f i n a l c o n c l u s i o n s on the s u b j e c t .  Although the anthrone  method o f e s t i m a t i n g l i v e r glycogen i s w i d e l y used, s e v e r a l disadvantages.  i t has  The d i l u t i o n s must be estimated on the  b a s i s o f the amount o f g l y c o g e n p r e s e n t , which v a r i e s w i d e l y depending on the drug used and when i t was a d m i n i s t e r e d ,  how  r e c e n t l y the animal has eaten or done any p h y s i c a l a c t i v i t y , and even from which section- of t h e l i v e r the sample i s taken.  In  s t u d i e s o f the glycogen content I n c a t t l e l i v e r , K u s i n and Pupin (83) noted v a r i a t i o n s o f 10 - 1J4.O mg % i n samples o b t a i n e d s i m u l t a n e o u s l y from d i f f e r e n t s e c t i o n s o f the same organ.  Also,  dust and l i n t which a c c i d e n t a l l y enter the s o l u t i o n w i l l cause a darker c o l o r f o r m a t i o n .  S o k a l and Sarcione  (81+) s t a t e d that a  change o f 20$ was not s i g n i f i c a n t i n t h i s type o f  experiment.  A l l these f a c t o r s make i t d i f f i c u l t f o r even a h i g h l y t r a i n e d t e c h n i c i a n to obtain reproducable r e s u l t s .  Therefore,  although a g r e a t d e a l o f time was spent on t h i s p a r t o f the work, the outcome was d i s c o u r a g i n g . I t was noted i n the l i t e r a t u r e than an "auto a n a l y z e r " has been /set up f o r the purpose of l i v e r glycogen determinations (85). F u r t h e r developments along t h i s l i n e w i l l be i n t e r e s t i n g t o note. The  s t u d i e s on growth were an outcome o f o b s e r v a t i o n s d u r i n g  the a n t i - i n f l a m m a t o r y experiments.  I t had been noted that r a t s  35  g i v e n s e l a c h y l DHSS i n t r a p e r i t o n e a l l y l o s t w e i g h t , certain  cases subcutaneous  weight.  injections  whereas i n  caused a s l i g h t  increase i n  F o r example, t h e y o u n g r a t s u s e d i n t h e c o t t o n p e l l e t  t e s t a t e 1 6 . 1 $ more f o o d t h a n t h e i r w e i g h t , w i t h a dose I t was f o u n d t h a t  controls  a n d g a i n e d 23.7$ more  o f 30 mg/Kg o f t h e s u c c i n a t e .  i n normal  rats,  (Table VIII)  s e l a c h y l DHSS, a t a dose o f  50 mg/Kg, a f f e c t s g r o w t h o n l y b y t h e i n t r a p e r i t o n e a l r o u t e where it  causes  a r e t a r d a t i o n o f weight  d r i n k i n g water  h a v e no e f f e c t .  gain.  The i n t r a p e r i t o n e a l g r o u p s  a l s o n o t e d t o h a v e e a t e n somewhat l e s s The  V e r y l o w amounts i n  food than t h e i r  r a t h e r unusual property o f having d i f f e r e n t  to a difference  of the succinate. is  i n the type o f l o c a l  controls.  physiological  e f f e c t s b y d i f f e r e n t r o u t e s o f a d m i n i s t r a t i o n might due  p o s s i b l y be  a b s o r p t i o n and metabolism  S i n c e i t h a s b e e n shown t h a t  chimyl  alcohol  e x t e n s i v e l y metabolized during a b s o r p t i o n through the  intestinal  mucosa  same p r o p e r t y .  (I|.9,50), p e r h a p s  In this  s e l a c h y l DHSS p o s s e s s e s  way, a s l i g h t l y  different  affect  growth.  could  However, t h e mechanism c o u l d a l s o be much s i m p l e r ,  such a s a n i r r i t a n t  effect.  A c u r i o u s phenomenon was n o t e d when h i g h d o s e s  s u c h as 600  mg/Kg o f s e l a c h y l DHSS were a d m i n i s t e r e d s u b c u t a n e o u s l y . twenty-four hours observed.  this  t y p e o f com-  p o u n d c o u l d be f o r m e d b y t h e i n t r a p e r i t o n e a l r o u t e , w h i c h  was  were  an a p p a r e n t l y l a r g e i n c r e a s e i n weight  I t was n o t due t o n o r m a l  growth,  was  s i n c e t h e weight  t h r e e o r f o u r t i m e s a s much a s n o r m a l l y r e c o r d e d .  a p p e a r e d v e r y s l u g g i s h a n d p u f f y , w h i l e a p o s t mortem revealed a thick layer  Within  gain  The r a t s examination  o f g e l a t i n - l i k e m a t e r i a l p r e s e n t next, t o  36  the subcutaneous abdominal t i s s u e .  T h i s was  not a l o c a l  effect,  s i n c e the i n j e c t i o n s were g i v e n on the back of the animal, and such l a y e r formed i n t h i s area. t h i s phenomenon was  observed,  no  In s e v e r a l experiments where  i n c l u d i n g the p a i r e d f e e d i n g s t u d i e s ,  no i n c r e a s e I n f o o d or water consumption o c c u r r e d . I t i s hoped t h a t f u r t h e r work w i l l be done on the aspects of growth and weight w i t h r e s p e c t t o the During  t h i s p r e l i m i n a r y study on some o f the  e f f e c t s o f the AGE,  pharmacological  the most n o t a b l e o b s e r v a t i o n was  u n i f o r m i t y i n the r e s u l t s . significant  AGE.  a lack of  F a c t o r s which appeared to cause a  change I n the outcome o f experiments Include the  age  o f the animals, the l e n g t h of time they were housed i n new q u a r t e r s b e f o r e the commencement o f the study, and the r o u t e o f a d m i n i s t r a t i o n of the compounds.  A complete l a c k o f l i n e a r  r e l a t i o n s h i p between dose and response  was  apparent.  a l s o been n o t i c e d i n p r e v i o u s s t u d i e s on AGE and appears to have been encountered compounds (60, may  possess  61+,  67).  had  i n this laboratory,  by others working with  these  I t i s p o s s i b l e t h a t s e l a c h y l DHSS  a maximum e f f e c t i v e dose, above which i t s p h y s i o l o g i c a l  p r o p e r t i e s may Due  63,  This  be l e s s e n e d or r e v e r s e d .  t o the p r e l i m i n a r y nature o f the work done f o r t h i s  t h e s i s , a great deal of time was  spent on each s e c t i o n to  determine u s e f u l doses and methods o f a d m i n i s t r a t i o n . work along these l i n e s c o u l d now  Further  be c a r r i e d out i n a more  exhaustive manner to c l e a r up the many q u e s t i o n s which a r i s e from the above r e s u l t s .  37 SUMMARY  1.  No a n t i - i n f l a m m a t o r y a c t i v i t y was e l i c i t e d by s e l a c h y l  DHSS o r s e l a c h y l a l c o h o l u s i n g the granuloma pouch method. 2. S e l a c h y l  DHSS showed a n t i - i n f l a m m a t o r y a c t i o n i n mature  r a t s with cotton p e l l e t  granulomas.  3» L i v e r g l y c o g e n d e p o s i t i o n  i n r a t s was i n c r e a s e d by  s e l a c h y l DHSS, b u t t h i s may be due t o i n c r e a s e d  f o o d i n t a k e or  utilization. 1+. I n t r a p e r i t o n e a l  i n j e c t i o n s of s e l a c h y l DHSS caused a  decrease i n weight o f the animals as compared t o c o n t r o l groups.  38 BIBLIOGRAPHY  1.  Tsujimoto, M., and Toyama, Y., The u n s a p o n i f i a b l e c o n s t i t u e n t s (higher a l c o h o l s ) o f the l i v e r o i l o f sharks and r a y s . Chem.'Urnschau. 22:27-9, 1+3-5 through Chem. Abs., 1 6 : 1 5 1 2  (1922).  (1922),  I960.  2.  Western Chemical I n d u s t r i e s L t d . , T e c h n i c a l B u l l e t i n ,  3.  Schmidt-Nielsen, S., and A r t u n , T., Chemical s t u d i e s on the b l a c k shark. K g l . Norske Videnskab. S e l s k a b s , S k r i f t e r . , 2:1+5 (191+0).  i+.  K i n d , C.A., and Bergmann, W., Marine P r o d u c t s , X I . The Occurrence o f o c t a d e c y l a l c o h o l , b a t y l a l c o h o l and c e t y l palraitate i n g o r g o n i a s . J . Org. Chem.  o  2:l+2l+-7  5.  (191+2).  Bergmann, W., McLean, M . J . , and L e s t e r , D., C o n t r i b u t i o n s t o the s t u d i e s o f marine p r o d u c t s . XIV. A s t r o l . J . Org. Chem. 8:283-1+  (191+3).  6.  Ruiz, A.S., C o n s t i t u e n t s o f the u n s a p o n i f i a b l e f r a c t i o n of some l i p i d e s of vegetable and animal o r i g i n . Anales r e a l acad. farm., 3:201-31 (191+3), through Chem. Abs., 3 8 : 5 0 3 ° (191+1+7.  7.  Matsumoto, T., Yajima, M., and Yoyaraa, T., S t e r o l s and other u n s a p o n i f i a b l e substances i n the f a t s o f s h e l l f i s h e s , Crustacea, and JSchinodermata. V. The c r y s t a l l i n e f r a c t i o n of t h e u n s a p o n i f i a b l e substances i n the f a t o f A s t e r i a s r o l l e s t o h i and Cucumaria chronh.jelmi. J . Chem. Soc. Japan 6k:1203-i+ (191+3) through Chem. Abs., ]jJ..3307h T191+7).  8.  C a r r i e r e , M., and Bonnemaison, J.M., The u n s a p o n i f i a b l e f r a c t i o n i n the o i l o f C e n t r i n a c e n t r i n a . Bull, soc. chim., 1 2 , (191+5).  936-9  9.  10.  Cmelik, S., The a d r i a t i c Elasmobranchia l i v e r o i l s . IV. B i o c h e m i c a l study o f o i l s from the l i v e r and eggs of C e n t r i n a s a l v i a n i . A c t a A d r i a t . , 1+:3-23 (191+9), through Chem. Abs. i j l j . : 5 6 l 9 d ( 1 9 5 0 . Toyama, Y., and Tanaka, T., S t e r o l s and other u n s a p o n i f i a b l e substances i n the l i p i d e s o f s h e l l f i s h e s , crustaceans, and echinoderms. X I I . Occurrence of 7-cholestenol as a major component o f the s t e r o l mixture of c h i t o n . B u l l . Chem. Soc. Japan, 26:1+97-9 ( 1 9 5 3 ) , through Chem. Abst., l £ : 9 1 7 1 g TT955).  39  11.  Matsumoto, T., and Wainai, T., The u n s a p o n i f i a b l e matter o f the echinoderms. I A 7 - c h o l e s t e n o l i n the f a t of starfish. J . Chem. Soc. Japan, Pure Chem. S e c t . , 75:756-8, (19514-), through Chem. Abs. k8:13109e  (1955-).  12.  Monforte, P., and Penech, G., Components of the l i v e r o i l o f Hexanchus g r i s e u 3 . Ann. chim. (Rome), j_J_:80i|.-lij. (I95I4.), through Chem. Abs., j _ 9 : l l 4 2 7 5 f (1955).  13.  Toyama, Y.,  lij..  Karnovsky, M.L., J e f f r e y , S.S., Thompson, M.S., and Deane, H.W., A chemical and h i s t o c h e m i c a l study of the l i p i d e s o f the p y l o r i c cecum of the s t a r f i s h , Asterias forbesi. J . Biophys. Biochem. C y t o l . , 1:173-82 ( 1 9 5 5 ) .  15.  Toyama, Y.,  Tanaka, T., and Maeda, T., P a t t y o i l s o f " a q u a t i c i n v e r t e b r a t e s . X. P a t t y o i l s of Stichopus iaponicus. Mem. Pac. Eng., Nagoya Univ. 7 : l 5 l - 5 ( 1 9 5 5 ) , through Chem. Abs., __l:i|.737e (1957).  16.  Bergraann, W.,  C r e i g h t o n , S.M., and Stokes, W.M., Marine products. XL. Waxes and t r i g l y c e r i d e s o f sea anemones. J . Org. Chem., j _ l : 721-8 (1956).  17.  Toyama, Y.,  and Tanaka, T., P a t t y o i l s of a q u a t i c Invertebrates. X I I I . F a t t y o i l s o f Tachypleus t r i d e n t a t u s f a t . Mem. Pac. Eng., Nagoya Univ., 8 : 3 5 - 9 ( 1 9 5 6 ) , through Chem. Abs., __L:l5972h T l 9 5 7 ) .  18.  Mitsunaga, S., Shimamura, U., T s u j i , J . , and Okada, T., L i v e r o i l o f Squalus s u c k l e y i . I. Decomposition products formed by steam d i s t i l l a t i o n . Abura Kagaku, __:355-8 ( 1 9 5 6 ) , through Chem. Abs., £ l : l 5 9 7 3 b (1957).  19.  Tanaka, T.,  20.  K i t a , M.,  and Takagi, T., P a t t y o i l s of a q u a t i c i n v e r t e b r a t e s . V I I I P a t t y o i l s from Cucumaria chroh.jelmi, C o s c i n a s t e r i a s a c u t e s p i n a , and Comanthus .japonica with s p e c i a l r e f e r e n c e s to s t e r o l components. Nippon Kagaku Z a s s h i , 7_:2i|3-6 (1951*), through Chem. Abs., __L:l8656g U 9 5 7 ) .  and Toyama, Y., P a t t y o i l s o f a q u a t i c invertebrates.. XIX. Nonconjugated s t e r o l s and other u n s a p o n i f i a b l e components i n the f a t t y o i l from Tonna luteostoma. Mem. Pac. Eng., Nagoya Univ., 1 0 : 7 7 - 8 3 , ( 1 9 5 8 ) , through Chem. Abs., ___5:l85l5c T l 9 5 9 ) .  and Toyama, Y., S t e r o l s of t w e n t y - f i v e species o f marine i n v e r t e b r a t e s i n Japanese waters. Nippon Kagaku Z a s s h i , 8 l , I].85-9 ( I 9 6 0 ) , through Chem. Abs., __5:6711i T l 9 6 l ) .  1+6  21.  Bodman, J . , and M a i s i n , J.H.,The a l p h a - g l y c e r y l e t h e r s . C l i n . Chim. A c t a . , J:253-7l+ (1958).  22.  Karnovsky, M.L., Rapson, W.S., and Black, M., South A f r i c a n f i s h p r o d u c t s . XXIV. The occurrence b f a l p h a g l y c e r y l ethers i n the u n s a p o n i f i a b l e f r a c t i o n s of n a t u r a l f a t s . J . Soc. Chem. Ind. 65:1+25-8 U9l|i>).  23.  C a r t e r , H.E., Smith, D.B., and Jones, D.N., A new ethanolamine-containing l i p i d e from egg y o l k . J . B i o l . Chem., 232:681-91+ (1958).  2l+.  H a l l g r e n , B., and L a r s s o n , S.D., The g l y c e r y l ethers i n man and cow. J . L i p i d Research, ,3:39-1+3 (1962).  25.  P r e l o g , V., Ruzicka, L., and S t e r n , P., U n s a p o n i f i a b l e l i p i d e s o f hog s p l e e n . H e l v . Chim. A c t a . , 26:2205-21 (191+3).  26.  B r o h u l t , A., and Holmberg, J . , A l k o x y g l y c e r o l s i n the t r e a t ment o f l e u c o p e h i a caused by i r r a d i a t i o n . Nature, 17l+:1102 (1954).  27.  Hardegger, E., Ruzicka, L., and Tagmann, E . , Organ e x t r a c t s . I I I . U n s a p o n i f i a b l e l i p i d e s from a r t e r i o s c l e r o t i c a o r t a s . Helv. Chim. A c t a . , 26:2205-21 (191+3).  28.  M a r i n e t t i , G.V., E r b l a n d , J . , and S t o t z , E., S t r u c t u r e o f p i g - h e a r t plasmalogens. J . Am. Chem. S o c , 80:l62l+-8 (1958).  29.  Debuch, H., The p o s i t i o n o f the aldehyde i n colamine plasmalogen from b r a i n . Z. P h y s i o l . Chem., 3ll+:l+9-50 (1959).  30.  Klenk, E . , and Hendricks, U.W., An i n o s i t o l phosphatidec o n t a i n i n g carbohydrate i s o l a t e d from human b r a i n . Biochem. et Biophys. A c t a . , ^0:602-3 (I96I).  31.  Nakagawa, S., and McKibbin, J.M., D i s t r i b u t i o n o f a l p h a g l y c e r y l ethers i n animal t i s s u e s . Proc. Soc. E x p t l . B i o l . Med., lll:63l+-6 (1962).  32.  Emmerie, A., The paper chromatography of a l p h a - g l y c e r y l e t h e r s . Rec. t r a v . chim., J2:823-6 (1953).  33.  Blomstrand, R., and G u r t l e r , J . , S e p a r a t i o n of g l y c e r y l ethers by g a s - l i q u i d chromatography. A c t a Chem. Scand., 1^:11+66-7 ( 1 9 5 9 ) .  1+1 3k»  H a l l g r e n , B., and L a r s s o n , S.O., S e p a r a t i o n and i d e n t i f i c a t i o n of a l k o x y g l y c e r o l s . A c t a . Chem. Scand., 13, 211+7-8 (1959).  35.  H a l l g r e n , B., and L a r s s o n , S.O., The g l y c e r y l ethers i n the' l i v e r o i l s of elasmobranch f i s h . J . L i p i d Res., 2L> 31-8 (1962).  36.  M a l i n s , D . C , P a t t y a c i d s and g l y c e r o l ethers i n alkoxydiglycerides of dogfish l i v e r o i l . and Ind. (London) I960, 1359-60.  37.  Chem.  Adam, N.K., Evidence from s u r f a c e f i l m s on the c o n s t i t u t i o n of b a t y l and chimyl a l c o h o l s . J . Chem. Soc.  1933, 161+-5.  38.  Davies, W.H., H e i l b r o n , I.M., and Jones, W.E., U n s a p o n i f i a b l e matter from the o i l s o f elasmobranch f i s h . IX. The s t r u c t u r e of b a t y l and s e l a c h y l a l c o h o l s . J . Chem. S o c , 1933, 165-7.  39.  Baer, E., and F i s c h e r , H.O.L., Acetone g l y c e r a l d e h y d e and o p t i c a l l y a c t i v e g l y c e r i d e s . IX. C o n f i g u r a t i o n of the n a t u r a l b a t y l , chimyl, and s e l a c h y l alcohols. J . B i o l . Chem., 34o:397-l|.10 (191+1).  1+0.  Kornblum, N., and Holmes, H.N., B a t y l a l c o h o l .  J . Am. Chem.  S o c , .61+:30lf.5-6 (19l[2). 1+1.  Gupta, S . C , and Kuramerow, F.A., Improved procedure f o r p r e p a r i n g g l y c e r o l e t h e r s . J . Org. Chem., 2JJ.: 1+09-11 (1959).  1+2.  Kuwamura, T., P r e p a r a t i o n o f h i g h e r a l k y l and a l k y l p h e n y l l y c i d y l e t h e r s . Kogyo Kagaku Z a s s h i , 6^:59500 (I960), through Chem. Abs., £6:l+706f (1962).  1+3.  B a y l i s , R.L., Bevan, T.H., and M a l k i n , T., S y n t h e s i s o f c e p h a l i n (phosphatidylethanolamine) and b a t y l , c h i m y l , g l y c o l , and a l k y l analogs. J . Chem. S o c , 1958, 2962-8.  ?  1+1+. Bevan, T.H., and M a l k i n , T., S y n t h e s i s o f a b e t a - b a t y l ( g l y c e r o l 2 - o c t a d e c y l ether) analog of c e p h a l i n , and m e l t i n g p o i n t data f o r b a t y l , chimyl, b e t a b a t y l , and beta-ehimyl a l c o h o l s and t h e i r derivatives. J . Chem. S o c , I960, 350-3. 1+5.  Bigelow, W.C., P i c k e t t , D.L., and Zisman, W.A., Oleophoblc monolayers. I . F i l m s absorbed from s o l u t i o n I n nonpolar l i q u i d s . J . C o l l o i d . S c i . , 1:513-38 (191+6).  1(2 Nakajima, I . , and Toyama, Y., I n t e r f a c i a l a c t i v i t y o f a l p h a monostearin and b a t y l a l c o h o l . Nagoya Sangyo 'Kagaku Kenkyujo Kenkyu Hokoku,*9:l|6-50 ( 1 9 5 6 ) , through Chem. Abs., __I:8509f ( 1 9 5 7 ) . S a n d l e r , O.E., Some experimental s t u d i e s on the e r y t h r o p o i e t i c e f f e c t o f y e l l o w bone marrow e x t r a c t s and b a t y l a l c o h o l . A c t a . Med. Scand. Suppl. 2 2 5 , 1-72 (I9I4.9). Bergstrom, S., and Blomstrand, R., I n t e s t i n a l a b s o r p t i o n and metabolism o f chimyl a l c o h o l i n the r a t . P h y s i o l . Scand., __3:l66-72 ( 1 9 5 6 ) . Blomstrand,  R., D i g e s t i o n , a b s o r p t i o n , and metabolism of chimyl a l c o h o l f e d as f r e e a l c o h o l or as a l k o x y d i g l y c e r i d e . P r o c . Soc. E x p t l . B i o l . Med., J_02:662-5 (1959).  Blomstrand,  R., and Ahrens, E.H., A b s o r p t i o n o f chimyl a l c o h o l i n man. Proc. Soc. E x p t l . B i o l . Med., 100:802-5 (1959).  Acta.  Berger, P.M., and B r a d l e y , W., The pharmacological p r o p e r t i e s of a l p h a : beta dihydroxy-gamma-(2-methylphenoxy)propane. (Myanesin) B r . J . Pharmacol., 1 : 2 6 5 272 (I9I4.6). ~" Berger, P.M., R e l a t i o n s h i p between chemical s t r u c t u r e and c e n t r a l depressant a c t i o n o f a l p h a - s u b s t i t u t e d ethers o f g l y c e r o l . J . Pharmacol. E x p t l . Therap., __3:1170-81 (I9I4B). de G a e t a n i , G.P., and B a i o t t i , G., Hemopoietic a c t i v i t y o f batyl alcohol. B o l l . soc. i t a l . b i o l . sper., ___:1156 ( 1 9 5 9 ) , through Chem. Abs., j _ _ : l 6 8 l 5 a (196lX Linman, J.W., B e t h e l l , P.H., and Long, M.J., The erythropoietic stimulatory a c t i v i t y of b a t y l alcohol. J . Lab. C l i n . Med., __2:596-60i|. ( 1 9 5 8 ) . Linman, J.W., Long, M.J., K o r s t , D.B. and B e t h e l l , P.H., S t i m u l a t i o n o f hemopoiesis by b a t y l a l c o h o l . J . Lab. C l i n . Med., ___:335-J4-3 (1959). Linman, J.W., K o r s t , D.R., and B e t h e l l , P.H., S t i m u l a t i o n of e r y t h r o p o i e s i s by humoral f a c t o r s . Ann. N.Y. Acad. S c i . , 22»3:638-lj.9 ( 1 9 5 9 ) . Linman, J.W., Hemopoietic e f f e c t s of g l y c e r y l e t h e r s . I I I . I n a c t i v i t y of s e l a c h y a l c o h o l . P r o c . Soc. E x p t l . B i o l . Med., 10l|.:703-6 (I960).  1+3 58.  Edlund, T., P r o t e c t i v e e f f e c t o f d l - a l p h a - o c t a d e c y l g l y c e r o l ether i n mice g i v e n t o t a l body x - i r r a d i a t i o n . Nature, r7j}.:1102 (1951+) •  59.  Evans, W.C., Evans, I.A., Edwards, CM., and Thomas, A. J . , Bracken p o i s o n i n g o f c a t t l e : therapeutic t r e a t ment. Biochem. J . , 6 £ : 6 p (1957).  60.  S c h u l t z e , M.O., Perman, V., Bates, P.W., and S a u t t e r , J.H., F a i l u r e o f DL-batyl a l c o h o l t o prevent a p l a s t i c anemia i n c a l v e s . Proc. Soc. E x p t l . B i o l . Med.,  28:1+70-1+ (1958).  61.  Matsui, E., S t u d i e s on c a r c i n o s t a t i c substances. XXV. E f f e c t o f d l - o c t a d e c y l a l p h a - g l y c e r y l ether on experimental l e u c o p e n i a o f r a t induced by n i t r o g e n mustard N-oxide. Chem. and Pharm.'Bull. (Tokyo), 8:168-9 (I960), through Chem. Abs., ££:7280a (1961).  62.  Hasegawa, Y., Wakui, N., Shimada, A., Nakamura, S., and K u n i i , K., B a t y l A l c o h o l . Nippon Rinsho, 19:1793800 (1961), through Chem. Abs., £6:1968g (1962).  63.  Ghys, R., E f f e c t s o f a n a l k o x y g y c e r o l (Kaby 700) on l e u c o p e n i a r e s u l t i n g from r a d i o - t h e r a p y . med., JO:331-1+2 (I960).  61+.  B r o h u l t , A., A l k o x y g l y c e r o l e s t e r s i n i r r a d i a t i o n treatment. I n t e r n a t i o n a l Conference on R a d i o b i o l o g y , Proceedings o f the F i f t h Conference, Stockholm,  Laval  1957.  65.  R i l e y , R.F., Hokama, Y., a n d K r a t z , P., N o n s t e r o i d a l components of the u n s a p o n i f i a b l e f r a c t i o n o f human l i v e r . Cancer Research, 18:825-32 (1958).  66.  R i l e y , R.P., Hokama, Y., Dowdy, A.H., choladienate f o r cancer.  67.  Stowe, B.B., Growth promotion i n pea stem segments. I . S t i m u l a t i o n o f a u x i n and g i b b e r e l l i n a c t i o n by a l k y l l i p i d e s . P l a n t P h y s i o l . , ^ 2 6 2 - 9 (I960).  68.  B r o h u l t , A., A l k o x y g l y c e r o l s as growth-stimulating substances.  C o l v e r , V., Coleman, M.K., and Seroflocculant a c t i v i t y of ethyl and v a r i o u s a l c o h o l s I n the Perm t e s t Cancer Research, JL8:833-1+1 (1958).  Nature, 188:591-2 (I960).  69.  D'Arcy, P.P., Howard, E.M., Muggleton, P.W., and Townsend, S.B., The anti-inflammatory a c t i o n o f g r i s e o f u l v l n i n experimental animals. J . Pharm. and Pharmacol., .12:659-65 (I960).  70.  D'Arcy, P.P., A method f o r screening compounds f o r a d r e n o c o r t i c a l a c t i v i t y u s i n g the r e s i s t a n c e o f mice to t h e e f f e c t o f c o l d . J . E n d o c r i n . , l l j . : 9 - l 6 (1957).  kh 71.  Labor-it, H.,  Moynler, Rv, T r z e h s k i , A., G u i o t , G., and Baron, C., I n f l u e n c e de l a composition i o n i a u e du m i l i e u e x t r a - c e l l u l a l r e et i n f l u e n c e coraparee de l ' a e i d e a s p a r t i q u e , de l ' a s p a r t a t e de potassium et du glucose sur l'e*preuve de nage du Rat b l a n c . S o c i e t e de B i o l o g i e , Comptes Rendus, 151:1383-6  (1957) . 72.  Selye, H.,  Thymus and adrenals i n the response of the organism t o i n j u r i e s and i n t o x i c a t i o n s , B r i t . J . Exper. Path., j_7_:23M-8 (1936).  73*  Meier, R.,  S c h u l e r , ¥., and D e s a u l l e s , P., Zur Prage des Mechanismus der Hemmung des Bindegewebswachstums durch C o r t i s o n e . E x p e r i e n t i a 6:I).69-70 (1950).  7I4..  S e l y e , H.,  The Diseases of A d a p t a t i o n : I n t r o d u c t o r y remarks. Recent Progress i n Hormone Research,  8:117  (1953).  75*  Robert, A.,  and Nezamis, J.E., The granuloma pouch as a r o u t i n e assay f o r a n t i - p h l o g i s t i c compounds. A c t a E n d o c r i n . , ___:105-112 (1957).  76.  Wilhelm, D.L., The m e d i a t i o n of i n c r e a s e d v a s c u l a r p e r m e a b i l i t y i n inflammation. P h a r m a c o l o g i c a l Reviews, ll_,2:25l-280 (1962).  77.  Cohen, Y., and,Got, P., E f f e c t o f ambient temperature on the inflammation o f r a t paws and on the a n t i inflammatory e f f e c t s of phenylbutazone. Compt. Rend., 2_5__:2l|.21-3 (1961).  78.  H a l l i d a y , J.E., unpublished r e s u l t s .  79.  Saxena, P.N., E f f e c t of drugs on e a r l y inflammatory r e a c t i o n . A r c h . i n t . pharmacodyn., C X X V T , 2 : 2 2 8 -  237 (I960).  80.  P r i e d , J . , and Borman, A., S y n t h e t i c d e r i v a t i v e s o f c o r t i c a l hormones. Vitamins and Hormones. XVI, 303-37I4. (1958) , Academic Press Inc., Pub., New York.  81.  Colowick, S.P., and Kaplan, N.O., D e t e r m i n a t i o n of glycogen by m o d i f i e d P f l u g e r method. Methods i n Enzymology I I I , 3i|.-36 (1957), Academic Press Inc., Pub.  82.  C a r r o l l , N.V., Longley, R.W., and Roe, J.H., The d e t e r m i n a t i o n of glycogen i n l i v e r and muscle by use of anthrone reagent. J . B i o l . Chem. 220:583-93 (1956)  83.  Kusen, S.I., and Pupin, I.G., Rhythmic f l u c t u a t i o n s o f the glycogen content i n the l i v e r of c a t t l e . Dopovidi Acad. Nauk. R.S.R., 1958,977-9. through Chem.  <#  Abs., ___:7356b  (1959).  8)4..  S o k a l , J.E., and S a r c i o n e , E . J . , E f f e c t o f epinephrine on glycogen s t o r e s . Am. J . o f P h y s i o l . 1 9 6 : 1 2 5 3 - 7 :  (1959).  85.  S i n g e r , P.M., Borman, A., and L e r n e r , L . J . , Automation of the l i v e r glycogen d e t e r m i n a t i o n . Ann. N.Y. Ac. of S c i . , 102,1:118-126 (1962).  

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