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UBC Theses and Dissertations

Some nutritional studies on the naturally occurring alpha-glyceryl ethers Carlson, Walter Eric 1966

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SOME NUTRITIONAL STUDIES ON THE NATURALLY  OCCURRING  ALPHA-GLYCERYL ETHERS  by WALTER ERIC CARLSON B.S.A., U n i v e r s i t y o f B r i t i s h Columbia, 1964  A T h e s i s Submitted i n P a r t i a l of the Requirements  Fulfilment  f o r the Degree of  MASTER OF SCIENCE IN AGRICULTURE i n the D i v i s i o n o f Animal S c i e n c e  We a c c e p t t h i s t h e s i s as conforming t o the standard required  from c a n d i d a t e s f o r the  Degree o f Master o f S c i e n c e i n A g r i c u l t u r e  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1966  In the  requirements  British  for  Columbia,  available mission  for  for  purposes his  presenting  may  I  an  reference  extensive be  without  of  this  my w r i t t e n  that  and  by  It for  the  Is  the  of  Head  / l a j f  /r,  I  of  understood  financial  Department  pete  Columbia  /fa  partial at  the  Library  this  permission.  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  in  degree  study.  copying  granted  thesis  thesis  advanced  agree  representatives.  cation  this  thesis  University  of  make  agree for  freely per-  scholarly  copying  shall  it  that  Department  that  gain  of  shall  further  my  fulfilment  not  or or  be  by publi-  allowed  ii  ABSTRACT  A p o s s i b l e n u t r i t i o n a l r o l e f o r c e r t a i n of the n a t u r a l l y o c c u r r i n g a - g l y c e r y l e t h e r s has been i n v e s t i g a t e d .  Batyl,  selachyl  and c h i m y l a l c o h o l s have been a d m i n i s t e r e d per orum f o r extended p e r i o d s to growing if  dogs and r a t s a t v a r i o u s dosage l e v e l s t o a s c e r t a i n  these compounds produce  i n i m i c a l or f a v o u r a b l e e f f e c t s i n terms of  growth and h a e m a t o p o i e t i c r e s p o n s e s . Groups of male and female dogs and r a t s m a i n t a i n e d on  normal  r a t i o n s were g i v e n d a i l y doses of each of the t h r e e a l c o h o l s a t a l e v e l of 6 mgs  per k i l o g r a m of body weight  f o r 180 days.  There was  e v i d e n c e to suggest t h a t these compounds produced h a r m f u l  effects.  H i s t o p a t h o l o g i c a l study of the major t i s s u e system confirmed conclusion.  noted i n the female r a t s t h a t r e c e i v e d the  No e v i d e n c e o f a h a e m a t o p o i e t i c e f f e c t was In a second experiment,  addendum t o a normal  selachyl  obtained.  s e l a c h y l a l c o h o l was  o f f e r e d as an  r a t i o n , to both dogs and r a t s a t l e v e l s  ensured a d a i l y consumption weight  this  A f a v o u r a b l e response i n the form of a s l i g h t l y i n c r e a s e d  growth r a t e was alcohol.  no  o f 600 and 2400 mg  that  per k i l o g r a m o f body  of t h i s a l c o h o l f o r a p e r i o d of s i x t y days.  Other groups  b o t h s p e c i e s r e c e i v e d b a t y l a l c o h o l a t the h i g h e r dosage l e v e l per k i l o g r a m of body weight)  d a i l y f o r the same time p e r i o d .  o b t a i n e d suggest t h a t both compounds when f e d a t the 2400 mg  of  (2400 mg The  results  per k i l o g r a m  l e v e l i n t e r f e r e d w i t h the d i g e s t i b i l i t y of the r a t i o n o f f e r e d and, i n  iii  so d o i n g , reduced  the growth r a t e of the e x p e r i m e n t a l dogs.  c o r r e s p o n d i n g e f f e c t d i d not o c c u r i n the r a t s .  A  Both a l c o h o l s , when  a d m i n i s t e r e d a t the h i g h e s t dosage l e v e l , induced an i n c r e a s e i n the r e t i c u l o c y t e count i n the b l o o d of the dogs and an i n c r e a s e i n the percentage prepared  of n u c l e a t e d r e d b l o o d c e l l s i n the bone marrow smears  from the  rats.  A g l y c e r y l e t h e r - f r e e s y n t h e t i c r a t i o n supplemented w i t h 0.5,  5 and 50 mg  prepared and  of b a t y l a l c o h o l per k i l o g r a m of body weight  was  then o f f e r e d to groups of young growing r a t s f o r a p e r i o d  of f i v e weeks.  No growth response was  o b t a i n e d a t any  level,  suggesting  t h a t t h i s compound does not have a n u t r i t i o n a l f u n c t i o n or t h a t the r a t i s a b l e t o s y n t h e s i z e the compound a t a r a t e t h a t i s adequate to permit near maximum growth.  I t i s a l s o p o s s i b l e t h a t the animals  had  s u f f i c i e n t r e s e r v e s of compounds of t h i s type t o permit growth a t the measured  rate.  iv  TABLE OF CONTENTS i  Page ABSTRACT TABLE OF CONTENTS  . .  i i  '. . . . . . . . . . . . .  iv  LIST OF TABLES . . . . . . . . .  . . . . . . . .  viii  LIST OF FIGURES  ix  ACKNOWLEDGEMENTS  .  INTRODUCTION  . . . . . .  xi  . . . . .  1  PART I - LITERATURE REVIEW A.  The c h e m i c a l nature o f the a - g l y c e r y l  B.  Occurrence of the g l y c e r y l e t h e r s  C.  The c o m p o s i t i o n o f t h e g l y c e r y l e t h e r s  D.  The mesenchymal  E.  B i o s y n t h e s i s of the g l y c e r y l e t h e r s  F.  D i g e s t i o n and metabolism of a - g l y c e r y l  G.  a-glyceryl  H.  Summary  ethers,  . . . . . . .  o r i g i n of the a - g l y c e r y l  e t h e r s and haemopoiesis  .  2 4  . . . .  13  ethers  16 16  ethers  .  . . . . . .  18 20  .  26  PART I I - EXPERIMENTAL A.  PRELIMINARY EVALUATION - TRIAL I . . . . . .  .  33  . . . . . . . . . .  33  . . . . . .  35  3. Animals  . . . . . . . . . . . . . . . . . .  36  4. R a t i o n s  . . . . . . . . . . . . .  36  5. Housing  . . . . . . .  37  1. Preamble 2. E x p e r i m e n t a l d e s i g n  V  TABLE OF CONTENTS  (cont'd) Page  6. A d m i n i s t r a t i o n of g l y c e r y l e t h e r s 7. R e s u l t s  . . . . . . . . . . . . . . . . .  8. C o n c l u s i o n  B.  . . . . .  2. Design  . . .  37  . .  37  .  42  „ . . . „ . . . . . .  PRELIMINARY EVALUATION - TRIAL I I 1. Preamble  . . . . . .  . . . . . . .  43  . . . . . . . . . .  43  . .  43  3. Animals . . . . . . . . . . . . . . . . . . .  45  4. Housing  45  . . . . . . . . . . . . . .  5. R a t i o n s and a d m i n i s t r a t i o n of g l y c e r y l e t h e r s  45  6. D i g e s t i b i l i t y procedures  47  . . .  (a) D i g e s t i b i l i t y of the e x p e r i m e n t a l r a t i o n s  47  (i)  Rats  47  (ii)  Dogs  48  (b) I s o l a t i o n and d e t e r m i n a t i o n of the g l y c e r y l ethers (i)  48  E x t r a c t i o n of l i p i d s from feed and f a e c e s  (ii)  of n e u t r a l l i p i d s  48  . .  49  ( i i i ) D e t e r m i n a t i o n of the u n s a p o n i f i a b l e lipid fraction  49  (iv)  Determination  . . . . . . . . .  Q u a l i t a t i v e and q u a n t i t a t i v e d e t e r m i n a t i o n of g l y c e r y l e t h e r s  7. D i s c u s s i o n of f e e d - i n t a k e , and weight g a i n . . . .  digestibility . . . . . . . . .  8. G l y c e r y l e t h e r s and d i g e s t i b i l i t y 9. R e s u l t s from c e l l  counts  50  .  51  . . . . . .  60  . . . . . . . . . .  65  vi  TABLE OF CONTENTS (cont'd) Page C.  FEEDING RESPONSE EXPERIMENT 1. Preamble  - TRIAL I I I . . .  .  68  . . . . . . . . . . . . . . . . . .  68  2. D e s i g n . . . . . . . . . . . . . . . . . . .  68  3. Animals  . . . . . . . . . . . .  4. Housing  . . . .  . . . . .  .  69  . . . . . . . .  69  5. R a t i o n . . . . . . . . . . . . . . . . . . .  69  6. Feed i n t a k e - g a i n r e l a t i o n s h i p s  . . . . . . .  70  . . . . . . . . . . . . . . .  71  7. D i g e s t i b i l i t y  8. R e s u l t s o f b l o o d and bone marrow c e l l counts  72  9. C o n c l u s i o n s  72  . . . . . . . . . .  PART I I I - GENERAL DISCUSSION  BIBLIOGRAPHY  „ .  . . . . . . . . . . . . . . .  75  . . . . . . . . . . . . . . . .  79  APPENDICES I II III IV  Composition of r a t i o n s used i n T r i a l s  I and I I  Mean d a i l y feed i n t a k e f o r T r i a l I r a t s Mean weekly weights i n T r i a l I  89  . . . . . . .  90  . . . . . . . . . . .  92  I . . . . . . . . . . . . . . .  .  94  V  Weight d a t a f o r animals i n T r i a l I I . . . . . . . .  .  99  VI  R e s u l t s o f r a t b l o o d taken one month from the b e g i n n i n g of T r i a l I I . . . . . . . . . . . . . .  .  102  R e s u l t s of r a t b l o o d taken two months from the b e g i n n i n g of T r i a l I I . . . . . . . . . . . . . .  .  105  Blood count d a t a from dogs one month from the beginning of T r i a l I I . . . . . . . . . . . . . .  .  108  VII  VIII  Blood counts i n T r i a l  . . .  vii  TABLE OF CONTENTS (cont'd) Page IX  X  XI  Blood count d a t a from dogs two months from the b e g i n n i n g of T r i a l I I . . . . . . . . . . . . . .  .  I l l  D i f f e r e n t i a l counts of f e m o r a l bone marrow of r a t s i n T r i a l I I . . . . . . . . . . . . . . .  .  113  .  117  Differential  counts of f e m o r a l bone marrow of  dogs i n T r i a l I I XII  . . . . . .  . . . . . . . . . . . . . . . .  121  XIII  Blood counts of r a t s i n T r i a l I I I . . . . . . . . . .  122  XIV  P e r c e n t d i f f e r e n t i a l of l e u c o c y t e s i n b l o o d smears from T r i a l I I I r a t s . . . . . . . . . . . .  123  XV  Percentages of c e l l s i n the femoral bone marrow of T r i a l I I I r a t s . . . . . . . . . . . . . . . . .  125  Standard methods used f o r c o u n t i n g and differentiating cells . . . . . . . . . . . . . . .  127  XVI  Mean weights i n grams f o r T r i a l I I I r a t s  viii:'.  LIST OF TABLES TABLE I II III  IV  V  Page M e l t i n g p o i n t and  . .  2  . .  6  G l y c e r y l e t h e r s 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 of n e u t r a l l i p i d s from s e v e r a l mammalian t i s s u e s . . .  9  Percentage of g l y c e r y l e t h e r s i n l i p i d s from some human and animal sources . . . . . . . . . . . . . .  9  Occurrence of g l y c e r y l e t h e r s  Composition fish  VI VII  s t r u c t u r e of the g l y c e r y l e t h e r s  of the n o n s a p o n i f i a b l e matter i n  liver oils  Liver oils  . . . .  . . . .  IX X XI XII  .  from Elasmobranch f i s h by  .  a - G l y c e r y l e t h e r content  . . . . . . . .  of dog  tissues  12  . .  12  of the a l k o x y g l y c e r o l s . . . . . . . . . of g l y c e r y l e t h e r s from s p l e e n , and human m i l k . .  13  Recovery and d i s t r i b u t i o n of r a d i o a c t i v i t y i n lymph l i p i d s a f t e r feeding C ^ - l a b e l l e d chimyl d i o l e a t e 1  XIII  XIV  XVI  B i o l o g i c a l experiments i n v o l v i n g the use  Representative  v a l u e s of important  XVIII  Schedule of b l o o d  19  of  vitamins  Amount of g l y c e r y l e t h e r a d m i n i s t e r e d  14  23  . . . . . . . . . . . . . . . . . .  the v i t a m i n requirements XVII  .  A l k o x y g l y c e r o l e s t e r s f e d to r a t s r e c e i v i n g t o t a l body X - i r r a d i a t i o n . . . . . . . . ...  g l y c e r y l ethers XV  11  . .  a - G l y c e r y l e t h e r s of mammalian y e l l o w bone marrow The percentage c o m p o s i t i o n from l i v e r o i l s . . . . The percentage c o m p o s i t i o n human bone marrow, human  10 10  A summary of the e x t e n s i v e t a b l e g i v e n Karnovsky and Rapson  VIII  . . . . . . . . .  i n milk  27 .  34  compared w i t h  of the a l b i n o l a b o r a t o r y r a t  34  counts . . . . . . . . . . . . . . .  36  Summary of mean body weight g a i n and mean f e e d - i n t a k e data f o r male r a t s . . . . . . . . . . .  38  ix  LIST OF TABLES  (cont'd)  TABLE XIX  Page Summary o f mean body weight g a i n and mean f e e d - i n t a k e data f o r female r a t s  XX XXI XXII XXIII  . . . . . . . . . .  Summary o f body weight g a i n f o r dogs  38  . . . . . . . . .  Summary of b l o o d counts taken i n T r i a l I . . . . . .  .  Grouping and dosage l e v e l s used i n T r i a l I I  XXV  Summary o f weight g a i n and f e e d - i n t a k e o f dogs  XXVII  XXVIII  XXIX XXX XXXI XXXII XXXIII  52  Summary o f f e e d e f f i c i e n c i e s  .  The r e l a t i o n s h i p between d i g e s t i b i l i t y efficiencies  XXVI  41 45  in Trial II XXIV  38  f o r the r a t s i n T r i a l I I  Recovery of g l y c e r y l e t h e r s from determination of d i g e s t i b i l i t y f o r the dogs i n T r i a l I I Recovery o f g l y c e r y l e t h e r s from determination of d i g e s t i b i l i t y f o r the r a t s i n T r i a l I I Significant differences  f a e c e s and of the e t h e r s f a e c e s and of the e t h e r s . . . . . . . . .  i n c e l l counts  Feed i n t a k e - g a i n r e l a t i o n s h i p s Digestibility  and f e e d  f o r a model dog . . . . . .  Feed i n t a k e - g a i n r e l a t i o n s h i p s  of experimental  57 60  62  63 65  f o r rats i n T r i a l I I I . rations  56  in Trial III . .  Summary o f the b l o o d and bone marrow data from T r i a l I I I . . . . . . . . . . . . . . . . . . . . E r y t h r o c y t e counts o f f o u r c o n t r o l r a t s fed b a t y l a l c o h o l a t the end o f the experimental period . . . . . . . . .  71 71  73  73  X  LIST OF FIGURES TABLE I II  III  Page Growth curves f o r female r a t s i n T r i a l I . . . . . .  .  39  The mean growth r a t e s of the two dogs from each group m a i n t a i n e d on the c o n t r o l r a t i o n f o l l o w i n g removal from t h e i r e x p e r i m e n t a l d i e t s . „ . . . „ .  58  D i g e s t i b i l i t y of g l y c e r y l e t h e r s  64  . . . . . . . . . . .  xi  ACKNOWLEDGEMENTS  I wish t o thank Dr. B.A. E a g l e s , Dean o f : t h e F a c u l t y o f A g r i c u l t u r e and Chairman o f t h e D i v i s i o n o f Animal S c i e n c e , f o r h i s p e r m i s s i o n t o undertake t h i s p r o j e c t and f o r the use o f departmental facilities.  My thanks a r e extended t o those a t the U n i v e r s i t y of  V i c t o r i a who p e r m i t t e d the c o m p l e t i o n o f t h i s study a t t h a t institution. To Dr. A . J . Wood, Dean o f A r t s and S c i e n c e a t the U n i v e r s i t y of  Victoria  ( f o r m e r l y P r o f e s s o r o f Animal S c i e n c e a t the U n i v e r s i t y o f  B r i t i s h Columbia)  I wish t o express my s i n c e r e a p p r e c i a t i o n f o r h i s  u n f a i l i n g i n t e r e s t , d i r e c t i o n and encouragement. I wish t o thank Dr. W. Chalmers and the s t a f f a t Western Chemical I n d u s t r i e s L i m i t e d , Vancouver, B . C . , f o r t h e i r guidance and r  for  t h e use o f t h e i r l a b o r a t o r y  facilities.  The t e c h n i c a l a s s i s t a n c e o f Miss Karen F o r g e r o n and Miss Heather McConnell i s g r a t e f u l l y I acknowledge Foundation.  acknowledged.  the generous f i n a n c i a l support o f the L a b a t t  PART  I:  LITERATURE REVIEW  1  INTRODUCTION  The agents  a - g l y c e r y l e t h e r s have been examined as p o s s i b l e t h e r a p e u t i c  f o r more than t e n y e a r s .  I n 1957, Evans, e_t al_ (33), r e p o r t e d on the  use o f one of t h e s e , b a t y l a l c o h o l , i n the treatment of bracken p o i s o n i n g i n the b o v i n e and s i n c e then o t h e r r e p o r t s have been p u b l i s h e d i n d i c a t i n g v a r i o u s degrees o f success i n the use of b a t y l a l c o h o l f o r the treatment of c e r t a i n blood dyscrasias.  The a - g l y c e r y l e t h e r s have aroused renewed  interest  w i t h the f i n d i n g t h a t they n o r m a l l y e x i s t i n a l l animals i n s m a l l q u a n t i t i e s . T h i s might  imply t h a t they possess v i t a m i n - l i k e  properties.  An e x t e n s i v e l i t e r a t u r e review has been c a r r i e d out c o v e r i n g the o c c u r r e n c e , c o m p o s i t i o n and c h e m i c a l n a t u r e of these e t h e r s .  The l i t e r a t u r e  c o n c e r n i n g the b i o l o g i c a l e f f e c t s of these compounds has been examined and found t o be wanting out under the " i l l  i n that much o f the e x p e r i m e n t a l work has been c a r r i e d d e f i n e d " c o n d i t i o n s o f the c l i n i c .  The p r e s e n t study was designed t o determine  i f c e r t a i n of the  a - g l y c e r y l e t h e r s can be c o n s i d e r e d t o be e s s e n t i a l n u t r i e n t s . end, a s e r i e s of experiments  To t h i s  have been c a r r i e d out u s i n g the dog and the  a l b i n o r a t as e x p e r i m e n t a l s u b j e c t s .  The work has, o f n e c e s s i t y ,  s c r e e n i n g assays t o a s c e r t a i n i f the compounds s e l e c t e d would  produce  i n i m i c a l e f f e c t s i n mammalian systems when a d m i n i s t e r e d p e r orum. b a s i s of t h i s p r e l i m i n a r y work, a f e e d i n g response experiment was u s i n g s y n t h e t i c a - g l y c e r y l f r e e b a s a l d i e t s , to determine  involved  On the conducted,  i f the compounds  possess growth promoting p r o p e r t i e s or d e s i r a b l e e f f e c t s on the h a e m a t o p o i e t i c system.  2  A.  THE CHEMICAL NATURE OF THE a-GLYCERYL ETHERS  The t h r e e most common, n a t u r a l l y o c c u r r i n g , g l y c e r y l e t h e r s a r e : a-hexadecylglyceryl  ether  (chimyl a l c o h o l ) , a - o c t a d e e y l g l y c e r y l  ( b a t y l a l c o h o l ) , and a - , 9 - o c t a d e c e n y l g l y c e r y l  TABLE I :  ether  ether  (selachyl alcohol).  MELTING POINT AND STRUCTURE OF THE GLYCERYL ETHERS  G l y c e r y l Ether  Melting Point  Structure .CH(OH) .CH 0H  60° - 61.5°C. (28)  CH (CH ) i CH .0.CH  batyl alcohol  70° - 71°C.  C H ( C H ) i 6 CH .0.CH .CH(OH) .CH 0H  selachyl alcohol  8-9  chimyl  alcohol  The e t h e r  (28)  C. (16)  l i n k a g e was f i r s t  3  3  2  1  +  2  2  CH (CH ) CH 3  2  2  7  2  2  2  2  CH(CH ) 2  E  i  . 0. CH 2.CH(OH).CH 0H 2  shown to be of the a form by Weidemann (106) i n  1926 when he demonstrated t h a t h y d r o i o d i c a c i d a c t s on b a t y l a l c o h o l t o produce methyl i o d i d e . a methoxy group  T h i s e s t a b l i s h e d t h a t the t h i r d oxygen i s p r e s e n t as  and, i n t u r n , i n d i c a t e s t h a t an e t h e r  the s t e a r y l a l c o h o l and the g l y c e r o l moiety.  l i n k a g e e x i s t s between  In 1930, Knight  (58) r e p o r t e d  the s u r f a c e f i l m a c t i v i t y of the s y n t h e t i c a form t o be i d e n t i c a l w i t h  that  of the n a t u r a l e t h e r s , b u t that the a c t i v i t y of the g form was d i f f e r e n t . The m e l t i n g  p o i n t of b a t y l a l c o h o l i s 60° - 61.5°; t h i s i s the same as t h a t  found f o r the a form.  The B o c t a d e c y l ether has a m e l t i n g  p o i n t of  62° - 63°C. ( 2 6 ) . A m i x t u r e of the B form w i t h n a t u r a l b a t y l a l c o h o l causes a marked m e l t i n g  point depression,  whereas the m i x t u r e of a - o c t a d e c y l  e t h e r w i t h n a t u r a l b a t y l produces only a s l i g h t d e p r e s s i o n . explained  T h i s can be.  on the b a s i s of the o p t i c a l a c t i v i t y of the g l y c e r y l e t h e r s .  3  B a t y l a l c o h o l belongs to the d - s e r i e s whereas s y n t h e t i c o c t a d e c y l g l y c e r y l ether  i s a racemic m i x t u r e and  different  expected to have s l i g h t l y  of b a t y l a l c o h o l w i t h l e a d t e t r a a c e t a t e y i e l d s g l y c o l  aldehyde o c t a d e c y l (28)  ether  (m.p.  51°C.) and  formaldehyde.  that there  and  1  i s no a l t e r n a t i v e other  optical activity  (9) shows that the e t h e r  (8)  molecule and  On  free  f a c t t h a t the g l y c e r y l e t h e r s  hence an a l i n k a g e must be  observed that b a t y l a l c o h o l i n h i g h gradually diminishing,  reaction  than to assume that g l y c e r y l e t h e r s  F i n a l l y , the v e r y  (7)  This  to  B p o s i t i o n s of the g l y c e r o l moiety must be  are of the same a type.  asymetric  According  t h i s i s a s p e c i f i c r e a c t i o n f o r an c ^ S - g l y c o l .  i n d i c a t e s t h a t the a and  can be  properties.  Oxidation  Criege  therefore  concentrations  l i n k a g e must form  an  involved.  been  I t has  shows a n e g a t i v e  to d i s a p p e a r e n t i r e l y at a c o n c e n t r a t i o n  further d i l u t i o n dextrorotation i s displayed  (Toyama and  rotation,  of 10 per  Isikawa)  i n e i t h e r the c i s or t r a n s  form; the n a t u r a l compound has  c o n f i g u r a t i o n , g i v i n g i t the low m e l t i n g trans  form, which has  been s y n t h e s i z e d  p o i n t of 8° - 9°C.  (8)  (9) has  a melting  cent.  (16).  S e l a c h y l a l c o h o l , having a double bond i n i t s long s i d e c h a i n , exist  have  can  the c i s .  While  the  point  of  48.5° - 49.5°C. In f u r t h e r i n v e s t i g a t i o n of the o p t i c a l p r o p e r t i e s , Baer and (6)  (9) s y n t h e s i z e d  1- and  d- forms of b a t y l , c h i m y l ,  and  Fischer  selachyl alcohols.  They found the p h y s i c a l p r o p e r t i e s of the d-forms to be i d e n t i c a l w i t h those of the n a t u r a l compounds, w h i l e the 1-forms d i f f e r e d s l i g h t l y .  4  B.  OCCURRENCE OF THE GLYCERYL ETHERS  G l y c e r y l e t h e r s have been found occurrence;  they e x i s t almost  from t i s s u e with  the l i p i d  t o be q u i t e wide-spread i n t h e i r  e n t i r e l y i n an e s t e r f i e d form and a r e e x t r a c t e d  fraction.  M a l i n s and Mangold  some of the f r e e form i n r a t f i s h l i v e r o i l and b a s k i n g Their properties are l i p i d - l i k e  (48) r e p o r t  finding  shark l i v e r o i l .  i n nature s i n c e the s t r u c t u r e of the  g l y c e r y l e t h e r s i s i d e n t i c a l t o the g l y c e r i d e s w i t h the e x c e p t i o n t h a t possess  an e t h e r bond i n s t e a d of an e s t e r bond. 0 CH 0CCH R | 0 CH0CCH R | 0 CH OCCH R 2  H CH OCCH R | 0 CH0CCH R | 0 CH 0CCH R 2  2  2  2  2  2  2  A g l y c e r y l ether was f i r s t  (m.p.  2  2  triglyceride  (10).  they  2  diesterfied glyceryl  ether  d e s c r i b e d i n 1915 by K o s s e l and E d l a c h e r  They a s s i g n e d the name " a s t r o l " t o the a l c o h o l t h a t they  isolated  71°C.) from the u n s a p o n i f i a b l e f r a c t i o n of the f a t of a s t a r f i s h .  L a t e r , Tsujimoto  and Toyama (28) r e p o r t e d what they b e l i e v e d to be the f i r s t  i s o l a t i o n o f the g l y c e r y l e t h e r s .  I n a subsequent communication Toyama  e s t a b l i s h e d the s t r u c t u r e of the component groups.  In 1943, Bergman and  co-workers (10) showed " a s t r o l " to be i d e n t i c a l with b a t y l a l c o h o l ,  thus  e s t a b l i s h i n g K o s s e l and E d l a c h e r as the o r i g i n a l d i s c o v e r e r s of the g l y c e r y l ethers.  I n 1941, Holmes e t a l (49) r e p o r t e d the f i r s t  i s o l a t i o n of b a t y l  a l c o h o l from a source other than the marine organisms; they b a t y l a l c o h o l from the y e l l o w bone marrow o f c a t t l e ,  isolated  thus opening the  p o s s i b i l i t y t h a t t h i s compound might have v a l u e as a n u t r i e n t .  S i n c e 1941,  5  g l y c e r y l e t h e r s have been i s o l a t e d  from many other  (1958) (23) i s o l a t e d and i d e n t i f i e d f o r the f i r s t containing phospholipid. phospholipid  fractions  bovine e r y t h r o c y t e s phospholipids Table  from many s o u r c e s .  time a g l y c e r y l  ether-  In at l e a s t two t i s s u e systems,  (44) and r e d bone marrow (101) g l y c e r y l  ether-containing  of the p h o s p h o l i p i d  a summary of the.|many n a t u r a l sources  e t h e r s have been i s o l a t e d .  Carter, et a l  Since 1948 g l y c e r y l e t h e r s have been found i n the  account f o r over 10 mole percent  I I provides  tissues.  fraction.  from which the g l y c e r y l  TABLE I I :  Tissue  OCCURRENCE OF GLYCERYL ETHERS  Content  Reference  some b a t y l a l c o h o l  1962 Helmy and Hack (47)  atherosclerotic a o r t a s (human)  285 mg. b a t y l a l c o h o l from 3,450 gm. f a t from 370 a o r t a s (0.008% of o i l )  1943  atherosclerotic aortas  0.0782 wet 0.0802 wet  1964 M i l l e r , eX a l  beef  65mg./Kg. t e t r a d e c a n o l  1960  Schogt, et_ a l (91)  bone marrow, y e l l o w (cattle) bone marrow ( c a t t l e ) bone marrow, r e d ( p i g epiphyses)  batyl alcohol  1941  Holmes, e_t a l (49)  0.2 - 0.7% G.E. ( f r e e form) l e c i t h i n f r a c t i o n has 20% G.E. d e r i v a t i v e  1957  Brohult  1962 P i e t r u s z k o  (81)  bone marrow (human)  9 - 16% of p h o s p h o l i p i d s are g l y c e r y l e t h e r s  1965  Wajda  (105)  brain  e t h e r - c o n t a i n i n g phosphol i p i d s range from 1 t o 2.5% o f the t o t a l l i p i d s  1960  Svennerholm and T h o r i n  (96)  Klenk and Hendicks  (57)  A n s e l l and Spanner  (3)  A n s e l l and Spanner  (3)  Omelik  (78)  amniotic  brain  fluid  (human)  t a l l o w (P.L. f r e e )  (calf)  (human)  ) ) ) )  b r a i n (human) cephalin fraction  microMole/lOOgm. tissue microMole/lOOgm. tissue  i n o s i t o l phosphate i s o l a t e d c o n t a i n e d 8.4% b a t y l - c h i m y l mixture  1961  Hardegger, et a l  (45)  (75)  (17)  G.E. p h o s p h o l i p i d i s 1.8% of t o t a l p h o s p h o l i p i d s  1963  G.E. p h o s p h o l i p i d i s 3.1% of t o t a l p h o s p h o l i p i d s  1963  Centrina s a l v i a n i ( l i v e r and eggs)  contains g l y c e r y l ethers  1949  dog t i s s u e s , w i t h the e x c e p t i o n of marrow  see Table  1962 Nakagawa, et a l  (77)  egg y o l k  g l y c e r y l ether c o n t a i n i n g phospholipids  1958 C a r t e r  (23)  brain  brain  (young r a t )  (adult r a t )  VIII  •  7  TABLE I I  Content  Tissue  Elasmobranch  (cont'd)  see T a b l e V  fish  Reference  1962  erythrocytes (bovine)  ethanolamine l i p i d s make up 30% of t o t a l phosphatides  erythrocytes (bovine)  75% of p h o s p h a t i d y l e t h a n o l a m i n e 1963 f r a c t i o n i s g l y c e r y l ethers  Halobacterium cutirubrum  93% of l i p i d s a r e p h o s p h o l i p i d s 73% of phosphatides are g l y c e r y l ethers  h e a r t muscle (human)  heart heart  liver  H a l l g r e n and Larsson  (41)  1961 Hanahan and Watts  (44)  Hanahan, e t a l  (43)  1962  Sehgal, et a l  (94)  mono- and d i - e t h e r s occur presumably as t h e i r phosphate e s t e r s  1965  Popovie  (83)  (ox)(P.L. f r e e ) (ox)  lOOOmg. t e t r a d e c a n o l / k g . g l y c e r y l ether d e r i v a t i v e forms 2% of the l e c i t h i n fraction  1960 1962  Schogt, e t a l Pietruszko  (91) (82)  (human)  contains g l y c e r y l ethers  1958  R i l e y , et a l  (86)  1948  Karnovsky,et  1948  Karnovsky, et a l (53)  1948  Karnovsky, e t a l (54)  1948  Karnovsky, et a l (54)  Manforte Fenech  l i v e r o i l of: - s o u p f i n shark (Galeorhemis rond.) - s e v e n - g i l l e d shark (Heptranchias pectorosus) -basking shark ( C e t o r h i n u s maximus) - s p i n y shark (Echinorhinus spinosus)  all contain glyceryl ethers  a l (51)  l i v e r o i l , cow shark (Hexanchus g r i s e n )  16.8% s e l a c h y l a l c o h o l ) 2.38% b a t y l a l c o h o l ) some c h i m y l a l c o h o l )  1954  liver oils (elasmobranch  see T a b l e VI  1956  Hilditch  (48)  16 - 17% of o i l (expressed as the f r e e form)  1957  Brohult  (17)  and (69)  fish)  l i v e r o i l (shark)  8  TABLE I I  Tissue  (cont'd)  Content  Reference  l i v e r o i l (dog-fish)  contains g l y c e r y l  liver o i l (ratfish and b a s k i n g shark)  f r e e g l y c e r y l e t h e r s as w e l l as g l y c e r y l e t h e r d i e s t e r s  ethers  1960 M a l i n s 1960 Mangold and Malins  liver  (dogfish)  g l y c e r y l ethers  (65)  (70)  found 1963 Hanahan, e t a l  (43)  see Table IX  1964 Todd and R i z z i  (104)  see Table IV  1962 H a l l g r e n and Larsson  (42)  1960 Schogt, e t a l  (91)  a l l are r i c h i n g l y c e r y l ethers  1965 Thompson and Lee  (102)  reef b u i l d i n g c o r a l (Plexaura flexuoso)  b a t y l a l c o h o l found  1942 K i n d and Bergman (56)  sea  anemones  batyl alcohol isolated  1956 Bergman, e t a l  (11)  spleen (pig)  contains b a t y l a l c o h o l  1943 P r e l o g , e t a l  (84)  starfish fat  astrol  1915 K o s s e l and Edlbacher  (10)  mammalian t i s s u e and s t a r f i s h man and cow  M i l k f a t (phosphol i p i d free) Phylum M o l l u s c a -chiton (Catherina tunicata) -marine (Thais Lamellosa) -clam ( P r o t o t h a c a staminea) -octopus (Octopus d o f l e i n i )  starfish fat  50 mg.  tetradecanol/Kg.  isolated  b a t y l a l c o h o l found  amounts to more than 25 t e r r e s t i a l slugs (Arian ater) mole per cent of t o t a l ( A r i o l i m a x columbianus) phospholipids  1943 Matsumoto, e t a l (74) 1954 Matsumoto and Wainai (73) 1963 Thompson and Hanahan  (99)  various  see Table V I I  1946 Karnovsky,et a l  (52)  various  see Table I I I  1963 G i l b e r t s o n and Karnovsky  (37)  TABLE I I I :  GLYCERYL ETHERS IN THE UNSAPONIFIABLE FRACTION OF NEUTRAL LIPIDS FROM SEVERAL MAMMALIAN TISSUES (taken from G i l b e r t s o n and Karnovsky)(37))  Total l i p i d as % of f r e s h wt.  Tissue  diverticulum 5.7 (starfish) 7.4 b r a i n (beef) 88.9 bone marrow (beef) 5.1 heart ( c a l f ) adipose t i s s u e ( r a t ) 76.8 43.1 brown f a t ( r a t ) leucocytes (polymorphs) (guinea p i g ) chylomicrons (human) beta-lipoprotein (human)  TABLE IV:  Neutral Phosphatide l i p i d s as residual % o f T.L. % o f N.L.  Total G.E. % o f N.L.  72.50 43.95 99.97 62.61 98.91 78.40  0.19 0.10 0.00 0.42 0.01 0.04  16.1 1.9 2.4 2.4 1.3 0.4  31.9 63.1 61.2 31.2 82.0 54.3  51.92  0.00  2.9  65.8  85.90  -  0.1  78.6  -  -  0.6  29.3  PERCENTAGE OF GLYCERYL ETHERS IN LIPIDS FROM SOME HUMAN AND ANIMAL SOURCES (taken from H a l l g r e n and L a r s s o n ) ( 4 2 )  G l y c e r y l E t h e r s as % of L i p i d s  Source  human bone marrow ( r e d ) human s p l e e n human r e d b l o o d c e l l s human m i l k cow bone marrow (yellow) cow m i l k egg y o l k l i v e r o i l of elasmobranch  V i n y l ethers molar % o f t o t a l G.E.  less  fish  than  0.2 0.05 0.01 0.1 0.01 0.01 none found 10 - 30  TABLE V:  COMPOSITION OF THE NONSAPONIFIABLE MATTER IN FISH LIVER OILS (taken from H a l l g r e n and Larsson) (41)  Species  % unsap. i n o i l  Chimaera monstrosa Somniosus m i c r o c e p h a l u s Squalus a c a n t h i a s  TABLE V I :  33 8 7  Composition of unsap. almost e x c l u s i v e l y G.E. 90% G.E. and 8% c h o l e s t e r o l 84% G.E. and 13% c h o l e s t e r o l  LIVER OILS FROM ELASMOBRANCH FISH (taken from H i l d i t c h ) (48)  % G.E.*  Composition o f u n s a p o n i f i a b l e s  skate angel f i s h t h r e s h e r shark spotted dogfish grey d o g f i s h  0.3 1.5 1.8 2.0 10.0  ratfish  37.0  shark s p e c i e s  50 - 80  mainly c h o l e s t e r o l mainly c h o l e s t e r o l mainly c h o l e s t e r o l mainly c h o l e s t e r o l mainly s e l a c h y l , some c h i m y l and b a t y l almost w h o l l y s e l a c h y l , o n l y some c h i m y l and b a t y l l a r g e amounts of squalene, some s e l a c h y l , c h i m y l and b a t y l  L i v e r f a t from  * G l y c e r y l Ethers  TABLE V I I : A SUMMARY OF THE EXTENSIVE TABLE GIVEN BY KARNOVSKY AND RAPSON (51)  G l y c e r y l e t h e r content of o i l c a l c u l a t e d as s e l a c h y l d i o l e a t e  pecies 25 s p e c i e s l i s t e d under Elasmobranchii  amount v a r i e s from n i l i n the l i v e r of the man e a t i n g shark to 77.5% i n the l i v e r of the s i x - g i l l e d shark  21 s p e c i e s l i s t e d Teleostomi  under  amount v a r i e s from n i l i n the b l u e h o t t e n t o t to 7.3% i n the l i v e r of the stone bass  6  species l i s t e d Mollusca  under  q u a n t i t i e s v a r y from 0.7% i n the Limpet t o 8.0% i n the v i s c e r a of the rock octopus  1  Arthropoda  1  Amphibia  1  Reptillia  1  Aves (domestic fowl)  0.1% i n the l i v e r viscera  6  s p e c i e s of Mammalia; 3 whales, 1 r a t , 1 cow and 1 man  q u a n t i t i e s range from n i l i n the l i v e r of a man to 2.5% i n the faeces o f the rat  4  vegetable o i l s investigated  o n l y tung nut o i l showed any g l y c e r y l e t h e r s ( l e s s than 0.1%)  q u a n t i t i e s v a r y from 0.9% i n the i n t e s t i n e t o 6.0% i n the f l e s h  (crayfish)  (cape clawed  (mole  toad)  snake)  q u a n t i t i e s v a r y from n i l i n the l i v e r to .4% i n the body o i l 0.3% i n the t o t a l v i s c e r a t o 1.2% i n the body t o 0.3% i n the  TABLE V I I I :  a-GLYCERYL ETHER CONTENT OF DOG TISSUES (taken from Nakagawa, e_t a l ) (77)  T i s s u e and animal no.  Molar r a t i o to l i p i d P  Lung 1 Lung 2 Kidney 1 Kidney 2 Liver 1 Liver 2 Liver 2 Spleen 1 Intestine 1 Heart 1 Aorta 1 S k e l e t a l muscle 1 S k e l e t a l muscle 1 Bone marrow ( r a b b i t ) 1 Bone marrow ( r a b b i t ) 2  TABLE IX:  Source  rabbit cow sheep Pig rat man  microMoles/gm. d r y l i p i d free tissue  .0027 .0043 .0015 .0086 .0026 .0077 .0071 .0048 .0102 .0069 .0039 .0157 .0159 .1570 .3600  .41 .70 .29 .80 .31 1.16 1.07 .67 1.35 .59 .20 1.02 1.03 8.73 14.10  a-GLYCERYL ETHERS OF MAMMALIAN YELLOW BONE MARROW (taken from Todd and R i z z i ) (104)  NS* as % wt. of marrow  G.E.** as % wt. of NS*  0.2 0.2 - 0.4 0.36 0.5 - 0.6  13 12 - 25 11 4 - 8 0 11.3 - 16.6  -  *nonsaponifiables * * g l y c e r y l ethers  G.E. c o m p o s i t i o n as % of t o t a l g l y c e r y l ethers 18:0 16:0 18:1 0 50  100 50  0 0  25  50  25  -  -  -  C.  THE COMPOSITION OF THE GLYCERYL ETHERS  For a good many years only the g l y c e r y l e t h e r s b a t y l , and  c h i m y l were mentioned i n the l i t e r a t u r e .  s e v e r a l r e p o r t s of g l y c e r y l e t h e r s having  Recently  selachyl,  t h e r e have been  the c h a i n l e n g t h o f the f a t t y  a l c o h o l moiety v a r y i n g from 12 t o 24 carbons,  i n c l u d i n g some odd c h a i n  l e n g t h s and some c h a i n s having more than one double bond (38, 40, 41, 42, 65).  The f i r s t  Larsson  (40).  of these r e p o r t s was p u b l i s h e d i n 1959 by H a l l g r e n and The f o l l o w i n g i s a t a b l e of t h e i r p u b l i s h e d  TABLE X:  Alkoxyglycerol 12:0 14:0 15:0 16:0 16:1 17:0 18:0 18:1 18:2 18:3 19:1 20:1 21:0 22:1  (chimyl)  (batyl) (selachyl)  results.  THE PERCENT COMPOSITION (WT) OF THE ALKOXYGLYCEROLS FROM LIVER OILS*  Grey d o g f i s h Squalus acanthias trace 5.7 1.9 13.2 10.6 3.0 3.4 47.8 2.4 trace 1.2 8.0 trace 2.7  Greenland shark Somniosus' microcephalus trace 2.0 0.7 9.1 10.8 3.6 2.8 59.4 1.6  Ratfish Chimaera monstrosa trace 1.7 1.1 10.4 9.1 4.7 6.7 53.6 2.5 ?  1.5 6.2 ?  2.2  *The a l k o x y g l y c e r o l s a r e r e p r e s e n t e d by the long c h a i n p a r t of the molecule. i  2.4 6.4 ? 1.0  14  H a l l g r e n and L a r s s o n p u b l i s h e d two f u r t h e r papers i n 1962, one (42) of which d e s c r i b e s the g l y c e r y l e t h e r s o c c u r r i n g i n man and i n the b o v i n e . T a b l e XI summarizes t h e i r c o m p o s i t i o n  TABLE X I :  data on t h e g l y c e r y l  ethers.  PERCENTAGE COMPOSITION (WT/WT) OF GLYCERYL ETHERS FROM HUMAN BONE MARROW, HUMAN SPLEEN, AND HUMAN MILK ( H a l l g r e n and L a r s s o n ) ( 4 2 )  Long-chain component i n g l y c e r y l ethers  Human bone marrow  unidentified components 16:0 16:1 17* 18:0 18:1 18:2 19* 20:0 20:1 22:0 22:1  Human spleen  -  Human milk  3.8 29.4  33.0  7.6 24.6 16.7  1.0 25.8 27.6  6.1 2.9 3.2  ) ) 7.3  2.0 23.9 trace 3.6 22.8 33.8 1.4 2.4 1.6 2.3  0.7 5.1  ) ) 5.2  0.7 3.4 2.1  24:0  *Normal and branched  In 1962, Guyer ( 3 8 ) , u s i n g gas chromatographic techniques g l y c e r y l ethers i s o l a t e d had  from d o g f i s h l i v e r o i l , showed  on the  t h a t the components  c h a i n l e n g t h s v a r y i n g from 12 t o 20 carbon atoms. Although  g l y c e r y l ether-containing phospholipids are.present i n  bone marrow they a r e v i r t u a l l y absent  from the e r y t h r o c y t e s , i n f a c t  human e r y t h r o c y t e s have no d e t e c t a b l e g l y c e r y l e t h e r s , b u t they do c o n t a i n  plasmalogens.  Young bovine  e r y t h r o c y t e s which s t i l l possess  complement of n u c l e i , m i t o c h o n d r i a , i n the m i t o c h o n d r i a n all  a  full  and microsomes have g l y c e r y l  ethers  f r a c t i o n ; whereas i n the o l d e r e r y t h r o c y t e s , where  i n t r a c e l l u l a r o r g a n e l l e s have a t r o p h i e d i n the c e l l ,  the  glyceryl  e t h e r c o n t a i n i n g p h o s p h o l i p i d s are l o c a l i z e d i n the c e l l membrane S t u d i e s of the occurrence  of g l y c e r y l e t h e r s i n v a r i o u s t i s s u e s  show t h a t bone marrow i s the r i c h e s t s i t e , w i t h l e s s e r amounts.  Todd and  with r a b b i t t i s s u e , found blood  the lowest.  Rizzi  (101).  (1964)(104),  the other t i s s u e s having  on the other hand, working  i n t r a p e r i t o n e a l f a t to be  the r i c h e s t  source  and  In the same paper, Todd and R i z z i r e p o r t a comparison  of mammalian y e l l o w bone marrow from the r a b b i t , the cow,  the sheep, the  p i g , the r a t , and man.  I t may  ( D e t a i l s are g i v e n i n Table IX.)  from t h i s data t h a t the r a b b i t has  only c h i m y l a l c o h o l , and  appears to have no g l y c e r y l e t h e r s i n i t s bone marrow. g l y c e r y l e t h e r s from s t a r f i s h show the b a t y l and present  and  the s e l a c h y l to be absent  (104);  A n a l y s i s of  t h i s i s i n c o n t r a s t to the ether.  s p e c i e s of t e r r e s t i a l s l u g , A r i a n a t e r  of the t o t a l p h o s p h o l i p i d m i x t u r e .  i s an absence of u n s a t u r a t i o n w i t h the major g l y c e r y l e t h e r b e i n g a l c o h o l , a sharp  the  c h i m y l a l c o h o l s to be  and A r i o l i m a x Columbians, c o n t a i n a - g l y c e r y l ether p h o s p h o l i p i d s f o r more than 25 mole percent  noted  t h a t the r a t  f i s h o i l s which have a h i g h p r o p o r t i o n of s e l a c h y l as a g l y c e r y l I t i s of i n t e r e s t t h a t two  be  c o n t r a s t to the f i n d i n g s w i t h other  organisms.  accounting There chimyl  16  D.  THE MESENCHYMAL ORIGIN OF THE ot-GLYCERYL ETHERS  Bodman and Maison (16) r e p o r t , perhaps w i t h i n s u f f i c i e n t evidence,  t h a t the a - g l y c e r y l e t h e r s a r e found p r e s e n t i n those  supporting tissues  c o n t a i n i n g c e l l s o f mesenchymal o r i g i n , 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 .  They support  t h i s by r e f e r r i n g t o  the a s s o c i a t i o n o f a - g l y c e r y l e t h e r s w i t h s a t u r a t e d f a t t y a c i d s 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 and the a s s o c i a t i o n between squalene and a-glyceryl ethers.  Squalene r i c h o i l has been found  the shark and i n the faeces of l a n d animals; to be p r e s e n t along w i t h the squalene  i f we assume g l y c e r y l e t h e r s  i n these c a s e s , then i t i s r e a s o n a b l e  to assume t h a t the gut mucosa i s concerned  E.  i n the stomach o f  with a - g l y c e r y l ether s y n t h e s i s .  BIOSYNTHESIS OF THE GLYCERYL ETHERS  In 1955, Karnovsky and Brumm (50), u s i n g the s t a r f i s h , r e p o r t e d the i n c o r p o r a t i o n o f r a d i o c a r b o n i n t o a - g l y c e r y l e t h e r m o i t i e s a t r a t e s comparable to those observed  i n the more commonly s t u d i e s l i p i d  fractions.  T h i s may  i n d i c a t e t h a t the s t a r f i s h does n o t simply accumulate these compounds from its  food. Data presented by Thompson and Hanahan (1962) (100), o b t a i n e d  experiments u s i n g C  llt  from  l a b e l l e d g l u c o s e , i n d i c a t e t h a t the g l y c e r y l moiety  of the g l y c e r y l e t h e r s can be formed from t h i s sugar i n bovine marrow. d i s t r i b u t i o n of C  1 L f  would s t r o n g l y suggest  d i r e c t l y i n v o l v e d i s 1-a-glycerophosphate. proposed  t h a t the g l u c o s e  The  metabolite  T h i s i s s i m i l a r t o the pathway  i n the b i o s y n t h e s i s of p h o s p h a t i d i c a c i d , or some o t h e r  17  m e t a b o l i c a l l y analogous compound. same authors The  I n a more r e c e n t paper (1963) t h e  d e s c r i b e i n v i t r o bone marrow i n c u b a t i o n experiments  r a d i o a c t i v i t y from g l u c o s e - 6 - C  t r i t i a t e d water were i n c o r p o r a t e d bone marrow e x t r a c t s .  (101).  , sodium p a l m i t a t e - l - C ^ and  1  1  i n t o g l y c e r y l e t h e r p h o s p h o l i p i d s by  At the end of the i n c u b a t i o n times used, r a d i o -  a c t i v i t y of t h e g l y c e r y l - e t h e r p h o s p h o l i p i d s nonglyceryl-ether phospholipids.  was l e s s than t h a t of the  Almost a l l of t h e r a d i o a c t i v i t y o f the  s y n t h e s i z e d g l y c e r y l ethers was l o c a t e d on the alpha carbon of the g l y c e r y l moiety, a g a i n s u g g e s t i n g derived precursor. ethers.  that a-glycerophosphate may be a d i r e c t  Both ethanolamine and c h o l i n e phosphatides c o n t a i n the  There appears t o be a m e t a b o l i c  ethers and plasmalogens. be s y n t h e s i z e d  lies  r e l a t i o n s h i p between g l y c e r y l  One p o s s i b l e r o u t e by which g l y c e r y l e t h e r s may  i n the f o r m a t i o n  by enzymatic hydrogenation  of a plasmalogen type m o l e c u l e  of the v i n y l e t h e r double bond.  H HG-0-CH-CH(CH )i3CH3  I 0  2  H HC-0-CH (CH )n CH  I  HC-O-C—R j 0 HC-0-P-O-CH CH-^H H OH 2  a plasmalogen  2  glucose-  enzymatic hydrogenation 2  0  2  2  f  3  HC-O-C-R  I  0  HC-C-P-C-CH CH - « H H OH 2  a - g l y c e r y l ether phospholipid  2  2  containing  followed  F.  DIGESTION AND  METABOLISM OF  a-GLYCERYL ETHERS  Much as d i e t a r y g l y c e r i d e s are d i g e s t e d by c r e a t i c l i p a s e present partially  i n the i n t e s t i n e , to g l y c e r o l , f a t t y a c i d s ,  s p l i t p r o d u c t s such as monoglycerides and  e s t e r f i e d g l y c e r y l ethers  f a t t y a c i d s , with  and  d i g l y c e r i d e s ; the  are broken down from g l y c e r y l e t h e r d i e s t e r s to  g l y c e r y l e t h e r monoesters, f r e e g l y c e r y l e t h e r s  mono- and  the a c t i o n of the pan-  and  fatty acids.  Then  the  the a i d of the b i l e s a l t s , c a r r y the g l y c e r y l e t h e r  d i e s t e r s and  the s m a l l i n t e s t i n e .  the f r e e g l y c e r y l e t h e r s  i n t o the mucosal c e l l s  Once w i t h i n the mucosal c e l l s ,  be broken to g i v e f r e e g l y c e r o l and  the e t h e r  of  linkage  can  free fatty acids.  S e v e r a l papers have been p u b l i s h e d which g i v e data about d i g e s t i o n and  to some e x t e n t ,  the metabolism of g l y c e r y l e t h e r s .  known about the r o l e of g l y c e r y l e t h e r s and  Blomstrand  (1956)(12), Blomstrand  i n c e l l metabolism.)  (1959((15) and  (1959)(14) r e p o r t data on the a b s o r p t i o n  of chimyl  Blomstrand and Ahrens (1959)(14) r e p o r t data M a l i n s , j2t al In man  and  That which passed i n t o the faeces only t h i n g t h a t happened w h i l e  little  and  t r a c e d i n the c h y l e had  Ahrens  alcohol i n rats; i n man  and  i n the rainbow t r o u t .  of f e d g l y c e r y l e t h e r s were absorbed. retained i t s ether linkage i n t a c t ,  i n the i n t e s t i n e was  the h y d r o l y s i s  r e s y n t h e s i s of the e s t e r l i n k a g e s on the g l y c e r y l e t h e r m o l e c u l e . other hand, the g l y c e r y l e t h e r s  is  Bergstrom  Blomstrand and  on a b s o r p t i o n  (1964)(66) r e p o r t data on a b s o r p t i o n  the r a t , w e l l over 90%  (Very  t h a t were absorbed i n t o the mucosal  t h e i r ether  t a b l e i s taken from Blomstrand's paper  l i n k a g e s broken. (1959)(15):  The  the  and On  the  cells  following  19  TABLE X I I :  RECOVERY AND DISTRIBUTION OF RADIOACTIVITY IN LYMPH LIPIDS AFTER FEEDING C ^-LABELLED CHIMYL DIOLEATE* 1  % of a d m i n i s t e r e d a c t i v i t y absorbed  % of absorbed activity recovered i n lymph l i p i d s  free  esterfied  6 5  t h i s work done by Blomstrand  i n lymph l i p i d s r e c o v e r e d a s :  chimyl a l c o h o l  60 45  90 85  All  % of a c t i v i t y  triglyceride  phospholipid  fatty acids  fatty acids  18 20  74 73  2 2  and h i s co-workers w i t h the r a t and the  human i s s u b s t a n t i a t e d by M a l i n s , e t a l (66) i n h i s work w i t h the rainbow trout  (Salmo g a i r d n e r i ) .  ethers.  The t r o u t were f e d b a t y l a l c o h o l and C ^ - g l y c e r y l 1  At the end of a f e e d i n g p e r i o d the l i p i d s of the i n t e s t i n a l  were a n a l y s e d ; although  t h e r e was simultaneous  contents  e s t e r f i c a t i o n and h y d r o l y s i s  i n the lumen of the i n t e s t i n e , t h e r e was no cleavage of the e t h e r bond.  No  a p p r e c i a b l e amounts of g l y c e r y l e t h e r s were found d e p o s i t e d i n the f l e s h of the f i s h which suggests m e t a b o l i c breakdown of the g l y c e r y l e t h e r s i n the f i s h as w e l l as i n r a t s and i n man. S w e l l , e t a l (1965)(98) r e p o r t on the comparative a- and 3-octadecyl  g l y c e r y l ethers.  r a t i o n s c o n t a i n i n g 35 mg  0  Lymph f i s t u l a t e d r a t s were f e d t e s t  of a- or 3-octadecyl  found  t h a t 3 - g l y c e r y l e t h e r was absorbed  (96%)  than the a - g l y c e r y l e t h e r  (76%).  i n d i c a t e d t h a t 37 to 48 percent of the C C ^-labelled 1  glyceryl C  l t f  a b s o r p t i o n of  glyceryl-l-C  1  ether.  They  i n t o the lymph t o a g r e a t e r extent F r a c t i o n a t i o n of the lymph l i p i d s 1£+  a c t i v i t y was i n the form of  f a t t y a c i d s , and from 55 to 63 percent as f r e e and e s t e r f i e d  -ethers.  The a - g l y c e r y l e t h e r was s p l i t  i n the i n t e s t i n e to a  *The c h i m y l d i o l e a t e was d i s s o l v e d i n o l i v e o i l and f e d to 2 r a t s u s i n g a t h o r a c i c duct f i s t u l a .  greater  e x t e n t than the B - g l y c e r y l e t h e r .  lymph l i p i d s was i n the a l k o x y d i g l y c e r i d e  Most of the C ^ a c t i v i t y of the 1  fraction.  The 3 - g l y c e r y l e t h e r  was more e f f i c i e n t l y converted t o the a l k o x y d i g l y c e r i d e a - g l y c e r y l ether. the  than the  More alkoxymonoglyceride appeared i n the lymph when  a - g l y c e r y l e t h e r was f e d than when the g - g l y c e r y l e t h e r was a d m i n i s t e r e d .  G.  As  a-GLYCERYL ETHERS AND  HAEMOPOIESIS  e a r l y as 1949 Sandler (90) suggested t h a t g l y c e r y l e t h e r s , i n  p a r t i c u l a r b a t y l a l c o h o l , had the a b i l i t y man.  to stimulate  Then i n 1951 A r t u r s o n and Lindback (4) r e p o r t e d  reticulocytosis i n that b a t y l  alcohol  g i v e n a t a dosage l e v e l o f 34 and 70 mg/Kg o f body weight t o male white mice caused a s i g n i f i c a n t i n c r e a s e little  i n r e t i c u l o c y t e s while red c e l l  a f f e c t e d by the treatment.  counts were  A s l i g h t c o n t r a d i c t i o n t o these p o s i t i v e  e f f e c t s i s g i v e n by E v e n s t e i n , ejt a_l (34) who dosed r a t s a t l e v e l s of 2 20 mg/Kg o f body weight, without s i g n i f i c a n t e f f e c t on the r e d b l o o d count, r e t i c u l o c y t e number, h e m a t o c r i t or hemoglobin v a l u e s . Linman, _e_t a l (61) r e p o r t e d  the subcutaneous i n j e c t i o n of female r a t s w i t h  s i g n i f i c a n t response i n e r y t h r o p o i e s i s ,  thrombopoiesis and  response appeared t o be one o f a c c e l e r a t e d  Linman, e_t a l (62) p u b l i s h e d  cell  I n 1958  150 and 300 mg of b a t y l a l c o h o l p e r Kg of body weight, and o b t a i n i n g  The  to  granulopoiesis.  cellular division.  another paper r e p o r t i n g  a  I n 1959  the a d m i n i s t r a t i o n ,  by  stomach tube, o f approximately 30 mg/Kg of b a t y l a l c o h o l t o r a t s ; the r e s u l t s were:  e r y t h r o c y t i c response, p r o d u c t i o n o f m i c r o c y t e s , r e t i c u l o c y t o s i s ,  thrombocytosis and l e u c o c y t o s i s . e f f e c t i v e o r a l l y and p a r e n t e r a l l y ,  Although Linman (62) found b a t y l  alcohol  independent of o p t i c a l a c t i v i t y , he  21  found s e l a c h y l a l c o h o l  ( n a t u r a l and s y n t h e t i c )  d e v o i d of a c t i v i t y .  The  s e l a c h y l was a d m i n i s t e r e d over a t e n day p e r i o d by g a s t r i c i n t u b a t i o n t o normal r a t s a t a dosage r a t e o f 50 mg/rat/day (312 - 356 mg/Kg.)  The  i n a c t i v i t y of s e l a c h y l a l c o h o l was confirmed by Osmond, e_t a l (79) i n t h e i r experiments u s i n g  guinea p i g s .  Both s e l a c h y l and b a t y l  alcohols  were g i v e n a t a dosage l e v e l o f a p p r o x i m a t e l y 10 mg/Kg of body weight. The  r e s u l t s suggest t h a t the b a t y l a l c o h o l may be an e r y t h r o p o i e t i c  stimulant  i n the guinea p i g whereas s e l a c h y l a l c o h o l i s not.  Negative  r e s u l t s were a l s o o b t a i n e d by Penny, et_ a l (80) working w i t h sheep, c a t t l e , and  mice.  Sheep were dosed i n t r a v e n o u s l y  a t a r a t e of 2 and 4 mg o f b a t y l  a l c o h o l p e r Kg o f body weight f o r 21 days w i t h no response. ment, sheep were dosed i n t r a v e n o u s l y  I n one e x p e r i -  w i t h 10 mg o f b a t y l a l c o h o l p e r Kg o f  body weight f o r 4 days which r e s u l t e d i n a r i s e i n polymorphonuclear leucocytes,  but no i n c r e a s e  1 gm o f d - b a t y l a l c o h o l  i n white b l o o d c e l l  count.  Two h e i f e r s  (3 mg/Kg o f body weight) i n t r a v e n o u s l y  y i e l d e d no h a e m a t o p o i e t i c response.  Mice were g i v e n  a s l i g h t decrease i n t h e r e d c e l l ,  f o r four  days  a p p r o x i m a t e l y 110 mg/Kg  of body weight subcutaneously y e t they showed no s t i m u l a t i o n , was  given  i n fact,  there  r e t i c u l o c y t e and marrow e r y t h r o b l a s t  counts. Under s p e c i f i c c o n d i t i o n s g l y c e r y l ethers  i n general  of d i s e a s e  and s t r e s s t o the body,  and b a t y l a l c o h o l i n p a r t i c u l a r s t i m u l a t e the  f o r m a t i o n of b l o o d c e l l s and appear to a l l e v i a t e some of t h e t o x i c effects.  The f i r s t  reported  use o f bone marrow was i n 1930 by G i f f i n and  Watkins (36) i n the treatment o f secondary anemia.  They used normal bone  marrow i n the hope that some f a c t o r i n i t would s t i m u l a t e the d i s e a s e d  side  bone marrow i n secondary anemia.  In general,  or a l l e v i a t e the bone marrow  proved  to be u n s a t i s f a c t o r y i n the treatment  i n one  case of a g r a n u l o c y t o s i s .  of secondary  Over the years 1930  anemia,  to 1938,  except  Giffin  and Watkins t r e a t e d 24 cases of a g r a n u l o c y t o s i s w i t h bone marrow, and o b t a i n e d a f a v o u r a b l e response  i n a l l but t h r e e c a s e s .  work, G i f f i n and Watkins a d m i n i s t e r e d of body weight) to t h e i r p a t i e n t s .  about 0.4  In 1936,  In the  original  grams of marrow (60 mg/Kg  Marberg and W i l e s  reported  the use of a c o n c e n t r a t e of the u n s a p o n i f i a b l e p o r t i o n of bone marrow d i s s o l v e d i n a b l a n d o i l ( e q u i v a l e n t to 2 grams of marrow per drop) administered t h e r e was  to p a t i e n t s s u f f e r i n g  from a g r a n u l o c y t o s i s .  As a r e s u l t ,  i n most cases a r i s e i n the number of g r a n u l o c y t e s which began  w i t h 24 to 36 hours and v a l u e s r e t u r n e d to normal i f the c a u s a t i v e i n f e c t i o n p e r s i s t e d d u r i n g the p e r i o d of One treatment  of the p r o m i s i n g uses of g l y c e r y l e t h e r s i s s a i d to be f o r the  of l e u c o p e n i a caused  and Holmberg  (the c o n c e n t r a t e s had  tions) .  Of 36 human cases  responded to the treatment In 9 cases decrease  by i r r a d i a t i o n .  In 1954,  a paper by B r o h u l t  (22) d e s c r i b e s the s u c c e s s f u l use of the n o n - s a p o n i f i a b l e  p o r t i o n of bone marrow f a t and esters  treatment.  there was  c o n c e n t r a t e s of a l k o x y g l y c e r o l s and  a h i g h e r potency  suffering  than the bone marrow p r e p a r a -  from i r r a d i a t i o n l e u c o p e n i a , 25  by an immediate  In 1957,  cases  increase i n the white c e l l count.  no f u r t h e r decrease w h i l e two  i n l e u c o c y t e counts.  their  showed a  continued  B r o h u l t p u b l i s h e d another  paper  w i t h s i m i l a r r e s u l t s , when she used a l k o x y - g l y c e r o l e s t e r s a d m i n i s t e r e d 100  patients suffering  from i r r a d i a t i o n l e u c o p e n i a .  dosage of 1.2  grams of o i l per day  e t h e r s per Kg  of body weight) and an a c t u a l decrease  She  ( e q u i v a l e n t to 2.74  mg  (17) to  r e p o r t s an optimum of f r e e  glyceryl  i n l e u c o c y t e numbers  at dosage l e v e l s above 2.5 of f r e e g l y c e r y l e t h e r s  grams of o i l per  per Kg  day  (equivalent  of body w e i g h t ) .  Brohult  to 5.70  mg  (1958)(18) used  a l k o x y g l y c e r o l e s t e r s f o r the p r o p h y l a c t i c treatment of r a t s t h a t were subsequently exposed to t o t a l body X - i r r a d i a t i o n . count of both megakaryocytes and  nucleated  of the p r o p h y l a c t i c a l l y t r e a t e d animals. r e s u l t s obtained  by  A significant  c e l l s was  higher  found i n the marrow  T a b l e XIII g i v e s a summary of  the  Brohult.  Mizuno, et^ a l (76)  t r e a t e d c a l v e s , exposed to whole body  i r r a d i a t i o n , w i t h autologous bone marrow. a severe hematologic  TABLE X I I I :  Nine out  of 15 r e c o v e r e d  crisis.  ALKOXYGLYCEROL ESTERS FED TO RATS RECEIVING TOTAL BODY X-IRRADIATION (taken from B r o h u l t ) (18)  Number of rats  E x p e r i m e n t a l group  70 87  Normal Control P r o p h y l a c t i c groups Shark l i v e r o i l Shark l i v e r o i l Shark l i v e r o i l Pure e s t e r s Batyl alcohol Selachyl alcohol Selachyl alcohol Selachyl alcohol  *0.15 mg i s e q u i v a l e n t body weight.  (mg/day)*  18 9 23 3 11 8 6 12  to  **Megakaryocyte count (M)  25 mg  (M%)** 100 19  0.25 0.40 0.50 0.25 0.14 0.12 0.14 0.18  26 33 27 29 32 61 49 27  of f r e e g l y c e r y l e t h e r s per Kg  i  i n )I of the megakaryocytes f o r normal :  after  24  There have been other r e p o r t s  of the t h e r a p e u t i c  e t h e r s i n the treatment o f i r r a d i a t i o n s i c k n e s s . treated  X-irradiated  use o f g l y c e r y l  Rusanov, e t a l (87)  r a t s w i t h b a t y l a l c o h o l and o b t a i n e d an a c t i v a t i o n  of b l o o d p r o d u c t i o n as w e l l as a f a s t e r r e c o v e r y from the r a d i a t i o n Prokhonchukov and P a n i k a r o v s k i i and  (85) used both s e l a c h y l and b a t y l  found b a t y l t o g i v e a s a t i s f a c t o r y t h e r a p e u t i c  gave an i n s i g n i f i c a n t response. therapeutic  Sviridov,  alcohols selachyl  e_t a l (97) s t u d i e d t h e  e f f e c t o f s e l a c h y l a l c o h o l a d m i n i s t e r e d t o dogs, r a b b i t s and  r a t s that had been exposed to X - i r r a d i a t i o n . gravity  response, w h i l e  sickness.  of the r a d i a t i o n s i c k n e s s ,  increased  The a l c o h o l a l l e v i a t e d the s u r v i v a l r a t e , normalized  tissue  r e s p i r a t i o n i n the b r a i n and myocardium, and somewhat s t i m u l a t e d the haemopoietic organs (the  f o l l i c u l a r appendages of the lymph nodes and the  spleen).  o f the use of b a t y l a l c o h o l  Another r e p o r t  i s g i v e n by Dudin  (29),  where p r i m a g r a v i d r a t s on the 15th day o f pregnancy were i r r a d i a t e d and injected intravenously  w i t h cystamine (65 mg/Kg) and b a t y l a l c o h o l  (0.5 mg/Kg)  d a i l y f o r 10 days a f t e r exposure t o X-rays.  There was no i n f l u e n c e  gravity  50 p e r c e n t o f the young were  of the X-ray s i c k n e s s .  In c o n t r o l s ,  on the  born dead; w i t h the combined treatment of b a t y l and cystamine o n l y 22 p e r c e n t of the f o e t i were born dead and, o f those born a l i v e , the development o f anemia and l e u c o p e n i a was completely p r e v e n t e d . B a t y l a l c o h o l has been, e f f e c t i v e i n s t i m u l a t i n g alleviating  c e r t a i n conditions  poisoning.  Sandler  marrow e x t r a c t s specific  of t o x i c i t y .  (1949) (90),  One of these i s i n benzene  examined the p o s s i b i l i t y  might have an a n t i t o x i c e f f e c t r a t h e r  leucopoietic  principle.  haemopoiesis and  that y e l l o w bone  than c o n t a i n i n g  a  He i n j e c t e d , subcutaneously, benzene and  25  preparations  made from n o n s a p o n i f i a b l e  y e l l o w bone marrow e x t r a c t s  group of benzene d e p l e t e d  rats.  i n j e c t e d w i t h benzene and  batyl alcohol.  benzene s o l e l y and  served as c o n t r o l s .  a l c o h o l therapy i n c r e a s e d to benzene n e c r o s i s De  G a e t a n i and  at the  Baiotti  restores  The  (1959)(27) r e p o r t  the  decreased the  tendency  lowered m o r t a l i t y .  r e l i e v e s i n t o x i c a t i o n , prolongs  the a c t i v i t y of bone marrow.  given  batyl  that b a t y l a l c o h o l given  osmotic r e s i s t a n c e of red c e l l s ,  that b a t y l a l c o h o l had  bone marrow and  s i t e of i n j e c t i o n , and  one  r a t s were  A number of animals were  the number of e r y t h r o c y t e s ,  w i t h acute benzene p o i s o n i n g , increases  Another group of d e p l e t e d  to  to r a t s  life,  f a v o u r s r e t i c u l o c y t o s i s , and  Hasegawa, e_t a l (1961) (46) , r e p o r t  e f f e c t i v e haemopoietic a c t i v i t y i n r a b b i t s s u f f e r i n g  from e x p e r i m e n t a l anemia caused by N i t r o m i n i n j e c t i o n or s o l v e n t i n t o x i c a t i o n . However, i n the case of induced a p l a s t i c anemia i n c a l v e s trichloroethylene-extracted  caused by  soybean o i l meal, b a t y l a l c o h o l f a i l e d  development of anemia (92) .  B a t y l a l c o h o l treatment was  tried  been used w i t h v a r y i n g  of bracken p o i s o n i n g .  et a l again reported recovered. claims  The  treatment of bracken p o i s o n i n g  the use  of b a t y l a l c o h o l ; 23  v a l i d i t y of these r e p o r t s  that normal c a t t l e may  4000 c e l l s per leucocyte  cmm  during  count r e p o r t e d  by  and  Evans was  from day 1000  out  haematology.  and  successful i n 1958,  use  Evans,  of 31 p o i s o n e d c a t t l e  count as much as  to day.  The  to 2000 c e l l s per  a l s o t r e a t e d c a t t l e a f f e c t e d w i t h bracken p o i s o n i n g , e f f e c t on the  the  treatment  i s q u e s t i o n e d by D a l t o n (25)  vary t h e i r leucocyte  the day  in cattle.  degrees of success i n the  Evans, e_t a l (1957) (33), r e p o r t  of b a t y l a l c o h o l i n the  to prevent  because  a p l a s t i c anemia so produced i s v e r y s i m i l a r to bracken p o i s o n i n g B a t y l a l c o h o l has  feeding  but  1000  increase cmm.  found no  who  in  Dalton obvious  to  26.  H.  SUMMARY  These n a t u r a l o c c u r r i n g appear  to be u s e f u l and perhaps  They may  compounds are of i n t e r e s t because  they  e s s e n t i a l to a l l animals i n c l u d i n g  man.  be u s e f u l as a s t i m u l a n t ,  a drug, or as a n u t r i e n t .  n o r m a l l y found i n the a n i m a l body, they are absorbed and m e t a b o l i z e d w i t h i n  the body.  They are  from the i n t e s t i n e  These compounds a r e not t o x i c , a l t h o u g h  there has been some r e p o r t of t o x i c i t y i n e a r l y work done by Agduhr (1934) (21), Berger  (1948) ( 2 1 ) , and Emmerie, e t a l (1952) (31).  t h a t the t o x i c i t y r e p o r t e d t h a t were used.  r e s u l t e d from i m p u r i t i e s  i n the  It i s possible alkoxyglycerols  TABLE XIV:  Glyceryl Ether  BIOLOGICAL EXPERIMENTS INVOLVING THE USE OF GLYCERYL ETHERS  Animal  Condition  Dosage  Response  b a t y l and selachyl  rats  transplanted malignant tumors  1 ml o f preparat i o n i n sunseed oil/day for a month  these i n j e c t i o n s caused the tumors t o develop slower i n the t r e a t e d r a t s ; s e l a c h y l gave the greatest i n h i b i t i o n  Abaturova and Shubina  batyl (purity unknown)  mice  normal  .01 t o .048 mg/Kg/day 160 - 210 days  lesions of heart, kidneys and l i v e r  Agduhr (1934) (cited i n Brohult)  alkoxyglycerols  mice  radiated  590 mg/Kg i n food  both g l y c e r y l e t h e r e s t e r s and e s s e n t i a l f a t t y a c i d s promote longer l i f e  Alexander, et a l  (1959)  34 mg/Kg and both l e v e l s gave a 70 mg/Kg s i g n i f i c a n t increase i n ( i n t r a p e r i t o n e a l ) r e t i c u l o c y t e s ; red blood c e l l counts remained normal  A r t u r s o n and Lindback  (1951)  batyl  mxce  normal  Reference  (1964)  various  mxce  normal  large (subcutaneous)  LD50 f o r b a t y l a l c o h o l was determined t o be 3 gm/Kg  Berger (1948) (cited i n Brohult)  C  rats  normal  5 mg by stomach tube  a b s o r p t i o n and breakdown s t u d i e d  Bergstrom and Blomstrand (1959)  11+  -chimyl  TABLE XIV (cont'd)  Glyceryl Ether  Animal  Condition  Dosage  Response  C  rats  normal  .5 ml of a 2% s o l u t i o n by stomach tube  a b s o r p t i o n and breakdown s t u d i e d  Blomstrand  li+  -chimyl  Reference (1956)  C ^-chimyl  man  25 mg by mouth  a b s o r p t i o n and breakdown s t u d i e d  Blomstrand and Ahrens (1959)  alkoxy exters  man  various  2.74 mg gave the optimum 5.7 mg and h i g h e r gave r e s u l t s lower than the controls  Brohult  (1958)  alkoxyglycerols including selachyl  man  25 mg/Kg w i t h meals  of some v a l u e where leuko- or thrombo-cytopenia has a l r e a d y o c c u r r e d  Brohult  (1958)  alkoxyglycerols  rats  the growth response was as Brohult f o l l o w s : normal, 1.7 gm/day; i r r a d i a t e d and t r e a t e d , 1.3 gm/day; i r r a d i a t e d without treatment, 1.1 gm/day  (1960)  alkoxyglycerols  bacteria  marked growth response  Brohult  (1960)  Brohult  (1962)  1  alkoxyglycerol esters  300 cancer of the cervix patients  radiated  20 mcgm/ml of media 2 mcgm/ml of media radiated  observable growth response  white c e l l and thrombocyte counts were h i g h e r f o r the t r e a t e d p a t i e n t s  TABLE XIV (cont'd)  Glyceryl Ether  Animal  Condition  Dosage  Response  Reference  batyl  cattle  normal  .5 mg/Kg on a l t e r n a t e days  no h a e m a t o l o g i c a l response  Dalton  (1964)  batyl  pregnant rats  radiated  .5 mg/Kg w i t h and w i t h o u t cystamine  l e s s s t i l l b o r n , anemia and l e u c o p e n i a prevented  Dud i n  (1961)  batyl  mice  radiated  10 mg every other day  l e t h a l i t y i s lower i n the t r e a t e d group  Edlund  (1954)  unsaponifiables  bacteria  1 mcgm/ml  inhibited marked  Emmerie, et al(1952)  batyl  cattle  bracken poisoning  .5 t o 1 mg  promoted r e c o v e r y from  Evans, e t a l (1958)  batyl  cattle  bracken poisoning  1 mg and 2 mg  gave a l e u c o c y t e response  Evans, e t a l (1957)  batyl  rats  normal  0.5 mg f o r 11 t h e r e was no response days; 1 mg f o r i n the b l o o d p i c t u r e 20 days, f o l l o w e d by 5 mg f o r 10 days (subcutaneous)  batyl  rats  benzene poisoning  relieves  Kaby 700  man  radiated  batyl  rabbit  anemia by Nitromin  growth,  Evenstein  (1958)  de G a e t a n i  (1959)  response i n o n l y 4 out of 15 cases  Ghys  (1960)  a haemopoietic response  Hasegawa  (1961)  toxicity  TABLE XIV (cont'd)  Glyceryl Ether  Animal  Condition  Dosage  Response  Reference  batyl  rats  normal  12.5 and 25 mg  response i n e r y t h r o c y t e s , thrombopoiesis, and granulopoiesis  Linman  (1958)  batyl  rats  normal  50 mg  a haemopoietic response  Linman  (1959)  batyl  rats  normal  165 - 330 mg  a haemopoietic response  Linman  (1960)  selachyl  rats  normal  312 - 356  no a c t i v i t y  Linman  (1960)  glyceryl ethers  man  effective i n treating wounds caused by X-ray burns  Maisen, ^ t a l (1959)  glyceryl ethers  rainbow trout  normal  d i g e s t i o n and metabolism data obtained  M a l i n s , eit a l (1964)  granulocytopenia  favourable r e s u l t s  Marberg  Melander (1954) (cited i n Brohult)  unsaponifiables of bone marrow  man  alkoxy esters  mice  normal  193 mg/Kg  four d i e d w i t h c a t a r r h a l e n t e r i t i s , survivors showed no path, changes  alkoxy e s t e r s (mainly selachyl)  mice  normal  167 mg/Kg  no deaths; weight g a i n normal  batyl  mice  normal  73 mg/Kg  no deaths, weight g a i n normal  (1936)  CO  o  TABLE XIV (cont'd)  Glyceryl Ether  Animal  Condition  Dosage  Response  alkoxy esters (mainly selachyl)  mice  normal  533 mg/Kg 1467 mg/Kg  no deaths; weight g a i n normal  b a t y l and selachyl  guinea  normal  10 mg/Kg  b a t y l a l c o h o l gave s i g n i f i c a n t changes i n both bone marrow and b l o o d ; s e l a c h y l showed no changes  Osmond  batyl  cattle  normal  3.1 mg/Kg intravenous  no response  Penny, e t a l (1964)  batyl  sheep  normal  2 t o 10 mg/Kg intravenous  no response  batyl  mice  normal  114 mg/Kg subcutaneous  no response  selachyl  rats  radiated  ineffective  Prokhonchukov and Panikarovskii (1963)  batyl  rats  radiated  s a t i s f a c t o r y therap e u t i c response  Prokhonchukov and Panikarovskii (1963)  batyl  rats  radiated  a c t i v a t i o n of b l o o d production  Rusanov, e_t a l (1962)  chimyl  blood cells  inhibited lysolecithin hemolysis  Safanda and Holecek  Pig  Reference  (1963)  (1965)  TABLE XIV  Glyceryl Ether  Animal  Condition  batyl  rats  benzene depleted  batyl  man  normal  batyl  calves  trichloroethylene extracted soybean meal  selachyl  pea  selachyl  dogs, rabbits, rats  radiated  a and B batyl  rat  normal  bone marrow  man  granulocytopenia  C  lh  Dosage  0.3 to 0.65 mg/Kg  stems  35 mg f e d  (cont'd)  Response  Reference  r i s e i n e r y t h r o c y t e s and a decrease i n n e c r o s i s  Sandler  (1949)  reticulocytosis  Sandler  (1949)  no response  S c h u l t z e , e_t a l (1958)  s t i m u l a t e s a u x i n and g i b e r e l l i n action  Stove  alleviated  S v i r i d o v and Abaturova (1964)  sickness  (1960)  d i g e s t i o n and metabolism studied  S w e l l , e t al(1965)  favourable  Watkins (1933) ( c i t e d i n Marberg)  results  PART I I :  EXPERIMENTAL  33  A.  PRELIMINARY EVALUATION - TRIAL I  1„ Preamble The  e x p e r i m e n t a l work c a r r i e d out as a p a r t of t h i s study can  c o n v e n i e n t l y be d i v i d e d i n t o two s e c t i o n s ; p r e l i m i n a r y t r i a l s and nutritional investigations. was n e c e s s a r y  P r i o r t o any n u t r i t i o n a l i n v e s t i g a t i o n s , i t  t o a s c e r t a i n i f the a - g l y c e r y l e t h e r s would produce any  d e l e t e r i o u s e f f e c t s on animal growth and development.  I n the absence o f  q u a n t i t a t i v e i n f o r m a t i o n on the dose-response  r e l a t i o n s h i p of the a - g l y c e r y l  e t h e r s f o r mammalian systems i t was n e c e s s a r y  t o e s t a b l i s h a more or l e s s  a r b i t r a r y dosage l e v e l w i t h which t o b e g i n the p r e l i m i n a r y t r i a l s .  The  amounts o f these compounds i n v a r i o u s t i s s u e systems i s i n the range n o r m a l l y expected  f o r c e r t a i n of the v i t a m i n s .  H a l l g r e n and L a r s s o n  bovine m i l k t o have 0.01 p e r c e n t o f i t s l i p i d  (42) r e p o r t  f r a c t i o n as g l y c e r y l e t h e r s .  A c a l c u l a t i o n f o r m i l k c o n t a i n i n g f o u r p e r c e n t f a t suggests 400 meg o f g l y c e r y l e t h e r s p e r 100 grams of m i l k .  a f i g u r e of  T h i s l e v e l i s of the same  order o f magnitude as t h a t n o r m a l l y r e c o r d e d f o r a number of the v i t a m i n s present i n milk  (see T a b l e XV).  On the b a s i s o f the above, a dosage l e v e l o f 6 mg p e r k i l o g r a m o f body weight p e r day was a r b i t r a r i l y s e l e c t e d f o r p r e l i m i n a r y t o x i c i t y trials.  T a b l e XVI a f f o r d s a comparison o f t h i s dosage l e v e l w i t h recommended  l e v e l s f o r s e v e r a l of the v i t a m i n s .  Both s e t s of v a l u e s a r e expressed as  meg p e r k i l o c a l o r i e of d i g e s t i b l e energy comparison.  i n the d i e t t o permit  direct  These v a l u e s a r e a l l computed f o r r a t s ; s i m i l a r e s t i m a t e s can  be made f o r the dog. d i g e s t i b l e energy  I n these c a l c u l a t i o n s i t was assumed t h a t the  o f the feed was 3800 k i l o c a l o r i e s per k i l o .  The  TABLE XV:  REPRESENTATIVE VALUES OF IMPORTANT VITAMINS IN MILK (from R u s o f f ) (88)  Vitamins  meg p e r 100 gm o f m i l k  Fat s o l u b l e v i t a m i n s vitamin A a c t i v i t y vitamin D vitamin E  42 - 63 0.045 - 0.058 60  Water-soluble thiamine riboflavin n i c o t i n i c acid pantothenic acid pyridoxine f o l i c acid biotin cobalamin vitamin C choline inositol (total)  TABLE XVI:  40 45 150 200 100 80 400 350 48 25 0.1 - 0.23 2.0 - 3.5 0.3 - 0.5 2000 13000 13000  AMOUNT OF GLYCERYL ETHER ADMINISTERED COMPARED WITH THE VITAMIN REQUIREMENTS OF THE ALBINO LABORATORY RAT  meg per k i l o c a l o r i e o f d i g e s t i b l e energy dosage o f g l y c e r y l e t h e r per k i l o g r a m o f body weight 6 mgs  11  Vitamin A cobalamin riboflavin choline chloride niacin p y r i d o x i n e HC1 t h i a m i n HC1  1.58 0.0013 0.76 197 4 0.32 0„66  apparent d i g e s t i b l e energy i n t a k e (A.D.E.I.) i n k i l o c a l o r i e s per day f o r the  r a t may  be e s t i m a t e d from the e x p r e s s i o n (Cheeke)  A.D.E.I.  =  6.82  W°  , l + 5 1  where W r e p r e s e n t s body weight i n grams. a 100 gram r a t would day.  (24)  On the b a s i s of t h i s e q u a t i o n ,  i n g e s t 54 k i l o c a l o r i e s of d i g e s t i b l e feed energy per  I n o r d e r t h a t t h i s r a t may  r e c e i v e 6 mg  of a g l y c e r y l e t h e r per  gram of body weight per day, i t s r a t i o n would have to p r o v i d e 11 meg  kiloof the  e t h e r per k i l o c a l o r i e of r a t i o n d i g e s t i b l e energy (54 times 11 equals 594 meg  per 100 gram r a t which i s a p p r o x i m a t e l y 6 mg  per k i l o g r a m of r a t ) c  2. E x p e r i m e n t a l D e s i g n E i g h t y male and female r a t s were randomly a l l o t t e d i n t o f o u r w i t h e q u a l numbers of each sex i n each group. f i v e dogs were used. to  groups  Four groups each c o n t a i n i n g  Each of the f o u r groups of r a t s and dogs were a l l o c a t e  a treatment as f o l l o w s :  one c o n t r o l group and one group f o r each o f the  t h r e e important n a t u r a l l y o c c u r r i n g g l y c e r y l e t h e r s .  These animals were f e d  n o r m a l l y and p r e s e n t e d w i t h t h e i r r e s p e c t i v e doses d a i l y f o r a p e r i o d of 6 months.  The r a t s were weighed  F r i d a y ) ; the dogs were weighed  t h r e e times weekly  once each week.  r e c o r d e d f o r the r a t s , but not f o r the dogs.  (Monday, Wednesday, and  D a i l y feed i n t a k e d a t a were Blood samples were o b t a i n e d  from both the dogs and the r a t s a c c o r d i n g t o the s c h e d u l e g i v e n i n T a b l e XVII.  E r y t h r o c y t e s and l e u c o c y t e s were counted u s i n g s t a n d a r d  methods (see Appendix XVI). each group were k i l l e d .  A f t e r 180 days a l l the r a t s and one dog from  Gross p a t h o l o g y was  r e c o v e r e d i n methanal a c e t a t e .  noted and t i s s u e s were  S e c t i o n s from the h e a r t , l u n g s ,  liver,  k i d n e y s , a d r e n a l s , i n t e s t i n e and s p l e e n were p r e p a r e d f o r h i s t o l o g i c a l study  TABLE XVII:  SCHEDULE OF BLOOD COUNTS  Time of b l o o d sampling* Rats erythrocytes l e u c o c y t e counts  180 90 and 180  Dogs erythrocytes l e u c o c y t e count  110 180  * Days from the b e g i n n i n g o f the t r i a l  3. Animals A l b i n o r a t s of the UBC W i s t a r s t r a i n and Labrador dogs, both r e a r e d at the u n i v e r s i t y animal u n i t s , were used i n t h i s experiment.  The r a t s were  p l a c e d on experiment a t a body weight of a p p r o x i m a t e l y 90 grams and the dogs at 10 k i l o g r a m s .  The d e t a i l s o f the weight changes i n b o t h groups of  e x p e r i m e n t a l animals a r e g i v e n i n Appendix I I .  4. R a t i o n The r a t s were f e d UBC s t o c k r a t r a t i o n i n ground the measurement o f feed i n t a k e . spillage.  A c o r r e c t i o n was n o t a p p l i e d f o r feed  The dogs were f e d UBC s t o c k dog crumbles.  dogs were f e d aid l i b i t u m . g i v e n i n Appendix I .  form t o f a c i l i t a t e  Both the r a t s and the  The c o n s t i t u e n t c o m p o s i t i o n of these r a t i o n s i s  37  5.  Housing The  r a t s were housed i n d i v i d u a l l y i n w i r e cages i n a room w i t h  c o n t r o l l e d ambient temperature and h u m i d i t y .  The dogs were housed i n  i n d i v i d u a l c o n c r e t e b l o c k pens u s i n g wood shavings f o r l i t t e r . exercised  They were  outdoors from 10:00 a.m. t o 4:00 p.m. each day, weather  permitting.  6. A d m i n i s t r a t i o n The  of g l y c e r y l e t h e r s  r a t s were dosed w i t h each of the t h r e e g l y c e r y l e t h e r s  suspended i n a s a f f l o w e r diluent.  oil/safflower ethyl esters/oleic acid  The c o n t r o l r a t s r e c e i v e d  the b a t y l and c h i m y l a l c o h o l s  the d i l u e n t  only.  (55/40/5)  The dogs  received  i n t a b l e t form, the s e l a c h y l a l c o h o l was  suspended i n the above d i l u e n t .  As was the case w i t h the r a t s , the c o n t r o l  dogs r e c e i v e d  The g l y c e r y l e t h e r s ,  the d i l u e n t o n l y .  i n diluent  suspension,  were a d m i n i s t e r e d p e r orum w i t h a s y r i n g e .  Those dogs r e c e i v i n g  their  g l y c e r y l e t h e r s i n t a b l e t form, were f o r c e d  to swallow t h e i r d a i l y dose.  7. R e s u l t s All  o f the p e r t i n e n t  d a t a c o n c e r n i n g feed  intake,  body weight  changes and b l o o d counts a r e g i v e n i n Appendices I I , I I I and IV. considering  a l l the d a t a c o l l e c t e d d u r i n g the 180 day t r i a l  c o l l e c t e d a t the time o f s l a u g h t e r , deleterious  T a b l e X V I I I , XIX and XX summarize  the data g i v e n i n the Appendices I I and I I I .  the  e f f i c i e n c i e s of the t r e a t e d  same as the c o n t r o l s .  and the data  t h e r e appeared t o be no evidence of  e f f e c t s from the treatments.  g a i n s and feed  After  They r e v e a l  that  the weight  dogs and r a t s were e s s e n t i a l l y  A " t " test indicates  that  the r a t e o f g a i n o f  TABLE X V I I I :  SUMMARY OF MEAN BODY WEIGHT GAIN AND MEAN FEED-INTAKE DATA FOR MALE RATS  Selachyl f i n a l weight i n i t i a l weight t o t a l weight g a i n gain/day t o t a l feed consumed feed i n t a k e / d a y gm o f feed/gm o f g a i n  TABLE XIX:  (gm) (gm) (gm) (gm) (gm) (gm)  421.5 ' 95.1 326.4 1.79 3,925 21.6 12.0  Batyl  Chimyl  Control  413.9 94.4 319.5 1.76 3,736 20.5 11.6  412.8 94.0 318.8 1.75 3,666 20.1 11.5  .418.9 88.7 330.2 1.81 3,829 21.0 11.6  SUMMARY OF MEAN BODY WEIGHT GAIN AND MEAN FEED-INTAKE DATA FOR FEMALE RATS  Selachyl f i n a l weight i n i t i a l weight t o t a l weight g a i n gain/day t o t a l feed consumed feed i n t a k e / d a y gm of feed/gm o f g a i n  TABLE XX:  (gm) (gm) (gm) (gm) (gm) (gm)  267.3 85.9 181.4 1.0 3,263 17.9 18.0  255.4 86.4 169.0 0.93 3,041 16.7 18.0  Chimyl 253.3 88.1 165.2 0.91 3,132 17.2 19.0  Control 256.4 85.7 170o7 0.94 2,930 16.1 17 o 2  SUMMARY OF BODY WEIGHT GAIN FOR DOGS*  Selachyl f i n a l weight i n i t i a l weight t o t a l weight g a i n weight gain/week weight gain/dog/week  Batyl  (Kg) (Kg) (Kg) (Kg) (Kg)  109.7 51.6 58.1 2.23 0.45  Batyl 103,,7 53.,6 50,,1 1..93 0.,39  *There were f i v e dogs i n each of these groups  Chimyl 115.0 58.1 56.9 2.19 0.44 •  Control 104.0 47.6 56.4 2.17 0.43  FIGURE I :  GROWTH CURVES FOR FEMALE RATS  30 Cf  o UJ  250f-  •  >-  Q o  •  «  •  •  $  8  *  • • • •  —' w 1  CO  CD  •  •  20C4-  50  <  I50f-  X X X  • • • 0 0 0 lOOf  O O O  CONTROL SELACHYL BATYL CHIMYL  1 10 WEEKS  20 ON  EXPERIMENT  25  40  the female  r a t s In the s e l a c h y l group i s s i g n i f i c a n t l y h i g h e r than t h a t  of the c o n t r o l female r a t s . response  However, the s i g n i f i c a n c e of t h i s growth  i s q u e s t i o n e d because i t d i d not occur i n the male r a t s on  s e l a c h y l treatment.  The b a t y l and  c h i m y l t r e a t e d groups of female  have i d e n t i c a l growth curves t o the c o n t r o l group.  the rats  The growth curve  ( F i g u r e I) f o r the female r a t s t h a t r e c e i v e d the s e l a c h y l a l c o h o l was i d e n t i c a l w i t h t h a t of the o t h e r groups to a mean body weight of about 200  grams.  Above t h i s body weight t h i s group gained weight a t a  slightly  f a s t e r r a t e and hence reached a s l i g h t l y h e a v i e r weight a t the end of the experiment. groups was  T h i s i n c r e a s e i n body weight above the o t h e r significant  u n t i l a f t e r puberty.  ( a t the .05  level).  experimental  T h i s i n c r e a s e d i d not  occur  I t d i d not appear i n the male r a t s r e c e i v i n g  these  alcohols. The growth curves of the male r a t s and of the dogs a r e not  presented  here because the d i f f e r e n t e x p e r i m e n t a l groups d i s p l a y e d i d e n t i c a l growth responses. T a b l e XXI g i v e s a summary of the b l o o d counts which were  taken  d u r i n g t h i s t r i a l and which a r e g i v e n i n d e t a i l i n Appendix IV.  There  a l a r g e v a r i a t i o n i n l e u c o c y t e counts between animals  group  i n any  one  ( e s p e c i a l l y i n the case of the r a t s ) , but v e r y s m a l l d i f f e r e n c e s i n counts between the c o n t r o l and t r e a t e d groups.  significantly  (.05)  from a low l e v e l  U s i n g the " t " t e s t on the l e u c o c y t e count  at 90 days, r e v e a l e d t h a t female  r a t s g i v e n the s e l a c h y l treatment  h i g h e r counts.  average  This v a r i a b i l i t y i n leucocyte  count p r o b a b l y a r o s e because the r a t s were s u f f e r i n g c u r r e n t lung i n f e c t i o n .  was  At v a r i a n c e w i t h t h i s r e s u l t ,  inter-  data, had the  TABLE XXI:  SUMMARY* OF BLOOD COUNTS TAKEN IN TRIAL I  Selachyl  Batyl  Chyml  Control  18,340 18,550  15,440 17,665  15,690 22,110  14,600 19,800  15,560 18,200  16,010 19,340  14,510 19,540  14,320 17,560  9.11  8.82  9.58  8.87  0.28  0.39  0.52  0.46  17,200  17,200  16,900  17,500  Rats leucocytes a f t e r 90 days female male leucocytes a f t e r 180 days female male erythrocytes after 180 days male Dogs erythrocytes after 110 days leucocytes a f t e r 180 days  *For r a t s , these a r e means o f 10 animals and f o r dogs these a r e means of 5 animals. Leucocytes a r e g i v e n as number o f c e l l s p e r cmm o f b l o o d and e r y t r o c y t e s i n m i l l i o n s o f c e l l s per cmm of b l o o d .  42  l e u c o c y t e counts f o r the male r a t s a t 90 days showed t h a t both the s e l a c h y l and b a t y l groups had s i g n i f i c a n t l y animals.  (.05) lower counts than the c o n t r o l  A t 180 days the " t " t e s t d i d not r e v e a l any s i g n i f i c a n t  differences,  p o s i t i v e o r n e g a t i v e , between the c o n t r o l and t r e a t e d male and female The  rats.  l e u c o c y t e counts taken o f the dogs' b l o o d a f t e r 180 days d i d not show  any s i g n i f i c a n t  (.05) d i f f e r e n c e s .  D e s p i t e the f a c t t h a t  statistically  s i g n i f i c a n t d i f f e r e n c e s have been noted i n a few comparisons  i n r e l a t i o n to  time o f examination and treatment, the o v e r a l l p a t t e r n o f the r e s u l t s does not permit the d e r i v a t i o n o f any f i n a l  conclusions.  E r y t h r o c y t e counts showed much l e s s v a r i a t i o n between i n d i v i d u a l s than d i d the l e u c o c y t e counts, b u t the d i f f e r e n c e s between groups was quite small.  still  S t a t i s t i c a l a n a l y s i s o f e r y t h r o c y t e counts from both dogs and  r a t s r e v e a l e d a s i g n i f i c a n t d i f f e r e n c e i n o n l y one case, i n which  the c h i m y l  group o f male r a t s showed h i g h e r counts than the c o n t r o l s . The h i s t o p a t h o l o g i c a l p r e p a r a t i o n s from the v a r i o u s t i s s u e  systems  of the r a t s and the dogs r e v e a l e d no apparent d i f f e r e n c e s between the treatment and the c o n t r o l  groups.  8. C o n c l u s i o n G l y c e r y l e t h e r s a d m i n i s t e r e d a t a l e v e l o f 6 mg p e r k i l o g r a m o f body weight over p r o l o n g e d p e r i o d s o f time a r e not d e l e t e r i o u s t o the growth of r a t s or of dogs. demonstrate systems  With the methods used h e r e , i t was n o t p o s s i b l e t o  any e f f e c t o f the a - g l y c e r y l e t h e r s on the h a e m a t o p o i e t i c  of either  species.  43  B.  1.  PRELIMINARY EVALUATION - TRIAL I I  Preamble Having e s t a b l i s h e d t h a t the a - g l y c e r y l e t h e r s j when f e d a t a  l e v e l comparable to demonstrated v i t a m i n r e q u i r e m e n t s , produced no d e l e t e r i o u s e f f e c t s on the r a t or dog, i t was  c o n s i d e r e d n e c e s s a r y to  a s c e r t a i n i f these n a t u r a l compounds had such e f f e c t s a t much h i g h e r levels.  dosage  For t h i s purpose an upper l e v e l of 2400 mg per k i l o g r a m of body  weight per day was Trial I).  selected  ( t h i s i s 400 times the dosage l e v e l used i n  Because of the c o n s i d e r a b l e expense a s s o c i a t e d w i t h these compounds  and w i t h t r i a l s w i t h dogs, o n l y b a t y l and s e l a c h y l a l c o h o l s were i n t h i s phase of the work.  Chimyl a l c o h o l was  examined  eliminated since i t s l e v e l  i n t i s s u e r e l a t i v e to the o t h e r s i s c o m p a r a t i v e l y low.  Batyl alcohol i s a  s u i t a b l e r e p r e s e n t a t i v e of the group of s a t u r a t e d g l y c e r y l e t h e r s and s e l a c h y l of the u n s a t u r a t e d e t h e r s . One group of r a t s and one group of dogs were o f f e r e d a r a t i o n c o n t a i n i n g s e l a c h y l a l c o h o l a t a l e v e l which p r o v i d e d a d a i l y i n t a k e of 600 mg per k i l o g r a m . i n t e r m e d i a t e dosage  T h i s was  done to p r o v i d e data on the e f f e c t s of an  level.  2. D e s i g n F o r t y r a t s were randomized i n t o f o u r groups of t e n animals cont a i n i n g f i v e males and f i v e females.  S i x t e e n dogs were d i v i d e d  randomly  i n t o f o u r groups of f o u r , w i t h two males and two females i n each group. The treatment of each of the f o u r groups from each s p e c i e s was  as f o l l o w s :  a c o n t r o l group, two groups f e d s e l a c h y l a l c o h o l and a group f e d b a t y l  44  alcohol.  The d e t a i l s of animal grouping and dosage schedule a r e g i v e n i n  T a b l e XXII.  The g l y c e r y l e t h e r s were i n c o r p o r a t e d i n t o the feed t o f a c i l i t a t e  administration. of  Feed i n t a k e was r e s t r i c t e d t o ensure complete  a l l the feed o f f e r e d each day.  consumption  The r a t s were o f f e r e d feed a t a l e v e l  c a l c u l a t e d t o be 80 p e r c e n t o f maximum i n t a k e t o a body weight then 90 p e r c e n t of maximum i n t a k e t o a body weight  of 160 grams,  of 190 grams and, f i n a l l y ,  100 p e r c e n t o f the c a l c u l a t e d maximum feed i n t a k e t o the end o f the 60 day t r i a l period. full  The dogs were f e d 80 p e r c e n t of maximum f e e d i n t a k e f o r the  two month p e r i o d .  The r a t s were weighed t h r e e times per week (Monday,  Wednesday, and F r i d a y ) ; the dogs were weighed once per week.  A t the end of  one month and a g a i n a t the end of two months, b l o o d samples were taken from all  of the animals; r e d b l o o d c e l l counts, white b l o o d c e l l counts, and  differential  counts o f the white b l o o d c e l l s were made.  Feed and f a e c e s  samples were taken every two weeks d u r i n g the two month e x p e r i m e n t a l p e r i o d . A f t e r two months a l l o f the r a t s and o n e - h a l f of the dogs (two from group) were k i l l e d , smears made.  each  posted, gross pathology noted, and femur bone marrow  The remaining e i g h t dogs were t r e a t e d to a l l e v i a t e an i n t e s t i n a l  p a r a s i t e i n f e s t a t i o n and were then p l a c e d on the c o n t r o l r a t i o n f o r one month.  Subsequently  they were k i l l e d ,  p o s t e d , gross p a t h o l o g y noted, and  femur bone marrow smears made.  In a d d i t i o n , t i s s u e s e c t i o n s were taken  from each animal; h e a r t , l i v e r ,  s p l e e n , k i d n e y , a d r e n a l and i n t e s t i n e .  45  TABLE XXII:  Group*  S  B S C  k  0  D a i l y Dose  °  2.4  1 0 0  0  gm  Rats male female  selachyl/Kg  2.4 gm b a t y l / K g 0.6 gm s e l a c h y l / K g 2.4 gm s a f f l o w e r o i l / K g  4 0 0  h  GROUPING AND DOSAGE LEVELS USED IN TRIAL I I  °  Dogs male female  5  5  2  2  5 5 5  5 5 5  2 2 2  2 2 2  * The s u p e r s c r i p t and the s u p e r s c r i p t r e f e r t o 400 times and 100 times the 6 mg dosage l e v e l used i n T r i a l I . 0  0  i  U  U  3. Animals A l b i n o r a t s of the UBC W i s t a r s t r a i n and Labrador dogs, both r e a r e d in  the u n i v e r s i t y animal u n i t s , were used i n t h i s experiment.  The r a t s were  p l a c e d on experiment a t a weight of a p p r o x i m a t e l y 80 grams and the dogs a t approximately 12 k i l o g r a m s .  The a c t u a l i n i t i a l weights a r e g i v e n i n  Appendix V.  4. Housing The r a t s were housed c o n t r o l l e d ambient  i n d i v i d u a l l y , i n w i r e cages, i n a room w i t h  temperature and h u m i d i t y .  The dogs were housed i n  i n d i v i d u a l c o n c r e t e b l o c k pens u s i n g wood shavings f o r l i t t e r .  The dogs  were e x e r c i s e d from 10:00 a.m. t o 4:00 p.m. every day i n an o u t s i d e r u n , weather  permitting.  5. R a t i o n and a d m i n i s t r a t i o n of g l y c e r y l  ethers  Both the dogs and the r a t s were f e d the s t o c k UBC dog crumbles (UBC r a t i o n No. 14 - 63 g i v e n i n Appendix arrangements  used i n the f i r s t  trial  I).  This d i f f e r s  from the r a t i o n  i n which the r a t s r e c e i v e d UBC r a t i o n  46  No.  10 - 63.  The a p p r o p r i a t e amounts of g l y c e r y l e t h e r s , and f o r the  c o n t r o l group, s a f f l o w e r o i l were i n c o r p o r a t e d fed  as a d r y mash.  A regression equation  i n t o the dog crumbles and  r e l a t i n g maximum feed i n t a k e t o  body weight was c a l c u l a t e d from feed i n t a k e v a l u e s used i n T r i a l I .  A corresponding  recorded  r e g r e s s i o n equation  f o r the r a t s  f o r dogs was c a l c u l a t e d  from data c o l l e c t e d d u r i n g a s i x day p r e l i m i n a r y f e e d i n g t r i a l when d r y dog crumbles were f e d to 16 dogs having (8.2  t o 14.1 k i l o g r a m s ) .  lower f a t content  a body weight range of 18 t o 31 pounds  The r a t i o n used t o e s t a b l i s h feed i n t a k e was o f  than t h a t used i n t h i s experiment, hence the standard  s e l e c t e d was reduced t o 80 p e r c e n t , by weight, of p r e d i c t e d f u l l intake.  The a d d i t i o n of 5.8 p e r c e n t  feed-  g l y c e r y l e t h e r and, i n the case of the  controls, safflower o i l ,  t o the e x p e r i m e n t a l  content by approximately  7 percent  r a t i o n s i n c r e a s e d the energy  so the dogs being  f e d 80 p e r c e n t  maximum c a l c u l a t e d feed i n t a k e were a c t u a l l y r e c e i v i n g 87 p e r c e n t maximum energy i n t a k e .  The r e g r e s s i o n equations  so o b t a i n e d  of t h e i r  of t h e i r  are given  below:  The  rats -  grams of feed i n t a k e  =  6.68W " gm  dogs -  grams of feed i n t a k e  =  57.79W ' Kg  0  0  157  955  exponent 0.956 f o r the dogs was not s i g n i f i c a n t l y d i f f e r e n t  hence the feed i n t a k e standard the weight range used.  from u n i t y  f o r t h i s s p e c i e s was taken t o be l i n e a r  The animals were f e d l e s s than t h e i r maximum  c a l c u l a t e d feed i n t a k e t o ensure t h a t a l l feed o f f e r e d was eaten; it  p o s s i b l e t o add s u f f i c i e n t g l y c e r y l ethers  the S  4 0 0  , B  4 0 0  and C ^  0 0  over  t h i s made  t o the r a t i o n t o ensure t h a t  groups r e c e i v e d 2.4 gm/Kg and S  1 0 0  , 0.6 gm/Kg of  47  body weight, d a i l y . was  The b l e n d i n g o f the g l y c e r y l e t h e r s i n t o the r a t i o n  a d j u s t e d a f t e r each weighing  o f the r a t s t o keep the l e v e l o f  a d m i n i s t r a t i o n a t the p r e s c r i b e d p o i n t .  F o r the dogs, the g l y c e r y l  ethers  formed 5.7 p e r c e n t o f t h e i r r a t i o n and f o r the r a t s i t v a r i e d from 1.7 p e r c e n t at the b e g i n n i n g end.  of the experiment t o 3.5 percent  T h i s was n e c e s s a r y  o f t h e i r r a t i o n a t the  i n the case o f the r a t s s i n c e feed i n t a k e p e r u n i t  of body weight decreases markedly as body weight i n c r e a s e s .  By i n c r e a s i n g  the r e l a t i v e l e v e l o f g l y c e r y l e t h e r s i n the r a t i o n as body weight i n c r e a s e d , i t was p o s s i b l e t o ensure t h a t the d a i l y i n t a k e o f the e t h e r s p e r u n i t weight of animal remained a t the p r e s c r i b e d l e v e l . The was  found  r e g r e s s i o n e q u a t i o n p r e d i c t i n g maximum feed i n t a k e f o r the r a t s  t o be i n e r r o r , as the i n i t i a l r e s t r i c t e d l e v e l o f 80 p e r c e n t had  to be r a i s e d t o 90 p e r c e n t a t the end o f 30 days and t o 100 p e r c e n t a t the end of 45 days, i n order t o m a i n t a i n a r e a s o n a b l e (approximately  2.3 grams/day).  r a t e o f growth  With the dogs, f e e d i n g a t 80 p e r c e n t of  the p r e d i c t e d maximum l e v e l was s u f f i c i e n t p e r i o d t o cause the c o n t r o l animals  throughout  the 60 day f e e d i n g  t o grow a t a r a t e of 0.78 kilograms  per week.  6. D i g e s t i b i l i t y  procedures  (a) D i g e s t i b i l i t y o f the e x p e r i m e n t a l r a t i o n s (i)  Rats The  feed i n t a k e was recorded  f o r the r a t s each day. T o t a l  48 hour f e c a l c o l l e c t i o n s were made a t two week i n t e r v a l s .  The  d i g e s t i b i l i t y v a l u e s c a l c u l a t e d from t h i s data a r e presented i n T a b l e XXVIII.  ( i i ) Dogs Chromic oxide (0.1%) was  added to the dog r a t i o n and  f e c a l samples were c o l l e c t e d from the i n d i v i d u a l dogs a t two week i n t e r v a l s .  In the d e t e r m i n a t i o n s , the C  were t r e a t e d as a whole, w h i l e the B  400  400  and S 400  and S 100  groups  groups were  t r e a t e d a c c o r d i n g to whether they a t e the same amount as the c o n t r o l s or n o t .  The d i g e s t i b i l i t y  of the dog r a t i o n s  was  determined by the i n c r e a s e i n the c o n c e n t r a t i o n of chromic oxide i n faeces over t h a t i n the f e e d , f o l l o w i n g the method proposed by Schiirch, et_ a l ( 9 3 ) .  The d i g e s t i b i l i t y  v a l u e s so  o b t a i n e d are used i n T a b l e XXVII.  (b)  I s o l a t i o n and d e t e r m i n a t i o n of g l y c e r y l e t h e r s * (i)  E x t r a c t i o n of l i p i d s  from feed and f a e c e s * *  F i r s t l y , samples of the m a t e r i a l to be e x t r a c t e d were oven d r i e d a t 110 degrees c e n t i g r a d e f o r two h o u r s .  These samples  were subsequently used f o r chromate d i g e s t i b i l i t y d e t e r m i n a tions.'  Another r e p r e s e n t a t i v e sample (5 gm on a dry weight  b a s i s ) of the m a t e r i a l was weighed i n t o a 16 ounce j a r . c a l c u l a t e d amount of water was of water to 80 p e r c e n t . be  unity.  added t o i n c r e a s e the percentage  This t o t a l  sample s i z e was  An e q u a l amount of c h l o r o f o r m and twice  amount of methanol was  added.  A  T h i s m i x t u r e was  taken to this  then b l e n d e d  * These are the methods approved and employed by Western Chemical I n d u s t r i e s L i m i t e d o f Vancouver. * * T h i s i s a m o d i f i e d B l i g h and Dyer procedure (13) t h a t was used f o r l i p i d e x t r a c t i o n of f i s h .  with a p r o p e l l e r - t y p e s t i r r e r a t about 2000 rpm Then one u n i t of c h l o r o f o r m was minute. for  T h i s was  one minute.  f o r 5 minutes.  added and blended  f o r one  f o l l o w e d by one u n i t of water blended The whole m i x t u r e was  a s t a i n l e s s s t e e l f u n n e l and  then t r a n s f e r r e d to  the s o l v e n t f i l t e r e d  into a  250 ml c y l i n d e r u s i n g a s l i g h t n e g a t i v e p r e s s u r e . was to  washed w i t h a f u r t h e r one and g i v e a mixture  residue  of s o l v e n t s w i t h the f i n a l c o m p o s i t i o n  The  T h i s mixture  the t o t a l l i p i d  The  of  separated  l i p i d - c o n t a i n i n g c h l o r o f o r m phase  t r a n s f e r r e d to an e v a p o r a t i n g f l a s k . s t r i p p e d o f f , and  The  o n e - h a l f u n i t s of c h l o r o f o r m  2/2.5/1.8 (chloroform/methanol/water). i n t o two phases.  in  chloroform  was  was  r e s i d u e d r i e d to a c o n s t a n t  weight i n a vacuum oven a t 60 degrees c e n t i g r a d e . (ii)  Determination The  acetone and  total lipid  f r a c t i o n was  per gram of l i p i d s .  the stoppered  centigrade.  taken up  i n 100 ml  of  Four ml of e t h y l e t h e r was  added  f l a s k p l a c e d i n running water a t 14 degrees  The p r e c i p i t a t e d p h o s p h o l i p i d s were removed by  f i l t r a t i o n and off  of n e u t r a l l i p i d s  the acetone  the n e u t r a l l i p i d s were r e c o v e r e d by  stripping  and d r y i n g the r e s i d u e to a constant weight  under vacuum a t 60 degrees c e n t i g r a d e . ( i i i ) Determination  of the u n s a p o n i f i a b l e l i p i d  fraction  The n e u t r a l l i p i d s were s a p o n i f i e d a c c o r d i n g to the f o l l o w i n g scheme.  T h i r t y ml of e t h a n o l and  50 p e r c e n t potassium  hydroxide were added to  t h r e e ml  of  approximately  one  gram o f n e u t r a l l i p i d s and r e f l u x e d f o r o n e - h a l f  Then t h i r t y ml o f water were added and the m i x t u r e to  cool.  ether  T h i s mixture  (30 ml each t i m e ) .  T h i s e x t r a c t was washed a l t e r n a t e l y  f i n a l l y w i t h water u n t i l  phenolphthalein.  allowed  was e x t r a c t e d f o u r times w i t h e t h y l  w i t h 20 ml o f water and 20 ml of 0.5 N potassium and  hour.  hydroxide  the washings were c l e a r t o  The e t h e r was then s t r i p p e d o f f and the  u n s a p o n i f i a b l e m a t e r i a l d r i e d t o a c o n s t a n t weight i n a vacuum oven a t 60 degrees c e n t i g r a d e . (iv)  Q u a l i t a t i v e and q u a n t i t a t i v e d e t e r m i n a t i o n ethers  of g l y c e r y l  Q u a l i t a t i v e p r e - e l u t e d 10 by 20 cm t h i n l a y e r p l a t e s (250 m i c r o n l a y e r o f s i l i c a g e l ) were s c o r e d w i t h 6 t o 7 l a n e s . About 100 gamma of g l y c e r y l e t h e r r e s i d u e was a p p l i e d t o a lane.  I d e n t i f i c a t i o n o f the components i n the r e s i d u e was made  by a p p l y i n g s u i t a b l e r e f e r e n c e compounds to c o r r e s p o n d i n g  lanes  These p l a t e s were e l u t e d w i t h 50/50/1 ( C H C l / C H O H / a c e t i c  acid)  3  Upon the completion 20 p e r c e n t for  3  of the e l u t i o n the p l a t e s were sprayed  with  s u l p h u r i c a c i d and c h a r r e d a t 150 degrees c e n t i g r a d e  20 minutes or u n t i l  the spots were s h a r p l y c h a r r e d .  The  amount o f g l y c e r y l ether i n the g l y c e r y l e t h e r r e s i d u e was determined by comparison of the spot s i z e w i t h the spot s i z e of known amounts of r e f e r e n c e g l y c e r y l e t h e r s . of  The a c t u a l weight  g l y c e r y l e t h e r s i n the g l y c e r y l ether r e s i d u e from the  p r e p a r a t o r y p l a t e was determined by u s i n g the c o r r e c t i o n f a c t o r determined from the q u a l i t a t i v e t h i n - l a y e r p l a t e .  51  7. D i s c u s s i o n of f e e d - I n t a k e , d i g e s t i b i l i t y and weight g a i n The  c o n s i d e r a t i o n of the e x p e r i m e n t a l r e s u l t s from the dogs  g i v e s r i s e to a number of problems. in  the i n i t i a l  weights  Firstly,  of the animals.  I t was  v a r i a t i o n s i n c e a l i m i t e d number of dogs was e x p e r i m e n t a l groups c o u l d be c o n s t i t u t e d . from some two of  dozen weanlings.  the s m a l l e s t and  i n weight from The of  the prepared  c o n s i d e r a t i o n was  rations.  one  necessary  to a c c e p t  c a r r i e d out by  The  elimination  s i x t e e n so s e l e c t e d  this f i r s t  varied  kilograms).  c o n c e r n i n g the  acceptability  A l l the dogs were fed the s t o c k dog  ration for  a f o u r week p e r i o d p r i o r to the commencement of the e x p e r i m e n t a l During  this  a v a i l a b l e from which the  S e l e c t i o n was  the l a r g e s t a n i m a l s .  a large variation  S i x t e e n pups were s e l e c t e d  18 to 31 pounds (8.2 to 14.1  second  there was  trial.  p e r i o d a l l of the dogs a t e and gained n o r m a l l y .  After  the dogs were p l a c e d on the e x p e r i m e n t a l r a t i o n s , i t became apparent a l l r a t i o n s were not e q u a l l y a c c e p t a b l e to the animals.  The  C^  the B  groups c o n t i n u e d to eat n o r m a l l y , but  0 0  S *  00  to  eat a l l the feed t h a t they were o f f e r e d .  1  r a t i o n s d i d not eat w i t h r e l i s h .  the B * 1  00  those o f f e r e d  S  that  and  1 0 0  4 0 0  and  In f a c t , some of the dogs r e f u s e d In the case of two  dogs i n  group, the s e l f - i m p o s e d f e e d - i n t a k e r e s t r i c t i o n a c t u a l l y l e d to  a body weight l o s s . r a t i o n s was  The a c c e p t a b i l i t y of the S ^  markedly l e s s than t h a t of the S  1 0 0  00  and B *  and C ^  1  00  experimental  00  rations.  Glyceryl  e t h e r s i n c o n t a c t w i t h water form complexes which adhere t o a d j a c e n t surfaces.  Thus the r a t i o n s h i g h i n g l y c e r y l e t h e r s tended  w i t h the p r e h e n s i o n and  i n g e s t i o n of the f e e d .  to i n t e r f e r e  Furthermore',  l e v e l s of g l y c e r y l e t h e r s i n the r a t i o n s gave them an unusual may  have been r e p e l l e n t to the  animals.  the h i g h odor which  52  TABLE X X I I I :  SUMMARY OF THE WEIGHT GAIN AND FEED INTAKE DATA OF DOGS  S * 1  00  I n d i v i d u a l Animals  #25  #10  #9  #23  Mean  f i n a l weight (Kg) 12.7 15. 9 12. 7 12. 3 13. 4 i n i t i a l weight (Kg) 10.9 14. 5 12. 3 10. 0 11. 9 t o t a l gain (Kg) 1.8 1. 4 0. 4 2. 3 1. 5 gain/Kg of weight 1.5 0. 9 (Kg) 0. 03 0. 21 0. 66 gain/week 0.200 0. 155 0. 044 (Kg) 0. 255 0. 164 feed o f f e r e d 38,955 30,380 (gm) 29,715 28,910 31,990 feed r e f u s e d 2,940 10,805 1; 095 (gm) nil 5,210 feed consumed 28,150 (gm) 26,775 23,285 28,910 26,780 feed consumed/week (gm) 2,975 3,128 3,212 2,587 2,975 gm feed/gm g a i n 14.9 20. 2 58. 8 12. 6 26. 6 gm feed/gm gain/Kg wt 1.26 1. 33 4. 70 2. 11 1. 13 gm feed/gm gain/W°' 7 3 2.46 2. 77 9. 30 2. 16 4. 17 1  B  H U U  I n d i v i d u a l Animals  #20 f i n a l weight  (Kg)  9. 5  i n i t i a l weight t o t a l gain  (Kg) (Kg)  10. 5 - 1.0  gain/Kg o f weight  (Kg)  - 0.1  gain/week  (Kg)  - 0.11  feed o f f e r e d feed refused feed consumed feed consumed/week gm feed/gm g a i n  (gm) 26,530 8,835 (gm) 17,695 (gm) 1,966 (gm)  gm feed/gm gain/Kg wt gm feed/gm gain/W°' 7 3  #34*  #14  14.1 10. 9 (15.0) 12.3 8. 6 1.8 2. 3 (2.7) 0.13 0. 26 (0.20) 0.20 0. 26 (0.30) 35,595 24,780 nil nil 35,595 24,780 3,955 2,753 19.8 10. 8 (13.2) 1.5 1. 1 (0.97) 3.0 2. 1 (2.0)  #12  Mean  12. 7  11.8 (12.1) 11.2 0.65 (0.88) 0.06 (0.08) 0.07 (0.10) 29,969 4,574 25,395 . 2,822  13. 2 - 0.5 - 0.04 - 0.06 32,970 9,460 23,510 2,612  *Dog #34 was s i c k the l a s t week of the e x p e r i m e n t a l p e r i o d and, as a r e s u l t , l o s t weight. I f he had gained n o r m a l l y then we would expect those f i g u r e s which appear i n b r a c k e t s .  53  TABLE X X I I I  S i  f i n a l weight  (Kg)  i n i t i a l weight t o t a l gain  (Kg) (Kg)  gain/Kg body weight(Kg) gain/week  (Kg)  feed o f f e r e d feed r e f u s e d feed consumed feed consumed/week gm feed/gm g a i n  (gm) (gm) (gm) (gm)  gm feed/gm gain/Kg wt gm feed/gm gain/W°'• 7 3  (cont'd)  Group I n d i v i d u a l Animals #26*  #33  19.5 15.9 (16.8) 13.2 11.4 4.5 6.3 (5.4) 0.39 0.33 (0.38) 0.7 0.5 (0.6) 34,860 40,915 nil nil 34,860 40,915 4,546 3,873 6.49 7.75 (6.46) 0.40 0.57 (0.40) 0.84 1.15 (1.12)  #21  #13  15.0  18.7  9.5 5.5  12.7 6.0  0.45  0.38  0.61  0.67  30,835 nil 30,835 3,426 5.62  39,690 nil 39,690 4,410 6.58  0.46  0.42  0.91  0.88  Mean 17.3 (17.5) 11.7 5.6 (5.8) 0.39 (0.40) 0.62 (0.65) 36,575 nil 36,575 4,068 6.61 (6.29) 0.46 (0.42) 0.95 (0.94)  *Dog #26 was s i c k d u r i n g the l a s t week of the e x p e r i m e n t a l p e r i o d and, as a r e s u l t , l o s t weight. I f he had gained n o r m a l l y then we would expect those f i g u r e s which appear i n b r a c k e t s .  C'+OO i n d i v i d u a l Animals #11 24. 5 f i n a l weight (Kg) 15. 5 i n i t i a l weight (Kg) 9. 0 weight g a i n (Kg) 0. 45 gain/Kg body weight:(Kg) gain/week 1. 00 (Kg) feed o f f e r e d (gm) 50,330 feed r e f u s e d (gm) n i l feed consumed (gm) 50,330 feed consumed/week (gm) 5,592 5. 59 gm feed/gm g a i n 0. 28 gm feed/gm gain/Kg wt 73 0. 63 gm feed/gm gain/W  #19 16 .4 10 .5 5 .9 0 .44 0 .66 33,110 65 33,045 3,672 5 .50 0 .41 0 .82  #22 16. 8 10. 9 5. 9 0. 43 0. 66 35,805 850 34,955 3,884 5. 92 0. 43 0. 87  #17 18. 2 10. 9 7. 3 0. 50 0. 81 36,611 415 36,196 4,022 4. 96 0. 34 0. 70  Mean 19. 0 12. 0 7. 0 0. 46 0. 78 38,964 333 38,632 4,293 5. 49 0. 37 0. 76  54  The t h i r d c o n s i d e r a t i o n concerns the presence o f p a r a s i t e s i n the e x p e r i m e n t a l dogs.  intestinal  During the course of the p r o j e c t  d e f i n i t e s i g n s of a s c a r i a s i s appeared, but no c o r r e c t i v e measures were taken f o r f e a r of confounding  the e x p e r i m e n t a l d e s i g n .  Post mortem of  those dogs k i l l e d - a f t e r n i n e weeks r e v e a l e d a v e r y heavy i n f e s t a t i o n o f ascarids  (Toxocara c a n i s ) and whipworms ( T r i c h u r i s v u l p i s ) .  i n f e s t a t i o n , coupled w i t h the p h y s i c a l d i f f i c u l t i e s  This i n t e s t i n a l  of i n g e s t i n g f e e d con-  t a i n i n g h i g h l e v e l s of the e t h e r s , p r o b a b l y accounts f o r the feed r e f u s a l s noted. The e x p e r i m e n t a l r e s u l t s show l a r g e v a r i a t i o n s i n the response to the treatments. the animals s e p a r a t e l y .  individual  T h i s makes i t n e c e s s a r y to c o n s i d e r each of  Feed i n t a k e and body weight  gain relationships  have been c a l c u l a t e d f o r each dog to f a c i l i t a t e c o n s i d e r a t i o n of the  findings  (see Table XXIII f o r a summary of the r e s u l t s ) . A comparison  of the r e s u l t s from the S  1 0 0  group w i t h those  from  the c o n t r o l animals shows a s i g n i f i c a n t l y slower growth r a t e and a lower feed e f f i c i e n c y .  The d i f f e r e n c e i n the energy  l e v e l of the two r a t i o n s  be enough to account f o r the d i f f e r e n c e i n growth r a t e between the The  r a t i o n had 5.8  w h i l e the S  1 0 0  percent s e l a c h y l a l c o h o l .  at the i n d i v i d u a l r e s u l t s from the animals i n the S apparent t h a t dog #26  groups.  p e r c e n t s a f f l o w e r o i l mixed i n the b a s i c dog  r a t i o n had o n l y 1.45  1 0 0  group  may  ration,  Looking  closer  i t becomes  had a s i g n i f i c a n t l y slower growth r a t e than the o t h e r  t h r e e animals i n the group.  T h i s can be e x p l a i n e d by examination of the  growth data p r e s e n t e d i n Appendix V.  I t may  be noted t h a t dog #26  o n e - h a l f a k i l o g r a m of body weight d u r i n g the l a s t week of the  lost  experiment  55  w h i l e the other dogs of the group gained from 0.5 is  suggested  to 0.9  kilograms.  t h a t the p a r a s i t e i n f e s t a t i o n i n t h i s animal  (#26)  It  had  reached  a l e v e l t h a t i n t e r f e r e d w i t h feed u t i l i z a t i o n and hence growth r a t e . A c l o s e examination the B  1 + 0 0  group shows t h a t two  of the i n d i v i d u a l r e s u l t s of the animals i n of the animals  q u i t e d i f f e r e n t from the other p a i r . of  responded  Dogs #20  and #12  the f e e d t h a t they were o f f e r e d and as a r e s u l t  the two month t r i a l .  The o t h e r two  dogs (#34  i n a manner t h a t i s  and  r e f u s e d to eat a l l  l o s t some weight d u r i n g #14)  i n t h i s group gained  s l o w l y and hence, because they consumed t h e i r a l l o t t e d amount of f e e d , y i e l d a feed e f f i c i e n c y r a t i o t h a t i s approximately from the c o n t r o l group. l e v e l of 2400 mgs  T h i s suggests  o n e - h a l f t h a t of the  t h a t the b a t y l a l c o h o l when f e d a t a  per k i l o g r a m of body weight i n t e r f e r e d w i t h the  Evidence  animals  of  the f e e d .  in  the data on g l y c e r y l e t h e r d i g e s t i b i l i t y  digestion  f o r t h i s i n t e r f e r e n c e w i t h d i g e s t i o n w i l l be (see Tables XXVII and  found  XXVIII).  T h i s aspect w i l l be e l a b o r a t e d upon l a t e r . Group S  4 0 0  may  be s i m i l a r l y d i v i d e d ; dogs #25  n e a r l y a l l the feed t h a t was for  these two  o f f e r e d to them.  animals were 14.9  / 1 and  12.6  The  and  #23  a t e a l l or  feed c o n v e r s i o n r a t i o s  / 1, or about o n e - h a l f of t h a t  o b t a i n e d f o r the c o n t r o l group. The and  l a r g e v a r i a t i o n i n feed e f f i c i e n c y between the c o n t r o l  c e r t a i n of the animals  animals  i n the e x p e r i m e n t a l groups (see T a b l e XXIV) can  p r o b a b l y be e x p l a i n e d on the b a s i s of the e f f e c t of the e t h e r s on  the  d i g e s t i b i l i t y of the v a r i o u s e x p e r i m e n t a l r a t i o n s .  low feed  conversion e f f i c i e n c i e s  f o r the second  the n e g a t i v e v a l u e s f o r the f i r s t  and  and  t h i r d dog  The  extremely  i n the S  f o u r t h dogs i n the B  4 0 0  4 0 0  group  group i s  and  56  TABLE XXIV:  Group Dog  SUMMARY OF  S  k  0  FEED EFFICIENCIES  B *  °  1  S  00  C  1 0 0  k  0  °  No.  1 2 3 4  14.9 20.2 58.8 12.6  -« 13.2 10.8 -»  6.46 6.49 5.62 6.58  5.59 5.50 5.92 4.96  I n e v i t a b l e s i n c e they r e f u s e d to eat a l l the feed t h a t was T h i s l e d to very  low  or n e g a t i v e weight g a i n s , hence the feed u t i l i z e d  u n i t of weight gained becomes i n f i n i t e l y group and schedule controls.  dogs #2  and  o f f e r e d to them.  #3  i n the B ^  0 0  large.  Dogs #1 and  group a t e a c c o r d i n g  #4  i n the  f o l l o w i n g model shows how  can markedly change feed c o n v e r s i o n  S^  00  to the same f e e d i n g  as the c o n t r o l animals y e t t h e i r g a i n f e l l w e l l below t h a t of The  per  the  a small v a r i a t i o n i n d i g e s t i b i l i t y  efficiency.  MODEL In t h i s model both body weight and while  feed i n t a k e are h e l d  the d i g e s t i b i l i t y of the r a t i o n i s v a r i e d from 48 to 66  constant,  percent.  T h i s model c o u l d be a t y p i c a l animal from the c o n t r o l group i n t h i s In t h i s model the f o l l o w i n g assumptions are made: body weight  15  kilograms  feed-intake/day 620 grams gain/day 110 grams energy content of g a i n 3 kcal/gram m e t a b o l i z a b l e energy of r a t i o n 3 kcal/gram  experiment.  57  I f we  accept  a d a i l y i n t a k e of 1860  k c a l of m e t a b o l i z a b l e  d i g e s t i b i l i t y of the feed i s 60 p e r c e n t , values  energy when the  i t i s p o s s i b l e to compute  the  g i v e n :i n the f o l l o w i n g; t a b l e :  TABLE XXV:  % Digestibility  THE AND  RELATIONSHIP BETWEEN DIGESTIBILITY FEED EFFICIENCIES FOR A MODEL DOG  Metabolizable energy k c a l  66 64 62 60 58 56 54 52 50 48  Energy content of g a i n k c a l  2,046 1,984 1,922 1,860 1,798 1,736 1,674 1,612 1,550 1,488  Grams of gain  516 454 392 330 268 206 144 82 20 - 42  Grams feed gram g a i n  172 151 131 110 89 69 48 27 6.7 negative  3.6 4.1 4.7 5.6 7.0 9.0 12.9 22.9 92.5 gain  These c a l c u l a t i o n s show t h a t i f the d i g e s t i b i l i t y of the r a t i o n f a l l s 60  to 54 p e r c e n t ,  the feed e f f i c i e n c y r a t i o changes from 5.6  of feed per gram of g a i n .  Thus a s m a l l change i n the d i g e s t i b i l i t y  ration results i n a relatively At  the end  l a r g e change i n the feed e f f i c i e n c y  of the e x p e r i m e n t a l  group'were s l a u g h t e r e d  p e r i o d two  as o u t l i n e d e a r l i e r .  s e l e c t e d at random from the C groups, the dogs t h a t had  4 0 0  and  S  1 0 0  The  of  the  ratio.  dogs to be h e l d over were  groups, but  i n the B  been r e f u s i n g feed were s e l e c t e d .  The  grams  of the f o u r dogs i n each  dogs were a l l f e d the c o n t r o l r a t i o n at a r a t e of 80 p e r c e n t c a l c u l a t e d maximum i n t a k e .  to 12.9  from  4 0 0  and  S  These e i g h t of  their  f i n d i n g of the heavy i n f e s t a t i o n of  4 0 0  FIGURE I I :  THE MEAN GROWTH RATES OF THE TWO DOGS FROM EACH GROUP MAINTAINED ON THE CONTROL RATION FOLLOWING REMOVAL FROM THEIR EXPERIMENTAL DIETS  BODY  WEIGHT IN KILOGRAMS  59  I  i n t e s t i n a l p a r a s i t e s i n the dogs t h a t were k i l l e d remaining dogs a l l be  t r e a t e d f o r worms.  ment appeared i n the c o n t r o l group. both to the worming and same r a t e as the C  4 0 0  The  S  1 0 0  dogs.  B  and  4 0 0  4  From t h i s response i t seems no  the  logical  l a s t i n g e f f e c t s on  A l l of the e x p e r i m e n t a l dogs so t r e a t e d , i n c l u d i n g  c o n t r o l s , showed an immediate response by than b e f o r e  S ° ° dogs gave a response  They grew at or near  to conclude t h a t the e x p e r i m e n t a l treatments had t r e a t e d animals.  the  A response to the worm t r e a t -  to the c o n t r o l r a t i o n .  and  d i c t a t e d that  the treatment.  the  the  g a i n i n g weight at a h i g h e r  rate  T h i s response suggests (1) t h a t a l l of the  dogs,  i n c l u d i n g the c o n t r o l s , were s u f f e r i n g from the worm i n f e s t a t i o n to a v a r i a b l e extent had  apparently  t h a t impaired s u f f e r e d no  growth .rate, and  (2) t h a t the t r e a t e d dogs  l a s t i n g e f f e c t s from the a l c o h o l s s i n c e  responded immediately when p l a c e d  on the c o n t r o l r a t i o n .  Figure  they  II  presents  the average growth curve from 4 weeks p r i o r to the e x p e r i m e n t a l p e r i o d 4 weeks a f t e r f o r the  two  dogs h e l d over from each group.  The  f i g u r e shows  the response to worming i n a l l the groups as w e l l as the response of B  4 0 0  and  S  4 0 0  the  dogs to the c o n t r o l r a t i o n .  T a b l e XXVI e x p l o r e s  the  feed i n t a k e - g a i n  relationships for  e x p e r i m e n t a l groups of r a t s .  No  f a c t t h a t the S  1 0 0  a s l i g h t l y lower feed e f f i c i e n c y than  c o n t r o l can be  explained  The  to  r a t i o n had  s i g n i f i c a n t d i f f e r e n c e s are r e v e a l e d . the  on the b a s i s of the energy l e v e l of the r a t i o n .  p h y s i c a l nature of the a - g l y c e r y l e t h e r s  e f f e c t on the feed consumption of the r a t s .  d i d not  appear to have  I t should  however, t h a t the r a t i o n o f f e r e d to the dogs c o n t a i n e d much of the e t h e r s  the  be p o i n t e d  any  out,  almost twice as  per u n i t weight as t h a t o f f e r e d to the r a t s .  The  60  TABLE XXVI:  FEED INTAKE-GAIN RELATIONSHIPS* FOR  S  f i n a l weight (gm) i n i t i a l weight (gm) t o t a l gain (gm) gain/gm of weight (gm) gain/day (gm) feed o f f e r e d (gm) feed r e f u s e d (gm) feed consumed (gm) feed consumed/day (gm) gm feed/gm g a i n gm feed/gm gain/Kg; wt gm feed/gm gain/W • 73  8. G l y c e r y l  are the mean f o r 10  E t h e r s and  B  RATS IN TRIAL I I  400  s  227,8 79.7 148.1 .96 2.43 827 14 813 13.3 5.58 36.2 22.5  216.1 79.6 136.5 .92 2.24 825 30 795 13.0 5.82 39.3 24o9  u  *These f i g u r e s  400  THE  C  212 o 6 83.6 129.0 .87 2.11 822 8 814 13.3 6.31 42.6 28.2  218.1 80.7 137.4 .92 2.25 824 19 805 13.2 5.86 39.3 24.9  Digestibility  of the t r i a l were a n l y z e d f o r the presence  s i x weeks a f t e r the of g l y c e r y l e t h e r s .  average feed i n t a k e f o r the week p r i o r to the sampling  and  a t by the chromic oxide method i n the dog  beginning Using  the  and by  total  of the g l y c e r y l e t h e r s was  I n these. d e t e r m i n a t i o n s  1  the dogs i n the S *  00  and B * 4  00  T a b l e XXVII.  A s i m i l a r summary f o r the r a t s  v a l u e s f o r the d i g e s t i b i l i t i e s the r a t s  The  are presented  those t h a t  r e s u l t s are summarized i n  i s g i v e n i n T a b l e XXVIII.  of the g l y c e r y l e t h e r s i n both  the dogs  i n the form of a bar graph i n F i g u r e I I I .  d i g e s t e d the g l y c e r y l e t h e r s more completely  determined.  groups were s e p a r a t e d  i n t o those which a t e a c c o r d i n g to the f e e d i n g standard and r e s t r i c t e d t h e i r feed i n t a k e .  the  digestibility  c o l l e c t i o n i n the r a t , the d i g e s t i b i l i t y  voluntarily  400  animals.  Faeces samples taken two weeks and  values a r r i v e d  ioo  than d i d the dogs.  The Batyl  The and rats  61  a l c o h o l was l e s s d i g e s t i b l e than s e l a c h y l i n both the dogs and the r a t s . In the r a t s s e l a c h y l a l c o h o l f e d a t the l e v e l o f 2.4 grams per k i l o g r a m of body weight was d i g e s t e d e q u a l l y as w e l l as was s e l a c h y l f e d a t the 0.6 grams p e r k i l o o f body weight l e v e l , whereas i n the dogs t h e r e was a marked d i f f e r e n c e i n the d i g e s t i b i l i t y two  dosage l e v e l s .  g l y c e r y l ethers  o f s e l a c h y l a l c o h o l between  This i s quite reasonable  i n the S  4 0 0  s i n c e the p e r c e n t  dog r a t i o n was 5.8 w h i l e  the S  4 0 0  those  of the rat ration  was i n the range o f 1.7 t o 3.5 p e r c e n t . The  dogs e a t i n g t o the f e e d i n g standard  of the g l y c e r y l e t h e r s t h e i r own feed i n t a k e . digestibility f o r the S  1 0 0  o f the S  i n g e s t e d than d i d the dogs which v o l u n t a r i l y T h i s , then, 4 0 0  and control.  and B  4 0 0  r a t i o n s f o r the dogs must be lower than  I t i s reasonable  to assume t h a t the g l y c e r y l  S  1 0 0  and B  4 0 0  of the whole r a t i o n was lowered  dogs a t e the same amount o f feed as the  and the c o n t r o l groups, but demonstrated l i t t l e  in digestibility body weight  o f 6 t o 10 p e r c e n t  or no g a i n .  A change  from the normal i s enough t o prevent  gain.  One p o i n t of note i s the f i n d i n g of g l y c e r y l e t h e r s  i n small  q u a n t i t i e s i n both the c o n t r o l feed and the faeces o f the c o n t r o l The  ethers  enzymatic a c t i o n on some of the feed  i n the d i g e s t i v e t r a c t and the d i g e s t i b i l i t y 4 0 0  restricted  i s a p a r t i a l e x p l a n a t i o n why the  c o a t i n g the feed p a r t i c l e s prevented  to a p o i n t where the S  digested a smaller portion  animals.  t o t a l amount i n the faeces was g r e a t e r than t h a t found i n the c o n t r o l  f e e d , thus i t seems t e n a b l e  that s y n t h e s i s of g l y c e r y l e t h e r s  occurred i n  the i n t e s t i n e or t h a t they were s e c r e t e d i n t o the i n t e s t i n e from the t i s s u e s . The  presence o f g l y c e r y l ethers  s i n c e the r a t i o n c o n t a i n e d  i n the c o n t r o l r a t i o n i s n o t unexpected  10 p e r c e n t  h e r r i n g meal.  TABLE XXVII:  RECOVERY OF GLYCERYL ETHERS FROM FAECES AND DETERMINATION OF DIGESTIBILITY OF THE ETHERS FOR DOGS IN TRIAL I I  % of Feed Digested  Feed/Day/Dog Dry wt % G • E. G.E. Wt (Grams) (Grams)  Samples 481 413 270 364 286 465  0.048 5.75 5.75 5.80 5.80 1.33  taken two weeks from the b e g i n n i n g  56.5 55.5 54.1 55.4 55.4 59.2  0.231 23.74 15.52 21.11 16.59 6.18  Faeces/Dog/Day Dry Wt** % G.E.* G.E. Wt (Grams) (Grams)  209.2 183.8 123.9 162.3 127.6 189.7  0.13 6.41 4.75 7.87 3.59 0.27  11.78 5.89 12.77 4.58 0.51  Samples taken s i x weeks from the b e g i n n i n g 598 437 271 423 361 550 a b Note:  0.048 5.75 5.75 5.80 5.80 1.33  dogs #14 and dogs #12 and  #34 #20  0.287 25.13 15.58 24.53 20.94 7.32  55.1 42.0 56.9 54.4 57.8 60.4 c d  268.5 253.5 116.8 192.9 152.3 217.8  0.126 6.05 4.43 6.29 4.52 0.39  0.34 15.34 5.17 12.13 6.88 0.85  G.E. Absorbed (Grams)  % G.E. Digested  Dog Weight (Kg)  G.E. Absorbed mgm/Kg/day  of the t r i a l 11.96 9.63 8.34 12.01 5.67  50.4 62.1 39.5 72.4 91.7  13.3 11.4 11.8 10.9 13.2 13.0  1.05 0.82 0.77 0.91 0.44  16.5 11.8 11.1 11.4 13.2 15.1  0.83 0.94 1.09 1.06 0.43  of the t r i a l 9.79 10.41 12.40 14.05 6.47  39.0 66.8 50.5 67.1 •88 o 4  dogs #23 and #25 dogs #9 and #10  The f e e d per day d a t a i s c a l c u l a t e d from the feed consumed f o r the week p r i o r to the f a e c e s sampling. The % G.E. ( g l y c e r y l e t h e r s ) i n the feed f o r a l l the groups has 0.05 s u b t r a c t e d from the e x p e r i m e n t a l v a l u e s and the % G.E. i n the faeces has 0.13 s u b t r a c t e d from the e x p e r i m e n t a l v a l u e s . ( T h i s has been done t o c o r r e c t f o r the g l y c e r y l e t h e r s found i n the c o n t r o l r a t i o n . )  * As determined  i n the f e e d by a m o d i f i c a t i o n o f the method of B l i g h and Dyer (13) .  ** As computed from r a t i o n d i g e s t i b i l i t y  u s i n g a chromic oxide method (93).  0 to  TABLE XXVIII:  Feed/Day/Group /o G • E a G.E. Wt Dry wt (Grams) (Grams)  RECOVERY OF GLYCERYL ETHERS FROM FAECES AND DETERMINATION OF DIGESTIBILITY OF THE ETHERS FOR THE RATS IN TRIAL I I  % of* Feed Digested  Faeces/Day/Group % G.E. G.E. Wt Dry Wt (Grams) (Grams)  G.E. Absorbed (Grams)  % G.E. Digested  Group Wt (Kg)  G.E. Absorbed mgm/Kg  Sample taken two weeks from the b e g i n n i n g of the t r i a l 105.3 104.4 105.3 105.3  0.048 2.27 2.29 0.50  0.05 2.37 2.41 0.53  71.5 72.2 72.1 72.2  30. 29. 29. 29,  0.26 1.55 0.15 nil  0.078 0.450 0.044 nil  1.92 2.37 0.53  81.0 98.2 100.0  1.176 1.181 1.196 1.189  1.63 1.98 0.44  1.841 1.898 1.856 1.798  1.495 1.93 0.454  Sample taken s i x weeks from the beginning of the t r i a l 128.5 120.1 127.6 128.3 Note:  0.048 2.85 2.87 0.64  0.06 3.42 3.66 0.82  72.3 72.7 75.4 70.9  35.6 32.8 31.4 37.3  0.19 1.78 0.27 0.01  0.068 0.58 0.085 0.004  2.84 3.58 0.82  83.0 97.8 99.5  The f e e d p e r day d a t a i s c a l c u l a t e d from the feed consumed f o r the week p r i o r to the faeces sampling. The % G.E. ( g l y c e r y l e t h e r s ) i n the feed has 0.05 s u b t r a c t e d from a l l the e x p e r i m e n t a l v a l u e s ; % G.E. of the f a e c e s taken two weeks from the b e g i n n i n g o f the t r i a l has 0.26 s u b t r a c t e d from a l l the e x p e r i m e n t a l v a l u e s , and % G.E. of the faeces samples taken s i x weeks from the b e g i n n i n g of the t r i a l has 0.19 s u b t r a c t e d . (These c o r r e c t i o n s a r e made because of the g l y c e r y l e t h e r s found i n the c o n t r o l f e e d and f a e c e s . )  * D i g e s t i b i l i t y by t o t a l  collection  FIGURE I I I :  DIGESTIBILITY OF GLYCERYL ETHERS  PERCENTAGE  OF  o  O  0017*  o CO  m m CO  1  oot^a  '  00i?  P  00t>  s  s  001 »  OO^  9  OOfr  9  CO  O m m  ^  0017  P  00*  S  o  CO  to  £  001  >  o  CO  m m  0017  s  001*  CO  OOfr* CO  x m m CO  3)  OOt^ 00l  ;  GLYCERYL  ETHERS O  DIGESTED CD  o  o o  65  9. R e s u l t s from c e l l  counts  The f o l l o w i n g t a b l e l i s t s the  the s i g n i f i c a n t d i f f e r e n c e s found i n  c e l l counts d e r i v e d i n t h i s t r i a l .  They i n c l u d e e r y t h r o c y t e ,  leucocyte  and r e t i c u l o c y t e counts of b l o o d , a d i f f e r e n t i a l of l e u c o c y t e s i n the b l o o d and a d i f f e r e n t i a l of bone marrow c e l l s .  A l l of these counts were made a f t e r  one month of treatment and a g a i n a t the end o f two months w i t h the e x c e p t i o n of the bone marrow d i f f e r e n t i a t i o n which was  TABLE XXIX:  n Group  c Sex  done when the animals were k i l l e d .  SIGNIFICANT (0.05 LEVEL) DIFFERENCES IN CELL COUNTS  o • Species  T>I j T> ™ r.n Blood or Bone Marrow C e l l s  Higher or Lower ° „ Counts  Counts Taken A f t e r One Month  400  s s  k00  female  rats dogs  l e u c o c y t e counts r e t i c u l o c y t e count Counts Taken A f t e r Two  ioo 400  s  s  s  -00  s  ioo  B  4 0 0  ioo  s  B  4 0 0  ioo  s  B  4 0  0  ioo  s  female female female  male male male male female female  rats rats rats dogs dogs dogs rats rats rats rats rats rats  lower higher Months  erytrocytes leucocytes reticulocytes reticulocytes reticulocytes reticulocytes polymorphs polymorphs lymphocytes lymphocytes lymphocytes lymphocytes  higher lower higher higher higher higher lower lower higher higher higher higher  D i f f e r e n t i a l of Rat Bone Marrow  ioo  s  S s  4 0 0  ioo  h00  s  B «t00 4 0 0  s  female male male male male female  promyelocytes myelocytes myelocytes metamyelocytes and ' r i n g c e l l s ' mature g r a n u l o c y t e s mature g r a n u l o c y t e s  higher lower lower lower lower lower  66  TABLE XXIX  o  n  Group  o  Sex  •  r  Species  .  i  (cont'd)  J  T  .  Differential gHUU glOO S * B^ S g'+OO B' °° 1  female female male male male female female  00  0 0  1 0 0  4  n  Higher or Lower ° „ Counts  r  Blood or Bone Marrow C e l l s of Rat Bone Marrow  mature g r a n u l o c y t e s mature g r a n u l o c y t e s nucleated red blood nucleated red blood nucleated red blood nucleated red blood nucleated red blood Differential  lower lower higher higher higher higher higher  cells cells cells cells cells  of Dog Bone Marrow  No s i g n i f i c a n t d i f f e r e n c e s were found i n the bone marrow smears of the dogs  Although  some s t a g g e r e d  a l l of the b l o o d counts  of the r a t s ,  the case of lymphocytes. o n l y the S control.  1 0 0  females  Similarly,  l e u c o c y t e count,  significant  t h e r e appears a d e f i n i t e  o n l y the S * 1  t h i s count b e i n g  00  the lymphocyte percentage 0 0  and S  1 0 0  trend only i n  show a s i g n i f i c a n t l y h i g h e r e r y t h r o c y t e count  S * ) showed a s i g n i f i c a n t l y  B^  throughout  Of the t h r e e groups of t r e a t e d r a t s , male and  00  females lower.  s i g n i f i c a n t l y h i g h e r r e t i c u l o c y t e count. 1  d i f f e r e n c e s occur  Only  the B ^ 1  0  females  of polymorphs.  over the  different showed a  Two groups of male r a t s  lower percentage  (B^  In four  of the white b l o o d c e l l s was s i g n i f i c a n t l y  groups (male and f e m a l e ) .  i n the t r e a t e d animals  show a s i g n i f i c a n t l y  female,  0 0  and  cases higher;  A l l other c a t e g o r i e s of l e u c o c y t e s  d i d not d i f f e r from the c o n t r o l .  67  The  dogs showed no s i g n i f i c a n t  differentials  except  d i f f e r e n c e s i n b l o o d counts or  i n the case of r e t i c u l o c y t e s where a l l the t r e a t e d  dogs had s i g n i f i c a n t l y h i g h e r counts  of r e t i c u l o c y t e s than had the c o n t r o l  animals. The definite  d i f f e r e n t i a l counts  of the r a t bone marrow shows o n l y two  t r e n d s , the one may be the r e s u l t  are s i g n i f i c a n t l y  lower i n the B ^  0 0  of the o t h e r .  males and the t h r e e t r e a t e d groups of  females, w h i l e n u c l e a t e d r e d b l o o d c e l l s a r e s i g n i f i c a n t l y the groups of male and female r a t s except No s i g n i f i c a n t in  female  rats.  i n the d i f f e r e n t i a l of c e l l s  response,  high l e v e l s ,  then, t o the a - g l y c e r y l e t h e r s , i s relatively  insignificant,  except  the case of the r a t s , where n u c l e a t e d r e d b l o o d c e l l s i n the bone marrow  are s i g n i f i c a n t l y  red  1 (  higher i n a l l  the bone marrow smears of the dogs.  a d m i n i s t e r e d a t extremely  in  the S ^  d i f f e r e n c e s were found  The haematopoietic  in  Mature g r a n u l o c y t e s  h i g h e r and i n the case of the dogs, where r e t i c u l o c y t e s  the b l o o d are s i g n i f i c a n t l y h i g h e r . b l o o d c e l l s i s not r e f l e c t e d  T h i s suggests or the l i f e  T h i s s t i m u l a t i o n of the immature  i n an i n c r e a s e i n e r y t h r o c y t e numbers.  t h a t e i t h e r the l i f e  o f the immature e r y t h r o c y t e i s extended  of the mature r e d b l o o d c e l l i s shortened.  68  C.  1.  FEEDING RESPONSE EXPERIMENT - TRIAL I I I  Preamble The  revealed  a n a l y s i s of the c o n t r o l feed used i n the p r e l i m i n a r y  an amount of g l y c e r y l e t h e r s  sufficient  g l y c e r y l e t h e r s possess v i t a m i n - l i k e p r o p e r t i e s . t h a t the a d d i t i o n of g l y c e r y l e t h e r s  to e l i c i t  a response i f  Hence i t i s p o s s i b l e  to t h i s r a t i o n ( T r i a l s I and  c o u l d produce l i t t l e or no p o s i t i v e response i n the e x p e r i m e n t a l The  hypothesis  t h a t the a d d i t i o n of g l y c e r y l e t h e r s  the g l y c e r y l e t h e r of c h o i c e  s i n c e i t was  r e a d i l y a v a i l a b l e and  selachyl i n alleviating P a n i k a r o v s k i i , 1963).  trial.  appears to have been more a c t i v e than  A l e v e l of 5 mg dosage.  the preamble to T r i a l I . one  tenth  (0.5 mg)  another which was  Batyl  i n t h i s f e e d i n g response experiment  per k i l o g r a m  T h i s was  of body weight  considered  Two  as  per k i l o g r a m  ten times as g r e a t  (50  was  discussed  more dosage l e v e l s were chosen:  of the 5 mg  and  an adequate dosage  l e v e l s i n c e i t i s s i m i l a r to recommended l e v e l s of v i t a m i n s  was  animals.  c e r t a i n c o n d i t i o n s of s t r e s s (Prokhonchukov  chosen as an a p p r o p r i a t e  in  II)  to a g l y c e r y l e t h e r  f r e e r a t i o n would show a response i s t e s t e d i n t h i s t h i r d a l c o h o l was  trials  one  of body weight l e v e l  which and  mg).  2. Design F o r t y female r a t s were p l a c e d at random i n t o f o u r groups of a c o n t r o l group and  three groups r e c e i v i n g 0.5,  a l c o h o l per k i l o g r a m  of body weight per day.  i n t o the feed t o f a c i l i t a t e a d m i n i s t r a t i o n . 5 weeks.  Cheeke (24)  has  5, and  50 mg  of b a t y l  B a t y l a l c o h o l was The  experimental  ten;  incorporated  period  was  suggested t h a t the apparent d i g e s t i b l e energy  69  i n t a k e o f the a l b i n o l a b o r a t o r y r a t over the body weight range, 50 t o 175 grams, be p r e d i c t e d from the f o l l o w i n g ADE  =  6.82 W * gm 0  relationship:  451  T h i s f e e d i n g s t a n d a r d was accepted as v a l i d  f o r t h i s experiment and the  animals were f e d 80 p e r c e n t o f t h i s t h e o r e t i c a l maximum i n t a k e . A l l animals were weighed samples  of feed and f a e c e s were taken t h r e e times d u r i n g the f i v e week  experimental period. all  t h r e e times each week. R e p r e s e n t a t i v e  During the f i f t h week b l o o d samples were taken from  the animals; r e d b l o o d c e l l counts and white c e l l counts were made,  smears were made f o r r e t i c u l o c y t e counts and l e u c o c y t e At  differentiation.  the end of the f i v e week p e r i o d a l l the animals were s a c r i f i c e d ,  organs  were i n s p e c t e d , and femur bone marrow smears were made.  3. Animals A l b i n o Sprague-Dawley (SPF) female r a t s were used as e x p e r i m e n t a l animals.  They were p l a c e d on the experiment a t a weight o f a p p r o x i m a t e l y  60 grams.  The a c t u a l weights a r e g i v e n i n Appendix I I I .  4. Housing The r a t s were housed were s u p p l i e d i n p o r c e l a i n  i n i n d i v i d u a l w i r e cages.  Feed and water  jars.  5. R a t i o n A g l y c e r y l e t h e r f r e e ration- was made up as f o l l o w s :  70  56 percent 25 10 4 2 3  sucrose casein safflower o i l m i n e r a l mix v i t a m i n mix alphacel To b a l a n c e t h i s r a t i o n w i t h r e s p e c t  to m i n e r a l s  and amino a c i d s the  f o l l o w i n g a d d i t i o n s were made: 11.7 mg 13.8 mg 773 mg The  CuSO^ per k i l o g r a m o f b a s i c r a t i o n ZnCL,2 per k i l o g r a m of b a s i c r a t i o n c y s t i n e per k i l o g r a m of b a s i c r a t i o n  i n g r e d i e n t s used i n t h i s s y n t h e t i c r a t i o n were shown to be f r e e of  g l y c e r y l ethers  by a n a l y s i s .  The s u c r o s e ,  c a s e i n , m i n e r a l mix and a l p h a c e l  were a l l f r e e of l i p i d m a t e r i a l and t h e r e f o r e were f r e e of g l y c e r y l e t h e r s . A n a l y s i s of the v i t a m i n mix and the s a f f l o w e r glyceryl  o i l revealed  no t r a c e of  ethers. T h i s b a s i c r a t i o n was f e d to a l l the r a t s w i t h the a p p r o p r i a t e  amounts of b a t y l a l c o h o l added to the r a t i o n f o r each of the t r e a t e d  groups.  6. Feed i n t a k e - g a i n r e l a t i o n s h i p s T a b l e XXX summarizes the feed i n t a k e - g a i n r a t s used i n t h i s t r i a l .  r e l a t i o n s h i p s f o r the  No d i f f e r e n c e s were found i n the r a t e o f g a i n or  feed e f f i c i e n c i e s between the groups.  The response of the b a t y l a l c o h o l  t r e a t e d groups was e s s e n t i a l l y the same as the c o n t r o l animals on the g l y c e r y l ether  free diet.  71  TABLE XXX:  FEED INTAKE-GAIN RELATIONSHIPS* FOR RATS IN TRIAL I I I  Control f i n a l weight (gm) i n i t i a l weight (gm) t o t a l gain (gm) gain/gm weight** (gm) gain/day (gm) feed o f f e r e d (gm) feed r e f u s e d (gm) feed consumed (gm) feed consumed/day gram feed/gram g a i n  169.3 57.5 111.8 0.99 3.4 371.8 18.3 353.5 10.7 3.16  B - 0.5 170.4 57.3 113.1 0.99 3.4 370.3 18.1 352.2 10.7 3.11  * a l l f i g u r e s a r e the mean of ten r a t s * * t h i s weight i s the mean of the i n i t i a l and weights f o r each group  B - 5  B - 50  169.5 57.6 111.9 0.99 3.4 370.9 20.4 350.5 10.6 3.13  167.8 56.3 111.4 0.99 3.4 369.5 20.3 349.2 10.6 3.13  final  7. D i g e s t i b i l i t y The d i g e s t i b i l i t y v a l u e s i n T a b l e XXXI a r e based on t o t a l c o l l e c t i o n s taken over 72 and 96 hours. between groups.  They show no s i g n i f i c a n t  faecal differences  The extremely s m a l l amount of f a e c e s e x c r e t e d by these  animals i s a r e s u l t o f the h i g h l y d i g e s t i b l e n a t u r e of the s y n t h e t i c e x p e r i m e n t a l r a t i o n used i n t h i s  TABLE XXXI:  trial.  DIGESTIBILITY OF EXPERIMENTAL  Group 2 weeks (72 h r ) 3 weeks (96 h r ) 4 weeks (72 h r )  Control 93.4 93.4 93.4  RATIONS IN TRIAL I I I  B - 0.5 93.9 93.2 94,2  B-5 94.5 93.5 93.6  B-50 94.9 93.7 93.6  72  8„ R e s u l t s  of b l o o d  and bone marrow c e l l  These r e s u l t s are l i s t e d  counts  i n Appendices X I I I , XIV  all  summarized i n T a b l e XXXII.  A n a l y s i s of these  any  s i g n i f i c a n t d i f f e r e n c e s between the e x p e r i m e n t a l  Large v a r i a t i o n s e x i s t e d between i n d i v i d u a l  and  XV  results failed groups and  The  are  to show the c o n t r o l .  animals.  A r b i t r a r y c a t e g o r i e s of bone marrow c e l l s were made to differentiation.  and  facilitate  s l i d e s were p o o r l y s t a i n e d so t h a t the c l a s s i c a l  types were not always r e c o g n i z a b l e .  The  cell  c a t e g o r i e s chosen showed no  s i g n i f i c a n t d i f f e r e n c e s between groups.  9.  Conclusion B a t y l a l c o h o l added to a g l y c e r y l e t h e r - f r e e s y n t h e t i c r a t i o n  fed f o r 5 weeks d i d not appear to a f f e c t production  of b l o o d  There was,  the r a t e of body growth or  the  c e l l s i n normal growing female a l b i n o r a t s . however, some s i g n of e r y t h r o c y t e s t i m u l a t i o n i n f o u r  of the c o n t r o l r a t s t h a t were f e d the B - 50 r a t i o n f o r 4 days a f t e r end  of the experiment.  Table XXXIII g i v e s the e r y t h r o c y t e counts.  the A "t"  t e s t i n d i c a t e s a s i g n i f i c a n t i n c r e a s e i n the number of e r y t h r o c y t e s . this  l i m i t e d experimental  d i v e r s i o n suggests f u r t h e r n u t r i t i o n a l  where g l y c e r y l e t h e r - d e p r i v e d administered  and  glyceryl  r a t s or other e x p e r i m e n t a l  animals  Thus,  trials are  ethers.  That b a t y l a l c o h o l s t i m u l a t e s haemopoiesis i n normal r a t s , i s suggested i n e x p e r i m e n t a l l e v e l used was kilogram  work done by Linman, e t a l , 1959.  50 mg/rat/day.  T h i s would e q u a l approximately  of body weight or 6 times the h i g h e s t l e v e l used by  The  dosage  300 mg  per  the author  73  TABLE XXXII:  A SUMMARY* OF THE BLOOD AND BONE MARROW DATA FROM TRIAL I I I  B - 0.5  Control  Group Erythrocytes ( m i l l i o n s p e r cmm) Leucocytes ( c e l l s p e r cmm)  B - 5  B - 50  7.60  7.26  7.44  7.60  11,200  12,800  12,400  12,400  D i f f e r e n t i a l of Leucocytes (percentage of l e u c o c y t e s ) 12.5 14„4 Polymorphs 17.1 0.6 0.6 Eosinophils 1.5 81.0 81.6 85.4 Lymphocytes 2.2 2.1 2.0 Monocytes  15.2 1.1 81.2 2.2  D i f f e r e n t i a l o f bone marrow c e l l 4.84 Category** 1 2 5.86 15.32 3 8.56 4 19.52 5 6 34.30 11.12 7 8 0.20  3.66 5.24 16.47 8.58 14.94 36.16 13.01 0.39  (percentage o f c e l l s ) 5.02 5.48 4.39 5.85 13.22 14.33 9.12 11.25 17.15 17.13 34.67 34.36 12.60 12.46 0.63 0.41  *These a r e averages o f t e n animals except i n the case o f t h e d i f f e r e n t i a l of the bone marrow of the c o n t r o l animals where the numbers a r e averages of f i v e animals. **The e i g h t c a t e g o r i e s o f bone marrow c e l l s a r e e x p l a i n e d i n Appendix XV.  TABLE XXXIII:  Rat No. 7 8 9 10 Average  ERYTHROCYTE COUNTS (MILLIONS PER CMM) OF FOUR CONTROL RATS FED BATYL ALCOHOL AT THE END OF THE EXPERIMENTAL PERIOD  End o f Experiment 6.50 7.01  7.93 7.43 7.60  Four Days on Feed 8.08 9.80 7.83 8.56 8.57  74  i n T r i a l IIIo t h a t he f e d .  Linman does not mention  the age of the r a t s nor the r a t i o n  The b a t y l a l c o h o l used was  a racemic s y n t h e t i c m a t e r i a l  and not the o p t i c a l l y a c t i v e n a t u r a l form used i n t h i s study. of the b a t y l a l c o h o l may  The  form  have some e f f e c t on the a c t i v i t y of the compound,  s i n c e dosage l e v e l s as h i g h as  2400 mg per k i l o g r a m o f body weight were  a d m i n i s t e r e d to animals i n t h i s p r o j e c t , w i t h o u t c a u s i n g a s i g n i f i c a n t haemopoietic  effects  T h i s apparent i n c o n s i s t e n c y between the e x p e r i m e n t a l  r e s u l t s o b t a i n e d by Linman and those o b t a i n e d by the author suggest a comparative study of the s y n t h e t i c form w i t h the n a t u r a l  form.  PART I I I :  GENERAL DISCUSSION  75  GENERAL DISCUSSION  The main c o n c l u s i o n s to be drawn from these experiments are summarized below: 1. Dogs and r a t s fed c h i m y l , s e l a c h y l and b a t y l a l c o h o l s a t l e v e l s of 6, 600 any  and  2400 mg  per k i l o g r a m of body weight d i d not show  s i g n s of i n t o x i c a t i o n , 2. Except  a t the 6 mg  i n the case of female r a t s r e c e i v i n g s e l a c h y l a l c o h o l  l e v e l f o r a p e r i o d of s i x months, t h e r e was  the growth r a t e between e x p e r i m e n t a l 3. Chimyl, l e v e l of 6 mg failed  and  control  animals.  s e l a c h y l and b a t y l a l c o h o l s a d m i n i s t e r e d  per k i l o g r a m of body weight to dogs and  to i n i t i a t e any d e f i n i t e haematopoietic 4.  no d i f f e r e n c e i n  r a t s f o r s i x months  response.  S e l a c h y l a l c o h o l a d m i n i s t e r e d at l e v e l s of 600  per k i l o g r a m of body weight and b a t y l a l c o h o l a d m i n i s t e r e d 2400 mg  o r a l l y at a  and  2400 mg  a t a l e v e l of  per k i l o g r a m of body weight f o r a p e r i o d of two months produced  r e l a t i v e l y i n s i g n i f i c a n t haematopoietic the r a t s .  The  o n l y i n d i c a t i o n s of haematopoietic  r e t i c u l o c y t e response percentage  responses  i n the b l o o d of the dogs and  of n u c l e a t e d red b l o o d c e l l s  i n both  the dogs and  s t i m u l u s were a a response  i n the  i n the bone marrow of the  rats.  5. B a t y l a l c o h o l added to a g l y c e r y l e t h e r - f r e e s y n t h e t i c r a t i o n and  f e d f o r a f i v e week p e r i o d d i d not appear to a f f e c t the growth r a t e  or the p r o d u c t i o n of blood c e l l s i n normal growing female a l b i n o r a t s .  76  Throughout  t h i s whole s e r i e s of experiments no s i g n of  appeared i n the e x p e r i m e n t a l a n i m a l s .  Gross p a t h o l o g y , growth  h i s t o p a t h o l o g y of t i s s u e s e c t i o n s from the h e a r t , l i v e r ,  toxicity r a t e and  lungs, spleen,  k i d n e y s , a d r e n a l s and i n t e s t i n e showed no i n d i c a t i o n of an  abnormal  condition. Any p i c t u r e of the response to the a d m i n i s t r a t i o n of n a t u r a l o c c u r r i n g a - g l y c e r y l e t h e r s i s obscured by the i n c o n s i s t e n c i e s o f the results.  There appears to be no growth response due  of g l y c e r y l e t h e r s , except i n the case of growing dosage  of s e l a c h y l a l c o h o l .  to the a d m i n i s t r a t i o n  female r a t s g i v e n a low  These r a t s have a growth curve i d e n t i c a l w i t h  that of the o t h e r groups of female r a t s to a mean body weight of about 200 grams.  Above t h i s body weight, the s e l a c h y l females grew a t a s l i g h t l y  f a s t e r r a t e to a f i n a l weight which was the o t h e r females.  s i g n i f i c a n t l y h i g h e r than t h a t of  The h a e m a t o p o i e t i c responses were a l s o  For example, i n the low dosage  inconsistent.  group of s e l a c h y l - f e d r a t s , a t 90 days the  females had s i g n i f i c a n t l y h i g h e r l e u c o c y t e counts, w h i l e the males had s i g n i f i c a n t l y lower l e u c o c y t e counts and a t 180 days no d i f f e r e n c e s were measured between e i t h e r the female or male and the c o n t r o l a n i m a l s . There appeared  to be some s t i m u l a t i o n i n e r y t h r o c y t e p r o d u c t i o n  when s e l a c h y l a l c o h o l was  f e d at 600 and 2400 mg  weight and b a t y l a l c o h o l a t 2400 mg  per k i l o g r a m o f body  per k i l o g r a m of body weight.  The  i n c r e a s e s were r e s t r i c t e d m a i n l y t o r e t i c u l o c y t e s i n the b l o o d o f the dogs and to n u c l e a t e d r e d b l o o d c e l l s i n the bone marrow of the r a t s . T h i s i n c r e a s e i n immature forms of e r y t h r o c y t e s was o v e r a l l i n c r e a s e i n the red b l o o d c e l l count.  not accompanied  Thus i t would  appear  by an that  77  the normal sequence of events i n the l i f e of an e r y t h r o c y t e was T h i s may  mean t h a t the immature p e r i o d  p e r i o d of a red blood red b l o o d  i s lengthened and  c e l l i s decreased.  altered.  t h a t the mature  T h i s suggests t h a t the  average  c e l l i s more y o u t h f u l and more capable of f u l f i l l i n g i t s r o l e  as an e r y t h r o c y t e ,  or that the mature e r y t h r o c y t e  removed from c i r c u l a t i o n The  i s more f r a g i l e and  is  sooner.  short experimental t r i a l using  r a t i o n showed no change i n e r y t h r o c y t e  a g l y c e r y l ether-free  synthetic  numbers over a f i v e week p e r i o d .  However, f o u r of the c o n t r o l r a t s t h a t were fed a g l y c e r y l e t h e r - f r e e r a t i o n r e c e i v e d b a t y l a l c o h o l i n t h e i r feed termination response.  of the experiment and  the s m a l l number of animals i n v o l v e d and i t does serve  s i g n i f i c a n t by  i t s e l f because of  because of the l a c k of c o n t r o l  as an i n d i c a t i o n t h a t another experiment  be done i n which r a t s are d e p l e t e d  of g l y c e r y l e t h e r s  of time and measure the response to a d m i n i s t e r e d blood  the  they showed an encouraging e r y t h r o p o i e t i c  T h i s i s o l a t e d response i s not  a n i m a l s , but  f o r f o u r days a f t e r  for varying  could  lengths  g l y c e r y l ethers.  If  c e l l counts do not decrease when the animals are f e d a g l y c e r y l  e t h e r - f r e e r a t i o n , y e t i n c r e a s e over normal values are a d m i n i s t e r e d  to g l y c e r y l e t h e r - d e p l e t e d  of i n t e r e s t to the b i o c h e m i s t and  when g l y c e r y l e t h e r s  r a t s , then the problem becomes  the p h y s i o l o g i s t as w e l l as to  the  nutritionist. In any  comparison of the e x p e r i m e n t a l r e s u l t s of t h i s p r o j e c t  w i t h those r e p o r t e d important factor» d-configuration.  i n the The  l i t e r a t u r e , o p t i c a l isomerism becomes an  n a t u r a l occurring a - g l y c e r y l ethers  Much of the e x p e r i m e n t a l work r e p o r t e d  are of  i n the  the  78  l i t e r a t u r e makes use o f s y n t h e t i c racemic m i x t u r e s .  From our knowledge  of enzymes we know t h a t they a r e most commonly enantiomer  specific.  T h i s means t h a t we might expect an e n t i r e l y d i f f e r e n t p h y s i o l o g i c a l response from a racemic mixture than we would from e i t h e r emantiomer. An  experiment s p e c i f i c a l l y designed t o compare pure 1- and d-forms o f  a - g l y c e r y l ethers Since  i s i n d i c a t e d here.  i t i s now e s t a b l i s h e d t h a t the n a t u r a l l y o c c u r r i n g  a - g l y c e r y l ethers  a r e not t o x i c and s i n c e the response i s v a r i a b l e and  i n c o n s i s t e n t i n the r a t and the dog, i t may be worth w h i l e t o use other species  as e x p e r i m e n t a l s u b j e c t s .  as models o f mammals i n g e n e r a l ; guinea p i g or c a t or some other g l y c e r y l ethers.  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R a t i o n No. 10-63 Rat  Pellets  Ground b a r l e y Ground wheat Wheat bran Wheat germ meal Ground oat g r o a t s Fishmeal (73%) Soyabean meal (44%) O i l c a k e meal Vitagrass Skim meal powder (spray) Brewers y e a s t I r r a d i a t e d dry yeast S t e r i l i z e d bone meal Stabilized f a t Molasses I r o n oxide Iodized s a l t h l b dry vitamin A  300 400 200 100 200 250 100 50 100 100 24 2 10 50 100 4 10 2000  90  APPENDIX I I  Mean D a i l y Feed Intake In Grams A.  Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27  Selachyl 14.1 16.8 19.4 18.0 19.5 19.8 17.6 19.6 20.4 19.3 21.5 22.4 21.5 24.7 23.0 24.3 25.9 25.2 24.7 26.3 25.0 23.5 23.0 21.5 20.8 20.9 22.2  Male Rats  Batyl 14.1 15.6 18.4 18.5 18.3 18.0 15.4 18.7 19.9 18.5 20.5 21.1 20.1 22.4 22.4 22.9 23.2 23.4 24.4 23.6 23.1 23.1 24.2 22.2 19.5 20.0 20.6  Chimyl 14.7 17.3 18.5 19.0 17.7 18.8 18.0 19.6 18.8 18.3 19.4 19.9 19.5 22.9 22.3 23.8 23.5 22.6 21.5 22.8 22.1 20.5 21.1 20.0 19.4 20.8 20.1  Control 14.2 17.2 18.5 18.3 20.4 20.0 19.7 20.5 19.9 18.6 20.5 21.1 20.9 22.8 22.9 24.0 23.4 23.1 22.7 25.3 22.4 22.2 24.8 21.7 20.6 20.6 21.4  APPENDIX I I (cont'd)  B.  Week 1 2 3 4. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27  Female Rats  Selachyl  Batyl  Chimyl  Control  13.3 14.8 15.7 16.0 16.2 17.2 16.2 17.5 17.3 15.2 17.4 18.6 18.6 20.1 18.1 19.2 19.6 18.9 20.0 19.4 .18.8 21.6 19.4 19.0 18.9 18.0 18.8  13.7 16.0 15.8 16.8 16.4 16.3 14.6 16.7 15.3 14.5 16.0 16.9 15.5 18.6 18.2 18.8 16.9 17.8 18.0 18.1 17.8 18.6 17.7 16.6 15.6 15.9 17.8  13.8 15.8 16.3 15.8 16.5 16.7 15.8 17.4 15.5 14.6 16.4 16.8 15.9 18.8 17.9 18.9 20.0 19.0 19.5 20.8 19.4 17.7 18.3 16.6 15.3 17.4 17.1  13.3 14.7 14.3 15.5 15.9 15.9 15.3 16.4 15.2 14.0 15.7 16.1 15.9 17.4 16.9 17.5 17.4 17.5 17.7 18.8 17.0 16.6 17.2 15.6 14.6 15.7 16.3  APPENDIX I I I  Mean Weekly Weights A.  Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27  Male R a t s *  Selachyl  Batyl  Chimyl  Control  102.3 132.2 164.3 200.1 226.4 249.4 261.4 276.4 294.8 314.2 329.6 338.9 347.3 358.3 362.6 373.2 380.9 382.7 387.0 391.1 398.7 400.6 403.2 405.8 410.1 410.7 421.5  99.5 119.5 149.8 184.3 201.8 228.0 237.3 254.3 274.1 288.0 307.4 319.4 329.6 338.7 347.4 356.8 364.3 368.8 374.0 379.7 390.9 391.8 402.9 407.0 410.0 410.9 413.9  99.0 131.7 161.5 187.0 210.4 230.6 247.2 263.9 270.0 295.0 308.7 317.7 326.1 334.2 341.8 353.6 363.1 370.2 378.1 376.4 387.4 390.1 401.7 403.1 407.3 409.3 412.8  94.3 124.6 150.4 183.1 212.8 230.2 243.4 262.1 277.3 291.8 303.1 316.8 329.3 339.7 348.9 362.2 369.4 373.6 375.2 377.9 383.4 393.3 398.4 406.1 409.5 413.4 418.9  * A l l weights a r e i n grams and a r e the mean of t e n a n i m a l s .  APPENDIX I I I  B.  Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27  (cont'd)  Female R a t s *  Selachyl  Batyl  Chimyl  Control  91.3 112.5 130.7 150.4 163.0 175.0 181.4 191.6 199.6 208.1 214.5 219.7 223.9 229.1 229.8 236.2 240.7 243.3: 243.2 247.9 252.7 254.6 253.3 258.2 262.8 264.0 267.3  90.9 113.7 133.4 151.6 164.9 175.9 183.1 192.3 202.8 206.1 211.6 213.6 216.5 220.4 224.7 229.0 232.6 234.4 238.8 238.6 241.4 241.5 245.9 248.6 250.4 251.3 255.4  93.3 115.8 134.9 151.8 166.1 175.6 181.8 191.1 196.3 201.4 206.5 212.7 216.2 220.2 222.7 228.8 232.7 238.0 237.5 239.2 241.9 246.9 246.0 247.9 248.3 250.0 253.3  94.3 114.3 133.7 151.2 165.0 175.9 183.7 194.3 198.8 204.6 208.2 213.8 219.0 221.0 224.1 228.8 232.6 235.4 237.2 238.6 242.4 242.9 246.3 248.2 249.7 251.5 256.4  * A l l weights a r e g i v e n i n grams and a r e the mean o f t e n a n i m a l s .  APPENDIX I I I  C.  (cont'd)  Dogs*  Week  Selachyl  Batyl  Chimyl  Control  1 2 3 4 5 6 . 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27  10.3 12.4 13.3 12.7 13.9 14.8 15.5 16.3 16.9 16.6 17.6 18.4 18.4 19.4 19.5 20.1 20.7 21.0 21.6 22.1 21.7 22.0 21.8 22.2 22.2 22.6 21.9  10.7 12.6 13.7 14.0 14.2 15.4 16.3 16.7 17.3 17.4 17.9 18.7 18.9 19.5 19.5 20.0 20.7 20.8 21.2 21.1 21.6 20.8 21.0 20.2 20.8 20.6 20.7  11.6 13.1 14.2 14.7 14.9 15.6 16.6 17.2 17.3 17.7 18.3 19.1 19.3 20.0 19.7 20.5 21.1 21.2 21.8 21.5 21.9 21.7 22.0 22.3 22.6 22.7 23.0  9.5 10.9 11.6 12.3 12.9 13.4 14.4 15.1 15.6 15.4 16.2 17.3 17.1 18.1 18.0 18.3 19.2 19.4 20.0 19.7 20.3 19.9 19.8 20.4 20.8 20.2 20.8  * A l l weights are i n k i l o g r a m s and are the mean of the f i v e  animals.  94  APPENDIX IV  Blood Counts A.  Rat  L e u c o c y t e Counts* of Rats Taken N i n e t y Days From The Beginning of The T r i a l  Selachyl  Batyl  Chimyl  Control  1 2 3 4 5 6 7 8 9 10  18,700 20,500 17,200 19,150 16,500 20,300 15,200 16,250 18,600 21,000  11,300 11,450 21,400 10,750 20,700 18,950 17,250 6,900 15,050 20,650  16,300 15,900 14,800 10,550 11,150 20,950 15,800 12,400 16,200 22,850  19,100 13,050 16,150 15,650 11,550 14,650 12,450 16,600 15,050 11,750  Mean  18,340  15,440  15,690  14,600  1,880  4,840  3,720  2,280  1 2 3 4 5 6 7 8 9 10  23,500 22,150 13,400 14,450 16,700 17,100 19,750 18,600 18,000 16,900  19,750 19,600 20,950 17,800 18,000 20,900 13,000 14,700 16,950 15,000  23,700 16,400 23,700 19,500 27,700 19,650 26,300 21,200 19,650 23,300  19,700 24,300 21,450 15,450 15,750 17,800 27,850 21,300 13,050 21,350  Mean  18,055  17,665  22,110  19,800  2,970  2,600  3,290  4,313  female  S.D.** male  S.D.**  * These counts a r e numbers of l e u c o c y t e s p e r cmm of b l o o d . ** Standard D e v i a t i o n .  APPENDIX IV (cont'd)  B.  Leucocyte Counts* of Rats Taken 180 Days From The B e g i n n i n g of t h e T r i a l  Selachyl  Batyl  Chimyl  Control  1 2 3 4 5 6 7 8 9 10  17,400 18,900 12,400 21,100 14,600 9,000 13,700 17,800 16,800 13,900  11,500 12,100 20,600 17,800 18,000 18,000 19,800 12,500 15,000 14,800  15,000 22,100 15,000 10,000 14,800 10,300 11,300 12,000 21,800 12,800  18,300 16,600 18,400 13,800 11,400 12,000 10,900 11,500 14,600 15,700  Mean  15,560  16,010  14,510  14,320  3,350  3,110  4,110  2,710  1 2 3 4 5 6 7 8 9 10  21,500 17,400 25,600 20,500 12,300 13,300 16,100 20,000 12,300 23,100  26,000 26,300 27,500 26,700 16,800 15,000 18,700 13,100 11,100 12,200  13,000 20,500 20,400 15,400 15,000 15,900 17,500 28,300 20,500 28,900  15,500 12,500 17,500 19,400 27,800 16,300 16,400 17,100 16,200 16,900  Mean  18,200  19,340  19,540  17,560  4,440  6,300  5,150  3,800  Rat female  S.D.** male  S.D.**  * These counts a r e number o f l e u c o c y t e s per cmm. ** Standard D e v i a t i o n  APPENDIX IV  C.  (cont'd)  E r y t h r o c y t e Counts* o f Male Rats Taken 180 Days From The Beginning o f the T r i a l  Rat  Selachyl  Batyl  Chimyl  Control  1 2 3 4 5 6 7 8 9 10  9.96 9.16 9.69 8.5 9.3 9.7 8.8 8.4 8.3 9.3  10.2 9.4 9.1 8.6 7.9 7.2 8.8 8.9 9.3 8.8  8.3 8.7 9.5 8.9 10.2 8.2 10.0 8.9 8.7 10.4  7.3 8.9 8.7 9.0 8.5 10.1 9.6 9.5 8.2 8.9  Mean  9.11  8.82  9.58  8.87  S.D.  .56  0.78  0.85  0.73  * The e r y t h r o c y t e counts a r e recorded of b l o o d .  i n m i l l i o n s o f c e l l s p e r cmm  APPENDIX IV (cont'd)  D.  E r y t h r o c y t e Counts* of Dogs Taken 110 Days from The Beginning of the T r i a l  Selachyl  Batyl dog it  dog #  count  59 60a 74 72 65  5,22 5.52 5.60 4.80 5.40  Mean  5.31  5.70  S.D.  0.28  0.39  60 69 66 70 64  Chimyl dog #  *  count 6.08 5.39 5.83 5.10 6.10  Control count  dog # 75 76 68 77 62  count  67 NT 58 71 63  6.29 6.68 5.25 5.54 6.20  5.08 5.40 6.38 5.72 5.24  Mean  5.99  5.56  S.D.  0.52  0.46  The e r y t h r o c y t e counts a r e r e c o r d e d i n m i l l i o n s of ce per cmm of b l o o d .  APPENDIX IV  E.  (cont'd)  Leucocyte Counts* of Dogs Taken 180 Days From The B e g i n n i n g of the T r i a l  Selachyl  Batyl  dog #  count  dog //  59 60a 74 72 65  15,300 16,100 20,300 14,900 19,300  Mean  17,200  17,200  S.D.  2,200  2,170  60 69 66 70 64  Chimyl dog #  count 16,200 15,500 17,300 15,100 21,100  Control count  dog #  67 NT 58 71 63  15,300 19,900 17,000 16,900 15,200  Mean  16,900  17,500  S.D.  1,700  2,360  *  75 76 68 77 62  count 17,200 22,200 18,100 15,700 15,200  These counts a r e numbers of l e u c o c y t e s p e r cmm  of blood.  APPENDIX V  Weight Data f o r A.  Initial 1 2 3 4 5 6 7 8 9 10 11 12 13  Initial 1 2 3 4 5 6 7 8 9 10 11 12 13  Group  1 0 0  #26  #33  #21  #13  11.4 10.9 12.3 12.7 13.6 13.6 14.5 15.5 16.4 15.9  13.2 13.6 14.5 15.0 15.9 16.4 16.8 17.7 18.6 19.5.  9.5 10.0 10.9 11.4 11.8 12.3 12.7 13.6 14.5 15.0 15.0 15.9 16.8 17.7  12.7 13.6 14.1 14.5 15.5 15.4 16.4 17.3 18.2 18.7 17.7 19.1 19.4 20.9  C  Week  T r i a l II  Weekly Weights i n K i l o g rams f o rDogs  S Week  Animals In  k  0  °  Group  #11  #19  #22  #17  15.5 16.4 17.3 18.2 19.5 20.0 21.4 22.7 23.6 24.5  10.5 10.9 11.4 12.3 13.2 13.2 13.6 14.5 15.5 16.4 16.4 17.3 18.2 19.1  10.9 11.4 12.3 12.7 14.1 14.1 15.0 16.4 17.3 16.8 17.3 17.7 19.5 20.5  10.9 11.4 12.3 12.7 14.1 15.0 15.9 16.8 17.7 18.2  APPENDIX V (cont'd)  S  Week Initial 1 2 3 4 5 6 7 8 9 10 11 12 13  h  0  Initial 1 2 3 4 5 6 7 8 9 10 11 12 13  Group  #25  #10  #9  #23  10.9 11.4 10.9 11.4 11.4 11.4 11.4 11.8 12.3 12.7  14.5 15.4 15.0 15.0 15.0 14.5 14.5 15.4 15.5 15.9 15.9 17.7 19.1 20.0  12.3 11.8 11.4 11.4 11.4 10.9 11.8 11.8 12.3 12.7 12.7 13.6 14.1 15.5  10.5 10.5 10.9 10.9 11.4 11.4 11.4 11.8 11.8 12.3  B^°° Week  °  Group  #20  #34  #14  #12  10.5 10.9 10.0 10.5 10.5 10.0 10.0 10.0 10.0 9.5 10.0 10.0 10.9 11.8  12.3 13.2 13.6 13.6 14.5 14.1 13.6 14.1 14.5 14.1  8.6 8.6 9.1 9.1 9.5 9.5 10.0 10.5 10.9 10.9  13.2 13.2 13.6 12.3 12.7 12.3 12.3 12.3 12.7 12.2 13.6 13.6 14.1 15.0  APPENDIX V (cont'd)  B.  Day  Mean Weights* i n Grams f o r Rats  S  4 0 0  B  k  0  °  S  1 0 0  C  k  0  °  1 3 5  79.6 90.1 97.6  79.7 92.5 101.5  83.6 95.0 104.6  80.7 92.8 102.9  8 10 12  105.3 113.7 119.6  108.6 112.5 118.1  110.0 114.9 118.9  109.2 112.5 117.6  15 17 19  125.4 134.0 137.1  124.3 131.1 134.2  123.8 127.9 131.3  123.0 128.2 132.3  22 24 26  143.5 148.1 153.4  140.0 145.6 149.9  137.3 143.0 146.8  138.9 146.2 149.7  29 31 33  158.5 162.5 163.5  156.8 160.4 160.4  152.0 155.9 155.1  154.6 160.2 159.5  36 38 40  174.8 177.3 . 181.4  172.1 176.7 185.9  165.8 169.6 174.1  170.5 173.2 177.9  43 45 47  185.6 191.9 191.7  189.8 195.6 196.1  179.8 185.2 185.7  184.1 191.0 192.2  50 52 54  202.1 207.4 208.7  210.3 215.8 220.1  196.2 202.9 205.7  201.8 208.0 211.6  57 59 61  215.6 216.8 216.1  222.8 225.9 227.8  211.9 211.2 212.6  220.1 217.0 218.1  * mean of t e n r a t s  APPENDIX VI  R e s u l t s o f Rat Blood Taken One Month From The Beginning of T r i a l I I A.. E r y t h r o c y t e Counts ( m i l l i o n s o f cells/cmm)  400  ,400  • 100  10.7 8.1 9.2 9.0 8.4  10.0 7.4 8.0 8.1 8.7  8.5 8.9 7.8 8.3 9.0  7.9 8.7 8.9 8.5 7.9  9.1  8.5  8.5  8.4  7.6 8.1 7.0 7.7 7.6  7.8 8.1 7.3 7.7 9.1  9.2 8.0 7.6 9.5 8.7  8.2 7.3 8.5 7.0 7.1  7.6  8.0  8.6  7.6  :  ^400  Male 1 2 3 4 5 average Female 1 2 3 4 5 average  B.  Leucocyte Counts (cells/cmm)  ;400  ,400  • 100  >400  Male 16,100 9,700 7,500 9,300 10,000  8,800 15,700 12,600 6,000 9,900  8,200 17,100 5,900 11,800 8,000  21,500 6,800 5,900 6,600 6,300  average  10,500  10,600  10,200  9,400  Female 1 2 3 4 5  10,700 13,100 11,900 8,800 14,200  19,000 9,900 15,300 13,000 7,300  11,000 13,900 14,000 11,700 15,300  15,700 19,900 13,100 19,500 10,500  average  11,700  12,900  13,200  15,700  1 2 3 4 5  103  APPENDIX VI  (cont'd)  C. R e t i c u l o c y t e s (percent o f e r y t h r o c y t e s )  :  400  .400  • 100  ^400  Male 1 2 3 4 5 average  4.7 4.1 3.9 6.9 3.7  3.0 3.8 4.6 4.2 3.2  3.1 4.6 2.9 3.8 3.9  4.6 4.2 2.8 4.4 3.5  4.6  3.8  3.7  3.9  3.0 3.6 4.4 3.8 4.8  8.2 4.0 5.4 3.9 3.6  4.0 3.2 4.8 4.5 6.2  3, 2. 2. 3. 3,  3.9  5.2  4.5  3.2  Female 1 2 3 4 5 average D.  Polymorphs  D i f f e r e n t i a l Leucocyte  S  4 0 0  B  4 0 0  Counts (percent o f l e u c o c y t e s )  S  1 0 0  C  4 0 0  Male 1 2 3 4 5 average  16 25 27 18 26  14 21 24 14 17  16 23: 22 22 24  31 22 24 22 14  22.4  18.0  21.4  22.6  17 17 26 16 21  16 19 24 21 24  23 18 19 18 14  26 16 27 23 15  19.4  20.8  18.4  21.4  Female 1 2 3 4 5 average  APPENDIX VI (cont'd)  Lymphocytes  S  400  B  H 0 0  s  ioo  H 0 0  C  Male 1 2 3 4 5 average  81 75 70 79 68  76 73 74 82 81  81 75 71 76 75  66 69 74 74 85  74. 6  77.2  75.6  73.6  78 77 74 81 74  82 80 73 74 68  71 75 80 77 80  72 75 69 76 83  76. 8  75.4  76.6  75.0  Female 1 2 3 4 5 average  Eosinophils and Monocytes  S  400  s  h00  ioo  c  Male 1 2 3 4 5 averages Female 2 3 4 5 averages  2 0 1 1 6  1 0 2 2 0  6 4 2 0 1  4 2 0 4 1  3 2 6 2 0  2. 0  1.0  2..6  2.2  2. 6  3 2 0 1 5  2 3 0 2 0  1 0 2 4 6  1 1 1 1 2  5 5 1 3. 5  2. 2  1.4  2. 6  1.2  3. 8  0 0 1 0 1 0.4 1 2 0 2 1 1.2  2  1 5 1 3 1  4 1 1 0  2. 2  1.6  1 8 2 1 1 2. 6  1 1 0 0 1 0.6  APPENDIX V I I R e s u l t s o f Rat Bood Taken Two Months From The Beginning of T r i a l I I A.  ;  E r y t h r o c y t e Counts ( m i l l i o n s of cells/cmm)  400  ,400  .100  9.9 8.8 9.7 9.1 8.3  10.8 8.6 9.0 9.2 9.4  9.2 9.1 9.2 8.6 9.0  8.8 8.2  9.2  9.4  9.0  8.6  8.3 9.0 8.6 9.4 8.8  10.9 8.6 8.6 9.4  9.2 8.9 8.4 9.9 9.4  8.7 7.8 8.8 7.5 7.7  8.8  9.4  9.2  8.1  -.400  Male 1 2 3 4 5 average Female 1 2 3 4 5 average  B.  :  Leucocyte Counts (cells/cmm)  400  ,400  .100  ^400  Male 1 2 3 4 5  18,200 13,300 9,900 12,500 11,800  10,300 14,300 10,100 5,800 9,600  7,800 23,000 7,900 15,200 9,200  22,600 9,900 8,600 10,200 9,100  average  13,100  10,000  12,600  12,100  13,000 14,300 10,700 11,200 13,200  17,900 8,900 19,000 15,000  9,800 13,200 18,700 11,300 19,300  15,700 20,800 15,100 23,200 11,600  12,500  15,200  14,500  17,300  Female 1 2 3 4 5 average  106  APPENDIX V I I C.  (cont'd)  R e t i c u l o c y t e s (percent o f e r y t h r o c y t e s )  :H-00  >400  .100  6.0 4.2 9.8 7.2 5.1  5.1 5.8 5.1 5.0 4.4  3.7 4.9 4.2 4.6 4.1  4.8 4.4 2.5 4.7 3.7  6.5  5.1  4.3  4.0  3.7 4.9 5.3 4.3 5.1 ~4T7~  7.9 4.9 5.8 5.2  4.1 3.6 4.7 4.8 6.1 4.7  3.3 2.9 2.3 3.4 3.3 3.0  ^00  Male  1 2 3 4 5 average Female  1 2 3 4 5 average  ~6~70~  D i f f e r e n t i a l Leucocyte  Counts (percent o f l e u c o c y t e s )  >400  .100  ^i+00  18 36 27 26 27  17 16 24 24 16  17 19 23 18 17  31 24 31 28 26  26.8  19.4  18.8  28  14 16 22 18 23  14 18 17 17  23 18 19 21 19  24 23 27 19 15  18.6  16.5  20.0  21.6  Polymorphs Male  1 2 3 4 5 average Female  1 2 3 4 5 average  APPENDIX V I I (cont'd)  Lymphocytes  S  400  400  B  s  ioo  C  400  Male 1 2 3 4 5 average  80 62 70 70 68  78 78 72 75 80  74 77 74 80 80  67 74 66 69 69  70. 0  76.6  77.0  69.0  81 79 73 76 73  82 81 78 80  74 78 77 75 76  72 67 67 72 81  76. 4  80.2  76.0  71.8  Female 1 2 3 4 5 average  Eosinophils and Monocytes  -  chQO  B  400  s  ioo  C  400  Male 1 2 3 4 5 average  1 0 1 2 3  1 2 2 2 2  3 2 2 1 1  2 3 2 0 3  8 2 2 0 2  1 2 1 2 1  0 1 0 3 2  2 1 3 0 1  1.4  1.8  1.8  2.0  2.8  1.4  1.4 l . i  1 1 3 2 3  3 4 2 4 1  2 1 3 2  1 0 1 1  2 1 2 3 4  1 2 2 1 1  2 3 5 4 1  2 7 1 4 1  2.0  2.8  2.0  0.8  2.4  1.4  3.0  3.0  Female 1 2 3 4 5 average  -  108  APPENDIX'VIII  Blood Count Data from Dogs Taken One Month From The Beginning o f T r i a l I I  Identification  of dogs  The dogs w i l l be i d e n t i f i e d i n t h i s s e t of d a t a by numbers 1, 2, 3 and 4 as f o l l o w s : S 1 2 3 4  4 0 0  dog  B  25 10 9 23  A.  4 0 0  dog  S  20 34 14 12  1 0 0  dog  C  26 33 21 13  4 0 0  dog  11 19 22 17  E r y t h r o c y t e Counts ( m i l l i o n s o f cells/cmm)  k00  S  B  400  s  ioo  k00  C  Dog 1 2 3 4 average  5.6 6.0 5.3 5.7  7.2 5.4 5.0 4.9  6.5 5.2 6.3 5.5  6.2 6.1 5.5 5.6  5.7  5.6  5.9  5.9  B.  S  Leucocyte: Counts  400  B  400  (cell/cmm)  s  ioo  C  400  Dog 1 2 3 4 average  13,300 8,600 14,600 14,800  16,200 14,100 17,500 12,300  14,600 11,900 17,000 13,000  9,400 10,700 12,600 10,000  12,800  15,000  14,100  10,800  109  APPENDIX V I I I  C„  S  400  (cont'd)  R e t i c u l o c y t e s ( p e r c e n t of e r y t r o c y t e s )  B  M-00  S  100  QM-OO  Dog 1 2 3 4 average  D.  Polymorphs  1.3 1.4 1.6 1.1  2.2 1.1 2.6 0.8  1.5 1.3 0.7 1.0  1.1 1.5 0.6 0.9  1.4  1.7  1.1  1.0  D i f f e r e n t i a l Leucocyte  S  4 0 0  B  4 0 0  Counts (percent o f l e u c o c y t e s )  S  1 0 0  C  4 0 0  Dog 1 2 3 4 average  Lymphocytes  67 57 65 72  66 69 72 81  50 69 65 59  59. 65 71 63  65.3  72.8  61.8  64.5  S  4 0 0  B  4 0 0  S  1 0 0  C  4 0 0  Dog 1 2 3 4 average  32 36 34 20  30 21 27 18  39 25 34 37  23 32 26 33  30.5  24.0  33.8  28.5  110  APPENDIX V I I I  Eosinophils and Monocytes  g  4  0  0  k  Q  (cont'd)  Q  l  Q  Q  k  Q  Q  Dog 1 2 3 4 average  1 0 6 1 0 1 _j8_ _ 0 _ 3.8  0.5  0 1 10 0 0 1 0_ _ 1 _ 2.5  0.8  10 1 0 2 0 0 3_ _ 1 _ 3.3  1.0  16  0_  2 3 1 4  4.5  2.5  0 2  APPENDIX IX  Blood  Counts Data Taken From Dogs* Two Months From The B e g i n n i n g Of T r i a l I I A.  k00  s  E r y t h r o c y t e Counts ( m i l l i o n s o f cells/cmm)  B  400  s  ioo  C  400  Dog 1 2 3 4 average  6.2 6.6 6.5 7.0  7.9 6.6 5.7 5.3  7.4 6.5 6.3 6.3  6.8 6.4 5.9 5.8  6.6  6.4  6.6  6.2  B.  S  H00  Leucocyte Counts (cells/cmm)  B  H00  s  ioo  C  400  Dog 1 2 3 4 average  15,000 10,800 13,900 18,900  18,900 12,400 18,100 10,700  12,800 10,800 21,400 14,300  15,400 11,100 11,800 12,300  14,700  15,000  14,800  12,700  C.  S  400  Reticulocytes  B  H 0 0  (percent of e r y t h r o c y t e s )  s  ioo  C  400  Dog 1 2 3 4 average  1.8 2.0 1.9 1.7  2.5 1.4 2.9 1.7  1.8 1.7 1.3 1.5  1.4 1.4 0.7 0.9  1.9  2.1  1.6  1.1  * The numbering scheme used here i d e n t i f i e s the dog as s e t out i n Appendix V I I I .  112  APPENDIX IX  D.  (cont'd)  D i f f e r e n t i a l Leucocyte  Polymorphs  S  H00  B  Counts (percent o f l e u c o c y t e s )  400  s  ioo  C  400  Dog 1 2 3 4 average  Lymphocytes  S  66 73 65 73  80 73 69 78  57 76 70 67  62 68 74 67  69.3  75.0  67.5  67.8  400  B  400  s  ioo  C  400  Dog 1 2 3 4 average  Eosinophils and Monocytes  26 25 33 23  20 20 26 21  32 23 28 32  30 29 20 26  26.8  21.8  28.8  26.3  S  400  B  400  clOO  nkOO  Dog 1 2 3 4 average  8 1 1 4  0 1 1 0  0 7 5 0  0 0 0 1  3.5  0.5  3.0  0.3  11 1 1 1 3.5  0 0 0 0  7 0 6 5  1 3 0 1  0  4.5  1.3  APPENDIX X Differential  Counts* of Femoral Bone Marrow Of Rats i n T r i a l I I 1.  Group  ;400  Myeloblasts  >400  .100  '400  Male 1 2 3 4 5 average  0.7 1.0 0.7 0.7 1.3  0.7 1.0 1.0 1.3  0.3 0.7 1.0 0.7 0.3  1.0 0.7 0.7 0.3  0.88  0.80  0.60  0.54  1.3 1.0 1.7 1.0 0.0  1.3 1.0 1.3 0.7 0.3  0.7 0.3 0.3 1.0 1.3  0.0 0.7 0.0 1.0 0.7  1.0  0.92  0.72  0.48  Female 1 2 3 4 5 average  2.  Group  .400  Promyelocytes  ,400  • 100  ^400  Male 1 2 3 4 5 average  2.0 2.7 2.0 1.7 3.7  2.7 2.7 1.0 1.7 3.0  1.3 2.3 2.0 2.0 1.7  3.0 3.7 4.3 1.7 1.0  2.42  2.22  1.86  2.74  2.0 1.7 2.0 2.3 2.3  2.0 0.7 1.3 1.0  2.54  2.3 2.7 2.0 1.3 1.0 1.86  2.06  1.0  Female 1 2 3 4 5 average  These v a l u e s a r e a l l percentages of 300 d i f f e r e n t i a t e d  cells.  114  APPENDIX X (cont'd)  3.  ;400  Group  Myelocytes  ,400  -100  ^400  Male 1 2 3 4 5 average  2,3 2.7 2.3 2.3 4.0  3.0 5.0 2.3 4.3 3.7  3.7 3.3 2.7 3.7 3.3  4.0 5.7 5.3 4.0 2.3  2.72  3.66  3.34  4.26  4.3 3.0 5.0 3.0 2.7  3.3 5.0 4.0 3.0 3.3  3.3 3.3 4.3 4.3 2.7  4.3 3.0 2.3 2.0 3.0  3.60  3.72  3.58  2.92  Female 1 2 3 4 5 average  4.  Group  Metamyelocytes and "Ring C e l l s "  :400  .400  • 100  ^400  9.7 10.0 11.0 9.7 11.0  14.3 13.3 11.7 13.0 13.3  12.3 12.7 11.3 12.0 11.0  12.0 16.0 13o7 9.7 10.3  10.28  13.12  11.0  12.34  11.3 10.7 12.3 11.7 12.7  14.0 13.3 15.3 10.3 10.0  13.7 12.0 11.0 14.3 11.7  12.3 14.3 12.3 13.0 11.0  11.74  12.58  12.54  12.58  Male 1 2 3 4 _5 average Female 1 2 3 4 5 average  115  APPENDIX X 5.  Group  (cont'd)  Mature G r a n u l o c y t e s  ;400  .400  .100  ^400  29.7 30.7 23.3 24.0 22.7  25.3 20.7 27.3 25.0 24.7  35.0 32.7 30.7 28.0 28.0  30.3 33.3 28.7 35.0 37.0  26.08  24.60  30.88  32.8  28.0 27.7 20.3 26.0 26.7  19.0 21.3 19.3 29.3 31.0  28.0 32.0 28.3 28.7 30.7  37.3 35.3 36.0 35.0 35.7  25.74  23.98  29.54  35.14  Male 1 2 3 4 5 average Female 1 2 3 4 5 average  6.  Group  .400  Lymphocyte-Like C e l l s  ,400  ,100  ^400  7.7 7.0 9.7 10.7 12.0  10.7 7.3 9.3 7.3 9.7  Male 1 2 3 4 5 average  4.0 8.0 5.3 9.0 9.0  7.7 8.0 9.0 7.3 9.7  7.06  8.34  9.42  8.86  Female 1 2 3 4 5 average  10.0 8.7 9.3 7.7 7.7 8.68  7.3 9.0 9.0 8.7 10.7 8.94  13.3 9.7 11.7 8.7 7.0 10.08  8.7 7.0 10.7 7.7 8.0 8.42  116  APPENDIX X (cont'd)  7.  Group  N u c l e a t e d Red Blood  Cells  • 100  .400  ,400  49.0 41.7 50.7 48.3 43.3  44.3 46.0 43.0 43.3 40.7  37.0 38.3 37.3 38.7 37.7  36.0 31.0 35.0 37.7 35.0  46.60  43.46  37.80  34.94  38.3 43.7 42.7 44.0 43.0  47.7 41.3 43.0 42.3 37.7  34.7 36.3 37.0 37.3 39.3  35.0 34.0 34.0 36.7 36.0  42.34  42.40  36.92  34.14  >H00  Male 1 2 3 4 5 average Female 1 2 3 4 5 average  8.  Megakaryocytes, Lymphocytes, Plasma C e l l s and Monocytes  Group  ;400  ,400  .100  ^400  Male 1 2 3 4 5 average  2.6 3.1 3.1 4.3 3.3  2.0 3.3 5.6 4.3 3.7  2.0 3.6 4.7 4.4 5.9  3.3 2.0 3.0 4.4 5.0  3.28  3.78  4.12  3.54  3.7 3.3 4.6 4.4 5.7  5.0 6.2 6.1 4.3 6.0  4.3 4.7 5.4 3.3 5.0  3.0 3.6 4.1 3.3 4.6  4.34  5.52  4.55  3.72  Female 1 2 3 4 5 average  APPENDIX XI  D i f f e r e n t i a l Counts* o f Femoral Bone Marrow Of Dogs i n T r i a l I I 1.  Group  S  Myeloblasts  B  4 0 0  4 0 0  S  1 0 0  C^  0 0  Dog.No. 1 2 3 4 average  1.7 0.7 0.7 0.7  0.7 0.7 1.7 1.0  1.3 1.0 0.7 1.3  0.7 1.0 1.3 0.7  0.95  1.03  1.08  0.93  2.  Group Dog  S  k  0  B  °  average  S  1 0 0  C  4 0 0  2.7 1.3 1.0 3.0  1.7 2.7 2.7 1.7  3.3 2.0 1.7 1.7  2.3 2.3 1.7 2.3  2.00  2.20  2.18  2.15  3.  Group  1 2 3 4  4 0 0  No.  1 2 3 4  Dog  Promyelocytes  S  k  0  °  Myelocytes  B  4 0 0  S  1 0 0  C  4 0 0  No. 5.0 4.0 3.0 5.7  2.3 4.7 4.7 2.3  5.6 3.7 3.7 3.0  6.6 3.3 3.0 4.3  4.40  3.50  4.00  3.55  * These v a l u e s a r e a l l percentages o f 300 d i f f e r e n t i a t e d c e l l s . The numbering scheme used i s e x p l a i n e d i n Appendix V I I I .  118  APPENDIX XI (cont'd)  4.  Group  Metamyelocytes and Stem C e l l s  S  4 0 0  B  4 0 0  S  1 0 0  C  k  0  °  Dog. No. 1 2 3 4 average  23.6 23.7 22.3 22.0  25.0 22.3 20.3 25.3  22.3 24.0 23.7 23.0  25.0 22.6 22.6 24.0  22.90  23.23  23.25  23.55  S  C  5.  Group  Mature G r a n u l o c y t e s  S  4 0 0  B^  0 0  1 0 0  4 0 0  Dog.No. 1 2 3 4 average  18.0 18.6 20.7 16.4  20.6 20.3 18.6 17.0  19.0 19.6 19.6 19.0  22.3 18.0 21.4 21.0  18.43  19.13  19.30  20.68  S  C  6.  Group  Proerythroblasts  S  k  0  °  B  4 0 0  1 0 0  4 0 0  Dog No. 1 2 3 4  3.3 2.7 2.7 3.7  2.3 3.7 3.7 3.3  3.3 2.0 2.3 3.0  2.3 2.7 2.3 2.3  3.10  3.25  2.65  2.4  119  APPENDIX XI (cont'd)  7.  Group  S  Normoblasts  B  4 0 0  u 0  °  S  1 0 0  C * 1  00  Dog No. 1 2 3 4 average  37.3 36.3 36.0 36.7  36.3 36.3 38.3 35.7  36.3 36.0 35.7 35.7  34.3 36.3 35.3 34.3  36.58  36.65  35.93  35.05  S  C"  8.  Group  S^  Lymphocytes  B  0 0  4 0 0  1 0 0  0 0  Dog No. 1 2 3 4  7.7 9.7 10.3 9.0  average  9.3 8.0 8.3 10.0  9.18  9.  Group  S  hQ0  8.90  7.7 9.7 10.3 9.3  8.3 10.7 9.3 10.0  9.25  9.58  Monocytes  B  4 0 0  S  1 0 0  C  k  0  °  Dog No. 1 2 3 4 average  0 0.7 1.0 0.7  0.7 1.0 0.3 1.3  0 0.7 1.0 1.3  0.7 0.7 1.3 0.3  0.60  0.83  0.75  0.75  120  APPENDIX XI (cont'd)  10.  (roup  S  k  0  Megakaryocytes  °  B  h  Q  0  S  1 0 0  C  4 0 0  Dog No. 1 2 3 4 average  0.7 1.3 1.3 0.7  0.7 0.3 0.7 1.3  0.3 0 1.0 1.3  0.3 1.3 1.0 0.3  1.25  0.85  0.58  0.83  11.  Group  S  4 0 0  Plasma  Cells  B  4 0 0  S  1 0 0  C  4 0 0  Dog No. 1 2 3 4 average  0 1.0 1.0 0.3  0.3 0 0.7 0.7  1.0 1.3 1.3 0.7  0 1.0 0.7 0.3  0.58  0.43  1.08  0.50  121  APPENDIX X I I  Mean Weights* I n Grams F o r T r i a l I I I Rats  Day Initial 1 3 5  Control  B - 0.5  57.5 60.5 69.5 76.6  57.3 60.7 68.4 75.6  B - 5 57.6 61.4 69.8 78.0  B-50 56.3 59.5 68.3 76.6  8 10 12  90.5 101.1 110.1  89.9 100.0 108.4  91.3 102.4 111.7  90.1 100.5 109.5  15 17 19  123.0 130.4 135.8  121.1 128.7 134.2  122.3 129.0 134.6  121.4 127.7 132.9  24 26  144.2 148.7  143.2 148.3  143.7 148.1  142.4 147.4  29 31 33  158.3 163.9 169.3  159.1 164.1 170.4  158.1 163.7 169.5  157.4 163.1 167.8  * mean o f t e n r a t s  122 APPENDIX X I I I  Blood  Counts Of Rats I n T r i a l I I I  A.  E r y t h r o c y t e counts ( m i l l i o n s p e r cmm)  Rat  Control  B-0.5  1 2 3 4 5 6 7 8 9 10  8.04 9.40 8.34 6.80 7.09 7.44 6.50 7.01 7.93 7.43  7.72 7.00 7.70 5.80 6.64 7.31 7.03 6.74 8.76 7.91  7.61 7.72 8.02 7.97 6.50 8.03 6.71 7.06 7.97 6.77  7.91 8.11 8.19 7.13 10.05 6.30 7.18 8.16 6.20 6.72  Mean  7.60  7.26  7.44  7.60  S.D.  0.81  0.77  0.59  1.09  B.  (cells  Leucocytes  B - 5  B-50  p e r cmm)  Rat  Control  B-0.5  B - 5  B-50  1 2 3 4 5 6 7 8 9 10  12,000 10,400 9,700 9,500 8,700 12,000 14,600 9,600 14,300 11,300  7,900 16,300 11,500 12,100 13,100 14,300 14,100 10,300 17,600 11,000  13,200 12,900 9,700 12,800 10,700 11,700 13,200 15,700 14,100 9,700  11,700 8,900 14,100 15,700 11,100 12,300 8,400 16,400 13,000 12,700  Mean  11,200  12,800  12,400  12,400  S.D.  1,920  2,670  1,830  2,460  123  APPENDIX XIV  P e r c e n t D i f f e r e n t i a l o f L e u c o c y t e s i n Blood Smears From Group I I I Rats  1.  Polymorphs Rat  Eosinophils  Lymphocytes  Monocytes  83 89 82 73 77 92 92 75 72 83  1  1 6 7 3  81.6  2.2  No.  1 2 3 4 5 6 7 8 9 10  16 11 15 23 22 8 7 19 9 14  average  14.4  1 3  11 1.5  2.  Polymorphs Rat  Control  2 1 1  B - 0.5  Eosinophils  Lymphocytes  Monocytes  No.  11 12 13 14 15 16 17 18 19 20 average  12 12 24 29 21 21 12 4 14 22  1  17.1  0.6  1 3 1  87 88 74 69 77 76 84 92 84 75 81.0  1 3 3 1 4 4 2 3 2.1  APPENDIX XIV (cont'd)  3.  Polymorphs Rat  B - 5  Eosinophils  Monocytes  No.  21 22 23 24 25 26 27 28 29 30  23 9 15 5 24 11 7 1 23 7  average  2 3 1  12.5  0.6  4.  Polymorphs Rat  Lymphocytes  75 91 80 94 77 83 90 96 75 93 85.4  1 2 1 6 3 3 4 2  B - 50  Eosinophils  Lymphocytes  Monocytes  No.  31 32 33 34 35 36 37 38 39 40 average  16 9 16 21 10 13 23 9 8 27 15.2  3  8  1.1  85 90 81 77 87 86 68 78 90 70  1 1 2 1 2 6 5 2 2  81.2  2.2  125  APPENDIX XV  Percentages o f C e l l s i n the A r b i t r a r y C a t e g o r i e s * of C e l l s i n the Femur Bone Marrow C o n t r o l Group  Category  1  2  4  3  5  6  7  8  Rat No. 1 2 3 4 5  4.3 5.3 4.8 3.5 6.3  average  4.84  4.9 10.0 6.9 3.8 3.7 5.86  5.9 11.0 10.7 8.2 7.0  20.3 13.7 13.4 17.6 11.6 15.32  22.0 15.7 18.3 15.4 26.2  31.8 39.7 34.8 37.0 28.2  9.8 4.7 10.3 13.5 17.3  0.33  8.56  19.52  34.30  11.12  0.20  4  5  6  7  8  -  0.34 0.31  -  B - 0.5  Category  1  2  3  -  Rat No. 11 12 13 14 15 16 17 18 19 20 average  6.6 5.9 3.7 5.3 3.3 6.5 4.1 4.1 7.2 3.5  3.0 5.3 4.9 2.3 7.7 5.3 4.8 3.2 6.5 0.9  15.0 19.8 10.4 13.0 11.3 8.5 12.4 11.7 16.6 13.5  12.3 6.3 12.3 17.3 7.0 6.3 9.9 13.6 17.8 9.6  16.9 10.2 17.2 19.3 18.3 10.3 18.2 20.6 18.5 22.0  34.9 36.3 35.6 31.9 32.3 43.3 35.0 35.7 24.0 37.7  9.6 14.5 8.9 10.6 19.7 18.7 15.6 9.2 7.6 11.6  0.66 0.33 0.92 0.33 0.70 0.33  5.02  4.39  13.22  11.25  17.15  34.67  12.60  0.63  1.30 1.10 0.63  ^Categories: 1. E o s i n o p h i l s - 10 - 15 m i c r o n s , l a r g e r e d g r a n u l e s c l o s e l y packed i n cytoplasm, n u c l e u s ; b l u e , round, kidney o r l o b e d . 2. Doughnut c e l l s - ( i ) 15 - 20 microns, b l u e w i t h r e d h o l e . ( i i ) 30 microns, l a r g e b l u e r i n g w i t h l i g h t coloured nucleus. 3. Myelocytes - ( i ) 25 microns, l i g h t - c o l o u r e d l a r g e nucleus (takes up most of the c e l l ) and a l i g h t b l u e cytoplasm (ring effect) ( i i ) 15 m i c r o n s , p u r p l e c o l o u r e d m o t t l e d c e l l  126  APPENDIX XV  (cont'd)  B - 5  Category  1  2  3  4  5  6  7  8  Rat No. 21 22 23 24 25 26 27 28 29 30 average  8.7 3.6 8.0 5.6 6.6 4.3 3.0 7.0 4.4 3.6 5.48  3.1 5.3 8.7 3.8 7.0 5.0 3.3 5.4 5.9 11.0  16.1 17.2 6.0 17.9 16.2 15.6 13.3 10.2 13.1 17.7  5.85  14.6 12.5 0.7 11.9 15.9 10.3 5.3 7.9 8.4 3.7  14.33  9.12  19.6 16.5 0.7 19.7 23.8 23.9 10.3 19.4 24.4 13.0  29.2 30.4 62.0 28.8 21.5 26.2 38.3 37.8 33.4 36.0  7.8 11.9 14.0 11.0 7.9 10.6 24.7 11.7 10.3 14.7  0.33 0.33  17.13  34.36  12.46  0.41  6  7  1.7 1.7  B - 50  Category  1  2  3  4  5  8  Rat No. 31 32 33 34 35 36 37 38 39 40 average  4.3 3.5 1.7 1.6 4.8 3.4 4.5 5.7 4.7 2.4  4.3 5.4 5.0 5.7 2.3 5.6 6.1 5.7 8.7 3.6  14.1 19.6 13.3 14.1 18.9 20.9 13.7 12.3 15.3 22.5  3.66  5.24  16.47  5.3 16.7 7.0 6.1 8.3 8.4 8.9 4.3 9.7 11.1 8.58  23.7 14.5 9.3 19.8 16.3 14.3 22.3 10.6 9.3 9.3  40.8 26.8 41.0 35.5 33.3 28.3 32.8 50.3 35.7 37.1  6.9 12.3 12.3 16.9 14.7 15.3 9.6 11.0 17.0 14.1  14.94  36.16  13.01  0.33 0.63 0.33 0.32 0.31 1.6 0.33  0.39  C a t e g o r i e s (cont'd) 4. E r y t h r o b l a s t s - 7 - 10 microns, l i g h t cytoplasm w i t h dark b l u e n u c l e u s . 5. Mature g r a n u l o c y t e s - 10 - 15 microns, p i n k g r a n u l a r cytoplasm w i t h n u c l e u s v a r y i n g from band t o kidney. 6. Lymphocyte-like c e l l s - 10 m i c r o n s , s m a l l c l e a r - c o l o u r e d c e l l s w i t h no n u c l e u s . 7. N u c l e a t e d r e d b l o o d c e l l s - 8 - 12 m i c r o n s , s m a l l c l e a r - c o l o u r e d c e l l s w i t h a s t r a n d i n the cytoplasm. 8. Megakaryocytes.  127  APPENDIX XVI  Standard Method Used f o r Counting  1. Red b l o o d c e l l  and D i f f e r e n t i a t i n g  Cells  counts  E r y t h r o c y t e s were counted u s i n g Hayem's d i l u e n t , Thoma r e d c e l l d i l u t i n g p i p e t t e s and d i s c u s s e d i n Wintrobe 2. White b l o o d c e l l  an improved Neubauer c o u n t i n g chamber as  (107).  counts  The procedure  as o u t l i n e d by Wintrobe was  followed.  l e u c o c y t e s were counted u s i n g a d i l u t i n g p i p e t t e marked 0.5, 11 and  The 1 and  g i v i n g a d i l u t i o n of 1 i n 20, an improved Neubauer c o u n t i n g  chamber and  the f o l l o w i n g d i l u e n t : g l a c i a l acetic acid 2 ml d i s t i l l e d water 98 ml a few drops of methylene b l u e  3. D i f f e r e n t i a l of l e u c o c y t e s Two  b l o o d smears were made from each a n i m a l .  The  were s t a i n e d w i t h Wright  s t a i n a c c o r d i n g t o Wintrobe and  b l o o d c e l l s were counted  on each  slides  100  white  slide.  4. R e t i c u l o c y t e counts Blood f i l m s were made by mixing f o l l o w i n g s o l u t i o n and  (50:50) b l o o d w i t h  the  then l e t stand f o r ten minutes b e f o r e making  smears on g l a s s s l i d e s . 100 ml 0.4 gm 1 gm  0.85% s a l i n e sodium c i t r a t e b r i l l i a n t c r e s y l blue  The number of r e t i c u l o c y t e s were counted  per 1000  red blood  cells.  APPENDIX XVI (cont'd)  D i f f e r e n t i a l o f bone marrow c e l l s Bone marrow was removed from femurs when the animals were killed.  An e q u a l q u a n t i t y o f homoserum was mixed w i t h the marrow  and s l i d e s were made of the m i x t u r e .  The s l i d e s were then s t a i n e d  w i t h Wright s t a i n and a t o t a l o f 300 c e l l s  differentiated.  

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