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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., University of B r i t i s h Columbia, 1964 A Thesis Submitted i n P a r t i a l Fulfilment of the Requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURE i n the D i v i s i o n of Animal Science We accept t h i s thesis as conforming to the standard required from candidates for the Degree of Master of Science i n Agri c u l t u r e THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1966 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r -m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t I s u n d e r s t o o d t h a t c o p y i n g o r p u b l i -c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a pete / l a j f /r, /fa i i ABSTRACT A possible 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 occurring a - g l y c e r y l ethers has been investigated. B a t y l , s e l a c h y l and chimyl alcohols have been administered per orum for extended periods to growing dogs and rats at various dosage l e v e l s to a s c e r t a i n i f these compounds produce i n i m i c a l or favourable e f f e c t s i n terms of growth and haematopoietic responses. Groups of male and female dogs and rats maintained on normal rations were given d a i l y doses of each of the three alcohols at a l e v e l of 6 mgs per kilogram of body weight for 180 days. There was no evidence to suggest that these compounds produced harmful e f f e c t s . H i s t o p a t h o l o g i c a l study of the major tissue system confirmed t h i s conclusion. A favourable response i n the form of a s l i g h t l y increased growth rate was noted i n the female rats that received the s e l a c h y l alcohol . No evidence of a haematopoietic e f f e c t was obtained. In a second experiment, s e l a c h y l alcohol was offered as an addendum to a normal r a t i o n , to both dogs and rats at l e v e l s that ensured a d a i l y consumption of 600 and 2400 mg per kilogram of body weight of t h i s alcohol for a period of s i x t y days. Other groups of both species received b a t y l alcohol at the higher dosage l e v e l (2400 mg per kilogram of body weight) d a i l y f o r the same time period. The r e s u l t s obtained suggest that both compounds when fed at the 2400 mg per kilogram l e v e l i n t e r f e r e d with the d i g e s t i b i l i t y of the r a t i o n offered and, i n i i i so doing, reduced the growth rate of the experimental dogs. A corresponding e f f e c t did not occur i n the r a t s . Both alcohols, when administered at the highest dosage l e v e l , induced an increase i n the r e t i c u l o c y t e count i n the blood of the dogs and an increase i n the percentage of nucleated red blood c e l l s i n the bone marrow smears prepared from the r a t s . A g l y c e r y l ether-free synthetic r a t i o n supplemented with 0.5, 5 and 50 mg of b a t y l alcohol per kilogram of body weight was prepared and then offered to groups of young growing rats f o r a period of f i v e weeks. No growth response was obtained at any l e v e l , suggesting that t h i s compound does not have a n u t r i t i o n a l function or that the r a t i s able to synthesize the compound at a rate that i s adequate to permit near maximum growth. I t i s also possible that the animals had s u f f i c i e n t reserves of compounds of t h i s type to permit growth at the measured rate. i v TABLE OF CONTENTS i Page ABSTRACT . . i i TABLE OF CONTENTS ' . . . . . . . . . . . . . i v LIST OF TABLES . . . . . . . . . . . . . . . . . v i i i LIST OF FIGURES i x ACKNOWLEDGEMENTS . . . . . . . x i INTRODUCTION . . . . . 1 PART I - LITERATURE REVIEW A. The chemical nature of the a- g l y c e r y l ethers, . 2 B. Occurrence of the g l y c e r y l ethers . . . . . . . 4 C. The composition of the g l y c e r y l ethers . . . . 13 D. The mesenchymal o r i g i n of the a-g l y c e r y l ethers 16 E. Biosynthesis of the g l y c e r y l ethers 16 F. Digestion and metabolism of a- g l y c e r y l ethers . 18 G. a-glyceryl ethers and haemopoiesis . . . . . . 20 H. Summary . 26 PART II - EXPERIMENTAL A. PRELIMINARY EVALUATION - TRIAL I . . . . . . . 33 1. Preamble . . . . . . . . . . 33 2. Experimental design . . . . . . 35 3. Animals . . . . . . . . . . . . . . . . . . 36 4. Rations . . . . . . . . . . . . . 36 5. Housing . . . . . . . 37 V TABLE OF CONTENTS (cont'd) Page 6. Administration of g l y c e r y l ethers . . . . . . 37 7. Results . . . . . . . . . . . . . . . . . . . 37 8. Conclusion . . . . . „ . . . „ . . . . . . . 42 B. PRELIMINARY EVALUATION - TRIAL I I . . . . . . . 43 1. Preamble . . . . . . . . . . . . . 43 2. Design . . 43 3. Animals . . . . . . . . . . . . . . . . . . . 45 4. Housing . . . . . . . . . . . . . . 45 5. Rations and administration of g l y c e r y l ethers 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 experimental rations 47 (i) Rats 47 ( i i ) Dogs 48 (b) I s o l a t i o n and determination of the g l y c e r y l ethers 48 ( i ) E x traction of l i p i d s from feed and faeces . . . . . . . . . 48 ( i i ) Determination of neutral l i p i d s . . 49 ( i i i ) Determination of the unsaponifiable l i p i d f r a c t i o n 49 (iv) Q u a l i t a t i v e and quantitative determination of g l y c e r y l ethers 50 7. Discussion of feed-intake, d i g e s t i b i l i t y and weight gain . . . . . . . . . . . . . . 51 8. G l y c e r y l ethers and d i g e s t i b i l i t y . . . . . . 60 9. Results from c e l l counts . . . . . . . . . . 65 v i TABLE OF CONTENTS (cont'd) Page C. FEEDING RESPONSE EXPERIMENT - TRIAL I I I . . . . 68 1. Preamble . . . . . . . . . . . . . . . . . . 68 2. Design . . . . . . . . . . . . . . . . . . . 68 3. Animals . . . . . . . . . . . . . . . . . . 69 4. Housing . . . . . . . . . . . . 69 5. Ration . . . . . . . . . . . . . . . . . . . 69 6. Feed intake-gain r e l a t i o n s h i p s . . . . . . . 70 7. D i g e s t i b i l i t y . . . . . . . . . . . . . . . 71 8. Results of blood and bone marrow c e l l counts 72 9. Conclusions . . . . . . . . . . „ . 72 PART I I I - GENERAL DISCUSSION . . . . . . . . . . . . . . . 75 BIBLIOGRAPHY . . . . . . . . . . . . . . . . 79 APPENDICES I Composition of rations used i n T r i a l s I and II . . . 89 II Mean d a i l y feed intake for T r i a l I rats . . . . . . . 90 III Mean weekly weights i n T r i a l I . . . . . . . . . . . 92 IV Blood counts i n T r i a l I . . . . . . . . . . . . . . . . 94 V Weight data for animals i n T r i a l II . . . . . . . . . 99 VI Results of rat blood taken one month from the beginning of T r i a l II . . . . . . . . . . . . . . . 102 VII Results of rat blood taken two months from the beginning of T r i a l II . . . . . . . . . . . . . . . 105 VIII Blood count data from dogs one month from the beginning of T r i a l II . . . . . . . . . . . . . . . 108 v i i TABLE OF CONTENTS (cont'd) Page IX Blood count data from dogs two months from the beginning of T r i a l II . . . . . . . . . . . . . . . I l l X D i f f e r e n t i a l counts of femoral bone marrow of rats i n T r i a l II . . . . . . . . . . . . . . . . 113 XI D i f f e r e n t i a l counts of femoral bone marrow of dogs i n T r i a l II . . . . . . . . . . . . . . . . . 117 XII Mean weights i n grams for T r i a l I I I rats . . . . . . 121 XIII Blood counts of rats i n T r i a l I I I . . . . . . . . . . 122 XIV Percent d i f f e r e n t i a l of leucocytes i n blood smears from T r i a l I I I rats . . . . . . . . . . . . 123 XV Percentages of c e l l s i n the femoral bone marrow of T r i a l I I I rats . . . . . . . . . . . . . . . . . 125 XVI Standard methods used for counting and d i f f e r e n t i a t i n g c e l l s . . . . . . . . . . . . . . . 127 v i i i : ' . LIST OF TABLES TABLE Page I Melting point and structure of the g l y c e r y l ethers . . 2 II Occurrence of g l y c e r y l ethers . . 6 I I I G l y c e r y l ethers i n the unsaponifiable f r a c t i o n of neutral l i p i d s from several mammalian tissues . . . 9 IV Percentage of g l y c e r y l ethers i n l i p i d s from some human and animal sources . . . . . . . . . . . . . . 9 V Composition of the nonsaponifiable matter i n f i s h l i v e r o i l s . . . . . . . . . . . . . . . . . . 10 VI L i v e r o i l s from Elasmobranch f i s h 10 VII A summary of the extensive table given by Karnovsky and Rapson . . . . . . . . . 11 VIII a-Glyceryl ether content of dog tissues . . 12 IX a-Glyceryl ethers of mammalian yellow bone marrow . . 12 X The percentage composition of the alkoxyglycerols from l i v e r o i l s . . . . . . . . . . . . . 13 XI The percentage composition of g l y c e r y l ethers from human bone marrow, human spleen, and human milk . . 14 XII 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 1 ^ - l a b e l l e d chimyl d i o l e a t e . 19 XIII Alkoxyglycerol esters fed to rats 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 . . . . . . . . . . . 23 XIV B i o l o g i c a l experiments in v o l v i n g the use of g l y c e r y l ethers . . . . . . . . . . . . . . . . . . 27 XV Representative values of important vitamins i n milk . 34 XVI Amount of g l y c e r y l ether administered compared with the vitamin requirements of the albino laboratory r a t 34 XVII Schedule of blood counts . . . . . . . . . . . . . . . 36 XVIII Summary of mean body weight gain and mean feed-intake data for male rats . . . . . . . . . . . 38 i x LIST OF TABLES (cont'd) TABLE Page XIX Summary of mean body weight gain and mean feed-intake data for female rats . . . . . . . . . . 38 XX Summary of body weight gain for dogs . . . . . . . . . 38 XXI Summary of blood counts taken i n T r i a l I . . . . . . . 41 XXII Grouping and dosage l e v e l s used i n T r i a l II 45 XXIII Summary of weight gain and feed-intake of dogs i n T r i a l II 52 XXIV Summary of feed e f f i c i e n c i e s . 56 XXV 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 and feed e f f i c i e n c i e s for a model dog . . . . . . 57 XXVI Feed intake-gain r e l a t i o n s h i p s f o r the rats i n T r i a l II 60 XXVII Recovery of g l y c e r y l ethers from faeces and determination of d i g e s t i b i l i t y of the ethers for the dogs i n T r i a l II 62 XXVIII Recovery of g l y c e r y l ethers from faeces and determination of d i g e s t i b i l i t y of the ethers for the rats i n T r i a l II . . . . . . . . . 63 XXIX S i g n i f i c a n t differences i n c e l l counts 65 XXX Feed intake-gain r e l a t i o n s h i p s for rats i n T r i a l III . 71 XXXI D i g e s t i b i l i t y of experimental rations i n T r i a l I I I . . 71 XXXII Summary of the blood and bone marrow data from T r i a l I I I . . . . . . . . . . . . . . . . . . . . 73 XXXIII Erythrocyte counts of four control rats fed b a t y l alcohol at the end of the experimental period . . . . . . . . . 73 X LIST OF FIGURES TABLE Page I Growth curves for female rats i n T r i a l I . . . . . . . 39 II The mean growth rates of the two dogs from each group maintained on the con t r o l r a t i o n following removal from t h e i r experimental di e t s . „ . . . „ . 58 I I I D i g e s t i b i l i t y of g l y c e r y l ethers . . . . . . . . . . . 64 x i ACKNOWLEDGEMENTS I wish to thank Dr. B.A. Eagles, Dean of:the Faculty of Agr i c u l t u r e and Chairman of the D i v i s i o n of Animal Science, for h i s permission to undertake t h i s project and for the use of departmental f a c i l i t i e s . My thanks are extended to those at the Univ e r s i t y of V i c t o r i a who permitted the completion of t h i s study at that i n s t i t u t i o n . To Dr. A.J. Wood, Dean of Arts and Science at the University of V i c t o r i a (formerly Professor of Animal Science at the Univ e r s i t y of B r i t i s h Columbia) I wish to express my sincere appreciation for 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 to thank Dr. W. Chalmers and the s t a f f at Western Chemical Industries Limited, Vancouver, B.C., rfor t h e i r guidance and for the use of th e i r laboratory f a c i l i t i e s . The technical assistance of Miss Karen Forgeron and Miss Heather McConnell i s g r a t e f u l l y acknowledged. I acknowledge the generous f i n a n c i a l support of the Labatt Foundation. PART I: LITERATURE REVIEW 1 INTRODUCTION The a-glyceryl ethers have been examined as possible therapeutic agents for more than ten years. In 1957, Evans, e_t al_ (33), reported on the use of one of these, b a t y l alcohol, i n the treatment of bracken poisoning i n the bovine and since then other reports have been published i n d i c a t i n g various degrees of success i n the use of b a t y l alcohol for the treatment of c e r t a i n blood dyscrasias. The a-g l y c e r y l ethers have aroused renewed i n t e r e s t with the finding that they normally e x i s t i n a l l animals i n small q u a n t i t i e s . This might imply that they possess v i t a m i n - l i k e properties. An extensive l i t e r a t u r e review has been c a r r i e d out covering the occurrence, composition and chemical nature of these ethers. The l i t e r a t u r e concerning 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 to be wanting i n that much of the experimental work has been ca r r i e d out under the " i l l defined" conditions of the c l i n i c . The present study was designed to determine i f c e r t a i n of the a - g l y c e r y l ethers can be considered to be e s s e n t i a l nutrients. To th i s end, a serie s of experiments have been ca r r i e d out using the dog and the albino rat as experimental subjects. The work has, of necessity, involved screening assays to ascertain i f the compounds selected would produce i n i m i c a l e f f e c t s i n mammalian systems when administered per orum. On the basis of t h i s preliminary work, a feeding response experiment was conducted, using synthetic a - g l y c e r y l free basal d i e t s , to determine i f the compounds possess growth promoting properties or desirable e f f e c t s on the haematopoietic system. 2 A. THE CHEMICAL NATURE OF THE a-GLYCERYL ETHERS The three most common, n a t u r a l l y occurring, g l y c e r y l ethers are: a-hexadecylglyceryl ether (chimyl a l c o h o l ) , a-octadeeylglyceryl ether (batyl a l c o h o l ) , and a-,9-octadecenylglyceryl ether (selachyl a l c o h o l ) . TABLE I: MELTING POINT AND STRUCTURE OF THE GLYCERYL ETHERS Gl y c e r y l Ether Melting Point Structure chimyl alcohol 60° - 61.5°C. (28) CH 3(CH 2) 1i +CH 2.0.CH 2 .CH(OH) .CH20H b a t y l alcohol 70° - 71°C. (28) CH 3(CH 2)i6 CH2.0.CH2 .CH(OH) .CH20H sela c h y l alcohol 8 - 9 C. (16) CH 3(CH 2) 7CH CH(CH 2) E i . 0. CH 2 .CH(OH).CH20H The ether linkage was f i r s t shown to be of the a form by Weidemann (106) i n 1926 when he demonstrated that hydroiodic acid acts on b a t y l alcohol to produce methyl iodide. This established that the t h i r d oxygen i s present as a methoxy group and, i n turn, indicates that an ether linkage exists between the s t e a r y l alcohol and the g l y c e r o l moiety. In 1930, Knight (58) reported the surface f i l m a c t i v i t y of the synthetic a form to be i d e n t i c a l with that of the natural ethers, but 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 melting point of b a t y l alcohol i s 60° - 61.5°; t h i s i s the same as that found f o r the a form. The B octadecyl ether has a melting point of 62° - 63°C. (26). A mixture of the B form with natural b a t y l alcohol causes a marked melting point depression, whereas the mixture of a-octadecyl ether with natural b a t y l produces only a s l i g h t depression. This can be. explained on the basis 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 ethers. 3 B a t y l alcohol belongs to the d-series whereas synthetic octadecyl g l y c e r y l ether i s a racemic mixture and therefore can be expected to have s l i g h t l y d i f f e r e n t properties. Oxidation of b a t y l alcohol with lead tetraacetate y i e l d s g l y c o l aldehyde octadecyl ether (m.p. 51°C.) and formaldehyde. According to Criege (28) t h i s i s a s p e c i f i c reaction for an c^S-glycol. This reaction indicates that the a 1 and B positions of the g l y c e r o l moiety must be free and that there i s no a l t e r n a t i v e other than to assume that g l y c e r y l ethers are of the same a type. F i n a l l y , the very f a c t that the g l y c e r y l ethers have o p t i c a l a c t i v i t y (7) (8) (9) shows that the ether linkage must form an asymetric molecule and hence an a linkage must be involved. I t has been observed that b a t y l alcohol i n high concentrations shows a negative r o t a t i o n , gradually diminishing, to disappear e n t i r e l y at a concentration of 10 per cent. On further d i l u t i o n dextrorotation i s displayed (Toyama and Isikawa) (16). Selachyl alcohol, having a double bond i n i t s long sidechain, can e x i s t i n e i t h e r the c i s or trans form; the natural compound has the c i s . configuration, giving i t the low melting point of 8° - 9°C. While the trans form, which has been synthesized (8) (9) has a melting point of 48.5° - 49.5°C. In further i n v e s t i g a t i o n of the o p t i c a l properties, Baer and Fischer (6) (9) synthesized 1- and d- forms of b a t y l , chimyl, and selachyl alcohols. They found the physical properties of the d-forms to be i d e n t i c a l with those of the natural compounds, while 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 Gl y c e r y l ethers have been found to be quite wide-spread i n t h e i r occurrence; they e x i s t almost e n t i r e l y i n an e s t e r f i e d form and are extracted from tissue with the l i p i d f r a c t i o n . Malins and Mangold (48) report finding some of the free form i n r a t f i s h l i v e r o i l and basking shark l i v e r o i l . Their properties are l i p i d - l i k e i n nature since the structure of the g l y c e r y l ethers i s i d e n t i c a l to the glycerides with the exception that they possess an ether bond instead of an ester bond. 0 H 2 CH20CCH2R CH2OCCH2R | 0 | 0 CH0CCH2R CH0CCH2R | 0 | 0 CH2OCCH2R CH20CCH2R t r i g l y c e r i d e d i e s t e r f i e d g l y c e r y l ether A g l y c e r y l ether was f i r s t described i n 1915 by Kossel and Edlacher (10). They assigned the name " a s t r o l " to the alcohol that they i s o l a t e d (m.p. 71°C.) from the unsaponifiable f r a c t i o n of the f a t of a s t a r f i s h . Later, Tsujimoto and Toyama (28) reported what they believed to be the f i r s t i s o l a t i o n of the g l y c e r y l ethers. In a subsequent communication Toyama established the structure 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 alcohol, thus est a b l i s h i n g Kossel and Edlacher as the o r i g i n a l discoverers of the g l y c e r y l ethers. In 1941, Holmes et a l (49) reported the f i r s t i s o l a t i o n of b a t y l alcohol from a source other than the marine organisms; they i s o l a t e d b a t y l alcohol from the yellow bone marrow of c a t t l e , thus opening the p o s s i b i l i t y that t h i s compound might have value as a nutrient. Since 1941, 5 g l y c e r y l ethers have been i s o l a t e d from many other t i s s u e s . Carter, et a l (1958) (23) i s o l a t e d and i d e n t i f i e d for the f i r s t time a g l y c e r y l ether-containing phospholipid. Since 1948 g l y c e r y l ethers have been found i n the phospholipid fract i o n s from many sources. In at least two tissue systems, bovine erythrocytes (44) and red bone marrow (101) g l y c e r y l ether-containing phospholipids account for over 10 mole percent of the phospholipid f r a c t i o n . Table II provides a summary of the.|many natural sources from which the g l y c e r y l ethers have been i s o l a t e d . TABLE I I : OCCURRENCE OF GLYCERYL ETHERS Tissue Content Reference amniotic f l u i d (human) some b a t y l alcohol 1962 Helmy and Hack (47) a t h e r o s c l e r o t i c 285 mg. b a t y l alcohol from 1943 Hardegger, aortas (human) 3,450 gm. f a t from 370 et a l (45) aortas (0.008% of o i l ) a t h e r o s c l e r o t i c 0.0782 microMole/lOOgm. 1964 M i l l e r , eX a l (75) aortas wet tissue 0.0802 microMole/lOOgm. wet ti s s u e beef tallow (P.L. free) 65mg./Kg. tetradecanol 1960 Schogt, et_ a l (91) bone marrow, yellow b a t y l alcohol 1941 Holmes, e_t a l (49) (c a t t l e ) bone marrow (cattle) 0.2 - 0.7% G.E. (free form) 1957 Brohult (17) bone marrow, red l e c i t h i n f r a c t i o n has (pig epiphyses) 20% G.E. deri v a t i v e 1962 Pietruszko (81) bone marrow (human) 9 - 16% of phospholipids 1965 Wajda (105) are g l y c e r y l ethers brain ( c a l f ) ) ether-containing phospho- 1960 Svennerholm ) l i p i d s range from 1 to and Thorin (96) ) 2.5% of the t o t a l l i p i d s b r ain (human) ) brain (human) i n o s i t o l phosphate i s o l a t e d 1961 Klenk and cephalin f r a c t i o n contained 8.4% batyl-chimyl Hendicks (57) mixture brain (young rat) G.E. phospholipid i s 1.8% 1963 A n s e l l and of t o t a l phospholipids Spanner (3) brain (adult rat) G.E. phospholipid i s 3.1% 1963 A n s e l l and of t o t a l phospholipids • Spanner (3) Centrina s a l v i a n i contains g l y c e r y l ethers 1949 Omelik (78) ( l i v e r and eggs) dog tissues, with the see Table VIII 1962 Nakagawa, exception of marrow et a l (77) egg yolk g l y c e r y l ether containing 1958 Carter (23) phospholipids 7 TABLE II (cont'd) Tissue Content Reference Elasmobranch f i s h erythrocytes (bovine) erythrocytes (bovine) Halobacterium cutirubrum heart muscle (human) heart (ox)(P.L. free) heart (ox) l i v e r (human) l i v e r o i l of: -soupfin shark (Galeorhemis rond.) -seven-gilled shark (Heptranchias  pectorosus) -basking shark (Cetorhinus maximus) -spiny shark (Echinorhinus spinosus) l i v e r o i l , cow shark (Hexanchus grisen) see Table V ethanolamine l i p i d s make up 30% of t o t a l phosphatides 75% of phosphatidylethanolamine f r a c t i o n i s g l y c e r y l ethers 93% of l i p i d s a r e phospholipids 73% of phosphatides are g l y c e r y l ethers mono- and di-ethers occur presumably as t h e i r phos-phate esters lOOOmg. tetradecanol/kg. 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 f r a c t i o n contains g l y c e r y l ethers a l l contain g l y c e r y l ethers l i v e r o i l s (elasmobranch f i s h ) l i v e r o i l (shark) 16.8% selachyl alcohol ) 2.38% b a t y l alcohol ) some chimyl alcohol ) see Table VI 16 - 17% of o i l (expressed as the free form) 1962 Hallgren and Larsson (41) 1961 Hanahan and Watts (44) 1963 Hanahan, et a l (43) 1962 Sehgal, et a l (94) 1965 Popovie (83) 1960 Schogt, et a l (91) 1962 Pietruszko (82) 1958 R i l e y , et a l (86) 1948 Karnovsky,et a l (51) 1948 Karnovsky, et a l (53) 1948 Karnovsky, et a l (54) 1948 Karnovsky, et a l (54) 1954 Manforte and Fenech (69) 1956 H i l d i t c h (48) 1957 Brohult (17) 8 TABLE II (cont'd) Tissue Content Reference l i v e r o i l (dog-fish) l i v e r o i l ( r a t f i s h and basking shark) l i v e r (dogfish) mammalian tissue and s t a r f i s h man and cow Milk f a t (phospho-l i p i d free) Phylum Mollusca -chiton (Catherina tunicata) -marine (Thais Lamellosa) -clam (Protothaca staminea) -octopus (Octopus d o f l e i n i ) reef b u i l d i n g c o r a l (Plexaura flexuoso) sea anemones spleen (pig) s t a r f i s h f a t s t a r f i s h f a t t e r r e s t i a l slugs (Arian ater) contains g l y c e r y l ethers free g l y c e r y l ethers as well as g l y c e r y l ether diesters g l y c e r y l ethers found see Table IX see Table IV 50 mg. tetradecanol/Kg. a l l are r i c h i n g l y c e r y l ethers b a t y l alcohol found b a t y l alcohol i s o l a t e d contains b a t y l alcohol a s t r o l i s o l a t e d b a t y l alcohol found amounts to more than 25 mole per cent of t o t a l (Ariolimax columbianus) phospholipids various various see Table VII see Table III 1960 Malins (65) 1960 Mangold and Malins (70) 1963 Hanahan, et a l (43) 1964 Todd and R i z z i (104) 1962 Hallgren and Larsson (42) 1960 Schogt, et a l (91) 1965 Thompson and Lee (102) 1942 Kind and Bergman (56) 1956 Bergman, et a l (11) 1943 Prelog, et a l (84) 1915 Kossel and Edlbacher (10) 1943 Matsumoto, et a l (74) 1954 Matsumoto and Wainai 1963 Thompson and Hanahan (73) (99) 1946 Karnovsky,et a l (52) 1963 Gilbertson and Karnovsky (37) TABLE I I I : GLYCERYL ETHERS IN THE UNSAPONIFIABLE FRACTION OF NEUTRAL LIPIDS FROM SEVERAL MAMMALIAN TISSUES (taken from Gilbertson and Karnovsky)(37)) Total l i p i d Neutral as % of l i p i d s as Tissue fresh wt. % of T.L. Phosphatide Total V i n y l ethers r e s i d u a l G.E. % molar % of % of N.L. of N.L. t o t a l G.E. diverticulum ( s t a r f i s h ) 5.7 72.50 0.19 16.1 31.9 brain (beef) 7.4 43.95 0.10 1.9 63.1 bone marrow (beef) 88.9 99.97 0.00 2.4 61.2 heart ( c a l f ) 5.1 62.61 0.42 2.4 31.2 adipose tissue (rat) 76.8 98.91 0.01 1.3 82.0 brown f a t (rat) 43.1 78.40 0.04 0.4 54.3 leucocytes (poly-morphs) (guinea pig) - 51.92 0.00 2.9 65.8 chylomicrons (human) 85.90 - 0.1 78.6 be t a - l i p o p r o t e i n (human) - - - 0.6 29.3 TABLE IV: PERCENTAGE OF GLYCERYL ETHERS IN LIPIDS FROM  SOME HUMAN AND ANIMAL SOURCES (taken from Hallgren and Larsson)(42) Source G l y c e r y l Ethers as % of Li p i d s human bone marrow (red) human spleen human red blood c e l l s human milk cow bone marrow (yellow) cow milk egg yolk l i v e r o i l of elasmobranch f i s h l e s s 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 Hallgren and Larsson) (41) Species % unsap. i n o i l Composition of unsap. Chimaera monstrosa 33 almost e x c l u s i v e l y G.E. Somniosus microcephalus 8 90% G.E. and 8% c h o l e s t e r o l Squalus acanthias 7 84% G.E. and 13% c h o l e s t e r o l TABLE VI: LIVER OILS FROM ELASMOBRANCH FISH (taken from H i l d i t c h ) (48) Liv e r f a t from % G.E.* Composition of unsaponifiables skate 0.3 mainly c h o l e s t e r o l angel f i s h 1.5 mainly c h o l e s t e r o l thresher shark 1.8 mainly c h o l e s t e r o l spotted dogfish 2.0 mainly c h o l e s t e r o l grey dogfish 10.0 mainly s e l a c h y l , some chimyl and b a t y l r a t f i s h 37.0 almost wholly s e l a c h y l , only some chimyl and b a t y l shark species 50 - 80 large amounts of squalene, some sel a c h y l , chimyl and b a t y l * G l y c e r y l Ethers TABLE VII: A SUMMARY OF THE EXTENSIVE TABLE GIVEN BY KARNOVSKY AND RAPSON (51) Glyc e r y l ether content of o i l pecies calculated as se l a c h y l dioleate 25 species l i s t e d under Elasmobranchii amount varies from n i l i n the l i v e r of the man eating 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 species l i s t e d under Teleostomi amount varies from n i l i n the blue hottentot to 7.3% i n the l i v e r of the stone bass 6 species l i s t e d under Mollusca quantities vary from 0.7% i n the Limpet to 8.0% i n the v i s c e r a of the rock octopus 1 Arthropoda (crayfish) quantities vary from 0.9% i n the in t e s t i n e to 6.0% i n the f l e s h 1 Amphibia (cape clawed toad) quantities vary from n i l i n the l i v e r to .4% i n the body o i l 1 R e p t i l l i a (mole snake) 0.3% i n the t o t a l v i s c e r a to 1.2% i n the body 1 Aves (domestic fowl) 0.1% i n the l i v e r to 0.3% i n the vi s c e r a 6 species of Mammalia; 3 whales, 1 r a t , 1 cow and 1 man quantities range from n i l i n the l i v e r of a man to 2.5% i n the faeces of the rat 4 vegetable o i l s investigated only tung nut o i l showed any g l y c e r y l ethers (less than 0.1%) TABLE VIII: a-GLYCERYL ETHER CONTENT OF DOG TISSUES (taken from Nakagawa, e_t al) (77) Tissue and Molar r a t i o microMoles/gm. dry animal no. to l i p i d P l i p i d free tissue Lung 1 .0027 .41 Lung 2 .0043 .70 Kidney 1 .0015 .29 Kidney 2 .0086 .80 Liv e r 1 .0026 .31 Liv e r 2 .0077 1.16 Li v e r 2 .0071 1.07 Spleen 1 .0048 .67 Intestine 1 .0102 1.35 Heart 1 .0069 .59 Aorta 1 .0039 .20 Ske l e t a l muscle 1 .0157 1.02 Skeletal muscle 1 .0159 1.03 Bone marrow (rabbit) 1 .1570 8.73 Bone marrow (rabbit) 2 .3600 14.10 TABLE IX: a-GLYCERYL ETHERS OF MAMMALIAN YELLOW BONE MARROW (taken from Todd and R i z z i ) (104) Source NS* as % wt. of marrow G.E.** as % wt. of NS* G.E. composition as % of t o t a l g l y c e r y l ethers 18:0 16:0 18:1 rabbit 0.2 13 0 100 0 cow 0.2 - 0.4 12 - 25 50 50 0 sheep 0.36 11 - - -Pig 0.5 - 0.6 4 - 8 - - -r a t - 0 - - -man - 11.3 - 16.6 25 50 25 *nonsaponifiables * * g l y c e r y l ethers C. THE COMPOSITION OF THE GLYCERYL ETHERS For a good many years only the g l y c e r y l ethers b a t y l , s e l a c h y l , and chimyl were mentioned i n the l i t e r a t u r e . Recently there have been several reports of g l y c e r y l ethers having the chain length of the f a t t y alcohol moiety varying from 12 to 24 carbons, including some odd chain lengths and some chains having more than one double bond (38, 40, 41, 42, 65). The f i r s t of these reports was published i n 1959 by Hallgren and Larsson (40). The following i s a table of t h e i r published r e s u l t s . TABLE X: THE PERCENT COMPOSITION (WT) OF THE ALKOXYGLYCEROLS FROM LIVER OILS* Grey dogfish Greenland shark R a t f i s h Squalus Somniosus' Chimaera Alkoxyglycerol acanthias microcephalus monstrosa 12:0 trace trace trace 14:0 5.7 2.0 1.7 15:0 1.9 0.7 1.1 16:0 (chimyl) 13.2 9.1 10.4 16:1 10.6 10.8 9.1 17:0 3.0 3.6 4.7 18:0 (batyl) 3.4 2.8 6.7 18:1 (selachyl) 47.8 59.4 53.6 18:2 2.4 1.6 2.5 18:3 trace ? 19:1 1.2 1.5 2.4 20:1 8.0 6.2 6.4 21:0 trace ? ? 22:1 2.7 2.2 1.0 *The alkoxyglycerols are represented by the long chain part of the molecule. i 14 Hallgren and Larsson published two further papers i n 1962, one (42) of which describes the g l y c e r y l ethers occurring i n man and i n the bovine. Table XI summarizes t h e i r composition data on the g l y c e r y l ethers. TABLE XI: PERCENTAGE COMPOSITION (WT/WT) OF GLYCERYL ETHERS FROM HUMAN BONE MARROW, HUMAN SPLEEN, AND HUMAN MILK (Hallgren and Larsson)(42) Long-chain component i n Human bone Human Human g l y c e r y l ethers marrow spleen milk u n i d e n t i f i e d components 3.8 - 2.0 16:0 29.4 33.0 23.9 16:1 trace 17* 7.6 1.0 3.6 18:0 24.6 25.8 22.8 18:1 16.7 27.6 33.8 18:2 1.4 19* 6.1 2.4 20:0 2.9 ) 1.6 20:1 3.2 ) 7.3 2.3 22:0 0.7 ) 0.7 22:1 5.1 ) 5.2 3.4 24:0 2.1 *Normal and branched In 1962, Guyer (38), using gas chromatographic techniques on the g l y c e r y l ethers i s o l a t e d from dogfish l i v e r o i l , showed that the components had chain lengths varying from 12 to 20 carbon atoms. Although g l y c e r y l ether-containing phospholipids are.present i n bone marrow they are v i r t u a l l y absent from the erythrocytes, i n fact human erythrocytes have no detectable g l y c e r y l ethers, but they do contain plasmalogens. Young bovine erythrocytes which s t i l l possess a f u l l complement of n u c l e i , mitochondria, and microsomes have g l y c e r y l ethers i n the mitochondrian f r a c t i o n ; whereas i n the older erythrocytes, where a l l i n t r a c e l l u l a r organelles have atrophied i n the c e l l , the g l y c e r y l ether containing phospholipids are l o c a l i z e d i n the c e l l membrane (101). Studies of the occurrence of g l y c e r y l ethers i n various tissues show that bone marrow i s the r i c h e s t s i t e , with the other tissues having l e s s e r amounts. Todd and R i z z i (1964)(104), on the other hand, working with rabbit t i s s u e , found 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 blood the lowest. In the same paper, Todd and R i z z i report a comparison of mammalian yellow bone marrow from the rabbit, the cow, the sheep, the p i g , the r a t , and man. (Details are given i n Table IX.) I t may be noted from t h i s data that the rabbit has only chimyl alcohol, and that the rat appears to have no g l y c e r y l ethers i n i t s bone marrow. Analysis of the g l y c e r y l ethers from s t a r f i s h show the b a t y l and chimyl alcohols to be present and the selachyl to be absent (104); t h i s i s i n contrast to the f i s h o i l s which have a high proportion of selachyl as a g l y c e r y l ether. It i s of i n t e r e s t that two species of t e r r e s t i a l slug, Arian ater and Ariolimax Columbians, contain a - g l y c e r y l ether phospholipids accounting for more than 25 mole percent of the t o t a l phospholipid mixture. There i s an absence of unsaturation with the major g l y c e r y l ether being chimyl alcohol, a sharp contrast to the findings with other organisms. 16 D. THE MESENCHYMAL ORIGIN OF THE ot-GLYCERYL ETHERS Bodman and Maison (16) report, perhaps with i n s u f f i c i e n t supporting evidence, that the a-glyceryl ethers are found present i n those tissues containing c e l l s of mesenchymal o r i g i n , from which tissue c e l l s are continually shed into a body cav i t y . They support t h i s by r e f e r r i n g to the association of a-glyceryl ethers with saturated f a t t y acids i n the unsaponifiable f r a c t i o n and the asso c i a t i o n between squalene and a - g l y c e r y l ethers. Squalene r i c h o i l has been found i n the stomach of the shark and i n the faeces of land animals; i f we assume g l y c e r y l ethers to be present along with the squalene i n these cases, then i t i s reasonable to assume that the gut mucosa i s concerned with a-glyceryl ether synthesis. E. BIOSYNTHESIS OF THE GLYCERYL ETHERS In 1955, Karnovsky and Brumm (50), using the s t a r f i s h , reported the incorporation of radiocarbon into a-glyceryl ether moities at rates comparable to those observed i n the more commonly studies l i p i d f r a c t i o n s . This may indicate that the s t a r f i s h does not simply accumulate these compounds from i t s food. Data presented by Thompson and Hanahan (1962) (100), obtained from experiments using C l l t l a b e l l e d glucose, indicate that the g l y c e r y l moiety of the g l y c e r y l ethers can be formed from t h i s sugar i n bovine marrow. The d i s t r i b u t i o n of C 1 L f would strongly suggest that the glucose metabolite d i r e c t l y involved i s 1-a-glycerophosphate. This i s si m i l a r to the pathway proposed i n the biosynthesis of phosphatidic acid, or some other 17 metabolically analogous compound. In a more recent paper (1963) the same authors describe i n v i t r o bone marrow incubation experiments (101). The r a d i o a c t i v i t y from glucose-6-C 1 , sodium palmitate-l-C 1 ^  and t r i t i a t e d water were incorporated into g l y c e r y l ether phospholipids by bone marrow extracts. At the end of the incubation times used, radio-a c t i v i t y of the glyceryl-ether phospholipids was less than that of the nonglyceryl-ether phospholipids. Almost a l l of the r a d i o a c t i v i t y of the synthesized g l y c e r y l ethers was located on the alpha carbon of the g l y c e r y l moiety, again suggesting that a-glycerophosphate may be a d i r e c t glucose-derived precursor. Both ethanolamine and choline phosphatides contain the ethers. There appears to be a metabolic r e l a t i o n s h i p between g l y c e r y l ethers and plasmalogens. One possible route by which g l y c e r y l ethers may be synthesized l i e s i n the formation of a plasmalogen type molecule followed by enzymatic hydrogenation of the v i n y l ether double bond. H HG-0-CH-CH(CH2)i3CH3 I 0 HC-O-C—R enzymatic j 0 hydrogenation HC-0-P-O-CH 2 CH 2-^H 2 H OH H HC-0-CH 2(CH 2)n f CH 3 I 0 HC-O-C-R I 0 HC-C-P-C-CH 2 CH 2-«H 2 H OH a plasmalogen a-glyceryl ether containing phospholipid F. DIGESTION AND METABOLISM OF a-GLYCERYL ETHERS Much as dietary glycerides are digested by the action of the pan-c r e a t i c l i p a s e present 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 acids, and p a r t i a l l y s p l i t products such as monoglycerides and d i g l y c e r i d e s ; the e s t e r f i e d g l y c e r y l ethers are broken down from g l y c e r y l ether diesters to g l y c e r y l ether monoesters, free g l y c e r y l ethers and f a t t y acids. Then the f a t t y acids, with the aid of the b i l e s a l t s , carry the g l y c e r y l ether mono- and diesters and the free g l y c e r y l ethers into the mucosal c e l l s of the small i n t e s t i n e . Once within the mucosal c e l l s , the ether linkage can be broken to give free g l y c e r o l and free f a t t y acids. Several papers have been published which give data about digestion and to some extent, the metabolism of g l y c e r y l ethers. (Very l i t t l e i s known about the r o l e of g l y c e r y l ethers i n c e l l metabolism.) Bergstrom and Blomstrand (1956)(12), Blomstrand (1959((15) and Blomstrand and Ahrens (1959)(14) report data on the absorption of chimyl alcohol i n r a t s ; Blomstrand and Ahrens (1959)(14) report data on absorption i n man and Malins, j2t al (1964)(66) report data on absorption i n the rainbow trout. In man and the r a t , well over 90% of fed g l y c e r y l ethers were absorbed. That which passed into the faeces retained i t s ether linkage i n t a c t , the only thing that happened while i n the i n t e s t i n e was the hydrolysis and resynthesis of the ester linkages on the g l y c e r y l ether molecule. On the other hand, the g l y c e r y l ethers that were absorbed into the mucosal c e l l s and traced i n the chyle had t h e i r ether linkages broken. The following table i s taken from Blomstrand's paper (1959)(15): 19 TABLE XII: RECOVERY AND DISTRIBUTION OF RADIOACTIVITY IN LYMPH LIPIDS AFTER FEEDING C1^-LABELLED CHIMYL DIOLEATE* % of administered a c t i v i t y absorbed % of absorbed a c t i v i t y re-covered i n lymph l i p i d s % of a c t i v i t y i n lymph l i p i d s recovered as: chimyl alcohol t r i g l y c e r i d e f a t t y acids phospholipid f a t t y acids free e s t e r f i e d 90 85 60 45 6 5 18 20 74 73 2 2 A l l t h i s work done by Blomstrand and h i s co-workers with the rat and the human i s substantiated by Malins, et a l (66) i n h i s work with the rainbow trout (Salmo g a i r d n e r i ) . The trout were fed b a t y l alcohol and C 1 ^ - g l y c e r y l ethers. At the end of a feeding period the l i p i d s of the i n t e s t i n a l contents were analysed; although there was simultaneous e s t e r f i c a t i o n and hydrolysis i n the lumen of the i n t e s t i n e , there was no cleavage of the ether bond. No appreciable amounts of g l y c e r y l ethers were found deposited i n the f l e s h of the f i s h which suggests metabolic breakdown of the g l y c e r y l ethers i n the f i s h as well as i n rats and i n man. Swell, et a l (1965)(98) report on the comparative absorption of a- and 3-octadecyl g l y c e r y l ethers. Lymph f i s t u l a t e d rats were fed test rations containing 35 mg0 of a- or 3-octadecyl g l y c e r y l - l - C 1 ether. They found that 3-glyceryl ether was absorbed into the lymph to a greater extent (96%) than the a-glyceryl ether (76%). Fractionation of the lymph l i p i d s indicated that 37 to 48 percent of the C 1 £ + a c t i v i t y was i n the form of C 1 ^ - l a b e l l e d f a t t y acids, and from 55 to 63 percent as free and e s t e r f i e d g l y c e r y l C l t f-ethers. The a- g l y c e r y l ether was s p l i t i n the i n t e s t i n e to a *The chimyl dioleate was dissolved i n o l i v e o i l and fed to 2 rats using a thoracic duct f i s t u l a . greater extent than the B-glyceryl ether. Most of the C 1^ a c t i v i t y of the lymph l i p i d s was i n the alkoxydiglyceride f r a c t i o n . The 3-glyceryl ether was more e f f i c i e n t l y converted to the alkoxydiglyceride than the a-glyceryl ether. More alkoxymonoglyceride appeared i n the lymph when the a-glyceryl ether was fed than when the g-glyceryl ether was administered. G. a-GLYCERYL ETHERS AND HAEMOPOIESIS As early as 1949 Sandler (90) suggested that g l y c e r y l ethers, i n p a r t i c u l a r b a t y l alcohol, had the a b i l i t y to stimulate r e t i c u l o c y t o s i s i n man. Then i n 1951 Arturson and Lindback (4) reported that b a t y l alcohol given at a dosage l e v e l of 34 and 70 mg/Kg of body weight to male white mice caused a s i g n i f i c a n t increase i n r e t i c u l o c y t e s while red c e l l counts were l i t t l e affected by the treatment. A s l i g h t c o ntradiction to these p o s i t i v e e f f e c t s i s given by Evenstein, ejt a_l (34) who dosed rats at l e v e l s of 2 to 20 mg/Kg of body weight, without s i g n i f i c a n t e f f e c t on the red blood c e l l count, r e t i c u l o c y t e number, hematocrit or hemoglobin values. In 1958 Linman, _e_t a l (61) reported the subcutaneous i n j e c t i o n of female rats with 150 and 300 mg of b a t y l alcohol per Kg of body weight, and obtaining a s i g n i f i c a n t response i n erythropoiesis, thrombopoiesis and granulopoiesis. The response appeared to be one of accelerated c e l l u l a r d i v i s i o n . In 1959 Linman, e_t a l (62) published another paper reporting the administration, by stomach tube, of approximately 30 mg/Kg of b a t y l alcohol to r a t s ; the r e s u l t s were: erythrocytic response, production of microcytes, r e t i c u l o c y t o s i s , thrombocytosis and leucocytosis. Although Linman (62) found b a t y l alcohol e f f e c t i v e o r a l l y and parenterally, independent of o p t i c a l a c t i v i t y , he 21 found selachyl alcohol (natural and synthetic) devoid of a c t i v i t y . The sela c h y l was administered over a ten day period by g a s t r i c intubation to normal rats at a dosage rate of 50 mg/rat/day (312 - 356 mg/Kg.) The i n a c t i v i t y of selachyl alcohol was confirmed by Osmond, e_t a l (79) i n th e i r experiments using guinea pigs. Both selachyl and b a t y l alcohols were given at a dosage l e v e l of approximately 10 mg/Kg of body weight. The r e s u l t s suggest that the b a t y l alcohol may be an erythropoietic stimulant i n the guinea pig whereas selachyl alcohol i s not. Negative r e s u l t s were also obtained by Penny, et_ a l (80) working with sheep, c a t t l e , and mice. Sheep were dosed intravenously at a rate of 2 and 4 mg of b a t y l alcohol per Kg of body weight for 21 days with no response. In one experi-ment, sheep were dosed intravenously with 10 mg of b a t y l alcohol per Kg of body weight for 4 days which resulted i n a r i s e i n polymorphonuclear leucocytes, but no increase i n white blood c e l l count. Two h e i f e r s given 1 gm of d-batyl alcohol (3 mg/Kg of body weight) intravenously for four days yielded no haematopoietic response. Mice were given approximately 110 mg/Kg of body weight subcutaneously yet they showed no stimulation, i n f a c t , there was a s l i g h t decrease i n the red c e l l , r e t i c u l o c y t e and marrow erythroblast counts. Under s p e c i f i c conditions of disease and stress to the body, g l y c e r y l ethers i n general and b a t y l alcohol i n p a r t i c u l a r stimulate the formation of blood c e l l s and appear to a l l e v i a t e some of the toxic side e f f e c t s . The f i r s t reported use of bone marrow was i n 1930 by G i f f i n and Watkins (36) i n the treatment of secondary anemia. They used normal bone marrow i n the hope that some factor i n i t would stimulate or a l l e v i a t e the diseased bone marrow i n secondary anemia. In general, the bone marrow proved to be unsatisfactory i n the treatment of secondary anemia, except i n one case of agranulocytosis. Over the years 1930 to 1938, G i f f i n and Watkins treated 24 cases of agranulocytosis with bone marrow, and obtained a favourable response i n a l l but three cases. In the o r i g i n a l work, G i f f i n and Watkins administered about 0.4 grams of marrow (60 mg/Kg of body weight) to t h e i r patients. In 1936, Marberg and Wiles reported the use of a concentrate of the unsaponifiable portion of bone marrow dissolved i n a bland o i l (equivalent to 2 grams of marrow per drop) administered to patients s u f f e r i n g from agranulocytosis. As a r e s u l t , there was i n most cases a r i s e i n the number of granulocytes which began with 24 to 36 hours and values returned to normal i f the causative i n f e c t i o n p ersisted during the period of treatment. One of the promising uses of g l y c e r y l ethers i s said to be for the treatment of leucopenia caused by i r r a d i a t i o n . In 1954, a paper by Brohult and Holmberg (22) describes the successful use of the non-saponifiable portion of bone marrow fat and concentrates of alkoxyglycerols and t h e i r esters (the concentrates had a higher potency than the bone marrow prepara-tions) . Of 36 human cases s u f f e r i n g from i r r a d i a t i o n leucopenia, 25 cases responded to the treatment by an immediate increase i n the white c e l l count. In 9 cases there was no further decrease while two showed a continued decrease i n leucocyte counts. In 1957, Brohult published another paper (17) with s i m i l a r r e s u l t s , when she used alkoxy-glycerol esters administered to 100 patients s u f f e r i n g from i r r a d i a t i o n leucopenia. She reports an optimum dosage of 1.2 grams of o i l per day (equivalent to 2.74 mg of free g l y c e r y l ethers per Kg of body weight) and an actual decrease i n leucocyte numbers at dosage l e v e l s above 2.5 grams of o i l per day (equivalent to 5.70 mg of free g l y c e r y l ethers per Kg of body weight). Brohult (1958)(18) used alkoxyglycerol esters for the prophylactic treatment of rats that were subsequently exposed to t o t a l body X - i r r a d i a t i o n . A s i g n i f i c a n t higher count of both megakaryocytes and nucleated c e l l s was found i n the marrow of the p r o p h y l a c t i c a l l y treated animals. Table XIII gives a summary of the r e s u l t s obtained by Brohult. Mizuno, et^ a l (76) treated calves, exposed to whole body i r r a d i a t i o n , with autologous bone marrow. Nine out of 15 recovered a f t e r a severe hematologic c r i s i s . TABLE XIII: ALKOXYGLYCEROL ESTERS FED TO RATS  RECEIVING TOTAL BODY X-IRRADIATION (taken from Brohult) (18) Number of Experimental group rats (mg/day)* (M%)** Normal 70 100 Control 87 19 Prophylactic groups Shark l i v e r o i l 18 0.25 26 Shark l i v e r o i l 9 0.40 33 Shark l i v e r o i l 23 0.50 27 Pure esters 3 0.25 29 Batyl alcohol 11 0.14 32 Selachyl alcohol 8 0.12 61 Selachyl alcohol 6 0.14 49 Selachyl alcohol 12 0.18 27 *0.15 mg i s equivalent to 25 mg of free g l y c e r y l ethers per Kg i body weight. **Megakaryocyte count (M) i n ) I of the megakaryocytes for normal : 24 There have been other reports of the therapeutic use of g l y c e r y l ethers i n the treatment of i r r a d i a t i o n sickness. Rusanov, et a l (87) treated X - i r r a d i a t e d rats with b a t y l alcohol and obtained an a c t i v a t i o n of blood production as well as a fast e r recovery from the r a d i a t i o n sickness. Prokhonchukov and Panikarovskii (85) used both s e l a c h y l and b a t y l alcohols and found b a t y l to give a s a t i s f a c t o r y therapeutic response, while selachyl gave an i n s i g n i f i c a n t response. Sviridov, e_t a l (97) studied the therapeutic e f f e c t of sel a c h y l alcohol administered to dogs, rabbits and rats that had been exposed to X - i r r a d i a t i o n . The alcohol a l l e v i a t e d the gravity of the r a d i a t i o n sickness, increased s u r v i v a l rate, normalized tissue r e s p i r a t i o n i n the brain and myocardium, and somewhat stimulated the haemopoietic organs (the f o l l i c u l a r appendages of the lymph nodes and the spleen). Another report of the use of b a t y l alcohol i s given by Dudin (29), where primagravid rats on the 15th day of pregnancy were i r r a d i a t e d and injecte d intravenously with cystamine (65 mg/Kg) and b a t y l alcohol (0.5 mg/Kg) d a i l y for 10 days a f t e r exposure to X-rays. There was no influence on the gravity of the X-ray sickness. In controls, 50 percent of the young were born dead; with the combined treatment of b a t y l and cystamine only 22 percent of the f o e t i were born dead and, of those born a l i v e , the development of anemia and leucopenia was completely prevented. Batyl alcohol has been, e f f e c t i v e i n stimulating haemopoiesis and a l l e v i a t i n g c e r t a i n conditions of t o x i c i t y . One of these i s i n benzene poisoning. Sandler (1949) (90), examined the p o s s i b i l i t y that yellow bone marrow extracts might have an a n t i t o x i c e f f e c t rather than containing a s p e c i f i c leucopoietic p r i n c i p l e . He in j e c t e d , subcutaneously, benzene and 25 preparations made from nonsaponifiable yellow bone marrow extracts to one group of benzene depleted r a t s . Another group of depleted rats were injected with benzene and b a t y l alcohol. A number of animals were given benzene s o l e l y and served as controls. The bone marrow and the b a t y l alcohol therapy increased the number of erythrocytes, decreased the tendency to benzene necrosis at the s i t e of i n j e c t i o n , and lowered m o r t a l i t y . De Gaetani and B a i o t t i (1959)(27) report that b a t y l alcohol given to rats with acute benzene poisoning, r e l i e v e s i n t o x i c a t i o n , prolongs l i f e , increases osmotic resistance of red c e l l s , favours r e t i c u l o c y t o s i s , and restores the a c t i v i t y of bone marrow. Hasegawa, e_t a l (1961) (46) , report that b a t y l alcohol had e f f e c t i v e haemopoietic a c t i v i t y i n rabbits s u f f e r i n g from experimental anemia caused by Nitromin i n j e c t i o n or solvent 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 calves caused by feeding trichloroethylene-extracted soybean o i l meal, b a t y l alcohol f a i l e d to prevent development of anemia (92) . Batyl alcohol treatment was t r i e d because a p l a s t i c anemia so produced i s very s i m i l a r to bracken poisoning i n c a t t l e . Batyl alcohol has been used with varying degrees of success i n the treatment of bracken poisoning. Evans, e_t a l (1957) (33), report the successful use of b a t y l alcohol i n the treatment of bracken poisoning and i n 1958, Evans, et a l again reported the use of b a t y l alcohol; 23 out of 31 poisoned c a t t l e recovered. The v a l i d i t y of these reports i s questioned by Dalton (25) who claims that normal c a t t l e may vary t h e i r leucocyte count as much as 1000 to 4000 c e l l s per cmm during the day and from day to day. The increase i n leucocyte count reported by Evans was 1000 to 2000 c e l l s per cmm. Dalton also treated c a t t l e affected with bracken poisoning, but found no obvious e f f e c t on the haematology. 26. H. SUMMARY These natural occurring compounds are of i n t e r e s t because they appear to be useful and perhaps e s s e n t i a l to a l l animals including man. They may be u s e f u l as a stimulant, a drug, or as a nu t r i e n t . They are normally found i n the animal body, they are absorbed from the i n t e s t i n e and metabolized within the body. These compounds are not t o x i c , although there has been some report of t o x i c i t y i n early work done by Agduhr (1934) (21), Berger (1948) (21), and Emmerie, et a l (1952) (31). I t i s possible that the t o x i c i t y reported resulted from impurities i n the alkoxyglycerols that were used. TABLE XIV: BIOLOGICAL EXPERIMENTS INVOLVING THE USE OF GLYCERYL ETHERS Gl y c e r y l Ether Animal Condition Dosage Response Reference b a t y l and rats s e l a c h y l b a t y l (purity mice unknown) alkoxyglycerols mice transplanted malignant tumors normal radiated b a t y l mxce normal 1 ml of prepara-t i o n i n sunseed oil/day for a month .01 to .048 mg/Kg/day 160 - 210 days 590 mg/Kg i n food 34 mg/Kg and 70 mg/Kg these in j e c t i o n s caused the tumors to develop slower i n the treated r a t s ; selachyl gave the greatest i n h i b i t i o n lesions of heart, kidneys and l i v e r both g l y c e r y l ether esters and e s s e n t i a l f a t t y acids promote longer l i f e both levels gave a s i g n i f i c a n t increase i n (intraperitoneal) r e t i c u l o c y t e s ; red blood c e l l counts remained normal Abaturova and Shubina (1964) Agduhr (1934) (cited i n Brohult) Alexander, et a l (1959) Arturson and Lindback (1951) various C 1 1 +-chimyl mxce rats normal normal large (subcutaneous) 5 mg by stomach tube LD50 for bat y l alcohol was determined to be 3 gm/Kg absorption and break-down studied Berger (1948) (cited i n Brohult) Bergstrom and Blomstrand (1959) TABLE XIV (cont'd) G l y c e r y l Ether Animal Condition Dosage Response Reference C l i +-chimyl C 1^-chimyl rats man normal alkoxy exters man alkoxyglycerols man including s e l a c h y l alkoxyglycerols rats alkoxyglycerols b a c t e r i a radiated .5 ml of a 2% so l u t i o n by stomach tube 25 mg by mouth various 25 mg/Kg with meals 20 mcgm/ml of media 2 mcgm/ml of media absorption and break- Blomstrand (1956) down studied absorption and break-down studied Blomstrand and Ahrens (1959) 2.74 mg gave the optimum Brohult 5.7 mg and higher gave res u l t s lower than the controls of some value where Brohult leuko- or thrombo-cyto-penia has already occurred the growth response was as Brohult follows: normal, 1.7 gm/day; ir r a d i a t e d and treated, 1.3 gm/day; i r r a d i a t e d without treatment, 1.1 gm/day marked growth response Brohult observable growth response (1958) (1958) (1960) (1960) alkoxyglycerol esters 300 cancer radiated of the cerv i x patients white c e l l and thrombocyte counts were higher for the treated patients Brohult (1962) TABLE XIV (cont'd) G l y c e r y l Ether Animal Condition Dosage Response Reference b a t y l b a t y l b a t y l c a t t l e normal pregnant radiated rats mice unsaponifiables bacteria b a t y l b a t y l b a t y l c a t t l e c a t t l e rats b a t y l Kaby 700 ba t y l rats man rabbit radiated bracken poisoning bracken poisoning normal benzene poisoning radiated anemia by Nitromin .5 mg/Kg on alternate days .5 mg/Kg with and without cystamine 10 mg every other day 1 mcgm/ml .5 to 1 mg 1 mg and 2 mg no haematological response less s t i l l b o r n , anemia and leucopenia prevented l e t h a l i t y i s lower i n the treated group i n h i b i t e d growth, marked promoted recovery from gave a leucocyte response 0.5 mg for 11 there was no response days; 1 mg for i n the blood picture 20 days, followed by 5 mg for 10 days (subcutaneous) re l i e v e s t o x i c i t y response i n only 4 out of 15 cases Dalton Dud i n Edlund (1964) (1961) (1954) Emmerie, et al(1952) Evans, et a l (1958) Evans, et a l (1957) Evenstein (1958) de Gaetani (1959) Ghys (1960) a haemopoietic response Hasegawa (1961) TABLE XIV (cont'd) G l y c e r y l Ether Animal Condition Dosage Response Reference b a t y l b a t y l b a t y l s e l a c h y l g l y c e r y l ethers g l y c e r y l ethers rats rats rats rats man rainbow trout unsaponifiables man of bone marrow alkoxy esters mice alkoxy esters mice (mainly selachyl) b a t y l mice normal normal normal normal normal granulo-cytopenia normal normal normal 12.5 and 25 mg 50 mg 165 - 330 mg 312 - 356 193 mg/Kg 167 mg/Kg 73 mg/Kg response i n erythrocytes, thrombopoiesis, and granulopoiesis a haemopoietic response a haemopoietic response no a c t i v i t y e f f e c t i v e i n treating wounds caused by X-ray burns digestion and metabolism data obtained favourable r e s u l t s four died with ca t a r r h a l e n t e r i t i s , survivors showed no path, changes no deaths; weight gain normal no deaths, weight gain normal Linman (1958) Linman (1959) Linman (1960) Linman (1960) Maisen, ^ t a l (1959) Malins, eit a l (1964) Marberg (1936) Melander (1954) (c i t e d i n Brohult) CO o TABLE XIV (cont'd) G l y c e r y l Ether Animal Condition Dosage Response Reference alkoxy esters (mainly selachyl) b a t y l and sel a c h y l b a t y l b a t y l b a t y l selachyl b a t y l b a t y l mice guinea Pig sheep mice rat s rats rats normal normal c a t t l e normal normal normal radiated radiated radiated 533 mg/Kg 1467 mg/Kg 10 mg/Kg 3.1 mg/Kg intravenous 2 to 10 mg/Kg intravenous 114 mg/Kg subcutaneous no deaths; weight gain normal bat y l alcohol gave s i g n i f i c a n t changes i n both bone marrow and blood; selachyl showed no changes no response no response no response i n e f f e c t i v e s a t i s f a c t o r y thera-peutic response a c t i v a t i o n of blood production Osmond (1963) Penny, et a l (1964) Prokhonchukov and Panikarovskii (1963) Prokhonchukov and Panikarovskii (1963) Rusanov, e_t a l (1962) chimyl blood c e l l s i n h i b i t e d l y s o l e c i t h i n hemolysis Safanda and Holecek (1965) TABLE XIV (cont'd) G l y c e r y l Ether Animal Condition Dosage Response Reference b a t y l b a t y l b a t y l s e l a c h y l s e l a c h y l Clh a and B b a t y l rats man calves pea stems dogs, ra b b i t s , rats rat bone marrow man benzene depleted normal t r i c h l o r o -ethylene extracted soybean meal radiated normal granulo-cytopenia 0.3 to 0.65 mg/Kg 35 mg fed r i s e i n erythrocytes and a decrease i n necrosis r e t i c u l o c y t o s i s no response stimulates auxin and g i b e r e l l i n action a l l e v i a t e d sickness digestion and metabolism studied favourable r e s u l t s Sandler (1949) Sandler (1949) Schultze, e_t a l (1958) Stove (1960) Sviridov and Abaturova (1964) Swell, et al(1965) Watkins (1933) (ci t e d i n Marberg) PART I I : EXPERIMENTAL 33 A. PRELIMINARY EVALUATION - TRIAL I 1„ Preamble The experimental work c a r r i e d out as a part of t h i s study can conveniently be divided into two sections; preliminary t r i a l s and 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 . P r i o r to 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 was necessary to asce r t a i n i f the a-glyceryl ethers would produce any deleterious e f f e c t s on animal growth and development. In the absence of quantitative information on the dose-response r e l a t i o n s h i p of the a-glyceryl ethers for mammalian systems i t was necessary to e s t a b l i s h a more or less a r b i t r a r y dosage l e v e l with which to begin the preliminary t r i a l s . The amounts of these compounds i n various tissue systems i s i n the range normally expected f o r c e r t a i n of the vitamins. Hallgren and Larsson (42) report bovine milk to have 0.01 percent of i t s l i p i d f r a c t i o n as g l y c e r y l ethers. A c a l c u l a t i o n for milk containing four percent f a t suggests a figure of 400 meg of g l y c e r y l ethers per 100 grams of milk. This l e v e l i s of the same order of magnitude as that normally recorded for a number of the vitamins present i n milk (see Table XV). On the basis of the above, a dosage l e v e l of 6 mg per kilogram of body weight per day was a r b i t r a r i l y selected for preliminary t o x i c i t y t r i a l s . Table XVI affords a comparison of th i s dosage l e v e l with recommended le v e l s for several of the vitamins. Both sets of values are expressed as meg per k i l o c a l o r i e of d i g e s t i b l e energy i n the di e t to permit d i r e c t comparison. These values are a l l computed for r a t s ; s i m i l a r estimates can be made for the dog. In these c a l c u l a t i o n s i t was assumed that the d i g e s t i b l e energy of 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 Rusoff) (88) Vitamins meg per 100 gm of milk Fat soluble vitamins vitamin A a c t i v i t y vitamin D vitamin E 42 - 63 0.045 - 0.058 60 Water-soluble thiamine r i b o f l a v i n n i c o t i n i c acid pantothenic acid pyridoxine f o l i c acid b i o t i n cobalamin vitamin C choline i n o s i t o l ( t o t a l ) 40 150 80 350 25 45 200 100 400 48 0.1 - 0.23 2.0 - 3.5 0.3 - 0.5 2000 13000 13000 TABLE XVI: 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 of d i g e s t i b l e energy dosage of g l y c e r y l ether per kilogram of body weight 6 mgs 11 Vitamin A cobalamin r i b o f l a v i n choline chloride n i a c i n pyridoxine HC1 thiamin 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 intake (A.D.E.I.) i n k i l o c a l o r i e s per day for the rat may be estimated from the expression (Cheeke) (24) A.D.E.I. = 6.82 W ° , l + 5 1 where W represents body weight i n grams. On the basis of t h i s equation, a 100 gram rat would ingest 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 day. In order that t h i s rat may receive 6 mg of a g l y c e r y l ether per k i l o -gram of body weight per day, i t s r a t i o n would have to provide 11 meg of the ether 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 rat which i s approximately 6 mg per kilogram of r a t c ) 2. Experimental Design Eighty male and female rats were randomly a l l o t t e d into four groups with equal numbers of each sex i n each group. Four groups each containing f i v e dogs were used. Each of the four groups of rats and dogs were a l l o c a t e to a treatment as follows: one co n t r o l group and one group for each of the three important n a t u r a l l y occurring g l y c e r y l ethers. These animals were fed normally and presented with t h e i r respective doses d a i l y for a period of 6 months. The rats were weighed three times weekly (Monday, Wednesday, and Friday); the dogs were weighed once each week. Daily feed intake data were recorded for the r a t s , but not for the dogs. Blood samples were obtained from both the dogs and the rats according to the schedule given i n Table XVII. Erythrocytes and leucocytes were counted using standard methods (see Appendix XVI). Af t e r 180 days a l l the rats and one dog from each group were k i l l e d . Gross pathology was noted and tissues were recovered i n methanal acetate. Sections from the heart, lungs, l i v e r , kidneys, adrenals, i n t e s t i n e and spleen were prepared for h i s t o l o g i c a l study TABLE XVII: SCHEDULE OF BLOOD COUNTS Time of blood sampling* Rats erythrocytes 180 leucocyte counts 90 and 180 Dogs erythrocytes 110 leucocyte count 180 * Days from the beginning of the t r i a l 3. Animals Albino rats of the UBC Wistar s t r a i n and Labrador dogs, both reared at the u n i v e r s i t y animal u n i t s , were used i n th i s experiment. The rats were placed on experiment at a body weight of approximately 90 grams and the dogs at 10 kilograms. The d e t a i l s of the weight changes i n both groups of experimental animals are given i n Appendix I I . 4. Ration The rats were fed UBC stock rat r a t i o n i n ground form to f a c i l i t a t e the measurement of feed intake. A cor r e c t i o n was not applied f o r feed s p i l l a g e . The dogs were fed UBC stock dog crumbles. Both the rats and the dogs were fed aid lib i t u m . The constituent composition of these rations i s given i n Appendix I. 37 5. Housing The rats were housed i n d i v i d u a l l y i n wire cages i n a room with c o n t r o l l e d ambient temperature and humidity. The dogs were housed i n i n d i v i d u a l concrete block pens using wood shavings for l i t t e r . They were exercised outdoors from 10:00 a.m. to 4:00 p.m. each day, weather permitting. 6. Administration of g l y c e r y l ethers The rats were dosed with each of the three g l y c e r y l ethers suspended i n a safflower o i l / s a f f l o w e r e t h y l e s t e r s / o l e i c acid (55/40/5) diluent. The con t r o l rats received the diluent only. The dogs received the b a t y l and chimyl alcohols i n tablet form, the se l a c h y l alcohol was suspended i n the above di l u e n t . As was the case with the r a t s , the control dogs received the diluent only. The g l y c e r y l ethers, i n diluent suspension, were administered per orum with a syringe. Those dogs receiving t h e i r g l y c e r y l ethers i n tablet form, were forced to swallow t h e i r d a i l y dose. 7. Results A l l of the pertinent data concerning feed intake, body weight changes and blood counts are given i n Appendices I I , I I I and IV. A f t e r considering a l l the data c o l l e c t e d during the 180 day t r i a l and the data c o l l e c t e d at the time of slaughter, there appeared to be no evidence of deleterious e f f e c t s from the treatments. Table XVIII, XIX and XX summarize the data given i n the Appendices II and I I I . They reveal that the weight gains and feed e f f i c i e n c i e s of the treated dogs and rats were e s s e n t i a l l y the same as the controls. A " t " test indicates that the rate of gain of TABLE XVIII: SUMMARY OF MEAN BODY WEIGHT GAIN AND MEAN FEED-INTAKE DATA FOR MALE RATS Selachyl Batyl Chimyl Control f i n a l weight (gm) 421.5 413.9 412.8 .418.9 i n i t i a l weight (gm) ' 95.1 94.4 94.0 88.7 t o t a l weight gain (gm) 326.4 319.5 318.8 330.2 gain/day (gm) 1.79 1.76 1.75 1.81 t o t a l feed consumed (gm) 3,925 3,736 3,666 3,829 feed intake/day (gm) 21.6 20.5 20.1 21.0 gm of feed/gm of gain 12.0 11.6 11.5 11.6 TABLE XIX: SUMMARY OF MEAN BODY WEIGHT GAIN AND MEAN FEED-INTAKE DATA FOR FEMALE RATS Selachyl Batyl Chimyl Control f i n a l weight (gm) 267.3 255.4 253.3 256.4 i n i t i a l weight (gm) 85.9 86.4 88.1 85.7 t o t a l weight gain (gm) 181.4 169.0 165.2 170o7 gain/day (gm) 1.0 0.93 0.91 0.94 t o t a l feed consumed (gm) 3,263 3,041 3,132 2,930 feed intake/day (gm) 17.9 16.7 17.2 16.1 gm of feed/gm of gain 18.0 18.0 19.0 17 o 2 TABLE XX: SUMMARY OF BODY WEIGHT GAIN FOR DOGS* Selachyl Batyl Chimyl Control f i n a l weight (Kg) 109.7 103, ,7 115.0 104.0 i n i t i a l weight (Kg) 51.6 53. ,6 58.1 47.6 t o t a l weight gain (Kg) 58.1 50, ,1 56.9 56.4 weight gain/week (Kg) 2.23 1. .93 2.19 2.17 weight gain/dog/week (Kg) 0.45 0. ,39 0.44 • 0.43 *There were f i v e dogs i n each of these groups FIGURE I: GROWTH CURVES FOR FEMALE RATS 30 Cf o 250f -UJ >-Q o CO 20C4-CD 50 < I50f-lOOf • • 1—' w • • • • « $ 8 * X X X • • • 0 0 0 O O O CONTROL SELACHYL BATYL CHIMYL • • • • 1 10 20 25 WEEKS ON EXPERIMENT 40 the female rats In the selachyl group i s s i g n i f i c a n t l y higher than that of the c o n t r o l female r a t s . However, the s i g n i f i c a n c e of t h i s growth response i s questioned because i t did not occur i n the male rats on the s e l a c h y l treatment. The b a t y l and chimyl treated groups of female rats have i d e n t i c a l growth curves to the c o n t r o l group. The growth curve (Figure I) f o r the female rats that received the s e l a c h y l alcohol was i d e n t i c a l with that of the other groups to a mean body weight of about 200 grams. Above t h i s body weight t h i s group gained weight at a s l i g h t l y f a s t e r rate and hence reached a s l i g h t l y heavier weight at the end of the experiment. This increase i n body weight above the other experimental groups was s i g n i f i c a n t (at the .05 l e v e l ) . This increase did not occur u n t i l a f t e r puberty. I t did not appear i n the male rats receiving these alcohols. The growth curves of the male rats and of the dogs are not presented here because the d i f f e r e n t experimental groups displayed i d e n t i c a l growth responses. Table XXI gives a summary of the blood counts which were taken during t h i s t r i a l and which are given i n d e t a i l i n Appendix IV. There was a large v a r i a t i o n i n leucocyte counts between animals i n any one group ( e s p e c i a l l y i n the case of the r a t s ) , but very small differences i n average counts between the co n t r o l and treated groups. This v a r i a b i l i t y i n leucocyte count probably arose because the rats were s u f f e r i n g from a low l e v e l i n t e r -current lung i n f e c t i o n . Using the " t " test on the leucocyte count data, at 90 days, revealed that female rats given the s e l a c h y l treatment had s i g n i f i c a n t l y (.05) higher counts. At variance with t h i s r e s u l t , the TABLE XXI: SUMMARY* OF BLOOD COUNTS TAKEN IN TRIAL I Selachyl Batyl Chyml Control Rats leucocytes a f t e r 90 days female 18,340 15,440 15,690 14,600 male 18,550 17,665 22,110 19,800 leucocytes a f t e r 180 days female 15,560 16,010 14,510 14,320 male 18,200 19,340 19,540 17,560 erythrocytes a f t e r 180 days male 9.11 8.82 9.58 8.87 Dogs erythrocytes a f t e r 110 days 0.28 0.39 0.52 0.46 leucocytes a f t e r 180 days 17,200 17,200 16,900 17,500 *For r a t s , these are means of 10 animals and for dogs these are means of 5 animals. Leucocytes are given as number of c e l l s per cmm of blood and erytrocytes i n m i l l i o n s of c e l l s per cmm of blood. 42 leucocyte counts for the male rats at 90 days showed that both the se 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 (.05) lower counts than the con t r o l animals. At 180 days the " t " test did not reveal any s i g n i f i c a n t d i f f e r e n c e s , p o s i t i v e or negative, between the con t r o l and treated male and female r a t s . The leucocyte counts taken of the dogs' blood a f t e r 180 days did not show any s i g n i f i c a n t (.05) di f f e r e n c e s . Despite the f a c t that s t a t i s t i c a l l y s i g n i f i c a n t differences have been noted i n a few comparisons i n r e l a t i o n to time of examination and treatment, the o v e r a l l pattern of the r e s u l t s does not permit the de r i v a t i o n of any f i n a l conclusions. Erythrocyte counts showed much less v a r i a t i o n between i n d i v i d u a l s than did the leucocyte counts, but the differences between groups was s t i l l quite small. S t a t i s t i c a l analysis of erythrocyte counts from both dogs and rats revealed a s i g n i f i c a n t difference i n only one case, i n which the chimyl group of male rats showed higher counts than the controls. The h i s t o p a t h o l o g i c a l preparations from the various tis s u e systems of the rats and the dogs revealed no apparent differences between the treatment and the control groups. 8. Conclusion G l y c e r y l ethers administered at a l e v e l of 6 mg per kilogram of body weight over prolonged periods of time are not deleterious to the growth of rats or of dogs. With the methods used here, i t was not possible to demonstrate any e f f e c t of the a-g l y c e r y l ethers on the haematopoietic systems of either species. 43 B. PRELIMINARY EVALUATION - TRIAL II 1. Preamble Having established that the a-glyceryl ethersj when fed at a l e v e l comparable to demonstrated vitamin requirements, produced no deleterious e f f e c t s on the rat or dog, i t was considered necessary to ascertain i f these natural compounds had such e f f e c t s at much higher dosage l e v e l s . For t h i s purpose an upper l e v e l of 2400 mg per kilogram of body weight per day was selected ( t h i s i s 400 times the dosage l e v e l used i n T r i a l I ) . Because of the considerable expense associated with these compounds and with t r i a l s with dogs, only b a t y l and selachyl alcohols were examined i n t h i s phase of the work. Chimyl alcohol was eliminated since i t s l e v e l i n tissue r e l a t i v e to the others i s comparatively low. Batyl alcohol i s a su i t a b l e representative of the group of saturated g l y c e r y l ethers and selachyl of the unsaturated ethers. One group of rats and one group of dogs were offered a r a t i o n containing selachyl alcohol at a l e v e l which provided a d a i l y intake of 600 mg per kilogram. This was done to provide data on the e f f e c t s of an intermediate dosage l e v e l . 2. Design Forty rats were randomized into four groups of ten animals con-taining f i v e males and f i v e females. Sixteen dogs were divided randomly into four groups of four, with two males and two females i n each group. The treatment of each of the four groups from each species was as follows: a control group, two groups fed selachyl alcohol and a group fed b a t y l 44 alcohol. The d e t a i l s of animal grouping and dosage schedule are given i n Table XXII. The g l y c e r y l ethers were incorporated into the feed to f a c i l i t a t e administration. Feed intake was r e s t r i c t e d to ensure complete consumption of a l l the feed offered each day. The rats were offered feed at a l e v e l calculated to be 80 percent of maximum intake to a body weight of 160 grams, then 90 percent of maximum intake to a body weight of 190 grams and, f i n a l l y , 100 percent of the calculated maximum feed intake to the end of the 60 day t r i a l period. The dogs were fed 80 percent of maximum feed intake for the f u l l two month period. The rats were weighed three times per week (Monday, Wednesday, and Friday); the dogs were weighed once per week. At the end of one month and again at the end of two months, blood samples were taken from a l l of the animals; red blood c e l l counts, white blood c e l l counts, and d i f f e r e n t i a l counts of the white blood c e l l s were made. Feed and faeces samples were taken every two weeks during the two month experimental period. After two months a l l of the rats and one-half of the dogs (two from each group) were k i l l e d , posted, gross pathology noted, and femur bone marrow smears made. The remaining eight dogs were treated to a l l e v i a t e an i n t e s t i n a l parasite i n f e s t a t i o n and were then placed on the control r a t i o n for one month. Subsequently they were k i l l e d , posted, gross pathology noted, and femur bone marrow smears made. In addition, tissue sections were taken from each animal; heart, l i v e r , spleen, kidney, adrenal and i n t e s t i n e . 45 TABLE XXII: GROUPING AND DOSAGE LEVELS USED IN TRIAL II Group* Daily Dose Rats Dogs male female male female S k 0 ° 2.4 gm selachyl/Kg 5 5 2 2 B 4 0 0 2.4 gm batyl/Kg 5 5 2 2 S 1 0 0 0.6 gm selachyl/Kg 5 5 2 2 C h 0 ° 2.4 gm safflower oil/Kg 5 5 2 2 * The superscript 0 0 and the superscript i U U r e f e r to 400 times and 100 times the 6 mg dosage l e v e l used i n T r i a l I. 3. Animals Albino rats of the UBC Wistar s t r a i n and Labrador dogs, both reared i n the u n i v e r s i t y animal u n i t s , were used i n th i s experiment. The rats were placed on experiment at a weight of approximately 80 grams and the dogs at approximately 12 kilograms. The actual i n i t i a l weights are given i n Appendix V. 4. Housing The rats were housed i n d i v i d u a l l y , i n wire cages, i n a room with con t r o l l e d ambient temperature and humidity. The dogs were housed i n i n d i v i d u a l concrete block pens using wood shavings for l i t t e r . The dogs were exercised from 10:00 a.m. to 4:00 p.m. every day i n an outside run, weather permitting. 5. Ration and administration of g l y c e r y l ethers Both the dogs and the rats were fed the stock UBC dog crumbles (UBC r a t i o n No. 14 - 63 given i n Appendix I ) . This d i f f e r s from the r a t i o n arrangements used i n the f i r s t t r i a l i n which the rats received UBC r a t i o n 46 No. 10 - 63. The appropriate amounts of g l y c e r y l ethers, and for the control group, safflower o i l were incorporated into the dog crumbles and fed as a dry mash. A regression equation r e l a t i n g maximum feed intake to body weight was calculated from feed intake values recorded f o r the rats used i n T r i a l I. A corresponding regression equation for dogs was calculated from data c o l l e c t e d during a s i x day preliminary feeding t r i a l when dry dog crumbles were fed to 16 dogs having a body weight range of 18 to 31 pounds (8.2 to 14.1 kilograms). The r a t i o n used to es t a b l i s h feed intake was of lower fat content than that used i n th i s experiment, hence the standard selected was reduced to 80 percent, by weight, of predicted f u l l feed-intake. The addition of 5.8 percent g l y c e r y l ether and, i n the case of the controls, safflower o i l , to the experimental rations increased the energy content by approximately 7 percent so the dogs being fed 80 percent of t h e i r maximum calculated feed intake were a c t u a l l y receiving 87 percent of t h e i r maximum energy intake. The regression equations so obtained are given below: rats - grams of feed intake = 6.68W 0" 1 5 7 gm dogs - grams of feed intake = 57.79W 0' 9 5 5 Kg The exponent 0.956 for 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 from unity hence the feed intake standard for th i s species was taken to be l i n e a r over the weight range used. The animals were fed less than t h e i r maximum calculated feed intake to ensure that a l l feed offered was eaten; t h i s made i t possible to add s u f f i c i e n t g l y c e r y l ethers to the r a t i o n to ensure that the S 4 0 0 , B 4 0 0 and C^ 0 0 groups received 2.4 gm/Kg and S 1 0 0 , 0.6 gm/Kg of 47 body weight, d a i l y . The blending of the g l y c e r y l ethers into the r a t i o n was adjusted a f t e r each weighing of the rats to keep the l e v e l of administration at the prescribed point. For the dogs, the g l y c e r y l ethers formed 5.7 percent of t h e i r r a t i o n and for the rats i t varied from 1.7 percent at the beginning of the experiment to 3.5 percent of t h e i r r a t i o n at the end. This was necessary i n the case of the rats since feed intake per unit of body weight decreases markedly as body weight increases. By increasing the r e l a t i v e l e v e l of g l y c e r y l ethers i n the r a t i o n as body weight increased, i t was possible to ensure that the d a i l y intake of the ethers per unit weight of animal remained at the prescribed l e v e l . The regression equation pr e d i c t i n g maximum feed intake f o r the rats was found to be i n error, as the i n i t i a l r e s t r i c t e d l e v e l of 80 percent had to be raised to 90 percent at the end of 30 days and to 100 percent at the end of 45 days, i n order to maintain a reasonable rate of growth (approximately 2.3 grams/day). With the dogs, feeding at 80 percent of the predicted maximum l e v e l was s u f f i c i e n t throughout the 60 day feeding period to cause the control animals to grow at a rate 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 of the experimental rations (i) Rats The feed intake was recorded for the rats each day. Total 48 hour f e c a l c o l l e c t i o n s were made at 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 values calculated from t h i s data are presented i n Table 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 at two week i n t e r v a l s . In the determinations, the C 400 and S 100 groups were treated as a whole, while the B 400 and S 400 groups were treated according to whether they ate the same amount as the controls or not. The d i g e s t i b i l i t y of the dog rations was determined by the increase i n the concentration of chromic oxide i n faeces over that i n the feed, following the method proposed by Schiirch, et_ a l (93). The d i g e s t i b i l i t y values so obtained are used i n Table XXVII. (b) I s o l a t i o n and determination of g l y c e r y l ethers* (i) Extraction of l i p i d s from feed and faeces** F i r s t l y , samples of the material to be extracted were oven dried at 110 degrees centigrade f or two hours. These samples were subsequently used for chromate d i g e s t i b i l i t y determina-tions.' Another representative sample (5 gm on a dry weight basis) of the material was weighed into a 16 ounce j a r . A calculated amount of water was added to increase the percentage of water to 80 percent. This t o t a l sample s i z e was taken to be unity. An equal amount of chloroform and twice t h i s amount of methanol was added. This mixture was then blended * These are the methods approved and employed by Western Chemical Industries Limited of Vancouver. **This i s a modified Bligh and Dyer procedure (13) that was used for l i p i d e xtraction of f i s h . with a propeller-type s t i r r e r at about 2000 rpm for 5 minutes. Then one unit of chloroform was added and blended for one minute. This was followed by one unit of water blended i n for one minute. The whole mixture was then transferred to a s t a i n l e s s s t e e l funnel and the solvent f i l t e r e d into a 250 ml cylinder using a s l i g h t negative pressure. The residue was washed with a further one and one-half units of chloroform to give a mixture of solvents with the f i n a l composition of 2/2.5/1.8 (chloroform/methanol/water). This mixture separated into two phases. The l i p i d - c o n t a i n i n g chloroform phase was transferred to an evaporating f l a s k . The chloroform was stripped o f f , and the t o t a l l i p i d residue dried to a constant weight i n a vacuum oven at 60 degrees centigrade. ( i i ) Determination of neutral l i p i d s The t o t a l l i p i d f r a c t i o n was taken up i n 100 ml of acetone per gram of l i p i d s . Four ml of e t h y l ether was added and the stoppered f l a s k placed i n running water at 14 degrees centigrade. The p r e c i p i t a t e d phospholipids were removed by f i l t r a t i o n and the neutral l i p i d s were recovered by s t r i p p i n g o f f the acetone and drying the residue to a constant weight under vacuum at 60 degrees centigrade. ( i i i ) Determination of the unsaponifiable l i p i d f r a c t i o n The neutral l i p i d s were saponified according to the following scheme. T h i r t y ml of ethanol and three ml of 50 percent potassium hydroxide were added to approximately one gram of neutral l i p i d s and refluxed f o r one-half hour. Then t h i r t y ml of water were added and the mixture allowed to cool. This mixture was extracted four times with e t h y l ether (30 ml each time). This extract was washed a l t e r n a t e l y with 20 ml of water and 20 ml of 0.5 N potassium hydroxide and f i n a l l y with water u n t i l the washings were clear to phenolphthalein. The ether was then stripped o f f and the unsaponifiable material dried to a constant weight i n a vacuum oven at 60 degrees centigrade. (iv) Q u a l i t a t i v e and quantitative determination of g l y c e r y l ethers  Q u a l i t a t i v e pre-eluted 10 by 20 cm thi n layer plates (250 micron layer of s i l i c a gel) were scored with 6 to 7 lanes. About 100 gamma of g l y c e r y l ether residue was applied to a lane. I d e n t i f i c a t i o n of the components i n the residue was made by applying suitable reference compounds to corresponding lanes These plates were eluted with 50/50/1 (CHCl 3/CH 3OH/acetic acid) Upon the completion of the e l u t i o n the plates were sprayed with 20 percent sulphuric acid and charred at 150 degrees centigrade for 20 minutes or u n t i l the spots were sharply charred. The amount of g l y c e r y l ether i n the g l y c e r y l ether residue was determined by comparison of the spot si z e with the spot s i z e of known amounts of reference g l y c e r y l ethers. The actual weight of g l y c e r y l ethers i n the g l y c e r y l ether residue from the preparatory plate was determined by using the c o r r e c t i o n factor determined from the q u a l i t a t i v e thin-layer plate. 51 7. Discussion of feed-Intake, d i g e s t i b i l i t y and weight gain The consideration of the experimental r e s u l t s from the dogs gives r i s e to a number of problems. F i r s t l y , there was a large v a r i a t i o n i n the i n i t i a l weights of the animals. I t was necessary to accept t h i s v a r i a t i o n since a l i m i t e d number of dogs was a v a i l a b l e from which the experimental groups could be constituted. Sixteen pups were selected from some two dozen weanlings. Selection was c a r r i e d out by elimination of the smallest and the largest animals. The sixteen so selected varied i n weight from 18 to 31 pounds (8.2 to 14.1 kilograms). The second consideration was one concerning the a c c e p t a b i l i t y of the prepared r a t i o n s . A l l the dogs were fed the stock dog r a t i o n for a four week period p r i o r to the commencement of the experimental t r i a l . During this f i r s t period a l l of the dogs ate and gained normally. A f t e r the dogs were placed on the experimental r a t i o n s , i t became apparent that a l l rations were not equally acceptable to the animals. The S 1 0 0 and C^ 0 0 groups continued to eat normally, but those offered the B 4 0 0 and S 1* 0 0 rations did not eat with r e l i s h . In f a c t , some of the dogs refused to eat a l l the feed that they were offered. In the case of two dogs i n the B 1* 0 0 group, the self-imposed feed-intake r e s t r i c t i o n a c t u a l l y led to a body weight l o s s . The a c c e p t a b i l i t y of the S^ 0 0 and B 1* 0 0 experimental rations was markedly less than that of the S 1 0 0 and C^ 0 0 r a t i o n s . G l y c e r y l ethers i n contact with water form complexes which adhere to adjacent surfaces. Thus the rations high i n g l y c e r y l ethers tended to i n t e r f e r e with the prehension and ingestion of the feed. Furthermore', the high l e v e l s of g l y c e r y l ethers i n the rations gave them an unusual odor which may have been repellent to the animals. 52 TABLE XXIII: SUMMARY OF THE WEIGHT GAIN AND FEED INTAKE DATA OF DOGS  S 1* 0 0 Individual 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 (Kg) 1.5 0. 9 0. 03 0. 21 0. 66 gain/week (Kg) 0.200 0. 155 0. 044 0. 255 0. 164 feed offered (gm) 29,715 38,955 30,380 28,910 31,990 feed refused (gm) 2,940 10,805 1; 095 n i l 5,210 feed consumed (gm) 26,775 28,150 23,285 28,910 26,780 feed consumed/week (gm) 2,975 3,128 2,587 3,212 2,975 gm feed/gm gain 14.9 20. 2 58. 8 12. 6 26. 6 gm feed/gm gain/Kg wt 1.26 1. 33 4. 70 1. 13 2. 11 gm feed/gm gain/W°' 1 7 3 2.46 2. 77 9. 30 2. 16 4. 17 B H U U Individual Animals #20 #34* #14 #12 Mean f i n a l weight (Kg) 9. 5 14.1 (15.0) 10. 9 12. 7 11.8 (12.1) i n i t i a l weight (Kg) 10. 5 12.3 8. 6 13. 2 11.2 t o t a l gain (Kg) - 1. 0 1.8 (2.7) 2. 3 - 0. 5 0.65 (0.88) gain/Kg of weight (Kg) - 0. 1 0.13 (0.20) 0. 26 - 0. 04 0.06 (0.08) gain/week (Kg) - 0. 11 0.20 (0.30) 0. 26 - 0. 06 0.07 (0.10) feed offered (gm) 26,530 35,595 24,780 32,970 29,969 feed refused (gm) 8,835 n i l n i l 9,460 4,574 feed consumed (gm) 17,695 35,595 24,780 23,510 25,395 . feed consumed/week (gm) 1,966 3,955 2,753 2,612 2,822 gm feed/gm gain 19.8 (13.2) 10. 8 gm feed/gm gain/Kg wt 1.5 1. 1 gm feed/gm gain/W°' 7 3 (0.97) 3.0 (2.0) 2. 1 *Dog #34 was sick the l a s t week of the experimental period and, as a r e s u l t , l o s t weight. I f he had gained normally then we would expect those figures which appear i n brackets. 53 TABLE XXIII (cont'd) S Group Individual Animals i #26* #33 #21 #13 Mean f i n a l weight (Kg) 15.9 19.5 15.0 18.7 17.3 (16.8) (17.5) i n i t i a l weight (Kg) 11.4 13.2 9.5 12.7 11.7 t o t a l gain (Kg) 4.5 6.3 5.5 6.0 5.6 (5.4) (5.8) gain/Kg body weight(Kg) 0.33 0.39 0.45 0.38 0.39 (0.38) (0.40) gain/week (Kg) 0.5 0.7 0.61 0.67 0.62 (0.6) (0.65) feed offered (gm) 34,860 40,915 30,835 39,690 36,575 feed refused (gm) n i l n i l n i l n i l n i l feed consumed (gm) 34,860 40,915 30,835 39,690 36,575 feed consumed/week (gm) 3,873 4,546 3,426 4,410 4,068 gm feed/gm gain 7.75 6.49 5.62 6.58 6.61 (6.46) (6.29) gm feed/gm gain/Kg wt 0.57 0.40 0.46 0.42 0.46 gm feed/gm gain/W°' • 7 3 (0.40) (0.42) 1.15 0.84 0.91 0.88 0.95 (1.12) (0.94) *Dog #26 was sic k during the l a s t week of the experimental period and, as a r e s u l t , l o s t weight. I f he had gained normally then we would expect those figures which appear i n brackets. C'+OO i n d i v i d u a l Animals #11 #19 #22 #17 Mean f i n a l weight (Kg) 24. 5 16 .4 16. 8 18. 2 19. 0 i n i t i a l weight (Kg) 15. 5 10 .5 10. 9 10. 9 12. 0 weight gain (Kg) 9. 0 5 .9 5. 9 7. 3 7. 0 gain/Kg body weight :(Kg) 0. 45 0 .44 0. 43 0. 50 0. 46 gain/week (Kg) 1. 00 0 .66 0. 66 0. 81 0. 78 feed offered (gm) 50,330 33,110 35,805 36,611 38,964 feed refused (gm) n i l 65 850 415 333 feed consumed (gm) 50,330 33,045 34,955 36,196 38,632 feed consumed/week (gm) 5,592 3,672 3,884 4,022 4,293 gm feed/gm gain 5. 59 5 .50 5. 92 4. 96 5. 49 gm feed/gm gain/Kg wt 0. 28 0 .41 0. 43 0. 34 0. 37 gm feed/gm gain/W 73 0. 63 0 .82 0. 87 0. 70 0. 76 54 The t h i r d consideration concerns the presence of i n t e s t i n a l parasites i n the experimental dogs. During the course of the project d e f i n i t e signs of a s c a r i a s i s appeared, but no co r r e c t i v e measures were taken for fear of confounding the experimental design. Post mortem of those dogs k i l l e d - a f t e r nine weeks revealed a very heavy i n f e s t a t i o n of ascarids (Toxocara canis) and whipworms (T r i c h u r i s v u l p i s ) . This i n t e s t i n a l i n f e s t a t i o n , coupled with the phy s i c a l d i f f i c u l t i e s of ingesting feed con-taini n g high l e v e l s of the ethers, probably accounts for the feed refusa l s noted. The experimental r e s u l t s show large v a r i a t i o n s i n the i n d i v i d u a l response to the treatments. This makes i t necessary to consider each of the animals separately. Feed intake and body weight gain r e l a t i o n s h i p s have been calculated for each dog to f a c i l i t a t e consideration of the findings (see Table XXIII for 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 with those from the control animals shows a s i g n i f i c a n t l y slower growth rate and a lower feed e f f i c i e n c y . The difference i n the energy l e v e l of the two rations may be enough to account for the difference i n growth rate between the groups. The r a t i o n had 5.8 percent safflower o i l mixed i n the basic dog r a t i o n , while the S 1 0 0 r a t i o n had only 1.45 percent s e l a c h y l alcohol. Looking c l o s e r 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 1 0 0 group i t becomes apparent that dog #26 had a s i g n i f i c a n t l y slower growth rate than the other three animals i n the group. This can be explained by examination of the growth data presented i n Appendix V. I t may be noted that dog #26 l o s t one-half a kilogram of body weight during the l a s t week of the experiment 55 while the other dogs of the group gained from 0.5 to 0.9 kilograms. It i s suggested that the parasite i n f e s t a t i o n i n t h i s animal (#26) had reached a l e v e l that i n t e r f e r e d with feed u t i l i z a t i o n and hence growth rate. A close examination of the i n d i v i d u a l r e s u l t s of the animals i n the B 1 + 0 0 group shows that two of the animals responded i n a manner that i s quite d i f f e r e n t from the other p a i r . Dogs #20 and #12 refused to eat a l l of the feed that they were offered and as a r e s u l t l o s t some weight during the two month t r i a l . The other two dogs (#34 and #14) i n t h i s group gained slowly and hence, because they consumed t h e i r a l l o t t e d amount of feed, y i e l d a feed e f f i c i e n c y r a t i o that i s approximately one-half that of the animals from the control group. This suggests that the b a t y l alcohol when fed at a l e v e l of 2400 mgs per kilogram of body weight i n t e r f e r e d with the digestion of the feed. Evidence for t h i s interference with digestion w i l l be found i n the data on g l y c e r y l ether d i g e s t i b i l i t y (see Tables XXVII and XXVIII). This aspect w i l l be elaborated upon l a t e r . Group S 4 0 0 may be s i m i l a r l y divided; dogs #25 and #23 ate a l l or nearly a l l the feed that was offered to them. The feed conversion r a t i o s for these two animals were 14.9 / 1 and 12.6 / 1, or about one-half of that obtained for the c o n t r o l group. The large v a r i a t i o n i n feed e f f i c i e n c y between the control animals and c e r t a i n of the animals i n the experimental groups (see Table XXIV) can probably be explained on the basis of the e f f e c t of the ethers on the d i g e s t i b i l i t y of the various experimental r a t i o n s . The extremely low feed conversion e f f i c i e n c i e s for the second and t h i r d dog i n the S 4 0 0 group and the negative values for the f i r s t and fourth dogs i n the B 4 0 0 group i s 56 TABLE XXIV: SUMMARY OF FEED EFFICIENCIES Group S k 0 ° B 1* 0 0 S 1 0 0 C k 0 ° Dog No. 1 14.9 -« 6.46 5.59 2 20.2 13.2 6.49 5.50 3 58.8 10.8 5.62 5.92 4 12.6 -» 6.58 4.96 Inevitable since they refused to eat a l l the feed that was offered to them. This led to very low or negative weight gains, hence the feed u t i l i z e d per unit of weight gained becomes i n f i n i t e l y large. Dogs #1 and #4 i n the S^ 0 0 group and dogs #2 and #3 i n the B^ 0 0 group ate according to the same feeding schedule as the control animals yet t h e i r gain f e l l w e l l below that of the controls. The following model shows how 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 can markedly change feed conversion e f f i c i e n c y . MODEL In t h i s model both body weight and feed intake are held constant, while the d i g e s t i b i l i t y of the r a t i o n i s varied from 48 to 66 percent. This model could be a t y p i c a l animal from the control group i n t h i s experiment. In t h i s model the following assumptions are made: body weight 15 kilograms feed-intake/day 620 grams gain/day 110 grams energy content of gain 3 kcal/gram metabolizable energy of r a t i o n 3 kcal/gram 57 I f we accept a d a i l y intake of 1860 k c a l of metabolizable energy when the d i g e s t i b i l i t y of the feed i s 60 percent, i t i s possible to compute the values given : in the following ; table: TABLE XXV: THE RELATIONSHIP BETWEEN DIGESTIBILITY AND FEED EFFICIENCIES FOR A MODEL DOG % Metabolizable Energy content Grams of Grams feed D i g e s t i b i l i t y energy k c a l of gain k c a l gain gram gain 66 2,046 516 172 3.6 64 1,984 454 151 4.1 62 1,922 392 131 4.7 60 1,860 330 110 5.6 58 1,798 268 89 7.0 56 1,736 206 69 9.0 54 1,674 144 48 12.9 52 1,612 82 27 22.9 50 1,550 20 6.7 92.5 48 1,488 - 42 negative gain These c a l c u l a t i o n s show that 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 from 60 to 54 percent, the feed e f f i c i e n c y r a t i o changes from 5.6 to 12.9 grams of feed per gram of gain. Thus a small change i n the d i g e s t i b i l i t y of the r a t i o n r e s u l t s i n a r e l a t i v e l y large change i n the feed e f f i c i e n c y r a t i o . At the end of the experimental period two of the four dogs i n each group'were slaughtered as outlined e a r l i e r . The dogs to be held over were selected at random from the C 4 0 0 and S 1 0 0 groups, but i n the B 4 0 0 and S 4 0 0 groups, the dogs that had been refusing feed were selected. These eight dogs were a l l fed the control r a t i o n at a rate of 80 percent of t h e i r calculated maximum intake. The f i n d i n g of the heavy i n f e s t a t i o n of 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 I 59 i n t e s t i n a l parasites i n the dogs that were k i l l e d dictated that the remaining dogs a l l be treated for worms. A response to the worm t r e a t -ment appeared i n the control group. The B 4 0 0 and S 4°° dogs gave a response both to the worming and to the control r a t i o n . They grew at or near the same rate as the C 4 0 0 and S 1 0 0 dogs. From t h i s response i t seems l o g i c a l to conclude that the experimental treatments had no l a s t i n g e f f e c t s on the treated animals. A l l of the experimental dogs so treated, including the controls, showed an immediate response by gaining weight at a higher rate than before the treatment. This response suggests (1) that a l l of the dogs, including the controls, 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 that impaired growth .rate, and (2) that the treated dogs had apparently suffered no l a s t i n g e f f e c t s from the alcohols since they responded immediately when placed on the control r a t i o n . Figure II presents the average growth curve from 4 weeks p r i o r to the experimental period to 4 weeks a f t e r for the two dogs held over from each group. The f i g u r e shows the response to worming i n a l l the groups as well as the response of the B 4 0 0 and S 4 0 0 dogs to the control r a t i o n . Table XXVI explores the feed intake-gain r e l a t i o n s h i p s for the experimental groups of r a t s . No s i g n i f i c a n t differences are revealed. The fa c t that the S 1 0 0 r a t i o n had a s l i g h t l y lower feed e f f i c i e n c y than the control can be explained on the basis of the energy l e v e l of the r a t i o n . The p h y s i c a l nature of the a-glyceryl ethers did not appear to have any e f f e c t on the feed consumption of the r a t s . I t should be pointed out, however, that the r a t i o n offered to the dogs contained almost twice as much of the ethers per unit weight as that offered to the r a t s . 60 TABLE XXVI: FEED INTAKE-GAIN RELATIONSHIPS* FOR THE RATS IN TRIAL II S400 B 4 0 0 s i o o C 4 0 0 f i n a l weight (gm) 216.1 227,8 212 o 6 218.1 i n i t i a l weight (gm) 79.6 79.7 83.6 80.7 t o t a l gain (gm) 136.5 148.1 129.0 137.4 gain/gm of weight (gm) .92 .96 .87 .92 gain/day (gm) 2.24 2.43 2.11 2.25 feed offered (gm) 825 827 822 824 feed refused (gm) 30 14 8 19 feed consumed (gm) 795 813 814 805 feed consumed/day (gm) 13.0 13.3 13.3 13.2 gm feed/gm gain 5.82 5.58 6.31 5.86 gm feed/gm gain/Kg ; wt 39.3 36.2 42.6 39.3 gm feed/gm gain/W u • 73 24o9 22.5 28.2 24.9 *These figures are the mean for 10 animals. 8. G l y c e r y l Ethers and D i g e s t i b i l i t y Faeces samples taken two weeks and s i x weeks a f t e r the beginning of the t r i a l were anlyzed for the presence of g l y c e r y l ethers. Using the average feed intake for the week p r i o r to the sampling and the d i g e s t i b i l i t y values arr i v e d at by the chromic oxide method i n the dog and by t o t a l 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 of the g l y c e r y l ethers was determined. In these. determinations the dogs i n the S 1* 0 0 and B 4* 0 0 groups were separated into those which ate according to the feeding standard and those that v o l u n t a r i l y r e s t r i c t e d t h e i r feed intake. The r e s u l t s are summarized i n Table XXVII. A s i m i l a r summary for the rats i s given i n Table XXVIII. The values for the d i g e s t i b i l i t i e s of the g l y c e r y l ethers i n both the dogs and the rats are presented i n the form of a bar graph i n Figure I I I . The rats digested the g l y c e r y l ethers more completely than did the dogs. Batyl 61 alcohol was less 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 rats s e l a c h y l alcohol fed at the l e v e l of 2.4 grams per kilogram of body weight was digested equally as well as was selachyl fed at the 0.6 grams per k i l o of body weight l e v e l , whereas i n the dogs there was a marked difference i n the d i g e s t i b i l i t y of se l a c h y l alcohol between those two dosage l e v e l s . This i s quite reasonable since the percent of the g l y c e r y l ethers i n the S 4 0 0 dog r a t i o n was 5.8 while the S 4 0 0 rat r a t i o n was i n the range of 1.7 to 3.5 percent. The dogs eating to the feeding standard digested a smaller portion of the g l y c e r y l ethers ingested than did the dogs which v o l u n t a r i l y r e s t r i c t e d t h e i r own feed intake. This, then, i s a p a r t i a l explanation why the d i g e s t i b i l i t y of the S 4 0 0 and B 4 0 0 rations for the dogs must be lower than for the S 1 0 0 a n d c o n t r o l . I t i s reasonable to assume that the g l y c e r y l ethers coating the feed p a r t i c l e s prevented enzymatic action on some of the feed i n the digestive t r a c t and the d i g e s t i b i l i t y of the whole r a t i o n was lowered to a point where the S 4 0 0 and B 4 0 0 dogs ate the same amount of feed as the S 1 0 0 and the control groups, but demonstrated l i t t l e or no gain. A change i n d i g e s t i b i l i t y of 6 to 10 percent from the normal i s enough to prevent body weight gain. One point of note i s the find i n g of g l y c e r y l ethers i n small quantities i n both the control feed and the faeces of the con t r o l animals. The t o t a l amount i n the faeces was greater than that found i n the control feed, thus i t seems tenable that synthesis of g l y c e r y l ethers occurred i n the i n t e s t i n e or that they were secreted into the i n t e s t i n e from the ti s s u e s . The presence of g l y c e r y l ethers i n the control r a t i o n i s not unexpected since the r a t i o n contained 10 percent herring meal. TABLE XXVII: RECOVERY OF GLYCERYL ETHERS FROM FAECES AND DETERMINATION OF DIGESTIBILITY OF THE ETHERS FOR DOGS IN TRIAL II Feed/Day/Dog Dry wt (Grams) % G • E. G.E. Wt (Grams) % of Feed Digested Faeces/Dog/Day Dry Wt** (Grams) % G.E.* G.E. Wt (Grams) G.E. Absorbed (Grams) % G.E. Digested Dog Weight (Kg) G.E. Absorbed mgm/Kg/day Samples taken 481 413 270 364 286 465 598 437 271 423 361 550 0.048 5.75 5.75 5.80 5.80 1.33 0.048 5.75 5.75 5.80 5.80 1.33 0.231 23.74 15.52 21.11 16.59 6.18 two weeks from the beginning of the t r i a l 56.5 209.2 0.13 55.5 183.8 6.41 54.1 123.9 4.75 55.4 162.3 7.87 55.4 127.6 3.59 59.2 189.7 0.27 11.78 5.89 12.77 4.58 0.51 11.96 9.63 8.34 12.01 5.67 50.4 62.1 39.5 72.4 91.7 Samples taken s i x weeks from the beginning of the t r i a l 0.287 25.13 15.58 24.53 20.94 7.32 55.1 268.5 0.126 42.0 253.5 6.05 56.9 116.8 4.43 54.4 192.9 6.29 57.8 152.3 4.52 60.4 217.8 0.39 0.34 15.34 5.17 12.13 6.88 0.85 9.79 10.41 12.40 14.05 6.47 39.0 66.8 50.5 67.1 •88 o 4 13.3 11.4 11.8 10.9 13.2 13.0 16.5 11.8 11.1 11.4 13.2 15.1 1.05 0.82 0.77 0.91 0.44 0.83 0.94 1.09 1.06 0.43 a b Note: dogs #14 and #34 dogs #12 and #20 c dogs #23 and #25 d dogs #9 and #10 The feed per day data i s c a l c u l a t e d from the feed consumed for the week p r i o r to the faeces sampling. The % G.E. ( g l y c e r y l ethers) i n the feed for a l l the groups has 0.05 subtracted from the experimental values and the % G.E. i n the faeces has 0.13 subtracted from the experimental values. (This has been done to correct f or the g l y c e r y l ethers found i n the control ration.) * As determined i n the feed by a modification of the method of Bligh 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 using a chromic oxide method (93). 0 to TABLE XXVIII: RECOVERY OF GLYCERYL ETHERS FROM FAECES AND DETERMINATION OF DIGESTIBILITY OF THE ETHERS FOR THE RATS IN TRIAL II Feed/Day/Group Dry wt (Grams) /o G • E a G.E. Wt (Grams) % of* Feed Digested Faeces/Day/Group Dry Wt (Grams) % G.E. G.E. Wt (Grams) G.E. Absorbed (Grams) % G.E. Digested Group Wt (Kg) G.E. Absorbed mgm/Kg Sample taken two weeks from the beginning of the t r i a l 105.3 104.4 105.3 105.3 128.5 120.1 127.6 128.3 0.048 2.27 2.29 0.50 0.048 2.85 2.87 0.64 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 n i l 0.078 0.450 0.044 n i l 1.92 2.37 0.53 81.0 98.2 100.0 Sample taken s i x weeks from the beginning of the t r i a l 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 1.176 1.181 1.196 1.189 1.841 1.898 1.856 1.798 1.63 1.98 0.44 1.495 1.93 0.454 Note: The feed per day data i s calculated from the feed consumed f or the week p r i o r to the faeces sampling. The % G.E. ( g l y c e r y l ethers) i n the feed has 0.05 subtracted from a l l the experimental values; % G.E. of the faeces taken two weeks from the beginning of the t r i a l has 0.26 subtracted from a l l the experimental values, and % G.E. of the faeces samples taken six weeks from the beginning of the t r i a l has 0.19 subtracted. (These corrections are made because of the g l y c e r y l ethers found i n the co n t r o l feed and faeces.) * D i g e s t i b i l i t y by t o t a l c o l l e c t i o n FIGURE I I I : DIGESTIBILITY OF GLYCERYL ETHERS PERCENTAGE OF GLYCERYL ETHERS DIGESTED O o O CD o o o 0017* 1 oot^a o m m CO CO ' 00i?s P 00t>s 001 » OO^9 OOfr9 CO m m to O o CO ^ 0017 S P 0 0 * £ 001 > o CO 0017 s m m CO 001* OOfr* CO x 3) OOt^  m m CO 00l; 65 9. Results from c e l l counts The following table l i s t s the s i g n i f i c a n t differences found i n the c e l l counts derived i n t h i s t r i a l . They include erythrocyte, leucocyte and r e t i c u l o c y t e counts of blood, a d i f f e r e n t i a l of leucocytes i n the blood 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 again at the end of two months with the exception of the bone marrow d i f f e r e n t i a t i o n which was done when the animals were k i l l e d . TABLE XXIX: SIGNIFICANT (0.05 LEVEL) DIFFERENCES IN CELL COUNTS n c o • T>I j T> ™ r . n Higher or Lower Group Sex Species Blood or Bone Marrow C e l l s ° „ Counts s 4 0 0 female rats sk00 dogs s i o o female rats s 4 0 0 female rats female rats s-00 dogs dogs s i o o dogs B 4 0 0 male rats s i o o male rats B 4 0 0 male rats s i o o male rats B 4 0 0 female rats s i o o female rats s i o o female S 4 0 0 male s i o o male sh00 male B 4 0 0 male s«t00 female Counts Taken After One Month leucocyte counts lower r e t i c u l o c y t e count higher Counts Taken A f t e r Two Months erytrocytes higher leucocytes lower r e t i c u l o c y t e s higher r e t i c u l o c y t e s higher r e t i c u l o c y t e s higher r e t i c u l o c y t e s higher polymorphs lower polymorphs lower lymphocytes higher lymphocytes higher lymphocytes higher lymphocytes higher D i f f e r e n t i a l of Rat Bone Marrow promyelocytes higher myelocytes lower myelocytes lower metamyelocytes and 'ring c e l l s ' lower mature granulocytes lower mature granulocytes lower 66 TABLE XXIX (cont'd) o n o • r . i J T . n r Higher or Lower Group Sex Species Blood or Bone Marrow C e l l s ° „ Counts D i f f e r e n t i a l of Rat Bone Marrow gHUU female mature granulocytes lower glOO female mature granulocytes lower S 1* 0 0 male nucleated red blood c e l l s higher B ^ 0 0 male nucleated red blood c e l l s higher S 1 0 0 male nucleated red blood c e l l s higher g'+OO female nucleated red blood c e l l s higher B ' 4 ° ° female nucleated red blood c e l l s higher D i f f e r e n t i a l of Dog Bone Marrow No s i g n i f i c a n t differences were found i n the bone marrow smears of the dogs Although some staggered s i g n i f i c a n t differences occur throughout a l l of the blood counts of the r a t s , there appears a d e f i n i t e trend only i n the case of lymphocytes. Of the three groups of treated r a t s , male and female, only the S 1 0 0 females show a s i g n i f i c a n t l y higher erythrocyte count over the cont r o l . S i m i l a r l y , only the S 1* 0 0 females show a s i g n i f i c a n t l y d i f f e r e n t leucocyte count, t h i s count being lower. Only the B 1^ 0 females showed a s i g n i f i c a n t l y higher r e t i c u l o c y t e count. Two groups of male rats ( B ^ 0 0 and S 1* 0 0) showed a s i g n i f i c a n t l y lower percentage of polymorphs. In four cases the lymphocyte percentage of the white blood c e l l s was s i g n i f i c a n t l y higher; B^ 0 0 and S 1 0 0 groups (male and female). A l l other categories of leucocytes i n the treated animals did not d i f f e r from the con t r o l . 67 The dogs showed no s i g n i f i c a n t differences i n blood counts or d i f f e r e n t i a l s except i n the case of r e t i c u l o c y t e s where a l l the treated dogs had s i g n i f i c a n t l y higher counts of r e t i c u l o c y t e s than had the con t r o l animals. The d i f f e r e n t i a l counts of the rat bone marrow shows only two d e f i n i t e trends, the one may be the r e s u l t of the other. Mature granulocytes are s i g n i f i c a n t l y lower i n the B^ 0 0 males and the three treated groups of females, while nucleated red blood c e l l s are s i g n i f i c a n t l y higher i n a l l the groups of male and female rats except the S 1 (^ female r a t s . No s i g n i f i c a n t differences were found i n the d i f f e r e n t i a l of c e l l s i n the bone marrow smears of the dogs. The haematopoietic response, then, to the a - g l y c e r y l ethers, administered at extremely high l e v e l s , i s r e l a t i v e l y i n s i g n i f i c a n t , except i n the case of the r a t s , where nucleated red blood c e l l s i n the bone marrow are s i g n i f i c a n t l y higher and i n the case of the dogs, where r e t i c u l o c y t e s i n the blood are s i g n i f i c a n t l y higher. This stimulation of the immature red blood c e l l s i s not r e f l e c t e d i n an increase i n erythrocyte numbers. This suggests that either the l i f e of the immature erythrocyte i s extended or the l i f e of the mature red blood c e l l i s shortened. 68 C. FEEDING RESPONSE EXPERIMENT - TRIAL III 1. Preamble The analysis of the control feed used i n the preliminary t r i a l s revealed an amount of g l y c e r y l ethers s u f f i c i e n t to e l i c i t a response i f g l y c e r y l ethers possess vitam i n - l i k e properties. Hence i t i s possible that the addition of g l y c e r y l ethers to t h i s r a t i o n ( T r i a l s I and II) could produce l i t t l e or no p o s i t i v e response i n the experimental animals. The hypothesis that the addition of g l y c e r y l ethers to a g l y c e r y l ether free r a t i o n would show a response i s tested i n t h i s t h i r d t r i a l . Batyl alcohol was the g l y c e r y l ether of choice i n t h i s feeding response experiment since i t was r e a d i l y a v a i l a b l e and appears to have been more active than se l a c h y l i n a l l e v i a t i n g c e r t a i n conditions of stress (Prokhonchukov and Panikarovskii, 1963). A l e v e l of 5 mg per kilogram of body weight was chosen as an appropriate dosage. This was considered an adequate dosage l e v e l since i t i s s i m i l a r to recommended l e v e l s of vitamins as discussed i n the preamble to T r i a l I. Two more dosage l e v e l s were chosen: one which was one tenth (0.5 mg) of the 5 mg per kilogram of body weight l e v e l and another which was ten times as great (50 mg). 2. Design Forty female rats were placed at random into four groups of ten; a c o n t r o l group and three groups receiving 0.5, 5, and 50 mg of b a t y l alcohol per kilogram of body weight per day. Batyl alcohol was incorporated into the feed to f a c i l i t a t e administration. The experimental period was 5 weeks. Cheeke (24) has suggested that the apparent d i g e s t i b l e energy 69 intake of the albino laboratory rat over the body weight range, 50 to 175 grams, be predicted from the following r e l a t i o n s h i p : ADE = 6.82 W0*451 gm This feeding standard was accepted as v a l i d for t h i s experiment and the animals were fed 80 percent of th i s t h e o r e t i c a l maximum intake. A l l animals were weighed three times each week. Representative samples of feed and faeces were taken three times during the f i v e week experimental period. During the f i f t h week blood samples were taken from a l l the animals; red blood c e l l counts and white c e l l counts were made, smears were made for r e t i c u l o c y t e counts and leucocyte d i f f e r e n t i a t i o n . At the end of the f i v e week period a l l the animals were s a c r i f i c e d , organs were inspected, and femur bone marrow smears were made. 3. Animals Albino Sprague-Dawley (SPF) female rats were used as experimental animals. They were placed on the experiment at a weight of approximately 60 grams. The actual weights are given i n Appendix I I I . 4. Housing The rats were housed i n i n d i v i d u a l wire cages. Feed and water were supplied i n porcelain j a r s . 5. Ration A g l y c e r y l ether free ration- was made up as follows: 70 sucrose 56 percent casein safflower o i l mineral mix vitamin mix alphacel 25 10 4 2 3 To balance t h i s r a t i o n with respect to minerals and amino acids the following additions were made: 11.7 mg CuSO^ per kilogram of basic r a t i o n 13.8 mg ZnCL,2 per kilogram of basic r a t i o n 773 mg cystine per kilogram of basic r a t i o n The ingredients used i n t h i s synthetic r a t i o n were shown to be free of g l y c e r y l ethers by analysis. The sucrose, casein, mineral mix and alphacel were a l l free of l i p i d material and therefore were free of g l y c e r y l ethers. Analysis of the vitamin mix and the safflower o i l revealed no trace of g l y c e r y l ethers. This basic r a t i o n was fed to a l l the rats with the appropriate amounts of b a t y l alcohol added to the r a t i o n for each of the treated groups. 6. Feed intake-gain r e l a t i o n s h i p s Table XXX summarizes the feed intake-gain r e l a t i o n s h i p s for the rats used i n t h i s t r i a l . No differences were found i n the rate of gain 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 alcohol treated groups was e s s e n t i a l l y the same as the control animals on the g l y c e r y l ether free d i e t . 71 TABLE XXX: FEED INTAKE-GAIN RELATIONSHIPS* FOR RATS IN TRIAL III Control B - 0.5 B - 5 B - 50 f i n a l weight (gm) 169.3 170.4 169.5 167.8 i n i t i a l weight (gm) 57.5 57.3 57.6 56.3 t o t a l gain (gm) 111.8 113.1 111.9 111.4 gain/gm weight** (gm) 0.99 0.99 0.99 0.99 gain/day (gm) 3.4 3.4 3.4 3.4 feed offered (gm) 371.8 370.3 370.9 369.5 feed refused (gm) 18.3 18.1 20.4 20.3 feed consumed (gm) 353.5 352.2 350.5 349.2 feed consumed/day 10.7 10.7 10.6 10.6 gram feed/gram gain 3.16 3.11 3.13 3.13 * a l l figures are * * t h i s weight i s the mean the mean of ten rats of the i n i t i a l and f i n a l weights f o r each group 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 values i n Table XXXI are based on t o t a l f a e c a l c o l l e c t i o n s taken over 72 and 96 hours. They show no s i g n i f i c a n t differences between groups. The extremely small amount of faeces excreted by these animals i s a r e s u l t of the highly d i g e s t i b l e nature of the synthetic experimental r a t i o n used i n t h i s t r i a l . TABLE XXXI: DIGESTIBILITY OF EXPERIMENTAL RATIONS IN TRIAL III Group Control B - 0.5 B - 5 B - 5 0 2 weeks (72 hr) 93.4 93.9 94.5 94.9 3 weeks (96 hr) 93.4 93.2 93.5 93.7 4 weeks (72 hr) 93.4 94,2 93.6 93.6 72 8„ Results of blood and bone marrow c e l l counts These r e s u l t s are l i s t e d i n Appendices XIII, XIV and XV and are a l l summarized i n Table XXXII. Analysis of these r e s u l t s f a i l e d to show any s i g n i f i c a n t differences between the experimental groups and the c o n t r o l . Large v a r i a t i o n s existed between i n d i v i d u a l animals. A r b i t r a r y categories of bone marrow c e l l s were made to f a c i l i t a t e d i f f e r e n t i a t i o n . The s l i d e s were poorly stained so that the c l a s s i c a l c e l l types were not always recognizable. The categories chosen showed no s i g n i f i c a n t differences between groups. 9. Conclusion Batyl alcohol added to a g l y c e r y l ether-free synthetic r a t i o n and fed for 5 weeks did not appear to a f f e c t the rate of body growth or the production of blood c e l l s i n normal growing female albino r a t s . There was, however, some sign of erythrocyte stimulation i n four of the control rats that were fed the B - 50 r a t i o n for 4 days a f t e r the end of the experiment. Table XXXIII gives the erythrocyte counts. A " t " test indicates a s i g n i f i c a n t increase i n the number of erythrocytes. Thus, this l i m i t e d experimental diversion suggests further n u t r i t i o n a l t r i a l s where g l y c e r y l ether-deprived rats or other experimental animals are administered g l y c e r y l ethers. That b a t y l alcohol stimulates haemopoiesis i n normal r a t s , i s suggested i n experimental work done by Linman, et a l , 1959. The dosage l e v e l used was 50 mg/rat/day. This would equal approximately 300 mg per kilogram of body weight or 6 times the highest l e v e l used by the author 73 TABLE XXXII: A SUMMARY* OF THE BLOOD AND BONE MARROW DATA FROM TRIAL I II Group Control B - 0.5 B - 5 B - 50 Erythrocytes (millions per cmm) 7.60 7.26 7.44 7.60 Leucocytes ( c e l l s per cmm) 11,200 12,800 12,400 12,400 D i f f e r e n t i a l of Leucocytes (percentage of leucocytes) Polymorphs 14„4 17.1 12.5 15.2 Eosinophils 1.5 0.6 0.6 1.1 Lymphocytes 81.6 81.0 85.4 81.2 Monocytes 2.2 2.1 2.0 2.2 D i f f e r e n t i a l of bone marrow c e l l (percentage of c e l l s ) Category** 1 4.84 5.02 5.48 3.66 2 5.86 4.39 5.85 5.24 3 15.32 13.22 14.33 16.47 4 8.56 11.25 9.12 8.58 5 19.52 17.15 17.13 14.94 6 34.30 34.67 34.36 36.16 7 11.12 12.60 12.46 13.01 8 0.20 0.63 0.41 0.39 *These are averages of ten animals except i n the case of the d i f f e r e n t i a l of the bone marrow of the cont r o l animals where the numbers are averages of f i v e animals. **The eight categories of bone marrow c e l l s are explained i n Appendix XV. TABLE XXXIII: ERYTHROCYTE COUNTS (MILLIONS PER CMM) OF FOUR CONTROL  RATS FED BATYL ALCOHOL AT THE END OF THE EXPERIMENTAL PERIOD Rat No. End of Experiment Four Days on Feed 7 6.50 8.08 8 7.01 9.80 9 7.93 7.83 10 7.43 8.56 Average 7.60 8.57 74 i n T r i a l IIIo Linman does not mention the age of the rats nor the r a t i o n that he fed. The b a t y l alcohol used was a racemic synthetic material and not the o p t i c a l l y active natural form used i n t h i s study. The form of the b a t y l alcohol may have some e f f e c t on the a c t i v i t y of the compound, since dosage l e v e l s as high as 2400 mg per kilogram of body weight were administered to animals i n t h i s project, without causing a s i g n i f i c a n t haemopoietic effects This apparent inconsistency between the experimental r e s u l t s obtained by Linman and those obtained by the author suggest a comparative study of the synthetic form with the natural form. PART I I I : GENERAL DISCUSSION 75 GENERAL DISCUSSION The main conclusions to be drawn from these experiments are summarized below: 1. Dogs and rats fed chimyl, selachyl and b a t y l alcohols at l e v e l s of 6, 600 and 2400 mg per kilogram of body weight did not show any signs of i n t o x i c a t i o n , 2. Except i n the case of female rats r e c e i v i n g s e l a c h y l alcohol at the 6 mg l e v e l for a period of s i x months, there was no difference i n the growth rate between experimental and c o n t r o l animals. 3. Chimyl, selachyl and b a t y l alcohols administered o r a l l y at a l e v e l of 6 mg per kilogram of body weight to dogs and rats for s i x months f a i l e d to i n i t i a t e any d e f i n i t e haematopoietic response. 4. Selachyl alcohol administered at l e v e l s of 600 and 2400 mg per kilogram of body weight and b a t y l alcohol administered at a l e v e l of 2400 mg per kilogram of body weight for a period 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 responses i n both the dogs and the r a t s . The only indications of haematopoietic stimulus were a r e t i c u l o c y t e response i n the blood of the dogs and a response i n the percentage of nucleated red blood c e l l s i n the bone marrow of the r a t s . 5. Batyl alcohol added to a g l y c e r y l ether-free synthetic r a t i o n and fed for a f i v e week period did not appear to a f f e c t the growth rate or the production of blood c e l l s i n normal growing female albino r a t s . 76 Throughout t h i s whole series of experiments no sign of t o x i c i t y appeared i n the experimental animals. Gross pathology, growth rate and histopathology of tissue sections from the heart, l i v e r , lungs, spleen, kidneys, adrenals 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 picture of the response to the administration of natural occurring a - g l y c e r y l ethers i s obscured by the inconsistencies of the r e s u l t s . There appears to be no growth response due to the administration of g l y c e r y l ethers, except i n the case of growing female rats given a low dosage of s e l a c h y l alcohol. These rats have a growth curve i d e n t i c a l with that of the other groups of female rats to a mean body weight of about 200 grams. Above t h i s body weight, the selachyl females grew at a s l i g h t l y f a s t e r rate to a f i n a l weight which was s i g n i f i c a n t l y higher than that of the other females. The haematopoietic responses were also inconsistent. For example, i n the low dosage group of selachyl-fed r a t s , at 90 days the females had s i g n i f i c a n t l y higher leucocyte counts, while the males had s i g n i f i c a n t l y lower leucocyte counts and at 180 days no differences were measured between eit h e r the female or male and the control animals. There appeared to be some stimulation i n erythrocyte production when selachyl alcohol was fed at 600 and 2400 mg per kilogram of body weight and b a t y l alcohol at 2400 mg per kilogram of body weight. The increases were r e s t r i c t e d mainly to r e t i c u l o c y t e s i n the blood of the dogs and to nucleated red blood c e l l s i n the bone marrow of the r a t s . This increase i n immature forms of erythrocytes was not accompanied by an o v e r a l l increase i n the red blood c e l l count. Thus i t would appear that 77 the normal sequence of events i n the l i f e of an erythrocyte was a l t e r e d . This may mean that the immature period i s lengthened and that the mature period of a red blood c e l l i s decreased. This suggests that the average red blood c e l l i s more youthful and more capable of f u l f i l l i n g i t s r o l e as an erythrocyte, or that the mature erythrocyte i s more f r a g i l e and i s removed from c i r c u l a t i o n sooner. The short experimental t r i a l using a g l y c e r y l ether-free synthetic r a t i o n showed no change i n erythrocyte numbers over a f i v e week period. However, four of the control rats that were fed a g l y c e r y l ether-free r a t i o n received b a t y l alcohol i n t h e i r feed for four days a f t e r the termination of the experiment and they showed an encouraging erythropoietic response. This i s o l a t e d response i s not s i g n i f i c a n t by i t s e l f because of the small number of animals involved and because of the lack of control animals, but i t does serve as an i n d i c a t i o n that another experiment could be done i n which rats are depleted of g l y c e r y l ethers for varying lengths of time and measure the response to administered g l y c e r y l ethers. If blood c e l l counts do not decrease when the animals are fed a g l y c e r y l ether-free r a t i o n , yet increase over normal values when g l y c e r y l ethers are administered to g l y c e r y l ether-depleted r a t s , then the problem becomes of i n t e r e s t to the biochemist and the physiologist as well as to the n u t r i t i o n i s t . In any comparison of the experimental r e s u l t s of t h i s project with those reported i n the l i t e r a t u r e , o p t i c a l isomerism becomes an important factor» The natural occurring a - g l y c e r y l ethers are of the d-configuration. Much of the experimental work reported i n the 78 l i t e r a t u r e makes use of synthetic racemic mixtures. From our knowledge of enzymes we know that they are most commonly enantiomer s p e c i f i c . This means that 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 ei t h e r emantiomer. An experiment s p e c i f i c a l l y designed to compare pure 1- and d-forms of a-g l y c e r y l ethers i s indicated here. Since i t i s now established that the n a t u r a l l y occurring a - g l y c e r y l ethers are not toxic and since the response i s v a r i a b l e and inconsistent i n the rat and the dog, i t may be worth while to use other species as experimental subjects. The dog and the rat were used p r i m a r i l y as models of mammals i n general; i t may well be that we should use the guinea pig or cat or some other animal to e s t a b l i s h the importance of g l y c e r y l ethers. I t may even be expedient to experiment with sheep or c a t t l e or even man with respect to the g l y c e r y l ethers as n u t r i t i o n a l e n t i t i e s . This thesis has established the non-toxic nature of the natural occurring a - g l y c e r y l ethers and has indicated the d i r e c t i o n i n which further research might proceed. BIBLIOGRAPHY 79 BIBLIOGRAPHY 1. Abaturova, E.A., and A.V. Shubina. O v l i y o n i i batilovogo i selak-hovogo sp i r t o v na rost zlokachestvennykh opukholei u krys (E f f e c t of b a t y l and selachyl alcohols on the growth of malignant tumors i n r a t s ) . Buyl Eksp B i o l . Med. 57(6): 81-83, 1964. B i o l . Absts. Feb. 15, 1965, 16020. 2. Alexander, P., D.I. Connell, A. Brohult and S. Brohult. Reduction of r a d i a t i o n induced shortening of l i f e span by die t augmented with alkoxy g l y c e r y l esters and f a t t y acids. Gerontologia 3:147-152, 1959. 3. A n s e l l , G.B. and S. Spanner. The probable occurrence of long-chain ether analoques of phosphatidylethanolamine i n br a i n t i s s u e . Biochem. J. 81:36P-37P, 1961. 4. Arturson, G. and M. Lindback. Experiments on the e f f e c t of b a t y l alcohol on the number of erythrocytes and r e t i c u l o c y t e s i n white mice. Acta Soc. Med. upsalien. 56:19-26, 1951. Nutr. Absts. Rev. 21(2):374, 1951. 5. Ashikawa, J.K. The l i p i d syndrome i n mammalian radiobiology with emphasis on p o s t i r r a d i a t i o n protection of animals with l i p i d s . Doctorate Thesis, University of C a l i f o r n i a , 1961. 6. Baer, E. and H.O.L. Fischer. Synthesis of o p t i c a l l y a c t i v e glycerides from d(+)-acetone glyceride. J . B i o l . Chem. 128:475-489, 1939. 7. Baer, E. and H.O.L. Fischer. Configuration of the natural b a t y l , chimyl, and selachyl alcohols. J . B i o l . Chem. 140:397-410, 1941. 8. Baer, E. and H.O.L. Fischer. Naturally occurring g l y c e r y l ethers. I I I . Selachyl alcohol and i t s geometrical isomer. J. B i o l . Chem. 170:337-342, 1947. 9. Baer, E., L.J. Rubin and H.O.L. Fischer. Naturally occurring g l y c e r y l ethers. I I . Synthesis of se l a c h y l alcohol. J. B i o l . Chem. 155:447, 1944. 10. Bergmann, W. and H.A. Stansbury. Contributing to the study of marine products. XIV. A s t r o l . J. Org. Chem. 8:283-284, 1943. 11. Bergmann, et a l . Marine Products. XL. Waxes and t r i g l y c e r i d e s of sea anemones. J. Org. Chem. 21:721-728, 1956. 80 12. Bergstrb'm, S. and R. Blomstrand. The i n t e s t i n a l absorption and metabolism of chimyl alcohol i n the r a t . Acta P h y s i o l . Scand. 38:166, 1956. 13. B l i g h , E.G. and W.J. Dyer. A rapid method of t o t a l l i p i d e xtraction and p u r i f i c a t i o n . Can. J . Biochem. Ph y s i o l . 37:911, 1959. 14. Blomstrand, R. and E.H. Ahrens, J r . Absorption of chimyl alcohol i n man. Soc. Exp. B i o l . Med. Proc. 100:802-805, 1959. 15. Blomstrand, R. Digestion, absorption and metabolism of chimyl alcohol fed as free alcohol or as alkoxydiglyceride. Soc. Exp. B i o l . Med. Proc. 102:662-665, 1959. 16. Bodman, J . and J.H. Maisin. The alpha-glyceryl ethers. C l i n . Chem. Acta 3:253-274, 1958. 17. Brohult, A. Alkoxy-glycerol esters i n i r r a d i a t i o n treatment. International conference on radiobiology. Proc. 5th conference, Stockholm 1956: 241-247, 1957. 18. Brohult, A. E f f e c t s of alkoxyglycerols and e s p e c i a l l y selachyl alcohol on the bone marrow i n connection with i r r a d i a t i o n t r e a t -ment and i n leukaemia therapy. Nature 181:1485, 1958. 19. Brohult, A. Alkoxyglycerols as growth-stimulating substances. Nature 188:591-592, 1960. 20. Brohult, A. Alkoxyglycerols i n i r r a d i a t i o n treatment. Nature 193:1304, 1962. 21. Brohult, A. Alkoxyglycerols and t h e i r use i n r a d i a t i o n treatment. Acta Radiol. Supp. 223:1-99, Stockholm, 1963. 22. Brohult, A. and J . Holmberg. Alkoxyglycerols i n the treatment of leukopaenia caused by i r r a d i a t i o n . Nature 174:1102, 1954. 23. Carter, H.E., D.B. Smith and D.N. Jones. A new ethanolamine-containing l i p i d from egg yolk. J . B i o l . Chem. 232:681-694, 1958. 24. Cheeke, P.R. The changes of the serum protein-bound iodine during growth i n the Hols t e i n c a l f and the Wistar albino r a t . Masterate Thesis, University of B r i t i s h Columbia, 1965. 25. Dalton, R.G. The e f f e c t of b a t y l alcohol on the hematology of c a t t l e poisoned with bracken. Vet. Rec. 76(15):411-416, 1964. 26. Davies, W.H., I.M. Heilbron and W.E. Jones. The structure of b a t y l and selachyl alcohols. J . Chem. Soc. 1933: 165-167. 81 27. de Gaetani, G.F. and G. B a i o t t i . Hemopoietic a c t i v i t y of b a t y l alcohol. B o l l . soc. i t a l . b i o l . sper. 35:1156, 1959. Chem. Absts. 55:16815a, 1961. 28. Deuel, H.J., J r . The l i p i d s . Interscience Publishers, New York, 1951. Vol. I. 29. Dudin, V.N. P r o f i l a k t i k a i lechenie luchevoi bolezni beremennykh zhivotnykh i ikh plodov tsistaminom i batilovym spirtom. (Prophylaxis and medical treatment of X-ray sickness i n pregnant animals and t h e i r f e t i with cystamine and b a t y l alcohol.) Med. Radiol. 6(ll):82-84, 1961. Referat. Zhur. B i o l . , 1962. B i o l . Absts. 42:297, 1963. 30. Edlund, T. Protective e f f e c t of d-, 1-alpha-octadecylglycerol ether i n mice given t o t a l body X - i r r a d i a t i o n . Nature 174:1102, 1954. 31. Emmerie,.A., C. Engel and W. K l i p . The tuberc u l o s t a t i c action i n v i t r o of the unsaponifiable f r a c t i o n of c o d - l i v e r o i l . J. S c i . Food Agric. 3:264-268, 1952. 32. Evans, I.A., A.J. Thomas, W.C. Evans and CM. Edwards. Studies on bracken poisoning i n c a t t l e . B r i t . Vet. J . 114:253-267, 1958. 33. Evans, W.C, I.A. Evans, CM. Edwards and A.J. Thomas. Bracken poisoning of c a t t l e ; therapeutic treatment. Biochem. J . 65:6P, 1957. 34. Evenstein, D., A.S. Gordon and M. E i s l e r . Lack of influence of b a t y l alcohol on erythropoiesis i n r a t s . Anat. Rec. 132:435, 1958. 35. Ghys, R. E f f e c t s of alkoxyglycerol (Kaby 700) on leucopenia r e s u l t i n g from radiotherapy. Laval Med. 30:331-342, 1962. 36. G i f f i n , H, and C.H. Watkins. Treatment of secondary anemia. J. Am. Med. Assoc. 95:587-592, 1930. 37. Gilbertson, J.R. and M.L. Karnovsky. Nonphosphatide f a t t y a c y l esters of alkenyl and a l y l ethers of g l y c e r o l . J. B i o l . Chem. 238(3):893-897, 1963. 38. Guyer, K.E. Studies on the i s o l a t i o n and analysis of g l y c e r y l ethers. Doctorate Thesis, Ohio State U n i v e r s i t y , 1962. Di s s e r t a t i o n Absts. 24(1):57, 1963. 39. Guyer, K.E. Studies on the composition of g l y c e r y l ethers and the i r preparation from d i a c y l g l y c e r y l ethers i n l i v e r o i l s . J . L i p i d Res. 4:385-391, 1963. 82 40. Hallgren, B. and S.O. Larsson. Separation and i d e n t i f i c a t i o n of alkoxyglycerols. Acta Chem. Scand. 13:2147-2148, 1959. 41. Hallgren, B. and S.O. Larsson. The g l y c e r y l ethers i n the l i v e r o i l s of elasmobranch f i s h . J . L i p i d Res. 3:31-38, 1962. 42. Hallgren, B. and S.O. Larsson. The g l y c e r y l ethers i n man and cow. J. L i p i d Res. 3:39-43, 1962. 43. Hanahan, D.J., J. Ekholm and CM. Jackson. Studies on the structure of g l y c e r y l ethers and the g l y c e r y l ether phospholipids of bovine erythrocytes. Biochem. 2(4):630-640,1963. 44. Hanahan, D.J. and R. Watts. The i s o l a t i o n of an a-alkoxy-3-acyl a-glycerophosphorylethanolamine from bovine erythorcytes. J. B i o l . Chem. 236 :PC59-PC60, 1961. 45. Hardegger, E., L. Ruzicka and E. Tagmann. Helv. Chem. Acta 26:2205, 1943. Cited by Hallgren, B. and S.O. Larsson. The g l y c e r y l ethers i n man and cow. Jo L i p i d Res. 3:39-43, 1962. 46. Hasegawa, Y., et a l . Batyl alcohol. Nippon Rinsho 19:1793-1800, 1961. Chem. Absts. 56-.1968G, 1962. 47. Helmy, F.M. and M.H. Hack. Comparison of the l i p i d s i n maternal and cord blood and of human amniotic f l u i d . Proc. Soc. Exp. B i o l . Med. 110:91-94, 1962. 48. H i l d i t c h , T.P. The chemical c o n s t i t u t i o n of natural f a t s . London, Chapman and H a l l , 1956. 49. Holmes, H.N., R.E. Corbet, W.B. Ceiger, N. Kornblum and W. Alexander. The i s o l a t i o n and i d e n t i f i c a t i o n of b a t y l alcohol and cho l e s t e r o l from yellow bone marrow. Amer. Chem. Soc. J. 63:2607-2609, 1941. 50. Karnovsky, M.L. and A„F. Brumm. Studies on n a t u r a l l y occurring alpha-glyceryl ethers. J . B i o l . Chem. 216:689-701, 1955. 51. Karnovsky, M.L., A.W. Lategan, W.S. Rapson and H.M. Schwartz. South A f r i c a n f i s h products. Part XXIX. The composition of the l i v e r o i l of the soupfin shark. (Galeorhenus, Rond.) J. Soc. Chem. Ind. 67:193-196, 1948. 52. Karnovsky, M.L., W.S. Rapson and M. Black. South A f r i c a n f i s h products. Part XXIV. The occurrence of alpha-glyceryl ethers i n the unsaponifiable f r a c t i o n s of natural f a t s . J. Soc. Chem. Ind 0 65:425, 1946. 83 53. Karnovsky, M.L., W.S. Rapson and H.M. Schwartz. South A f r i c a n f i s h products. Part XXVIII. The composition of the l i v e r o i l of the seven-gilled shark. (Heptranchias pectorosus) (Garman). J. Soc. Chem. Ind. 67:144-147, 1948. 54. Karnovsky, M.L., W.S. Rapson, H.M. Schwartz, M. Black and N.J. van Rensburg. South A f r i c a n f i s h products. Part XXVII. The composition of the l i v e r o i l s of the basking shark (Cetorhinus maximus, Gunner) and the spiny shark (Echinorhinus  spinosus, Gmelin). J . Soc. Chem. Ind. 67:104-107, 1948. 55. Kates, M., L.S. Yengoyan and P.S. Sastry. A diether analog of phosphatidyl glycerophosphate i n Halobacterium cutirubrum. Biochem. Biophys. Acta 98:252-268, 1965. 56. Kind, C.A. and Bergman, The occurrence of octadecyl alcohol, b a t y l alcohol, and c e t y l palmitate i n Gorgonias. J, Org. Chem. 7:424, 1942. 57. Klenk, E. and U.W. Hendricks. An i n o s i t a l phosphate containing carbohydrate i s o l a t e d from human brain. Biochem. Biophys. Acta 50:602-603, 1961. 58. Knight, B.C.J.G. Surface films of b a t y l , chimyl and sel a c h y l alcohols. Biochem. J. 24:257-261, 1930. 59. Linman, J.W. Hemopoietic e f f e c t s of g l y c e r y l ethers. I I I . I n a c t i v i t y of se l a c h y l alcohol. Proc. Soc. Exp. B i o l . 104:703-706, 1960. 60. Linman, J.W. and F.H. B e t h e l l . Observations on c i r c u l a t i n g erythrocytes and bone marrow of rats receiving protein-free extracts of rabbit plasma. Blood 11:310-323, 1956. 61. Linman, e_t a l . The erythropoietic stimulating a c t i v i t y of b a t y l a l c o h o l . J. Lab. C l i n . Med. 52:596-604, 1958. 62. Linman, e_t a l . Studies on the stimulation of hemopoiesis by b a t y l alcohol. J . Lab. C l i n . Med. 54:335-343, 1959. 63. Lovern, J.A. S p e c i f i c p e c u l i a r i t i e s i n depot f a t composition. Biochem. J. 31:755-763, 1937. 64. Maisin, J.H., J . Keusters, H. Guidetto and G. Lambert. The e f f e c t i v e use of g l y c e r y l ethers i n tre a t i n g X-ray burn. J. de Radiologie d"Electrologie et Medicine Nucleaire 40:454-460, 1959. 65. Malins, D.C. Fatty acids and g l y c e r y l ethers i n alkoxyglycerides of dog-fish l i v e r o i l . Chem. Ind. 38D:1359-1360, 1960. 84 66. Malins, D.C., J.C. Wekell and C.R. Houle. The metabolic fate of ingested g l y c e r y l ethers i n rainbow trout (Salmo g a i r d n e r i ) . F i r s t World Fat Congress, Hamburg, Germany. Oct., 1964. 67. Malins, D.C., J.C. Wekell and C.R. Houle. Composition of d i a c y l g l y c e r y l ethers and t r i g l y c e r i d e s of the f l e s h and l i v e r of the dogfish (Squalus acanthias). J . L i p i d Res. 6(1):100-105, 1965. 68. Malkins, T. Recent work i n the phospholipid f i e l d . Chem. Ind. 39B:605-611, 1961. 69. Manforte, F. and G. Fenech. Components of the l i v e r o i l of Hexanchus griseu. Ann. Chim. 44:804-814, 1954. 70. Mangold, H.K. and D.C. Malins. Fractionation of f a t s , o i l s and waxes on thi n layers of s i l i c i c a c i d . J . Am. O i l Chem. Soc. 37:383-385, 1960. 71. Maqsood, M. and J.K. Ashikawa. P o s t - i r r a d i a t i o n protection and recovery. I. E f f e c t s of l i p i d s on hematopoietic organs of X-ir r a d i a t e d male mice. Internat. J . Rad. B i o l . 4:521-531, 1961. 72. Marberg, CM. and H.O. Wiles. Yellow bone marrow extracts i n granulocytopenia. J . Am. Med. Assoc. 109:1965-1966, 1936. 73. Matsumoto, T. and T. Wainai. The saponifiable matter of the Echinoderms. I. A 7 - C h o l e s t e r o l i n the fat of s t a r f i s h . J . Chem. Soc. Japan, Pure Chem. Sect. 75:756-758, 1954. 74. Matsumoto, T., ^ t a l . Sterols and other unsaponifiable substances i n the fats of s h e l l f i s h e s , Crustacea and Echinodermata. J. Chem. Soc. Japan 64:1203-1204, 1943. 75. M i l l e r , B., C.E. Anderson and C. Piantadosi. Plasmalogen and g l y c e r y l ether concentrations i n normal and a t h e r o s c l e r o t i c a o r t i c t i s s u e . J. Gerontol. 19(4):430-434, 1964. 76. Mizuno, N.S., V. Perman, D.D. J o e l , F.W. Bates, J.H. Sautter and M.O. Schultze. Survival of calves treated with autologous bone marrow a f t e r exposure to l e t h a l dose of whole body i r r a d i a t i o n . Proc. Soc. Exp. B i o l . Med. 105(2):317-320, 1960. 77. Nakagawa, Sachiko and J.M. McKibbin. D i s t r i b u t i o n of alpha g l y c e r y l ethers i n animal tissue s . Pro. Soc. Exp. B i o l . Med. 111(3):634-636, 1962. 85 78. Omelik, S. The a d r i a t i c elasmobranch l i v e r o i l s . IV. Biochemical study of o i l s from the l i v e r and eggs of Centrina s a l v i a n i . Juguslavije Acta A d r i a t . 4:3-23, 1949. 79. Osmond, D.G., P.J. Raylance, A.J. Webb and J.N. Yoffey. The action of b a t y l alcohol and selachyl on the bone marrow of the guinea pig. Acta Haematol. 29(3):180-186, 1963. 80. Penny, R.H.C., A.J. Wright and J.W. Stoker. Observations on the re l a t i o n s h i p between b a t y l alcohol and haemopoiesis i n c a t t l e , sheep and mice. B r i t . Vet. J . 120:286-293, 1964. 81. Pietruszko, R. Li p i d s of red bone marrow from pig epiphyses. Biochem. Biophys. Acta 64:562-564, 1962. 82. Pietruszko, R. and G.M. Gray. The products of mild a l k a l i n e and mild acid hydrolysis of plasmalogens. Biochem. Biophys. Acta 56:232-239, 1962. 83. Popovie, M. fiber die glycerinather des menschlichen herzmuskels Z. Physiol. Chem. 340:18-23, 1965. 84. Prelog, V.L., Ruzicka, L., F. Steinmann. The i s o l a t i o n of chimyl alcohol from testes extract and i t s i d e n t i f i c a t i o n with t e s t r i o l . Helv. Chem. Acta 27:674, 1944. 85. Prokhonchukov, A.A. and V.V. Panikarovskii. The condition of the maxillodental system during the experimental therapy of ra d i a t i o n sickness. Teoriya i Prakt. Stomatol., Sb. 6:61-63, 1963. Chem. Absts. Biochem. Sections 63(7):8706H, 1965. 86. R i l e y , R.F., Y. Hokama and P. Kratz. Nonsteroidal components of the unsaponifiable f r a c t i o n of human l i v e r . Cancer Res. 18:825-832, 1958. 87. Rusanov, A.M., E.N. Mosharova and R.S. Komarova. Therapy with chemical compounds of hematopoiesis i n h i b i t i o n caused by i o n i z i n g i r r a d i a t i o n . Med. Radiol. 7(2):42-9, 1962. Chem. Absts. Biochem. Sect. 63(6):7325C, 1965. 88. Rusoff, L.L. The r o l e of milk i n modern n u t r i t i o n . Borden's Rev. Nutr. Res. 25:2-3, 1964. 89. Safanda, J. and V. Holecek. Inhibitory e f f e c t of chimyl alcohol on l y s o l e c i t h i n hemolysis. F o l i a Haematol. 83(2):171-175, 1965. Chem. Absts. Biochem. Sect. 63(10):13865G, 1965. 90. Sandler, O.E. Some experimental studies on the erythropoietic e f f e c t of yellow bone marrow extracts and b a t y l a lcohol. Acta Med. Scan. Supp. 225:1-99, 1949. 86 91. Schogt, J.CM., P. Haverkamp Begemann and J . Koster. Nonphosphatide aldehydrogenic l i p i d s i n milk f a t , beef tallow and ox heart. J. L i p i d Res. 1:446-449, 1960. 92. Schultze, M.O., V. Perman, F.W. Bates and J.H. Saulter. F a i l u r e of DL-batyl alcohol to prevent a p l a s t i c anemia i n calves. Proc. Soc. Exp. B i o l . Med. 98(3) :470-474, 1958. 93. Schiirch, A.F., L.E. Lloyd and E.W. Crampton. The use of chromic oxide as an index f or determining the d i g e s t i b i l i t y of a d i e t . J . Nutr. 41:629-636, 1950. 94. Sehgal, S.N., M. Kates and N.E. Gibbons. L i p i d s of Halobacterium cutirubrum. Can. J . Biochem. Ph y s i o l . 40:69-81, 1962. 95. Stove, B.B. Growth promotion i n pea stem segments. I. Stimulation of auxin and g i b b e r e l l i n action by a l k y l l i p i d s . Plant Ph y s i o l . 35:262-269, 1960. 96. Svennerholm, L. and H. Thorin. I s o l a t i o n of "Kepalin B" from cerebral l i p i d s . Biochem. Biophys. Acta 41:371-372, 1960. 97. Sviridov, N.K., E.A. Abaturova, A.V. Shubina and G.N. Elpat'evskaya. Therapeutic e f f e c t of s e l a c h y l alcohol on experimental r a d i a t i o n sickness. Moscow Med. Sb. 1964 254-258. Chem. Absts. Biochem. Sect. 63(10):13676C, 1965. 98. Swell, L., M.D. Law and C.R. Treadwell. Absorption of a- and 3-octadecyl g l y c e r y l ethers. Arch. Biochem. Biophys. 110(2):231-236, 1965. Chem. Absts. Biochem. Sect. 63(1):1014D, 1965. 99. Thompson, G.A. , J r . and D.J. Hanahan. I d e n t i f i c a t i o n of g l y c e r y l ether phospholipids as major l i p i d constituents i n two species of t e r r e s t i a l slug. J. B i o l . Chem. 238(8):2628-2631, 1963. 100. Thompson, G.A. and D.J. Hanahan. Origin of the g l y c e r y l component of alpha g l y c e r y l ethers. J . Biochem. Biophys. 96:671-672, 1962. 101. Thompson, G.A., J r . and D.J. Hanahan. Studies on the nature and formation of alpha-glyceryl ether l i p i d s i n bovine bone marrow. Biochem. 2(4):641-646 , 1963. 102. Thompson, G.A. and P. Lee. Studies of the alpha-glyceryl ether l i p i d s occurring i n Molluscan tissu e s . Biochem. Biophys. Acta 98:151-159, 1965. 87 103. T i e t z , A., M. Lindberg and E.P. Kennedy. The oxidation of g l y c e r y l ethers: A new enzyme system requiring a p t e r i d i n e cofactor. Fed. Proc. 22:296, 1963, 104. Todd, D. and G.P. R i z z i . Biochemistry of the alpha-glyceryl ethers. I. D i s t r i b u t i o n i n mammalian tissues and i n s t a r f i s h . Proc. Soc. Exp. B i o l . Med. 115:218-222, 1964. 105. Wajda, M. L i p i d composition of human bone marrow. Biochem. J . 95:252-255, 1965. 106. Weidemann, G. Studies on the chemical nature of the unsaponifiable f r a c t i o n of f i s h o i l . Biochem. J . 20:685-691, 1926. 107. Wintrobe, M.M. C l i n i c a l Hematology. Philadelphia, Lea and Febiger, 1952. APPENDICES APPENDIX I A. Composition of U.B.C. Ration No. 14-63 Dog Crumbles Tomato pomace 100 Ground wheat 590 Wheat bran 100 Wheat germ meal 65 Oat groats 150 Fis h meal (70%) 200 Soya meal (50%) 200 Linseed o i l meal 100 Vitagrass 100 Skim milk powder 100 Brewers yeast 20 Steamed bone meal 20 Iodized s a l t 5 S t a b i l i z e d f a t 150 Molasses (cane) 100 2000 Dry vitamin A 1 mi Dry vitamin D 150,' B. Composition of U.B.C. Ration No. 10-63 Rat P e l l e t s Ground barley 300 Ground wheat 400 Wheat bran 200 Wheat germ meal 100 Ground oat groats 200 Fishmeal (73%) 250 Soyabean meal (44%) 100 Oilcake meal 50 Vitagrass 100 Skim meal powder (spray) 100 Brewers yeast 24 Irradiated dry yeast 2 S t e r i l i z e d bone meal 10 S t a b i l i z e d f a t 50 Molasses 100 Iron oxide 4 Iodized s a l t 10 2000 h lb dry vitamin A 90 APPENDIX II Mean Daily Feed Intake In Grams A. Male Rats Week Selachyl Batyl Chimyl Control 1 14.1 14.1 14.7 14.2 2 16.8 15.6 17.3 17.2 3 19.4 18.4 18.5 18.5 4 18.0 18.5 19.0 18.3 5 19.5 18.3 17.7 20.4 6 19.8 18.0 18.8 20.0 7 17.6 15.4 18.0 19.7 8 19.6 18.7 19.6 20.5 9 20.4 19.9 18.8 19.9 10 19.3 18.5 18.3 18.6 11 21.5 20.5 19.4 20.5 12 22.4 21.1 19.9 21.1 13 21.5 20.1 19.5 20.9 14 24.7 22.4 22.9 22.8 15 23.0 22.4 22.3 22.9 16 24.3 22.9 23.8 24.0 17 25.9 23.2 23.5 23.4 18 25.2 23.4 22.6 23.1 19 24.7 24.4 21.5 22.7 20 26.3 23.6 22.8 25.3 21 25.0 23.1 22.1 22.4 22 23.5 23.1 20.5 22.2 23 23.0 24.2 21.1 24.8 24 21.5 22.2 20.0 21.7 25 20.8 19.5 19.4 20.6 26 20.9 20.0 20.8 20.6 27 22.2 20.6 20.1 21.4 APPENDIX II (cont'd) B. Female Rats Week Selachyl Batyl Chimyl Control 1 13.3 13.7 13.8 13.3 2 14.8 16.0 15.8 14.7 3 15.7 15.8 16.3 14.3 4. 16.0 16.8 15.8 15.5 5 16.2 16.4 16.5 15.9 6 17.2 16.3 16.7 15.9 7 16.2 14.6 15.8 15.3 8 17.5 16.7 17.4 16.4 9 17.3 15.3 15.5 15.2 10 15.2 14.5 14.6 14.0 11 17.4 16.0 16.4 15.7 12 18.6 16.9 16.8 16.1 13 18.6 15.5 15.9 15.9 14 20.1 18.6 18.8 17.4 15 18.1 18.2 17.9 16.9 16 19.2 18.8 18.9 17.5 17 19.6 16.9 20.0 17.4 18 18.9 17.8 19.0 17.5 19 20.0 18.0 19.5 17.7 20 19.4 18.1 20.8 18.8 21 .18.8 17.8 19.4 17.0 22 21.6 18.6 17.7 16.6 23 19.4 17.7 18.3 17.2 24 19.0 16.6 16.6 15.6 25 18.9 15.6 15.3 14.6 26 18.0 15.9 17.4 15.7 27 18.8 17.8 17.1 16.3 APPENDIX I I I Mean Weekly Weights A. Male Rats* Week Selachyl Batyl Chimyl Control 1 102.3 99.5 99.0 94.3 2 132.2 119.5 131.7 124.6 3 164.3 149.8 161.5 150.4 4 200.1 184.3 187.0 183.1 5 226.4 201.8 210.4 212.8 6 249.4 228.0 230.6 230.2 7 261.4 237.3 247.2 243.4 8 276.4 254.3 263.9 262.1 9 294.8 274.1 270.0 277.3 10 314.2 288.0 295.0 291.8 11 329.6 307.4 308.7 303.1 12 338.9 319.4 317.7 316.8 13 347.3 329.6 326.1 329.3 14 358.3 338.7 334.2 339.7 15 362.6 347.4 341.8 348.9 16 373.2 356.8 353.6 362.2 17 380.9 364.3 363.1 369.4 18 382.7 368.8 370.2 373.6 19 387.0 374.0 378.1 375.2 20 391.1 379.7 376.4 377.9 21 398.7 390.9 387.4 383.4 22 400.6 391.8 390.1 393.3 23 403.2 402.9 401.7 398.4 24 405.8 407.0 403.1 406.1 25 410.1 410.0 407.3 409.5 26 410.7 410.9 409.3 413.4 27 421.5 413.9 412.8 418.9 * A l l weights are i n grams and are the mean of ten animals. APPENDIX III (cont'd) B. Female Rats* Week Selachyl Batyl Chimyl Control 1 91.3 90.9 93.3 94.3 2 112.5 113.7 115.8 114.3 3 130.7 133.4 134.9 133.7 4 150.4 151.6 151.8 151.2 5 163.0 164.9 166.1 165.0 6 175.0 175.9 175.6 175.9 7 181.4 183.1 181.8 183.7 8 191.6 192.3 191.1 194.3 9 199.6 202.8 196.3 198.8 10 208.1 206.1 201.4 204.6 11 214.5 211.6 206.5 208.2 12 219.7 213.6 212.7 213.8 13 223.9 216.5 216.2 219.0 14 229.1 220.4 220.2 221.0 15 229.8 224.7 222.7 224.1 16 236.2 229.0 228.8 228.8 17 240.7 232.6 232.7 232.6 18 243.3: 234.4 238.0 235.4 19 243.2 238.8 237.5 237.2 20 247.9 238.6 239.2 238.6 21 252.7 241.4 241.9 242.4 22 254.6 241.5 246.9 242.9 23 253.3 245.9 246.0 246.3 24 258.2 248.6 247.9 248.2 25 262.8 250.4 248.3 249.7 26 264.0 251.3 250.0 251.5 27 267.3 255.4 253.3 256.4 * A l l weights are given i n grams and are the mean of ten animals. APPENDIX III (cont'd) C. Dogs* Week Selachyl Batyl Chimyl Control 1 10.3 10.7 11.6 9.5 2 12.4 12.6 13.1 10.9 3 13.3 13.7 14.2 11.6 4 12.7 14.0 14.7 12.3 5 13.9 14.2 14.9 12.9 6 . 14.8 15.4 15.6 13.4 7 15.5 16.3 16.6 14.4 8 16.3 16.7 17.2 15.1 9 16.9 17.3 17.3 15.6 10 16.6 17.4 17.7 15.4 11 17.6 17.9 18.3 16.2 12 18.4 18.7 19.1 17.3 13 18.4 18.9 19.3 17.1 14 19.4 19.5 20.0 18.1 15 19.5 19.5 19.7 18.0 16 20.1 20.0 20.5 18.3 17 20.7 20.7 21.1 19.2 18 21.0 20.8 21.2 19.4 19 21.6 21.2 21.8 20.0 20 22.1 21.1 21.5 19.7 21 21.7 21.6 21.9 20.3 22 22.0 20.8 21.7 19.9 23 21.8 21.0 22.0 19.8 24 22.2 20.2 22.3 20.4 25 22.2 20.8 22.6 20.8 26 22.6 20.6 22.7 20.2 27 21.9 20.7 23.0 20.8 * A l l weights are i n kilograms and are the mean of the f i v e animals. 94 APPENDIX IV Blood Counts A. Leucocyte Counts* of Rats Taken Ninety Days From The Beginning of The T r i a l Rat Selachyl Batyl Chimyl Control female 1 18,700 11,300 16,300 19,100 2 20,500 11,450 15,900 13,050 3 17,200 21,400 14,800 16,150 4 19,150 10,750 10,550 15,650 5 16,500 20,700 11,150 11,550 6 20,300 18,950 20,950 14,650 7 15,200 17,250 15,800 12,450 8 16,250 6,900 12,400 16,600 9 18,600 15,050 16,200 15,050 10 21,000 20,650 22,850 11,750 Mean 18,340 15,440 15,690 14,600 S.D.** 1,880 4,840 3,720 2,280 male 1 23,500 19,750 23,700 19,700 2 22,150 19,600 16,400 24,300 3 13,400 20,950 23,700 21,450 4 14,450 17,800 19,500 15,450 5 16,700 18,000 27,700 15,750 6 17,100 20,900 19,650 17,800 7 19,750 13,000 26,300 27,850 8 18,600 14,700 21,200 21,300 9 18,000 16,950 19,650 13,050 10 16,900 15,000 23,300 21,350 Mean 18,055 17,665 22,110 19,800 S.D.** 2,970 2,600 3,290 4,313 * These counts are numbers of leucocytes per cmm of blood. ** Standard Deviation. APPENDIX IV (cont'd) B. Leucocyte Counts* of Rats Taken 180 Days From  The Beginning of the T r i a l Rat Selachyl Batyl Chimyl Control female 1 17,400 11,500 15,000 18,300 2 18,900 12,100 22,100 16,600 3 12,400 20,600 15,000 18,400 4 21,100 17,800 10,000 13,800 5 14,600 18,000 14,800 11,400 6 9,000 18,000 10,300 12,000 7 13,700 19,800 11,300 10,900 8 17,800 12,500 12,000 11,500 9 16,800 15,000 21,800 14,600 10 13,900 14,800 12,800 15,700 Mean 15,560 16,010 14,510 14,320 S.D.** 3,350 3,110 4,110 2,710 male 1 21,500 26,000 13,000 15,500 2 17,400 26,300 20,500 12,500 3 25,600 27,500 20,400 17,500 4 20,500 26,700 15,400 19,400 5 12,300 16,800 15,000 27,800 6 13,300 15,000 15,900 16,300 7 16,100 18,700 17,500 16,400 8 20,000 13,100 28,300 17,100 9 12,300 11,100 20,500 16,200 10 23,100 12,200 28,900 16,900 Mean 18,200 19,340 19,540 17,560 S.D.** 4,440 6,300 5,150 3,800 * These counts are number of leucocytes per cmm. ** Standard Deviation APPENDIX IV (cont'd) C. Erythrocyte Counts* of Male Rats Taken 180 Days From The Beginning of the T r i a l Rat Selachyl Batyl Chimyl Control 1 9.96 10.2 8.3 7.3 2 9.16 9.4 8.7 8.9 3 9.69 9.1 9.5 8.7 4 8.5 8.6 8.9 9.0 5 9.3 7.9 10.2 8.5 6 9.7 7.2 8.2 10.1 7 8.8 8.8 10.0 9.6 8 8.4 8.9 8.9 9.5 9 8.3 9.3 8.7 8.2 10 9.3 8.8 10.4 8.9 Mean 9.11 8.82 9.58 8.87 S.D. .56 0.78 0.85 0.73 * The erythrocyte counts are recorded i n m i l l i o n s of c e l l s per cmm of blood. APPENDIX IV (cont'd) D. Erythrocyte Counts* of Dogs Taken 110 Days from  The Beginning of the T r i a l Selachyl Batyl dog # count dog it count 59 5,22 60 6.08 60a 5.52 69 5.39 74 5.60 66 5.83 72 4.80 70 5.10 65 5.40 64 6.10 Mean 5.31 5.70 S.D. 0.28 0.39 Chimyl Control dog # count dog # count 67 6.29 75 5.08 NT 6.68 76 5.40 58 5.25 68 6.38 71 5.54 77 5.72 63 6.20 62 5.24 Mean 5.99 5.56 S.D. 0.52 0.46 * The erythrocyte counts are recorded i n m i l l i o n s of ce per cmm of blood. APPENDIX IV (cont'd) E. Leucocyte Counts* of Dogs Taken 180 Days From  The Beginning of the T r i a l Selachyl Batyl dog # count dog // count 59 15,300 60 16,200 60a 16,100 69 15,500 74 20,300 66 17,300 72 14,900 70 15,100 65 19,300 64 21,100 Mean 17,200 17,200 S.D. 2,200 2,170 Chimyl Control dog # count dog # count 67 15,300 75 17,200 NT 19,900 76 22,200 58 17,000 68 18,100 71 16,900 77 15,700 63 15,200 62 15,200 Mean 16,900 17,500 S.D. 1,700 2,360 * These counts are numbers of leucocytes per cmm of blood. APPENDIX V Weight Data for Animals In T r i a l II A. Weekly Weights i n Kilog rams for Dogs S 1 0 0 Group Week #26 #33 #21 #13 I n i t i a l 11.4 13.2 9.5 12.7 1 10.9 13.6 10.0 13.6 2 12.3 14.5 10.9 14.1 3 12.7 15.0 11.4 14.5 4 13.6 15.9 11.8 15.5 5 13.6 16.4 12.3 15.4 6 14.5 16.8 12.7 16.4 7 15.5 17.7 13.6 17.3 8 16.4 18.6 14.5 18.2 9 15.9 19.5. 15.0 18.7 10 15.0 17.7 11 15.9 19.1 12 16.8 19.4 13 17.7 20.9 C k 0 ° Group  Week #11 #19 #22 #17 I n i t i a l 15.5 10.5 10.9 10.9 1 16.4 10.9 11.4 11.4 2 17.3 11.4 12.3 12.3 3 18.2 12.3 12.7 12.7 4 19.5 13.2 14.1 14.1 5 20.0 13.2 14.1 15.0 6 21.4 13.6 15.0 15.9 7 22.7 14.5 16.4 16.8 8 23.6 15.5 17.3 17.7 9 24.5 16.4 16.8 18.2 10 16.4 17.3 11 17.3 17.7 12 18.2 19.5 13 19.1 20.5 APPENDIX V (cont'd) S h 0 ° Group  Week #25 #10 #9 #23 I n i t i a l 10.9 14.5 12.3 10.5 1 11.4 15.4 11.8 10.5 2 10.9 15.0 11.4 10.9 3 11.4 15.0 11.4 10.9 4 11.4 15.0 11.4 11.4 5 11.4 14.5 10.9 11.4 6 11.4 14.5 11.8 11.4 7 11.8 15.4 11.8 11.8 8 12.3 15.5 12.3 11.8 9 12.7 15.9 12.7 12.3 10 15.9 12.7 11 17.7 13.6 12 19.1 14.1 13 20.0 15.5 B^°° Group  Week #20 #34 #14 #12 I n i t i a l 10.5 12.3 8.6 13.2 1 10.9 13.2 8.6 13.2 2 10.0 13.6 9.1 13.6 3 10.5 13.6 9.1 12.3 4 10.5 14.5 9.5 12.7 5 10.0 14.1 9.5 12.3 6 10.0 13.6 10.0 12.3 7 10.0 14.1 10.5 12.3 8 10.0 14.5 10.9 12.7 9 9.5 14.1 10.9 12.2 10 10.0 13.6 11 10.0 13.6 12 10.9 14.1 13 11.8 15.0 APPENDIX V (cont'd) B. Mean Weights* i n Grams for Rats Day S 4 0 0 B k 0 ° S 1 0 0 C k 0 ° 1 79.6 79.7 83.6 80.7 3 90.1 92.5 95.0 92.8 5 97.6 101.5 104.6 102.9 8 105.3 108.6 110.0 109.2 10 113.7 112.5 114.9 112.5 12 119.6 118.1 118.9 117.6 15 125.4 124.3 123.8 123.0 17 134.0 131.1 127.9 128.2 19 137.1 134.2 131.3 132.3 22 143.5 140.0 137.3 138.9 24 148.1 145.6 143.0 146.2 26 153.4 149.9 146.8 149.7 29 158.5 156.8 152.0 154.6 31 162.5 160.4 155.9 160.2 33 163.5 160.4 155.1 159.5 36 174.8 172.1 165.8 170.5 38 177.3 . 176.7 169.6 173.2 40 181.4 185.9 174.1 177.9 43 185.6 189.8 179.8 184.1 45 191.9 195.6 185.2 191.0 47 191.7 196.1 185.7 192.2 50 202.1 210.3 196.2 201.8 52 207.4 215.8 202.9 208.0 54 208.7 220.1 205.7 211.6 57 215.6 222.8 211.9 220.1 59 216.8 225.9 211.2 217.0 61 216.1 227.8 212.6 218.1 * mean of ten rats APPENDIX VI Results of Rat Blood Taken One Month From  The Beginning of T r i a l II A.. Erythrocyte Counts (m i l l i o n s of cells/cmm) : 400 ,400 • 100 ^400 Male 1 2 3 4 5 average Female 1 2 3 4 5 average 10.7 8.1 9.2 9.0 8.4 9.1 7.6 8.1 7.0 7.7 7.6 7.6 10.0 7.4 8.0 8.1 8.7 8.5 7.8 8.1 7.3 7.7 9.1 8.0 8.5 8.9 7.8 8.3 9.0 8.5 9.2 8.0 7.6 9.5 8.7 8.6 7.9 8.7 8.9 8.5 7.9 8.4 8.2 7.3 8.5 7.0 7.1 7.6 B. Leucocyte Counts (cells/cmm) ;400 ,400 • 100 >400 Male 1 2 3 4 5 average 16,100 9,700 7,500 9,300 10,000 10,500 8,800 15,700 12,600 6,000 9,900 10,600 8,200 17,100 5,900 11,800 8,000 10,200 21,500 6,800 5,900 6,600 6,300 9,400 Female 1 2 3 4 5 average 10,700 13,100 11,900 8,800 14,200 11,700 19,000 9,900 15,300 13,000 7,300 12,900 11,000 13,900 14,000 11,700 15,300 13,200 15,700 19,900 13,100 19,500 10,500 15,700 103 APPENDIX VI (cont'd) C. Reticulocytes (percent of erythrocytes) :400 .400 • 100 ^400 Male 1 2 3 4 5 average 4.7 4.1 3.9 6.9 3.7 4.6 3.0 3.8 4.6 4.2 3.2 3.8 3.1 4.6 2.9 3.8 3.9 3.7 4.6 4.2 2.8 4.4 3.5 3.9 Female 1 2 3 4 5 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, average 3.9 5.2 4.5 3.2 D. D i f f e r e n t i a l Leucocyte Counts (percent of leucocytes) Polymorphs S 4 0 0 B 4 0 0 S 1 0 0 C 4 0 0 Male 1 2 3 4 5 average 16 25 27 18 26 22.4 14 21 24 14 17 18.0 16 23: 22 22 24 21.4 31 22 24 22 14 22.6 Female 1 2 3 4 5 17 17 26 16 21 16 19 24 21 24 23 18 19 18 14 26 16 27 23 15 average 19.4 20.8 18.4 21.4 APPENDIX VI (cont'd) Lymphocytes S 4 0 0 B H 0 0 s i o o C H 0 0 Male 1 81 76 81 66 2 75 73 75 69 3 70 74 71 74 4 79 82 76 74 5 68 81 75 85 average 74. 6 77.2 75.6 73.6 Female 1 78 82 71 72 2 77 80 75 75 3 74 73 80 69 4 81 74 77 76 5 74 68 80 83 average 76. 8 75.4 76.6 75.0 Eosinophils S400 s i o o ch00 and Monocytes Male 1 2 1 6 4 3 0 1 2 2 0 0 4 2 2 0 5 4 3 1 2 2 0 6 1 1 1 4 1 2 0 4 2 0 3 1 5 6 0 1 1 0 1 1 0 averages 2. 0 1.0 2. .6 2.2 2. 6 0.4 2. 2 1.6 Female 3 2 1 1 5 1 1 1 2 2 3 0 1 5 2 8 1 3 0 0 2 1 1 0 2 0 4 1 2 4 1 3. 2 1 0 5 5 0 6 2 5 1 1 1 averages 2. 2 1.4 2. 6 1.2 3. 8 1.2 2. 6 0.6 APPENDIX VII Results of Rat Bood Taken Two Months From  The Beginning of T r i a l II A. Erythrocyte Counts (mil l i o n s of cells/cmm) ; 400 ,400 .100 -.400 Male 1 2 3 4 5 average 9.9 8.8 9.7 9.1 8.3 9.2 10.8 8.6 9.0 9.2 9.4 9.4 9.2 9.1 9.2 8.6 9.0 9.0 8.8 8.2 8.6 Female 1 2 3 4 5 average 8.3 9.0 8.6 9.4 8.8 8.8 10.9 8.6 8.6 9.4 9.4 9.2 8.9 8.4 9.9 9.4 9.2 8.7 7.8 8.8 7.5 7.7 8.1 B. Leucocyte Counts (cells/cmm) :400 ,400 .100 ^400 Male 1 2 3 4 5 average 18,200 13,300 9,900 12,500 11,800 13,100 10,300 14,300 10,100 5,800 9,600 10,000 7,800 23,000 7,900 15,200 9,200 12,600 22,600 9,900 8,600 10,200 9,100 12,100 Female 1 2 3 4 5 average 13,000 14,300 10,700 11,200 13,200 12,500 17,900 8,900 19,000 15,000 15,200 9,800 13,200 18,700 11,300 19,300 14,500 15,700 20,800 15,100 23,200 11,600 17,300 106 APPENDIX VII (cont'd) C. Reticulocytes (percent of erythrocytes) :H-00 >400 .100 ^00 Male 1 2 3 4 5 average 6.0 4.2 9.8 7.2 5.1 6.5 5.1 5.8 5.1 5.0 4.4 5.1 3.7 4.9 4.2 4.6 4.1 4.3 4.8 4.4 2.5 4.7 3.7 4.0 Female 1 2 3 4 5 average 3.7 4.9 5.3 4.3 5.1 ~4T7~ 7.9 4.9 5.8 5.2 ~6~70~ 4.1 3.6 4.7 4.8 6.1 4.7 3.3 2.9 2.3 3.4 3.3 3.0 D i f f e r e n t i a l Leucocyte Counts (percent of leucocytes) Polymorphs >400 .100 i^+00 Male 1 2 3 4 5 average 18 36 27 26 27 26.8 17 16 24 24 16 19.4 17 19 23 18 17 18.8 31 24 31 28 26 28 Female 1 2 3 4 5 14 16 22 18 23 14 18 17 17 23 18 19 21 19 24 23 27 19 15 average 18.6 16.5 20.0 21.6 APPENDIX VII (cont'd) Lymphocytes S400 B400 s i o o C400 Male 1 80 78 74 67 2 62 78 77 74 3 70 72 74 66 4 70 75 80 69 5 68 80 80 69 average 70. 0 76.6 77.0 69.0 Female 1 81 82 74 72 2 79 81 78 67 3 73 78 77 67 4 76 80 75 72 5 73 - 76 81 average 76. 4 80.2 76.0 71.8 Eosinophils chQO B400 s i o o C400 and Monocytes Male 1 1 1 3 2 8 1 0 2 2 0 2 2 3 2 2 1 1 3 1 2 2 2 2 1 0 3 4 2 2 1 0 0 2 3 0 5 3 2 1 3 2 1 2 1 average 1.4 1.8 1.8 2.0 2.8 1.4 1.4 l . i Female 1 1 3 2 1 2 1 2 2 2 1 4 1 0 1 2 3 7 3 3 2 3 1 2 2 5 1 4 2 4 2 1 3 1 4 4 5 3 1 - 4 1 1 1 average 2.0 2.8 2.0 0.8 2.4 1.4 3.0 3.0 108 APPENDIX'VIII Blood Count Data from Dogs Taken One Month From  The Beginning of T r i a l II I d e n t i f i c a t i o n 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 set of data by numbers 1, 2, 3 and 4 as follows: S 4 0 0 dog B 4 0 0 dog S 1 0 0 d o g C 4 0 0 dog 1 25 20 26 11 2 10 34 33 19 3 9 14 21 22 4 23 12 13 17 A. Erythrocyte Counts (mill i o n s of cells/cmm) Sk00 B 4 0 0 s i o o Ck00 Dog 1 5.6 7.2 6.5 6.2 2 6.0 5.4 5.2 6.1 3 5.3 5.0 6.3 5.5 4 5.7 4.9 5.5 5.6 average 5.7 5.6 5.9 5.9 B. Leucocyte : Counts (cell/cmm) S 4 0 0 B 4 0 0 s i o o C 4 0 0 Dog 1 13,300 16,200 14,600 9,400 2 8,600 14,100 11,900 10,700 3 14,600 17,500 17,000 12,600 4 14,800 12,300 13,000 10,000 average 12,800 15,000 14,100 10,800 109 APPENDIX VIII (cont'd) C„ Reticulocytes (percent of erytrocytes) S 4 0 0 BM-00 S 1 0 0 QM-OO Dog 1 1.3 2.2 1.5 1.1 2 1.4 1.1 1.3 1.5 3 1.6 2.6 0.7 0.6 4 1.1 0.8 1.0 0.9 average 1.4 1.7 1.1 1.0 D. D i f f e r e n t i a l Leucocyte Counts (percent of leucocytes) Polymorphs S 4 0 0 B 4 0 0 S 1 0 0 C 4 0 0 Dog 1 67 66 50 59. 2 57 69 69 65 3 65 72 65 71 4 72 81 59 63 average 65.3 72.8 61.8 64.5 Lymphocytes S 4 0 0 B 4 0 0 S 1 0 0 C 4 0 0 Dog 1 32 30 39 23 2 36 21 25 32 3 34 27 34 26 4 20 18 37 33 average 30.5 24.0 33.8 28.5 110 APPENDIX VIII (cont'd) Eosinophils g 4 0 0 k Q Q l Q Q k Q Q and Monocytes Dog 1 1 0 0 1 10 1 16 2 2 6 1 10 0 0 2 0 3 3 0 1 0 1 0 0 2 1 4 _j8_ _0_ 0_ _1_ 3_ _1_ 0_ 4 average 3.8 0.5 2.5 0.8 3.3 1.0 4.5 2.5 APPENDIX IX Blood Counts Data Taken From Dogs* Two Months  From The Beginning Of T r i a l II A. Erythrocyte Counts (mil l i o n s of cells/cmm) sk00 B 4 0 0 s i o o C 4 0 0 Dog 1 6.2 7.9 7.4 6.8 2 6.6 6.6 6.5 6.4 3 6.5 5.7 6.3 5.9 4 7.0 5.3 6.3 5.8 average 6.6 6.4 6.6 6.2 B. Leucocyte Counts (cells/cmm) S H 0 0 BH 0 0 s i o o C 4 0 0 Dog 1 15,000 18,900 12,800 15,400 2 10,800 12,400 10,800 11,100 3 13,900 18,100 21,400 11,800 4 18,900 10,700 14,300 12,300 average 14,700 15,000 14,800 12,700 C. Reticulocytes (percent of erythrocytes) S 4 0 0 B H 0 0 s i o o C 4 0 0 Dog 1 1.8 2.5 1.8 1.4 2 2.0 1.4 1.7 1.4 3 1.9 2.9 1.3 0.7 4 1.7 1.7 1.5 0.9 average 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 set out i n Appendix VIII. 112 APPENDIX IX (cont'd) D. D i f f e r e n t i a l Leucocyte Counts (percent of leucocytes) Polymorphs SH00 B400 s i o o C400 Dog 1 66 80 57 62 2 73 73 76 68 3 65 69 70 74 4 73 78 67 67 average 69.3 75.0 67.5 67.8 Lymphocytes S400 B400 s i o o C400 Dog 1 26 20 32 30 2 25 20 23 29 3 33 26 28 20 4 23 21 32 26 average 26.8 21.8 28.8 26.3 Eosinophils S400 B400 clOO nkOO and Monocytes Dog 1 8 0 0 0 11 0 7 1 2 1 1 7 0 1 0 0 3 3 1 1 5 0 1 0 6 0 4 4 0 0 1 1 0 5 1 average 3.5 0.5 3.0 0.3 3.5 0 4.5 1.3 APPENDIX X D i f f e r e n t i a l Counts* of Femoral Bone Marrow Of Rats i n T r i a l II 1. Myeloblasts Group ;400 >400 .100 '400 Male 1 2 3 4 5 average 0.7 1.0 0.7 0.7 1.3 0.88 0.7 1.0 1.0 1.3 0.80 0.3 0.7 1.0 0.7 0.3 0.60 1.0 0.7 0.7 0.3 0.54 Female 1 2 3 4 5 average 1.3 1.0 1.7 1.0 0.0 1.0 1.3 1.0 1.3 0.7 0.3 0.92 0.7 0.3 0.3 1.0 1.3 0.72 0.0 0.7 0.0 1.0 0.7 0.48 2. Promyelocytes Group .400 ,400 • 100 ^400 Male 1 2 3 4 5 average 2.0 2.7 2.0 1.7 3.7 2.42 2.7 2.7 1.0 1.7 3.0 2.22 1.3 2.3 2.0 2.0 1.7 1.86 3.0 3.7 4.3 1.7 1.0 2.74 Female 1 2 3 4 5 average 2.54 2.3 2.7 2.0 1.3 1.0 1.86 2.0 1.7 2.0 2.3 2.3 2.06 2.0 0.7 1.3 1.0 1.0 These values are a l l percentages of 300 d i f f e r e n t i a t e d c e l l s . APPENDIX X (cont'd) 3. Myelocytes 114 Group ;400 ,400 -100 ^400 Male 1 2 3 4 5 average 2,3 2.7 2.3 2.3 4.0 2.72 3.0 5.0 2.3 4.3 3.7 3.66 3.7 3.3 2.7 3.7 3.3 3.34 4.0 5.7 5.3 4.0 2.3 4.26 Female 1 2 3 4 5 average 4.3 3.0 5.0 3.0 2.7 3.60 3.3 5.0 4.0 3.0 3.3 3.72 3.3 3.3 4.3 4.3 2.7 3.58 4.3 3.0 2.3 2.0 3.0 2.92 4. Metamyelocytes and "Ring C e l l s " Group :400 .400 • 100 ^400 Male 1 2 3 4 _5 average 9.7 10.0 11.0 9.7 11.0 10.28 14.3 13.3 11.7 13.0 13.3 13.12 12.3 12.7 11.3 12.0 11.0 11.0 12.0 16.0 13o7 9.7 10.3 12.34 Female 1 2 3 4 5 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 average 11.74 12.58 12.54 12.58 APPENDIX X (cont'd) 5. Mature Granulocytes 115 Group ;400 .400 .100 ^400 Male 1 2 3 4 5 average 29.7 30.7 23.3 24.0 22.7 26.08 25.3 20.7 27.3 25.0 24.7 24.60 35.0 32.7 30.7 28.0 28.0 30.88 30.3 33.3 28.7 35.0 37.0 32.8 Female 1 2 3 4 5 average 28.0 27.7 20.3 26.0 26.7 25.74 19.0 21.3 19.3 29.3 31.0 23.98 28.0 32.0 28.3 28.7 30.7 29.54 37.3 35.3 36.0 35.0 35.7 35.14 6. Lymphocyte-Like C e l l s Group .400 ,400 ,100 ^400 Male 1 2 3 4 5 average 4.0 8.0 5.3 9.0 9.0 7.06 7.7 8.0 9.0 7.3 9.7 8.34 7.7 7.0 9.7 10.7 12.0 9.42 10.7 7.3 9.3 7.3 9.7 8.86 Female 1 2 3 4 5 10.0 8.7 9.3 7.7 7.7 7.3 9.0 9.0 8.7 10.7 13.3 9.7 11.7 8.7 7.0 8.7 7.0 10.7 7.7 8.0 average 8.68 8.94 10.08 8.42 APPENDIX X (cont'd) 7. Nucleated Red Blood C e l l s 116 Group .400 ,400 • 100 >H00 Male 1 2 3 4 5 average 49.0 41.7 50.7 48.3 43.3 46.60 44.3 46.0 43.0 43.3 40.7 43.46 37.0 38.3 37.3 38.7 37.7 37.80 36.0 31.0 35.0 37.7 35.0 34.94 Female 1 2 3 4 5 average 38.3 43.7 42.7 44.0 43.0 42.34 47.7 41.3 43.0 42.3 37.7 42.40 34.7 36.3 37.0 37.3 39.3 36.92 35.0 34.0 34.0 36.7 36.0 34.14 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 3.28 2.0 3.3 5.6 4.3 3.7 3.78 2.0 3.6 4.7 4.4 5.9 4.12 3.3 2.0 3.0 4.4 5.0 3.54 Female 1 2 3 4 5 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 average 4.34 5.52 4.55 3.72 APPENDIX XI D i f f e r e n t i a l Counts* of Femoral Bone Marrow  Of Dogs i n T r i a l II 1. Myeloblasts Group S 4 0 0 B 4 0 0 S 1 0 0 C^ 0 0 Dog.No. 1 1.7 0.7 1.3 0.7 2 0.7 0.7 1.0 1.0 3 0.7 1.7 0.7 1.3 4 0.7 1.0 1.3 0.7 average 0.95 1.03 1.08 0.93 2. Promyelocytes Group S k 0 ° B 4 0 0 S 1 0 0 C 4 0 0 Dog No. 1 2.7 1.7 3.3 2.3 2 1.3 2.7 2.0 2.3 3 1.0 2.7 1.7 1.7 4 3.0 1.7 1.7 2.3 average 2.00 2.20 2.18 2.15 3. Myelocytes Group S k 0 ° B 4 0 0 S 1 0 0 C 4 0 0 Dog No. 1 5.0 2.3 5.6 6.6 2 4.0 4.7 3.7 3.3 3 3.0 4.7 3.7 3.0 4 5.7 2.3 3.0 4.3 4.40 3.50 4.00 3.55 * These values are a l l percentages of 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 explained i n Appendix VIII. 118 APPENDIX XI (cont'd) 4. Metamyelocytes and Stem C e l l s Group S 4 0 0 B 4 0 0 S 1 0 0 C k 0 ° Dog. No. 1 23.6 25.0 22.3 25.0 2 23.7 22.3 24.0 22.6 3 22.3 20.3 23.7 22.6 4 22.0 25.3 23.0 24.0 average 22.90 23.23 23.25 23.55 5. Mature Granulocytes Group S 4 0 0 B^ 0 0 S 1 0 0 C 4 0 0 Dog.No. 1 18.0 20.6 19.0 22.3 2 18.6 20.3 19.6 18.0 3 20.7 18.6 19.6 21.4 4 16.4 17.0 19.0 21.0 average 18.43 19.13 19.30 20.68 6. Proerythroblasts Group S k 0 ° B 4 0 0 S 1 0 0 C 4 0 0 Dog No. 1 3.3 2.3 3.3 2.3 2 2.7 3.7 2.0 2.7 3 2.7 3.7 2.3 2.3 4 3.7 3.3 3.0 2.3 3.10 3.25 2.65 2.4 119 APPENDIX XI (cont'd) 7. Normoblasts Group S 4 0 0 B u 0 ° S 1 0 0 C 1* 0 0 Dog No. 1 37.3 36.3 36.3 34.3 2 36.3 36.3 36.0 36.3 3 36.0 38.3 35.7 35.3 4 36.7 35.7 35.7 34.3 average 36.58 36.65 35.93 35.05 8. Lymphocytes Group S^ 0 0 B 4 0 0 S 1 0 0 C" 0 0 Dog No. 1 7.7 9.3 7.7 8.3 2 9.7 8.0 9.7 10.7 3 10.3 8.3 10.3 9.3 4 9.0 10.0 9.3 10.0 average 9.18 8.90 9.25 9.58 9. Monocytes Group ShQ0 B 4 0 0 S 1 0 0 C k 0 ° Dog No. 1 0 0.7 0 0.7 2 0.7 1.0 0.7 0.7 3 1.0 0.3 1.0 1.3 4 0.7 1.3 1.3 0.3 average 0.60 0.83 0.75 0.75 120 APPENDIX XI (cont'd) 10. Megakaryocytes (roup S k 0 ° B h Q 0 S 1 0 0 C 4 0 0 Dog No. 1 0.7 0.7 0.3 0.3 2 1.3 0.3 0 1.3 3 1.3 0.7 1.0 1.0 4 0.7 1.3 1.3 0.3 average 1.25 0.85 0.58 0.83 11. Plasma C e l l s Group S 4 0 0 B 4 0 0 S 1 0 0 C 4 0 0 Dog No. 1 0 0.3 1.0 0 2 1.0 0 1.3 1.0 3 1.0 0.7 1.3 0.7 4 0.3 0.7 0.7 0.3 average 0.58 0.43 1.08 0.50 121 APPENDIX XII  Mean Weights* In Grams For T r i a l I I I Rats Day Control B - 0.5 B - 5 B - 5 0 I n i t i a l 57.5 57.3 57.6 56.3 1 60.5 60.7 61.4 59.5 3 69.5 68.4 69.8 68.3 5 76.6 75.6 78.0 76.6 8 90.5 89.9 91.3 90.1 10 101.1 100.0 102.4 100.5 12 110.1 108.4 111.7 109.5 15 123.0 121.1 122.3 121.4 17 130.4 128.7 129.0 127.7 19 135.8 134.2 134.6 132.9 24 144.2 143.2 143.7 142.4 26 148.7 148.3 148.1 147.4 29 158.3 159.1 158.1 157.4 31 163.9 164.1 163.7 163.1 33 169.3 170.4 169.5 167.8 * mean of ten rats 122 APPENDIX XIII Blood Counts Of Rats In T r i a l I I I A. Erythrocyte counts (mil l i o n s per cmm) Rat Control B - 0 . 5 B - 5 B - 5 0 1 8.04 7.72 7.61 7.91 2 9.40 7.00 7.72 8.11 3 8.34 7.70 8.02 8.19 4 6.80 5.80 7.97 7.13 5 7.09 6.64 6.50 10.05 6 7.44 7.31 8.03 6.30 7 6.50 7.03 6.71 7.18 8 7.01 6.74 7.06 8.16 9 7.93 8.76 7.97 6.20 10 7.43 7.91 6.77 6.72 Mean 7.60 7.26 7.44 7.60 S.D. 0.81 0.77 0.59 1.09 B. Leucocytes ( c e l l s per cmm) Rat Control B - 0 . 5 B - 5 B - 5 0 1 12,000 7,900 13,200 11,700 2 10,400 16,300 12,900 8,900 3 9,700 11,500 9,700 14,100 4 9,500 12,100 12,800 15,700 5 8,700 13,100 10,700 11,100 6 12,000 14,300 11,700 12,300 7 14,600 14,100 13,200 8,400 8 9,600 10,300 15,700 16,400 9 14,300 17,600 14,100 13,000 10 11,300 11,000 9,700 12,700 Mean 11,200 12,800 12,400 12,400 S.D. 1,920 2,670 1,830 2,460 123 APPENDIX XIV Percent D i f f e r e n t i a l of Leucocytes i n Blood  Smears From Group I I I Rats 1. Control Polymorphs Eosinophils Lymphocytes Monocytes Rat No. 1 16 83 1 2 11 89 3 15 1 82 2 4 23 3 73 1 5 22 77 1 6 8 92 7 7 92 1 8 19 75 6 9 9 11 72 7 10 14 83 3 average 14.4 1.5 81.6 2.2 2. B - 0.5 Polymorphs Eosinophils Lymphocytes Monocytes Rat No. 11 12 1 87 12 12 88 1 13 24 1 74 3 14 29 3 69 3 15 21 1 77 1 16 21 76 17 12 84 4 18 4 92 4 19 14 84 2 20 22 75 3 average 17.1 0.6 81.0 2.1 APPENDIX XIV (cont'd) 3. B - 5 Polymorphs Eosinophils Lymphocytes Monocytes Rat No. 21 23 2 75 1 22 9 91 23 15 3 80 2 24 5 1 94 1 25 24 77 26 11 83 6 27 7 90 3 28 1 96 3 29 23 75 4 30 7 93 average 12.5 0.6 85.4 2 4. B - 50 Polymorphs Eosinophils Lymphocytes Monocytes Rat No. 31 16 85 1 32 9 90 1 33 16 3 81 34 21 77 2 35 10 87 1 36 13 86 2 37 23 68 6 38 9 8 78 5 39 8 90 2 40 27 70 2 average 15.2 1.1 81.2 2.2 125 APPENDIX XV Percentages of C e l l s i n the A r b i t r a r y Categories* of C e l l s i n the Femur Bone Marrow Control Group Category 1 2 3 4 5 6 7 8 Rat No. 1 4.3 4.9 20.3 5.9 22.0 31.8 9.8 0.33 2 5.3 10.0 13.7 11.0 15.7 39.7 4.7 -3 4.8 6.9 13.4 10.7 18.3 34.8 10.3 0.34 4 3.5 3.8 17.6 8.2 15.4 37.0 13.5 0.31 5 6.3 3.7 11.6 7.0 26.2 28.2 17.3 -average 4.84 5.86 15.32 8.56 19.52 34.30 11.12 0.20 B - 0.5 Category 1 2 3 4 5 6 7 8 Rat No. -11 6.6 3.0 15.0 12.3 16.9 34.9 9.6 0.66 12 5.9 5.3 19.8 6.3 10.2 36.3 14.5 0.33 13 3.7 4.9 10.4 12.3 17.2 35.6 8.9 0.92 14 5.3 2.3 13.0 17.3 19.3 31.9 10.6 0.33 15 3.3 7.7 11.3 7.0 18.3 32.3 19.7 0.70 16 6.5 5.3 8.5 6.3 10.3 43.3 18.7 0.33 17 4.1 4.8 12.4 9.9 18.2 35.0 15.6 -18 4.1 3.2 11.7 13.6 20.6 35.7 9.2 1.30 19 7.2 6.5 16.6 17.8 18.5 24.0 7.6 1.10 20 3.5 0.9 13.5 9.6 22.0 37.7 11.6 0.63 average 5.02 4.39 13.22 11.25 17.15 34.67 12.60 0.63 ^Categories: 1. Eosinophils - 10 - 15 microns, large red granules c l o s e l y packed i n cytoplasm, nucleus; blue, round, kidney or lobed. 2. Doughnut c e l l s - ( i ) 15 - 20 microns, blue with red hole. ( i i ) 30 microns, large blue r i n g with l i g h t coloured nucleus. 3. Myelocytes - (i) 25 microns, light-coloured large nucleus (takes up most of the c e l l ) and a l i g h t blue cytoplasm (ring e f fect) ( i i ) 15 microns, purple coloured mottled c e l l 126 APPENDIX XV (cont'd) B - 5 Category 1 2 3 4 5 6 7 8 Rat No. 21 8.7 3.1 16.1 14.6 19.6 29.2 7.8 22 3.6 5.3 17.2 12.5 16.5 30.4 11.9 1.7 23 8.0 8.7 6.0 0.7 0.7 62.0 14.0 1.7 24 5.6 3.8 17.9 11.9 19.7 28.8 11.0 25 6.6 7.0 16.2 15.9 23.8 21.5 7.9 0.33 26 4.3 5.0 15.6 10.3 23.9 26.2 10.6 0.33 27 3.0 3.3 13.3 5.3 10.3 38.3 24.7 28 7.0 5.4 10.2 7.9 19.4 37.8 11.7 29 4.4 5.9 13.1 8.4 24.4 33.4 10.3 30 3.6 11.0 17.7 3.7 13.0 36.0 14.7 average 5.48 5.85 14.33 9.12 17.13 34.36 12.46 0.41 B - 50 Category 1 2 3 4 5 6 7 8 Rat No. 31 4.3 4.3 14.1 5.3 23.7 40.8 6.9 0.33 32 3.5 5.4 19.6 16.7 14.5 26.8 12.3 0.63 33 1.7 5.0 13.3 7.0 9.3 41.0 12.3 0.33 34 1.6 5.7 14.1 6.1 19.8 35.5 16.9 0.32 35 4.8 2.3 18.9 8.3 16.3 33.3 14.7 36 3.4 5.6 20.9 8.4 14.3 28.3 15.3 0.31 37 4.5 6.1 13.7 8.9 22.3 32.8 9.6 1.6 38 5.7 5.7 12.3 4.3 10.6 50.3 11.0 0.33 39 4.7 8.7 15.3 9.7 9.3 35.7 17.0 40 2.4 3.6 22.5 11.1 9.3 37.1 14.1 average 3.66 5.24 16.47 8.58 14.94 36.16 13.01 0.39 Categories (cont'd) 4. Erythroblasts - 7 - 10 microns, l i g h t cytoplasm with dark blue nucleus. 5. Mature granulocytes - 10 - 15 microns, pink granular cytoplasm with nucleus varying from band to kidney. 6. Lymphocyte-like c e l l s - 10 microns, small clear-coloured c e l l s with no nucleus. 7. Nucleated red blood c e l l s - 8 - 12 microns, small clear-coloured c e l l s with a strand i n the cytoplasm. 8. Megakaryocytes. 127 APPENDIX XVI Standard Method Used for Counting and D i f f e r e n t i a t i n g C e l l s 1. Red blood c e l l counts Erythrocytes were counted using Hayem's dil u e n t , Thoma red c e l l d i l u t i n g pipettes and an improved Neubauer counting chamber as discussed i n Wintrobe (107). 2. White blood c e l l counts The procedure as outlined by Wintrobe was followed. The leucocytes were counted using a d i l u t i n g pipette marked 0.5, 1 and 11 and giving a d i l u t i o n of 1 i n 20, an improved Neubauer counting chamber and the following d i l u e n t : g l a c i a l a c e t i c acid 2 ml d i s t i l l e d water 98 ml a few drops of methylene blue 3. D i f f e r e n t i a l of leucocytes Two blood smears were made from each animal. The s l i d e s were stained with Wright s t a i n according to Wintrobe and 100 white blood c e l l s were counted on each s l i d e . 4. Reticulocyte counts Blood films were made by mixing (50:50) blood with the following s o l u t i o n and then l e t stand for ten minutes before making smears on glass s l i d e s . 100 ml 0.85% s a l i n e 0.4 gm sodium c i t r a t e 1 gm 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 c e l l s . APPENDIX XVI (cont'd) D i f f e r e n t i a l of bone marrow c e l l s Bone marrow was removed from femurs when the animals were k i l l e d . An equal quantity of homoserum was mixed with the marrow and s l i d e s were made of the mixture. The s l i d e s were then stained with Wright s t a i n and a t o t a l of 300 c e l l s d i f f e r e n t i a t e d . 

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