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Excess vitamin A as a factor in alimentary cholesterolemia, blood coagulation and clot lysis in the chicken Woodward, William Douglas Hiram 1973

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EXCESS VITAMIN A AS A FACTOR IN ALIMENTARY CHOLESTEROLEMIA, BLOOD COAGULATION AND CLOT LYSIS IN THE CHICKEN BY BILL' WOODWARD A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of POULTRY SCIENCE We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1973 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the University of B r i t i s h Columbia, I agree that the l i b r a r y s h a l l make i t f r e e l y available for reference and study. I further agree that permission f o r extensive copying of this thesis f o r scholarly purposes may be granted by the Head of my Department or by h i s representatives. I t i s understood that copying or publication of this thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia Vancouver 8, Canada - i i -ABSTRACT The physiological effects of excess vitamin A are frequently studied i n the investigation of the systemic function of the vitamin. The l i v e r vitamin A storage ef-f i c i e n c y of dietary excesses of vitamin A and the eff e c t of excesses of the vitamin on alimentary cholesterolemia, the one-stage prothrombin time and the one-stage euglobulin l y s i s time were investigated i n several genetically d i s t i n c t populations of chickens. Birds of a New Hampshire (NH) l i n e were found to store vitamin A i n the l i v e r from the diet more e f f i c i e n t l y than birds of three White Leghorn (WL) stra i n s (D,K and B) and of one b r o i l e r s t r a i n . An excess of dietary vitamin A reduced alimen-tary cholesterolemia i n the NH chicks but increased alimen-tary cholesterolemia i n the D chicks. There was no eff e c t of hypervitaminosis A on the blood cholesterol concentration of either the D or the NH chicks receiving a low-cholesterol diet. No carryover effect of vitamin A upon removal of the excessive quantity of the vitamin from the d i e t , could be demonstrated on alimentary cholesterolemia i n either the D or the NH chicks. The e f f i c i e n c y of the i n t e s t i n a l absorption of - i i i -cholesterol was increased i n both the NH and the D chicks by an excess of vitamin A. The live r - t o - b l o o d p a r t i t i o n r a t i o of cholesterol was increased by excess vitamin A ad-ministration i n the NH chicks, but was decreased by excess vitamin A i n the D chicks. The one-stage prothrombin time of D,K,B, and NH chicks was unaffected by the dietary administration of large excesses of the vitamin from day-old to three weeks of age. Chicks of a Black Australorp (A) l i n e , however, responded to large excesses of the vitamin i n the d i e t with an increase i n one-stage prothrombin time. A single intramuscular i n j e c -tion of menadione (5 mg/kg body weight) brought the pro-longed prothrombin times of the A chicks to normal within twenty hours. The one-stage euglobulin l y s i s time of NH,D and A chicks was decreased by the i n c l u s i o n of large excesses of vitamin A i n the die t of the birds from day-old to six weeks of age. Intramuscular administration of menadione (5 mg/kg body weight) was without ef f e c t on the shortened l y s i s times within twenty-four hours. Removal of the ex-cessive quantities of vitamin A from the d i e t for s i x weeks f a i l e d to affect the shortened c l o t l y s i s times of the A hypervitaminotic chicks. vitamin K deficiency induced i n a Shaver s t r a i n of WL chicks by feeding the birds a K-defi c i e n t d i e t , had no effect on the euglobulin l y s i s time. - i v -The res u l t s of the present experiments extend knowledge of the effects of hypervitaminosis A on the i n -duction of alimentary cholesterolemia and on the blood coag-ulation mechanism i n the chicken. - V -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS LIST OF FIGURES v i i LIST OF TABLES v i i i ACKNOWLEDGEMENTS x i INTRODUCTION • x i i i REVIEW OF LITERATURE GENERAL 1 VITAMIN A AND THE METABOLISM OF CHOLESTEROL.. 9 VITAMIN A, BLOOD COAGULATION AND FIBRINOLYSIS 24 VITAMIN A AND ALIMENTARY CHOLESTEROLEMIA IN CHICKENS OF DIFFERENT GENETIC BACKGROUNDS Experiment 1: EFFICIENCY OF LIVER VITAMIN A STORAGE IN SEVERAL GENETICALLY DIFFERENT POP-ULATIONS OF BIRDS Materials and Methods 32 Results 39 Discussion 47 Experiment 2: THE EFFECT OF A MODERATE DIETARY EXCESS OF VITAMIN A ON ALIMENTARY CHOLESTEROL-EMIA Materials and Methods 52 Results 53 Discussion 59 Experiment 3: THE EFFECT ON ALIMENTARY CHOL-ESTEROLEMIA '• OF A MODERATE EXCESS OF DIETARY VITAMIN A FED PRIOR TO BUT NOT SIMULTANEOUSLY WITH CHOLESTEROL Materials and Methods 61 Results 62 Discussion 65 EXPERIMENTAL PART I - v i -Experiment 4: EFFECT OF EXCESS DIETARY AND OF EXCESS PARENTERALLY-ADMINISTERED VITAMIN A ON THE INTESTINAL ABSORPTION AND LIVER RE-TENTION OF A SINGLE ORAL DOSE OF LABELED CHOLESTEROL Materials and Methods 67 Results 72 Discussion.. 79 PART II THE EFFECT OF EXCESSES OF VITAMIN A ON THE ONE-STAGE PROTHROMBIN TIME AND THE ONE-STAGE EUGLOBULIN LYSIS TIME OF CHICKENS WITH DIF-FERENT GENETIC BACKGROUNDS Experiment 5: EFFECT OF A LARGE DIETARY EXCESS OF VITAMIN A ON THE ONE-STAGE PRO-THROMBIN TIMES OF THREE STRAINS OF WHITE LEGHORN CHICKENS Materials and Methods 89 Results 92 Discussion 92 Experiment 6: EFFECT OF MODERATE AND LARGE EXCESSES OF VITAMIN A INTAKE ON THE ONE-STAGE PROTHROMBIN TIMES AND THE EUGLOBULIN LYSIS TIMES OF THREE BREEDS OF CHICKENS Materials and Methods 96 Results 104 Discussion 122 Experiment 7: EFFECT OF VITAMIN K DEFICIENCY ON THE EUGLOBULIN LYSIS TIME Materials and Methods 133 Results 135 Discussion 135 SUMMARY AND CONCLUSIONS 142 BIBLIOGRAPHY 145 APPENDIX 166 - v i i -LIST OF FIGURES Figure Page 1 Percent l y s i s vs. incubation time at s i x weeks of age 110 2 Percent l y s i s vs. incubation time a f t e r i n -jections with menadione or soybean o i l 113 3 Percent l y s i s vs. incubation time.three breeds combined and i n j e c t i o n treatments of soybean o i l and menadione combined within " diets 117 4 L i v e r vitamin A concentration vs. dietary vitamin A l e v e l at seven weeks of age 121 5 Percent l y s i s vs. incubation time, J M H chicks and D chicks combined a f t e r six weeks being fed the b r o i l e r grower diet 123 6 Percent l y s i s vs. incubation time 138 7 Percent l y s i s vs. incubation time 139 - v i i i -LIST OF TABLES Table Page 1 Basal diet used i n experiments 1,2,3, and 4 34 2a Breeder diet 1 35 2b Breeder diet 2 36 2c Breeder diet 3 37 3 Average three-week body weights ( t standard deviation) and average feed consumption during the eighty-nine-hour test period .... 40 4a Average wet l i v e r weights (1 standard dev-iation) and the wet l i v e r weights as a per-cent of body weight ( t standard deviation) . 42 4b Average dry l i v e r weights as a percent of body weight ( 1 standard deviation) 43 5 Average l i v e r concentrations of vitamin A ( t standard deviation) and percentage of ingested vitamin A stored i n the livens of the birds receiving 22,000 I U and 100,000 IU of vitamin A per kilogram of diet 44 6 Average body weights ( t standard deviation), wet and dry l i v e r weights {t standard dev-ia t i o n ) and l i v e r wet weigaos as a percent-age of body weight ( 1 standard deviation) f o r the day-old NH and D chicks 45 7 Average l i v e r vitamin A concentration ( i standard deviation) and average l i v e r v i t a -min A content per b i r d and per gram of body weight ( t standard deviation) f o r the day-o l d D and NH chicks , 46 8 Average eight-week body weights ( t standard deviation) and average feed consumption during the three-day t e s t period 55 9 Average wet and dry l i v e r weights {- stand-ard deviation) and the wet l i v e r weights as a percent of body weight ( 4 standard devia-tion) 56 - i s -Tab! e Page 10 Average l i v e r c o n c e n t r a t i o n L O f vitamin A (1 standard deviation) and percentage of ingested vitamin A stored i n the l i v e r s of the birds receiving 22,000 IU of vitamin A per kilogram of diet 57 11 Average plasma cholesterol concentrations (1 standard deviation) before and aft e r feed-ing 0.5$ cholesterol f o r three days with di f f e r e n t l e v e l s of vitamin A and average increase i n plasma cholesterol concentra-tion (t standard deviation) r e s u l t i n g from the ingestion of the cholesterol diets 58 12 Average four-week body weights (t standard deviation) and average feed consumption dur-ing the three-day test period 63 13 Average plasma cholesterol concentrations (1 standard deviation) before and a f t e r feed-ing 0.5% cholesterol for three days with d i f -ferent l e v e l s of vitamin A 64 14 Wet and dry l i v e r weights as a percent of body weight ( i standard deviation) 73 15 Percent absorption of single o r a l dose of cholesterol (± standard deviation) and per-cent of absorbed cholesterol found i n hepatic pool (1 standard deviation) 74 16 Percent ( i standard deviation) of intravenously injected 4-14c-cholesterol found i n hepatic pool and b i l i a r y excretion of intravenously-injected 4 - 1 4 C - c h o l e s t e r o l as a percent (1 standard deviation) of ± 4 C l a b e l found i n hepatic pool within the f i r s t eighteen hours a f t e r i n j e c t i o n 75 17 Recovery of r a d i o a c t i v i t y from ce l l u l o s e test samples 78 18 Basal diet used i n experiments 5 and 6 90 19 Average three-week body weights ( t standard deviation) 93 20 Average one-stage prothrombin times ( t standard deviation) at three weeks of age ,, 94 X -Table Page 21 Guaranteed analysis of the b r o i l e r grower diet fed the NH and D chicks from eight to fourteen weeks of age 102 22 Average body weights ( t standard deviations) at one day, two weeks, f i v e weeks, six weeks and eight weeks of age 105 23 Average one-stage prothrombin times ( i stan-dard deviation) at three weeks of age 106 24 Average one-stage prothrombin times (1 stan-dard deviation) of the D and A chicks at. thirty-one days of age 108 25 Average one-stage prothrombin times (1 stan-dard deviation) of the A chicks at thirty-two days of age showing the effect of i n t r a -muscular menadione i n j e c t i o n on the birds re-ceiving 100,000 IU and 200,000 IU of vitamin A per kilogram of diet 109 26 Plasma fibrinogen concentrations (1 standard deviation) at eight weeks of age . 119 27 L i v e r vitamin A concentrations d standard deviation) at eight weeks of age 120 28 Plasma vitamin A and carotene concentrations (I standard deviation) of the D and NH chicks at fourteen weeks of age a f t e r being fed the b r o i l e r grower diet (table 21) f o r s i x weeks 124 29 Basal (vitamin K-deficient) diet 134 30 Average body weights (1 standard deviation) at two weeks and at four weeks of age 136 31 Average one-stage prothrombin times ( i stan-dard deviation) at two weeks of age 137 - x i -ACKNOWLEDGEMENTS The author g r a t e f u l l y acknowledges the encourage-ment, i n the form of a Postgraduate Scholarship, received from the iMational Research Council of Canada. Tfe§ Scholar-ship supported, i n f u l l , my two years of postgraduate work leading to the degree of Master of Science. I would l i k e to extend a f u l l measure of ap-preciation to the members of my Thesis Committee: Professor B.E. March Department of Poultry Science (chairman) Dr. W.D. K i t t s Chairman, Departments of Animal Science and Poultry Science Dr. D.B. Bragg Department of Poultry Science Dr. CR. Krishnamurti Department of Animal Science Dr. J.E. P h i l l i p s Department of Zoology Professor B.E. March and Dr. J". B i e l y (Research Professor, Department of Poultry Science) must receive special mention i n accord with t h e i r p a r t i c u l a r contribu-t i o n to my academic development. I would also l i k e to thank Dr. CW. Roberts and Dr. R.C Fitzsimmons, both of the Department of Poultry Science, f o r t h e i r i n t e r e s t i n my program and for the stim-u l a t i n g environment which they helped to provide. x i i -L a s t l y , i t i s my pleasure to include a note of appreciation to the technical s t a f f and farm operators f o r services so often and congenially rendered. x i i i INTRODUCTION A clear understanding i s emerging of the broade r aspects of vitamin A metabolism. Such i s not the case re-garding the biochemical function of the vitamin elsewhere than i n the v i s u a l pathway, nor i s i t true of the prophylactic or therapeutic role which may be played by supplements of the vitamin under a number of s t r e s s f u l or pathological condi-tions. The prophylactic value of vitamin A supplements to the development of experimental atherosclerosis Induced i n laboratory animals by the feeding of high-fat, high-choles-t e r o l diets has been examined by a number of investigators. Also, the therapeutic value of the vitamin against athero-s c l e r o s i s i n human subjects has been tested on a number o f occasions. The r e s u l t s of these studies have con f l i c t e d (both wi-thin and among species) i n s o f a r as the apparent a n t i -atherogenic e f f e c t s of dietary vitamin A supplements obtained by a number of authors are counterbalanced by the f a i l u r e of-others to f i n d any effect of the vitamin. Considering the "p o s i t i v e " nature of the contradiction i n the l i t e r a t u r e i t seemed worthwhile to investigate further the question of the eff e c t of dietary vitamin A supplements on the development of atherosclerosis. A study was therefore made of the effect o f vitamin A excesses on physiological parameters strongly i m p l i -cated i n the aetiology of atherosclerosis. The well-established r i s k - f a c t o r f o r atherosclerosis of alimentary cholesterolemia - x i v -(Mann, 1959; Hegsted et a l . , 1965; Pate _et a l . , 1965; Wilson and Lindsey, 1965; Brown, 1969) and the often-considered r i s k -f a c t o r of a blood coagulation or f i b r i n o l y t i c defect (Nitzberg et a l . , 1959; Kudrjashov et a l . , 1961; French, 1966; Farbis-zewski and Worowski, 1968; Pilgeram and Pickart, 1968; Jones, 1970) were selected as topics for investigation. In general, i t appears that dietary administration of members of the vitamin A series w i l l reduce alimentary cholesterolemia i n several species. Experiments i n our own laboratory, however, have sometimes shown a decrease, and on other occasions an increase, i n alimentary cholesterolemia following the in c l u s i o n of non-toxic excesses of vitamin A in. the d i e t . I t seemed of i n t e r e s t to t e s t the hypothesis that the inconsistency of the results obtained i n our laboratory might be related to a genetic difference i n the a b i l i t y of dif-ferent l i n e s of birds to absorb or to. store vitamin A. Further-more, no direct experiment appears to have been carried out testing the effect of excess dietary or parenterally-admini-stered vitamin A on the fate of dietary cholesterol. Consider-ing the i n d i r e c t nature of the evidence that vitamin A reduces the e f f i c i e n c y of cholesterol absorption from the small i n t e s -tine, i t seemed of in t e r e s t to conduct a cholesterol-absorp-tion experiment using the radioactively-labeled compound. Unpublished work i n t h i s laboratory has generally shown no e f f e c t of moderate and very large excesses of dietary vitamin A on the one-stage prothrombin - XV -time or on the incidence of spontaneous hemorrhages i n White Leghorn chickens. In one experiment, however, subcutaneous hemorrhages were seen i n White Leghorn X Black Australorp cockerel chicks fed a d i e t containing 20,000 int e r n a t i o n a l units: CUT) of vitamin A palmitate per pound. In view of the lack of data regarding the r e l a t i o n s h i p of vitamin A and the coagu-l a t i o n mechanism i n chickens, and i n view of the d i f f e r e n t genetic backgrounds of White Leghorn, New Hampshire, and Black Australorp breeds, i t seemed worthwhile to investigate a possible genetic difference i n the response of the coagu-l a t i o n mechanism i n these breeds to moderate and large dietary excesses of vitamin A. Furthermore, since no data concerning vitamin A and f i b r i n o l y s i s i n avian species was found i n the l i t e r a t u r e , i t seemed important to extend the investigation to include the thrombolytic aspect of blood coagulation which i s of such fundamental importance i n other species. - 1 -REVIEW OF LITERATURE GENERAL I t i s now recognized that the term "vitamin A" i s a generic term f o r a group of compounds (found, i n nature, only i n animal tissues) a l l of which have, to varying degrees, the b i o l o g i c a l a c t i v i t y of vitamin A%- alcohol (Dyke, 1965; Olson, 1967). This l a t t e r compound, also call e d a l l - t r a n s r e t i n o l , i s the parent compound of the group from the point of view both of chemical nomenclature (Olson, 1967) and of b i o l o g i c a l a c t i v i t y ( P i t t , 1965). I t s structure i s as shown below 1: . The only change i n the C-l to C-14 portion of the molecule which does not v i r t u a l l y eliminate biopotenoy i s further de-hydrogenation ( P i t t , 1965). The compounds of the vitamin Ag s e r i e s , i n which there i s a 3 - 4 desaturation, are the best-known examples of such a modification and are found mainly i n fresh-water f i s h (Dyke, 1965; Olson, 1967). Changes on C-15 have les s marked e f f e c t on vitamin A a c t i v i t y than those i n the C-l to C-14 region. In general i t seems that the v i t a -min A a c t i v i t y of a given compound may be dependent upon the a b i l i t y of an organism to convert i t to the alcohol form of The Merck Index, P.G. Stecher ed., M. Windholz and D.S. Leaky Assoc. eds., Merck Co. Inc., Rahway N.J., 1968 p l l l l . either vitamin Aj_ or vitamin A 2 ( P i t t , 1965). The outstanding counterexample to the l a t t e r generalization, however, i s r e t i n o i c acid which i s the C-15 carboxylie acid derivative of vitamin A-j_ alcohol. Preformed vitamin A occurs i n the diet mainly as a l l - t r a n s r e t i n y l esters, primarily of palmitic a c i d (Mahadevan and Ganguly, 1961; Plack, 1965; Olson, 1969; Moore, 197.0; P i t t , 1971). Both r e t i n o l and e s t e r f i e d vitamin A appear to reach the small i n t e s t i n a l lumen of monogastrics chemically unchanged (Thompson, 1949), although some cis-trans isomerization may occur i n the stomach as shown by Murray ejt a l . (1959) i n the ra t . In the small i n t e s t i n a l lumen vitamin A esters are hydrolyzed to r e t i n o l and free f a t t y acids (Gray e t - a l . , 1940; Eden and S e l l e r s , 1950; Mahadevan et„al., 1959; Mahadevan et a l . 1963 a,b), a process which may occur either at the surface of the bulk l i p i d phase or i n the m i c e l l a r phase, i t s e l f (Olson, 1968). Retinyl acetate, which i s not a normal dietary c o n s t i t -uent (Olson, 1968), may be absorbed i n t a c t into the mucosa at l e a s t to a certain degree both i n the rat (David and Ganguly, 1967) and i n the chick (Shellenberger _et a l . , 1964). The luminal vitamin A ester hydrolase i s a component of pancreatic juice and i s activated by conjugated b i l e acids (which are also required i n the formation of l i p i d micelles suitable f o r absorption) as shown i n both the rat (Mahadevan e t . a l . , 1961) and the chick (Nir ^ t a l . , 1967). Another hydrolase, probably-located i n the brush border of the i n t e s t i n a l mucosal c e l l s , may also be of s i g n i f i c a n c e as shown by Ganguly (1967) i n - 3 -rats. In v i t r o work suggests that the transfer of r e t i n o l across the mucosal c e l l membrane i n man and the rat i s by an active transport mechanism (Loran and Althausen, 1958; Mahadevan et a l . , 1959; Loran et a l . , 1961; Skala and Hruba, 1964). Newly-absorbed r e t i n o l i n the i n t e s t i n a l mucosa i s e s t e r i f i e d to long-chain fa t t y acids, mainly palmitic acid, and i s passed into the lymphatics i n the chylomicra (Gray et., a l . , 1940; Eden and S e l l e r s , 1950; Mahadevan and Ganguly, 1961; Huang and Goodman, 1965; Goodman et a l . , 1966). The importance of the lymphatic system i n the absorption of v i t a -min A was f i r s t indicated by the study of Drummond e t . a l . (1935) on the lymph f l u i d of a patient with Chylothorax. Con-firmation of the role of the lymphatics has since been obtained i n many species of animals including the rat (Popper and Volk, 1944; Eden and S e l l e r s , 1949; Thompson et_ al_., 1950; Huang and Goodman, 1965) , the guinea pig (Woytkiw and Esselbugh, 1951) , the bovine and the goat (Eden and S e l l e r s , 1948) , and the pig (Thompson et__ a l . , 1949; Thompson et a l . , 1950). In man (Goodman ejb a l . , 1966 a; Blomstrand and Werner, 1967) and the rat (Huang and Goodman, 1965) oyer 80$ of the lymph v i t a -min A i s found i n the chylomicra. S i g n i f i c a n t quantities of stearate, oleate, and l i n o l e a t e esters of r e t i n o l are released into the lymphatics i n man (Goodman et a l . , 1966) and i n the rat (Huang and Goodman, 1965). In the chicken, and presumably i n a l l avian species, the i n t e s t i n a l lymphatic system i s not as well-developed as that of mammals. Kiyasu (1955) found that blood c a p i l l a r i e s occupy almost the entire central core of the v i l l u s , and t h i s author obtained no evidence f o r the existence of a l a c t e a l . Noyan et a l . (1964) showed the portal system to be the main route f o r absorption of o r a l l y administered palmitic acid i n the chicken, and these workers suggested that such might be the case f o r the absorption of a l l l i p i d s i n this species. I t appears that, i n chickens, newly absorbed l i p i d s leave the mucosa as components of very low density lipoproteins (Noyan et >al., 1964), and that chylomicra may not be formed i n th i s species (Husbands, 1970). In the chicken, c i r c u l a t i n g vitamin A esters are associated with the B-lipoproteins (Scott et . a l . , 1969). In the r a t , chylomicra can be taken i n t a c t into the l i v e r (Belfrage et a l . , 1965), perhaps by the hepatocytes, themselves (Higgins and Green, 1966). As shown i n various f i s h , b i r d s , and mammals, vitamin A i n excess of the body's immediate need i s stored mainly i n the l i v e r e s t e r i f i e d to long-chain f a t t y acids, p a r t i c u l a r l y palmitic acid (Bacharach and Smith, 19£8; R e t i , 1935; Mahadevan and Ganguly, 1961; Rao et a l . , 1961; Futterman and Andrews, 1964; Mahadevan et a l . , 1964; Goodman e t _ a l . , 1965; Lawrence et a l . , 1966). In the rat l i v e r , 95% of the t o t a l vitamin A content was shown by Gray et a l . (1940) to be i n the e s t e r i f i e d form. The work of Futterman and Andrews (1964) showed s i g n i f i c a n t concentra-tions of r e t i n y l esters of myristate, pentadecanoate, palmi-toleate, heptadecanoate, stearate, oleate, and l i n o l e a t e i n - 5 -the l i v e r s of trout, frogs, rats, cats, calves, sheep, rabbits and man. In the chicken about 70% of the l i v e r vitamin A esters are of palmitic acid and most of the remaining 30% are of longer-chain f a t t y acids (Mahadevan and Ganguly 1961). There appears to be a steady turnover (hydrolysis and re-e s t e r i f i c a t i o n ) of stored vitamin A esters i n the l i v e r (Law-rence _et ,_al., 1966; Sewell _et _ a l . , 1967; B o l i n g . e i . a l . , 1969), and the enzyme a c t i v i t y responsible both f o r the hydrolysis (Ganguly and Devel, 1953) and f o r the r e - e s t e r i f i c a t i o n appears to be located i n the microsomal f r a c t i o n . Early fluorescence studies (Popper 1940 a, b; Popper and Greenberg, 1941) indicated the Kupffer c e l l s as a ma.jor s i t e of vitamin A storage i n the l i v e r . The concept was subsequently advanced that vitamin A ester i s stored i n the Kupffer c e l l s , and r e t i n o l i n the l i v e r parenchymal c e l l s (Glover and Morton, 1948; Krishnamurthy and Ganguly, 1956). More recent work with rats, however (Linder j t . a l . , 1971; Hori and Kitamura, 1972), has shown that the Kupffer c e l l s normally possess only a small f r a c t i o n of the t o t a l vitamin A of the l i v e r . Sherman (1969) has shown by autoradiography that vitamin A i s distributed d i f f u s e l y through-out the cytoplasm of the l i v e r parenchymal c e l l of the rat. This finding i s consistent with the early flourescence micro-scope work on the human and the rat l i v e r (Popper 1940 a,b, 1941; Popper and Greenberg, 1941), and also with the demonstra-tion of Krishnamurthy e_t a l . (1958) i n rats that l i v e r vitamin A ester i s found i n association with a very low-density l i p o -protein of an u l t r a centrifugation supernatant. A role of the Kupffer c e l l s i n the uptake of vitamin A into the l i v e r i s - 6 -e n t i r e l y possible (Popper and Greenberg, 1941; Krishnamurthy and Ganguly, 1956; Sherman, 1969; Linder et a l . , 1971). Of a l l the tissues i n the body which possess s i g n i f i c a n t concentrations of vitamin A, only the plasma of the post-absorptive animal, and the r e t i n a , contain the major portion of the vitamin i n the non-esterified form (Moore, 1957). At one time or another nearly every major electrophor-e t i c f r a c t i o n of plasma has. been named as the main c a r r i e r of vitamin A alcohol i n the blood (Olson, 1969). Retinol i n the plasma i s associated with e n t i r e l y d i f f e r e n t proteins from those associated with the c i r c u l a t i n g vitamin A esters as shown by Ganguly et a l . (195E) i n the chicken, the cow, and the pig and by Deshmukh et a l . (1964) i n man. Retinol i n human blood i s bound to a s p e c i f i c protein, r e t i n o l - b i n d e r protein (RBP) , which i s an oC^-globulin with a molecular weight of 21,000 to 22,000.RBP possesses a single binding s i t e for one molecule of r e t i n o l '(Kanai et; a l . , 1968; Smith et a l . , 1970). I t appears, furthermore, that RBP c i r c u l a t e s i n man as a 1:1 protein-protein complex with an acidic protein of molecu-l a r weight about 49,000, and that t h i s complex migrates electro-p h o r e t i c a l l y with the prealbumin f r a c t i o n (Kanai et.,al., 1968). This finding i s consistent with that of Alvsaker e t . a l . (1967) that only the prealbumin fraction, of human plasma possesses the fluorescence c h a r a c t e r i s t i c of r e t i n o l . In the rat (Gar-bers e t . a l . , 1960) and i n the bovine (Erwin et a l , , 1959), i t also seems that the -globulin f r a c t i o n c a r r i e s at l e a s t a large part of t h e ' c i r c u l a t i n g vitamin A, alcohol. The possible - 7 -rol e of EBP i n regulating the a v a i l a b i l i t y of vitamin A to the tissues, and perhaps to s p e c i f i c i n t r a c e l l u l a r l o c i , i s as yet unknown. Kanai e t , a l . (1968) pointed out that RBP serves to s o l u b i l i z e the water-insoluble r e t i n o l molecule and to protect i t from chemical degradation. Dingle et a l . (197-2) obtained evidence that RBP may protect tissues from toxic manifestations of the well-documented surface a c t i v i t y of r e t i n o l . In the rat, the vitamin A alcohol concentration i n the blood i s independent of the l i v e r vitamin A reserves (Ganguly and Krinsky, 1953). Not u n t i l the l i v e r i s v i r t u a l l y depleted of the vitamin does the plasma r e t i n o l concentration drop s i g n i f i c a n t l y , and i t i s only a f t e r the plasma concentra-tion f a l l s that c l i n i c a l signs of vitamin A deficiency develop (High and Wilson, 1956; Dowling and Wald, 1958). In human volunteers, only a few ind i v i d u a l s (presumed to have had lower l i v e r vitamin A reserves than t h e i r colleagues at the start of the tes t period) showed drops i n c i r c u l a t i n g vitamin A concentration, even a f t e r many months of dietary deprivation of the vitamin (Hume and Krebs, 1949). The concentration of r e t i n o l i n the blood appears, then, to be quite s t r i c t l y regulated. Recently Nyquist jet _al. (1971) shed some l i g h t onto the problem of the mechanism of this regulations. These workers obtained evidence that the l i v e r Golgi apparatus might be involved i n r a t s , since the concentration of vitamin A esters i n t h i s s u bcellular f r a c t i o n was p o s i t i v e l y correlated both to the l i v e r reserves of vitamin A and to the rate of - 8 -supply of vitamin A alcohol to the c i r c u l a t i o n . I t appears, i n the rat, that metabolites of r e t i n o l (Wolf et a l . , 1957; Roberts and Deluca, 1967) and of r e t i n o i c acid (Nath and Olson, 1967; Roberts and DeLuca, 1967) are excreted predominantly i n the feces. S i m i l a r l y , the b i l e appears to be a major route f o r the excretion of metabolistes of r e t i n o i c a c i d i n the rabbit (Chupanya and Olson, 1968) , and of both t e t i n o l and r e t i n o i c acid i n the guinea pig and the chick (Zachman et a l . , 1966 b) . Interesting autoradiographic work by Sherman (1969) provides d i r e c t , v i s u a l evidence for the b i l i a r y excretion of vitamin A derivatives by the r a t . The main b i l i a r y metabolites.of r e t i n o l (Lippel and Olson, 1968 a) and of r e t i n o i c a c i d (Dunagin et a l . . 1965, 1966; Lippel and Olson 1968 b) i n the rat and of r e t i n o l and r e t i n o i c acid i n the guinea pig and chick (Zachman jet a l . , 1966 b) are conjugates of g l u -curonic acid, being r e t i n y l B-glycosiduronate (an 0-ether) and r e t i n o l B-glucuronide, respectively. A s i g n i f i c a n t proportion of the f e c a l glucuronic acid conjugates may be hydrolyzed i n the i n t e s t i n a l tract by a b a c t e r i a l B-glucuronidase (Nath and Olson, 1963). An enterohepatic c i r c u l a t i o n of r e t i n o l has been shown i n rats (Zachman jet a l . , 1966 a) and i n sheep (Hume et a l . , 1961). This enterohepatic c i r c u l a t i o n , however, i s u n l i k l e y to he of physiological s i g -n i f i c a n c e i n conserving the vitamin (Nath and Olson, 1967). A s i g n i f i c a n t excretion of r e t i n o l and r e t i n o i c acid metabolites i s achieved i n the urine of the rat (Wolf et a l . , 1957; Nath and Olson, 1967; Roberts and DeLuoa, 1967; Sundaresan and T h e r r i a u l t , 1967), but these metabolites have not been well characterized. Some are glucuronic acid conju-gates (Emerick jet a l . , 1967; Nath and Olson, 1967) and a major fr a c t i o n appear to be products of side-chain decarboxylation (Roberts and DeLuca 1967; Sundaresan and Therriault 1967). Heywood (1967) showed i n several species of cats, and Lawrie et a l . (1941) showed i n man, rabbits, and rats that normal urine contains no Carr-Price reactive compounds. Possibly the dog i s an exception i n t h i s regard (Lawrie jet j a l . , 1941; Worden _et al,, 1955). VITAMIN A AND THE METABOLISM OF CHOLESTEROL Asada (1923) studied vitamin A-deficient young rat s , and Collazo and Bosch (1923) investigated severely vitamin A-deficient guinea pigs and dogs. In both reports the d e f i c i e n t animals showed higher blood concentrations of t o t a l f a t and of t o t a l cholesterol than the vitamin A-normal controls. Similar r e s u l t s were obtained i n rats by Smith (1933) except that i n severe deficiency, the animals had a lower blood cholesterol concentration than normal. R a l l i and Waterhouse (1933) noted that the addition of either cod l i v e r o i l or B-carotene to the diet of vitamin A-deficient female - 1 0 -dogs reduced t h e i r blood cholesterol concentration to the control range of values within one or two weeks. On the other hand, Sure jet j a l . ( 1 9 3 3 ) and Green _et J E L L . ( 1 9 5 5 ) observed no effect on the t o t a l blood cholesterol concentration i n rats at any stage of vitamin A deficiency. A decreased rate of cholesterol synthesis was observed by Migicovsky ( 1 9 5 5 ) i n l i v e r homogenates taken from vitamin A-deficient r a t s . Green jet j a l . ( 1 9 5 5 ) , however, ob-served no e f f e c t of severe vitamin A deficiency on the l i v e r cholesterol concentration of ra t s . The int e r p r e t a t i o n of both publications i s complicated by the i n a n i t i o n of the experi-mental animals. Gloor and Wiss ( 1 9 5 9 a,b), using mevalonate -2 - 1 4 C , found vitamin A deficiency to increase the incor-poration of r a d i o a c t i v i t y into rat l i v e r squalene and ubiqui-nones while decreasing i t s incorporation into cholesterol. The e f f e c t on ubiquinones appeared to be progressive with increasing vitamin A deficiency, whereas that on cholesterol was seen only i n severe deficiency. Diplock j e t . a l . ( 1 9 6 5 ) also reported an increase i n the rate of rat l i v e r ubiquinone synthesis a t t r i b u t a b l e to vitamin A deficiency, and Morton and P h i l l i p s ( 1 9 5 9 ) noted a progressive increase i n rat l i v e r ubiquinone concentration with increasing severity of vitamin A deficiency. Thompson et a l . ( 1 9 6 0 ) obtained an increase i n the concentration of l i v e r ubiquinones i n vitamin A - d e f i -cient rats, and these authors showed that vitamin A acid was as eff ectiveas the alcohol form i n maintaining a normal l i v e r concentration of the ubiquinones. P h i l l i p s ( 1 9 6 2 ) also showed - 11 -a large increase i n the t o t a l l i v e r ubiquinone concentration of vitamin A-deficient rats as compared to controls. Donchenko (1964) showed that the progressive increase i n l i v e r ubiquinone concentration with advancing vitamin A deficiency could be p a r t l y a ttributed to a progressive l i v e r shrinkage, a sugges-tion o r i g i n a l l y made by MOO re and Sharman (1960). In the study of Donchenko (1964) the t o t a l l i v e r ubiquinone content was nonetheless seen to increase throughout the development of vitamin A deficiency. In hindsight, the fact that a com-pound i n rat l i v e r with absorption maximum at 275 nm was f i r s t detected i n vitamin A-deficient l i v e r s (Moore and Rajagopol, 1940) and was seen .to increase i n concentration considerably as vitamin A deficiency became more severe (Lowe et, a l . , 1953) may be attributed both to an increased l i v e r concentration of ubiquinones and to a decreased spectrophotornetric i n t e r -ference from vitamin A (Heaton e t , a l . , 1957). Since adrenalectomy had no e f f e c t on the rat l i v e r ubiquinone concentration, P h i l l i p s and Morton (1959) concluded that the e f f e c t of vitamin A deficiency was not a r e s u l t of adrenal impairment. Gloor and Wiss (1960) showed that the uptake of r a d i o a c t i v i t y into rat l i v e r cholesterol, squalene and ubiquinones was brought to normal within hours of a single dose of vitamin A to, d e f i c i e n t animals. I t was subsequently shown (Gloor et a l . , 1961) that l i v e r homogenates from vitamin A-deficient rats which had received a single large oral dose of the vitamin also were normal within hours of the dosing except that no r a d i o a c t i v i t y could be detected i n the ubiquinone - 12 -f r a c t i o n . Although i t was concluded from these data that vitamin A plays a direot role i n the synthesis of cholesterol from squalene i n rat l i v e r (Wiss and Gloor 1960; Gloor et a l . , 1961). P h i l l i p s (1961), however, suggested that the effect on the incorporation of mevalonate into squalene, cholesterol and ubiquinones (which he further confirmed) was i n d i r e c t since i t was not rev e r s i b l e i n v i t r o by the addition of v i t a -min A to a homogenate of vitamin A-deficient rat l i v e r . Edwin .et .a l . (1962) also suggested that the eff e c t was i n d i r e c t . These authors postulated that the increased Uvea? concentra-tion of vitamin E consequent to the vitamin A deficiency might be more d i r e c t l y responsible f o r any e f f e c t on the rate of ubiquinone or ubichromenol synthesis. Diplock _et._al. (1965) further suggested that the eff e c t of vitamin A deficiency on rat l i v e r isoprenoid metabolism was i n d i r e c t . In contrast to e a r l i e r reports, j o s h i et a l . (1965) 1 observed no e f f e c t of vitamin A deficiency upon the incorpora-t i o n of r a d i o a c t i v i t y into r a t l i v e r s t e r o l s , j o s h i et a l . (1965) and j oshi and Kamasarma (1966) have suggested that the rate of ubiquinone catabolism i n vitamin A-deficient r a t l i v e r i s decreased r e l a t i v e to. that i n the normal l i v e r , and that the rate of synthesis may not be affected. Monitoring the incorporation of mevalonate - 2 - 1 4 ( J into l i v e r ubiqui-nones much e a r l i e r a f t e r dosing with the l a b e l than had pre-viously been done, j o s h i et a l . (1965) observed a much lower incorporation of r a d i o a c t i v i t y into the d e f i c i e n t than into the control l i v e r s four hours a f t e r dosing, and a la r g e r - 13 -amount of incorporated r a d i o a c t i v i t y retained by the d e f i c i e n t l i v e r s than by the controls after a prolonged time i n t e r v a l (seven ty-two hours a f t e r dosing). Joshi and Ramasarma (1966) subsequently showed that the l i v e r s of vitamin A-deficient rats catabolize an oral dose of ubiquinone - l 4 c much more slowly than do the l i v e r s of normal r a t s . I t was further shown that the administration of vitamin A to d e f i c i e n t rats would overcome the apparent catabolic block i n these animals as measured by the l i v e r retention curve f o r ubiquinone - l ^ c (Ramasarma, 1968). Most of the increase i n l i v e r ubiquinone content of vitamin A-deficient rat l i v e r i s att r i b u t a b l e to an increased content i n the microsomal and supernatant f r a c t i o n s (Henning et a l . , 1958; Aiyan and Sreenivasan, 1961). Also, vitamin A-deficient and normal rat l i v e r s show a s i m i l a r sub-c e l l u l a r d i s t r i b u t i o n of newly-synthesized ubiquinones (Joshi and Ramasarma, 1966). I t was therefore speculated (Joshi and Ramasarma, 1966) that impaired membrane turnover i n vitamin A-deficient rat l i v e r r e s u l t s i n an accumulation of membrane-found ubiquinones i n the cytoplasm. The effect of vitamin A deficiency on rat l i v e r ubiquinones seems quite well confirmed. Interestingly, i t seems that no other tissue i n the rat responds s i m i l a r l y (Heaton et a l , , 1957; Green _et a l . , 1960; Joshi and Ramasarma, 1966), although Edwin jet, a l . (1962) obtained an increase i n rat heart ubiquinone concentration i n mild vitamin A deficiency. Also, a species difference i s apparent. The l i v e r , kidney, and i n t e s t i n a l concentrations of ubiquinones i n chickens {Lowe - 14 -_et a l . , 1957) and the l i v e r concentration i n guinea pigs (Morton and P h i l l i p s , 1959; P h i l l i p s , 1961) do not appear to be affected by vitamin A deficiency. There i s some controversy over the guinea pig, however, since Cunningham (1956) reported that the l i v e r , kidney, and i n t e s t i n a l concentrations of ubiquinones i n this species are reduced by vitamin A deficiency. With regard to the work of Cunningham (1956), i t should be pointed out that P h i l l i p s (1961) obtained a s i m i l a r incorporation of mevalonate - 2 - 1 4 C into the l i v e r non-saponifiable material Of both vitamin A-deficient and normal guinea pigs, vitamin A deficiency, however, decreased the percentage of the l a b e l (from acetate - 1 - 1 4 C ) i n the guinea pig l i v e r unsaponi-f i a b l e matter which was located i n the hydrocarbon f r a c t i o n and i n the ubiquinones. ao e f f e c t was seen on the t o t a l l i v e r ubiquinone concentration. Furthermore, P h i l l i p s (1961) showed a decreased incorporation of mevalonate - 2 - l^C into squa-lene and an increased incorporation into s t e r o l s i n the l i v e r s of vitamin A-deficient guinea pigs. The e f f e c t of excesses of vitamin A on the metabolism of isoprene derivatives has also been investigated. Subba Rao and Olson (1967, 1967 a) studied several l e v e l s of vitamin A n u t r i t i o n i n the rat from the d e f i c i e n t state to the hyper-vitaminotic. These authors noted that the l i v e r ubiquinone concentration i n t h e i r rats was inversely proportional to the l i v e r vitamin A content. Also the rate of incorporation of acetate - 1 - benzoate - U - 14Q and mevalonate - 2 14Q into ubiquinone Q,g was inversely proportional to the l i v e r - 15 -vitamin A concentration. F i n a l l y , with mevalonate as the radioactive precursor, the squalene to cholesterol s p e c i f i c a c t i v i t y r a t i o was inversely proportional to the l i v e r vitamin A concentration. I t was postulated that vitamin A may exert control on squalene oxidocyclase or some closely related enzyme. Misra (1966) reported an increase i n the t o t a l chol-esterol content of the l i v e r s , small i n t e s t i n e s , brains, and kidneys of young male rats receiving d a i l y intramuscular i n -jections of 100,000 International Units (IU) of vitamin A alcohol f o r ten days. No effect was seen on the cholesterol content of the heart. A s i m i l a r experiment by Misra (1967), however, showed that the technique used by Misra (1966) to determine the cholesterol concentration of the various tissues was inadequate. Vitamin A produces interference with the color reagent used which was detectable at the wavelength employed f o r the cholesterol measurements. I t was shown (Misra 1967) that large parenteral doses of r e t i n o l have no e f f e c t on the concentration of t o t a l or free cholesterol i n the rat small i n t e s t i n e . Subsequent work c l e a r l y demon-strated an increase i n the concentration of l i v e r t o t a l chol-esterol and cholesterol esters i n young male rats which received daily intramuscular i n j e c t i o n s of 100,000 IU of vitamin A alcohol for thirteen days (Misra, 1968). Singh _et. a l . (1969) also noted an increase i n the l i v e r cholesterol concentration of young male rats fed 30,000 IU of r e t i n o l d a i l y f o r only two days. These authors made the s i g n i f i c a n t observation that the e f f e c t of vitamin A on the l i v e r cholesterol could not be obtained i n adrenalectomized r a t s . Lacord-Bonneau _et a l . (1972) - 16 -f e d male r a t s 60,000 IU o f v i t a m i n A by g a s t r i c tube d a i l y f o r seven weeks. These authors found that the c h o l e s t e r o l content o f the subcutaneous adipose t i s s u e markedly decreased i n the t e s t animals, whereas the c h o l e s t e r o l content o f the small i n t e s t i n e ( i n c o n t r a s t to M i s r a (1967)) and o f the l i v e r was i n c r e a s e d . A r e d i s t r i b u t i o n o f e x i s t i n g p o o l s o f ch o l e s -t e r o l was suggested as p a r t o f the e x p l a n a t i o n . I n t h i s exp-eriment, no e f f e c t o f v i t a m i n A supplementation was seen on the c h o l e s t e r o l content o f the c e n t r a l nervous system, the h e a r t , o r the kidney. In male chickens fed 157,000 to 273,000 IU o f vi t a m i n A p a l m i t a t e p e r k i l o g r a m o f d i e t from one day o f age to 60, 120, o r 170 days o f age, an i n c r e a s e i n both the t o t a l and the e s t e r i f i e d c h o l e s t e r o l c o n c e n t r a t i o n s was seen i n the kidney and the l i v e r ( K o r d y l a s , 1972 a ) . The same f i n d i n g was made i n the l i v e r s o f male r a t s f e d the h i g h v i t a -min A d i e t s f o r 60 and 130 days. A s i g n i f i c a n t but s m a l l e r i n c r e a s e i n l i v e r t o t a l and e s t e r i f i e d c h o l e s t e r o l concentra-t i o n s was a l s o seen i n chickens fed 94,000 to 164,000 ug o f car o t e n o i d s ( n a t u r a l form i n red palm o i l ) per k i l o g r a m o f d i e t f o r 60, 120, and 170 days. The t o t a l v i t a m i n A and t o t a l c h o l e s t e r o l c o n c e n t r a t i o n s o f r a t and chicken l i v e r and o f chicken kidney were p o s i t i v e l y c o r r e l a t e d . Furthermore, t h e r e was a steady i n c r e a s e i n the l i v e r c h o l e s t e r o l c o n c e n t r a t i o n o f the chickens f e d the excesses o f vi t a m i n A between 60 and 170 days o f supplementation. K o r d y l a s (1972 b) confirmed the f i n d i n g s o f K o r d y l a s (1972 a) r e g a r d i n g the e f f e c t o f l a r g e excesses o f v i t a m i n A p a l m i t a t e on chicken l i v e r and kidney t o t a l and e s t e r i f i e d c h o l e s t e r o l c o n c e n t r a t i o n s . The data - 17 -obtained with rats appeared, also, to confirm the e a r l i e r f i n d i n g on rat l i v e r cholesterol, but no s t a t i s t i c a l analysis was presented. The same eff e c t of red palm o i l was also seen on the chicken l i v e r t o t a l and free cholesterol concentration, but only a f t e r 170 days of feeding. Although Kordylas (1972 a, b) concludes that excessive dietary l e v e l s of vitamin A stim-ulate cholesterol synthesis i n the chicken and the r a t , the data presented by t h i s author does not appear to j u s t i f y such a statement. The i n t e r e s t i n g suggestion i s made, however, that the mobilization of free f a t t y acids (presumably from adipose tissue) which i s seen within days i n rats fed large excesses of vitamin A (Singh et . a l . , 1968; Singh e t - a l . , 1969) could provide acetate groups to enhance the synthesis of chol-esterol i n the l i v e r and other tissues. In v i t r o work with rat l i v e r homogenates has led Eskelson and Meets (1972) to conclude that vitamin A i n h i b i t s cholesterol synthesis between acetate and mevalonate ( i n contrast to the i n vivo finding of Subba Rao and Olson (1967)) but promotes cholesterol synthesis beyond mevalonate (i n agreement with Subba Rao and Olson (1967)). These authors also made the vague suggestion that vitamin A may be required for the synthesis of a protein needed i n the metabolism of cholesterol. Perhaps t h i s l a t t e r suggestion can be related to the finding of Weitzel et a l . (1956) i n o l d , atherosclerotic hens and of Kordylas (1972 a) i n male chickens and rats that the increase i n l i v e r cholesterol concentration r e s u l t i n g from the prolonged feeding of excessive quantities of vitamin A i s exclusively i n the cholesterol ester f r a c t i o n . Kordylas (1972 a) postulated that vitamin A might be required - 18 -at some point i n the cholesterol e s t e r i f i c a t i o n process. The c i t e d work c l e a r l y shows that, i r r e s p e c t i v e of the mechanism, large quantities of vitamin A and of the pro-vitamins A i n the diet cause an accumulation of cholesterol i n the l i v e r and perhaps i n other tissues of rats and chickens. There i s much contradiction i n the l i t e r a t u r e , however, as to the e f f e c t of dietary excesses of vitamin A on the vascular pool of cholesterol. Oppenheim and Bruger (1952) found that feeding 50,000 IU of vitamin A palmitate three times weekly, for fourteen weeks had no effect on the t o t a l cholesterol con-centration of the blood (or of the aorta) of adolescent male rabbits. The same r e s u l t was obtained by Kinley and Krause (1959) i n normal human subjects administered 100,000 IU of vitamin A acetate o r a l l y per day f o r four to six months. Patients with a history of coronary artery disease, however, showed a decrease i n serum cholesterol concentration i n res-ponse to the same l e v e l and duration of o r a l vitamin A dosing. This e f f e c t was due s o l e l y to a decrease i n the c i r c u l a t i n g cholesterol ester concentration. Bring et a l . (1965) obtained no effect on the serum or the l i v e r cholesterol concentration of weanling male rats by daily oral administration of 10, 150, or 1000 IU of vitamin A acetate f o r periods of seven, eighteen, and twnety-eight days. Kinley and Krause (1959) c i t e e a r l i e r work of Lasch (1934) and of Wendt (1936) i n which the blood cholesterol concentration of human subjects was apparently increased by large o r a l doses of vitamin A. I t i s pointed out that a f e r r i c - 19 -chloride color reagent was used i n the cholesterol determina-tions. Retinol, however, has been shown by Kinley and Krause (1958) to produce a color with f e r r i c chloride that can produce a spuriously high o p t i c a l density reading at the wavelength used f o r cholesterol measurements, van Bruggen and Strauam-f jord (1948) noted that daily administration of 100,000 I u of vitamin A by capsule (unspecified form) caused an increase i n the free and t o t a l c i r c u l a t i n g cholesterol concentrations of human subjects a f t e r twenty-four and t h i r t y - s i x months. Also, s i x months a f t e r the vitamin A supplementation was term-inated, the blood free and t o t a l cholesterol concentrations of the test subjects had returned to control values. S i g n i -f i c a n t l y , the cholesterol-determination procedure used by van Bruggen and Strauamfjord (1948) was a d i g i t o n i n - p r e c i p i t a t i o n method which was shown by Kinley and Krause (1958) to y i e l d results free of interference by vitamin A i n the l i p i d extract. Relevant, too, i s the work of Bring et a l . , (1955), using f i f -teen-and sixteen-year-old school children as subjects, which showed a p o s i t i v e correlation between the serum t o t a l and free cholesterol concentrations and the estimated dietary content of both vitamin A and B-carotene. S i m i l a r l y Hard and E s s e l -baugh (1960) found a p o s i t i v e correlation between the serum to t a l and free cholesterol concentrations and the estimated dietary vitamin A intake of fifteen-and sixteen-year-old boys. Both Bring et a l . (1955) and Hard and Esselbaugh (1960) used a d i g i t o n i n p r e c i p i t a t i o n method f o r the serum cholesterol measurements. - 20 -Interest has developed regarding the effect of dietary excesses of vitamin A and related compounds on a l i -mentary cholesterolemia. Oppenheim and Bruger (1953) found that feeding 50,000 IU of vitamin A palmitate three times weekly f o r fourteen weeks was without e f f e c t on the cholesterol-emia r e s u l t i n g i n adult male rabbits receiving l g of cholesterol o r a l l y three times each week. The test animals received the vitamin A and cholesterol supplements combined as a single dose. Si m i l a r l y Dua _et a l . (1967) obtained no ef f e c t on the alimen-tary cholesterolemia of l a y i n g hens receiving 2$ of cholesterol i n t h e i r diet by including either 22,000 IU of vitamin A (un-specified form) or 220,000 IU of vitamin A per kilogram i n t h e i r d i e t . Wood and B i e l y (1960 a) , however, showed that lingcod l i v e r o i l and h a l i b u t l i v e r o i l could reduce the chol-esterolemia induced i n male chicks fed a diet containing 1% of cholesterol from one week to three weeks of age. The hypo-cholesterol emic a c t i v i t y was subsequently located i n the unsap-o n i f i a b l e f r a c t i o n of lingcod l i v e r o i l (Wood and B i e l y , 1960 b) • This l a t t e r finding i s i n apparent contradiction with the report of de Groot and Reed (1959) who claimed that the unsaponifiable f r a c t i o n of cod l i v e r o i l was without effect on the cholesterolemia produced i n weanling rats fed a diet containing 1% of cholesterol f o r four weeks. In the experi-ment of de Groot and Reed (1959), whole cod l i v e r o i l was hypocholesterolemic. Wood (1960) separated lingcod l i v e r o i l into three f r a c t i o n s and showed that the portion containing the vitamin A a c t i v i t y also exerted the most strongly hypo-cholesterolemic e f f e c t when fed f o r one or two weeks to week-- 21 -old male chicks receiving lf0 of dietary cholesterol. This author also showed that vitamin A acetate fed f o r one week at l e v e l s ranging from 20,000 IU to 1,300,000 IU per kilogram of diet was e f f e c t i v e i n reducing alimentary cholesterolemia i n chicks receiving 1% of dietary cholesterol. I t was speculated (Wood 1960) that the vitamin A i n lingcod l i v e r o i l may be the primary hypocholesterolemic factor i n the o i l , and that the mechanism of the eff e c t may be a reduction i n the e f f i -ciency of cholesterol absorption from the i n t e s t i n e , perhaps by a decrease i n the rate of cholesterol e s t e r i f l c a t i o n i n the i n t e s t i n a l mucosa. Feeding diets containing 1% of chol-esterol f o r two weeks to chicks beginning at one week of age, Wood and T o p l i f f (1961) showed that the a b i l i t y of li n g c o d l i v e r o i l to reduce the cholesterolemia i n the chicks was pro-portional to the vitamin A a c t i v i t y of the o i l . Also, the hypocholesterolemic e f f e c t of vitamin A acetate-enriched corn o i l was proportional to the degree of enrichment of the o i l and corn o i l , alone, had no eff e c t on alimentary c h o l e s t e r o l -emia. Kuckle and Kruger (1961) fed rabbits d a i l y doses of l g cholesterol, 20,000 IU vitamin A and £0 mg vitamin E f o r twelve weeks. Compared to controls receiving l g of cholesterol, only, the animals receiving the vitamin supplements appeared to show a smaller cholesteroliamic response i n the f i r s t few weeks of the test. The vitamin-supplemented rabbits demonstrated no further increase i n blood cholesterol concentration a f t e r seven weeks whereas the controls increased s t e a d i l y to the end of the twelve-week period. The experiment i s d i f f i c u l t to in t e r p r e t , however, since no s t a t i s t i c a l analysis of the data - 22 -was presented, and no tests with only vitamin E and choles-t e r o l , or vitamin A and cholesterol were run. Wood (1962) published an i n t e r e s t i n g study using week-old male chicks fed a diet containing 1% of cholesterol f o r two weeks. Vitamin A acetate and alcohol and three stereoisomers of vitamin A alde-hyde a l l reduced the elevated l i v e r and blood cholesterol con-centrations r e l a t i v e to controls when fed simultaneously with cholesterol. The results of Lachance and Amen (1972) and of Erdman and Lachance (1973) appear to contradict those of Wood (1962) i n that d a i l y o r a l administration of B-carotene f o r twenty-eight days was shown to reduce the alimentary choles-terolemia of male weanling rats fed diets containing 1% of cholesterol. Using various l e v e l s of B-carotene supplementa-tion up to 1765 ug per day, Lachance and Amen (1972) showed that the hypocholesterolemic e f f e c t of the compound increased with the dosage l e v e l . The same authors obtained no ef f e c t on alimentary cholesterolemia of or a l doses of canthaxanthin (a carotenoid with no recognized vitamin A a c t i v i t y ) reaching the l e v e l of 29.4 mg per day. Neither B-carotene nor canthax-anthin affected the net accumulation of cholesterol i n the l i v e r s of the r a t s . An i n t e r e s t i n g result reported by Erdman and Lachance (1973) i s that oral administration of lycopene (an open-ring carotenoid with no vitamin A growth-promoting a c t i v i t y ) at l e v e l s ranging from 78 ug to 3813 ug per day elevated the alimentary cholesterolemia of male weanling rats receiving a diet containing 1% of cholesterol. Wood (1963) extended h i s studies to an experiment s i m i l a r to that of Wood (1962), and showed that l-apo^S'-carotenal w i l l decrease a l i -- 23 -mentary cholesterolemia i n chicks. The elongated side chain, however, seemed to reduce the hypocholesterolemic a c t i v i t y of the compound r e l a t i v e to vitamin A aldehyde. Beeler et..al. (1962) provided day-old male chicks f o r four to twenty weeks with a diet containing 1% of cholesterol. These authors re-ported that supplementation of the cholesterol diet with 42,000, 83,000, or 167,000 IU of vitamin A (unspecified form) per k i l o -gram f o r the entire feeding period reduced alimentary cholest-erolemia. Since i n c l u s i o n of 0.3% of sodium glycocholate i n the diets eliminated the e f f e c t of vitamin A, i t was concluded that the vitamin decreased the absorptive capacity of the birds for c holesterol. Daily o r a l administrations of 18,300 IU of vitamin A palmitate i n aqueous emulsion for two weeks to seven-week-old p u l l e t s reduced the alimentary cholesterolemia re-s u l t i n g from the the feeding of a diet containing 1% of chol-esterol over the same period, (March and B i e l y , 1963). Also, l i v e r and small i n t e s t i n a l cholesterol accumulation was re-duced r e l a t i v e to controls receiving cholesterol, only. In the same experiment, the hypercholesterolemia induced i n seven-week-old cockerels by d a i l y subcutaneous i n j e c t i o n s of 4 mg d i e t h y l s t i l b e s t r o l was unaffected by twice-daily oral admini-stration of 18,300 IU of vitamin A palmitate i n aqueous emul-sion f o r twelve days. I t was therefore concluded that the effect of dietary vitamin A on alimentary cholesterolemia i s to reduce the e f f i c i e n c y of cholesterol absorption from the i n t e s t i n a l lumen. Furthermore, since the high-cholesterol diet reduced l i v e r vitamin A stores, a mutual interference during absorption was hypothesized to exist between cholesterol - 24 -and vitamin A. Bring et a U (1965) showed that d a i l y oral administration of 150, or 1000 IU of vitamin A acetate decreased the elevation of serum and l i v e r cholesterol concentrations of weanling male rats provided with a diet containing 0.5fc of cholesterol for seven, eighteen, or twenty-eight days. S i m i l a r l y Erdman and Laehance (1973) showed that a daily oral dose of 6617 IU of r e t i n o l reduced the cholesterolemia of male weanling rats r e c e i v i n g 1% of cholesterol i n the i r diets for twenty-eight days. The extremely high l e v e l of 250 m i l l i o n IU of vitamin A acetate per kilogram of diet reduced the chol-esterolemic response of male rabbits during a one-year feeding experiment with a diet containing 0.2% of cholesterol (Bonner et a l . , 1973). VITAMIN A, BLOOD COAGULATION AND FIBRINOLYSIS The f i r s t evidence that vitamin A, i t s e l f , might be toxic was put forward by Takahashi ejt a l . (1925). These workers found that a daily oral dose of t h e i r semi-purified preparation ("Biosterin") providing ten thousand times the minimum vitamin A a c t i v i t y required f o r growth was l e t h a l to rats and mice within two weeks. Autopsy revealed, among many other findings, hemorrhages of the lungs and small i n -testines. Hartwell (1927) noticed that pregnant rats given diets containing large quantities of cod l i v e r o i l often died of profuse uterine hemorrhage at p a r t u r i t i o n . Harris and Moore (1928) reported that young, vitamin B complex-de-f i c i e n t rats of both sexes soon developed diffuse bleeding i n various areas of the skin when fed diets containing the un-- 25 -saponifiable matter of cod l i v e r o i l . Administration of B vitamins f a i l e d to r e l i e v e the condition. Bomskov and Seemann (1933) observed a prolonged c l o t t i n g time i n rabbits receiving large doses of vitamin A. A s i m i l a r observation was reported by Raoul and Ragheb-Hanna (1950) who were able to maintain normal c l o t t i n g times i n A-hypervitaminotic rabbits by the administration of large doses of vitamin D. Vedder and Rosen-berg (1938) observed ocular and nasal hemorrhaging i n >a p r o p o r t i o n o f young male rats receiving f o r one hundred days, 100,000 IU of vitamin A d a i l y i n a f i s h o i l concentrate. Massive oral doses of vitamin D appeared to have some amelio-r a t i n g effect on the production of hemorrhages and other symp-toms of hypervitaminosis A i n t h i s experiment. Rodahl and Moore (1943) presented results of a preliminary nature using adult rats receiving from 6000 IU-to 15,000 IU of vitamin A (in chopped bear l i v e r ) per day f o r periods up to three weeks. The rats developed profuse i n t e r n a l hemorrhages, especially subcutaneously and i n the pericardium. Also, one rat bled to death from a minor cut on i t s paw. These workers c i t e an e a r l i e r observation made i n t h e i r laboratory that the d a i l y administration to adult rats of 30,000 IU of vitamin A as halibut l i v e r o i l caused death a f t e r an unspecified supplemen-tation period from sudden and severe i n t e r n a l hemorrhaging. Light jet a l . (1944) demonstrated a pronounced increase i n the one-stage prothrombin times of fats after ten days of d a i l y oral supplementation with 15,000 IU, 35,000 IU, or 40,000 IU of r e t i n o l or r e t i n y l esters. I t was found that 9000 IU per day supplied as vitamin A alcohol, or 40,000 IU per day supplied 26 -as B-carotene were both without ef f e c t on the coagulation mechanism. Daily o r a l administration of 25 ug of vitamin prevented the increase i n prothrombin times i n the A-hyper-vitaminotic r a t s . In the f i r s t study using a highly p u r i f i e d form of the vitamin, Moore and Wang (1945) stated that the most c h a r a c t e r i s t i c lesions of hypervitaminosis A i n rats are skele t a l fractures and hemorrhages, the l a t t e r being " . . . f r e -quent although i r r e g u l a r i n incidence, d i s t r i b u t i o n , and sever-i t y . " . These authors showed that d a i l y administration of 10,000 ID" of vitamin A as the acetate (or as cod l i v e r o i l or hal i b u t l i v e r o i l ) would cause death of female rats at p a r t u r i t i o n i f the supplementation were begun within the f i r s t ten days of pregnancy. A f a t a l hemorrhage of the spleen was also reported i n a male rat receiving 36,000 IU of vitamin A acetate d a i l y for an unspecified length of time. Walker et a l . (1947) fed young rats of both sexes massive doses of vitamin A as halibut l i v e r o i l f o r ten to eighteen days. A prolonged one-stage prothrombin time and the incidence of numerous in t e r n a l hem-orrhages were reported i n the hypervitaminotic animals. Oral administration of 80 ug of menadione three times weekly pre-vented the increase i n prothrombin times of the A-hypervitamin-o t i c rats and l a r g e l y prevented the occurrence of hemorrhages. Maddook et a l . (1948) reported prolonged one-stage prothrombin times (more than double the control values) and a very high incidence of hemorrhages i n young male rats receiving d a i l y oral doses of 55,000 IU of vitamin A fo r two weeks. In a highly s i g n i f i c a n t series of tes t s , Poole (1958) administered d a i l y o r a l doses of 40,000 IU of vitamin A as the palmitate - 27 -or as a f i s h o i l concentrate to adult rats of both sexes. After eleven to fourteen days, the hypervitaminotic rats showed prolonged one-stage prothrombin times, plasma r e c a l c i f i c a t i o n times, and thrombin generation times. I t was concluded that a genuine hypoprothrombinemia had been induced i n the test ani-mals, a conclusion not possible from the data of e a r l i e r pub-lishe d reports. In the same experiment, the d a i l y subcutaneous i n j e c t i o n of 65 ug of vitamin K]_, or of 25 ug of menadione, concomitantly with the oral doses of vitamin A prevented the development of the coagulation defect. Isra e l s (1962) has extended the findings of Poole (1958) by showing a vitamin -preventable increase i n both the one-stage prothrombin times and the stypven times of rats receiving d a i l y o r a l doses of 70,000 IU of vitamin A f o r twenty-eight days. These re s u l t s were attributed both to a hypoprothrombinemia and to a decrease i n the plasma concentration of Factor VII. Furthermore, a decrease to 20% of normal was seen i n the serum thromboplastin generation time of the A-hypervitaminotic animals. Thompson and P i t t (1960) showed that 4 mg of sodium retinoate admini-stered d a i l y by mouth to young male rats caused bleeding around the eyes and nose a f t e r only four days. Retinol seemed l e s s potent since the f i r s t signs of s i m i l a r bleeding due to da i l y oral dosing with 7.5 mg (25,000IU) of t h i s compound appeared only a f t e r eighteen days. Matschiner and Doisy (1962) con-firmed t h i s f i n d i n g with regard to r e t i n o i c acid. Adult male rats, receiving 2.5 ug of vitamin K i diphosphate per gram of diet, maintained normal stypven times when fed 250 IU of v i t a -min A acetate per gram, but showed an increase i n stypven - 28 -time when fed 250 IU of vitamin A acid per gram. Interestingly , i n a control experiment, the e f f e c t on stypven times of feeding 500 IU of vitamin A acetate per gram of diet seemed to be more severe i n males than i n females. Of p a r t i c u l a r s i g n i f i c a n c e i n the report of Matschiner and Doisy (1962) i s the finding that the i n c l u s i o n of only 0.5 IU or 5 IU of vitamin A acetate per gram of a vitamin K-deficient, irradiated-beef diet caused an increase i n the stypven times of weanling male rats main-tained on the d i e t f o r eight weeks. The e f f e c t seen on the stypven times was progressive from zero to 500 IU of supple-mentation per gram of diet. In t h i s experiment, the animals receiving no dietary vitamin A appeared normal, gained weight s a t i s f a c t o r i l y throughout the feeding period, and showed normal stypven times at the end of the eight-week feeding period. The paper of Matschiner and Doisy (1962) indicates that the e f f e c t of vitamin A on blood coagulation i n the rat i s detectable at physiological l e v e l s of supplementation. The mechanism of the e f f e c t which appears to exist on the blood prothrombin concentration and possibly on the concentration of other c l o t t i n g factors i s unknown. An antagonism between vitamin K i and large oral doses of vitamin A acetate has been observed i n germfree weanling male rats (Wostmann and Knight, 1965). Also, the d a i l y intramuscular i n j e c t i o n of large doses of r e t i n o l into young male rats has been shown to cause the well-known nasal bleeding symptoms of hypervitaminosis A a f t e r ten to thirteen days (Misra 1967, 1968). I t would seem, there-fore, that the e f f e c t i n r a t s i s not on the i n t e s t i n a l synthesis - 29 -or absorption of vitamin K. In animals other than the rat, there i s very l i t t l e data concerning the effect of vitamin A on the c l o t t i n g mech-anism. Biful c o (1953) documented the case of a middle-aged woman who had taken a daily o r a l dose of 100,000 III of vitamin A (unspecified form) fo r more than four years. Immediate severe hemorrhaging resulted from the extraction of her upper teeth and bleeding continued even a f t e r the suturing of her gums. The administration of vitamin K following the operation was credited having stopped the bleeding. O l i v e r (1958) stated that bleeding i s not a prominent manifestation of hypervitamin-osis A i n children. This author cited oases i n which minor epistaxes have been demonstrated concomitantly with A-hyper-vitaminosis but pointed out that a causative r o l e of vitamin A has not established. Di Benedetto (1967) stated that only eleven cases of chronic human adult hypervitaminosi3 A had been reported up to the time of his a r t i c l e . Petechial hemor-rhages of the lower extremeties and moderate prolongation of prothrombin times are occassionally reported but are not promi-nent symptoms, according to t h i s author. Lane (1968) reported a prolonged prothrombin time (presumably a one-stage determina-tion) i n a twenty-four-year-old man who had consumed 50,000 IU to 5,000,000 IU of vitamin A d a i l y f o r f i v e years. Certainly c l i n i c a l case reports are of l i t t l e value i n establishing the mechanism of occurrence, or perhaps even the causative r e l a -tions, of the symptoms of hypervitaminosis A. Mason and Smith (1939) demonstrated extremely I - 30 -prolonged one-stage prothrombin times i n chicks maintained on a vitamin A depletion diet from one day of age to ten and t h i r -teen days of age. Importantly, these workers remarked that no spontaneous hemorrhaging was noticed; i n the d e f i c i e n t chicks. Quick and S t e f a n i n i (1948) observed no e f f e c t on the one-stage prothrombin times of chicks fed 10,000, 20,000, 30,000 or 50,000 IU of vitamin A (unspecified form) from one day of age u n t i l ten days of age. In t h i s experiment, the chicks were given daily o r a l doses of 2.5 ug of vitamin or 1.5 ug of menadione, both dosages being l e s s than the amount believed to be required to maintain the blood prothrombin concentration at a normal l e v e l . Gurcay _et a l . (1950) fed turkey poults 32,000 IU of vitamin A acetate per pound of diet f o r two weeks but reported no overt effects on the blood c l o t t i n g mechanism. S i m i l a r l y Castano jet a l . (1951) observed no gross e f f e c t s such as hemor-rhaging i n chicks fed 24,000 IU of vitamin A acetate f o r eight weeks. Only a decreased food intake and depressed growth rate were seen i n ducks fed up to 600,000 IU of vitamin A d a i l y by dropper (Wolbach and tiegsted, 1953). On the other hand, how-ever, Wolbach and Hegsted (1952) noticed inflammation of the noses, mouth and adjacent skin, and the skin of the feet i n chicks fed 50,000 IU of vitamin A by dropper d a i l y from one to three weeks of age. S i m i l a r l y Pudelkiewicz e_t al.. (1964) noticed that reddening of the eyelids and mouth occurred with-i n the f i r s t ten days of feeding young cockerels approximately 1.7 m i l l i o n IU of vitamin A acetate per kilogram of d i e t . The lesions cleared up spontaneously a f t e r ten days, however, and were not evident at any time with lower l e v e l s of vitamin - 31 -A supplementation. The author has found only two reports r e l a t i n g vitamin A and f i b r i n o l y s i s . Both are concerned with the effect of excesses of the vitamin on human c l o t l y s i s . Loscialpo (1959) administered a single o r a l dose of 30,000 IU of vitamin A (unspecified form) per kilogram of body weight to children aged s i x months to two years. Twanty-four hours l a t e r an increase i n the plasminogen act i v a t o r concentration of the blood was detected. This increase was not seen within eight hours of the vitamin A dosing, and the activator concentration had declined nearly to the pre-test l e v e l a f t e r forty-eight hours. T o r s e l l i n i and Lombardi (1971) provided i n v i t r o con-firmation f o r the results of Loscialpo (1959). The authors showed that the i n c l u s i o n of 60,000 IU of vitamin A (unspeci-f i e d form) per ml of incubation medium decreased the euglobulin l y s i s time of human blood clots i f leukocytes were also included i n the medium. The i n c l u s i o n of p l a t e l e t s i n the high vitamin A medium f a i l e d to shorten the l y s i s times and had no e f f e c t on the action of the leukocytes, i t was suggested that the release of plasminogen-activating enzymes from leukocyte lysosames i s effectedeby vitamin A. - 52 -PART I VITAMIN A AND ALIMENTARY CHOLESTEROLEMIA IN CHICKENS OF DIFFERENT GENETIC BACKGROUNDS Experiment 1: EFFICIENCY OF LIVER VITAMIN A STORAGE IN SEVERAL GENETICALLY DIFFERENT POPULATIONS OF BIRDS Experiments i n t h i s laboratory have shown both a decrease and an increase i n alimentary cholesterolemia of the chicken with the i n c l u s i o n of excess vitamin A i n the die t . The birds used d i f f e r e d i n genetic background among experiments. I t seemed possible that the incon-sistent r e s u l t s obtained i n our laboratory might r e f l e c t a genetic difference i n the a b i l i t y of the chickens used in d i f f e r e n t experiments to absorb vitamin A from the int e s t i n e or to store the vitamin i n the l i v e r . I t was therefore decided to measure the overall a b i l i t y to r e t a i n dietary vitamin A i n the l i v e r i n several g e n e t i c a l l y -d i s t i n c t populations of chickens maintained by thi s lab-oratory. Materials and Methods One strain of New Hampshire (NH) chicks, three strains (B,D, and K) of White Leghorn (WL) chicks, and one s t r a i n of b r o i l e r (Br) chicks were used. Eighty day-old Br cock-erels, 120 NH cockerels, and 120 of each of the three WL - 3 3 -strains were wing-banded, weighed and distributed into groups of twenty. The birds were housed i n elect r i c a l l y - h e a t e d battery brooders and were fed and watered ad libitum . The composition of the diet provided i s shown i n Table 1. The NRG (1971) minimal requirement of vitamin A was supplied and the calculated analysis of the diet i s shown as part of Table 1. The formula of the diet fed the dams of the Br and WL cockerels i s shown i n Table £a and that of the diet fed the NH dams i s shown i n Table 2b (c£ discussion pp 50-51). At two weeks of age the birds were vaccinated against Newcastle Disease. At three weeks of age groups of sixteen Br chicks and twenty-four of each of the dither types of b i r d were fed diets s i m i l a r to the basal, f o r which the composition i s shown i n Table 1, except that the diets contained either 22,000 IU or 100,000 IU of vitamin A per kilogram. The vitamin A, stored under nitrogen at -25°C i n corn o i l with BHA and BHT as a n t i -oxidants, was added to the basal i n s u f f i c i e n t soybean o i l to bring the c a r r i e r to 1% of the diet a s fed. Control birds received the basal diet (Table 1) with soybean o i l added to a t o t a l of 1%. Feed consumption was recorded over an eighty-nine-hour period and the birds were then weighed and k i l l e d by c e r v i c a l d i s l o c a t i o n . The l i v e r s were removed, blotted on paper towels, weighed, and pooled into groups of four i n p l a s t i c bags flushed with general-grade nitrogen. They were immediately frozen at -25°C and held at t h i s temperature u n t i l being freeze-dried. The dry l i v e r s were weighed and were ground - 34 -Table 1. Basal diet used i n experiments 1,8,3, and 4 Component % of diet around wheat (12.5% 65.7 Soybean meal (48.5% 26.0 D i s t i l l e r s ' dried solubles (27.0%) 2.0 Lard 2.0 Limestone 0.8 Bonemeal 3.0 Iodized s a l t (unknown hydration) 0.5 lTJoTo* Micronutrients per kg diet Manganese sulphate 0;.17g Vitamin D3 440 IU Vitamin E 11.0 IU Vitamin A palmitate 1400 IU Riboflavin 4.3 mg Pantothenic acid 14.0 mg F o l i c acid 1.3 mg Vitamin B12 0.01 mg Methionine (99%) 1.30 g Lysine-HCl (98%) 1.86 g Choline chloride (50%) 0.44 g Amprolium (coccidiostat) 0.15 g Calculated Analysis: % Protein = 21.2% Metabolizable energy = 3260 kcal/kg Total vitamin A (IU/kg) =1720 - 35 -Table 2a. Breeder diet 1 Component % of diet Ground wheat (19.5%) 71.0 Pulverized oats (11.0%) 10.0 D i s t i l l e r ' dried solubles (25.0%) 2.0 Dehydrated cereal grass (20.0%) 2.0 Herring meal (73.3%) 8.0 Soybean o i l 0.5 Bonemeal 1.2 Limestone 5.0 Iodized s a l t (unknown hydration) 0.3 100.0 Mi cronu t r i en ts Manganese sulpfeate Vitamin A palmitate Vitamin D3 Riboflavin per kg of diet 0.15 g 4400 IU 750 IU 4.4 mg - 36 -Table 2b. Breeder diet 2 Component % of diet #1 wheat screenings 45.8 Wheat 15.0 Oats 15.0 Soybean meal 13.0 D i s t i l l e r s ' dried solubles 2.0 Dehydrated cereal grass 1.0 Limestone 5.4 Tricalcium phosphate 1.8 Iodized s a l t 0.4 Feeding o i l (300 D, 1500 A) 0.5 Mi cronu t r i ents 0.1 1 0 0 . 0 Mi c ronu t r i en t s Manganese sulphate vitamin A palmitate Vitamin B^g Ribo f l a v i n per kg di e t 0.25 g 2500 IU 0.01 mg 2.5 mg Table 2c. Breeder diet 3 Component <f0 of diet #1 wheat screenings 68.4 Oats 9.9 Fishmeal 3.0 Meatmeal 3.0 Soybean meal 4.5 Dehydrated cereal grass 3.0 Limestone 5.0 Tallow 1.0 diCalcium phosphate 1.0 Iodized s a l t (unknown hydration) 0.3 Fermentation by-products 0.05 OBC premix 0.85 100.0 UBC premix per kg diet Manganese sulphate 0.20 g Zinc sulphate 80.6 mg Vitamin A palmitate 1683 IU Vitamin D 3 1128 IU Vitamin B^g 0.005 mg Riboflavin 4.5 mg Wheat middlings ( f i l l e r ) 7.0 g - 38 -to a powder i n a mortar and pestle under a p l a s t i c hood continually flushed with either G-grade nitrogen or carbon dioxide. The ground l i v e r s were stored under nitrogen at - 2 5 0 c i . awaiting vitamin A determination. For vitamin A analysis, 0.5 gram samples of the dried l i v e r s were extracted with diethyl ether f o r three hours i n a Soxhlet apparatus protected from l i g h t by an aluminum f o i l wrapping. Preliminary work indicated that recovery of vitamin A was not improved by increasing the extraction time. Most of the ether was then removed by f l a s h evaporation, and the extract was taken to dryness at room temperature with general-grade nitrogen. The residue was then saponified over a steam bath f o r twenty minutes i n 20 ml of 2N KOH i n 50$ ethanol. Nitrogen was continually flushed through the reflux apparatus during t h i s step. The nonsaponifiable f r a c t i o n was then extracted with successive^50 ml, 30 ml, and 30 ml portions of p e t r o l -eum ether (b.p. 40°-60°C). Emulsions were broken with solium chloride. The extracts were combined, washed to approximately pH 6, and dried with anhydrous c r y s t a l l i n e sodium s u l f a t e . Most of the solvent was removed by f l a s h -evaporation and the extract was taken to dryness under a stream of general grade nitrogen. The residue was then dissolved i n chloroform. A 1.5 ml sample of the chloro-form solution was pipetted into a cuvette and 1.5 ml of t r i f l u o r o a c e t i c acid (TFA) was added from a 2-ml syringe f i t t e d with a #18 needle aft e r the cuvette had been placed - 39 -into a Unlearn SP 800 Spectrophotometer. An o p t i c a l density reading was taken at 616 nm ten seconds a f t e r the addition of the color reagent, TFA. A l l determinations were per-formed i n duplicate. The o p t i c a l density readings were converted to international units of vitamin A a c t i v i t y per gram of dry l i v e r using the molecular extinction coe-f f i c i e n t of 145,000 f o r r e t i n o l with TFA which was found by Dugan e t . a l . (1964). A l l organic solvents used for the analyses were r e d i s t i l l e d using calcium hydride as the dry-ing agent. The determinations were carried out using either low-actinic glassware or glassware wrapped i n aluminum f o i l to protect the extracts from l i g h t . In a subsequent small experiment, twenty day-old D chicks and twenty day-old NH chicks of mixed sexes were used from another hatch. The formulas of the di e t s fed the dams are shown i n Tables 2a and 2b respectively. The chicks were weighed and t h e i r l i v e r s were removed, weighed, pooled into groups of four and analyzed f o r vitamin A as previously described. The determinations were performed on 0.1 g samples of the l y o p h i l i z e d ground l i v e r s . Results Table 3 shows the average three-week body weights of the birds i n each group and the t o t a l feed consumption per one hundred grams of body weight f o r the t e s t period of eighty-nine hours. The average wet and dry weights of the l i v e r s , and the l i v e r wet weights expressed as a per-- 40 -Table 3. Experiment li Average three-week body weights (1 standard deviation) and average feed consumption during the eighty-nine-hour test period Level of Dietary Vitamin A Supplied During Test Period (IU/kg) 1700 22,000 100,000 1700 22,000 100,000 Type Body Weight(g) Feed Consumption of (g/lOOg body weight) Chicks D 304±35 305130 307±34 42.7 41.6 41.5 K 305143 300131 291±28 41.3 40.3 39.5 B 298134 307138 298129 40.4 38.9 42.2 NH 314136 306132 293139 38.9 36.7 34.6 Br 555178 547+82 553197 35.1 34.2 37.7 - 41 -centage of body weight are shown i n Tables 4a and b. Presented i n Table 5 are the average vitamin A concentrations i n the l i v e r s of each group of birds expressed as international units per gram of dry l i v e r weight. Also shown i n Table 5 are the percentages of ingested vitamin A stored i n the l i v e r s of the birds receiving the supplemental l e v e l s of 22,000 IU and 100,000 IU of vitamin A per kilogram of die t . The NH chicks stored 45.0% of ingested vitamin A at the lower l e v e l of supplementation and 39.5% at the higher l e v e l of supplementation. The D chicks, representa-t i v e of the WL and Br st r a i n s , stored 29.5% of the ingested vitamin at the lower l e v e l of supplementation and 23.0% at the higher l e v e l s of supplementation. Table 5 ., shows that ttHe NH chicks receiving either the basal or the excess l e v e l s of dietary vitamin A had greater l i v e r vitamin A concentrations than did the corresponding D chicks (p^ DJXEL, appendix Table TV Also the NH chicks stored i n t h e i r l i v e r s a l a r g e r percentage of the excessive vitamin A doses than did the D chicks as shown by the s i g n i f i c a n t (p<0.001) i n t e r -action of breed with the dietary vitamin A l e v e l . For the day-old chicks tested, the average body weight, wet and dry l i v e r weights, and l i v e r wet weights as a percentage of body weight are shown i n Table 6. Table 7 shows the average l i v e r vitamin A concentrations of the chicks of each breed, and the average l i v e r vitamin A content per b i r d and per gram of body weight. The l i v e r vitamin A concentration of the NH chicks was greater than - 42 -Table 4a. Experiment 1: Average wet l i v e r weights as a percait of body weight (t standard deviation) Level of dietary vitamin A supplied during test period (IU/kg) 1700 22.000 100.000  L i v e r Weights Type g/100 g g/100 g g/100 g of g/bird body g/bird body g/bird body chicks w ei ght weight weight D 9.611.1 3.210.4 9.411.2 3.110.5 9.311.0 3.0l0.3 K 9.511.5 3.110.4 9.311.4 3.110.3 9.211.0. 3.2+0.3 B 9.511.1 3.210.3 9.. 3*1.4 3.0l0.3 9.511.0 3.2+0.1 NH 10.Oil.5 3.2+0.3 9.4+1.4 3.110.3 9.711.3 3.3+0.3 Br 17.913.3 3.2+0.5 17.6+3.1 3.2+0.9 17.713.8 3.210.3 - 43 -Table 4b. Experiment 1: Average dry l i v e r weights as a percent of body weight (* standard deviation) Level of dietary vitamin A supplied during test period (IU/kg) 1700 22,000 100,000 Type of chicks L i v e r Weights (g/pooled sample of 4 birds) D 9.910.6 9.810.6 9.8*0.7 K 1 0 . i l l . 1 9.1+1.0 9.7+0.4 B l O . l l l . l 10.3+0.9 10.5ll.O NH 10.110.9 10.0+0.7 9.9+0.5 Br 18.111.1 18.5+2.1 19.112.0 - 44 -Table 5. Experiment 1: Average l i v e r concentrations of vitamin A (1 standard deviation) and percentage of ingested vitamin A stored i n the l i v e r s of the birds receiv-ing 22,000 IU and 100,000 IU of vitamin A per k i l o -gram of diet Level of dietary vitamin A supplied during test period (IU/kg) 1700 22,000 100,000 22,000 100,000 Vitamin A concentration (IU/' g dry % of ingested vitamin A weight) stored i n l i v e r Type of chicks D 12.3+0.9 345153 12081163 29.3 23.0 B 11.521.2 378148 11431235 31.2 23.5 K 10.2±L.4 364140 11501237 30.4 24.0 NH 62.618.8 504150 1797ll78 45.0 39.5 Br 9.722.1 373147 9521190 32.8 21.8 - 45 -Table 6. Experiment 1: Average body weights (1 standard deviation), wet and dry l i v e r weights (t standard deviation) and l i v e r wet weights as a percentage of body weight (t standard deviation) f o r the day-old NH and D chicks Body weight Wet l i v e r s Dry l i v e r s (g/ (g) pooled sample g/bird g/100 g of 4 birds) Type of body weight chicks D 43±3 1.2t0.1 2.80±0.4 1.6to.l m 3 8 ± 4 l . l t O . l 2.80*0.3 1.6+0.1 - 46 -Table 7. Experiment 1: Average l i v e r vitamin A concentration (t standard deviation) and average l i v e r vitamin A content per b i r d and per gram of body weight ( t standard deviation) f o r the day-old D and NH chicks Vitamin A Total l i v e r Vitamin A concentration vitamin A content per (IU/g dry content per 100 g body Type of chicks l i v e r weight) b i r d (IU) weight D 180123 58ll0 168t31 NH 302+15 94 +8 295ll6 - 47 -that of the D chicks (p<0.05) as shown i n Table 2 of the appendix. Discussion In the conventional l i v e r vitamin A storage assay, as devised by Guggenheim and Koch (1944) aBud as subsequently employed and modified by numerous workers (eg. Foy and Morgareidge, 1948; Week and Sevigne 1949 a,b; March et a l . 1952; Ames and H a r r i s , 1956), young animals are f i r s t depleted of t h e i r l i v e r vitamin A stores. The purpose of the assay i n t h i s experiment was to measure the r e l a t i v e a b i l i t y of d i f f e r e n t strains of birds to store dietary vitamin A~in-,the l i v e r rather than to evaluate the b i o l o g i c a l vitamin A potency of a preparation. I t was therefore de-cided to eliminate thastress of a depletion period and to prepare the birds f o r the test by giving them the minimal WRC requirement (1971) of the vitamin i n t h e i r diet during the pre-test period. As can be seen i n Table 5, the cor-rection for the vitamin A content of the control l i v e r s i s r e l a t i v e l y small. In the conventional l i v e r assay, the vitamin i s administered i n one or two oral doses of o i l . v a k i l et a l . (1964), however, showed that mixing vitamin A acetate d i r e c t l y into the diet of young rats permitted better -intestinal absorption and l i v e r storage of the vitamin than did oral administration of single doses of si m i l a r quantities of the acetate i n corn o i l . I t was considered, therefore, that a more r e l i a b l e comparison of absorptive capacity would be made among the various - 4 8 -s t r a i n s of birds i n t h i s experiment by including the sup-plementary vitamin A with the d i e t . Although the day-old WH chicks had a greater carry-over of vitamin A from the egg than did the day-old D' chicks, the difference i n terms of international units of the vitamin (Table 7) i s ... too small to account f o r the results seen i n the l i v e r storage test (Table 5 ) . The same i s true i n comparing the U H chicks to the K, B, and Br chicks i n which l i v e r vitamin A carryover was not measured. Even i f the l i v e r vitamin A content at one day of age were zero i n these b i r d s , the difference i n carryover would be i n s u f f i c i e n t to explain the r e s u l t s of the l i v e r storage experiment. The reason f o r the difference between the WH chicks and the chicks of the WL and Br strains i n l i v e r vitamin A storage e f f i c i e n c y i s not apparent from the work reported here. I t i s important to point out that a difference i n i n t e s t i n a l absorptive capacity f o r the vitamin (whether at the stage of luminal ester hydrolysis or at some l a t e r stage) i s not the only p o s s i b i l i t y . Perhaps the l i v e r s of the NH birds are p a r t i c u l a r l y e f f e c t i v e i n the uptake and/or retention of the vitamin. Perhaps, also, degrada-ti v e l o s s of vitamin A i s slower i n the NH chicks than i n the WL and Br chicks. A small part of the explanation could be. that the iMH chicks have a lower metabolic re-quirement f o r vitamin A than do the other types of birds tested. - 49 -The r e s u l t s of s i m i l a r studies by Week and Sevigne (1949 a) i n adolescent chickens of both sexes, and by March jet a l . (1952) i n young cockerels suggest that the vitamin A storage e f f i c i e n c y of the NH chicks used i n the present experiment was high, rather than that of the WL and Br chicks being low. I t i s d i f f i c u l t , how-ever, to compare the re s u l t s of this experiment d i r e c t l y with the findings of other investigators. In the f i r s t place, the form of the vitamin administered affects i t s absorption e f f i c i e n c y i n birds (Biely e t . a l . , 1941; Nestler e t a l . , 1948; Week and Sevigne, 1949 a; March et a l . , 1952). Secondly, the c a r r i e r o i l has a marked e f f e c t on absorption e f f i c i e n c y of vitamin A. The l a t t e r e f f e c t may be due both to the protection afforded the vitamin by the tocoph-eral content of the o i l (Foy and Morgareidge, 1948) and to the presence of substances i n the o i l which may i n h i b i t vitamin A ester hydrolysis (Week and Sevigne, 1949 a,b; March et a l . 1952). Third l y , the age of the test subjects may be a factor as indicated i n humans (Lewis _et al.-, 1947) , rats (Moore 1957 a) and chickens (Shellenberger et a l . , 1964). Another important variable i s the composition of the basal diet used, p a r t i c u l a r l y with respect to i t s protein and fat content (Scott e t . a l . , 1969 b) . Week and Sevigne (1949 a) administered oral doses of natural vitamin A esters i n o i l to adolescent NH and WL chickens. These authors did not s t a t i s t i c a l l y compare the l i v e r vitamin A storage e f f i c i e n c i e s of the two breeds, - 50 -but i t appears that the NH may have been less e f f i c i e n t i n storing the vitamin than the WL i n the reported experiment. Also, SL fs, f o r unknown reasons, are generally better able to withstand n u t r i t i o n a l d e f i c i e n c i e s than are heavy breeds (Hutt, 1961). The greater l i v e r vitamin A storage e f f i c i e n c y of the WH chicks used i n the present experiment, as compared to the WL and Br chicks, i s therefore considered to be char-a c t e r i s t i c of the p a r t i c u l a r populations of birds rather than of the breeds used. The three-week body weights of the birds (Table 3) were normal (Morrison, 1959; NEC, 1971). Feed consumption expressed as g feed per 100 g body weight was s i m i l a r among the various groups of birds within each breed (Table 3). The difference i n the l i v e r storage e f f i c i e n c y of vitamin A between the NH chicks and the WL and Br chicks cannot be attributed to a difference i n the quantity of the test diets consumed. Furthermore, the s i m i l a r l i v e r sizes (Table 4a) and the s i m i l a r proportions of dry matter i n the l i v e r s (Table 4b) of the birds show that the difference i n vitamin A storage e f f i c i e n c y i s not simply a r e s u l t of a gross difference in the amount of tissue available for the storage of the vitamin. The diet provided the NH dams (Table 2b) was quite si m i l a r i n crude protein and f a t content to that provided the D dams (Table 2a) but was somewhat lower i n vitamin A content. I t would seem u n l i k e l y , therefore, that the greater l i v e r vitamin A content of the day-old NH chicks than of the day-old D chicks Gan be attributed to a larger vitamin A - 51 -content i n the NH eggs than i n the D eggs. Also, the day-old body weights and l i v e r sizes were si m i l a r between the two breeds (Table 6). I t i s an i n t e r e s t i n g p o s s i b i l i t y that the difference between the day-old NH and D chicks i n l i v e r v i t a -min A content may r e f l e c t i n the embryo, the same difference i n e f f i c i e n c y of vitamin A storage i n the l i v e r as was observed i n the birds at three weeks of age. In support of the l a t t e r p o s s i b i l i t y are a number of experiments i n d i c a t i n g that both the i n t e s t i n a l absorption of nutrients and the p a r t i c i p a -tion of the l i v e r i n l i p i d metabolism are o l physiologi-cal s i g n i f i c a n c e i n the chick embryo long before hatching (Rol»nik, 1970). The d i f f e r e n t n u t r i t i o n a l h i s t o r i e s of the dams constitute an uncontrolled f a c t o r i n t h i s experiment. Further-more, the vitamin A storage test using the NH chicks was conducted two weeks l a t e r than that using the WL and Br chicks. This experiment should, therefore, be repeated to eliminate the uncontrolled factors involved. - 52 -Experiment 2: THE EFFECT OF A MODERATE DIETARY EXCESS OF VITAMIN A ON ALIMENTARY CHOLESTEROLEMIA Materials and Methods Forty D and for t y NH cockerels were removed from the same populations of birds as were used i n experiment 1. The birds were fed and watered ad lib i t u m , and were vaccinated against Newcastle Disease as described i n exp-eriment 1. The composition of the diet provided the cock-erels up to eight weeks of age i s shown i n Table 1. At seven weeks of age the birds were removed from e l e c t r i c a l l y -heated battery brooders and were redistributed i n groups of f i v e into larger, unheated cages. When the cockerels were eight weeks old blood samples of 2 mis were obtained f o r t o t a l cholesterol determination. Immediately a f t e r the blood sampling, half the birds were placed on a diet (A+) which consisted of the basal (Table 1) supplemented both with cholesterol* (added to a l e v e l of 0.5$ of the diet) and with vitamin A (added to a l e v e l of 22,000 U per kilogram of diet) . The supplemental vitamin A was taken from the same concentrated corn o i l preparation of r e t i n y l palmitate as was used i n experiment 1. The c a r r i e r was soy-bean o i l added to a tota l of 1% of the di e t . An i d e n t i c a l diet containing 0.5$ of cholesterol, but only 1700 IU of vitamin A per kilogram, served as a control. Blood samples were again taken f o r t o t a l cholesterol determination afte r the birds had been fed the cholesterol-* C r y s t a l l i n e form, N u t r i t i o n a l Biochemicals Corporation. - 53 -containing diets f o r seventy-two hours. At t h i s time the birds were weighed and k i l l e d , and the l i v e r s were removed for determination of the vitamin A concentration. The l i v e r s from the f i v e birds of each pen were pooled. The blood samples, taken from the wing vein of each b i r d , were c o l l e c t e d i n s i l i c o n i z e d 10mm X 75 mm glass culture tabes. A solution of 0.1% heparin Sodium was used as the anticoagulant i n the proportion of 0.1 ml to 1.0 ml whole blood. Plasma was obtained from each sample of blood by centrifugation at 1200 X G f o r eight minutes. Care was taken to avoid hemolysis which could contribute red c e l l cholesterol to the plasma. The d i g i -tonin p r e c i p i t a t i o n method of Schoenheimer and Sperry (1934) as modified by Sperry and Webb (1950) and by Vahouny a t . a l . (1960) was used to measure the cholesterol concen-t r a t i o n of the plasma samples. Each sample was analyzed i n duplicate. A l l organic solvents used were r e d i s t i l l e d using calcium hydride as the drying agent. The procedure used to determine the l i v e r vitamin A concentrations was i d e n t i c a l to that used i n experiment 1. Duplicate samples of 0.5g of the dry, ground l i v e r s were analyzed. Results The average eight-week body weights and the average feed consumption of the birds per hundred grams of body weight over the three-day cholesterol feeding period are - 54 -shown i n Table 8. Table 9 shows, f o r each group, the wet and dry l i v e r weights and the wet l i v e r weights as percentages of body v/eight. The average l i v e r vitamin A concentration of each group along with the percentage of ingested v i t a -min A stored by the birds receiving the moderate excess of 22,000 IU of vitamin A per kilogram of diet are shown in Table 10. The table? shows that the NH chicks receiving the basal diet and those receiving the A+ diet (22,000 IU/kg) had greater l i v e r concentrations of vitamin A than did the D chicks fed the respective diets (p^O.OOl, appendix Table 3) . The NH chicks receiving the Af diet also stored a l a r g e r f r a c t i o n of ingested vitamin A (54.6$) than did the D chicks receiving the Al- diet (34.9$) (p<0.05). The average plasma cholesterol concentrations of the birds before and a f t e r they were fed cholesterol are shown i n Table 11. The analyses of variance presented i n Tables 4a and 4b of the appendix show that the pre-test plasma cholesterol concentrations of the Dekalbs i n the control and the At groups were not s i g n i f i c a n t l y d i f f e r e n t , but that a f t e r the cholesterol feeding period the birds receiving the A+ diet had a higher average c i r c u l a t i n g cholesterol concentration (147.0 mg$) than did the con-t r o l s (128.8 mg$) (p<0.05). In the case of the NH chicks, the absolute plasma cholesterol concentrations of the birds fed the control and the A+- diets were not d i f f e r e n t either before or a f t e r the cholesterol feeding period - 55 -Table 8: Experiment 2: Average eight-week body weights (1 standard deviation) and average feed consumption during the three-day test period Level of dietary vitamin A supplied during test period (IU/kg) 1700 22,000 1700 22,000 Type of Body weight (g) Feed consumption Chicks (g/l00g body weight) D 1063tl26 1124+116 32.2 33.1 NH 12801131 1238+.175 27.2 28.1 Table 9 . Experiment 2: Average wet and dry l i v e r weights ( l standard deviation) and the wet l i v e r weights as a percent of body weight (1 standard deviation) Level of dietary vitamin A supplied during t e s t period (IU/kg) 1700 22,000 Wet l i v e r s Dry l i v e r s Wet l i v e r s Dry l i v e r s Type g/bird g/100 g g/pooled g/birds g/100 g g/pooled of body sample of body sample of Chicks weight 5 birds weight 5 birds D 3313.8 3 . l l 0 . 4 46 .315 .1 35 .013 .2 3 . 1 - 0 . 3 4 9 . 1 - 2 . 3 NH 37 .714 .0 3 .010 .3 54.0J.2.5 39 .216 .5 3 . 2 1 0 . 3 54 .715 .2 - 57 -Table 10. Experiment 2: Average l i v e r concentration of vitamin A (- standard deviation) and percentage of ingested vitamin A stored i n the l i v e r s of the birds r e c e i v i n g 22,000 IU of vitamin A per kilogram of diet Legel of dietary vitamin A supplied during test period (IU/kg) 1700 22,000 Vitamin A con-centration Type of chicks (IU/g dry-l i v e r weight) Vitamin A concentration (IU/g dry l i v e r weight) <fo of ingested vitamin A stored i n l i v e r D NH 10.Oil.4 3 2 . 6 l l . l 300*57 404^49 34.9 54.6 - 58 -Table 11. Experiment 2: Average plasma cholesterol concentra-tions (I standard deviation) before and after feeding 0.5% cholesterol f o r three days with d i f f e r e n t l e v e l s of vitamin A and average increase i n plasma cholesterol concentration (± standard deviation) r e s u l t i n g from the ingestion of the cholesterol diets Level of dietary vitamin A supplied e/uring test period (IU/kgj 1700 22,000 Type of Chicks Plasma cholesterol Increase Plasma cholesterol Increase concentration (mg%) (mg%) (mg%) (mg%) Pre-test Test Pre-Test Test D NH 78.3±10.2 128.8±25.6 50.5127.1 83.926.6 144.6217.2 60.7±15.9 - 59 -(appendix, Table 5a and 5b). An analysis of variance on the increases i n plasma cholesterol concentration r e s u l t i n g from cholesterol ingestion, however, showed (appendix Table 5c) that the plasma l e v e l had increased more in the control birds (60.7 mgf0) than i n the A+ birds (47.4 mg$) (p<0.01). Discussion The e f f i c i e n c i e s of l i v e r storage of vitamin A obtained i n t h i s experiment (Table 10) appear to confirm the difference between the NH and the D chicles observed i n experi-ment 1. The body weights of the birds were normal f o r t h e i r age (Morrison, 1959, NRC, 1971). As i n experiment 1, the difference i n l i v e r storage e f f i c i e n c y of vitamin A between the NH and D chicks can be attributed neither to a difference i n the consumption of the test diets (Table 8) nor to a d i f -ference i n the l i v e r sizes of the two breeds (Table 9). The vitamin A storage e f f i c i e n c i e s of the birds i n the present experiment were greater than those of the bircfe receiving the same l e v e l of supplementary vitamin A i n the previous experiment. I t might have been expected, on the basis of e a r l i e r l i t e r a t u r e , that a smaller l i v e r storage e f f i c i e n c y would have been obtained i n the present experiment than i n experiment 1. Shellenberger _et a l . (1964) demonstrated a superior i n t e s t i n a l absorptive capacity f o r vitamin A acetate i n five-week-eid than In nine-week-old b i r d s . Also March and B i e l y (1963) reported i n p u l l e t s , and Green et a l . (1965) reported i n r a t s , that the i n c l u s i o n of cholesterol i n the diet depresses the l i v e r storage of normal and excessive quan-- 60 -t i t i e s of dietary vitamin A. Thorbjarnarson and Drummond (1938) , however, found that the i n c l u s i o n of cholesterol i n the diet of rats increased t h e i r l i v e r storage of vitamin A. The apparent depressing e f f e c t of a dietary excess of vitamin A on alimentary cholesterolemia i n the NH cockerels (Table 11) i s consistent with the l i t e r a t u r e . The author has found no report on any species, however, of an increasing effect of vitamin A on alimentary cholesterolemia such as was obtained with the D chicks i n th i s experiment (Table 11). No informa-t i o n i s provided by th i s work as to the mechanism of the diverg-ent effects of excess dietary vitamin A on alimentary chol-esterolemia i n the NH and D chicks. The explanation i s not to be found i n a difference between the two breeds i n the consump-tion of the cholesterol test d i e t (Table 8). The i n t e s t i n a l ab-sorption and/or the subsequent tissue d i s t r i b u t i o n of cholesterol could be affected d i f f e r e n t l y by excesses of vitamin A i n the two types of chickens. I t i s also possible that the NH and D chicks d i f f e r i n blood volume r e l a t i v e to body s i z e . With the r e l a t i v e l y long feeding period employed, the p o s s i b i l i t y of an effe c t on the blood cholesterol concentration through the r e d i s -t r i b u t i o n of endogenous cholesterol pools cannot be ignored. Differences i n the n u t r i t i o n a l h i s t o r y of the dams, and environmental v a r i a t i o n between the two tests prevented comparison of the pre-test plasma cholesterol concentrations i n the chicks of the two breeds although the NH cockerels ap-peared to be higher than the D cockerels. The experiment was accordingly repeated as part of experiment 3 i n order to elim-inate the uncontrolled factors involved. - 61 -Experiment 3: THE EFFECT ON ALIMENTARY CHOLESTEROLEMIA OF A MODERATE EXCESS OF DIETARY VITAMIN A FED PRIOR TO BUT NOT SIMULTANEOUSLY WITH CHOLESTEROL Materials and Methods F i f t y D and f i f t y NH day-old cockerels of a d i f -ferent hatch from the birds used i n experiments 1 and 2 were wing-banded and weighed. The birds were dis t r i b u t e d i n groups of ten i n e l e c t r i c a l l y - h e a t e d battery brooders and were provided with feed and water .&d libitum f o r four weeks. At two weeks of age the birds were vaccinated against Newcastle Disease. The composition of the diet fed the dams of the chicks of both breeds i s shown i n Table 2b. T h i r t y of the birds of each breed were fed the basal diet (Table 1) and were then used i n a test s i m i l a r to experiment 2. There were, accordingly, f i f t e e n A+ (group I) and f i f t e e n control birds of each breed. In a second part of t h i s experiment, twenty D and twenty NH chicks (group II) received, u n t i l four weeks of age, a die t i d e n t i c a l to the basal (Table 1) except that i t contained 22,000 IU of vitamin A per kilogram. The supplemental vitamin A was , from a s t a b i l i z e d concentrate of r e t i n y l palmitate*. The test d i e t , fed for seventy-two hours when the birds were four weeks old, consisted of the basal containing 0.5% of cholesterol. The c a r r i e r employed was soybean o i l added to a t o t a l of 1% of the di e t . The control birds from the f i r s t part of * Courtesy of noffmann-La Roche, Ltd., Montreal, Canada - 62 -t h i s experiment (which duplicated experiment 2) also served as controls f o r the second part since the two parts were conducted simultaneously. Immediately before placing the birds on the chol-esterol diets, a blood sample f o r t o t a l cholesterol determ-in a t i o n was taken from each b i r d as described i n experi-ment 2. S i m i l a r l y , blood samples were obtained f o r t o t a l cholesterol measurement immediately aft e r the seventy-two hour cholesterol feeding period. Feed consumption was recorded over the t e s t period. Blood samples were obtained, and the plasma chol-esterol concentration of ea£h sample was determined, as described i n experiment 2. Results The average four-week body weights of the birds are shown i n Table 12 together with the average feed con-sumption of the birds per hundred grams of body weight over the seventy-two hour te s t period. The average pre-test and test plasma cholesterol concentrations of the cockerels are shown i n Table 13. As shown i n Table 6a of the appendix, there was no difference, i n either breed, between the pre-test plasma cholesterol concentrations of the controls and those of either the group I or the group II b i r d s . A f t e r the cholesterol diets were fed, however, the group I D chicks showed a higher average plasma cholesterol concentration (167.9 mg$) than the - 63 -Table 12. Experiment 3: Average four-week body weights (I standard deviation) and average feed consumption dur-ing the three-day test period. Level of dietary vitamin A supplied during test period (IU/kg) ' 1700' 1700 22,000 1700 1700 22,000 (control) (groupll) (groupl) (control) (groupll) (groupl) Type of Chicks Body weight (g) Feed consumption (g/lOOg body weight) D 301+.25 298232 2952l8 38.3 37.9 40.1 NH 298229 3012 8 299230 35.1 34.2 35.8 - 64 -Table 13. Experiment 3: Average plasma cholesterol concentra-tions (2 standard deviation) before and after feeding 0.5% cholesterol for three days with d i f f e r e n t l e v e l s of vitamin A Level of dietary vitamin A supplied during test period (IU/kg) 1700 (control) 1700 (groupll) 22,000 (groupl) Plasma cholesterol Plasma cholesterol Plasma cholesterol concentration (mg%) concentration (mg%) concentration (mg%) Type of Pre-Test Test Pre-Test Test Pre-Test Test Chicks D 88.8+.10.1 151.1*-6.6 89.4-12.9 151.0-7.7 89.4+11.4 167.9*.10.2 NH 90.9+11.4 153.626.2 92.6+9.8 152.426.2 89.2+-10.8 137.8+10.5 - 65 -controls (151.1 mgf0) (p<0.001), and the group I NH chicks showed a lower average plasma cholesterol concentration (137.8 mg$) than the controls (152.4 mgf0) (p^O.001) (ap-pendix, Table 6b). In both breeds, the plasma cholesterol concentration of the group I I birds showed the same re-sponse to the cholesterol diets as did that of the controls birds (appendix, Table 6b). Discussion This experiment confirms the f i n d i n g of experiment 2 that a dietary excess of vitamin A increases alimentary cholesterolemia i n the D chicks and has the opposite e f f e c t i n the NH chicks (Table 13). As f o r the difference i n vitamin A storage observed i n experiment 1, these diverg-ent cholesterolemic responses are considered to be char-a c t e r i s t i c s of the st r a i n s of birds used rather than of the breeds. The responses obtained within each s t r a i n were consistent between the ages of four weeks and eight weeks. Since the group II and the control birds of both strains had s i m i l a r pre-test plasma cholesterol concentra-tions, there appears to be no net e f f e c t , through endogenous cholesterol pools, of a moderate dietary excess of vitamin A on the plasma cholesterol concentration. The removal of the excess vitamin A from the diet of the group I I birds resulted i n alimentary cholesterolemia s i m i l a r i n degree to that occurring with the controls (Table 13). - 66 -I t i s concluded that the effect of vitamin A on the ah sorption and/or the subsequent d i s t r i b u t i o n of dietary-cholesterol i s of short duration, requiring the simultan-eous presence i n the d i e t of vitamin A with the cholesterol. The mechanism of t h i s effect remains obscure. At four weeks of age there was no difference i n "normal" c i r c u l a t i n g cholesterol concentration (Table 13), between the D and the NH cockerels. The values obtained were higher than those of experiment 2. Seasonal variations in the size of various cholesterol pools including that of the plasma have been reported i n chickens (Whiteside and Fluckiger, 1965). I t may therefore be of significance that experiment 1 was conducted i n December and January, whereas experiment 2 was carried out i n June and J u l y . The cholesterolemic responses of the control birds of the two experiments were s i m i l a r (Tables 11 and 13). I t should be noted that no overt t o x i c i t y symptoms resulted i n the group II birds which received the A+ d i e t f o r four weeks. They were si m i l a r i n outward appearance to the control chicks and t h e i r average body weight at four weeks of age (Table 12) was equal to that of the control chicks. The body weights of the birds of the various treat-ments were normal f o r chicks four weeks of age (Morrison, 1959; NRC, 1971). - 67 -Experiment 4: EFFECT OF EXCESS DIETARY AND OF EXCESS PARENT-ERALLY-ADMINISTERED VITAMIN A ON THE INTESTINAL ABSORPTION AND LIVER RETENTION OF A SINGLE ORAL DOSE OF LABELED CHOLESTEROL Materials and Methods Seventy D and seventy NH day-old cockerels from the same hatch as the birds used i n experiment 3 were wing-banded and weighed. The birds were distributed i n groups of ten into e l e c t r i c a l l y - h e a t e d battery brooders and were pro-vided with feed and water _ad libiturn u n t i l they were used as experimental subjects between the ages of f i v e and seven weeks. The diet provided i s shown i n Table 1. The experiment was carried out over a period of fourteen days since seven t r i a l s were performed and a t r i a l was completed on every second day. The various treatments were conducted over the fourteen day period i n an attempt to avoid the introduction of confounding environmental and age factors. Each t r i a l required ten birds of each breed. The design of the experiment i s outlined be-low: Treatment No. of birds per breed Group 1: Oral dose 4 - 1 4 C - c h o l e s t e r o l * 10 Group 2: Oral dose 4 - 1 4 C - c h o l e s t e r o l + vitamin A 10 Group 3: Oral dose 4 - 1 4 c - c h o l e s t e r o l , intramus-cular vitamin A i n j e c t i o n s 10 * MaJLlinckrodt Radiochemical Department. Purity 98$ checked by TLC on S i l i o a Gel "GF". Sp. Ac. = 61 mCi/mM. ( - 68 -Treatment JMO. of birds per breed G-roup 4: Oral dose "cold" cholesterol, i n t r a -venous i n j e c t i o n of 4- 1 4 C - c h o l e s t e r o l 5 Group 5: Oral dose "cold" cholesterol + v i t a -min A, intravenous i n j e c t i o n of 4--L4C-cholesterol 5 Group 6: Oral dose "cold" cholesterol, i n t r a -muscular vitamin A i n j e c t i o n s , i n -travenous i n j e c t i o n of 4-^ 4C-choles-t e r o l 5 Group 7: Oral dose 4—-^C-cholesterol; i n t r a -venous i n j e c t i o n of saline-Tween 80-benzene 5 Group 8: Oral dose 4 - 1 4 C - c h o l e s t e r o l + v i t a -min A, intravenous i n j e c t i o n of sa-line-Tween 80-benzene 5 Group 9: Oral dose 4 - l 4 C - c h o l e s t e r o l , intramus-cular vitamin A i n j e c t i o n s ; intraven-ous i n j e c t i o n s of saline-Tween 80-benzene 5 Group 10: Oral dose 4 - 1 4 C - c h o l e s t e r o l , intramus-cular Tween-80-saline i n j e c t i o n s 5 Group 11: Oral dose "cold" cholesterol; i n t r a -muscular Tween-80-saline i n j e c t i o n s , intravenous i n j e c t i o n of 4— t l 4G-chol-esterol 5 The oral doses of cholesterol and vitamin A were given i n 1 ml of corn o i l by calib r a t e d pipette a f t e r a starvation period of eight hours. In each case 0.2 uci of labeled cholesterol was administered and was accompanied by 50 mg of "cold" chol-e s t e r o l . The corn o i l solutions were given at a temperature of about 45°C to maintain the cholesterol i n solution. The o r a l doses of vitamin A consisted of 2000 IU per b i r d from the concentrated preparation of r e t i n y l palmitate i n corn o i l used i n experiments 1,2, and 3. The doses of vitamin A - 69 -were given simultaneously with the cholesterol. The intramuscular i n j e c t i o n s of vitamin A consis-ted of 5000 IU of vitamin A palmitate (from the same concen-trated corn o i l preparation) i n j e c t e d into the breast muscle da i l y f o r one week p r i o r to the o r a l cholesterol dose. The c a r r i e r employed was 0.5% Tween-80 i n 0.9% NaCl, and the v o l -ume i n j e c t e d was 0.1 ml each time. A tuberculin syringe and a #27 gauge needle were used f o r the i n j e c t i o n s . Intravenous i n j e c t i o n s of 0.5 uCi of 4- 1 4C-chol-esterol were made int o the l e f t wing vein. The c a r r i e r was a m i c e l l a r mixture of 0.9% NaCl, 0.5% Tween-80, and 0.2% ben-zene, and the volume injected was 0.25 ml. The injections of 4- 1 4 C - c h o l e s t e r o l and of c a r r i e r without radioactive choles-t e r o l (groups 4-9 and 11) were made six hours following oral administration of "cold" cholesterol with or without vitamin A, depending on the group. A tuberculin syringe and a #26 gauge needle were used for the i n j e c t i o n s . The needle was held i n place u n t i l several seconds after each i n j e c t i o n was complete. Bleeding from the point of i n j e c t i o n was quickly stopped with a small quantity of ce l l u l o s e powder. After the various oral doses of cholesterol and vitamin A were administered, the birds were held i n i n d i v i d u a l cages f o r twenty-four hours. During t h i s period, feed and water was available ad li b i t u m . Except f o r groups 2,5, and 8, the diet provided was basal (Table 1) which the birds had been receiving since one day of age. Groups 2, 5, and 8 received, - 70 -f o r the twenty-four-hour period, a diet consisting of the basal to which was added s u f f i c i e n t s t a b i l i z e d vitamin A palmitate to provide 22,000 IU of vitamin A per kilogram. A f t e r the twenty-four hours, the birds were weighed and then were k i l l e d by c e r v i c a l d i s l o c a t i o n . The l i v e r s were removed, blotted and weighed. They were then freeze-dried, weighed, and ground i n a mortar and pestle. The large and small i n t e s t i n e s , the ceca and the g a l l bladder were excised with t h e i r contents and combined with the collected excreta and the ingesta present i n the digestive t r a c t from the crop to the gizzard i n c l u s i v e . This mixture, hereafter ref erred to as GIT+E, was f reeze-dried, weighed and ground. Total l i p i d s were extracted from the l i v e r s and from 5 g samples of the ground GIT + E. The extraction was carried out i n a soxhlet apparatus f o r sixteen hours using diet h y l ether. The ether was then flash-evaporated and the f i n a l traces of solvent removed with a stream of nitrogen. The residue from each extraction was dissolved i n 80 mis of acetone. The mixture was refluxed f o r 30 minutes during which time s u f f i c i e n t KMn04 was added to remove the yellow pigmen-ta t i o n . The acetone solution was s u c t i o n - f i l t e r e d through #42 grade f i l t e r paper to remove suspended MnOg and the f i l -t r a te was then transferred to a 250 ml separatory funnel. Transfer was effected with the aid of small amounts of p e t r o l -eum ether (b.p. 40°-60°). F i f t y ml of m e t a l - d i s t i l l e d water were added and the mixture was extracted with 50 ml of pet-roleum ether. The lower l a y e r was re-extracted with 50 ml - 71 -of petroleum ether and the two petroleum ether extracts were combined by suction f i l t r a t i o n through #42 grade f i l t e r paper and transferred quantitatively to a 250 ml round-bottom f l a s k . The solvent was removed by f l a s h evaporation followed by a nitrogen stream, and the residue was transferred to glass counting v i a l s i n several small aliquots t o t a l l i n g 20 ml of s c i n t i l l a t i o n f l u i d . The s c i n t i l l a t i o n f l u i d consisted of toluene containing 0.6$ of 2.5-diphenyloxazole (PPO) and 0.05$ of 1.4-Bis(2-(5-phenyloxazolyl))benzene (P0P0P). The amount of r a d i o a c t i v i t y i n the v i a l s was measured i n a ten-minute count using a Nuclear Chicago Isocap 300 l i q u i d s c i n t i l l a t i o n counter held at 7°C f o r approximately one hour p r i o r to being counted. A standard curve of counting e f f i c i e n c y versus 1 3 3 B a external standard ratiouwas plotted using a ^ 4C quench s e r i e s . From t h i s curve, the e f f i c i e n c y of counting was ob-tained f o r each sample so that the data could be converted from counts per minute to disintegrations per minute. To check the procedure used f o r recovering the r a d i o a c t i v i t y , aliquots of the 4- 1 4C-cholesterol dissolved i n benzene were mixed into 10 g samples of c e l l u l o s e powder. Duplicate 10 g samples containing 0.0001 u c i , 0.001 uCi or 0.01 uCi per gram were made. Five-gram portions of each of these six t e s t samples were extracted i n the Soxhlet apparatus and processed as described above. Background radiation was measured with a f o r t y -minute-count of a 20 ml sample of the s c i n t i l l a t i o n f l u i d . - 72 -Results Since the e f f e c t of each experimental treatment was examined at i n t e r v a l s over a period of fourteen days there was v a r i a b i l i t y i n the body weights of the birds used. For both breeds the weights within each group ranged from approximately 350 g to more than 600 g. Inspection of the data revealed no obvious correlation between body siz e and the d i s t r i b u t i o n of eitherorally-administered or intravenously-injected cholesterol. Presented i n Table 14 are the wet and dry l i v e r weights f o r the chicks of each breed and f o r each experimental treatment expressed as a percent of body weight. For the purposes of t h i s experiment, the quantity of excreted r a d i o a c t i v i t y was summed with the amount found i n the l i v e r s and termed the "hepatic pool" of r a d i o a c t i v i t y . Table 15 shows tooth the percent absorption of the cholesterol doses, and the percent of absorbed cholesterol found i n the hepatic pool i n a l l groups of birds receiving oral doses of 4- l 4 C - c h o l e s t e r o l . For each group of birds receiving i n t r a -venous in j e c t i o n s of radioactive cholesterol, the excreted l a b e l expressed as a percent of the r a d i o a c t i v i t y found i n the hepatic pool i s shown i n Table 16 together with the percent of the injected dose found i n the hepatixj pool. As shown i n Table 15, the parenterally-administered vitamin A s i g n i f i c a n t l y increased the eficiency of absorption of cholesterol from a value of 41% to a value of 56% i n the NH chicks (p<0.01, appendix Table 7d) and from a value of 46% to a - 73 -Table 14. Experiment 4: Wet and dry l i v e r weights as a percent of body weight ( t standard deviation) Group # D chicks NH chicks wet dry wet dry 1 3.5+0.5 1.0+0.2 3.2+0.6 i.oto.s -; 2 3.2+.0.6 0.9t0.2 3.0t0.4 0.9+0.1 3 3.4+0.4 1.0+0.1 3.5+0.4 l . i t o . i 4 3.4+0.5 1.0+0.2 3.4+-0.3 1.0+0.1 5 3.6t0.4 1.1+0.1 3.6+0.5 1.1+0.2 . 6 3.1+0.5 0.9+0.2 3.3+0.5 1.0t0.2 7 3.6+0.5 l. l t o . 2 3.3+-0.4 0.9to.l 8 3.3+0.5 1.0+0.2 3.1+0.6 0.9+0.2 9 3.3+0.3 1.0+0.1 5.5+0.6 l.lto.2 10 3.2+0.5 0.9+0.2 3.410.4 1.1+0.1 11 3.5+-0.4 1.1+0.1 3.6i0.5 l.ltO.2 - 74 -Table 15. Experiment 5: Percent absorption of single oral dose of cholesterol (t standard deviation) and percent of absorbed cholesterol found i n hepatic pool ( + standard deviation) Group# D chicks NH chicks fo absorption % i n Hep- fo absorption fo i n Hepatic a t i c pool pool 1 46tl0 53+9 41+10 5 l l l 0 2 5 O i l 2 49+12 45+9 60fl0 3 60 i n 44t9 56+11 69ll5 7: 48+11 54211 44+14 47+12 8 52+14 50±10 49+11 65+14 9 56+12 42+14 57±L2 71+15 10 49ll3 49*10 40*13 5 6 i l l - 75 -Table 16. Experiment 4: Percent ( + standard deviation) of i n t r a -venously-injected 4 - 1 4 C - c h o l e s t e r o l found i n hepatic pool and b i l i a r y excretion of intravenously-injected 4_14c-cholesterol as a percent (t standard deviation) of 1 4C l a b e l found i n hepatic pool within the f i r s t eighteen hours a f t e r i n j e c t i o n Group # D chicks NH chicks fo excreted % i n hepatic fo excreted % i n hepatic pool pool 4 16*3 42+6 14+3 40+7 5 13*4 39+8 17+4 45*9 6 16*5 31+9 20+5 58+11 11 15+4 35±7 15±4 38+9 - 76 -value of 60% i n the D chicks (p<0.01, appendix Table 7b). There was no s i g n i f i c a n t effect of orally-administered vitamin A upon cholesterol absorption i n cockerels of either breed, (appendix Tables 7a, c ) . As shown i n Table 15, the percent of absorbed cholesterol found i n the hepatic pool was increased from a l e v e l of 51% to a l e v e l of 60% by dietary vitamin A (p<0.05, appendix Table 8c) and from a l e v e l of 51% to a l e v e l of 69% by the par-en terally-administered vitamin (p<0.01, appendix Table 8d) i n the NH cockerels. The orally-administered vitamin A was without e f f e c t , however, i n the D chicks, and the parenterally-administered vitamin A decreased the percentage of absorbed cholesterol found i n the hepatic pool from a value of 53% to a value of 44% (p<0.05 appendix Table 8b) . In the case of the intravenously-injected 4- 1 4C-cholesterol (appendix, Table 9) the parenterally-administered vitamin A increased the percentage of injected l a b e l found i n the hepatic pool of the NH chicks (p<0.05), but decreased the percentage i n the D chicks (p«(0.05). The orally-administered vitamin A was without e f f e c t i n either breed. Table 16 shows that the excretion of l a b e l expressed as a percentage of r a d i o a c t i v i t y found i n the hepatic pool was increased i n the NH cockerels by the intramuscular i n j e c t i o n s of vitamin A (p<p.05, appendix Table lOd) but was not affected i n thi s breed by the dietary vitamin A (appendix Table 10c). No effe c t of the vitamin, either o r a l l y - or parenterally-administered, was seen on the excretion of cholesterol by the D cockerels (appendix Table 10 a, b). - 77 -The analyses of variance shown i n Table 11 of the appendix demonstrate, i n both breeds, that the d a i l y intramuscular i n j e c t i o n f o r one week of 0.1 ml of the Tween-80-saline c a r r i e r with no added vitamin A was without ef-fect on the absorption of cholesterol, the amount of ab-sorbed cholesterol found i n the hepatic pool, and the excre-ti o n of the cholesterol taken into the l i v e r . S i m i l a r l y , the analyses of variance shown i n Table 12 of the appendix show that the intravenous i n j e c t i o n of the saline-Tween 80-benzene mi c e l l a r mixture had no effect i n either breed on the absorption of cholesterol or the amount of absorbed cholesterol found i n the hepativ pool, .furthermore, (Table 13 of the appendix) the intravenous i n j e c t i o n of the m i c e l l a r c a r r i e r had no net influence i n either breed on the effect of dietary or parenterally-administered v i t a -min A on the absorption or the subsequent d i s t r i b u t i o n of cholesterol. The analysis of variance presented i n Table 14 of the appendix reveals a breed difference, independent of vitamin A treatment, i n the e f f i c i e n c y of cholesterol absorption (p<0.01). The NH chicks absorbed cholesterol less e f f i c i e n t l y than the D chicks (Table 15) . In Table 17 are presented the recoveries of radio-a c t i v i t y from the c e l l u l o s e test samples used to check the l i p i d extraction and de-pigmentation procedure. O v e r a l l , - 78 -Table 17. Experiment 4: Recovery of r a d i o a c t i v i t y from c e l l u -lose test s<amples Expected recovery (dpm) Actual recovery (dpm) $ of expected sample 1 sample 2 sample 1 sample 2 1,110 1081 1159 98 104 11,100 9557 10,724 86 97 111,000 106,426 109,952 96 99 - 79 -i n c l u d i n g the one poor recovery of 86%, the average recovery of r a d i o a c t i v i t y was 97% of the expected. Discussion The quantity of 1 4 c found i n the GIT + E of the birds of groups 4,5, and 6 was used to estimate the excretion rate of the control birds (group 1) the birds receiving oral doses of vitamin A (group £), and the birds receiving paren-t e r a l doses of vitamin A (group 3), respectively. Although the urine and feces were mixed i n the c o l l e c t i o n procedure employed, t h i s i s not a s i g n i f i c a n t source of error since the l o s s of the s t e r o l nucleus through the kidney i s n e g l i g i b l e (Hellman et a l . , 1958; A l l e n , 1970). The 4 - 1 * 0 - c h o l e s t e r o l was administered intravenously s i x hours following the oral dose of "cold" cholesterol i n an attempt to allow mixing of the labeled compound with newly-absorbed cold cholesterol i n the l i v e r . Six hours was chosen as the time i n t e r v a l since Borgstrom e_t aL. (1958) and I r i t a n i and Takeuchi (1969) have shown i n rats that the maximal l i v e r content of newly-absorbed cholesterol occurs three to nine hours a f t e r administering a single o r a l dose i n o i l . Also, Andrews _et a l . (1967) reported data on the laying hen suggesting that the maximal l i v e r con-tent of newly absorbed cholesterol occurs at some point (not discernible from the data reported) within the f i r s t twelve hours a f t e r the administration of a single oral dose of chol-esterol i n o i l . The intravenously-injected 4 - l 4 C - c h o l e s t e r o l was i n a Tween m i c e l l a r phase and not i n a physiological l i p o -protein form. There i s evidence, however, from studies with - 80 -rats (Nilsson and Z i l v e r s m i t , 1972) that the turnover rate of intravenously-injected cholesterol from the l i v e r into both the blood and the b i l e i s independent of the physical form i n which cholesterol i s administered. I t was not possible to obtain the same l e v -e l o f r a d i o a c t i v i t y i n the l i v e r s of the birds i n groups 4,5, and 6 as were obtained i n the l i v e r s of the corresponding birds i n groups 1,2, and 3. Tables 12 and 13 of the appendix show that the greater recovery of counts i n the l i v e r s of the birds i n groups 4-6 than i n the l i v e r s of the birds i n groups 1-3 does not r e f l e c t any difference i n the amount of newly-absorbed cholesterol i n the l i v e r s of the cockerels i n corres-ponding groups. The injected s t e r o l made a n e g l i g i b l e c o n t r i -bution to the t o t a l l i v e r cholesterol pool since only a tracer quantity of cholesterol was i n j e c t e d . The b i l i a r y excretion of newly-absorbed cholesterol by the birds i n groups 1,2, and 3 was therefore estimated by the following c a l c u l a t i o n : estimated disintegrations per minute (dpm) excreted = own l i v p r ( « r r m n 1 ? nr> c t w d p m GIT * E (group 4,5, or 6) dpm l i v e r (group 1,2 or 5 ) X d p m j . i v e r (group 4,b| or 6 ) — The e f f i c i e n c y of cholesterol absorption was found i n the birds given 4-14 C -cholesterol o r a l l y by subtracting the estimated excretion of l a b e l from the t o t a l r a d i o a c t i v i t y found i n the GIT 4- E. Nilsson and Zilversmit (1972) showed that the l i v e r , mostly by way of the Kupffer c e l l s , removes 75% to 85% of intravenously injected p a r t i c u l a t e labeled cholesterol i n the rat within t h i r t y minutes of i n j e c t i o n . ( I t has been shown - 81 -i n many animals that from 70% to v i r t u a l l y 100% of an i n t r a -venously-injected dose of p a r t i c u l a t e material i s removed "by the r e t i c u l o e n d o t h e l i a l c e l l s of the l i v e r (Benacerraf, 1964).) I t has also been demonstrated by G-ans (1968) i n dogs and by Nilsson and Zilversmit (1972) i n rats that engulfed radio-active cholesterol begins to reappear i n the blood soon a f t e r the i n i t i a l phagocytosis of the labeled compound, and that maximal s p e c i f i c a c t i v i t y i s achieved i n the blood ten to twenty-four hours aft e r the cholesterol i n j e c t i o n . Loss of r a d i o a c t i v i t y from the l i v e r into the blood probably accounts for the low values obtained f o r the percentage of injected dose of l a b e l found i n the hepatic pool eighteen hours a f t e r intravenous i n j e c t i o n of the cholesterol (Table 16). In measuring the excretion rate of injected l a b e l i t was assumed that no excreted r a d i o a c t i v i t y had been absorbed from the i n t e s t i n a l lumen during the eighteen hours of the test. The excretion rate of cholesterol may therefore have been under-estimated i n t h i s experiment. In t h i s regard, however, i t should be mentioned that the values obtained f o r the percentage of the injected 4-1*0-cholesterol which was excreted i n the b i l e within eighteen hours (Table 16) agree well, upon i n t e r p o l a t i o n , with the r e s u l t s of r a t experiments reported by Wilson (1962), I r i t a n i and Takeuchi (1957), and Borgstrom (1968). Since only the r a d i o a c t i v i t y i n t o t a l l i p i d ex-tracts was measured, no information was obtained as to the trans formations undergone by the absorbed cholesterol. The pur-- 82 -pose of t h i s experiment, however, was to monitor the i n t e s t i n a l absorption and the l i v e r retention of the orally-administered compound. Using cholesterol labeled i n the s t e r o i d nucleus, the problem of loss of r a d i o a c t i v i t y as 1 4C02 from within the tissues of the experimental animals was eliminated as shown i n rats by Chaikoff et _aL. (1952) and i n man by Hellman et a l . (1955). There remains the p o s s i b i l i t y of destruction of the s t e r o i d r i n g system by the i n t e s t i n a l microflora as i n d i -cated i n human subjects by Grundy et el. (1968). I f such destruction were s i g n i f i c a n t i t would reduce the estimate of absorption e f f i c i e n c y of cholesterol. In order to minimize possible l o s s of l a b e l caused by microbial degradation of the s t e r o i d nucleus, the GIT + E samples were obtained and frozen as quickly as possible. The birds were subjected to a starvation period immediately before the oral doses of cholesterol were given i n order to induce them to eat soon aft e r receiving the dose. I t was hoped, i n t h i s way, to allow mixing of the o i l and chol-esterol with the diet in the upper digestive t r a c t and thus to avoid the unphysiological presence of a large o i l y mass i n the small i n t e s t i n e . In view of the documented effects of prolonged f a s t i n g and subsequent refeeding upon hepatic chol-esterol synthesis i n the chicken (Goodridge, 1968; L e v e i l l e , 1969) the feed deprivation time employed i n t h i s experiment was kept as short as was thought would be e f f e c t i v e . I t was observed that most birds were eating within t h i r t y minutes of the oral dosing treatment. I t i s possible, however, that - 83 -absorption was hindered by t h i s method of administering the cholesterol as has been shown to be the case f o r vitamin A i n young chickens (Vakil jet a l . , 1964). I t i s considered, nonetheless, that a v a l i d comparison of absorptive e f f i c i e n c i e s of cholesterol i s possible among the various groups of birds even i f the absolute values obtained f o r the absorption e f f i -ciency have been depressed by the experimental procedure. The present experiment shows that an excess of v i t a -min A increases the i n t e s t i n a l absorption e f f i c i e n c y of chol-esterol i n both the D and the NH chicks. The l i v e r i s the main organ responsible f o r removing newly-absorbed cholesterol from the blood, the removal being v i r t u a l l y complete (Biggs, 1952; Borg-strom et a l . 1958; Byers, 1964). Within several hours a f t e r entering the l i v e r , newly-absorbed cholesterol has mixed homo-geneously with the endogenous hepatic pool (Byers, 1964) and i s therefore i n rapid exchange with the blood compartment (Landon and Greenburg, 1954; Andrews et a l . , 1968). The calculated values for the amount of absorbed cholesterol found i n the hepatic pool i n t h i s experiment, therefore, represent the net quantities of exogenous cholesterol which were held from the blood compartment within the twenty-four hour period of the test. Vitamin A increased the percentage of absorbed chol-esterol i n the hepatic pool of the NH chicks but decreased t h i s retention i n the hepatic pool of the D chicks. I t i s concluded that the divergent alimentary cholesterolamic responses of the two populations of birds to excesses of vitamin A re-s u l t , not from a d i f f e r e n t response i n the i n t e s t i n a l absorp-- 84 -t i o n of cholesterol, but from a d i f f e r e n t response i n the hep-atic-to-extrahepatic p a r t i t i o n r a t i o of cholesterol. This inte r p r e t a t i o n i s consistent with the suggestion of riyers and Friedman (1966) that an increase i n the plasma cholesterol concentration (and presumably, therefore, also a decrease i n the plasma cholesterol concentration) can r e s u l t purely from a s h i f t i n cholesterol p a r t i t i o n between the blood and the l i v e r . The increased b i l i a r y excretion of cholesterol seen i n the NH cockerels i n response to excess vitamin A may be simply a r e f l e c t i o n of the increase i n cholesterol retention i n the hepatic pool of this l i n e of birds i n response to excess vitamin A. Examination of Tables 15 and 16 reveals that, i n every instance, the values representing cholesterol absorption e f f i c i e n c y and d i s t r i b u t i o n of absorbed cholesterol f o r the birds receiving oral dose3 of vitamin A d i f f e r i n the same dir e c t i o n from the control values as those of the birds re-ceiving the intramuscular vitamin A i n j e c t i o n s . A f t e r enter-ing the c i r c u l a t i o n , vitamin A i s transported and distributed i d e n t i c a l l y whether administered o r a l l y or parenterally. I t i s considered, therefore, that the f a i l u r e of the birds receiv-ing the oral doses of vitamin A to show a difference from the controls may have resulted from i n s u f f i c i e n t time having been allowed f o r the vitamin to take e f f e c t . I t should be noted, also, that the dose administered o r a l l y was much smaller than that administered parenterally. Perhaps the s i g n i f i c a n t e f f e c t of the oral dose of vitamin A on the hepatic pool of choles-- 85 -t e r o l i n the NH cockerels (Appendix Table 8c) r e f l e c t s a greater vitamin A absorptive capacity i n these birds than i n the D chicks. This p o s s i b i l i t y i s suggested by the results of exp-eriment 1. which showed that the NH chicks are more e f f i c i e n t than the D chicks i n storing dietary excesses of vitamin A i n the l i v e r . Since only the parenteral doses of vitamin A affected the i n t e s t i n a l absorption e f f i c i e n c y of cholesterol, i t i s concluded that the effect occurs within the i n t e s t i n a l wall rather than i n the lumen of the i n t e s t i n e . The p o s s i b i l i t y -enhanced enterohepatic c i r c u l a t i o n of vitamin A i n the birds receiving parenteral doses of the vitamin i s considered un-l i k e l y to be of quantitative significance as a factor i n the lumen. Sylven and Bb'rgstrom (1968) have proposed that the rate-limiting step i n cholesterol transport from the i n t e s t -i n a l mucosa may be the formation of a cholesterol-containing l i p o p r o t e i n i n the mucosal c e l l . Most of the cholesterol i s e s t e r i f i e d i n t h i s process, primarily to unsaturated f a t t y acids (White et a l . , 1968; Treadwell and Vahouny, 1968). In t h i s regard, i t i s i n t e r e s t i n g that large parenteral doses of r e t i n o l have been shown to increase the turnover rate of free fa t t y acids i n the rat small i n t e s t i n e (Misra, 1965). Further-more, the rate of protein synthesis on the rough endoplasmic reticulum of the rat small i n t e s t i n e has been shown to be influenced by r e l a t i v e l y small changes i n the vitamin A status (DeLuca et a l . , 1969). This l a t t e r f i nding could implicate - 86 -vitamin A i n the synthesis of the l i p o p r o t e i n c a r r i e r f o r cholesterol. Furthermore, there i s accumulating evidence of a direct role f o r vitamin A i n the synthesis of some secre-tory glycoproteins of the rat i n t e s t i n a l mucosa (DeLuca et_ a l . , 1970; DeLuca et a l . 1970 a). The glycoproteins of the mucosal surface of the small i n t e s t i n e may be important i n sequestering cholesterol from the i n t e s t i n a l lumen f o r absorp-tion from the mi c e l l a r phase (Treadwell and Vahouny, 1968) and removal of cholesterol from the mi c e l l a r phase may be the r a t e - l i m i t i n g step i n cholesterol absorption from the i n t e s t -i n a l lumen (Sylven and Borgstrom, 1968). Experiment 3 showed that there i s no net choles-terolemic response i n either the D or the NH chicks to the ingestion, over an extended period of time, of excess vitamin A with a low-cholesterol d i e t . On the basis of the published l i t e r a t u r e concerning the e f f e c t of vitamin A excesses on the size of the hepatic cholesterol pool i n rats and chickens (see "Review"), i t i s reasonable to expect that the group II birds of experiment 3 experienced a small increase in l i v e r choles-t e r o l content during the four weeks of vitamin A feeding. I t i s therefore concluded, from the r e s u l t s of t h i s experiment and of experiment 3, that the D and NH chicks d i f f e r , when i n the A hypervitaminotic state, i n the hepatic-to-blood p a r t i t i o n r a t i o of large loads of exogenous or endogenous cholesterol. I t appears that normal loads of newly absorbed cholesterol are removed from the blood by the hepatic pare-chymal c e l l s (French e^,al_., 1958; Riggi and DeLuzio, 1962). - 87 -Kupffer c e l l involvement becomes s i g n i f i c a n t , however, when large quantities of cholesterol i n physiological l i p o p r o t e i n form and when even very small quantities of cholesterol i n a r t i f i c i a l emulsion must be removed from the blood by the l i v e r (Byers, 1964; Nilsson and Z i l v e r s m i t , 1972). A divergent effect of hypervitarainosis A was observed between the D and NH chicks both i n the percentage of newly-absorbed cholesterol found i n the hepatic pool, and i n the percentage of intraven-ously-injected cholesterol found i n the hepa-tic pool. The results suggest that the difference between the two l i n e s of birds may l i e i n t h e i r a b i l i t y to hold large amounts of chol-esterol i n the Kupffer c e l l s . I t was concluded i n the d i s -cussion of .experiment 3 that the e f f e c t of vitamin A on a l i -mentary cholesterolemia must be of short duration i n both the D and the NH chicks. Florsheim et a l . (1963) have shown, i n roosters, that the h a l f - l i f e of serum B-lipoproteins, the main cholesterol-carrying l i p o p r o t e i n s i n the blood, i s of the order of only a few hours. I t i s reasonable to speculate, therefore, that the two d i f f e r e n t types of b i r d tested respond oppositely to hypervitaminosis A i n either the synthesis or the secretion of lipoproteins by the Kupffer c e l l s . Newly-absorbed vitamin A i n normal amounts i s taken from the blood by the hepatic parenchymal c e l l s . Ab-normally large quantities of the vitamin, however, are removed and held by the Kupffer c e l l s (see "Review"). As i s the case fo r abnormally large quantities of cholesterol. In view of the r e s u l t s of the present experiment i t seems possible that - 88 -greater l i v e r storage of excessive dietary doses of vitamin A by the NH than by the D chicks (experiment 1) may r e f l e c t a generally greater a b i l i t y of birds of the NH l i n e than of birds of the D s t r a i n to retain l i p i d s i n the Kupffer c e l l s . - 89 -PART II THE EFFECT OF EXCESSES OF VITAMIN A ON THE ONE-STAGE PROTHROM-BIN TIME AND THE ONE-STAGE EUGLOBULIN LYSIS TIME OF CHICKENS WITH DIFFERENT GENETIC BACKGROUNDS Experiment 5: EFFECT OF A LARGE DIETARY EXCESS OF VITAMIN A ON THE ONE-STAGE PROTHROMBIN TIMES OF THREE STRAINS OF WHITE LEGHORN CHICKENS Materials and Methods Forty day-old chicks of mixed sexes of each of the three WL strains Dekalb, Kathline, and Babcock (D, K, and B) were weighed, wingbanded and distributed into groups of twenty. The birds were housed i n e l e c t r i c a l l y - h e a t e d battery brooders and were fed and watered ad libitum. Half the birds of each s t r a i n received the control diet for which the com-position i s shown i n Table 18. The diet contained 5000 IU of vitamin A per kilogram. The test (A+) chicks were fed the control diet with additional s t a b i l i z e d vitamin A palmitate s u f f i c i e n t to supply a t o t a l of 200,000 IU of vitamin A per kilogram. The formula of the diet fed the dams of the chicks i s shown i n Table 2a. At ten days of age the birds were vac-cinated against New castle Disease.; At eighteen days of age one-stage prothrombin times were measured on blood samples from ten birds of each s t r a i n and d i e t . The blood was taken from the wing vein as described i n experiments 2 and 3. The anticoagulant was 0.1 M sodium oxalate used i n the proportion of 0.1 ml to 1 ml of whole - 90 -Table 18. Basal diet used i n experiments 5 and 6 Component % of diet Ground wheat (12.5$) 29.2 Corn meal (9.0%) 37.8 Soybean meal (48.5$) 15.0 Herring meal (72.0^) 10.0 D i s t i l l e r ' dried solubles (25.0$) 3.0 Dehydrated cereal grass (20.0%) 2.0 Bonemeal " 1.5 Limestone 1.0 Iodized s a l t (unknown hydration) 0.5 1T3TJ70" Micronutrients per kg diet -Manganese sulphate - .- 0.24 g Vitamin A palmitate 4500 IU Vitamin D 3 440 IU Riboflavin 3.6 mg Amprolium 0.13 g Calculated Analysis: $ Protein = 22.7 Metabolizable Energy = 301-8 kcal/kg Total Vitamin A (lU/kg) z 5000 - 91 -blood. The c l o t t i n g agent used i n the prothrombin time determinations was prepared from the breast muscle of a control b i r d of the D s t r a i n which was k i l l e d by bleeding from the jugular vein. Five grams of the muscle was ground i n a mor-tar and pestle with Ottawa sand and about 10 mis. of 0.9% sodium chloride solution. The slurr y was then centrifuged at 3000 X G f o r ten minutes at 0 oto 4°C, and the supernatant was brought to a volume of 100 mis with 0.9% sodium chloride solution. Then 100 mis of 0.025 M calcium chloride was added and the cloudy suspension was mixed and allowed to warm for at l e a s t one hour i n a water bath regulated to 39°C. More con-s i s t e n t r e p l i c a t i o n s of prothrombin times were obtained i f the c l o t t i n g agent was permitted to stand f o r t h i s period i n the water bath. For the prothrombin time measurement, 0.1 ml aliquots of the oxalated whole blood were pipetted into pre-warmed, c y l i n d r i c a l , flat-bottomed v i a l s 15 mm X 50 mm. The v i a l s containing the blood samples were held i n the water bath f o r about ten minutes. To each v i a l , i n turn, 0.2 ml of the warmed c l o t t i n g agent was then added, and the v i a l was quickly shaken and t i l t e d back and f o r t h to an angle of about 45° at the rate of approximately one second per complete oycle. A stop-watch calibrated i n units of 0.01 min. was used to re-cord the time between the addition of the c l o t t i n g agent and the formation of a c l o t i n the bottom of the v i a l . Readings were done i n duplicate on each sample of blood and were i n t e r -- 92 -polated to the nearest 0.005 min. on the stop-watch. The con-centration of c l o t t i n g agent was adjusted so as to give con-t r o l times of twenty-five to t h i r t y seconds i n order to obtain a sensitive comparison among the various groups of b i r d s . Results The average three-week body weights of the birds are shown i n Table 19. The one-stage prothrombin times ob-tained at eighteen days of age are shown i n Table 20. There was no effect a t t r i b u t a b l e to the large dietary excess of vitamin A on the one-stage prothrombin times of any of the three strains of birds, (appendix, Table 15). Furthermore, no s t r a i n differences i n one-stage prothrombin times were evident when the values for the control birds of each s t r a i n were compared by analysis of variance (appendix, Table 15 d). Discussion The re s u l t s of t h i s experiment confirm the f i n d i n g of Quick and Stefanini (1948) that; one-stage prothrombin times of young chickens are not affected by large dietary excesses of vitamin A fed f o r a period of weeks. Biggs and Douglas (1953) have stated thatsimple hypoprothrombinemia may not always cause a prolongation of one-stage prothrombin times but that a hemorrhagic tendency i s l i k e l y to r e s u l t , JMO hemor-rhages were seen during the course of t h i s experiment even i n birds which were maintained on the diets up to twelve weeks of age. Furthermore, there was no apparent effect of the largs excesses of vitamin A on the a b i l i t y of the birds, f o r which - 93 -Table 19. Experiment 5: Average three-week body weights (+ standard deviation) Level of dietary vitamin A (IU/kg) 5000 - . 800,000 Type of Chicks Body weight (g) D K B 206220 195217 203120 197223 2002.25 201221 - 94 -Table 20. Experiment 5: Average one-stage prothrombin times (t standard deviation) at three weeks of age Level of dietary Vitamin A (IU/kg) 5000 200,000 Type of chicks One-stage prothrombin time (min.) D 0.51*0.05 0.55+0.10 K 0.5610.05 0.54±0.08 B 0.5ll0.04 0.54+0.07 - 95 -prothrombin times were measured, to form a c l o t i n the wound from which blood was obtained. Both diffuse hemorrhaging and an increased one-stage prothrombin time are c h a r a c t e r i s t i c of hypervitaminosis A i n r a t s , but are only occasionally seen i n man (see "Review"). Perhaps the chicken i s even more refractory than i s man to these p a r t i c u l a r symptoms of hypervitaminosis. Josephs (1944) has stated that rats and mice are p a r t i c u l a r l y susceptible to the effects of overdosage with vitamin A. In any case, no influence of large dietary excesses of vitamin A was seen on the effectiveness of either the i n t r i n s i c or the e x t r i n s i c c l o t t i n g mechanism of the birds i n th i s experiment, i t i s relevant, however, that i n each group of birds receiving the large excess of vitamin A, one or two had noticeably longer pro-thrombin times than did the others. This observation suggests that the response of the one-stage prothrombin time i n chick-ens to excess vitamin A may be the same as the response i n rats except that i t i s much l e s s severe. The c l o t t i n g agent used was a crude, unstandard-ized tissue extract. Therefore other sets of measurements such as those obtained i n experiments 6 and 7, or those re-ported i n the l i t e r a t u r e (eg. Quick and S t e f a n i n i , 1948; Stopforth, 1970) cannot be meaningfully compared i n absolute value to the prothrombin times reported i n t h i s experiment. I\io gross toxic e f f e c t of the large excess of v i t a -min A was evident at eighteen days of age. The body weights of a l l birds were normal (NRC, 1971). - 96 -Experiment 6: EFFECT OF MODERATE AND LARGE EXCESSES OF VITAMIN A INTAKE ON THE ONE-STAGE PROTHROMBIN TIMES AND THE EUGLOBULIN LYSIS TIMES OF THREE BREEDS OF CHICKENS Materials and Methods One hundred-twenty day-old chicks, of each of the WL (D s t r a i n ) , NH, and Black Australorp (A) breeds were wing-banded and weighed. The chicks, of mixed sexes, were held i n e l e c t r i c a l l y - h e a t e d battery brooders i n groups of t h i r t y , ten birds of each breed being held i n each compart-ment. Feed and water were available _ad libitum. At ten days of age the birds were vaccinated against Newcastle Disease. The formula of the control diet i s shown i n Table 18 and the diet contained 5000 IU of vitamin A per kilogram. The three test diets contained 35,000 IU (R2), 100,000 IU (R3), and 200,000 IU (R4) of vitamin A per kilogram of d i e t . Each of the four diets was fed to t h i r t y birds of each breed from one day of age. The diets fed the dams of the WL, NH, and A chicks are shown i n Tables 2a, 2b, and 2c respectively. When the birds were three weeks old, one-stage prothrombin time measurements were conducted, as described i n experiment 5, on ten chicks of each breed and d i e t . Ten days l a t e r s i m i l a r determinations were performed on seven A chicks r e c e i v i n g the control d i e t and on seven, each, receiv-ing the R3 and R4 diets. As a spot check, at t h i s time pro-thrombin time determinations were also carried out on three D - 97 -chicks receiving the control diet and on three receiving the R4 d i e t . Also, at t h i s same time, seven A chicks (four re-ceiving S3 and three receiving R4) were injected intramuscu-l a r l y with 5 mg of menadione i n soybean o i l per kilogram of body weight. S i m i l a r l y , seven chicks (four receiving R4 and three receiving R3) were injected with the quantity of c a r r i e r soybean o i l which they would have received had they been i n the group treated with menadione. Twenty hours aft e r the i n -jections were completed one-stage prothrombin determinations were conducted on each of the b i r d s . At the same time, simi-l a r determinations were carried out on seven A chicks receiv-ing the control diet, but which had received no intramuscular i n j e c t i o n of either menatione or pure c a r r i e r o i l . The i n j e c t i o n s of menadione were made into the breast muscle using a tuberculin syringe and a #27 gauge needle. The soybean o i l solution used contained 15 mg of menadione per ml so that each b i r d received a volume of ap-proximately 0.1 ml depending on i t s body weight. When the birds were s i x weeks of age one-stage euglobulin l y s i s time (ELT) measurements were conducted on blood samples from ten birds of each breed and d i e t . Due to the necessity of handling the blood quickly f o r the ELT measure-ments, the determinations were carried out over a period of three days, those of one breed being completed each day. Blood samples were obtained between 1:00 P.M. and 2:00 P.M. each day and t h i s procedure was adhered to f o r a l l subsequent ELT measurements i n t h i s and the next experiment. - 98 -Ten birds of each, breed and each diet were then injected with 5 mg of menadione per kilogram of body weight as described before, and ten were injected with the soybean o i l c a r r i e r , only. Twenty-four hours l a t e r ELT measurements were again conducted. As previously, i t was necessary to space the ELT determinations (and therefore, i n t h i s case, the i n j e c t i o n s as well) to allow rapid handling of the blood. In a single day only h a l f the measurements for the birds of any breed were completed. Determinations were conducted each day on f i v e menadione-treated birds from each d i e t , and on f i v e o i l - i n j e c t e d birds from each d i e t . The determinations therefore extended over a period of six days, and the birds were about seven weeks bid when t h i s part of the experiment was completed. As a control f o r the technique, euglobulin l y s i s times were also determined on the blood obtained from two sheep by bleeding from the jugular vein. The ELT procedure followed was a modification of that of Von Kau l l a (1963). One ml of blood was coll e c t e d from the wing vein of each b i r d as described i n experiments 2,3 and 5. P r e - c h i l l e d s i l i c o n i z e d tubes containing 0.1 ml of 0.1M solium oxalate were used. The tubes were held i n crushed i c e u n t i l the plasma was obtained by spinning the blood at 1000 X G- f o r f i v e minutes i n a pre-cooled, refrigerated centrifuge held at -2° to 0°C. Then 0.2 ml of plasma was diluted to 4.0 ml in g l a s s - d i s t i l l e d water which was pre-cooled to about 4°C. The tubes of diluted plasma were held i n crushed i c e . The euglobulin p r e c i p i t a t i o n was achieved - 99 -by adding 0.G6 ml of 0. 68% acetic acid to the diluted plasma and i n v e r t i n g each tube twice against a piece of parafilm. The pH of the mixture was about 5.5. The tubes were placed i n crushed ice f o r t h i r t y minutes a f t e r which time they were spun f o r four minutes at 800 X G i n an unrefrigerated c e n t r i -fuge. The supernatant was decanted and the tubes were allowed to drain at room temperature f o r two minutes a f t e r which time they were returned to the cur shed i c e . Two ml of phosphate-saline buffer, pH 7.4,were then added to each tube, and gentle shaking redissolved the euglobulin p r e c i p i t a t e without foaming. Bovine thrombin* was then added from a g l y c e r o l - s t a b i l i z e d preparation of 1400 N I H Units of thrombin per ml. Glass rods, 2mm i n diameter, were inserted into the thrombin solution to a depth of 5 mm and were then placed into the euglobulin solu-t i o n . A f t e r c l o t t i n g , which took from twenty to f o r t y minutes (except f o r the sheep euglobulin which clotted i n ten to f i f -teen minutes) each tube was placed i n a water bath regulated to 39 °c , and the time f o r l y s i s to occur was recorded. The endpoint was taken to be l i q u e f a c t i o n of the gel with f i n e white strands, presumably of f i b r i n , f l o a t i n g i n the f l u i d (the c r i t e r i o n of Simmons, 1968). A l l determinations were done i n duplicate, except those of the sheep blood which were done i n quadruplicate. The time elapsed from the blood c o l l e c -tion u n t i l the beginning of the incubation varied from one and one h a l f to two hours. * Parke - Davis - 100 -When the birds were eight weeks of age, the plasma fibrinogen concentration was determined on blood samples from f i v e birds of each breed and diet. Five mis of blood was taken from each b i r d by heart puncture and was placed i n tubes containing 0.5 ml of 0.1M sodium oxalate. The blood was held at room temperature u n t i l the plasma was recovered by c e n t r i -fugation at 1200 X G for f i v e minutes at 0° to 2°C. The plasma was then held at 2°C for two days before analysis. The procedure used to determine the plasma f i b r i n o -gen concentration was a modification of that of Ratnoff and Menzie (1951). Measurements on plasma samples from two sheep served as a control f o r the technique. To 25 ml of 0.9% sodium chloride solution i n a 50 ml p l a s t i c centrifuge tube were added 2 ml of 0.1% calcium chloride and 1 ml of plasma. Then 0.2 ml of a bovine thrombin solution (100 NTH Units per ml i n glycerol-saline) was added to each tube, the contents of which were mixed by inversion onto a piece of parafilm. The mixtures were allowed to stand f o r two hours and were then f i l t e r e d through plugs of glass wool approximately 1 cm i n diameter. The plugs were rinsed with two washes of 0.9% sod-ium chloride solution and were placed into 50 ml p l a s t i c cen-trif u g e tubes and dried at 40°C. Then 2 ml of 0.5% sodium deoxycholate and 8 ml of Gornall's reagent (Gornall e t . a l . 1949) were added and the contents of the tubes were sonicated fo r about one minute using a Bronwill Biosonik at a set t i n g of "60". The o p t i c a l density of the solutions was read at 540 nm t h i r t y to f o r t y minutes a f t e r the sonication, using a - 101 -Unicam SP 800 Spectrophotometer. A solution of 8 ml Gornall's reagent diluted with 2 ml of 0.5$ sodium deoxycholate served as a reference f l a s k . A l l measurements were carried out i n duplicate. As a blank f o r the entire method, the above pro-cedure was carried out on two samples of serum, one obtained from a D chick receiving the control diet and one obtained from a D chick receiving R4. A standard curve drawn from d i l u t i o n s of egg albumen i n 0.9$ NaCl was used to quantitate the o p t i c a l density readings into mg of protein per ml. of plasma. When the birds were eight weeks.of age, s i x males of each breed and diet were k i l l e d and t h e i r l i v e r s were re-moved and analyzed f o r vitamin A concentration as described i n experiment 1. In the case of the control A chicks and of the D chicks receiving R2, four males and two females were selected (since there was a shortage of male b i r d s ) , and i n the case of the A chicks receiving R3 and D and A chicks re-ceiving R4, f i v e males and one female were used. The remaining D and NH birds were then fed a com-mercial b r o i l e r grower d i e t for which the guaranteed analysis i s shown i n Table 21. Six weeks l a t e r ELT determinations were performed on f i v e NH and f i v e D birds of each former dietary l e v e l of vitamin A. The procedure followed was i d e n t i c a l to that described e a r l i e r except that 5 mis of blood was c o l -lected by heart puncture. The plasma samples remaining aft e r the completion of the ELT determinations performed on the fourteen-week-old - 102 -Table 21. Experiment 6: Guaranteed analysis of the b r o i l e r grower d i e t fed the NH and D chicks from eight to fourteen weeks of age Component % of diet Protein 23 (min.) Fat 3.5 (min.) Fibre 5.0 (max,) NaCl 0.45 (actual) Calcium 0.1 (actual) Phosphorus 0.75 (actual) Vitamin A 10000 IU/kg (actual) - 103 -D and NH b i r d s , were stored under nitrogen i n the dark at -25°C p r i o r to measurement of i t s vitamin A and t o t a l carotene con-centration. The procedure followed was b a s i c a l l y that of Nee3d and Pearson (1963), with modifications being made to solvent volumes and plasma d i l u t i o n . Duplicate measuremtnts were made on each b i r d using 1 ml of plasma f o r each determination. For each determination, 1 ml of plasma was pipetted into a 12 ml glass-stoppered centrifuge tube. The plasma proteins were precipitated with 1 ml of 95% ethanol. Next 3.5 ml of pet-roleum ether (b.p. 40° - 60°) was added and the tube was shaken, by hand f o r two minutes and was centrifuged at 1500 X G for three minutes. Using a syringe, 3.0 ml of the petroleum ether (top) layer was transferred to a quartz cuvette and the o p t i -cal density was read at 450 nm against a petroleum ether blank i n a Unican SP 800 spectrophotometer. The cuvette was than placed i n a water bath at 40°C and the solvent was evaporated to dryness under a stream of general grade nitrogen gas. Immediately, 0.15 ml of chloroform and 0.15 ml of acetic an-hydride were pipetted into the dry cuvette. A f t e r placing the cuvette into the spectrophotometer, 1.5 ml of a t r i f l u o r o -acetic acid-chloroform color reagent (1:2 v/v) was added from a syringe. T h i r t y seconds a f t e r the addition of the color reagent to the plasma extract, an op t i c a l density reading was taken at 616 nm against a blank consisting of the TFA-chloro-form solution. With the d i l u t i o n s employed, the following algebraic r e l a t i o n s were used to calculate the carotene (as B-carotene) and vitamin A concentrations of the plasma sam-pl es: - 104 -a) ug carotene/100 ml plasma = O.D. 450 X 1440 b) ug vitamin A/100 ml plasma = (O.D. 616 - (O.D. 450 X 0.3)) X 950.7 The correction f a c t o r of O.D. 450 X 0.3 was taken from the publication of Neeld and Pearson (1963). I t i s the absorb-ance at 616 nm which i s contributed by the carotene i n the plasma extract, and i s i n error insofar as i t i s taken from a standard absorbance curve of pure B-carotene with TFA i n chloro-form. Results Table 22 shows the average day-old, two-week, f i v e -week, six-week, and eight-week body weights of the chicks of each breed and diet. Growth depression was apparent by the eighth week i n the NH (p<0.01) and D (pO.05) chicks receiving R3 and R4 (appendix, Table 16). The growth rate of the A chicks receiving R3 and R4 was not d i f f e r e n t from that of the A chicks receiving either the control d i e t or R2 as i s clear upon inspection of Table 22. The prothrombin time measurements obtained on the birds at three weeks of age are presented i n Table 23 f o r each breed and d i e t . As shown i n Table 17 of the appendix, the A chicks receiving the R3 and R4 diets had prolonged one-stage prothrombin times (p<0.01) compared to the birds receiv-ing the control and R2 di e t s , but no such e f f e c t of large d i e t -ary excesses of vitamin A was seen i n either the D or the NH chicks. A breed difference also appeared i n prothrombin time - 105 -Table 22. Experiment 6: Average body weights (1 standard deviations) at one day, two weeks, f i v e weeks, s i x weets and eight weeks of age Level of dietary vitamin A (IU/kg) 5000 35000 100,000 200,000 Type of Body weight (g) Chi cks Age  1 day 43^3 43^4 43l3 4ll3 2 weeks 13lll3 13lll3 128ll9 131112 D 5 weeks 360155 353152 360148 351146 6 weeks 650lll5 663178 59ll40 581146 1 day 40l4 3913 37l3 38l4 2 weeks 129il7 127113 127ll0 130±13 NH 5 weeks 356154 359159 361162 367+58 6 weeks 56ll62 566174 546160 543152 8 weeks 824+76 824165 744190 725184 1 day 3513 34l2 35+3 35l3 2 weeks 114114 112+J.5 114ll2 m i n A 5 weeks 354137 357146 35ll46 339143 6 weeks 477144 482160 473154 483143 8 weeks 686164 675191 676158 675147 - 106 -Table 2 3 . Experiment 6 : Average one-stage prothrombin times (2 standard deviation) at three weeks of age Level of dietary vitamin A (IU/kg) 5000 35000 100,000 200,000 Type of chicks One-stage prothrombin time (min.) D 0.4120.03 0.4220.04 0.4520.06 0.4520.5 NH 0.4510.04 0.5020.06 0.5020.07 0.4820.07 A 0.4020.03 0.44±0.05 0.5610.10 0.5420.10 - 107 -(Table 18 of the appendix}. The NH chicks proved to have longer prothrombin times than both the D (p<0.05) and the A (p<0.05) chicks, when the control birds were compared. The prothrombin times of the A and D chicks at thirty-one days of age are shown i n Table 24. Presented i n Table 25 are the prothrombin times obtained on the A chicks twenty hours a f t e r they were injected with menadione. As showi i n Table 19 of the appendix, the prolongation of prothrombin time observed at three weeks of age i n the A chicks receiving the R3 and R4 diets, persisted to thirty-one days of age i n these birds (p<0.01). Again, no e f f e c t of excess vitamin A was apparent on the prothrombin times of the D chicks. Appendix Table 20 shows that the intramuscular menadione i n -jections reduced the prolonged prothrombin times of the A chicks receiving the R3 and R4 diets to the control value with-i n twenty hours. In Figures l a , b, and c are shown graphs of the percent c l o t l y s i s f o r the birds of each breed and die t at six weeks of age. Since the c l o t l y s i s times of the birds receiving the control and R2 diets were s i m i l a r , the results with these two groups were pooled as were the res u l t s with the birds receiving the R3 and R4 d i e t s . The values of a l l three breeds were pooled since no breed difference was evident, and the r e s u l t i n g curves are shown i n Figure Id. The pooled percent l y s i s data f o r the three breeds was analyzed by X 2 according to Snedecor (1946) at three, four, f i v e and six hours of incubation as shown i n Table 21 of the appendix. - 108 -Table 24. Experiment 6: Average (2 standard deviation) thirty-one days of age one-stage prothrombin times of the D and A chicks a t Level of dietary vitamin A (IU/kg) 5000 100,000 200,000 Type of Chicks One-stage prothrombin time (min.) D 0.5320.04 NOT TESTED 0.5420.06 A 0.5120.07 0.9620.15 1.082-0.25 - 109 -Table 25. Experiment 6: Average one-stage prothrombin times (1 standard deviation) of the A chicks at thrity-two days of age showing the eff e c t of intramuscular mena-dione i n j e c t i o n on the birds receiving 100,000 IU and 200,000 IU of vitamin A per kilogram of diet Level of dietary Vitamin A (IU/kg) Injection t r e a t -ment One-stage prothrombin time (min) 5000 none 0.39*0.02 100,000 and 200.000 (pooled sample) 5 mg menadione i n -tramuscularly/kg body weight 0.42+0.03 100,000 and 200.000 (pooled sample; soybean o i l i n tramu s cul a r l y 0.81+0.13 - 110 -Figure 1. Experiment 6: Percent l y s i s vs. incubation time at six weeks of age O - Control 4 R2 A - R3 • R4 incubation time (hrs.) incubation time (hrs.) - I l l -Figure 1. cont'd <f0 l y s i s incubation time (hrs.) d) Three breeds combined (numbers i n brackets re-f e r to the number of lysed clots out of 60) incubation time (hrs.) - 112 -A l a r g e r f r a c t i o n of the c l o t s from the birds receiving the large excesses of dietary vitamin A had shown complete l y s i s by four, f i v e and six hours of incubation than was the case for those from the birds receiving the control diet or the moderate excess of vitamin A (p<0.01). In Figures 2a, b, and c are shown graphs of per-cent c l o t l y s i s vs incubation time f o r the D, NH and A chicks, respectively, comparing those birds injected with menadione to those which received only the soybean o i l c a r r i e r , IMO e f f e c t of menadione on the E L T i s evident i n any breed or d i e t , and a graph combining the data from the three breeds, and making the same comparison as that made i n Figures 2a, b, and c, i s shown i n Figure 2d. In Figure 3 i s shown a graph of percent c l o t l y s i s vs incubation time which was made by combining the data f o r the three breeds and ignoring the i n j e c t i o n s of mena-dione and c a r r i e r o i l . Figures 3 and Id show very s i m i l a r curves. Table 22 of the appendix contains the re s u l t s of X 2 tests comparing the pooled c l o t l y s i s values, indicated i n Figure 3, of the birds receiving the control and R2 diets to the pooled c l o t l y s i s values of the birds receiving the R3 and R4 diets at three, four, f i v e , six and seven hours of incubation. The birds receiving the large excesses of dietary vitamin A showed a greater percentage of c l o t l y s i s at three, four, f i v e , and six hours of incubation (p<0.01) than did the birds receiving the control and R2 di e t s . A difference was also evident at seven hours (p<0.05) at which time a l l the .clots made from plasma samples of R3 and R4 birds had lysed. - 113 -Figure 2. Experiment 6: Percent l y s i s vs. incubation time a f t e r i n j e c t i o n s with menadione or soybean o i l O: soybean o i l - i n j e c t e d ^•meandione-injected a) D chicks i ) control 4 R2 fo l y s i s a J £ ~r I 7 J incubation time (hrs.) i i ) R3 *R4 fo l y s i s incubation time (hrs.) - 114 -Figure 2. cont'd b) NH chicks Q=. soybean oi l - i n . j ected ^ z menadione-in.j ected i ) control 4 R2 i i ) R3 f R4 incubation time (hrs.) - 115 -Figure 2. cont'd c) A chicks soybean o i l - i n j e c t e d menadione-injected i ) control 4 R2 % l y s i s incubation time (hrs.) i i ) R3 4 R4 incubation time (hrs.) - 116 -Figure E. cont'd d) Three breeds combined O = R3 R4 A-"control 4 R2 Numbers i n brackets r e f e r to the number of lysed c l o t s out of i menadione-injected birds incubation time (hrs.) i i ) soybean oil-in.]*ected birds incubation time (hrs.) - 117 -Figure 3: Experiment 6: Percent l y s i s vs. incubation time three breeds combined and i n j e c t i o n treatments of soybean o i l and menadione combined within diets (Numbers i n brackets r e f e r to the number of lysed cl o t s out of 60) Q- control 4 R2 ^ . R3 + H4 incubation time (hrs.) - 118 -The results of the plasma fibrinogen determinations, including those of the two sheep, are shown i n Table 26. An-a l y s i s of variance (appendix Table 23) shows a breed d i f f e r -ence i n plasma fibrinogen concentration, the A chicks having a higher concentration at a l l l e v e l s of vitamin A supplemen-tation than the D and the NH chicks, (p«0.05). Also the large excesses of vitamin A (R3 and R4) caused an increase i n plasma fibrinogen concentration i n a l l three breeds (p<0.001). The difference i n plasma fibrinogen concentration between the A chicks receiving the control diet and those receiving the R2 diet was not s t a t i s t i c a l l y s i g n i f i c a n t (appendix Table 24). The l i v e r vitamin A concentrations of the bir d s at eight weeks of age are shown i n Table 27. The analysis of variance shown i n Table 25 of the appendix revealed a s i g n i -f i c a n t effect on the liver- v i t a m i n A concentrations of both the l e v e l of dietary vitamin A intake (p<0.001) and the breed of chicken (p<0.001). An i n t e r a c t i o n of breed and l e v e l vitamin A supplementation also appeared (p<0.001). At the lower dietary l e v e l s of vitamin A (control and R2 diets) the NH chicks appeared to accumulate greater l i v e r concentrations of the vitamin than did the other two breeds. At the higher l e v e l s of vitamin A intake, the A chicks accumulated the l a r g e r l i v e r concentrations of the vitamin. The graphs of l i v e r concentration of vitamin A vs. dietary vitamin A l e v e l are shown for each breed i n Figure 4 and c l e a r l y show the d i f -ferences i n l i v e r vitamin A concentration and the in t e r a c t i o n - 119 -Table 26. Experiment 6: Plasma fibrinogen concentrations ( t standard deviation) at eight weeks of age Level of dietary vitamin A (IU/kg) 5000 35000 100,000 200,000 Type of Plasma fibrinogen concentration (mg/ml) Chicks  D 2.6*.0.5 3.1+.1.0 4.8+.1.9 5.0+-1.3 NH 2.8+0.6 3.3±0.9 4.4^1.3 4.7+.1.2 A 3.6^0.6 5 . i l l . 6 5.7*2.7 5.8+1.4 sheep: 3.310.6 mg/ml plasma - 120 -Table 27. Experiment 6: Liver vitamin A concentrations (1 standard deviation) at eight weeks of age Level of dietary vitamin A (IU/kg) 5000 ' 35000 lOO-gOO© 200,000 Type of Vitamin A concentration (IU/g dry l i v e r weight) Chicks  D 627*76 39571542 92831.923 10,888±1537 NH 7 9 8 l l l l 48701579 10675*579 14,502ll746 A 622193 4318J638 11666+1157 18,97212573 - 121 -Figure 4 . Experiment 6: L i v e r vitamin A concentration vs. dietary vitamin A l e v e l at seven weeks of age O - D chicks A - NH chicks Q- A chicks vitamin A l e v e l i n diet (IU/kg) - 122 -of breed with .level of vitamin A supplementation revealed by the analysis of variance. Figure 5 presents the graphs of euglobulin l y s i s time versus incubation time comparing the birds formerly re-ceiving either the control or the R2 diet to those formerly receiving either the R3 or the R4 diet a f t e r a l l the birds had been fed b r o i l e r grower f o r s i x weeks. The data for the D and NH chicks i s combined i n the plots shown since, as before, no breed difference was evident. The graphs are very s i m i l a r to those obtained from the same populations of birds six weeks e a r l i e r before they were fed the b r o i l e r grower d i e t . The birds formerly receiving the large excesses of dietary vitamin A showed a greater percentage of complete c l o t l y s i s by four, f i v e , and six hours of incubation than did the birds formerly receiving the control and R2 diets (p<0.01) as indicated by the X 2 values presented i n Table 26 of the appendix. In Table 28 are shown the plasma carotene and vitamin A concentrations of the birds at the time of the l a s t ELT determinations. JMo difference i n plasma vitamin A con-centration i s evident comparing the birds formerly receiving the control and R2 diets to those formerly receiving the large excesses of vitamin A (appendix, Table 27a). This i s alSO true of the plasma carotene concentrations (appendix Table 27b). Discussion The prothrombin time data taken at three weeks of age on the D and NH chicks and at thirty-one days of age - 123 -Figure 5. Experiment 6: Percent l y s i s vs. incubation time. NH chicks and D chicks combined a f t e r six weeks being fed the b r o i l e r grower d i e t . (Numbers i n brackets re f e r to the number of lysed clots out of 20) 0« control + R2 (formerly) 4*R3 4-R4 (formerly) Incubation time (hrs.) - 124 -Table 28. Experiment 6: Plasma vitamin A and carotene concen-trat i o n s ( + standard deviation) of the D and NH chicks at fourteen weeks of age a f t e r being fed the b r o i l e r grower diet (table 21) f o r six weeks Level of Plasma carotene con- Plasma vitamin A con-dietary centration (ug/lOOml) centration (ug/100 ml) vitamin A D chicks NH chicks D chicks NH chicks f o r f i r s t eight weeks (IU/kg) 5000 35,000 100,000 200,000 194122 183118 183t31 193+30 205125 200±32 191+20 197+25 37.4+9.8 40.9t7.0 39.0+8.4 36.217.7 40.2+8.6 42.7^9.1 35.7+8.4 41.9+10.1 - 125 -on the D chicks, confirm the results of experiment 5. The A chicks receiving the two highest l e v e l s of vitamin A, however, had prolonged one-stage prothrombin times at three weeks of age, and the e f f e c t of the excess vitamin A seemed to have become more pronounced by thirty-one days of age (Tables 22 and 23). i t must be remembered, however, that d i f f e r e n t c l o t t i n g agents were necessarily used for the determinations on the birds at trie two d i f f e r e n t ages, so that the values obtained are not d i r e c t l y comparable. The extract used for the determinations on the thirty-one-day-old birds was apparently les s active than that used for the three-week-old birds, as indicated by a comparison of the control prothrombin times obtained i n the two separate t e s t s . Examining the prothrombin time data of the i n -dividual birds, i t was evident that, at the two highest l e v e l s of vitamin A supplementation, one or two of the D and NH chicks from each diet had prolonged prothrombin times whereas seven or eight of the A chicks from each of the two diets were affected by the excess of vitamin A. From the r e s u l t s of t h i s and the previous experiment, i t i s suggested that the response i n one-stage prothrombin time to excesses of vitamin A i s the same i n the D,B,K and NH chicks as i t i s i n the A chicks, but that the response i s l e s s severe than i n the A chicks. Since the NH chicks had the longest control prothrombin times and the D and A control chicks did not d i f f e r i n prothrombin time, i t i s concluded that the response of prothrombin time to excess vitamin A i s not related to the normal time c h a r a c t e r i s t i c - 186 -of the population of chickens. i n mammals, a prolonged one-stage prothrombin time can r e s u l t from a decreased c i r c u l a t i n g concentration of prothrombin, Factor VII, or Factor V (Biggs, 1955: Q,uick, 1961). In birds, however, the amount of Factors V (Didisheim et a l . , 1959; Stopforth, 1970) and VII (Stopforth, 1970) i n the blood i s very small so that an increase i n the one-stage time can be interpreted unambiguously as having resulted from a decreased plasma concentration of functional prothrom-bin. I t i s therefore concluded that large dietary excesses of vitamin A caused a hypoprothrombinemia i n the A chicks used i n the present experiment. The rapid menadione-reversi-b i l i t y of the effect further confirms this conclusion. The mechanism of the eff e c t of excess vitamin A on the prothrombin time i s not elucidated by the results ob-tained i n t h i s experiment, but speculation i s possible. Pro-thrombin i s a secretory glycoprotein, vitamin A has recently been implicated i n the glycosylation steps of secretory glyco-protein synthesis ( a l b e i t i n the small i n t e s t i n e and not i n the l i v e r ) (DeLuca &t a l . , 1970, 1970a). In reviewing the l i t e r a t u r e to date, Suttie (1973) has pointed out that vitamin K-deficient c a t t l e can glycosylate and secrete into the blood an i n a c t i v e precursor of prothrombin. (Losito (1965), how-ever, could not f i n d a c i r c u l a t i n g preprothrombin i n the chick.) Furthermore, the vitamin K-dependent step i n the production of active prothrombin i s located p r i o r to the glycosylation steps (Suttie, 1973). Perhaps, then, excessive quantities - 127 -o f v i t a m i n A cause an i n c r e a s e i n the r a t e of g l y c o s y l a t i o n of i n a c t i v e prothrombin precursor, and t h i s molecule i s , i n e f f e c t , constructed and secreted so r a p i d l y i n hypervitaminosis A that a l a r g e p r o p o r t i o n of the molecules produced f a i l to be a c t i v a t e d by v i t a m i n K. T h i s suggestion i s reminiscent of the general conclusion drawn by Greaves (1972) t h a t a n t i -coagulants a n t a g o n i s t i c to v i t a m i n K f u n c t i o n by l i m i t i n g the a v a i l a b i l i t y of the v i t a m i n f o r i t s a c t i v e s i t e on the prothrombin precursor molecule. N e i t h e r hemorrhages nor n o t i c e a b l e inflammation were seen i n any of the b i r d s r e c e i v i n g the two highest l e v e l s of d i e t a r y v i t a m i n A. T h i s observation c l e a r l y separates the two phenomena of a c l o t t i n g defect and a defect i n vascu-l a r i n t e g r i t y . I t should be mentioned that the prolonged pro-thrombin times i n the A chicks r e f l e c t e d a r e a l defect i n the c l o t t i n g mechanism. Bl e e d i n g was d i f f i c u l t to stop upon ob-t a i n i n g blood samples from some of the b i r d s of t h i s breed which were r e c e i v i n g the l a r g e excesses of d i e t a r y vitamin A. In the ELT procedure of von K a u l l a (1963) the eu-g l o b u l i n p r e c i p i t a t e i s obtained by bubbling COg through the d i l u t e d plasma f o r t h i r t y seconds, t i e d l i n and Monkhouse (1969) have shown that the pji achieved i n the d i l u t e d plasma i s an important f a c t o r i n determining the composition of the eu-g l o b u l i n p r e c i p i t a t e . I t was f e l t that the d e s i r e d pH could be more un i f o r m l y achieved by using measured q u a n t i t i e s of a d i l u t e a c e t i c a c i d s o l u t i o n r a t h e r than by using COg gas as - 128 -suggested by vol K a u l l a (1963). The author was unable to f i n d published ELT values f o r any avian species, and for t h i s reason, the procedure used was checked using samples of sheep blood. The values of 2 and Z-\ hours obtained for the sheep are i n good agreement with those found by Irfan (1968) f o r thi s species. The clot l y s i s times of 4 to 8 hours obtained i n t h i s experiment for the control chickens, seem long i n comparison to the l y s i s times for other animals (Irfan, 1968: Hedlin et a l . , 1971) and man (Kowalski et. a l , 1959; Fearnley 1965; Hedlin et a l . , 1971). An uncontrolled difference between the procedure as run f o r the sheep and f o r the chickens i s i n the c l o t t i n g time of the euglobulin solution with bovine thrombin. Before the values obtained for the chickens can be accepted as true, i t must be shown that the long c l o t t i n g time was without s i g -n i f i c a n t e f f e c t on the r e s u l t s . Since c i r c u l a t i n g plasmino-gen activator i s unstable u n t i l adsorbed onto f i b r i n (Fearnley 1953, 1965) i t i s quite possible that a certain proportion of the activ a t o r recovered i n the euglobulin p r e c i p i t a t e was l o s t during the c l o t t i n g procedure. This p o s s i b i l i t y does not, however, negate the value of the data f o r making compari-sons among the birds receiving the various l e v e l s of vitamin A supplementation. The large excesses of vitamin A markedly decreased the ELT's i n a l l three populations of birds tested. This, i n general, confirms the i n vivo (Loscialpo, 1959) and i n v i t r o ( T o r s e l l i n i and Lombardi, 1971) findings i n human sub-- 129 -j e c t s . The present experiment also shows that the effect of vitamin A on the ELT i s not through an induced vitamin K de-f i c i e n c y and, furthermore, that the ELT of chickens i s inde-pendent of vitamin K status of the animals over a wide range. The l a t t e r f i n d i n g i s consistent with most of the l i t e r a t u r e on prothrombin antagonists i n man (Fearnley, 1965). A f t e r s i x weeks of feeding the b r o i l e r grower diet, the cl o t l y s i s times of the birds formerly receiving the two largest excesses of vitamin A had not increased to the control range. Since the plasma vitamin A concentrations of the A hypervitaminotic birds were not d i f f e r e n t from those of the control birds at t h i s time (and were within the normal range for domestic chickens according to Koch (1973), the effect of excess vitamin A on the ELT i s concluded to be at l e a s t p a r t l y through the accumulation of the vitamin i n some extravascular tissue or t i s s u e s . Furthermore, since the feeding of 35,000 IU of vitamin A per kilogram of diet f o r six weeks was without effect on the ELT, i t would seem that a threshold mechanism of some sort i s operative. Since the birds receiving 35,000 IU of vitamin A per kilogram of diet would be expected to have an elevated plasma vitamin A ester concentration, the present experiment also suggests that, unlike the conclusion indicated i n man by the work of Loscialpo (1959) and of T o r s e l l i n i and Lombardi (1971), the blood vitamin A ester and alcohol con-centration has no d i r e c t effect on the ELT i n chickens. The studies reported on human subjects have been of short duration and i t i s possible that there might be a long?term e f f e c t of - 130 -vitamin A on the ELT i n man s i m i l a r to that found i n the chicken i n t h i s experiment. Plasma fibrinogen concentrations were measured i n order to check the p o s s i b i l i t y that the effect of excess v i t a -min A was to decrease the quantity of substrate precipitated i n the euglobulin f r a c t i o n rather than to increase the a c t i v i t y of a part of the f i b r i n o l y t i c system. The importance of the fibrinogen concentration i n the one-stage ELT has been empha-sized by Hougie (1972). The data obtained (Table 26) show that the quantity of substrate was actually increased by the vitamin A excesses, assuming no e f f e c t of the vitamin on the recovery of fibrinogen i n the euglobulin p r e c i p i t a t e . I t remains possible that vitamin A might a f f e c t the process of f i b r i n formation, i t s e l f . Lorand and Jacobsen (1962) have shown f o r instance, that the presence during the formation of a clo t of i n h i b i t o r s of c r o s s - l i n k i n g accelerates the rate of c l o t l y s i s . I t should be mentioned that the values of f i b r i n -ogen concentration found for the control chickens and f o r the sheep were within the normal range f o r these species (Dukes, 1955). Singh _et _al. (1968 ; 1969) have shown i n rats that large excesses of vitamin A cause the mobilization of free f a t t y acids (presumably from adipose tissue) to the l i v e r . This could be the mechanism of the e f f e c t seen on the c i r c u -l a t i n g fibrinogen concentration since Pilgeram and Pickart (1968) have shown that saturated, long-chain free f a t t y acids - 131 -increase the rate of fibrinogen synthesis i n the l i v e r i n man. The results of the one-stage ELT procedure are p a r t i c u l a r l y sensitive to variations i n the blood plasminogen activator concentration, and are l a r g e l y independent of the i n h i b i t o r concentration and of the plasminogen or plasmin concentrations (Fearnley 1965; Pechet, 1965). Blood plasmino-gen activators are p r o t e o l y t i c enzymes (Mullertz, 1957; Fearn-l e y , 1965) . Excess vitamin A i s recognized to cause the re-lease of lysosomal enzymes from a great variety of c e l l types (Lucy, 1969). Neither i n this experiment nor i n the preced-ing work on human subjects, has the p o s s i b i l i t y been tested of a completely nonspecific e f f e c t of vitamin A on the ELT through the release of lysosomal enzymes. I t i s possible, fo r that matter, that blood plasminogen activators are of lysosomal o r i g i n ( T o r s e l l i n i and Lombardi, 1971). A further problem i n the in t e r p r e t a t i o n of the res u l t s of the present experiment arises from the f a c t that the f i r s t set of ELT measurements was carr i e d out just when the t o x i c i t y of the two l a r g e s t excessesoof vitamin A began to eff e c t growth rate i n the D and NH chicks (Table 22). The ef f e c t of excess vitamin A on the ELT was the same i n the A chicks however, and t h i s breed did not show growth retardation when receiving the R3 and R4 diets (Table 22). L a s t l y i t i s important to emphasize that ELT measurements are i n v i t r o determinations, the results of which are not necessarily i n d i c a t i v e of i n vivo f i b r i n o l y t i c a c t i v i t y (Bucknell, 1958; Kowalski et a l . , 1959; Hedlin et a l , - 132 -1971). The ELT measurements provide an i n d i c a t i o n of the events i n only a part of the f i b r i n o l y t i c system, and the re-sults obtained are highly s e n s i t i v e to s l i g h t modifications of technique. - 133 -Experiment 7: EFFECT OF VITAMIN K DEFICIENCY ON THE EUGLOBU-LIN LYSIS TIME Evidence was obtained i n experiment 6 that the effect of excess vitamin A on the ELT i n chickens i s not med-ia t e d through an induced vitamin K deficiency.. I t was con-sidered of i n t e r e s t to further investigate t h i s point using vitamin Ji-deficient chicks free of other n u t r i t i o n a l s t r e s s . Materials and Methods: Sixty day-old WL chicks of the Shaver s t r a i n were wing-banded, weighed and d i s t r i b u t e d into four groups of f i f -teen i n e l e c t r i c a l l y - h e a t e d battery brooders. The chicks were fed and watered ad l i b i t u m . T h i r t y b i r d s received the basal vitamin K-deficient diet f o r which the composition i s shown i n Table 29, and t h i r t y received'^the same diet to which was added 5.5 mg of menadione per kilogram. At ten days of age, euglobulin l y s i s time measurements were performed on ten birds of each d i e t , and three days l a t e r one-stage prothrombin time measurements were carried out on f i v e birds of each d i e t . Half the birds of each diet were then injected, as described i n experiment 6, with 5 mg of menadione per kilogram of body weight, and h a l f the birds were injected with the soybean o i l c a r r i e r , only. Sixteen hours after the i n j e c t i o n s were completed, one-stage prothrombin time determinations were conducted on eight birds of each diet (four having received menadione and, four, only the soybean o i l c a r r i e r ) . Twenty hours a f t e r the i n j e c t i o n s were completed, the remaining ten - 134 -> Table 29. Experiment 7: Basal (vitamin K-deficient) d i e t Component % of diet Ground wheat (12.5%) 82.5 Herring meal (72.0%) 15.0 Limestone 1.5 Iodized s a l t (unknown hydration) 0.5 Micronutrients 0.5 1UU7TJ Micronutrients per kg of d i e t Manganese sulphate 0.13 g Vitamin A palmitate 4400 IU Vitamin D 3 440 IU Vitamin E 18 IU Ribo f l a v i n 4.0 mg F o l i c acid 0.60 mg Pantothenic acid 9.2 mg Cornstarch 4.8 g Calculated Analysis: % Protein = 21.1 Metabolizable Energy = 3165 kcal/kg - 135 -birds of each diet and of each i n j e c t i o n treatment were tested f o r euglobulin l y s i s time. Results The body weights of the birds at two weeks and four weeks of age are shown i n Table 30. Graphs of percent l y s i s vs. incubation time f o r the ten-day-old chicks on the control and the vitamin K-deficient diets are shown in figures 6a and 6b, respectively. They appear i d e n t i c a l . The pro-thrombin time data taken on the birds at thirteen days of age i s presented i n Table 31, along with the times obtained on the following day, sixteen hours a f t e r the i n j e c t i o n s of mena-dione or soybean o i l . Table 28 of the appendix shows that the vitamin K-de f i c i e n t birds had prolonged prothrombin times as compared to the controls (p<0.01), and Table 29 i n the appendix shows that t h i s defect was corrected within sixteen hours afte r menadione i n j e c t i o n (p<0.01). Soybean o i l , alone, was i n e f f e c t i v e . In figures 7a and 7b are shown graphs of percent l y s i s against incubation time comparing the birds of both diets which were injected with menadione and with soybean o i l , only, respectively. The .graphs of the menadione-injected and the o i l - i n j e c t e d birds are very similar for each d i e t . Discussion The one-stage prothrombin time data presented i n Table 31 show that the birds receiving the vitamin K-def i c i e n t diet were, indeed, deficient i n vitamin K and were not re-ceiving adequate supplies of the vitamin from i n t e s t i n a l microorganisma. Neither vitamin K-deficiency nor correction of the deficiency have any ef f e c t - 136 -Table 30. Experiment 7: Average body w e i g h t s ( t standard dev-iation) at two weeks and at four weeks of age Level of dietary vitamin K -K 5.5 mg menadione /kg Age Body weight (g) two weeks 126*3.2 127+15 four weeks 201*29 200±31 - 137 -Table 31. Experiment 7: Average one-stage prothrombin times (t standard deviation) at two weeks of age One-stage prothrombin time (min) before i n j e c - A f t e r injection-treatment t i o n - t r e a t -ment Level of 5 mg menadione soybean o i l dietary intramuscu- intramuscularly vitamin K l a r l y per kg body weight -K 5.5 mg per kg 0.66t0.08 0.45+.0.05 0.48+.0.03 0.46+.0.07 0.76+0.12 0.46+0.06 - 138 -Figure 6. Experiment 7: Percent l y s i s vs. incubation b) Vitamin K - d e f i c i e n t diet incubation time (hrs.) - 139 -Figure 7. Experiment 7: Percent l y s i s vs. incubation time a) Menadione-inj" ected birds (5 mg/kg body weight) i) vitamin K - s u f f i c i e n t diet incubation time (hrs.) incubation time (hrs.) - 140 -Figure 7. cont»d b) Soybean o i l - i n j e c t e d birds i ) vitamin K - s u f f i c i e n t diet incubation time (hrs.) incubation time (hrs.) - 141 -on the ELT of chicks as shown i n Figures 6a and 6b and in Figures 7a and 7b respectively. The finding of experiment 6 that the e f f e c t of large dietary vitamin A excesses on the ELT of chicks i s through a mechanism other than the induction of a vitamin & deficiency i s thus confirmed by the present exper-iment. The c l o t l y s i s times of the Shaver chicks appear shorter than those of the NH, A, and D chicks used i n experi-ment 6. The Shaver chicks tested were younger than the birds tested i n the previous experiment and there i s the p o s s i b i l i t y of an age e f f e c t on the ELT (Buckell and E l l i o t , 1959). Also there could be a s t r a i n difference i n ELT between the Shaver and the 1MH, A, and D chicks. - 142 -SUMMARY AND CONCLUSIONS A two-part study,with several breeds and strains of chickens, was conducted concerning the e f f e c t s of excesses of vitamin A on factors considered to be involved i n athero-genesis. The f i r s t part of the study dealti.with the effect of excess vitamin A on alimentary cholesterolemia. In the second part, e f f e c t s of excess vitamin A on the coagulation mechanism were studied r e l a t i n g to the formation and the l y s i s of thrombi. By a .modification of the conventional l i v e r v i t a -min A storage t e s t , a NH l i n e of chickens was shown to be superior to three WL st r a i n s and a Br s t r a i n i n the a b i l i t y to accumulate vitamin A from the diet into the l i v e r . An excess of dietary vitamin A reduced alimentary cholesterolemia i n the NH chicks but increased alimentary cholesterolemia i n a WL s t r a i n of chicks (D s t r a i n ) . Feeding an excess of vitamin A for four weeks in a low-cholesterol diet had no e f f e c t on the blood cholesterol concentration of either the D or the NH chicks. When the excess of vitamin A was subsequently removed from the diet and 0 . 5 % of. cholesterol added to the d i e t , the degree of alimentary cholesterolemia induced was s i m i l a r i n the control birds and the birds pre-loaded with vitamin A. An experiment to study the effect of excess v i t a -min A on the i n t e s t i n a l absorption and l i v e r retention of - 143 -cholesterol was performed using 4 - l 4 C - c h o l e s t e r o l . The e f f i -ciency of absorption of cholesterol was increased by paren-t e r a l administration of an excess of vitamin A. The response to the excess vitamin i s therefore concluded to be mediated through an effect on the i n t e s t i n a l mucosa. In the D chicks the percentage of newly-absorbed cholesterol found i n the hepatic pool was reduced by an excess of vitamin A. In the NH chicks, on the other hand, the percentage of newly-absorbed cholesterol i n the hepatic pool was enhanced by excess vitamin A. The r e s u l t s indicate that the divergent effects of excess vitamin A on alimentary cholesterolemia i n the D and NH chicks are due to opposite effects on the l i v e r - t o - b l o o d p a r t i t i o n r a t i o of a large load of cholesterol. Studies on the effect of excess vitamin A on the blood coagulation mechanism showed that large dietary excesses of vitamin A (100,000 IU and £00,000 IU per kilogram of diet) caused a menadione-reversible increase i n the one-stage pro-thrombin times of the A chicks tested. A moderate excess of 35,000 IU of vitamin A per kilogram of diet had no e f f e c t i n the A chicks. None of the l e v e l s of vitamin A administered had a s i g n i f i c a n t e f f e c t on the prothrombin times of the NH, D, B, or K chicks. The feeding of large excesses of vitamin A (100 9000 IU and £00,000 IU per kilogram of diet) f o r six weeks caused a s i m i l a r decrease i n the one rstage euglobulin l y s i s times of NH, D, and A chicks. A moderate excess of 35,000 IU per kilogram was without e f f e c t . Since intramuscular administration - 144 -of menadione to the A hypervitaminotic chicks did not increase the shortened l y s i s times, and since vitamin K deficiency did not a l t e r the l y s i s time, i t i s concluded that the effect of excess vitamin A on the ELT was not due to an induced deficiency of vitamin &. Six weeks subsequent to removal of excess v i t a -min A from the d i e t , control and previously-treated hypervita-minotic chicks showed similar plasma vitamin A concentrations. The chicks formerly receiving the large excesses of vitamin A , however, s t i l l showed a shortened ELT. Accordingly, the e f f e c t of excess vitamin A on c l o t l y s i s i s concluded to be mediated through some ti s s u e or tissues which accumulates vitamin A. - 145 -BIBLIOGRAPHY Aiyar A.S., A. Sreenivasan, 1961. I n t r a c e l l u l a r d i s t r i b u t i o n of ubiquinone i n rat l i v e r under certain stress conditions. JMature 190: 344 All e n R.S.? 1970. " L i p i d Metabolism" i n Duke's Physiology of  Domestic Animals, M.J. Swenson ed., Cornell U n i v e r s i t y Press Ithaca, London, pp. 560-575 Alvsaker J.O., F.B. Haugli.., S.G. Laland, 1967. The presence of vitamin A i n human tryptophan-rich prealbumin. Bioc. J . 102: 362-366 Ames S.R., P.L. Harr i s , 1956. Liver-storage bioassay f o r vitamin A. Anal. Chem. 28: .874-878 Andrews J.W., J r . , R.K. Wagstaff, H.M. Edwards, j r . , 1967. Cholesterol metabolism i n the laying fowl. Am. J . Physiol. 214: 1078-1083 Asada K., 1923. ci t e d by M.E. Smith, 1933. The effect of vitamin A deficiency on the concentration of the blood l i p i d s of albino r a t s . j . Nut. 8: 675-687 Bacharach A.L., E.L. Smith, 1928. Some notes on the chemistry of the fat-soluble vitamins i n cod l i v e r o i l . Q.uart. J . Pharm. 1: 539-545 Bayer R.C., R.K. Ringer, E.A. Cogger, 1971. The influence of dietary vitamin A on atherogenesis i n Japanese Quail. Poul. sc. 50: 1551 (Abstr.) Beeler D.A., J.C. Rogler, F.W. Quackenbush, 1962. E f f e c t s of l e v e l s of certain dietary l i p i d s on plasma cholesterol and atherosclerosis i n the chick, j . Nut. 78: 184-188 Belfrage P., J . Elovson, T. Olivecrona, 1965. Radioactivity i n blood and l i v e r p a r t i a l glycerides, and l i v e r phospho-l i p i d s a f t e r intravenous administration to carbohydrate-fed rats of chyle containing double-labeled t r i g l y c e r i d e s . Bioc. Biop. Acta 106: 45-55 Benacerraf B., 1964. "Functions of the Kupffer C e l l s " i n The  L i v e r Morphology, Biochemistry, Physiology, "Vol. I I , C. R o u i l l e r ed., Adademic Press, New.York, London, pp 37-62 B i e l y J . , J . Prat t , W. Chalmers, 1941. Vitamin A Requirements of growing chicks. Poul. Sc. 20: 456 (abstr.) - 146 -B i f u l c o E., 1953. Vitamin A i n t o x i c a t i o n . Report of a Case i n an Adult. New Eng. J". Med. 248: 690-692 Biggs R., 1955. Assessment of c l o t t i n g e f f i c i e n c y . Br. Med. B u l l . 11: 5-10 Biggs R., AiS. Douglas,,. 1953. Measurement of prothrombin i n plasma.' A Case of Prothrombin Deficiency. J . C l i n . Path. 6: 15-22 Biggs M.W.j D. Kritchevsky, D. Colman, J.W. Gofman, H.B. Jones, F.T. Lmdgren, G. Hyde, T.P. Lyon ? 1952. Observations on the fate of ingested cholesterol i n man. C i r c . 6: 359-366 Blomstrand R., B. Werner, 1967. Studies on the I n t e s t i n a l absorption of radioactive B-carotene and vitamin A i n man. Conversion of B-carotene into vitamin A. Scand. J . C l i n . Invest. 19: 339-345 Boling J.A., G.E. M i t c h e l l , CO. L i t t l e , C L . F i e l d s , K.E. Webb, 1969. Mobilization of l i v e r vitamin A i n mature sheep. J . Nut. 99: 502-504 Bomskov C , G. Seemann, 1933. Z. Ges. Exp. Med. 89: 771 cited by C. Nieman and H.J. K l e i n 0bbink ? 1954. The biochemistry and pathology of hypervitaminosis A. V i t s . Horm. 12: 69-99 Bonner M.J., B.F. M i l l e r , H.V. Kothari, 1973. Influence of vitamin A on experimental atherosclerosis i n rabbits. Experientia 29: 187-188 Borgstrom B., B. Lindhe, P. Wlodawer. 1958. Absorption and d i s t r i b u t i o n of cholesterol -4- 1 4C i n the r a t . Proc. Soc. Exp. B i o l . Med. 99: 365-368 Bring S.V., C.A. Ricard, M.V. Zachringer, 1965. Relationship between cholesterol and vitamin A metabolism i n rats fed at d i f f e r e n t l e v e l s of vitamin A. J . Nut. 85: 400-406 Bring S.V., K.P. Warnick, E..,Woods, 1955. N u t r i t i o n a l status of school children 15 and 16 years of age i n three Idaho communities; blood biochemical tests. J . Nut. 57: 29-45 Brown H.B., 1970. "Diets that Lower Blood Cholesterol i n Man" i n Atherosclerosis: Proceedings of the Second Inter- national Symposium, R.J. Jones ed., Springer-Verlag, New York, Heidelberg, B e r l i n , pp 426-435 Buckell M., 1958. The e f f e c t of c i t r a t e on euglobulin methods of estimating f i b r i n o l y t i c a c t i v i t y . J . C l i n . Path. 11: 403-405 - 147 -Buckell M., F.A. E l l i o t , 1959. Diurnal fluctuation of plasma-f i b r i n o l y t i c a c t i v i t y i n normal males. Lancet i : 660-661 Byers S.O., 1964. "Liver Regulation of Plasma Cholesterol" i n Biochemical C l i n i c s 5. The L i v e r . Reuben H. Donnelley Corporation, New York, pp 157-168 Byers S.O., M. Friedman, 1966. Transport of cholesterol during phosphatide-induced hypercholesterolemia. Bioc. Biop. Acta 125: 157-165 Castano F.F., R.V. Boucher, E.W. Callenbach, 1951. U t i l i z a -tion by the chick of vitamin A from d i f f e r e n t sources. J . Nut. 45: 131-141 Chaikoff I.L., M.D. Si p e r s t e i n , W.G. Dauben, H.L. Bradlow, J . F. Eastham, G.M. Tomkins, J.R. Meier, R.W. Chen, S. Hotta, P.A. Srere, 1952. C 1 4 - c h o l e s t e r o l I I . Oxidation of carbons 4 and 26 to carbon dioxide by the i n t a c t r a t . J . B i o l , chem. 194: 413-416 Chupanya P., J.A. Olson, 1968. ci t e d by J.A. Olson, 1968. Some aspects of vitamin A metabolism. V i t . Horm. 26: 1-63 Collazo J.A., G. Bosch, 1923. ci t e d by M.E. Smith, 1933. The effect of vitamin A deficiency on the concentration o f the blood l i p i d s of albino rats. J . Nut. 8: 675-687 Cornwell D.G., F.A. Kruger, H.V. Robinson, 1962. Studies on absorption of beta-carotene and the d i s t r i b u t i o n of t o t a l carotenoid i n human serum li p o p r o t e i n s a f t e r oral admini-s t r a t i o n . J . L i p . Res. 3: 65-70 Cunningham N.F.,1956. Ph.D. Thesis University of Liverpool, cited by F.W. Hemming and J.F. Pennock, 1965. "Vitamins and Ubiquinone Status i n Animals" i n Biochemistry of  Quinones, R.A. Morton ed. Academic Press, London and New York, pp 287-315 David J.S., J . Ganguly, 1967. Further studies on the mechanisa of absorption of vitamin A and cholesterol. Ind. J . Bioc. 4: 14-17 DeLuca L., E.P. L i t t l e , G. Wolf, 1969. Vitamin A and protein synthesis by rat i n t e s t i n a l mucosa. J . B i o l . chem. 244: 701-708 DeLuca L., G. Rosso, G. Wolf, 1970. The biosynthesis of a mannolipid that contains a polar metabolite of Ib-^-^G-r e t i n o l . Bioc. Biop. Res. Comm. 41: 615-620 - 148 -DeLuca L., M. Schumacher, G. Wolf, 1970. Beosynthesis of a fucose-containing glycopeptide from rat small i n t e s t i n e i n normal and vitamin A-deficient conditions. J . B i o l , chem. 245: 4551-4558 Deshmukh D.S., P. Malathi, J . Ganguly, 1964. Studies on metab-olism of vitamin A-dietary protein content and metabolism of vitamin A. Bioc. J . 90: 98-104 Di Benedetto, 1967. Chronic hypervitaminosis A i n an adult. JAMA 201: 700-702 Didisheim P., K. H a t t o r i , J . Lewis, 1959. Hematologic and coagulation studies i n various animal species. J . Lat. C l i n . Med. 53: 866-875 Dingle J.T., H.B. F e l l , D.S. Goodman, 1972. The e f f e c t of r e t i n o l and of ret i n o l - b i n d i n g protein on embryonic skele-t a l tissue in organ culture. J . C e l l Sc. 11: 393-402 Diplock A.T.', J . Green, J . Bunyan, 1965. Vitamin A and i s o -prenoid synthesis i n the rat. Bioc. J . 95: 138-143 Donchenko G.V., 1964. Cited by J.G. B i e r i , 1966. Interre-l a t i o n s h i p s between ubiquinones and vitamins and hormones. V i t s . Horm. 24: 587-598 Donovan G.A., D.C.. Henderson, R.N. Luneau, 1961. Influence of high l e v e l vitamin A feeding on la y i n g hens. Poul. Sc. 40: 1395-1396, (abstr.) Dowling J.E., G. Wald, 1958. Vitamin A deficiency and night blindness. Proc. Soc. Nat. Acad. Sc. Wash. 44: 648-661 Drummond J . C , M.E. B e l l , E.T. Palmer, 1935. Observations on the absorption of carotene and vitamin A. Br. Med. J . 1: 1208-1210 Dua P.N.j B.C. Dilworth, E.J. Day, J.E. H i l l , 1967. E f f e c t of dietary vitamin A and cholesterol on cholesterol and carotenoid content of plasma and egg yolk. Poul. Sc. 46: 530-531 Dugan R.E., N.A. F r i g e r i o , J.M. Siebert, 1964. Colorimetric determination of vitamin A and i t s derivatives with t r i -f l u o r o a c e t i c acid. Anal. Chem. 36: 114-117 Dukes H.H., 1955. "Blood" i n The Physiology of Domestic Animals by H.H. Dukes, Cornell University Press, Ithaca, New York, pp 18-65 Dunagin P.E., J r . , E.H. Meadows? J r . , J.A. Olson, 1965. Retinoyl beta-glucuronic acid: A mo,jor metabolite of vitamin A i n rat b i l e . Science 148: 86-87 - 149 -Dunagin P.E., J r . , R.D. Zachman, J.A. Olson, 1966. The i d e n t i -f i c a t i o n of metabolites of r e t i n o l and r e t i n o i c acid i n rat b i l e . Bioc. Biop. Acta. 184: 71*85 Dyke, S.F., 1965. "The Vitamins A" i n The Chemistry of the  Vitamins. Interscience Publishers, London, New York, Sydney, pp 208-255 Eden E., K.C. S e l l e r s , 1948. The absorption of vitamin A. Bioc. J . 42: XLIX Eden E., K.C. S e l l e r s , 1949. The absorption of vitamin A i n ruminants and rats. Bioc. J . 44: 264-267 Eden E., K.C. S e l l e r s , 1950. Hydrolysis and e s t e r i f i c a t i o n of vitamin A during absorption. Bioc. J . 46: 261-266 Edwin E.E., J . Bunyan, J . Green, A.T. Diplock, 1962. The ef f e c t of vitamin A on ubiquinone and ubichromenol i n the rat, and i t s r e l a t i o n 46 the effect of vitamin E. Br. J . Nut. 16: 135-149 Emerick R.J., M. Z i l e , H.F. DeLuca, 1967. Formation tftf r e t i n o i c acid from r e t i n o l i n the r a t . Bioc. 0". 102: 606-611 Erdman J r . , J*.W., P.A. Lachance, 1973. The effect of lycopene upon serum, l i v e r , and i n t e s t i n a l cholesterol i n hyper-cholesterol emic rat s . Fed. Proc. 32: 904, (abstr.) Erwin T.S., T.R. V a r n e l l , H.M. Page, 1959. Relationship of vitamin A and carotene to bovine serum protein. Proc. Soc. Exp. B i o l . Med. 100: 373-375 Eskelson CD., L.A. Meeks, 1972. Vitamin A's influence on cholesterolgenesis. Adv. Exp. Med. B i o l . 26: 290-291 Farbiszewski R., K. Worowski, 1968. Enhancement of p l a t e l e t aggregation and adhesiveness by B-lipoprotein. J . Athero-s c l e r . Res. 8: 988-990 Fearnley G.R., 1953. F i b r i n o l y s i s by absorption. Nature 172: 544-545 Fearnley G.R., 1965. "Measurement of F i b r i n o l y s i s " i n F i b r i n o - l y s i s by G. R. Fearnley, 1965. Edward Arnold Publishers Ltd., London, pp:28-44 FidgeN.H., T. S h i r a t o r i , J . Ganguly, D.S. Goodman, 1968. Pathways of absorption of r e t i n o l and r e t i n o i c acid i n the r a t . J . L i p . Res. 9: 103-109 Florsheim W.H., M.A. F a i r c l o t h , D. Graff, N.S. Austin, S.M. V e l c o f f , 1963. The synthesis of the protein moiety of serum B-lipoprotein i n the rooster. Metab. 12: 598-607 - 150 -Foy J.R., K. Morgareidge, 1948. B i o l o g i c a l assay of vitamin A l i v e r storage test of Guggenheim and Koch. Anal. Chem. £0: 304-507 French J.E., 1966. Atherosclerosis i n r e l a t i o n to the struc-ture and function of the a r t e r i a l intima, with special reference to the endothelium. Int. Rev. Exp. Path. 5: £53-353 French J.E., B. Morris, D. Robinson, 1958. Removal of l i p i d s from the blood stream. Br. Med. B u l l . 14: £34-£38 Futterman S., J.S. Andrews, 1964. The composition of l i v e r vitamin A ester and the synthesis of vitamin A ester by l i v e r microsomes, <J. B i o l . Chem. £39: 4077-4080 Ganguly j . , 1960. Absorption, transport, and storage of v i t a -min A. V i t s . Horm. 18: 387-402 Ganguly J . , 1967. cited by G.A.J. P i t t , 1971. "Vitamin A" i n Carotensils, 0. I s l e r ed., Birkhauser verlag, Basle and Stuttgart, pp 717-742 Ganguly J . , H.J. Devel, dr., 1953. I n t r a c e l l u l a r d i s t r i b u -tion of vitamin A esterase a c t i v i t y i n rat l i v e r . Nature 172: 1£0-1£1 Ganguly J . , N.I. Krinsky, 1953. Absence of relat i o n s h i p be-tween vitamin A alcohol l e v e l s i n plasma and l i v e r of ra t s . Bioc. J . 54: 177-181 Ganguly J . , N.I. Krinsky, J.W. Mehl, H.J. Devel, J r . , 195£. Studies of the d i s t r i b u t i o n of vitamin A as ester and alcohol and of carotenoids i n plasma proteins of several species. Arch. Bioc. Biop. 38: £75-£8£ Gans J.H., 1968. The d i s t r i b u t i o n of 1 4 C - l a b e l e d cholesterol i n the dog: E f f e c t of long-term epinephrine administra-t i o n . L i p i d s 3 : 324-330 Garbers,G.E., 1958. Transport of vitamin A ester i n rat serum. Nature 182: 1018-1019 Garbers C.F., J . Gillman, M. Peisach, 1960. The transport of vitamin A i n rat serum with special reference to the oc-currence of unidentified metabolites of vitamin A i n the r a t . Bioc. J . 75: 124-132 Gloor U., 0. Wiss, 1959 a. Influence of vitamin A deficiency on the biosynthesis of cholesterol, squalene, and ubiqui-none. Bioc. Biop. Res. Comm. 1: 182-185 Gloor U., 0. Wiss, 1959 b. On the biosynthesis of ubiquinone (50). Arch. Bioc. Biop. 83: 216-222 - 151 -Gloor U., 0. Wiss, 1960. ubiquinones and solasenol i n human and rat tissues. Bioc. Biop. Res. Comm. 2: 222-225 Gloor U., F. Weber, 0. Wiss, 1961. "Vitamin A and the bio-synthesis of cholesterol" i n Drugs A f f e c t i n g L i p i d Metab- olism S. Garattini.iand R. Paoiexti eas. . E l s e v i e r ^ucusn-ing Company, Amsterdam, London, New York, Princeton, pp 80-82 Glover J . , R.A. Morton, 1948. The administration, storage and metabolism of vitamin A. Bioc. J . 43: XII-XIII Goodman D.S., R. Blomstrand, B. Werner, H.S. Huang, T. Shira-t o r i , 1966 a. The i n t e s t i n a l absorption and metabolism of vitamin A and B-carotene i n man. J . C l i n . Invest. 45: 1615-1623 Goodman D.S., H.S. Huang, T. S h i r a t o r i , 1966. Mechanism of the biosynthesis of vitamin A from B-carotene. J . B i o l . Chem. 241: 1929-1932 Goodman D.S., H.S. Huang, T. S h i r a t o r i , 1965. Tissue d i s t r i -bution and metabolism of newly absorbed vitamin A i n the rat . J . L i p . Res. 6: 390-396 uoodridge A.G., 1968. The effect of starvation and starvation followed by feeding on the enzyme a c t i v i t y and the metab-olism of (U-^ 4C). Glucose i n l i v e r from growing chicks. Bioc. J . 108: 667-673 S o m a l i A.G., C.J. Bardowill, M.M. David, 1949. Determina-ti o n of serum proteins by means of the biuret reaction. J . B i o l . Chem. 177: 751-766 Gray E.L., CD. Hickman, E.F. Brown, 1940. The state of v i t a -min A i n the l i v e r of the r a t a f t e r feeding various forms of the vitamin. J . Nut. 19: 39-45 Gray E.L., K. Morgareidge, J.D. Crawley, 1940. I n t e s t i n a l absorption of vitamin A i n the normal r a t . J . Nut. 20: 67-74 Greaves J . , 1972. How super rats survive. New S c i . 56: 156-158 Green J . , E.E. Edwin, A.T. Diplock, D. Mottale, 1960. The conversion of ubiquinone to ubichromenol. Bioc. Biop. Res. Comm. 2: 269-271 Green B., A.R. Horner, J.S. Lowe, R.A. Morton, 1957. Chol-esterol and l i v e r vitamin A i n male and female r a t s . Bioc. J . 67: 235-238 de Groot A.P., S.A. Reed, 1959. Influence of dietary cod-liver o i l and some fra c t i o n s of c o l - l i v e r o i l on serum cholesterol l e v e l of rats'. Nature 183: 1191 - 152 -Grundy S., E.H. Ahrens, J r . , G. Salen, 1968. Dietary B - s i t o -s t e r o l as an i n t e r n a l standard to correct f o r cholesterol losses i n s t e r o l balance studies. J . L i p . Res. 9: 374-387 Guggenheim K., W. Koch, 1944. A l i v e r storage test f o r the assessment of vitamin A. Bioc. J . 38: 256-260 Gurcay R., R.V. Boucher, E.W. Callenbach, 1950. U t i l i z a t i o n of vitamin A by turkey poults. J . Nut. 41: 565-582 Hard M.M., N.C. Esselbaugh, 1960. N u t r i t i o n a l status of ado-lescent children IV. Cholesterol relationships. Amer. J . C l i n . Nut. 8<: 346-352 Harris L.J., T. Moore, 1928. "Hypervitaminosis" and "Vitamin Balance" Bioc. J . 22: 1461-1477 Ha£twell.G.A., 1927. Growth and reproduction on synthetic diets I I . Bioc. J . 21: 1076-1086 Haust M.C., 1970. Atherosclerosis; Proceedings of the Second  International Sprposium. R.J. Jones ed., Springer-Verlag, New York, pp 12-20 Heaton F.W., J.S. Lowe, R.A. Morton, 1957. Aspects of vitamin A deficiency i n the r a t . Bioc. 67: 208-215 Hedlin A.M., F.C. Monkhouse, 1969. F i b r i n o l y t i c a c t i v i t i e s of euglobulins precipitated at pH 6.4, 6.0, and 5.3. Can. J . Physiol. Pharmacol. 47: 935-940 Hedlin A.M., F.C. Monkhouse, S.M. M i l o j e r i c , 1971. A com-parative study of f i b r i n o l y t i c a c t i v i t y i n human, r a t , rabbit and dog blood. Can. J . Physiol. Pharmacol. 50: 11-16 Hegsted D.M., R.B. McGandy, M.L. Myers, F.J. Stare, 1965. Quantitative effects of dietary f a t on serum cholesterol i n man. Amer. J . C l i n . Nut. 17: 281-295 Hellman L., R.S. Rosenfeld, M.L. E i d i n o f f , D.K. Fukushima, T. F. Gallagher, C. Wang, D. Adlersberg, 1955. Isotopic studies of plasma cholesterol of endogenous and exogenous o r i g i n s . J . C l i n . Invest. 34: 48-60 Hemming F.W., J.F. Pennock, R.A. Morton, 1958. I n t r a c e l l u l a r d i s t r i b u t i o n of SA and SC. Bioc. J . 68: 29p-30p Heywood R., 1967. Vitamin A i n the l i v e r and kidney of some fe l i d a e . Br. Vet. J . 123: 390-396 Higgins J.A.. C. Green, 1966. The uptake of l i p i d s by rat l i v e r cells, Bioc. J . 99: 631-639 - 153 -High E.G., S.S. Wilson, 1956. Studies on vitamin A ester, slcohol p a r t i t i o n between the l i v e r , kidneys and plasma of ra t s . Arch. Bioc. Biop. 62: 163-172 Hori S.H., T. Kitamura, 1972. The vitamin A content and r e t i n o l e s t e r i f y i n g a c t i v i t y of a Kupffer c e l l f r a c t i o n of rat l i v e r . J . Histochem. Cytochem. 20: 811-816 Horner A.A., R.A. Morton, 1960. Dietary cholesterol and l i v e r vitamin A i n normal and castrated male rats. Bioc. J". 74: 301-304 Hougie C , 1972. Euglobulin l y s i s time i n Hematology W.J. 'Williams, E. Beutler, A.J. E r s l e r , R.W. Rundles eds., McGraw-Hill Book Company, New York, St. Louis, San Fran-cisco, Dusseldorf, Johannesburg, Kuala Lumpur, London, Mexico, Montreal, New Delhi, Panama, Rio de Janeiro, Singapore, Sydney, Toronto, pp 1411-1412 Huang H.S.. D.S. Goodman, 1965. Vitamin A and carotenoids 1. i n t e s t i n a l absorption and metabolism of l 4 c - l a b e l e d v i t a -min A alcohol and B-carotene i n the r a t . J . B i o l . Chem. 240: 2839-2844 Hume E.M., H.A. Krebs, 1970. c i t e d by T. Moore, 1970. "The Biochemistry of Vitamin A i n the General System" i n Fat- Soluble Vitamins R.A. Morton ed., Oxford, London, Edinburgh, New York, Toronto, Sydney, P a r i s , Braunschweig, Pergaman Press, Med. Res. Council Spec. Rep. Series No. 264. H.M. Stationary O f f i c e , London, pp 223-265 Hume I.D., G.E. M i t c h e l l , J r . , R.E. Tucker, 1971. B i l i a r y and urinary excretion and enterohepatic r e c y c l i n g of vitamin A i n sheep J . Nut. 101: 1169-1176 Husbands D.R., 1971. cited by E.F. Annison, 1971. " L i p i d and Acetate Metabolism" i n Physiology and Biochemistry of the  Domestic Fowl, D.J. B e l l and B.M. Freeman eds., Academic"" Press London, New York, pp 321-337 Hutt F.B., 1961. N u t r i t i o n and genes i n the domestic fowl. Nut. Rev. 19: 225-227 Irfan M., 1968. F i b r i n o l y t i c a c t i v i t y i n animals of d i f f e r e n t species. Quart. J . Exp. Physiol. 53: 374-380 I r i t a n i N., N. Takeuchi, 1969. Influence of lactose feeding on cholesterol absorption and metabolism. J . Atheroscler. Res. 10: 207-216 I s r a e l i s L.G., J . Foerster, A. Zipursky, 1962. The coagulation defect i n hypervitaminosis A i n r a t s . Chem. Abstr. 57-384 7Jr - 154 -Josephs H.W., 1944. Hypervitaminosis A and carotenemia A.M. A. J . Diseases C h i l d . 67: 33-43 Joshi V.C., J . Jayaraman ? T. Ramasarma, 1965. Some observa-tions on the metabolism of coenzyme Q and ubichromenol i n rat tissues. Bioc. Biop. Res. Comm. 18: 108-114 Joshi V.C., T. Ramasarma, 1966. Biosynthesis of ubiquinone and ubichromenol i n vitamin A-deficient r a t s . Bioc. Biop. Acta 115: 294-305 Kanai M., A. Raz. D.S. Goodman, 1968. Retinol-binding protein: the transport protein f o r vitamin A i n human plasma. J . C l i n . Invest. 47: 2025-2044 Kinley L.J., R.F. Krause, 1958. Serum cholesterol determina-tions as affected by vitamin A. Proc. Soc. Exp. B i o l . Med. 99: 244-245 Kinley L.J., R.F. Krause, 1959. Influence of vitamin A on cholesterol blood l e v e l s . Proc. Soc. Exp. B i o l . Med. 102: 353-355 Kiyasu J.Y., 1955. Ph.D. t h e s i s , C a l i f o r n i a , cited by A. Noyal e t . a l . , 1964. J . L i p . Res. 5: 538-541 Koch T., 1973. "Circulatory System" In Anatomy of the chicken  and Domestic Birds B.H. Skold, L. Devrles eds., Iowa ' State University Press, Ames, Iowa, pp 101-118 Kon S.K., W.A. M c G i l l i v r a y , S.Y. Thompson, 1955. Metabolism of carotene and vitamin A given by mouth or vein i n o i l y solution on aaueous dispersion to calves, rabbits, and rats. Br. J . Nut. 9: 244-267 Kowalski E.£ M. Kopec, S. Niewiarowski, 1959. An evaluation of the euglobulin method f o r the determination of f i b r i n o -l y s i s . J . C l i n . Path. 12: 215-218 Krishnamurthy S., J . Ganguly, 1956. E f f e c t of blocking the r e t i c u l o e n d o t h e l i a l system on the storage of vitamin A ester and alcohol i n the l i v e r of the rat. Nature 177: 575-576 Krishnamurthy S., S. Mahadevan, J . Ganguly, 1958. Association of vitamin A ester and vitamin A alcohol with proteins i n rat l i v e r . J . B i o l . Chem. 233: 32-36 Kuchle H.J., R. Krueger, 1961. Investigations about the i n -fluence of heparin and of vitamin A and E on the sequelae of experimental hypercholesterolemia i n Drugs A f f e c t i n g  L i p i d Metabolism S. G a r a t t i n i and R. P a o l e t t i , eds., E l s e v i e r i-ublishing Co., Amsterdam, London, New York, Princeton, pp:'S18-322 - 155 -Kudjaskov B.A., G.G. Bozasian, N.P. Sytina, G.V. Andreenko, 1961. Experimental pre-thrombotic state of an organism r e s u l t i n g from the dysfunction of the physiological a n t i -coagulating system induced by an atherogenic diet . Nature 189: 67-68 Lachance P.A., R.J. Amen, 1972. The ef f e c t of B-carotene and canthaxanthin on cholesterol l e v e l s In the r a t . Fed. Proc. 31: 728- (abstr.) Lacord-Bonneau M., L. Dubemard, J . Picard, 1972. E f f e c t of vitamin A on l i p i d mobilization i n r a t . CR. Acad. S c i . Paris, 275D: 707-709 Landon E.J., D.M. Greenburg, 1954. Endogenous cholesterol metabolism i n the rat studied with C r 4 - l a b e l e d acetate, d. B i o l . Chem. 209: 493-502 Lane B.P., 1968. Hepatic microanatomy i n hypervitaminosis A i n man and rat . Am. J . Path. 53: 591-598 Lasch F., K l i n . Wochschr, 1934. 13: 1534 cited by L.J. Kinley and R.F. Krause, 1959. Influence of vitamin A on chol-esterol blood l e v e l s . Proc. Soc. Exp. B i o l . Med. 1025 353-355 Lawrence C.W., F.D. Crain, F.J. Lotspeich, R.F. Krause. 1966. Absorption, transport, and storage of r e t i n y l - 1 5 - 1 4 C palmitate-9, 10- 3H i n the r a t . J . L i p . Res. 7: 226-229 Lawrie N.R., T. Moore, K.R. Rajagopal, 1941. The excretion of vitamin A i n urine. Bioc. J . 35: 825-836 L e v e i l l e G.A., 1969. In vivo f a t t y acid and cholesterol syn-thesis i n fasted and fasted-refed chicks. J . Nut. 98: 367-372 Lewis J.M., 0. Bodansky, j . Birmingham, S.Q. Cohlan, 1947. Comparative absorption, excretion, and storage of o i l y and aqueous preparations of vitamin A. J . Pelat. 31: 496-508 Light R.F., R.P. Alscher, C N . Frey, 1944. vitamin A t o x i c i t y and hypoprothrombinemia. Science 100: 225-226 Linder M.C , H. Anderson, I. A s c a r e l l i , 1971. Quantitative d i s t r i b u t i o n of vitamin A i n Kupffer c e l l and hepatocyte populations of r a t l i v e r s . J . B i o l . Chem. 246: 5538-5540 Lippel K., J.A. Olson, 1968 a. Biosynthesis of beta-glucuron-ides of r e t i n o l and of r e t i n o i c acid i n Vivo and i n V i t r o . J . L i p . Res. 9: 168-175 - 156 -Lip p e l K., J.A. Olson, 1968 b. Origin of some derivatives of r e t i n o i c acid found i n rat b i l e , j . L i p / Res. 9: 580-586 Loran M.R., T.L. Althausen, 1958. Hypertrophy and changes i n cholinesterase a c t i v i t i e s of the i n t e s t i n e , erythrocytes and plasma a f t e r " P a r t i a l " resution of the small i n t e s t i n e of the r a t . Am. «T. Phy s i o l . 193: 516-520 Loran M.R., T.L. Althausen, F.W. Spicer, W.I. Goldstein, 1961. Transport of vitamin A across human i n t e s t i n e i n v i t r o . J . Lab. C l i n . Med. 58: 622-626 Lorand L., A. Jacobsen, 1965. Accelerated l y s i s of blood c l o t s . Nature 195: 911-912 Loscialpo D., 1959. Azione D e l i a vitamina A s u l l a f i b r i n o l i s i ematica. B o l l . Soc. I t a l . B i o l . 35: 310-312 Losito R., 1965. Investigations into the presence of a com-p e t i t i v e i n h i b i t o r (Preprothrombin) i n the plasma of chicks. Acta Chem. Scand. 19: 2229-2234 Lowe J.S.. R.A. Morton, N.F. Cunningham. J . Vernon ? 1957. Vitamin A d e f i c i e n c y i n the domestic fowl. Bioc. J . 67: 215-223 Lowe J.S., R.A. Morton, R.G. Harrison, 1953. Aspects of v i t a -min A deficiency i n r a t s . Nature 172: 716-719 Maddock C.L., S.G. Wolbach, D. Jjensen, 1948. Hypoprothrom-binemia with hemorrhage as a cause of death i n rats i n hypervitaminosis A. Fed. Proc. 7: 275 (Abstr.) Mahadevan S., D.S. Deshmukh, d. Ganguly, 1964. The fate of i n t r a c a r d i a l l y administered r e t i n y l esters i n the r a t . Bioc. J . 93: 499-503 Mahadevan S., J . Ganguly, 1961. Further studies on the absorp-tion of vitamin A. Bioc. J . 81: 53-58 Mahadevan S., S. Krishnamurthy, J . Ganguly, 1959. The mode of absorption of vitamin A across the i n t e s t i n e of r a t s . Arch. Bioc. Biop. 83: 371-375 Mahadevan S., S.K. Murthy, S. Krishnamurthy, d". Ganguly, 1961. Studies on vitamin A esterase 4. The hydrolysis and synthesis of vitamin A esters by rat i n t e s t i n a l mucosae. Bioc. J . 79: 416-424. Mahadevan S., P. Seshadri Sastry, J . Ganguly, 1963 a. Studies on metabolism of vitamin A 3. The mode of absorption of vitamin A esters i n the l i v i n g r a t . 'Bioc. J . 88: 531-534 - 157 -Mahadevan S., P. Seshadri Sastry, if, Ganguly, 1963 b. Studies on metabolism of vitamin A 4. Studies on the mode of absorption of vitamin A by rat in t e s t i n e i n v i t r o . Bioo. J . 88: 534-539 Mahadevan S., P. Seshadri Sastry, S.K. Murthy, d. Ganguly, 1960. Annual report submitted to the Indian council of medical research, New Delhi, October 1959, cited by d. Ganguly, 1960. Absorption, transport and Storage of v i t a -min A. V i t s . Horm. 18: 387-402 Mann G.V., 1959. Diet and coronary heart disease. Arch. Intern. Med. 104: 921-936 March B.E., J". B i e l y , 1963. Vitamin A and cholesterol absorp-tion i n the chicken. d. Nut. 79: 474-478 March B.E., V. Coates, C. Goudie, 1972. Delayed hatching time of chicks from dams-fed excess vitamin A and from eggs injected with vitamin A. Poul. Sc. 51: 891-896 March B.E., E. English, d. B i e l y , 1952. The eff e c t of c a r r i e r on the r e l a t i v e u t i l i z a t i o n of d i f f e r e n t forms of vitamin A by the chick. Arch. Bioc. 36: 259-268 Mason H.C., M.E. Smith, 1939. Delayed prothrombin c l o t t i n g time i n A vitaminosis A and p e l l a g r a - l i k e chicks. Proc. Soc. Exp. B i o l . Med. 41: 583-585 Matschiner J.T., E.A. Doisy, dr., 1962. Role of vitamin A i n induction of vitamin K deficiency i n the r a t . Proc. Soc. Exp. B i o l . Med. 109: 139-141 Migicovsky B.B., 1955. I n h i b i t i o n of cholesterol formation by rat l i v e r homogenates. Can. J . Bioc. P h y s i o l . 33: 135-138 Misra U.K., 1967. E f f e c t of hypervitaminosis A on i n t e s t i n a l l i p i d s of r a t s . Can. J . Bioc. 45: 1413-1418 Misra U.K., 1965. Hypervitaminosis A and tissue f a t t y acids Can. if, Bioc. 43: 1885-1886 Misra U.K., 1966. L i p i d metabolism i n hypervitaminosis A Nature 209: 910 Misra U.K., 1968. L i v e r l i p i d s of rats administered excessive amounts of r e t i n o l . Can. J . Bioc. 46: 697-701 Moffa D.F., F.J. Lotspeich, R.F. Krause, 1970. Preparation and properties of r e t i n a l - o x i d i z i n g enzyme from rat i n t e s -t i n a l mucosa. J . B i o l . Chem. 345: 439-447 Moore T. , 1957. "The Absorption of Preformed Vitamin A" i n Vitamin A by T. Moore, E l s e v i e r Publishing Co., Amsterdam, -L'bndbn, New York, Princeton, pp 192-207 - 158 -Moore T., 1970. "The Biochemistry of Vitamin A i n the General System" i n Fat-Soluble vitamins, R.A. Morton, ed. Pergamon Press Oxford, London, Edinburgh, New York, Toronto, Sydney, P a r i s , Braunschweig, pp 223-265 Moore T., 1957. "The Storage and D i s t r i b u t i o n of Vitamin A i n the Body" i n Vitamin A by T. Moore, E l s e v i e r Publishing Co., Amsterdam, London, New 'York, Princeton, pp 208-219 Moore T., KvR. Rajagopal, 1940. The spectroscopic detection of vitamin E i n the tissues of the r a t . Bioc. J . 34: 335-342 MooreT., I.M. Sharman, 1960. Ubiquinone i n rats d e f i c i e n t i n vitamin A. Br. J". Nut. 14: 473-483 Moore T., I.M. Sharman, R.J. Ward, 1951. The d i s t r i b u t i o n of vitamin A i n male and female rats at d i f f e r e n t l e v e l s of dosing. Bioc. 3-, 49: XXXIX-XI Moore T., Y.L.Wang, 1945. Mypervitaminosis A. Bioc. J . 39: 222-228 * Morton R.A., W.E.J. P h i l l i p s , 1959. Unsaponifiable c o n s t i t -uents of l i v e r , kidney and heart tissues from vitamin E-d e f i c i e n t rats compared "with L-tocopherol-supplemented ra t s . Bioc. J . 73: 427-430 MUllertz S., 1957. Activation of plasminogen. Am. N.Y. Acad. S c i . 68: 38-51 Murray T.K., D.W. Stainer, J.A. Campbell, 1959. Isomerization of vitamin A i n vivo. Can. J . Bioc. Physiol. 37: 1469-1474 NathK., J.A. Olson, 1967. Natural occurrence and b i o l o g i c a l a c t i v i t y of vitamin A derivatives i n rat b i l e . J . Nut. 93: 461-469 National Research Council, 1971. Nutrient requirements of domestic animals Number 1 Nutrient requirements of poultry by subcommittee on poultry n u t r i t i o n , committee on animal n u t r i t i o n , a g r i c u l t u r a l board, national research council. National Academy of Sciences, Washington, D.C. Neeld J.B., J r . , N.N. Pearson, 1963. Macro-and Micromethods for the determination of serum vitamin A using t r i f luoro-acetic acid. J . Nut. 79: 454-462 Nestler R.B., J.V. Derby, J.B. DeWitt, 1948. Storage by Bobwhite quail of vitamin A fed in various forms, d . Nut. 36; 323-329 Nilsson A., D.B. Zilversmit, 1972. Fate of intravenously administered p a r t i c u l a t e and l i p o p r o t e i n i n the r a t . d. L i p . Res. 13: 32-38 -^Morrison F.B. , 1959 ? "Feeding and Caring f o r Poultry" i n Feeds and Feeding. The Morrison Publishing Company, Clinton, Iowa"?"" pp. 968-995 - .159 -N i r I., I. Bruekental, I . A s c a r e l l i , 1967. vitamin A esterase a c t i v i t y i n various organs of the chick. Br. J . Nut. 21: 557-563 Nitzberg S.I., M.A. Peyman, R. Goldstein, S. Proger, 1959. Studies' of blood coagulation and f i b r i n o l y s i s i n patients with i d i o p a t h i c hyperlipemia and 1° hypercholesteremia before and afte r a f a t t y meal. C i r c . 19: 676-690 Noyan A., W.J. Lossow, N. Brot, I.C. Chaikoff, 1964. Pathway and form of absorption of palmitic acid i n the chicken d. L i p . Res. 5: 538-541 " Nyquist S.E., F.L. Crane, D.J. Morre, 1971. vitamin A : Con-centration i n the rat l i v e r golgi apparatus. Science 173: 939-941 -Oli v e r T.K., J r . , 1958. Chronic vitamin A int o x i c a t i o n A.M. A. J . Dis. C h i l d . 95: 57-68 Olson J.A., 1969. The alpha and omega of vitamin A metabolism. Amer. J . C l i n . Nut. 22: 953-962 Olson J.A., 1969. Metabolism and function of vitamin A. Fed. Proc. 28: 1670-1677 Olson, J.A., 1967. The metabolism of vitamin A. Pharm. Rev. 19: 559-596 Olson J.A., 1968. Some aspects of vitamin A metabolism. V i t . Horm. 26: 1-63 Oppenheim E., M. Bruger, 1952. Experimental cholesterol atherosclerosis XI. Studies with vitamin A. A.M.A. Arch. Path. 53: 520-522 Page I.H., J.G. Green, A.L. Robertson, 1965. The physician's incompleat guide to atherosclerosis. Ann. Int. Med. 64: 189- 203 Pechet L., 1965. F i b r i n o l y s i s . New Eng. J . Med. 273: 1024-1034 P h i l l i p s 'W.E.J., 1962. The ubiquinone and crude s t e r o l content of the l i v e r with variations i n the Intake of tocopherol i n the vitamin A d e f i c i e n t r a t . Can. J . Bioc. Physiol. 40: 1347-1352 P h i l l i p s W.E.J., 1961. vitamin A deficiency and isoprenoid metabolism. Can. J . Bioc. P h y s i o l . 39: 855-861 P h i l l i p s W.E.J., R.A. Morton. 1959. Ef f e c t s of vitamin A deficiency and adrenalectomy on ubiquinone and substance SC i n rat l i v e r . Bioc. J . 73: 430-433 - 160 -Pilgeram L.O., L.R. Pickart, 1968. Control of fibrinogen biosynthesis;: The role of FFA. J . Atheroscler. Res. 8: 155-166 P i t t , G.A.J., 1965. Chemical structure and vitamin A a c t i v i t y Proc. Nut. Soc. 24: 153-159 P i t t G.A.J., 1971. "Vitamin A" i n Carotenoids 0. I s l e r , ed. Birkhausen Verlag, Basle and Stuttgart, pp 717-748 Plack P.A., 1965. Occurrence, absorption and d i s t r i b u t i o n of vitamin A. Proc. Nut. Soc. 24: 146-153 Popper H., 1940 a. H i s t o l o g i c a l Demonstration of vitamin A i n the human l i v e r by means of fluorescence microscopy. Proc. Soc. Exp. B i o l . Med. 43: 234-236 Popper H., 1940 b. H i s t o l o g i c a l demonstration of vitamin A i n rats by means of fluorescence microscopy. Proc. Soc. Exp. B i o l . Med. 43: 133-136 Popper H., 1S41. H i s t o l o g i c d i s t r i b u t i o n of vitamin A i n human organs under normal and under pathologic conditions. Arch. Path. 31: 766-802 Popper H., R. Greenberg, 1941. v i s u a l i z a t i o n of vitamin A i n r a t organs by fluorescence microscopy. A.M.A. Arch. Path. 32: 11-32 Popper H., B.W. Volk, 1944. Absorption of vitamin A i n the rat A.M.A. Arch. Path. 38: 71-75 Poole J.C.F., 1958. The effect of hypervitaminosis A on blood coagulation i n the rat. Quart. J . Exp. Physiol. 43: 427-437 Pudelkiewicz, W.J., L. Webster, G. Olson, L.D. Matterson, 1964. Some physiological effects of feeding high l e v e l s of v i t a -min A acetate to chicks. Poul. Sc. 43: 1157-1164 Quick A.J., 1961. C l i n i c a l i n t e r p r e t a t i o n of the one-stage prothrombin time. C i r c . 24: 1422-1428 Quick A.J., M. Ste f a n i n i , 1948. Experimentally-induced changes i n the prothrombin l e v e l of the blood IV the r e l a t i o n of vitamin K deficiency to the i n t e n s i t y of dicumarol action and to the effe c t of excess vitamin A: With a s i m p l i f i e d method f o r vitamin A assay. J . B i o l . Chem. 175: 945-952 R a l l i E.P., A. Waterhouse, 1933. Blood cholesterol i n dogs on an A def i c i e n t d i e t . Roc. Soc. Exp. B i o l . Med. 30: 519-523 - 161 -Ramasarma T., 1968. L i p i d quinones. Adv. L i p . Res. 6: 107-180 Raoul Y., N. Ragheb-Hanna, 1950. Compt. Rend. Soc. B i o l . cited by C. Nieman and H.J. K l e i n Obbink, 1954, The b i o -chemistry and pathology of hypervitaminosis A. V i t s . Horm. 12: 69-99 Ratnoff O.D., C. Menzie, 1951. A new method for the determina-t i o n of fibrinogen i n small samples of plasma. J . Lab. C l i n . Med. 37: 316-320 Reti L., 1935. Sur 1'Etat de combinaison de l a vitamin A dans l e s h u i l e s to f o i e . C S . Soc. B i o l . 120: 577-580 Riggi S., N.R. DiLuzio, 1962. Removal of chylomicra and f a t emulsions by the r e t i c u l o endothelial system (RES): A new functional test f o r RE a c t i v i t y . Fed. Proc. 21: 279-(abstr.) Roberts A.B., H.F. DeLuca, 1969. ''Metabolism of r e t i n o l and r e t i n o i c acid" i n The Fat-Soluble Vitamins H.F. DeLuca and J.W. Suttie eds. The u n i v e r s i t y of Wisconsin Press, Madi son, Milwaukee, London, pp 227-240 Roberts A.B., H.F. DeLuca, 1967. Pathways of r e t i n o l and r e t -i n o i c acid metabolism i n the r a t . Bioc. J . 102: 600-605 Rodahl K., T. Moore, 1943. The vitamin A oontent and t o x i c i t y of bear and seal l i v e r . Bioc. J . 37: 166-168 Rol'nik V.V., 1970. "Development of the digestive Organs" i n B i r d Embryology by V.V. Rolc'nik,' I s r a e l Program for S c i e n t i f i c Translation, l t d . , Jerusalem, pp 179-188 Schettler F.G., R. Sanwold, 1969. Chapt. 10, section d (iv) "Vitamins" i n Atherosclerosis Pathology, Physiology, Aetiology, Diagnosis and C l i n i c a l Management F.G. Schettler ana c s . Boyd eds E l s e v i e r Publisnmg company, Amsterdam, London, New York, pp 891-899 Schoenheimer R., N.M. Sperry, 1934. A mioromethod f o r the determination of cholesterol and cholesterol esters. J . B i o l . Chem. 106: 745-760 Scott M.L., M.C Nesheim, R.J. Young, 1969 a. " I n t e r r e l a t i o n -ships a f f e c t i n g n u t r i t i o n a l Requirements" i n N u t r i t i o n  of the Chicken by M.L. Scott, M.C. Nesheim, and R.J. Young, M.L. Scott and Associates, Ithaca, New York, pp 372-390 Scott M.L., M.C. Nesheim, R.J. Young 1969 b. "The Vitamins" i n N u t r i t i o n of the Chicken by M.L. Scott, M.C. Nesheim, and R.J. Young, M.L. Scott and Associates, Ithaca, New York, pp 106-257 - 162 -Sewell H.B., G.E. M i t c h e l l , CO. L i t t l e , B.W. Hayes, 1967. Internet. Z. Vitaminforsch. 37: 301 cited i n "Vitamin A" by G.A.J. P i t t , 1971. i n Carotenoids, 0. I s l e r , ed., Birkhauser Verlag, Basle and Stuttgart, p 720 Shellenberger T.E., D.B. P a r r i s h , P.E. Sanford, 1964. Absorp tion of preformed vitamin A from l i g a t u r e d poultry i n t e s t i n a l sections. J . Nut. 82: 99-105 a. Sherman B.S., 1969. Autoradiographic l o c a l i z a t i o n of H-Vitamin A i n rat l i v e r . Int. J . V i t . Res. 39: 111-118 Simmons A., 1968. Technical Hematology, J.B. Lipp i n c o t t company, Philadelphia and Toronto, p 204 Singh V.N., M. Singh, T.A. Venkitasubramanian, 1969. Early effects of feeding excess vitamin A: Mechanism of f a t t y l i v e r production i n ra t s , d. L i p . Res. 10: 395-401 Singh M., V.N. Singh, T.A. Venkitasubramanian, 1968. Early E f f e c t s of Feeding Excess vitamin A: Hepatic glycogen, blood l a c t i c acid, Plasma NEFA, and glucose tolerance m rats. L i f e S c i . 7: 239-247 Skala I., F. Hruba, 1964. Accumulation of vitamin A by small i n t e s t i n e of the r a t i n v i t r o . Am. J . Physiol. 206: 458 460 Smith F.R., A. Raz, D.S. Goodman, 1970. Radioimmuno-assay of human plasma r e t i n o l - b i n d i n g protein. J . C l i n . Invest. 49: 1020-1032 Smith M.E., 1933. The effect of vitamin A deficiency on the concentration of the blood l i p i d s of albino r a t s . J . Nut. 8: 675-687 Sperry W.M., M. Webb., 1950. A r e v i s i o n of the Schoenheimer-Sperry method f o r cholesterol determination. J . B i o l . Chem. 187: 97-106 Snedecor G.W., 1946. S t a t i s t i c a l methods. Iowa state Colleg Press, Ames, Iowa, pp 206-208 Stopforth A., 1970. A study of coagulation mechanisms i n domestic chickens d. Comp. Path. 80: 525-533 Sturkie P.D., 1965. "Thyroids" i n Avian Physiology by P.D. Sturkie, Comstock Publishing Associates, Cornell Univer-s i t y Press, Ithaca, New York, pp'592-648 Subba Rao K., R.E. Olson, 1967. The effect of excessive vitamin A upon the metabolism of isoprenoid compounds i n rat l i v e r . Bioc. Biop. Acta 136: 406-409 - 163 -Subba Rao K., P. Seshari-Sastry, J . Ganguly, 1961. Fatty acid component of vitamin A ester i n sheep l i v e r . Arch. Bioc. Biop. 95: 285-889 Sundaresan P.R., D.G. T h e r r i a u l t , 1967. Matabolism of C 1 4 -r e t i n o i c acid i n the r e t i n o l - d e f i c i e n t rat. Fed. Proc. 26: 6354, (abstr.) Sure B., M.C. Kik, A.E. Church, 1933. Avitaminosis XIV E f f e c t of vitamin A deficiency on" concentration of blood l i p i d s of albino r a t . Proc. Soc. Exp. B i o l . Med. 30: 620-621 Suttie J.W., 1973. Vitamin K and prothrombin synthesis. Nut. Rev. 31: 105-109 Sylven C , B. Borgstrom, 1968. Absorption and lymphatic trans-port of cholesterol i n the rat". J . L i p . Res. 9: 596-601 Takahashi K., Z. Nakamiya, M. Kawakimi, T. Kitasato, 1925. S c i . Papers Inst. Phys. Chem. Res., Tokyo, 3: 81 ci t e d by T. Moore "Hypervitaminosis A" i n Vitamin A, 1957. by T. Moore, E l s e v i e r Publishing Company, .Amsterdam, London, New York, Princeton, pp 340-351 Thompson J.N. ; G.A.J. P i t t , 1960. vitamin A acid and hyper-vitaminosis A. Nature 188: 672-673. Thompson J.N., G.A.J. P i t t , R.A. Morton, 1960. vitamin A ac i d and l i v e r l e v e l s of ubiquinone and ubichromenol. B i o c . Biop. Acta. 45: 396-397 Thorbjarnarson T., J.C. Drummond, 1938. Conditions influencing the storage of vitamin A i n the l i v e r . Bioc. J . 32: 5-9 T o r s e l l i n i A., V. Lombardi, 1971. Modifioazioni D e l l ' a t t i v i t a f i b r i n o l i t i c a ematica indotte d a l l a vitamina A: Richerche i n v i t r o . Giorn. Geront. 19: 805-810 Treadwell C.R., G.V. Vahouny, 1968. "Cholesterol Absorption" i n Handbook of Physiology Section 6. Alimentary Canal  V o l . I l l i n t e s t i n a l ADsorption" C F . Code ed. , American Physiological society, wasnmgton, D.C pp 1407-1438 Vahouny G.V., CR. Borja, R.M. Mayer, CR. Treadwell, 1960. A rapid quantitive determination of t o t a l and free chol-esterol with enthrone reagent. Anal. Bioc. 1: 371-381 Va k i l U.K., O.A. Roels, M. Trout, 1964. Storage and transport ?I vi£??P-SoA i n r e l a t i o n to protein intake. Br. J . Nut. - 164 -Van Bruggen J.T., J.V. Strauamfjord, 1948. High vitamin A intake and blood l e v e l s of cholesterol, phospholipids, carotene, and vitamins C,A, and E. J . Lab. C l i n . Med. 33: 67-74 Vedder E.B., C. Rosenberg, 1938. Concerning the t o x i c i t y of vitamin A. J . Nut. 16: 57-68 von Kaulla, 1963, cited by G.A. Fearnley, 1965. "Measurement of F i b r i n o l y s i s " i n F i b r i n o l y s i s by G.R. Rearnley, Edward Arnold (Publishers) Ltd., London, pp:28-44 Walker S.E., E. Eylenburg, T. Moore, 1947. The action of vitamin K i n hypervitaminosis A. Bioc. J . 41: 575-580 Week E.F., F.J. Sevigne, 1949 a. vitamin A u t i l i z a t i o n studies I . The u t i l i z a t i o n of vitamin A alcohol, vitamin A ace-tate, and vitamin-A natural esters by the chick. J . Nut. 39: 233-250 Week E.F., F.J. Sevigne, 1949 b. vitamin A u t i l i z a t i o n studies I I . The u t i l i z a t i o n of vitamin A alcohol, vitamin A ace-tate, and vitamin A natural esters by the r a t . J . Nut. 39: 251-257 Weitzel G.. H. Schon, F.Z. Grey, 1956 a. Physiol. Chem. 305: 247 cited by J.M. Kordylas, 1972 a. Retinol i n chol-esterol biosynthesis. Glana Med. J . 11: 71-82 Wendt H., 1936. Dent. Med. Wochschr. 62: 1213 cited by L . J . Kinley and R.F. Krause, 1959. Influence of vitamin A on cholesterol blood l e v e l s . Proc. Soc. Exp. B i o l . Med. 102: 353-355 White A., P. Handler, E.L. Smith, 1968. " L i p i d Metabolism I I " i n P r i n c i p l e s of Biochemistry, McGraw-Hill Book Company, New York, Sydney, Toronto, London, pp 507-530 ** Wilson J.D., C.A. Lindsey, J r . , 1965. Studies on the influence of dietary cholesterol on cholesterol metabolism i n the i s o t o p i c steady state i n man. J . C l i n . Invest. 44: 1805-1814 Wiss 0., U. Gloor, 1960. vitamin A and l i p i d metabolism. V i t s . Horm. 18: 485-498 Wolbach S.B., D.M. Hegsted, 1952. Hypervitaminosis A and the skeleton of growing chicks. Arch. Path. 54: 30-38 Wolbach S.B., D.M. Hegsted, 1953. Hypervitaminosis A i n young ducks x.Arch. Path. 55: 47-54 **Whiteside, C.H., H.B. Fluckiger, 1965. Seasonal v a r i a t i o n i n the response of chicks to dietary cholesterol. Poul. Sc. 44: 257-259 - 165 -Wolf G., S.G. Kahir, B.C. Johnson, 1957. Metabolism studies with radioactive vitamin A. J . Am. Chem. Soc. 79: 1208-1812 Wood J.D., 1960. Dietary marine f i s h o i l s and cholesterol metabolism 2. The ef f e c t of vitamin A and lingcod l i v e r o i l components on the serum cholesterol l e v e l s i n chicks. Can. J". Bioc. Physiol. 38: 879-887 Wood J.D., 1963. The hypocholesterolemic a c t i v i t y of B-apo-8'-earotenal. -Can. J . Bioc. Physiol. 41: 1663-1665 Wood J.D. 1962. The hypocholesterolemic a c t i v i t y of vitamin A and related compounds as influenced by their chemical structure. Can. J . Bioc. Physiol. 40: 529-536 Wood J.D., J . B i e l y , 1960 a. The eff e c t of dietary marine f i s h o i l s on the serum cholesterol l e v e l s i n hyperchol-esterolemia chickens. Can. J . Bioc. Physiol. 38: 19-24 Wood J.D., J . B i e l y , 1960b. A substance i n lingcod l i v e r o i l which prevents hypercholesterolaemia i n cholesterol-fed chickens. Nature 185: 473-474 Wood J.D., J . T o p l i f f , 1961. Dietary marine f i s h o i l s and cholesterol metabolism 3. The comparative hypocholesterol-emic a c t i v i t i e s of f i s h o i l and vitamin A. J . Fis h e r i e s Res. Board Can. 18: 377-382 Worden A.N., 0". Bunyan, A.W. Danes, 1955. The urinary excre-tion of vitamin A by the dog. Bioc. d. 59: 527-528 Wostmann B.S., P.L. Knight, 1965. Antagonism between vitamins A and K i n the germfree r a t . J . Nut. 87: 155-160 Woytkiw L., N.C. Esselbugh, 1951. vitamin A and carotene absorptions i n the guinea pig. d. Nut. 43: 451-458 Zachman R.D., B.E. Dunagin, J.A. Olson, 1966a. Formation and enterohepatic c i r c u l a t i o n of metabolites of r e t i n o l and r e t i n o i c acid i n b i l e dust-cannulated rats, d. L i p . Res. 7: 3-9 Zachman R.D., j'.A. Olson, 1965. Uptake and metabolism of r e t -i n o l (vitamin A) i n the iso l a t e d perfused rat l i v e r , d. L i p . Res. 6: 27-32 . Zachman R.D., M.B. Singer, J.A. Olson, 1966b. B i l i a r y secre-tion of metabolites of r e t i n o l and of r e t i n o i c acid i n the guinea pig and chick, d. Nut. 88: 137-142 - 166 -APPENDIX - 167 -Table 1(A). Experiment L: Analysis of variance comparing the l i v e r vitamin A concentrations of the D and NH chicks a f t e r the eighty-nine hour test period Source of var i a t i o n df MS F P Breed 1 637,288.2 60.2 <0.001 vitamin A content of diet 2 65915,298.2 653.8 <0.001 breed X v i t a -min A 2 242,997.4 23.0 error 30 10,577.5 t o t a l 35 - 168 -Table 2(A). Experiment 1: Analysis of variance comparing the l i v e r vitamin A concentrations of the day-old D and NH chicks Source of var i a t i o n df MS F Breed 1 within breeds 8 t o t a l 9 36,880.1 377.4 9.8 <0.05 - 169 -Table 3 (A) . Experiment 2: Analysis of variance comparing the l i v e r vitamin A concentrations of the D and NH chicks fed for three days the Gontrol and the A* diets con-t a i n i n g 0.5% of cholesterol Source of vari a t i o n df MS F P Breed 1 431,160.3 304.9 0.001 vitamin A con-tent of diet 1 9866.3 7.0 0.05 breed X vitamin A 1 12,945.3 9.2 0.05 error 12 1414.2 t o t a l 15 - 170 -Table 4 (A) . Experiment 2: Analysis of variance comparing the pre-test plasma cholesterol concentrations of the D chicks which were then fed the control and the A+ diets-; containing 0.5% of cholesterol Source of vari a t i o n df MS E P Between pros-pective groups (control vs A-) 1' 273.6 3.08 ns within pros-pective groups 38 88,8 t o t a l 39 - 171 -Table 4(A) cont'd: Analysis of variance comparing the test plasma cholesterol concentrations of the D chicks fed f o r three days the control and the A* diets containing 0.5% of cholesterol Source of vari a t i o n df MS Vitamin A con-tent of diet 1 3327.0 5.52 0.05 within groups 38 602.7 t o t a l 39 - 172 -Table 5(A) . Experiment 2: a) Analysis of variance comparing the pre-test plasma cholesterol concentrations of the NH chicks which were then fed the control and the A+ diets containing 0.5% of cholesterol Source of vari a t i o n df MS F P Between pros-pective groups (control vs. A-f) 1 171.3 2.39 ns within pros-pective groups 38 71.6 t o t a l 39 b) Analysis of variance comparing the test plasma chol-esterol concentration of the NH chicks receiving f o r three days the control and A* the diets containing 0.5% of cholesterol Source of vari a t i o n df MS F P Vitamin A con-tent of diet 1 831.7 2.82 ns within groups 38 295.4 t o t a l 39 - 173 -Table 5(A) cont'd: c) Analysis of variance comparing the increases i n the plasma cholesterol concentrations of the NH chicks which were fed f o r three days the control and the A+ diets containing 0.5% of cholesterol. Source of vari a t i o n df MS F Vitamin A content of diet 1 1762.8 7.62 0.01 within groups 38 231.2 t o t a l 39 - 174 -Table 6 (A) * Experiment 3: Analyses of variance on the plasma cholesterol concentration data a) pre-test plasma cholesterol values: 1) D chicks groupl vs. controls Source of v a r i a t i o n df MS F P Vitamin A content of diet 1 2.6 0.02 ns within groups 28 116.0 t o t a l 29 2) D chicks groupll vs. controls Source of v a r i a t i o n df MS F P Vitamin A content of diet 1 3.2 0.02 ns within groups 33 138.4 t o t a l 34 3) NH chicks groupl vs. controls Source of v a r i a t i o n df MS F P Vitamin A content, of diet 1 20.4 0.17 ns within groups 28 122.2 t o t a l 29 - 1 7 5 -Table 6(A). Pre-test plasma cholesterol values (cont'd): 4) NH chicks groupll vs. controls Source of v a r i a t i o n df MS F P Vitamin A content of diet 1 2 4 . 6 0.22 within groups 33 109.7 t o t a l 34 ns - 176 -Table 6(A) . cont'd b) test plasma cholesterol values: 1) D chicks groupl vs. controls Source of variation df MS F P dietary vitamin A content 1 2130.0 28.9 <0.001 within groups 28 73.63 tot a l 29 2) D chicks groupll vs. controls Source of var i a t i o n df MS F P dietary vitamin A content 1 0 0 ns within groups 33 52.3 t o t a l 34 3) NH chicks groupl vs. controls Source of v a r i a t i o n df MS F P dietary vitamin A content 1 1882.0 25.4 <0.001 within groups 28 74.0 t o t a l 29 4) NH chicks groupll vs. controls Source of variation df MS F P dietary vitamin A content 1 12.5 0.35 ns within groups 33 37.9 t o t a l 34 - 177 -Table 7(A) . Experiment 4: Analysis of variance comparing the cholesterol absorption e f f i c i e n c i e s i n groups 1 and 2, and groups 1 and 3 a) D chicks groups 1 and 2 Source of va r i a t i o n df MS F P Quantity of o r a l vitamin A 1 96.4 2.1 ns within groups 18 46.0 t o t a l 19 b) D chicks groups 1 and 3 Source of va r i a t i o n df MS F P Parenteral dose of vitamin A 1 451.6 12.4 <0.01 within groups 18 36.4 t o t a l 19 c) NH chicks groups 1 and 2 Source of v a r i a t i o n df MS F P Quantity of oral vitamin A 1 451.6 12.4 <0.01 within groups 18 36.4 t o t a l 19 d) NH chicks groups 1 and 3 Source of v a r i a t i o n df MS F P Parenteral dose of vitamin A 1 421.2 12.0 <0.01 within groups 18 35.0 t o t a l 19 - 178 -Table 8(A). Experiment 4: Analyses of variance comparing the percent of absorbed cholesterol found i n the hepatic pool i n groups 1 and 2, and groups 1 and 3 a) D chicks groups 1 and 2 Source of va r i a t i o n df MS F P Quantity of o r a l vitamin A 1 108.6 2.1 ns within groups 18 50.6 t o t a l 19 b) D chicks groups 1 and 3 Source of var i a t i o n df MS F P Parenteral dose of vitamin A 1 382.8 7.0 <0.05 within groups 18 54.8 t o t a l 19 c) NH chicks groups 1 and 2 Source of variation df MS F P Quantity of oral vitamin A 1 250.0 4.9 <0.05 within groups 18 40.8 t o t a l 19 d) NH chicks groups 1 and 3 Source of v a r i a t i o n df MS F P Parenteral dose of vitamin A 1 794.8 13.0 <0.01 within groups 18 61.3 t o t a l 19 - 1 7 9 -Table 9(A) . Experiment 4 : Analyses of variance comparing the percent of intravenously-injected 4 - 1 4 c - c h o l e s t e r o l found i n the hepatic pool i n groups 4 and 5 , and groups 4 and 6 . a) D chicks groups 4 and 5 Source of va r i a t i o n df MS F P Quantity of oral vitamin A within groups t o t a l 1 8 9 5 2 . 9 1 7 . 1 3 . 1 ns b) D chicks groups 4 and 6 Source of v a r i a t i o n df MS F Parenteral dose of vitamin A within groups t o t a l 1 8 9 1 0 7 . 0 1 7 . 1 5.7 ^ 0 . 0 5 c) NH chicks groups 4 and 5 Source of v a r i a t i o n df MS Quantity of o r a l vitamin A within groups t o t a l 1 1 2 4 . 7 8 4 6 . 5 9 2.7 ns d) NH chicks groups 4 and 6 Source of v a r i a t i o n df MS Parenteral dose of vitamin A within groups t o t a l 1 4 7 7 . 5 8 5 4 . 0 9 8 . 8 < 0 . 0 5 - 180 -Table 10(A) . Experiment 4: Analyses of variance comparing the b i l i a r y excretion of l a b e l expressed as a percent of r a d i o a c t i v i t y found i n the hepatic pool i n groups 4 and 5, and groups 4 and 6. a) D chicks groups 4 and 5 Source of variation df MS F P Quantity of o r a l vitamin A 1 15.2 3.7 ns within groups 8 4.1 t o t a l 9 b) D chicks groups 4 and 6 Source of v a r i a t i o n df MS F P parenteral dose of vitamin A 1 0.1 0.02 ns within groups 8 4.8 t o t a l 9 c) NH chicks groups 4 and 5 Source of v a r i a t i o n df MS F P Quantity of oral vitamin A 1 14.3 4.1 ns within groups 8 3.5 t o t a l 9 d) NH chicks groups 4 and 6 Source of v a r i a t i o n df MS F P Parenteral dose of vitamin A 1 52.8 10.4 40.05 within groups 8 5.1 t o t a l 9 - 181 -Table 11(A). Experiment 4: Analyses of variance comparing the e f f i c i e n c y of cholesterol absorption and the percent of absorbed cholesterol found i n the hepatic pool i n groups 1 and 10, and also comparing the per-cent of intravenously-injected 4- 1 4C-cholesterol found i n the hepatic pool and the b i l i a r y excretion of l a b e l i n groups 4 and 11. a) D chicks cholesterol absorption groups 1 and 10 Source of v a r i a t i o n df MS F P Intramuscular i n j e c -t i o n treatment 1 within groups 1_3 t o t a l 14 11.6 40.6 0.3 ns b) NH chicks cholesterol absorption groups 1 and 10 Source of v a r i a t i o n df MS F P Intramuscular i n j e c -tion treatment 1 within groups 1J3 t o t a l 14 60.7 42.0 1.4 ns c) D chicks hepatic pool groups 1 and 10 Source of variation df MS F Intramuscular i n j e c -tion treatment 1 within groups 13 t o t a l 14 13.0 45.9 0.3 ns - 182 -Table 11(A). cont'd d) NH chicks hepatic pool groups 1 and 10 Source of v a r i a t i o n df MS F intramuscular i n j e c -tion treatment 1 within groups 13 t o t a l 14 21.1 27.7 0.8 ns e) D chicks hepatic pool groups 4 and 11 Source of v a r i a t i o n df MS F Intramuscular i n j e c -tion treatment within groups t o t a l 1 8 9 5.4 36.2 0.1 ns f) NH chicks hepatic pool croups 4 and 11 Source of v a r i a t i o n df MS F P Intramuscular i n j e c -t i o n treatment within groups t o t a l 1 8 9 10.2 41.9 0.2 - 183 -Table 11(A). cont'd g) D chicks excretion groups 4 and 11 Source of v a r i a t i o n df MS F Intramuscular i n j e c -t i o n treatment within groups t o t a l 1 8 9 2.5 5.0 0.5 ns h) NH chicks excretion groups 4 and 11 Source of v a r i a t i o n df MS F intramuscular i n j e c -tion treatment within groups to t a l 1 8 9 3.0 5.4 0.6 ns - 184 -Table 12(A) . Experiment 4: Analyses of variance comparing the e f f i c i e n c y of cholesterol absorption and the percent of absorbed cholesterol found i n the hep-a t i c pool i n groups 1 and 7 a) D chicks cholesterol absorption groups 1 and 7 Source of v a r i a t i o n df MS E P Intravenous i n j e c -t i o n treatment 1 14.1 0.5 ns within groups 13 27.3 t o t a l 14 b) NH chicks cholesterol absorption groups 1 and 7 Source of v a r i a t i o n df MS F P Intravenous i n j e c -tion treatment 1 30.5 0.8 ns within groups 2^5 37.1 t o t a l 14 c) D chicks hepatic pool groups 1 and 7 Source of variation kf MS F P Intravenous i n j e c -tion treatment 1 4.6 0.1 ns within groups 1_3 46.0 t o t a l 14 - 185 -Table 12(A) . cont'd d) NH chicks Source of v a r i a t i o n Intravenous i n j e c -tion treatment within groups t o t a l hepatic pool groups 1 and 7 df MS F 1 '44.1 1.2 13 37.9 14 - 186 -Table 13(A). Experiment 4: Analyses of variance comparing the e f f i c i e n c y of cholesterol absorption, and the per-cent of absorbed cholesterol found i n the hepatic pool i n groups 2 and 8, and i n groups 3 and 9 a) D chicks cholesterol absorption groups 2 and 8 Source of v a r i a t i o n df MS F P Intravenous i n j e c -t i o n treatment 1 31.0 0.7 ns within groups 13. 43.6 t o t a l 14 b) NH chicks cholesterol absorption groups 2 and 8 Source of v a r i a t i o n df MS F P Intravenous i n j e c -tion treatment 1 26.1 0.8 ns within groups 1_3 34.5 t o t a l 14 c) D chicks cholesterol absorption groups 3 and 9 Source of v a r i a t i o n df MS F P Intravenous i n j e c -tion treatment 1 34.6 0.6 ns within groups 13 54.7 t o t a l 14 - 187 -Table 13(A). cont'd d) NH chicks cholesterol absorption groups 3 and 9 Source of v a r i a t i o n df MS F P Intravenous i n j e c -t i o n treatment 1 60.7 1.4 ns within groups 13 42.0 t o t a l 14 e) D chicks hepatic pool groups 2 and 8 Source of v a r i a t i o n df MS F P Intravenous i n j e c -tion treatment 1 0.9 0.02 ns within groups 1_3 58v'4 t o t a l 14 f) NH chicks hepatic pool groups 2 and 8 Source of v a r i a t i o n df MS F P Intravenous i n j e c -t i o n treatment 1 45.7 0.8 ns within groups 13 58.8 t o t a l 14 g) D chicks hepatic pool groups 3 and 9 Source of v a r i a t i o n df MS F P Intravenous i n j e c -t i o n treatment 1 66.4 1.1 ns within groups 13 60.5 t o t a l 14 - 188 -Table 13(A) . cont'd h) NH chicks hepatic pool groups 3 and 9 Source of v a r i a t i o n df MS F Intravenous i n j e c -t i o n treatment within groups t o t a l 1 13 14 19.0 67.2 0.3 ns - 189 -Table 14(A). Experiment 4: Two-way analysis of variance on the percent cholesterol absorption data of groups 1 and 3 Source of v a r i a t i o n df MS F P Breed 1 -283.0 7.9 <0.01 vitamin A dose 1 1745.0 48.9 <0.001 breed X vitamin A 1 0.6 0.02 ns error 36 35.7 t o t a l 39 - 190 -Table 15(A). Experiment 5: Analyses of variance on the eighteen-day one-stage prothrombin time data a) D chicks Source of var i a t i o n df MS Vitamin A content of diet 1 within diets 18 t o t a l 19 0.001 0.007 1.3 ns b) K chicks Source of va r i a t i o n df MS Vitamin A content of diet 1 within diets 18 t o t a l 19 0.001 0.004 0.25 ns c) B chicks Source of v a r i a t i o n df MS Vitamin A content of diet 1 within diets 18 t o t a l 19 0.007 0.003 2.33 ns - 191 -Table 15(A). cont'd d) comparison of -the three s t r a i n s Source of var i a t i o n df Str a i n of b i r d 2 within s t r a i n 27_ t o t a l 29 e control prothrombin times among MS F P 0.008 2.67 ns 0.003 - 192 -Table 16(A) . Experiment 6: S t a t i s t i c a l analyses on the body weight data within each breed at six and eight weeks of age a) D chicks at s i x weeks Source of variation df MS F P Vitamin A content of di e t 3 3349 0.96 ns within groups 49 3470 t o t a l 52 b) NH chicks at six weeks Source of var i a t i o n df MS F P Vitamin A content of diet 3 1683 0.42 ns within group 48 3923 t o t a l 51 c) A chicks at s i x weeks Source of var i a t i o n df MS F P Vitamin A content of diet 3 333 0.13 ns within groups 50 2532 t o t a l 53 - 193 -Table 18(A). cont'd d) D chicks at eight weeks i) Analysis of variance: Source of va r i a t i o n df MS F P Vitamin A content of diet 3 22,958 4.35 <0.01 within groups 49 4266 t o t a l 52 i i ) Duncan's multiple range test: Level of d i e t -ary vitamin A (IU/kg) 35000 5000 100,000 200,000 body weight (g) 663 650 591 581 Duncan's com-parison a a b b 40.01 - 194 -Table 16(A). cont'd e) NH chicles at eight weeks i ) Analysis of variance: Source of var i a t i o n df MS F P Vitamin A content of diet 3 35,682 5.71 <0.01 within groups 48 6249 t o t a l 51 i i ) Duncan's multiple range test: l e v e l of d i e t -ary vitamin A (IU/kg) 5000 35,000 100,000 200,000 body weight 824 824 744 725 Duncan's comparison a a b b (0,01 f) A chicks at eight weeks Source of v a r i a t i o n df MS F P Vitamin A content of diet 3 372 0.08 ' ns within groups 50 4415 t o t a l 53 - 195 -Table 17(A). Experiment 6: S t a t i s t i c a l analysis of the three-week prothrombin times comparing the four d i f f e r e n t dietary vitamin A l e v e l s within each breed a) A chicks i ) Analysis of variance Source of variation df Vitamin A content of diet 3 within diets _36 t o t a l 39 i i ) Duncan's multiple range te-st: Level of diet-ary vitamin A (IU/kg) 5000 35000 200,000 100,000 One-stage pro-thrombin time (min) 0.40 0.44 0.54 0.56 Duncan's com-parison a a b b <0.01 MS E P 0.05783 11.08 <0.001 0.00522 - 196 -Table 17(A). cont'd b) D chicks Source of va r i a t i o n Vitamin A content of di et within diets t o t a l c) NH chicks Source of v a r i a t i o n Vitamin A content of diet within diets t o t a l df MS 3 0.00294 36 0.00210 39 df MS 3 0.00531 36 0.00217 39 F P 1.40 ns F P 2.44 ns - 197 -Table 18(A). Experiment 6: S t a t i s t i c a l comparison of the three-week prothrombin times of the IMH and D chicks and the NH and A chicks receiving the control diet a) NH chicks vs. D chicks i ) Analysis of variance: Source of v a r i a t i o n df MS F P Between breeds 1 0.0065 4.96 ^0.05 within breeds 18 0.0013 t o t a l 19 i i ) Duncan's multiple range test: Breed WL (D strain) New Hampshire One-stage prothrombin time (min) 0.41 .0.45 Duncan's comparison a b ^0.05 - 198 -Table 18(A). cont'd b) NH chicks vs. A chicks i ) Analysis of variance: Source of v a r i a t i o n df MS F P Between breeds 1 0.0125 11.4 <T0.01 within breeds 18 0.0011 t o t a l 19 i i ) Duncan's multiple range test: Breed A NH One-stage-pro-thrombin time (min) 0.40 0.45 Duncan's comparison a b <0.05 - 199 -Table 19(A). Experiment 6: S t a t i s t i c a l analysis comparing the prothrombin times of the A and D chicks receiv-ing the control diet to those receiving a large ex-cess of vitamin A i n t h e i r diets at thirty-one days of age a) A chicks i ) Analysis of variance: Source of variance df MS F P Level of dietary v i t a -min A 2 0.6278 20.79 40.001 within groups 18 0.0302 t o t a l 20 i i ) Duncan's multiple range test: Level of di etary vitamin A (IU/kg) 5000 100,000 200,000 One-stage-pro-thrombin time (min) 0.51 0.96 1.08 Duncan's compar-ison a b b 40.01 - 200 -Table 19(A). cont'd b) D chicks Source of v a r i a t i o n df MS Level of dietary-vitamin A within groups t o t a l 1 4 5 0.0004 0.0015 0.27 ns i - 201 -Table 20(A). Experiment 6: S t a t i s t i c a l analysis comparing the prothrombin times of the A chicks receiving R3 and R4 injected with menadione with those of the A chicks receiving the same diets injected only with the soybean o i l c a r r i e r and also with those of the controls which had received no i n j e c t i o n s of o i l or menadione i ) Analysis of variance: Source of v a r i a t i o n df MS E P Among groups within groups Total Level of di etary vitamin A (IU/kg) i n j ection treatment 2 18 20 0.3964 0.0059 67.3 i i ) Duncan's multiple range test: 5000 none One-stage pro-thrombin time 0.39 (min) Duncan's comparison 100,000 - 200,000 pooled 5 mg mena- soybean o i l dione per intramuscut kg body l a r l y weight i n -tramuscu-l a r l y 0.42 0.81 40.001 a <0.01 - 202 -Table 21(A). Experiment 6: Chi-square tests comparing the percent c l o t l y s i s of the control and R2 birds of a l l three breeds to the percent c l o t l y s i s of the R3 and R4 birds at three, four, f i v e , and six hours of incubation Incubation time (hrs) X^ p 3 1.1 ns 4 13.7 <0.01 5 6 40.0 28.0 .£0.01 <0.01 - 2G3 -Table 28(A). Experiment 6: Chi-square tests comparing the percent l y s i s of cl o t s from the control and R8 birds of a l l three breeds to the percent l y s i s of cl o t s from the R3 and R4 birds at three, four, f i v e , six and seven hours of incubation (After i n j e c t i o n with menadione or c a r r i e r o i l but ignoring these t r e a t -ments ). Incubation time (hrs) P 3 8 . 1 40.01 4 81.1 <0.01 5 83.1 < 0 . 0 1 6 1 8 . 0 <-0.01 7 6.3 < 0 . 0 5 - 204 -Table 23(A). Experiment 6: Two-way analysis of variance com-paring the concentration of plasma fibrinogen of the three breeds and four d i f f e r e n t l e v e l s of vitamin A intake. Source of va r i a t i o n df MS F P Breeds 2 9.. 741 5.09 <0.05 Level of dietary v i t a -min A 3 15.107 7.90 40.001 Breed X vitamin A l e v e l 6 0.502 0.26 ns error 48 t o t a l 59 - 205 Table 24(A). Experiment 6: S t a t i s t i c a l analysis comparing the plasma fibrinogen concentrations of the A chicks receiving the control diet to those of the A chicks receiving R2 Source of v a r i a t i o n df MS F P Vitamin A l e v e l i n diet 1 5.929 4.20 ns within diets 8 1.412 t o t a l 9 - 206 -Table 25(A) . Experiment 6: Analysis of variance on the l i v e r vitamin A concentration data obtained from the birds of each breed and diet at eight weeks of age Source of v a r i a t i o n df MS F P Breeds 2 44,146,962 43.9 <0.001 vitamin A content of diet 3 711,105,366 706.6 40.001 breed X vitamin A l e v e l 6 21,387,147 21.3 ^0.001 error 60 1,006,319 t o t a l 71 - 207 -Table 26(A). Experiment 6: Chi-square tests comparing the percent l y s i s of clots from the D and NH chicks formerly receiving either the control diet or R2 to the percent l y s i s of clots from birds formerly receiving either R3 or R4 at three, four, f i v e , and six hours of incubation. (Ar fourteen weeks of age after s i x weeks feeding on the b r o i l e r grower diet) Incubation time (hrs) X 2 P 3 1.1 ns 4 6.7 40.01 5 11.0 ^0.01 6 8.5 40.01 - 208 -Table 27(A). Experiment 6: a) Analysis of variance comparing the plasma vitamin A concentrations of the birds formerly receiving the control and R2 diets to those of the birds form-er l y receiving R3 and R4 a f t e r s i x weeks of receiv-ing the b r o i l e r grower diet (data f o r NH and D chicks combined) Source df MS F P Control-R2 vs. R3-R4 1 1.22 0.031 ns within groups 38 39,93 t o t a l 39 b) Analysis of variance comparing the plasma caro-tene concentrations of the birds formerly receiving the control and R2 diets to those of birds formerly receiving R3 and R4 afte r s i x weeks of receiving the b r o i l e r grower di e t , (data for NH and D chicks com-bined. ) Source df MS F P Control-R2 vs. R3-R4 1 15 0.028 ns within groups 38 541 t o t a l 39 - 209 -Table 28(A). Experiment 7: Analysis of variance comparing the one-stage prothrombin times of the birds on the vitamin K - s u f f i c i e n t and - d e f i c i e n t diets at t h i r - • teen days of age Source of v a r i a t i o n df MS F P Level of vitamin K i n diet 1 0.1040 23.64 <,0.01 with diets 8 0.0044 t o t a l 9 - 210 -Table 29 (A). Experiment 7: S t a t i s t i c a l analysis showing the eff e c t of menadione on the one-stage prothrombin time sixteen hours a f t e r i t s i n j e c t i o n intramuscu-l a r l y into chicks receiving vitamin K - s u f f i c i e n t and - d e f i c i e n t diets i ) Analysis of variance: Source of va r i a t i o n df MS F P Among groups within groups t o t a l 3 12 15 0.0835 0.0061 i i ) Duncan's multiple range test: Level of dietary vitamin K Injection treatment 13.6 ^0.001 5mg mena-dione per kg body weight intramus-cul a r l y One-stage prothrombin time (min) Duncan's comparison 0.46 -K 0.0055g menadione/kg Soybean 5mg mena-o i l i n t r a - dione per muscularly kg body weight intramus-cu l a r l y Soybean o i l i n t r a -muscularly 0.46 0.48 0.76 40.01 

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