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Some aspects of ketosis in domestic animals Shore, Alan W. 1948

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3 3 7 SOME ASPECTS OF KETOSIS IN DOMESTIC ANIMALS Alan W. Shore A Thesis submitted i n P a r t i a l Fulfillment of the Requirements for the Degree of Master of Soienoe i n Agrioulture in the Department of Animal Husbandry. THE UNIVERSITY OF BRITISH COLUMBIA OCTOBER , 1948 SOME ASPECTS OP KETOSIS IN DOMESTIC ANIMALS An abstract of the thesis submitted by Alan W. Shore. Department of Animal Husbandry, Faculty of Agriculture, University of British Columbia. October, 1948. SOME ASPECTS OF KETOSIS IN DOMESTIC ANIMALS Ketosis in dairy cattle is now universally recognized as a c l i n i c a l syndrome causing appreciable monetary loss to the dairy industry. It has been established as a definite pathological state within the area studied in the present work. No completely adequate specific therapy has been suggested for the treatment of the various recognized forms of ketosis. Present methods for the rapid detection of uncomplicated and complicated ketosis in the f i e l d are not entirely satis-factory. However, sufficient data has been published to pro-vide a reasonable picture of the blood and urine changes that occur in ketosis; and the necessary laboratory procedures are available for i t s detection. The present work has added to this information and has afforded a f i r s t comparison of .ketosis as recognized here, with the condition observed else-where. Information was obtained from the University herd of Ayrshire cattle permitting the establishment of normal levels for blood and urine constituents under winter and summer feeding conditions. It has been demonstrated that, under the feeding and management conditions prevailing in the University herd, a subclinical avitaminosis A exists not only in the winter, but probably in the summer months. From the information published elsewhere, as well as from the present data, i t would appear that the heavy drain on the maternal energy reserve and on certain of the vitamins follow-ing parturition, is sufficiently great to cause a physiological derangement of pathological significance. The possibility i 3 suggested that vi tamip. A might play a c r i t i c a l role, in the induction of this derangement. It seems likely that the fundamental metabolic disorder is concerned with the energy metabolism of the animal. It i s not yet•established whether this is solely within the carbohydrate cycle, the l i p i d cycle or within both. Nor is i t established whether vitamin A is involved in this derangement. In the present Investigation, no experimental work has been carried out relative to ketosis in other domestic animals. A review of the current literature concerning ketosis in other domestic animals has been made and the various conditions have been compared to the ketotic state in dairy cattle. These observations confirm, in part, the tentative conclusion that ketosis is a disturbance of energy metabolism. The mechanisms involved in normal energy metabolism have been' discussed in relation to the energy picture in the ketotic state. A brief outline has been given of the various theories which have been suggested to account for the form-ation of ketone bodies. The experimental work was planned on the assumption that the basic cause of ketosis is a deficiency of available carbohydrate and as a result the animal's energy requirements are not satisfied. This deficiency may arise through a diet-ary inadequacy or through the inability of the animal to metabolize carbohydrate arising from protein and l i p i d precursors. - A working hypothesis was advanced proposing that the inability of an animal to make use of i t s energy resources through normal pathways is due to a deficiency of vitamin A* On the basis of this hypothesis, i t was assumed that animals suffering from avitaminosi.s A would be unable to metabolize administered carbohydrate in the normal manner. The experimental results obtained indicate that in young animals deprived of vitamin A , a hypoglycemia of c l i n i c a l l y significant proportions develops as the vitamin A deficiency s progresses. The data suggests further that this hypoglycemia is one of the earliest symptoms of avitaminosis A and arises despite an adequate Intake of dietary energy. The development of a hypoglycemia on adequate energy intake followed by a characteristic loss in body weight is offered as supplementary evidence that vitamin A may have a role in carbohydrate metabolism. Other experiments have been conducted that demonstrate the inability of the vitamin A deficient animal to u t i l i z e glucose administered per os. Work is proceeding in order to confirm this finding. In conclusion, results have been presented to suggest that vitamin A may possess, in addition to i t s other established functions, a specific role in carbohydrate metabolism. The experimental data is not yet sufficiently complete to assign the properties of a coenzyme to vitamin A. Moreover, the possibility of vitamin A affecting the hormonal balance and thus i n d i r e c t l y , carbohydrate metabolism, cannot be precluded on the basis of the present i n v e s t i g a t i o n . F i n a l proof that vitamin A may possess t h i s coenzyme a c t i v i t y must await f u r t h e r s t u d i e s . I t i s suggested that subsequent work might w e l l e x p l o i t the advantages of " i n v i t r o " t i s s u e s l i c e techniques. Related experiments have been c a r r i e d out and are appended to the t h e s i s f o r future reference. AGKNOWLEDGEMENT I wish to thank Professor H . M. King for suggesting this problem and for providing the f a o i l i t i e s for the exeoution of this investiga-tion. To Dr. A. J . Wood I would l i k e to express my appreciation for his direction, assistanoe and oritioism. Gratitude i s also expressed to Dr. S. N. Wood for his interest i n this thesis1," and particularly for his help in obtaining samples from the University herd. 'TABLE OF CONTENTS Page I. KETOSIS IN DAIRY CATTLE 1 A. Historical Background 1 B. Economic Importance 1 C. Incidence 2 1. General 2 2. Geographical Distribution 3 3. Fie l d Survey in B r i t i s h Columbia 3 D. Symptoms 4 1. Types 4-2* Hematology 6 3. Urology 7 4. Biochemical Changes in the Milk 7 5* Histopathology 7 E. Diagnosis 8 F. Treatments 8 1. Carbohydrate 8 2. Sedatives 9 3. Hormones 9 4. Minerals 10 5. Vitamins 10 6. Experimental " 16 I. A Typical Case of Ketosis 16 II. Blood and Urine Histories of the University Herd - Pre and Post Parturient 18 III. Blood and Urine History of University Herd - Before and AfterPasture 21 G. Summary 27 II. KETOSIS IN OTHER DOMESTIC ANIMALS 28 A. Pregnancy Disease i n Sheep 28 B. Ketosis i n Swine 31 C. Ketosis in Goats 32 D. A l l i e d C l i n i c a l Conditions 33 1. Milk Fever 33 2. Baby Pig Disease — Acute Hypoglycemia 35 3. Diabetes 38 E. Summary 40 III. THE ETIOLOGY OF KETOSIS 42 A. ' Introduction 42 1. Normal Energy Metabolism 42 2. Metabolism in the Ketotic State -51 '3. Working Hypothesis 62 B. Experimental 65 IV. SUMMARY 88 1 SOME ASPECTS OF KETOSIS IN DOMESTIC ANIMALS, 1. KETOSIS IN DAIRY CATTLE A, Hiatorieal Background Ketosis is a metabolic disorder of animals characterized by an abnormal accumulation of ketone bodies in the tissues and body f l u i d s . Ketonemia and ketonuria signify the presence of excess amounts of ketones in the blood and urine'respectively. Although some confusion in the terminology relevant to this disease s t i l l exists in the current literature, i t is believed that the term "Ketosis" is preferable to "Acetonemia" because i t is more indicative of the complete syndrome. Udall (103) reports that a nervous form of the disease now known as ketosis of dairy eattle was reoognized as early as eighteen hundred and forty nine by Landel, who described it-under the name of "mania puerperalis". Fleming*s classic textbook "Veterinary Obstetrics" , refers to several reports of mania puerperalis. One of these aooounts was written by Robellet and later translated by Rolls (71). He describes in great detail an "affeotion i n a oow, simulating rabies* and cured by chloral". Fleming in commenting on this oase (29) was inclined to believe that this condition was caused by cerebral i r r i t a -tion from some specific physical influence such as indigestion. It is significant to note that he recommended chloral hydrate therapy, a treatment s t i l l extensively used today, B. Economio Importance The economic loss to the dairy industry caused by ketosis is d i f f i c u l t to assess beoause of the comparatively low mortality 2 rate; and because of the number of sub-olinioal oases that are probably never reported. Severely affected cows/ i f not given prompt treatment'^ may die; or i f they eventually reeover, they frequently f a i l to return to normal milk production levels. The greatest loss to the industry as a result of this disease comes from the lowered milk production which most frequently occurs i n high-producing animals. Magill (58) estimates that in cows suffering from ketosis the milk yield decreases from twenty-five to f i f t y percent. In addition to this lowered pro-duction, many animals eontinue to give off the repellent acetone odour in the milk for some time after the peak of the c l i n i c a l condition has been reached, thus making the milk unsuitable for human consumption (73). Cows with acute ketosis frequently have to be discarded because of extreme emaciation and low milk production. The recognition of the importance of this economic loss both to the producer and to the consumer has emphasized the necessity for extensive fundamental re-search into the etiology of this disease. C. Incidenoe 1. General Ketosis ooours in well-fed, high-producing oows and heifers of a l l ages (103). It occurs in each month of the year and i n both pastured and stabled animals. The highest inoidenee appears to be in the late winter, early spring, and when the animals are on dried-out pasture. Approximately f i f t y percent of the cases are parturient and the remainder are non-parturient. Of the parturient cases/ about one half develop within thirty days after parturition. Ketosis may 3 occur independently or as a complication of metritis^' parturient paresisf or indigestion. 2. Geographical Distribution. Ketosis is a disease of world-wide importance. Janssen (49) and Hupka (47) have desoribed the oondition as i t ocours in Germany. It has also been reported from Holland (92), Denmark and Sweden (103), Great Britain (7) and Canada (64). The earliest reports of ketosis i n the United States oame from Texas (41) and Mississippi (2). With the recognition of ketosis as a c l i n i c a l entity, i t soon became evident that this disease might be encountered in any area where dairying i s practised. It has been re-ported on the Atlantic Coast from New England to Georgia; in the Central United States; and i n the South. Cases have also been desoribed as occurring on the Paoifio Coast (31)• There appears to be some con-troversy about the extent to which geographic', climatic and cultural factors influence the incidence of ketosis (65) (75)• 3. Fi e l d Survey i n B r i t i s h Columbia. As far as can be ascertained from the present literature^ no oomplete survey of the inoidenoe and distribution of ketosis i n B r i t i s h Columbia has ever been attempted, despite i t s recognition as a pathological condition i n the province, for a number of years. With a view to obtaining an estimate of the extent of ketosis in B r i t i s h Columbia, advantage was taken of the proximity of the Fraser Valley Milk Shed to conduct a detailed survey of this area], The survey was instigated in January, 1948. The immediate objective was to find the number of oases of ketosis treated by veterinarians during the months of January to May/ nineteen hundred and forty-eight, - a period believed l i k e l y to show the highest inoidenoe i n B r i t i s h Columbia. The details of the survey procedure, together with the.results obtained are recorded in Appendix I. As may be noted from this data, the limited response precludes any estimate of the inoidenoe of ketosis during nineteen hundred and forty eight. From personal contact with the veterinarians surveyed, the impression was gained that the. incidence is considerably higher than suspected, despite the lack of positive returns from the survey. In addition to the eases reported to veterinarians, i t is l i k e l y that there are many sub-olinioal cases that are never discovered. Because of the small number of oases reported, no attempt was made to oonsider the inoidenoe data in relation to age and breed of animal.-The survey did reveal that no specific therapy was practiced. Individual praotitioners administer minerals and vitamins in varying combinations and with a wide range of results, whenever the dextrose or chloral hydrate f a i l s to e l i c i t the expected response. D. Symptoms. 1. Types Ketosis may arise in parturient or non-parturient animals. It i s usual to recognize three o l i n i o a l types: The milk fever syndrome, the digestive syndrome and the nervous form, manifested by excitation, delirium, motor i r r i t a t i o n and paresthesia. The majority of the parturient oases are associated with milk fever and some with metritis. Occasionally the digestive or nervous type is parturient. Two third*) of the non-parturient oases present the digestive syndrome^ while the remainder are distributed between the nervous type and ketosis as a complication of pneumonia and traumatio g a s t r i t i s . The milk fever syndrome is not usually found i n the non-parturient group. The digestive type of ketosis oocurs i n high-producing dairy cows from ten days to six or more weeks after parturition. The f i r s t sign of the disease is usually anorexia, followed by a rapid decrease i n condition and lowered milk production. The animal stands with i t s back arohed, head lowered, and eyes half-closed. The body temperature is usually normal and the pulse variable. Atony of the rumen and soanty evacuations are the rule/ but diarrhea is not unusual. Further physical signs include a staggering gait and possible paralysis.- The digestive type is often confused with traumatic gastritis/ traumatic perioarditis or enteritis. Anorexia, decreased milk flow, and run-down condition are com-bined with nervous symptoms in the nervous type of ketosis. The attack is usually more severe than i n the digestive type. Excitation is marked. Various forms of motor i r r i t a t i o n are present, such as: r o l l i n g of the eyes, spasms of the neok and back muscles, and con-vulsions. Paresis is often present, manifested by excessive salivation and staggering. In the milk fever type the symptoms of ketosis are super-imposed on those of milk fever. Ketosis should be suspected when nervous symptoms other than paralysis predominate. 2. Hematology In severe ketosis there may he a lowering in the carbon dioxide - combining power of the blood although in the majority of cases, values are within the normal range (23) (79)(92). Sometimes there i s an increase in blood cholesterol (86); but i n uncomplicated ketosis, normal values are usually found for oalcium^' inorganic phosphorous, magnesium,' chloride, and non-protein nitrogen (23) (76) (79) (92). The main blood changes i n ketosis'are hypoglycemia and ketonemia. The reported values of blood gluoose and blood ketones for normal oows and cows with ketosis are l i s t e d i n Table 1. TABLE 1 Investigator Blood Glucose mg# Blood Ketones mg]& Normal Ketotic Normal Ketotic A l l c r o f t (1) 41-70 - 0-10 10-20 Boddie (7) - - 1.91 9.01 Braun (9) 82.5 - - -Christalson (40) - - 4.28 -• ' Duncan (21) mm - 2.96 23.09 Eden (25) 60 - 3r8.1 — f Fincher (28) - 4.16 86.25 Hayden (40) 53.2 33,45 -Knodt (52) mm 2.66 - -L i t t l e (55) - - 1.25 m Lormore (57) 40-60 1§13 3r6 14.5 Patton (65) 56.9 34.1 2.0 43.7 Sampson n (76) 40-60 22-31 2r6 10-64 (73) 48.21 28.51 2,21 55,8-a (79) - - 3.3 44,56 Shaw (85) 42.5 24.4 5.14 56.34 (86) 48.5 27.63 24.4 37.77 Sjollema (91) - • - trace up to 100 Udall (103) - - 3 10 7 3. Urology The urine of ketotio cows is often quite acid because of the high ketone content. For the same reason there is usually a great i n -crease in the ammonia content of the urine from these cows (76).. Sjollema and Van der Zande (92) reported the presenoe of excessive amounts of oalcium in the urine of cows with ketosis; but Sampson (73) has observed that many ketotic cows excrete relatively small amounts of oalcium by this route. Ketonuria is the main change in the urine during ketosis (Table 2)* TABLE 2 Urine Ketones mg# Investigator Normal Ketotic Boddie ( 7) 3.-28 77.2 Duncan ( 21) 53.6 154.5 Finoher ( 28) 6.8 1025 L i t t l e ( 55) 20.5 -Lormore, ( 57) 3-15 i i 2 Patton ( 65) 37 "274 Sampson ( 76) '3-15 25-1209 n ( 79) 7.17 578.3 Shaw ( 52) 11.81 -Sjollema ( 92) . 10-70 1000-1300 Udall (103) 7.0 50-1250 • 4. Biochemical Changes in the Milk. Milk produced by ketotio cows oontains ketone bodies to the extent of 30 to 450 mg# (92). Sampson (73) finds the range to be 5.46 to 31.25 mg% in contrast to the milk of normal cows where only traces of ketones are found (23) (75) (52). 5. Histopathology. Because of the relatively low mortality in ketosis, few studies have been reported on the histopathology of this condition. 8 Any work that has been done has been confined to those organs which usually show gross changes, namely the l i v e r and kidneys. In one such case (92), the cow had died of ketosis ten days after parturition. The l i v e r was enlarged. Microscopic examination'showed some fatty degeneration in the l i v e r , heart and kidneys. Similar aocounts of the high l i p o i d content of livers from cows with ketosis have been reported by M o r r i l l (81), Mcintosh (64), and Roderick (70). E. Diagnosis Sinoe ketosis often occurs as a complication of milk fever, metritis, or atony of the rumen, dif f e r e n t i a l diagnosis is sometimes extremely d i f f i c u l t . The physical signs and general symptoms may often be masked by these other conditions. Most practicing veterinarians resort to Rothera's test, or a modification of this test (103)/ as an aid to diagnosis. For details of this test see Appendix II. Rothera's test is limited by the faot that i t is not quantitative. Its effectiveness is restricted by the fact that i t sometimes gives a positive reaction to urine from normal oows; and a positive test f a i l s to reveal the exact intensity of the ketonemia and ketonuria. A more sensitive test would be invaluable for f i e l d diagnosis, since f a c i l i t i e s are not usually available for a confirmatory quantitative analysis of the blood and urine. F. Treatments 1. Carbohydrate Prompt benefit usually results from the administration of dextrose (103). The usual recommendation is fiv e hundred co. of a 40$ 9 solution intravenously, given daily for one to five days (75) (50) (88). If milk fever is a possible complication, oalcium gluconate as well as glucose may be indicated (28). If ketosis is associated with metritis, indigestion or some other disorder, appropriate treatment for these conditions should supplement the carbohydrate therapy. 2. Sedatives Chloral hydrate was probably f i r s t used by Rob i l l e t a s reported by Rolls (71) and is s t i l l widely applied - especially i n treating the nervous type of ketosis (69). The usual dosage i s thir t y grams once or twice daily for six days (103). It is a hazardous form of therapy when ketosis i s complicated by milk fever (28) j and even i n uncomplicated ketosis i f the cow is weak or emaoiated (75)* It i s recognized that chloral hydrate does not treat the specific cause of ketosis but only serves to.reduce the nervous symptoms of the disease. 3. Hormones Sjollema (91) reports that i t is common practice i n Holland to add one hundred to one hundred and f i f t y units of insulin to two hundred grams of dextrose in one thousand oc. of water for administration to cases of ketosis. Smith and Soderholm (93) also recommend insulin it. »» therapy. Anterior Pituitary Lobe extract has been used with apparently excellent results (29). Shaw (87) reports on the therapeutio effect of Adrenal Cortical extracts on four oases of uncomplicated ketosis. Sub-cutaneous injection of these extracts improved the blood, glucose and ketone levels. Shaw postulates that an adrenal insufficiency may be involved in this disease. 10 4* Minerals Henderson (42) treated twelve oases of c l i n i c a l ketosis with a solution of two oz. of cobalt sulphate in one gallon of water* When one-third oz* per day of this mixture was administered, nine of the oases showed definite improvement* 5* Vitamins Various members of the Vitamin B. oomplex have been recommended for treating ketosis (64) (17). Shaw (85) however, reported that thiamine hydrochloride administered alone, (either orally or int r a -venously) or i n combination with nicotinio acid, calcium pantothenate, riboflavin, pyridoxine, para aminobenzoio aoid, inositol, choline, and biot i n was ineffective. The addition of any of the B vitamins to glucose therapy showed l i t t l e improvement. The only other vitamin to be used to any extent in treating ketosis has been vitamin A. The importance of ensuring an optimum intake of carotene has long been recognized in dairy cattle feeding(38 <\); but i t has not been u n t i l recent years that a relationship between vitamin A and the ketotic state was hypothesized. The value of vitamin A in the therapy of ketosis in.dairy cattle has been the subject of muoh controversy. Several workers have re-ported excellent results (10) (45) (84) (90) (99). Patton (65) (66) was one of the few veterinarians to publish quantitative data in this f i e l d . In nineteen hundred and forty-five, he reported (66) on one hundred and thirty-nine oases of ketosis. Vitamin A therapy alone was 11 used in one hundred and thirty-five of the eases. One f i f t h of the animals recovered in twelve hours; most of the others within three days. The type of ketosis referred to i n his work showed the following general signs and symptoms: a positive Rothera test, and aoetone odour of the breath; anorexia and suspended rumination; reduced milk pro-duction; oonstipation, unsteady gait, rapid loss of flesh, impaired vision, arched back with the spine kinked to one side, muscular tremors, grinding of the teeth and convulsions. He reported that ketosis occurs i n a l l parts of the country and that the incidence corresponds closely to a deficiency of vitamin A or carotene in the feed and to the requirements of the animal for i t . The symptoms approximate those of aoute uncomplicated vitamin A deficiency. He observed a lower vitamin A level in the blood of cows suffering from ketosis than normal cows as illustrated by the following table. ' TABLE 3 Effect of Ketosis on Blood and Urine of Dairy Cattle Blood Blood Blood Blood Urine Glucose Carotene Vitamin A Ketones Ketones mg$ mg$ jug/lOOoc mg# mg% Normal 56.9 0.405 14.91 2.0 37.0 Ketotic • 34.1 0.620 0.84 43.7 274.0 Patton noticed that oows with ketosis responded promptly and recovered completely when given vitamin A peros. There was a rapid and complete disappearance of the c l i n i c a l symptoms and a marked rise in blood glucose to within the normal range. Blood ketones decreased to normal and milk production started to r i s e . 1 12 TABLE 4 An Uncomplicated Case of Ketosis Time Blood Blood Urine in Glucose Ketones Ketones days Treatment 1 Million 0 I.U. — Vit.A 27.5 mm 116 1 1 Mil l i o n . .. -I.U. 37.0 64.6 126 2 1 Mil l i o n - I.U. - - 136 62 N i l 54.5 2.0 16 TABLE 5 A Case of Ketosis Complicated with Metritis Time Blood Blood Urine in Glucose KetoneB Ketones days Treatment mg% mg# mg# 0 500,000 I.U. Vit.A. / 345.0 62-7 1 1,000,000 I.U. Vit.A. - - 506 2 1,000,000 I.U. Vit.A. # - 313 5 N i l 52.7 37.0 155 7 1,000,000 I.U. Vit.A. 47.8 25.0 100 (/--1 oz. chloral hydrate given six hours prior to f i r s t treatment of vitamin A.) (#—25 mg. diethylstilbesterol administered) In subsequent publications, Patton amplified some of his earlier work. He noticed that (a) ketosis was more prevalent among cattle of the southern United States; (b) the incidence of ketosis was greatest in herds that were poorest fed in relation to their milk production; (c) below normal blood sugar and above normal blood ketones were common in southern cattle during the dry feeding period, and (d) low blood vitamin A and high carotene values among these oattle i n the early spring. IS After further sueoess with vitamin A therapy i n improving the condition of oattle with ketosis, restoring them to normal milk pro-duction and even increasing the production of cows on the borderline of a vitamin A deficiency, Patton concluded that this therapy brings about these improvements with greater celerity than any other treatment. He recommended as a standard treatment, the administration of one million I.TJ. of vitamin A per adult cow per day,for at least three days. Mcintosh (64) and Daugherty (flD,) both discount the vitamin A theory. Shaw (86) analyzed blood samples from normal cows and cows with ketosis. Of the eight animals a f f l i c t e d with ketosis, a l l showed normal blood carotene and blood vitamin A. Oral administration of four million I.TJ. had no beneficial effect. The condition was f i n a l l y cured by pasturing the animals on green grass (fable 6). He concluded that ketosis in dairy oattle is not caused by a vitamin A deficiency. TABLE 6 A Case of Uncomplicated Ketosis Time Whole Blood . Blood Plasma in Gluoose Ketones Carotene Vit.A days Treatment mg$ mg# mg% p%% 0 250,000 I.U. Vit.A intravenously 27.12 46,75 0.134 14,4 4 1,000,000 I.U. per oS 31.87 22.84 0.126 16.3 16 330,000 I.U. Carotene plus 2,500,000 I.U. Vit.A 30.28 50,93 26 5 days on pasture 50.62 3.35 TABLE 7 Blood Plasma from Eight Cases of Ketosis Blood Glucose 27.63 mg$ n Ketones 37,77 mg# " Carotene 0.325 mg$ 11 Vitamin A 23.2 jig% H Shaw states that the values 0.325 and 23.2 (Table 7) compare favorably with values for normal cows reported by other workers. Therefore, he concludes that these eight oases of ketosis were not oaused by a vitamin A deficiency. The negative results of the therapy would seem to confirm th i s . Later (85) he reported on five oases of ketosis that a l l recovered spontaneously without any treatment. The following data is indicative of the results obtained: TABLE 8 Time Blood Blood in Gluoose Ketones dayB mg$ mg# Remarks 1 - - Parturition 11© - - Inappetenoe 16 24.4 56.34 Deoreased milk production 17 31.6 61.1 21 33.2 49.88 Appetite improved 23 48.6 20.92 27 42.6 5.14 Normal Hayden, et a l (40) desoribed ten oases that were diagnosed as ketosis by the attending olinioian. Four of the ten represent l i t t l e ketosis. Two of these four reoovered with no treatment other than vitamin A (Table 9). TABLE 9 Digestive Type of Ketosis — Recovery by Vitamin A Therapy Time Blood Blood Urine in Gluoose Ketones Ketones days Treatment mg£ mg# 0 1 M i l l i o n I.U.Vit. 40.0 14.41 24.88 A every twelve hours — . 2 for five doses then . 35.35 18.96 37.49 four Mil l i o n I.U. 7 doses every twelve 35.6 12.65 17.93 hours. 15 It is doubtful whether there is any ketosis ! in this particular case, sihoe the blood ketone and urine ketone values are not much greater than those reported for normal cows* Moreover the blood gluoose level before treatment is within the normal range. The six oases which represent some degree of real ketosis as reported by Hayden, gave unsatisfactory responses to vitamin A therapy (Table 10). TABLE 10 A Marked Case of Uncomplicated Ketosis Time Blood Blood Urine in Glucose Ketones Ketones days Treatment mg% mg% ng% 0 1 Mil l i o n I.U.Vit. 33.45 25.76 286.28 A for five days - ineffective -so dextrose and yeast 4 for four days 25.32 ° 28.66 555.00 8 Recovery 53.2 13.68 The authors state that i t is not unreasonable to assume that vitamin A deficiency would be overoome by proper vitamin A therapy. They oonclude that none of the oases of ketosis that they treated were due to a vitamin A deficiency. The vitamin may have been of some help in these cases but i t had not been a specif io remedy. 16 6* Experimental In view of the encouraging results obtained by Patton in relieving ketosis with vitamin A therapy, an attempt was made to repeat his work using animals from the University herd of Ayrshire cattle. This particular herd is "barn-fed11 from September or October to May each year. The winter ration is usually low in carotene and i t was thought that with suoh a group of animals, bordering on a vitamin A deficiency, cases of ketosis might become quite prevalent. 1. A Typical Case of Ketosis. Only one case of ketosis developed during the period under observation. This particular oow (Table 11) was given vitamin A peros i n an attempt to reduce the ketoneraia and ketonuria and to i n -crease the blood gluoose. Analyses of blood and urine samples were made within thirty six hours of sampling. Details of analytical procedures used w i l l be found in Appendix III. TABLE 11 A case of Ketosis i n the University Herd (Marigold) Time Milk Blood Blood Blood Blood Urine in Prod. Gluoose Carotene V i t . A Ketones Ketones days # mg# mg# i.u.Aoo mg# 0 53.1 50,25 0.299 77.8 3.5 18,8 35.6 39.0 .280 58.6 5.0 -76.5 10 39,7 49.6 .310 60.0 4,8 69.5 11 39.5 33.0 .311 61.5 5.5 81.0 12 37.7 43.6 .307 64.0 5.0 79,5 13 35.0 36.0 .319 71,5 6,2 83.5 14 36.6 42.0 .310 -72.0 5.8 81,6 16X 37.6 39.0 .312 67.2 4,2 75.4 17X 38.8 39.0 .314 110.4 4.5 69.4 TABLE 11 (Cont'd) A oase of Ketosis in the University Herd (Marigold) (Cont'd) Time Milk Blood Blood Blood Blood Urine in Prod. Gluoose Carotene V i t . A Ketones Ketones days I.U./100 18X 40.6 45.0 .314 100.8 3.8 50,5 19 40.1 47.5 .307 103.2 3.2 35.8 23 40.5 44.3 .269 84.5 4.2 32.0 26 37.3 39.0 .264 82.9 4,5 34.5 34 39.3 50.3 .300 75.4 3.5 31,6 40 40.2 39.0 .285 78.3 4.1 32.6 61 38.5 39.0 .139 59.8 5.2 24.6 80 39.9 54.0 . .594 96.1 7.0 -# — 250 oo of a 40% gluoose solution intravenously. X — 1,000,000 I.U. vitamin A peros. The dextrose therapy f a i l e d to relieve the ketosis. The administration of vitamin A oaused a gradual decrease i n the ketonuria and a temporary increase in blood gluoose (Figure 2). A larger dose of vitamin A might have brought about a more marked improvement i n the hypoglycemia. Marigold was only a mild oase of ketosis as evidenced by the comparatively low blood ketone level at a l l times. However; the signs and symptoms were typical of the ketosis syndrome. The animal was l i s t l e s s , run-down in condition, suffering from anorexia and her milk production was low. Figure 1 illustrates the benefioial effeot of the therapy on milk production. Once again i t was f e l t that a more prolonged course of vitamin A therapy might have brought about an earlier return to normal production. The effectiveness of maintaining a therapeutic level of vitamin A in the blood after oral injection w i l l be discussed in Appendix IV, in relation to the poss i b i l i t y of using another route. 18 II. Blood and Urine Histories of the University Herd -Pre and Post Parturient. Sinoe the reported highest inoidenoe of ketosis occurs within three to six weeks after parturition, complete blood and urine histories of a l l pregnant cows in the herd were made for varying intervals before and after parturition. The object of this experiment was to be able to predict olinioal ketosis from the hematological data obtained and try to fore s t a l l the development of ketosis by administering vitamin A. In addition, colostrum samples were analyzed to assess the drain on the maternal vitamin A reserve following parturition. Blood, urine, and oolostrum data were obtained from the five cows that were i n gestation during the period of this experiment. The carotene and vitamin A oontent of the oolostrum samples were determined by the method of Boyer et a l (8). The results are tabulated i n Table 12. Additional data on the daily output of colostrum per oow from the herd during nineteen hundred and forty-six and nineteen hundred and forty-seven are presented i n Appendix V. TABLE 12 Time Blood Blood Blood Blood Urine Colostrum in Gluoose Carotene Vit.A. Ketones Ketones Wt. Carotene Vit.A. days mgjS mg% I.U./LOQte.mg^ mg# # I.U./gm Cow No. 1 (Nora) 0 45.0 0.314 96.4 3.10 6.-15 - mm 16 57.75 .272 85.8 4.25 7.60 mm - _ 19 65.75 .365 84.8 3.85 8.10 mm - -24 Parturition 25 17.5 0.89 1.89 26 30.0 27 33.9 0,10 0.51 28 38.2 0.08 0.40 29 - 40.9 0.18 0.58 43 44.25 .12 61.-8 4.25 8.6 52.7 - _ 62 50.25 .466 81.6 4.70 - 49.2 - -19 TABLE 12 (Cont'd) Time Blood Blood Blood Blood Urine Colostrum in Gluoose Carotene Vit.A. Ketones Ketones Wt. Carotene Vit.A. days mg# I.U./lOOee.mg^ mgjg # /iE-/gm I.U./gm Cow No. 2 (Octavia) 0 45.0 .480 79.2 3.45 6.1 28 57.75 .511 55.14 4.45 ' 8.24 43 52.25 .449 81.2 5.6 13.1 49 Parturition 37.5 0.65 0.88 50 37.0 0.48 0.51 51 40.4 0.21 0.17 52 46.7 53 42.5 54 48.0 0.14 0.15 62 45.0 .592 95.1 10.0 - 51.0 Cow No. 3 (Natalie) 0 48.4 .299 76.5 3.15 5.16 46 54.0 .360 51.5 3.0 6.24 61 62.3 .366 79.4 4.1 7.5 80 50.3 .360 67.2 7.5 mm 105 Parturition 33^2 3.91 2.77 106 36,4 2.81 1.71 107 50.0 - -108 63,3 - - . 109 66.0 0.86 1.65 110 44.4 1.14 101.2 - - 58.4 - -Cow No. 4 (Peggy) 0 39.0 0.511 87.4 7.44 32^0 20 45.0 1.90 100.6 - - • 24 Parturition 22.5 3.7 2.8-25 27.5 2.74 1.86 26 28.2 27 32.1 28 33,1 0.98 1.78 29 36.4 Cow No. 5 (Ona) 0 57.75 0.364 69.8 1.95 8.92 62 57.8 .586 89.1 7.40 mm 82 51.8 1.310 110.2 - -90 Parturition 91 27,1 2.85 3.83 92 29.5 - -93 35.0 94 42.5 1.42 1.89 95 4 38.8 — mm 20 Although the majority of the blood carotene and vitamin A levels are within the normal range, the colostrum values are definitely lower than those of eighteen to twenty I.U. per gram reported i n the l i t e r a -ture (54) (101). None of the cows in this experiment developed ketosis, although i t i s noticeable that there was a tendency for the animals to develop a slight ketonemia and ketonuria just prior to parturition. Cows No. 3, 4;and 5 had been approximately two weeks on pasture when the f i n a l blood samples were taken. This accounts for the increased blood carotene and vitamin A values shown for these animals. The data indicates quite clearly that there i s an intensive drain on the maternal vitamin A supply at parturition. This i s more clearly illustrated by Figure 3. The amount of vitamin A present i n the colostrum decreases quite rapidly following the f i r s t sample (the same is true for carotene). Considerably more carotene and vitamin A were found i n 00lostrum samples from cows that had access to green pasture (cows 3, 4 and 5 - Figure 3)• As an estimate of the extent of the withdrawal of vitamin A and oarotene from the 00lostrum at parturition, data w i l l be extrapolated for cows one and three to give an approximate total of vitamin A secreted in the colostrum for twenty suooessive milkings. Total Carotene Total Vitamin A Total Carotene & mg. I.U, vitamin A as I.U. • of vitamin A. Cow#l (No pasture) 30 108,000 158,000 Cow #2 (Pasture) 152 568,000 * 821,000 This calculation, even though i t is only an approximate one, does indioate quite olearly the enormous drain of vitamin A from the dam to the colostrum at parturition. - f l l i l i : i i l f f l i 21 III. Blood and Urine History of University Herd -Before and After Pasture. In order to obtain base values for blood glucose, ketones/ carotene and vitamin A,and urine ketones i n the University herd of dairy oattle, samples were taken when the oattle were being fed i n the barn on standard winter rations; and, later, after they had been on pasture for approximately two weeks. The f i r s t samples of blood and urine were taken at varying intervals from seventh January to eighteenth February, nineteen hundred and forty^eight. Blood samples were taken on Apr i l twentieth and again on May tenth. Data from this investigation are presented i n Table 13. Blood samples from several cows indicated slight hypoglycemia and some ketonemia. However, only one case (Marigold) developed o l i n i c a l symptoms. There does not appear to be any correlation be-tween: (a) blood carotene and vitamin A (Table 14) - although the average vitamin A /carotene ratios for the three periods indicate a greater increase in carotene than vitamin A when the animals had access to pasture. (b) Blood vitamin A and blood ketones or (o) between blood vitamin A and blood glucose. There is a tendency for the blood glucose level to decrease before the ketonemia and ketonuria develop. It is quite possible that tests for blood glucose might be a more rapid aid to diagnosis than the ketone body reaction. Pasturing the animals did not appear to influence the blood glucose values. As might be expected, the change to summer feed did affect quite markedly the carotene and vitamin A levels of the blood. The average value for thirty two cows showed an 22 .increase of almost f i f t y percent in blood carotene content after approximately two weeks on pasture. The rise in blood vitamin A eon-tent is only about twenty percent of the pre-pasture level* This might indicate that eaoh ruminant has an Individual threshold for converting carotene to vitamin A; and that i t is not necessary to reach the maximum daily intake for oarotene. Beyond the optimum level the animal i s unable to convert the oarotene to readily available vitamin A circulating i n the blood stream* TABLE 13 Blood and Urine History of University Herd -Before and after Pasture Name Blood Glucose mg# Blood Carotene ng% . cc Blood Vit.A IU/100 Blood Ketones me^ .. rag Urine Ketones of Age Before Pasture After Before After Before After Before Before Cow yra lbs, Jan April May Jan April May Jan April May Jan April Jan Heather 10 1216 53.65 _ 0.299 _ _ 77.2 mm mm 3.3 AM 6.25 Iona 9 1371 62.3 - - 0.290 - - 58.0 mm mm 4.0 - - 5.5 Janice 8 1237 65.00 50.25 45.0 0.311 0.586 0.940 52.8 91.2 103.4 3.25 6.0 •5,95 Joyoe 8 1228 66.8 57.8 44.0 0.315 0.578 0.891 64.7 , 91.-5 102.2 3.9 8.5 6,65 Laura 6 1165 51.6 49.0 57.8 0.466 0,585 0.821 68.2 89,1 94.3 3.8 8,9 9,65 Lenora 6 1125 42.63 45.0 40.3 0.311 0.591 1.120 79.5 105.2 108,2 4.8 5,6 5,96 Luoretia 6 1087 49.55 54.0 39.5 0.295 0.591 0.830 57.2 100.5 107.0 4.83 2.75 22,5 Lucy 6 1189 47.5 36.75 44.4 0.501 0.486 0.840 75.6 86.4 94.1 4.2 8.75 ' 5,65 Mable 5 1121 46.8 54.0 44.4 0,452 0.420 0.943 67.6 68.5 89.2 2,8 8.5 5.7 Marigold 5 1050 50.25 54.0 54.9 0.299 0.594 0.784 77.8 96.1 104,1 3.5 7.0 18.8 Midge 5 1219 62.25 - r 0.311 - 1.900 69.3 - 100.6 1.7 - 7,8 Mildred 5 1406 51.6 50.25 50.3 0.316 0.286 0.864 80.4 71.1 98.5 4.65 11.0 10.25 Moira 5 1255 54.0 48.0 50.0 0.304 0.510 1.020 72.5 81.2 100.8 1.70 7,60 8,10 Myra 5 1008 51.3 51.8 45.0 0.501 0.604 1.310 70.8 108.0 110,2 2,0 6.8 6.85 Nancy 4 1454 45.0 50.25 45.75 0.325 0.592 0,920 69.8 94.1 99,0 4,8 7,6 11.1 Nanette .4 1411 57.75 66.75 40.0 0.302 0.584 1.04 66.8 82.1 98,6 2.50 7,45 7.70 Naomi 4 1084 65.0 59.2 50.25 0.322 . 0.552 0.740 72,1 89.3 95.2 4.0 5.0 6,40 Natalie 4 1242 48.4 50.25 44.4 0.299 0.360 1.140 76.5 67.2 101.2 3,15 7.5 5,15 Nellie 4 1097 50.2 50.25 39.0 0.360 0.589 1.100 65.2 94.6 106.1 3.95 1.75 7,18 ^Nettie 4 1140 54.0 50.25 50.25 0.408 0.480 0.914 72.9 71.9 96,1 4.75 9,1 8.55 Nora 4 1090 45.0 50.25 45.0 0.314 0,466 1.140 76.4 81.6 95.0 3,1 4.75 6.15 Ootavia 3 1297 45.0 45.0 45.0 0.480 0.592 0.712 79.2 95.1 99.2 3.45 10.0 6,10 Olive 3 39.0 39.0 39.5 0.552 0,577 1.210 81.9 83.4 101.0 2.15 8.15 • 6.46 Olivia 3 1085 39.0 57.8 54.0 0.480 . 0.582 0.921 75.1 94.3 100.8 4.90 4.93 7.65 TABLE 1 5 (Cont'd) Name Blood Glucose Blood Carotene mg% Blood Vit.A IU/L88 Blood Ketonesmg% rag^ Urine Ketones of k Wt. Before Pasture After Before After Before After Before Before Cow yrs lbs. Jan April May Jan April May Jan April May Jan April Jan Ona 3 1 1 0 6 5 7 . 7 5 5 7 . 8 5 1 . 8 0 . 3 6 4 0 , 5 8 6 1 . 1 4 0 6 9 . 8 8 9 . 1 1 0 3 , 3 1 . 9 5 . 7 . 4 Bs92 Omega 3 1 2 4 4 3 9 . 0 4 5 . 0 4 5 . 0 0 . 3 8 4 0 . 5 9 6 0 . 8 5 0 8 2 . 1 8 9 . 0 9 4 . 5 2 . 0 3 . 9 7 . 9 5 Ophelia 3 1 1 7 5 5 0 . 3 5 0 . 2 5 5 5 . 4 0 . 6 1 0 0 . 2 4 0 1 . 1 4 7 1 . 5 7 1 . 5 9 8 . 9 3 , 8 1 5 . 1 9 , 8 6 Orchid 3 1 0 6 1 3 9 . 0 4 5 . 0 5 0 . 2 5 0 . 4 7 5 0 . 5 8 0 0 . 8 7 0 7 4 . 6 8 1 . 2 9 2 . 8 3 . 6 5 2 . 1 5 8 . 4 2 Pamela 2 1 0 3 5 5 7 . 7 5 4 5 . 0 5 9 . 2 0 . 2 8 7 0 . 5 9 8 1 . 0 0 6 0 . 0 10L .9 1 0 4 . 5 2 . 1 5 8 . 3 4 8 . 1 0 Patricia 2 9 8 0 5 0 . 2 5 3 9 . - 0 . 4 2 2 0 . 9 2 1 - 8 4 . 5 1 0 2 . 2 - 8 , 1 _ Peggy 2 9 6 5 - 3 9 . 0 4 5 . 0 - 0 . 5 1 1 1 . 1 3 0 -. 8 7 , 4 1 0 3 , 1 - 7 . 4 4 • Penelope 2 9 3 0 5 6 . 7 5 3 9 . 0 5 0 . 2 5 0 . 3 0 4 0 . 5 9 2 0 . 9 1 2 8 0 . 8 9 2 . 8 9 2 . 8 1 . 7 5 1 0 , 5 - 7 . 8 Precious 2 9 2 7 5 0 . 2 5 5 0 . 2 5 5 4 . 0 0 . 2 8 9 0 . 5 9 4 1 . 0 6 7 2 . 0 9 4 . 7 9 9 . 0 3 , 8 3 4 . 1 6 7 , 7 0 Primrose 2 9 5 5 6 5 . 0 5 4 . 0 5 1 . 8 0 . 3 1 6 0 . 4 8 1 1 . 2 0 7 0 . 5 8 1 . 7 1 0 6 , 1 2 , 5 9 . 1 2 7 , 0 5 Princess 2 9 4 4 6 0 . 8 7 5 7 . 8 3 9 . 0 0 . 3 0 4 0 . 5 8 5 0 . 9 4 0 6 8 . 4 9 1 . 4 9 9 . 6 . 2 . 0 5 . 1 5 5 . 1 5 Average 5 5 . 1 6 5 0 . 1 4 7 . 1 7 . 3 6 8 . 5 6 1 1 . 0 0 8 7 1 . 3 7 8 7 . 7 3 9 9 . 7 4 3 . 3 1 7 . 1 8 8 .12 ro TABLE 14 Vitamin A / Carotene Ratio -- University Herd Blood.Vitamin A / Blood Carotene Name of Before Pasture After Pas Cow January April May Heather 258 mm Iona 200 • - -Janice 169x I56x llOx Joyce 205 158 101 Laura 146 152 115 Lenora 256 178 107 Lucretia 194 169 129 Lucy 150x 178x 112X Mable 149 163 95 Marigold 360 157 122 Midge 223 - 53 Mildred 255 248 114 Moira 238x I59x 98x Myra 141 178x 85x Nancy 2l4x 159 107 Nanette 220 I40x 95x Naomi 222 161 129 Natalie 256x 187x 89x Nellie 181x 160 105 Nettie 179 150 105 Nora 243x 175 87 Ootavia 148x 145 87 Olive 156 162 llOx Olivia 165 160 139x Ona 192 152x 90x Omega 213 149 11© Ophelia 117 298 87 Orchid 157 141 107 Pamela 208 171 10 Sx Patrioia mm 200 l l l x Peggy - 171 91 Penelope 266 156 101 Precious 249 159 99 Primrose 223 169 89 Princess 225 156 106 Avge. 201 169 103 ' Avge. x 200 164 104 x - Non-laotating The gathering of this data, while i t served to isolate only one case of ketosis, did present an excellent opportunity to establish a parti a l blood and urine history of the entire milking herd. Several observations can be made from this information, but perhaps the most noteworthy i s that the herd as a whole is bordering a vitamin A deficient state during the months of winter feeding. This is evidenced by the marked increase i n blood carotene and vitamin A after being on pasture (table 13); and by the comparison of the pre-pasture levels with values reported i n the literature (table 15). For this reason; i t might be advisable to add some form of vitamin A or i t s precursors to the winter ration. TABLE 15 Blodd Carotene and Vitamin A Levels Source Carotene mg% Vit.A. I.U/lOOco (a) (b) Stall-fed — University of B.C. 0.368 71,4 Gillam (32) 0.400 61,9 Hibbs (43) 89.5 Patton (69) 0,406 71,0 Shaw (86) 0,325 110.0 Sutton (102) 0.6 to 0.9 86 to 90 Pasture-fed University of B.C. 1.01 99.7 Gillam (32) 1.11 138,1 Lord (56) 1.35 150.0 Sutton (102) 1.2 to 1.5 100 to 110 6. Summary During pregnancy and with the onset of milk production, there i s an increased demand for energy whioh rapidly drains the body reserves of carbohydrates. In the ketotic state, some derangement of carbohydrate metabolism oocurs producing a hypoglycemia; and therefore the animal turns to the oxidation of f a t to meet these increased energy requirements. Ketone bodies are the intermediary products formed during the oxidation of this f a t . It i s known that ketosis i s a metabolic disorder, but the fundamental cause of the disturbance i s s t i l l unknown. Present methods of treatment lo g i c a l l y favour the administration of glucose intravenously, and then supplementing the ration with molasses. Numerous other treatments, however^ have been attempted. For example, a recent review of the literature has revealed that many veterinarians, both i n Canada and the United States, have been advocat-ing vitamin A therapy. The therapeutic work done with the University Herd here is primarily a repetition on a limited scale of investigations oarried out elsewhere. However, the confirmatory results suggest new po s s i b i l i t i e s as to the fundamental cause of this disease. Before discussing the etiology in greater detail,' i t i s well to realize that ketosis occurs i n many domestic animals other than dairy cattle. The signs and symptoms are practically identical between species and closely parallel the symptoms of a l l i e d metabolic disorders such as diabetes mellitus and acute hypoglycemia.. These inter-28 relationships, as well as some aspects of the various diseases that are usually found as complications of c l i n i c a l ketosis. w i l l be pre-sented in the following section. II. KETOSIS IN OTHER DOMESTIC ANIMALS A. Pregnancy Disease in Sheep Many names have been assigned to "Ketosis" of sheep; but the fact that i t oocurs primarily during the gestation period accounts for the use of the name "Pregnanoy Disease" by most writers. The syndrome was probably recognized as a specific disease of sheep as early as eighteen hundred and ninety (70), despite the fact that i t has not been u n t i l recent years that information has become available about the etiology, pathologic physiology, and prevention of this disease. When pregnancy disease appears in a flock of breeding ewes",-mortality losses ranging from one to twenty-five percent may -be expected. Dimook, etal ( 2 2 ) reported a sixty percent loss in one flock of one hundred ewes. The total economic loss i s not solely due to the high mortality of ewes. Since most of the ewes affeoted carry more than one fetus (12) (70), the loss in lambs is quite important. Roderick and Harshfield (70), and Udall (103) state that pre-gnancy disease oocurs in most countries of the world. Reports from such widely separated states as New York and California indicate that ketosis of ewes oocurs in practically a l l the sheep-raising areas of the Americas. The general impression is that this disease is more 29 common in farm flooks than in sheep on the open range (59) (103). The highest incidence appears to be in the late winter and early spring for ewes in the last t h i r t y days of pregnancy. Most of the suggested causes, such as mineral deficiency/ toxic absorption from the uterus,- and lack of exercise, have been discarded (73) (103). Some of the others are considered as predisposing causes, Ketosis is essentially a disturbance of carbohydrate metabolism. The pathological state, in this case ketosis, leads to an anorexia, where the animal i s not interested i n oonsuming readily available carbohydrate, thus contributing to the heavy drain on the maternal energy reserve during the gestation period. It is quite possible that twin pregnancy imposes metabolic requirements beyond the capacity of the ewe. This oauses the li v e r glycogen reserve to be depleted i n order to maintain the blood sugar level. When this Occurs, carbohydrate metabolism i s interfered with in some manner so that oxidation of fats oannot be carried beyond the ketone body stage. Ketonemia and hypoglycemia are often as intense as in ketosis of dairy cattle (73). Table 16 indicates the comparative blood ketone levels of normal ewes and ewes with spontaneous ketosis. TABLE 16 Sampson (73) 2.04 43.3: Sampson & Boley (74) 3 to 10.5 41.0 Cameron (12) -Investigator 4.9 to 36.2 13. to 83.0 30 Other blood constituents are apparently normal (79) j exoept that Sampson etal (76) report a high concentration of non-protein nitrogen associated with the later stages of pregnancy disease. This might possibly be attributed to an increase in protein oatabolism. The concentration of ketone bodies in the urine of ewes with this condition is as high as i t is in the urine from cows with ketosis. Sampson (76) found 735.5 mgjS of total ketones in one particular ease. TJdall (103) states that the urine ketone content for pregnancy disease is in the range ten to three hundred mg# as compared to normal urine where only traoes of ketones are found. Considerably more work has been done on the histopathology of ketosiB in the ewe than in the oow. The primary pathological changes are in the liver, and kidneys. Roderick and Harshfield (70) describe these changes in d e t a i l . The l i v e r is yellow and friable indicating marked fatty degeneration. These fatty ohanges are emphasized by microscopic examination. L i t t l e damage to the nuolei is reported (7o)rjf but vacuolation of the cytoplasm is common. The glomeruli of the kidneys do not appear to be affected, but there is some swelling of the epithelium of the convoluted tubules. Fat globules are found in the epithelial cells of the ascending and descending limbs of Henle's loop at the edge of the cortex. Aggressive work is continuing on the histopathology of this disease. Complete agreement has not yet been reaohed among pathologists as to the extent and significance of the changes discovered to date. 31 The symptoms are- similar to the non-nervous type of ketosis. The disease is f i r s t recognized by increasing listlessness. . Anorexia, thirs t , and rapid breathing are other signs. Some motor i r r i t a t i o n i s usually evident and the animal may pass into a coma. As in the ease of ketosis of dairy cattle, a quantitative examina-tion of the blood and urine i s the best aid to diagnosis beyond the general symptoms observed. A daily intravenous injection of 500cc of a five percent dextrose solution i s the recommended treatment (103), although poor results with carbohydrate therapy have led several workers to. employ other treatments with even less success. Foss (30) has suggested the use of vitamin A therapy. Underwood, Curnow and Shier (104) found that the vitamin A oontent of livers from ketotic ewes was similar to those of healthy ones. They deemed i t unlikely that vitamin A deficiency is associated with pregnancy disease. However, in view of the successful results of vitamin A therapy with dairy cattle and the similarity of the two ofjiabolio conditions, further investigations into this relationship might prove worthwhile. B. Ketosis in Swine The realization that the poroine species was susceptible to ketosis is a development of the last few years. The f i r s t o linioal observation was made by Hull and Nolan (46) in nineteen hundred and forty at the Kentucky Agricultural Experiment•Station. Sinoe then, oases have been reported from the states of New York (17) and I l l i n o i s (78). Evidently the pig has a low susceptibility to ketosis and for 32 this reason few oases have been desoribed. The symptoms and results of chemical analyses on blood and urine samples are quite similar to those already known to characterize un-complicated ketosis i n the coir and ewe. The hypoglycemia does not appear to be as severe but the ketonemia can be as intensive as for dairy cattle (Table 17). TABLE 17 Total Ketone Bodies and Sugar in Blood of Healthy Sows and Sows Affected with C l i n i c a l Ketosis (73) Normal Ketotio Sow Ketones Sugar Sow Ketones . Sugar 1 trace 56.50mg# 5 68.2 mg% 61,02mg# 2 41 40.68 6 36.08 58,76 3 n 80.23 7 95,23 79.10 4 II 71.19 8 41.17 54.24 With reference to the etiology of ketosis in swine, Sampson and Hanawalt (78) state that the fundamental cause i s the same whether the ketosis occurs i n cows, ewes, or sows; namely, an insufficient intake of readily available carbohydrate or potential carbohydrate material. More information i s needed on therapeutic and prophylactic measures for this disease i n swine, but the limited evidenoe available (78) indioates that the same general therapy recommended for dairy cattle is applicable here. C. Ketosis in Goats Gilyard and Gilyard (33) have desoribed typioal c l i n i c a l ketosis in a herd of goats. They reported on twelve f a t a l oases of pregnant does. Paresis and ooma were characteristic symptoms. High concentrations of ketone bodies i n the urine were found on further examination* The ketonuria decreased after the injeotion of glucose, but was not effective i n saving the does* No further cases developed after adding molasses to the ration. Although analytical data were not reported for blood sugar and ketone levels, the general indications were that these eases were typical of spontaneous ketosis. D. A l l i e d C l i n i c a l Conditions 1. Milk Fever Several metabolic disorders are found as complications of simple ketosis. One of the most important of these diseases is milk fever or parturient paresis. Milk fever affeots cows i n the post-parturient stage causing paralysis and loss of consciousness leading to coma. There is a marked and rapid lowering of the blood calcium content so that the name parturient hypocalcemia is quite often appropriately used. Milk fever is one of the most common, and most serious of the acute af f l i c t i o n s of dairy oattle. Like ketosis i t affeots almost exclusively, the high-producing better nourished animals. Consequently^ the etiology of this disease has been the subject of intensive study. The f i r s t theory to be proposed was one of baoterial infection. In eighteen hundred and ninety^seven, Sohmidt (103), a Danish veterinarian, aoting on the hypothesis that milk fever resulted from an udder infection, experimented with injections of potassium iodide solution into the udders of a f f l i c t e d cows. This treatment proved successful under his direction but did not help to explain his infection 34 theory, because when ai r was introduced into the udder, together with KI, equally good results were obtained. Later i t was shown that inflating the udder with a i r alone proved superior to the.iodide treatment. Bacterial investigation proved conclusively that milk fever was not an infection of the udder. Thus i t was demonstrated that meohanioal distension of the udder effected a cure, but the cause of the disease remained a mystery. In nineteen hundred and twenty-five, Dryerre and Greig (20) proposed the theory that milk fever was an expression of an acute blood oalcium deficiency. In support of this theory they demonstrated hypo-calcemia in oases of milk fever, and showed that the intravenous i n -jection of oalcium gluoonate relieved the symptoms. Moreover there is a marked rise in the oaloium level of colostrum just after parturition. Greig explained the successful results from mechanical inflation by stating that the curative effect " i s a mechanical one i n that i t e l i o i t s mammary distension and so prevents the further interchange of oalcium from the blood to the gland acini"• In the normal animal, a l l of the various blood constituents are maintained at a very oonstant level through the proper oo-ordination of the body 18 complex and delicate glandular mechanism. The discovery of the changes i n the blood brought about by the disordered functioning of this mechanism during milk fever, has been of immense importance i n pointing the way to effeotive treatment; but the basic causes of the breakdown of the meohanism have not yet been f u l l y explained (108). Hibbs et a l (43) (44), are oonduoting a series of experiments on 35 the possible role of vitamin D i n milk fever* The results obtained so far indicate that pre-parturition treatment with vitamin D had no beneficial effect on the inoidenoe of this disease. Craigie (18) states that hypocalcemia is a characteristic symptom of milk fever but that i t is secondary to a parturient alkalosis. In their experiments (17), a r t i f i c i a l alkalization by the intravenous or oral injection of sodium carbonate into non-parturient cows/ reduced the blood calcium and increased the carbon dioxide capacity of the blood. A r t i f i c i a l acidification by oral administration of ehlorethamine to parturient oows with low blood caloium and phosphorus brought the levels back to normal. Chlorethamine had no effect in non-parturient oows with normal calcium and phosphorous. Their tentative explanation is that there is a sudden demand for aotive withdrawal of various ++ + electrolytes, especially Ca and H from the blood stream into the oolo-strum. The -developing alkalosis suppresses caloium mobilization causing milk fever. For treatment they recommend the standard dose of calcium gluoonate plus f i f t y gm. of chlorethamine every eight hours for twenty four hours. These recent experiments are shedding some light on the fundamental etiology of milk fever; but, as with the other metabolic disorders, the recommended therapy is not completely suocessful; and consequently, c l i n i c a l investigators (43) are turning to the research laboratory i n an effort to arrive at the speoifio cause of the c l i n i c a l condition. 2. Baby Pig Disease Acute Hypoglycemia v Since nineteen hundred and thirty-four the I l l i n o i s 36 Agricultural Experiment Station staff have been investigating a highly f a t a l disease of new-born pigs called acute hypoglycemia. In the typical syndrome (35), normal l i t t e r s at twenty-four to forty-eight hours of age show symptoms of shivering, dullness and inappetence, -often followed by death. No gross pathological lesions are in evidence at autopsy and no pathologio agents are recoverable. Normal amounts of caloium, phosphorous and ketones were found in blood samples. The only atypioal analysis was acute hypoglycemia. Graham and Sampson (35) found an average blood sugar level of twenty-six mg% in cases of this disease as compared to the normal range of one hundred to one hundred and thirty mg#. Glucose therapy proved successful only in the i n i t i a l stages of the disease. Beoause of the nature of the signs and symptoms, one might suspect that,the pregnant sows were inadequately fed. The authors report that as far as they could determine the rations were quite adequate, but they are continuing their investigations along this particular line, with a view to determining what influence the nutrition of the dam has on the progress of the disease. In further observations on acute hypoglycemia from Sampson* s laboratory (80), the results of blood analyses from hypoglycemic pigs consistently revealed the typical low blood sugar (Table 18). 37 TABLE 18 Blood Sugar mg# Liver Glycogen % Range Avge., Range Avge. Normal Pigs 12 to 48 hours old 75.6 to 149.2 114.5 2 - 3 2.5 Pigs with acute oli n i o a l . hypoglycemia 24 to 96 hours old 3.3 to 61 24.83 trace -Aoute experimental hypoglycemia in fasting pigs 36 to 96 hours old 9.04 to 42*94 30.43 trace The observation that the experimentally induced hypoglycemia reduced the l i v e r glycogen supply suggests that the new-born pig requires a period of several days for the regulatory meohanism i n -volved i n the production of l i v e r glycogen from non-carbohydrate material, such as tissue protein, to funotion effectively. If this meohanism is not ready and the absorbed sugar as well as the sugar from reserve glycogen f a i l to maintain a safe sugar level, then a f a t a l hypoglycemia beoomes imminent. Later (77), the I l l i n o i s workers studied the deleterious effects of severe and prolonged insulin hypoglycemia in young pigs. They observed that glucose injeotions hastened the reoovery from hypo-glycemia of short duration, but were of no value i n prolonged hypo-glycemia. Sampson states that the failure of many new-born pigs, affected with this disease, to respond favorably to injections of glucose might be due to an insulin influence. Hanawalt and Sampson (37) studied the effect of fasting and refeeding on the sugar and oertain other blood constituents of weanling pigs. Apparently a fast 38 of twenty-four days or twenty^eight days (water ad libitum) produced no pathologic ohanges in the ohemical components of the blood for which determinations were made. Only a traoe of ketones was found i n the blood of fasting pigs and none i n the controls. Blood sugar concen-trations decreased somewhat during fasting and then increased during re-feeding. In the later stage of fasting there was a rise in carbon dioxide combining power of the blood. Inanition gave rise to no significant alterations i n the concentration of inorganic phosphorous, hemoglobin, or blood creatinine. Prolonged fasting apparently gave rise to a temporary impairment of carbohydrate metabolism. This'recent work substantiates the earlier findings (80) of Sampson, et a l , that hypoglycemia was the primary blood change in the experimentally induced hypoglycemic pigs; but does not-assist i n explaining the etiology of the disease. 3. Diabetes Diabetes mellitus is a ohronio disease of metabolism that develops as the result of an insufficient supply of endogenous insulin. The greatest apparent disturbance oaused by this disease i s i n the u t i l i z a t i o n of carbohydrate. Since some of the blood and urine changes resemble those found i n o l i n i c a l ketosis of domestic animals, a brief review of the diabetio syndrome in humans is of some significance i n this discussion. If the insulin supply is inadequate, carbohydrate metabolism is disturbed. The concentration of sugar i n the blood increase?,and when the renal threshold for glucose is exceeded, sugar appears i n the 39 urine. As the amount of glycosuria increases, polyuria develops, a • great loss in body weight ensues, and other characteristic symptoms appear. Notable among these are: excessive t h i r s t (polydipsia)^ an excessive appetite (polyphagia), and general weakness. As the disease progresses, the patient develops a predisposition to infections, chronic degenerative changes, and ketosis. Unless the ketosis is curbed by i proper treatment, progress of the disorder is hastened by infection, toxemia and fever - leading to coma and death. As Long states in a recent review (21), "the fundamental dis-order known as diabetes mellitus rests on a disproportion between the requirement of the organism for insulin and the capacity of the Islets of Langerhans to meet this demand. There s t i l l exists a difference of opinion regarding the exact insulin mechanism and i t s relation to diabetes. Superficially everything would seem to point to insulin deficiency as the cause of the majority of cases; in spite of the failure to demonstrate the universal occurrence of lesions of the Islets of Langerhans, and the inconsistency of the type of Islets found. The modern view does not necessarily imply that the i s l e t s are destroyed but rather.that their capacity to produce insulin is i n -adequate to meet the circumstances under which the metabolism is working". According to this concept, there may be two types of insulin deficiency: (a) where there is an actual decrease i n the insulin- output of the pancreas due to a reduction in the quantity of Islet tissue. 40 (b) in which the insulin production may be normal or above normal, but owing to alterations i n the metabolism of the tissues, the amount produced is not adequate to supply the needs of the organism. The effect of insulin therapy is to reduce the hyperglycemia/ prevent the deposition of large amounts of f a t i n the liver.' and restore the blood level of ketone bodies to normal. In spite of a l l the experimental work done on diabetes mellitus, i t is s t i l l a c l i n i c a l disease of unknown etiology. Some of the essential characteristics of this syndrome are identioal with those found i n ketosis of other animals. These similarities/' together with the realisation that diabetes in humans is an endocrinological dis-order has led to a general comparison of this disease with ketosis. Although the fundamental disturbance underlying a l l ketosis is a relative or absolute laok of oarbohydrate i n the l i v e r leading to an excessive breakdown of fat (or a failure to oonvert free fatty acids to glycogen), the conditions leading up to this fundamental disturbance oan -be divided into three sub-groups: 1. Disturbances i n food intake 2. Impairment of l i v e r function ^ 3. Endocrine disorders Of the endocrine causes of ketosis, diabetes mellitus i s the only one of major c l i n i o a l signifioanoe. E. Summary In the previous discussion, ketosis of domestio animals has 41 been desoribed b r i e f l y and also compared to oertain a l l i e d meta-bolic disfunctions that either occur as complications of c l i n i c a l ketosis; or, like diabetes and acute hypoglycemia of baby pigs, possess similar symptoms* In a l l cases there was an impairment of normal carbohydrate metabolism. In some manner, the relationship be-tween blood glucose and liver glycogen was interfered with so that the animal turned to body fat as a source of energy and appeared to bypass the usual pathway through glycogen. When this took place, ketone bodies aooummulated in the blood and urine. In diabetes mellitus, the fundamental cause of the disturbance in the metabolism of carbohydrate is generally aooepted to be an endocrinological effect* Milk fever has been considered by several investigators to be an endocrine condition beoause of the suggested relation between calcium metabolism and the parathyroid gland* Pregnancy disease of ewes is probably due to some disturbance in the balance of feed intake and is accompanied by marked pathological ohanges i n the l i v e r * Ketosis i n dairy cattle and other domestic animals has been considered a disorder of carbohydrate metabolism for several years, but i t has not been u n t i l comparatively reoently that studies* have been made of the fundamental cause. It may be due to a hormonal influence as i n diabetes, although recent work has drawn attention to the possible effects of various nutritional factors i n relation to the etiology - in particular, aocessory food factors in their established role as essential catalysts i n carbohydrate meta-bolism. The fact that the highest incidence of ketosis occurs when t 42 the oattle are inadequately fed in relation to their increased meta-bolic requirements, lends support to these various nutritional hypotheses. The supposition that one of the food essentials might serve a catalytic role in carbohydrate metabolism and thus prevent the development of ketosis, makes i t desirable to study more closely the basic etiology. The relationships between carbohydrate and l i p i d metabolism in the ketotic state are extremely oomplex and thus necessitated an intensive review of the literature pertaining to the normal as well as the abnormal condition. III. THE ETIOLOGY OF KETOSIS A. Introduction 1. Normal Energy Metabolism (3) (5) (39) (67) (96) (loT) The animal body, like any other machine, must be provided with fuel in order to function. The supply must be continuous because the various metabolic processes continue even when the animal is at rest in a thermoneutral environment. The work of circulating the body fl u i d s , stimulating respiratory exchanges, manufacturing enzymes and hormones, and breaking down and resynthesizing the products of metabolism must continue i f the animal is to survive. The work efficiency of the body compares favorably with a typical gasoline engine. Approximately thir t y peroent of the total energy is converted to work, the remainder changed to heat and used to maintain body temperature. Fuel for the l i v i n g machine is provided by food which is oonverted 43 to heat and mechanical energy by the oxidation of this food to carbon dioxide and water.' The three basic food constituents - protein, carbohydrate and fat, were formerly believed to follow separate metabolic pathways and perform separate physiological functions. It is now known that the intermediary metabolism of a l l three, once having passed a certain stage, gives rise to a number of identical intermediate products (96)• These produots evidently constitute a dynamic pool into which degrada-tion products of both food and tissue breakdown are merged and from which the body can withdraw any one or any combination of these to synthesize new tissue constituents or metabolic substances. This dynamic concept assists i n explaining the supposed inter-convertibility of one food constituent to another. Whereas protein is directly convertible to oarbohydrate, i t is also ultimately inter-convertible with a l l of the foodstuffs because of their common products of 'intermediate metabolism. In the body, the conversion of the chemical energy resulting from the oxidation of food, to mechanical energy; takes place i n muscle tissue, whereas heat may be produced i n any tissue of the body. One of the constituents of musole cells i s the protein "myosin". The contraction of whole muscle is due to a summation of individual contractions of a l l the myosin particles. Metabolic energy i s required to stretch the.myofibrils, while the external force exerted by the contracting muscle is a result of the contraction of the 44 stretched f i b r i l s . The study of the chemical source of the energy expended when muscle does i t s work and the transformation of this energy into mechanical energy of contraction is one of the most complex problems i n the fields of biochemistry and physiology. i Adenosine triphosphate is suggested as the immediate source of the chemical energy converted by myosin into meohanioal energy. The energy required to form this ATP from adenylic acid is obtained by the oxidation of carbohydrate derivatives suoh as phosphopyruvic acid and glyceraldehyde. Proteins, fats, and carbohydrates are a l l potential sources of energy for muscular a c t i v i t y . They may serve as a direct source i n that they are involved i n the .formation of ATP. Indirectly, proteins and fats may serve as a souroe of muscle energy by means of a conversion to gluoose or glyoogen. . Sinoe their i n i t i a l metabolic pathways dif f e r , and because each is preferentially adapted to f u l f i l l certain physiological functions*' proteins, oarbohydrates, and fats w i l l be considered individually in relation to their role in metabolism. Amino acids from the digestion of protein are used for growth/' repair, and maintenance of the structural and catalytic machinery of li v i n g c e l l s . Amino acids that are not used i n the synthesis of protein, together with those derived from tissue breakdown, are deaminized; and the non-nitrogenous portion oxidized. It has been shown experimentally that some of the amino acids are glucogenic/ some are ketogenie, and some are neither. ' This i s one example of the merging of protein and carbohydrate metabolisms i n a common, reversible pathway. The remainder of the energy value of proteins obtained by the oxidation of the non-nitrogenous portion is u t i l i z e d for heat production. Figure 1. (67) • LIVER BLOOD MUSCLE at « TGlyoogen 1 Glucose — G l y c o g e n o Fructose Phosphate Carl L actic •« Lactic < — Lactio Acid Acid Acid C() & HgO Figure 1 represents schematically some general aspects of carbohydrate metabolism. Hepatio glyoogen i s converted to gluoose and transported by the blood to the muscles where i t is again converted to glyoogen. It is then broken down anaerobically to laotic acid and carried by the blood to the l i v e r for reconversion to glycogen. In the presence of oxygen, glycogen may be transformed to other intermediate products leading to oxidative combustion to carbon dioxide and water, thus providing energy. The anaerobic oycle of muscular carbohydrate metabolism serves as an energy meohanism to meet the short periods of exercise so severe that the respiratory and circulatory systems are unable to yie l d to the muscles enough oxygen to operate the oxidative cycle. The early work i n the anaerobic f i e l d was instigated by Fletcher and Hopkins in nineteen hundred and seven who showed that muscle can contract in the abscenes of oxygen; that laotic acid is produced r during anaerobic contraction and accumulates u n t i l the muscle i s 46 fatigued; and that i f this fatigued muscle is then put into oxygeri, i t recovers - is able to contract again, and the laotic acid disappears* Meyerhoff was later able to show that this l a c t i c aoid was produced quantitatively from glycogen, and that the energy expended in muscular contraction comes from this glyoolytio reaction. Phosphooreatine (phosphagen or creatine phosphate) was discovered to be present in striated, smooth, and cardiac muscles of vertebrates. Invertebrate muscle contained phosphoarginine. Both substances contain energy, rich phosphate bonds. I t developed that creatine phosphate played an important role in muscular contraction. It breaks down during activity and is resynthesized during rest. These early developments together with the work of Lundsgaapd and others established that the process of glycolysis i n the anaerobic metabolism (Figure 2) of carbohydrate was very similar to alcoholic fermentation. 47 Figure 2. [ i Anaerobic Carbohydrate Metabolism muscle Glyoogen + Inorganic Phosphate ^ alpha glucose-1-phosphate Phosphorylase phospho-glucomutase gluoo s e-6-pho.spha t e ^/y^ oxoisomerase Fructose-6-phosphate phosphohexokinase & ATP ^ Fructose 1:6 diphosphate & ADP zymohexase ^ phospho dihydroxyaoetone ^ 3 phospho glyoeraldehyde phospho-triose isomersse ^ Inorganic phosphate 1:3 diphosphoglyceraldehyde |^triose phosphate dehydrogenase 1:3 diphosphoglyceric acid ^^phosphokinase & ADP 3 phbsphoglycerio acid & ATP pho spho -gly o e romuta 2 phosphoglyoerie acid enolase phosphoenolpyruvic aoid ADP pyruvic acid & ATP phosphokinase / / coenzyme I. 2H / Lactic dehydrogenase Lactic Acid & Coenzyme I* 1 Bix carbon unit of glycogen — 2 moles Lactic Acid 48 The i n i t i a l energy source for this reaction comes from the Lohmann Reaction: ADP. + C P . ^ ATP +• C. The main energy comes from the new energy rich bonds generated by the formation of three moles of ATP. (net gain). The l a s t resource of energy comes from: 2 ADP Myokinase •>ATP + Adenylic Acid It must be realized that glucose does not l i e on the main pathway of glycolysis. It must be phosphorylated before i t oan be stored as glycogen. This is illustrated by Figure 3. Liver Phosphatase Glucose' Figure 3. Liver Glycogen Phosphorylase + Inorganic Phosphate Glucose - 1 - Phosphate Phospho glucomuta se I f ATP hexokinase Glucose - 6 - Phosphate Fructose - 6 - Phosphate Glycolysis The tissues take up the free glucose from the blood stream and phosphorylate i t into glucose - 6 - phosphate which is changed to alpha gluoose 1-phosphate and hence into glycogen by tissue phos-phorylase. This glycogen is then held there u n t i l i t is needed. 49 Although aerobic oxidation provides most of the energy for metabolism, small amounts of l a c t i c acid do exist i n the blood and these amounts inorease with exercise. In mild exercise^ oxygen is brought into the cells fast enough to reoxidize. the reduced coenzyme as rapidly as i t is formed, so that no l a c t i c acid is formed and pyruvic acid i n -stead of being reduced is completely oxidized. I f exertion i s increased"/ glycogen w i l l be more rapidly broken down and the coenzyme more rapidly reduced. Eventually a point is reached at which the oxygen supplied, by the circulatory apparatus can only just keep up with reduction of coenzyme. However the muscle can s t i l l work harder by using oxygen as fast as i t is made available by the circulation and re-oxidizing any coenzyme that s t i l l remains in the reduoed form by using the anaerobic device of laotic acid formation. As far as the aerobio mechanism for the oxidation of carbo-hydrate is concerned, glucose and glycogen are oxidized by way of the usual glycolytic reactions yielding pyruvio acid. I t is generally accepted that the pyruvic acid is then completely oxidized by way of the tri-oarboxylio aoid cycle (Figure 4). 50 Figure 4. Tri-Carboxylio Acid Cycle Aspartic Acid i 2H Keto oxaloacetic Acid i! Enol Oxaloacetic Acid *co 2 ^ ^ » Pyruvio Acid Alanine*"'' / / K+%0 // Glyoogen 4 Glucose -HO Malic Acid=-*Fumaric Acid Fumerase \[ tZE Succinic Acid Alpha keto Glutamic glutaric Acid Acid CO* Oxalosuccinic Aoid Acetic Acid • i Fatty Acids *2H Isocitric Acid Aconitase Aconitic Aoid C i t r i c Acid 1 Mole Pyruvic Acid +-50 3 C0„ +• 2H 0 2 2 This scheme is well established and explains the complete oxidation of pyruvate, acetate, and of any substance that yields pyruvate or acetate. It accounts for the complete oxidation of any substance lying directly on the cycle and of any substance that yields a compound on the 51 cycle. It also provides a link between the metabolism of carbohydrate and protein, and further establishes the hypothesis of a common metabolic pathway. Following digestion, absorption, and transportation via the blood stream; fat, removed from the blood, may be hydrolyzed then oxidized with the evolution Of heat; or i t may be stored for future us.e as fuel. Oxidation of the glycerol portion of the fat molecule yields metabolic intermediates similar to those found i n carbohydrate metabolism. These may be u t i l i z e d directly as a source of energy or for the synthesis of glucose and glycogen (107) . Oxidation of the fatty acid portion may also yield intermediates for glucose synthesis or may give rise to ATP for direct energy. As an example of the former possibility, acetoaoetic acid (derived from fatty acid metabolism) can react with pyruvic aoid derivatives (whether derived originally from protein or carbohydrate sources) thereby merging, as did the proteins and oarbphydrates, in a common f i n a l metabolic pathway. 2. Metabolism i n the Ketotic State The essential cause of ketosis is unknown but is commonly believed to arise when oarbohydrate metabolism i s deranged. This derangement affects the normal course of fat oxidation and as a result there is an accumulation of ketone bodies (acetone, aceto-acetio acid, and beta hydroxy butyric aoid) in the blood and urine. Although these.ketone bodies are considered to be normal produots of fa t metabolism, an exoess in the blood and tissues cannot be tolerated. An accumulation of acetoacetic acid and beta hydroxy butyric acid 52 upsets the acid-base balance, depleting the a l k a l i reserve and causing acidosis. Besides i t s acidic properties aoeto-aoetic aoid is a toxic substance (4). Beta hydroxy butyric aoid is relatively non-toxio but acetone is quite poisonous (4). The toxioity of aoetone was confirmed as indicated by the t o x i c i t y - t r i a l data outlined i n Appendix VI. Ketogenesis refers to the formation of ketones bodies. Their site of formation is the l i v e r . The known precursors of these substances are: (a) partial oxidation of fatty aoids in the l i v e r (b) certain ketogenio amino acids, such as: leucine, 1 iso-leucine, tyrosine and phenylalanine (o) a third possible source is the oxidation of pyruvic acid to give aoeto-aoetic acid (15), Fatty aoids are the main source of ketone bodies. Normally the acids are oxidized to oarbon dioxide and water. Under certain conditions this catabolic process is interrupted and ketone bodies are formed in abnormal amounts. Beta hydroxybutyrio acid and aoeto-aoetic aoid are believed to be the par t i a l oxidation products of butyrio aoid. Acetone is thought to be a derivative of aceto-acetio acid by the loss of carbon dioxide (38). aceto-acetio aoid acetone +• CO spontaneous decarboxylation beta hydroxy butyrio acid 55 What is the metabolic process that gives rise to ketone bodies in the organism? If carbohydrate metabolism is sub-normal, more fat has to be metabolized, forming larger amounts of ketones. In starva-tion, the l i v e r glycogen supply is exhausted and ketones accumulate. The injeotion of phloridzin depletes the glyoogen of the l i v e r and leads to the formation of ketone bodies. In diabetes, the liver's a b i l i t y to store and mobilize glyoogen is impaired and consequently ketones accumulate. The f i r s t theory to be developed regarding the metabolio pro-oess behind ketone body formation was the theory of "Koto-lysis" (6) (82)• Fat oatabolism instead of going to completion to carbon dioxide and water, stops at the ketone stage whenever there is a deficiency of carbohydrate in the metabolio mixture. Carbohydrate metabolism oontinues the u t i l i z a t i o n of the ketone bodies formed during f a t oatabolism. "Fats burn i n the flame of oarbohydrates". The second, and at present the more generally accepted point of view is the theory of "Anti-ketogenesis" (13) (24). Optimum carbo-hydrate metabolism inhibits ketosis not by increasing the u t i l i z a t i o n of ketones, but by keeping their production down to a minimum. Somogyi (95) suggests that "ketolysis" has been invalidated as an explanation of the mechanism by which the metabolism of carbohydrate oauses a lowering in ketonuria. Since carbohydrate is the preferential source of energy, ketones are produced in exoessive amounts only when li v e r glyoogen is exhausted and fat must be used as an alternative energy souroe. When the hyper-ketonemia so produced exceeds the u t i l i z a t i o n of ketone bodies by peripheral tissues, ketonuria results. 54 However i t is d i f f i c u l t to interpret the experiments of Bobbii and Deuel (6) i n the light of the anti-keto-genesis theory* They immersed rat l i v e r slices i n a butyrate medium* Less ketone bodies were reoovered from the l i v e r i f glycogen had also been added; and the amount of butyrate that disappeared was always greater* If glucose is the preferred substrate, then either the butyrio aoid shouldn't have been metabolized to ketones when glycogen is present; or i f oxidized to ketones, they should accumulate and be quantitatively recoverable* The faot that glycogen whioh is non-permeable to c e l l membranes, is active; while glucose whioh is permeable, is inactive, is d i f f i o u l t to explain* Possibly hexose phosphate is the ketolytically active intermediate and i t cannot be formed from glycogen. The d i f f i c u l t y in phosphorylation of glucose may account for the failure of gluoose to be changed to glycogen in l i v e r s l i c e s . It seems to be well established that the chief source of ketone bodies is f a t . Exactly how these substanoes are produoed from fat in the l i v e r i s a more complex problem. Metabolism of f a t " i n vivo" apparently begins with the hydrolysis of the f a t molecule to glyoerol and fatty aoids* Glycerol-probably gives rise to glyoogen i n the l i v e r (38). The ohanges involved i n the oxidation of the fatty aoid fraotion have been more d i f f i o u l t to follow. The "Beta Oxidation" theory of fatty acid oatabolism was originated by Knoop in nineteen hundred and four (53). At each step i n the de-gradation of fatty acids containing an even number of carbon atoms (those present i n natural fats), oxidation occurs at the carbon atom 55 in the beta position to the carboxyl group. By this process two carbon atoms are s p l i t off in each step u n t i l the four carbon stage is reaohed where aceto-acetio aoid is one of the products. This aoid is then converted to acetic acid whioh undergoes further changes to carbon dioxide and water. A series of omega-phenyl fatty aoids were administered to dogs. Samples of urine were collected and found to contain phenylated oompounds: (a) administration of fatty aoids with an even number of oarbon atoms - e.g. phenylbutyric aoid — p h e n y l acetic acid —> phenaoeturio aoid (b) administration of fatty aoids with an odd number of carbon atoms - e.g. phenylpropionio aoid —•> benzoic acid ^ hippurio aoid The conclusion made from this data was that the organism cannot remove carbon atoms one at a time from the chain or phenylacetio acid would be converted to benzoio aoid. Therefore the oarbon atoms must s p l i t off in pairs. As an example of Knoop's theory, l e t us consider the oxidation of palmitic acid (C H COOH): 1 5 3 1 I CHg - (CH 2) 1 2 - CHg - CHg - COOH CH - (CH ) - G* /CH -COOH 3 2712 / 2 CH - (CH ) 3 2 ] OH-#-H 2 8 - CH - CH - COOH 4- CH - COOH I 2 2 3 - CH - CH - COOH 4- CH - COOH 2 / 2 3 H f t . O 3 . 0 * t & A T « O N 56 C H - ( C H ) -CH - CH - COOH +- C H - OOOH 3 ' 2 6 2 | 2 3 C H - ( C H ) -CH - C H - COOH + C H - COOH 3 2 4 2 | 2 3 C H - C H C H - CH - C H - COOH +• C H - COOH 3 2 2 2 2 3 C H - C H - C H - COOH + C H - COOH 3 E 2 2 3 butyric aoid C H - C — - C H - C O O H 3 2 aceto-acetic aoid 1 mole Palm.itio acid +• 7 moles 0-*l mole aoetoaoetio acid 2 + 6 moles acetic aoid 6 moles acetio acid 4- 12 moles OL — 9 CO + 1 0 2 z 2 19 moles oxygen per mole aoetoaoetio acid The acetio aoid molecules are removed by oxidation as soon as they are formed. Ketones11; we re formed from fatty acids with an even number of carbon atoms, but not from those with an odd number. This was con-firmed by Embden's experiments on perfused l i v e r s . I f the l i v e r was perfused with an even numbered fatty acid there was an inorease i n ketones. If perfused with a fatty aoid with an odd number of carbon atoms, there was no inorease. The reason advanoed for this phenomenon was the lack of a four carbon stage with odd numbered fatty acids. Propionio acid is formed instead of b u t y r i c Fropionio is a glucose 57 former and ketone body formation is always less when carbohydrate material is being metabolized. One of the main criticisms of this theory has been the i n a b i l i t y to find any butyrio acid or acetic acid i n the l i v e r during active ketogenesis. Probably the acetic aoid i s immediately oxidized to carbon dioxide and water. Clutterbuck (14) and Witzemann (106) claimed that oxygen could be attached at the alpha and gamma positions on the side chain. Butts et a l (11) reported that some fatty acids yielded more than one mole of aoetoaoetio acid for each mole of fatty acid oxidized. They found that ootanoio aoid, for example, gave rise to two moles of aoetoaoetio acid. According to the beta-oxidation theory, ootanoio acid would be oxidized to butyric acid and y i e l d one mole of acetoaoetio aoid. Jowett and Quastel (50) found that the amounts of ketones formed by l i v e r slices "invitro" could not be accounted for on the assumption that only the last four carbon atoms of each fa t t y aoid moleoule yielded a ketone body. The work of Stadie et a l (97), showed that the molecular ratio of oxygen consumed to ketones produced from fatty acids by the liver'was 1.25. Therefore the higher fatty aoids produoe not one but four moles of ketones per mole of fatty acid oxidized. Edson (26) and MacKay (63) c r i t i c i z e d Snoop's theory when they found that certain of the odd numbered fatty acids l i k e valeric and heptylio oould give rise to ketone bodies. The next theory to be proposed was the theory of Multiple Alternate Oxidation. Hurtley (48) and Jowett and Quastel (50) suggested that the intact fatty aoid chain i s f i r s t oxidized by a 58 common enzyme at each alternate oarbon atom and then the chain s p l i t s into fragments of four carbons each, e.g. Palmitio Acid O O I O O J 0 O i o CH CH CH CH ', CH CH CH CH « CH CH CH CH ! CH CH CH COOH 3 2 2 2' 2 2 2 21 2 2 2 21 2 2 2 4r CH COCH GOOH + CH COCH COOH 4-CH COCH COOH +CH COCH COOH 3 2 3 2 3 2 3 2 C H 0 + 70 —*4 CH CO CH COOH •*• 4H 0 16 32 2 3 2 2 1.75 moles of oxygen per mole acetoaoetic acid. This helps to explain the greater than 1:1 ratio of ketogenesis from the higher fatty acids; the lower oxygen consumption than that expeoted from the 1:1 ratio; and the formation of ketone bodies from odd numbered fatty acids. However i t is d i f f i c u l t to explain the selective splitting of the molecule at every second keto group i n -stead of at every keto group. How could fatty acids with oarbon atoms not an even multiple of four be entirely converted into ketones without assuming that the extra carbon units were converted into ketones? MacKay (62) i n his beta oxidation - condensation theory maintains that a l l fatty aoid chains are oxidized at each alternate oarbon atom/ the molecule s p l i t t i n g at each keto group to form molecules of aoetio acid except where a three carbon chain remains. Such a chain forms propionio acid whioh can be converted to glucose. Two molecules of acetic aoid oondense to form aoetoaoetio acid. Some of the preliminary facts leading up to MacKay1s explanation a re: (l) Valeric acid (5 oarbon atoms) —* Ketones Hexanoic aoid — * more ketones, than butyrio acid 59 Since valeric acid produces sugar through propionic acid one can . account for the ketone body formation only by assuming a condensation of a two carbon atom fragment from one mole of valeric aoid with a similar two-oarbon fragment from another molecule. The condensation of suoh two-carbon atom fragments (aoetio acid) could also account for the greater ketone body formation from hexanoic than from butyrio acid. (2) The feeding of propionic aoid to animals led to an accumula-tion of glyoogen i n the l i v e r without the formation of ketones. Feed-ing valerio acid yielded an accumulation of glyoogen and formation of ketones. When heptanoio aoid was fed, glyoogen accumulated and more ketones were produced than with valeric acid.. These factors led MacKay and his co-workers to believe that oxidation proceeds simultaneously along the whole chain, of the fatty acid at alternate carbon atoms with the ultimate formation of aceto-acetic acid. e.g. Palmitio acid 0 * 0 1 o 1 o 1 0 1 o' 1 6 1 o' CH CH 1 CH CH iCH CH j CH CH iCH CH ;CH<CH ;CH CH • CH COOH 3 2! 2 2 - 2 2' 2 2' 2 2' 2 2' 2 2* 2 CH COOH CH COOH • CH COOH CH COOH' CH COOH CH COOH ' CH COOH CH COOH 3 3 I 3 3 3 3 3 3 • . I CH COCH COOH 1 CH COCH COOH ' CH COCH COOH J CH COCH COOH 3 2 . 3 2 ''3 2 ' 3 2 C H 0 + 7 0 — * 8 C H 0 -* 4 CH COCH COOH +- 4 H, 0 16 32 2 2 2 4 2 3 2 1.76 moles oxygen per mole acetoacetic acid. 60 Baldwin (3) i n his latest book sets forth three hypotheses to summarize the present knowledge of fatty acid oatabolism; (a) The two carbon fragments removed by beta oxidation consist of acetic aoid or some highly reactive derivative of acetio acid. (b) These two oarbon units are oxidized by way of the tri-oarboxylio aoid oyole. (c) When they are not oxidized as fast as they appear, these units condense to form acetoacetate and other ketone bodies. ' The actual oxidation takes plaoe on the fatty aoid chain by means of dehydrogenation and hydrolysis. ~CH CH CH -COOH 2 2 2 --CH CH r CH COOH 2 +1^ 0 V —CH CHOH —CH COOH —9 2H —> —CH GO CH COOH 2 2 2 2 The classical view of the meohanism whereby fatty acids were then s p l i t into two carbon fragments and fatty acids with two carbons less was one of simple hydrolysis: -CH -COklH 4J00H 2 / 2 OH,1H ~ C H COOH ¥ CH COOH 2 3 Baldwin objects to this approach on the grounds that beta-keto acids are not prone to this kind of hydrolysis at physiologioal pH; and that the biologioal formation of aoetio aoid from aeetoacetio acid or any other beta-ketonic aoid has never been demonstrated. (According 61 to Enoop the acetate was removed as fast as i t was formed). Reoent discoveries with the breakdown of carbohydrate show that the i n i t i a l stages involve a priming of the substrate by the trans-ference to i t of energy rioh phosphate radioals from adenosine t r i -phosphate. He states that evidence is mounting to show that some-thing similar may take place i n the breakdown of fatty acids. In other words, phosphorylation may be a preliminary stage to beta oxidation. -CH CH CH COOH *ATP -CH CH CH C-oJg)/ >ADP 2 2 2 If one assumes that the phosphorylated fatty aoid undergoes beta oxidation to give the corresponding beta-ketonio aoids, then how does the two-oarbon fragment"split off? Reoent work suggests that this splitting off is phosphorylytio rather than hydrolytic: -CH COfCH -CO -0 ^ P 2 ' 2 | +H0-® -CH GO - O^P + CH-CO- 0«® 2 3 The aoetyl phosphate units, oan then either undergo oxidation or condense to form acetoaoetio acid: 2 CH - C -0&) 3 tHO-CB CH -CO -CH CO - 0 * ® ^ / ADP 3 2 CH -CO -CH COOH kT ^ATP 3 2 62 3. Working Hypothesis Patton (65) (66) was able to alleviate the c l i n i c a l symptoms of ketosis i n dairy oattle by the oral administration of vitamin A. This therapy reduced the ketonemia and ketonuria and restored the blood glucose level to normal. On a limited scale, the same results were obtained using oattle from the University herd. On the basis of these results and the positive therapeutic-reports on the use of vitamin A by other clinicians (10) (45) (84) (90) (99), i t was hypo-thesized that ketosis might be due to a failure of the conversion of glyoogen to gluoose i n the l i v e r when there is a rapid inorease i n carbohydrate requirements and a sudden drain of stored vitamin A from the body to the colostrum as at parturition. The definite increase in blood sugar after the administration of vitamin A tends to sub-stantiate this p o s s i b i l i t y . On the other hand, since there is a depleted storage of hepatic glyoogen i n ketosis/ the cause may be due to a failure of the formation of glyoogen from gluoose. It was reoognized from the discussion of the normal and ketotic metabolic states i n parts one and two of this section that the mechanism of the glucose - glycogen conversion and the subsequent glycolysis reactions are considerably more complicated than represented by the soheme in Figure 5. 63 Carbohydrate (Diet) Glyoerol from fat (Diet and Body) Glycogenic Amino Acids (Diet and Body) Figure 5. (37) Liver ^ Glyoogen Blood Gluoose Blood Lactic Acid Muscle • Glyoogen Lactic Acid However, i t was believed that for the reasons outlined above'y vitamin A might play an activating role at some stage of carbohydrate meta-bolism. t L i t t l e i s known about the catalytic significance i n oxido-reduotion of the fat - soluble vitamins. On the other hand, most of the water soluble vitamins of the bios-type appear to be almost universally distributed and involved i n basic oxido-reductions. Thiamine exists as a oomponent of carboxylase and co-carboxylasey oatalyzing the decarboxylation of pyruvic acid and the carboxylation i n the assimilation of carbon dioxide. The riboflavin-protein enzymes are exemplified by the yellow enzyme, dinucleotides, oytoohrome-o reductase, and many others. Nicotinic acid exists in the form of two coenzymes: I and II. Several other vitamins from this group (e.g. pyridoxins and nicotinic acid) appear to be re-lated funotionally as well as structurally. After the hypothesis had been constructed, suggesting the possibility of a catalytic role for vitamin A in carbohydrate 64 metabolism, the expressions of several other workers on this subject were discovered. Morton (60)', in a recent review, wonders i f the findings relating to the water soluble vitamins have any bearing on the mode of action of the fat!.soluble vitamins lik e A and D. "There has so far been no clear evidence that these vitamins have any connection with energy metabolism, no evidence of union with phos-phate groups, nor of co-enzyme function, nor of oxidation-reduction " i n vivo". Yet i t is pretty obvious that the vitamins A and D exhibit interlocking roles. Their existenoe together i n f i s h l i v e r o i l s is not necessarily significant, but the fact that D regulates the laying down of calcium and phosphorous while A controls the osteoclastic removal of Calcium phosphate to determine the shape of the growing bone is "prima facie" evidenoe. He goes on to describe b r i e f l y some aspects of the biochemistry of vitamin A, and concludes with the following statements: "The mode of action of vitamin A thus presents some fundamentally novel characteristics but the broad conclusions to be drawn from the pre-sent state of knowledge are: (a) the role of vitamins or their derivatives in acting as oo-enzymes makes their modes of action oentral to the study of metabolism and to the rational develop-ment of chemotherapy. (b) the isolated and chemically characterized "vitamin" need not necessarily be the functioning catalyst; e.g. just as oarotene is a provitamin A and ergosterol a provitamin D, the vitamins themselves may be pre-cursors of the effective entities. (p). the gut, both as the "home" of characteristic microflora using and providing essential nutrients*;' and i n respect of i t s mucosal and submucosal layers an effective locus of chemical change, acquires an enhanced physiological importance." 65 Green, although writing at an earlier date (36), suggested much the same thing. He developed the enzyme - trace substance theory which predicted that any substance necessary in the diet in trace amounts must be an essential part of some enzyme system. The theory was not meant to imply that substances required i n higher concentra-tions cannot be essential parts of enzymes. "On the basis of the enzyme - trace substance theory we may confidently expeot i n the near future the identification of bio t i n , pantothenic acid,' f o l i c acid, vitamin A, vitamin K, and vitamin D with essential parts of new prosthetic groups." In order to test the soundness of the hypothesis proposed earlier that vitamin A was involved in some enzymatic role i n metabolism/ fundamental studies were instigated on the relationship of vitamin A to the ketotic state. It was. recognized at the outset of the experi-mental work that the most desirable animal, to study would be one of the larger domestic animals l i k e the dairy oow, which i s quite sus-ceptible to ketosis. This plan was obviously impractical for any large scale investigation. It was decided, therefore, that the more basic aspects of this problem could best be oarried out using laboratory animals. Later, perhaps, i t would be possible to extrapolate the early findings to the larger animal. B. Experimental 1. In this f i r s t experiment, which was oarried out from the twenty-f i r s t of August, nineteen hundred and forty-seven to twenty seventh of December, nineteen hundred and forty-seven, four groups of white Wistar rats, bred i n the Animal Nutrition Laboratory under controlled breeding and'management conditions, from twenty-eight to thirty-three days old, were placed, on a synthetic vitamin A deficient ration manufactured by the General Biochemicals Inc. (for composition of this diet see Appendix VII (a). Pour groups of white rats of comparable age, breeding and body weights, were used as controls/ receiving an oral vitamin A supplement (USP vitamin A Reference Standard: 2 I.U. per rat per day i n sunflower seed o i l ) . At the end of the experimental period, when the deficient rats showed v i s i b l e signs of vitamin A deficiency, a l l the animals were sacrificed to permit determination of blood vitamin A; glucose", and ketones; l i v e r vitamin A and glyoogen; and urine glucose and ketones. Details of the analytical procedures used are given in Appendix III. A l l food was weighed to determine the level of consumption per group (Figure 5 (a) and (b) ). The rats were weighed individually at three day intervals. The vitamin A supplement was administered orally to the control groups at the same time (deficient groups received straight sunflower seed o i l ) . A summary of the average body weights at weekly intervals is presented i n Table 19 and growth curves i n Figure 4, (a) and (b)• At the end of the depletion time, a l l the female rats were bred to the experimental males. It was hoped that this would not only further deplete the maternal supply of vitamin A^ but also i n -crease the extent of the ketonemia. Experiment 1. TABLE 19 Average Body Weight (gms) rH o I I 00 00 CM rH o> O CM r-i U3 CM CO CO t o o CO 1 1 1 1 rH rH CM H IO CM ! CO C-t o O C - 00 1 1 1 I rH rH CM rH CO rH CM t o CO rH t- CO 1 1 1 1 f-l rH CM rH CM <* rH CO rH t- CO 1 1 1 1 rH rH CM rH CO CM (O a> t o . CO (O CO t- c? » o f-l rH CM rH rH rH CM CM CO <6 t o CO t o -<* t o t o o a> O ft rH CM rH rH rH CM CM t o o> rH CM CD CO CO OS IO 6- o CO Oi rH rH CM rH rH rH CM ft O o> ^< o> en o> ft IO CO ^ CO O CO o> ft rH CM rH rH rH CM ft o> GO CO t o . CO IO <tf ft e - t o CO t o rH t - Oi rH rH CM rH rH rH CM t-t CM CO o t o t- t o ft >* 00 CM t o to CO CO ft H CM rH rH rH CM ft e - (O CM C- 3* 00 t- CO 6- r-i CO rH rH CM rH rH rH CM ft rH O CO O CO CO t o . CO 6- 0> CO ^ t o . CO rH rH rH rH rH rH CM rH t o • t f t o CO t o OV t o CM t o tO t> CM OS CM . CO rH rH rH rH rH rH CM ft IO rH t- IA Q CO CO ft CM lO rH © O t o rH rH rH rH CM ft CM ft 00 rH CM rH o> Q CO CM CM rH CO o CO o> rH rH H rH rH rH rH ft CO t o CO t o t o CM IO ft o a> rH en c - CO t> CO rH rH rH rH ft rH IA o> H O a o> CO 1 1 t o CM •f ft rH rH ft ft CO ft IO Q 1 I ' 1 1 O o> O o> rH rH 1 i 1 1 t o CO CO CO co 1 i + +• 1 1 + + < < < <5 •< < fe fe fe a fe IO CO IO CO IO t o IO t o rH H M t> > H ft M H H rH |> M ft H ft 68 Observations: (a) . As ean be seen by the graphs i n Figures 4 (a) and (b), a l l four groups of the vitamin A deficient rats showed a general loss in body weight. This loss i n weight beoame quite evident for groups I and II after six to seven weeks on the diet. Groups 7 and VI were one week younger at the start of the experiment and con-sequently began to show the typical loss i n body weight after five weeks on the experimental diet. A l l the control rats grew normally, and their growth ourves are comparable to those reported i n the literature (39). (b) Groups V and VI showed v i s i b l e signs of vitamin A deficiency i n the form of an incrustation of the eyes after the seventh weeky and groups I and II after the eighth week of the experiment. The oontrol rats showed no signs of vitamin A deficiency. (o) The 11A deficient" females (groups II and VI) did not conceive j whereas the female rats receiving the vitamin A supplement conceived and raised normal l i t t e r s (six to ten rats per l i t t e r ) . (d) The vitamin A deficient rats showed a significant hypoglycemia (Table 20). (e) The level of ketone bodies i n the blood and urine was slightly,but not significantly,higher i n the deficient rats. (f) The hepatic glycogen supply was subnormal in groups I, II, V, and VI, the deficient groups. •BLOOD No. > Gluoose 'Acetone Group Rats Sex Diet mg# mg% I 5 M A-Range Avge. 53.1-66.8 58.19 0.0-3.0 1.875 V 5 M A- Range Avge. 50*3-71.3 62.60 0.0-2.9 1.63 II 5 P A- Range Avge. 57.8-66.8 61.55 1.38-3.5 2.69 VI 4 F A- Range Avge. 53.1-62.3 56.60 0.0-2.3 1.47 III 5 M A* Range. Avge. 83.3-103.5 83.94 0-1.5 0.58 VII 5 M A + Range Avge. 79.5-119.3 97.99 0.0 0.0 IV 5 F A + Range Avge. 92.3-107.3 99.78 0.0-0.88 0.18 VIII 4 F A +• Range Avge. 92.3-107.3 100.70 0.0-0.88 0.41 Normal Range 80 to 129 0 to 5 URINE LIVER •Vit.A. IUAOOCC Gluoose Acetone mg# mg# i Glyoogen % Vit.A. IU/gm 0.0 0,0 0.0-1.2 0.97-3:.28 0,0 0.0 0.0 0.75 1.96 0.0 0.0 0.0 0 -1.25 1.1-2.73 0.0 0.0 0.0 0.25 1,71 0.0 0.0 0.0 0 -2.5 0.77-2.88 0,0 0.0 0.0 1.55 1.50 6.0 0.0 0,0 0.0-2.2 0.91-2.18 O'Jo 0.0 0.0 1.21 1.38 0.0 25-38.4 0.0 0-1.3 3.21-4.91 18.13r36.5 32.44 0.0 0.25 4.06 24.04 15.4-35.7 0,0 0.0-1.5 2.98-4.76 0.0-35.0 27.83 0.0 0.51 4.03 22.02 19.4-41.4 0,0 0,0 2.63-4.54 0.0-21.97 27.56 0.0 0.0 3.46 12.19 25.6-35.3 0,0 0,0 2.91-3.15 25.1^44. 5 29.15 0.0 0.0 2.97 34.30 30 to 112 0.0 0.0 2 to 3.6 0 to 135 a I' CO w <D a C r-1 ta o I M O CO CO (g) There was no vitamin A in the blood or l i v e r of the defioient animals* (h) Blood and l i v e r vitamin A levels of the oontrol groups are slightly lower than normally found, indicating that the dietary level of vitamin A may have been too low. (i) There was no glycosuria i n any of the rats. (j) As far as oan be ascertained from the analytical results, there was no variation that could be attributed to a difference i n sex. (k) Figures 5 (a) and (b) indioate a wide daily fluctuation in food intake. Some of this variation was caused by wastage due to the nature of the food and the type of feed con-tainer. Allowances were made for some spillage, and s t i l l the daily-feed consumption was within the daily recommenda-tion of ten to fifteen grams per rat. Conclusions: (a) The diminished glucose and glyoogen levels are not due to simple inanition sinoe the vitamin A depleted rats appeared to be eating the special depletion diet at an iso-caloric level with controls reoeiving a vitamin A supplement. (b) Since a l l the rats on the vitamin A depleted diet showed reduced blood glucose and liver glycogen levels,' the animal body evidently was unable to produce sufficient glyoogen from glucose to maintain the normal levels of glycogen in the l i v e r and consequently glucose i n the blood. 71 (c) There is some evidence of a slight ketosis i n the deficient rats which would suggest the possibility of vitamin A i n -' fluencing the formation of ketone bodies i n the l i v e r for exoretion via the blood and urine. The extent of ketosis would of course be limited i n these rats because they had very l i t t l e adipose tissue. 2. This experiment was so designed as to follow the pattern of the f i r s t experiment and thereby attempt to duplicate the hypo-glycemia shown by the vitamin A deficient rats in the previous work. Unfortunately, the deficiency developed more rapidly than expected; with the result that the analytical work could not be completed i n the time available. The data was incorporated into a laboratory exercise for the course i n Animal Nutrition (Animal Husbandry 322) to demonstrate the conditions of vitamin A deficiency i n rats. The results of this work are presented in Appendix VII (a). 3. Experiment three was planned along the same general lines as the f i r s t two experiments. In an effort to reduce the operational cost, an attempt was made to find a cheaper diet than the synthetic General Biochemioal Inc., product. The composition of the seleoted diet i s given i n Appendix VII (b). The rats did not show any signs of a vitamin A deficiency after thirteen weeks on this diet, so the form of the experiment was altered. The females were bred and then used for experiment No. 7. The l i t t e r s were raised to wean at twenty eight days and then placed on a vitamin A synthetio 72 ration, ^ Appendix VII (a)). After a suitable depletion time^ these rats were then used for subsequent experiments. Further details of Experiment 3 are summarized i n Appendix VII (b). ' 4. Since the f i r s t experimental results with laboratory animals indicated a possible relationship between ketosis and the glucose — glycogen mechanism i n vitamin A depleted rats, i t was deemed advisable, to study this association as i t is found i n normal adult rats. At what period after starvation does the l i v e r glycogen supply reach i t s lowest point? Does starvation influence the hepatic vitamin A reserve? Does the injection of gluoose restore the glyoogen supply of the l i v e r to normal? If so, which is the most* efficient route; the oral or intraperitoneal? Ten large male rats (white Wistar) were selected from the main laboratory breeding colony. They were of approximately the same age and had been, fed the standard colony diet: Purina Fox Chow. Two rats were k i l l e d immediately to determine base values for blood gluoose, l i v e r vitamin A, and glycogen. Four of the remaining rats were starved for varying periods. Two were sacrificed at twenty-four hours and two at fonrty-eight hours. Blood glucose and ketones; and l i v e r vitamin A and glyoogen values were determined. The last four rats were starved for foHrty-«eight hours. Two of the four received glucose per os and two intraperitoneally. Once again the blood and l i v e r values for the above constituents were deter-mined. The orally-treated rats were k i l l e d two hours post injection 73 and the intraperitoneally-injected one hour post injection. Observations (Tables 21 and 22): (a) The control rats exhibited normal blood and l i v e r values except for blood gluoose which was somewhat below the normal range of eighty to one hundred mg%. (b) The two rats that were starved for twenty four hours showed normal blood gluoose and l i v e r vitamin A; slightly lower l i v e r glycogen, and some ketonemia. (c) When starved for forty eight hours, the ketonemia inoreased and hypoglycemia was in evidence. Liver glyoogen supply was markedly reduced but the hepatic vitamin A reserve was not signifioantly affected. (d) Both the oral administration and the intraperitoneal injection of gluoose relieved the hypoglycemia, reduoed the ketonemia, but did not influence the l i v e r vitamin A storage. In both cases the glyoogen oontent of the l i v e r was restored to almost the same level - but not as high as was found for the control animals. (e) Table 22 indicates the variation i n analysis found when determin-ing hepatio glyoogen from the same lobe of a rat's l i v e r . This variation has also been reported by Oomori and Goldner for the rabbit (34). Body Liver Blood 'Blood Liver 'Liver Group Rat No. Wt. gms. Wt. gms. Glucose mg% Ketones Vit.A. IU/gm. Glycogen % (a) Controls (killed 1. 290 8.5 65.8 81.5 2.8 immediately) 2. 296 9.0 57.5 mm mm 68.7 2.8 (b) 1. Starved 24 hrs. 1. 213 6.0 54.0 12.6 87.2 1.5 2. 238 7.0 50.3 14.1 81.2 1.4 2. Starved 48 hrs. 1. 242 5.5 27.0 22.4 75. i 0.4 2. 232 5.6 10 min.. 33.0 10 min. 2 hrs. 19.5 71.6 0.5 (c) 1;: Starved 48 hrs. 200 rag Gluoose per os. 1. 242 5.5 pre-in-jection 33.0 post-in-jeotion '45.0 post-in-jection 87.8 8.2 75.9 1.9 2. 258 6.0 39.0 50.3 83.3 1 hour 9.4 78.3 2.4 2. 200 mg Glucose intra-peritoneally 1. 275 6.0 45.0 50.3 post-in--jeetion 92.3 8.6 75.2 1.4 2. 290 7.5 39.0 45.0 75.0 9.4 83.3 2.4 75 TABLE 22 Experiment 4. Liver Glycogen — Simultaneous Samples from the same Lobe of Rat's Liver. Rat No. Liver Sample No. 1. Z 3 4 Average 2.75 2.69 2.81 2.84 2.77 2.48 2.84 3.07 2.91 2.83 Conclusions: (a) Forty eight hours would appear to be near the optimum period required to induce hypoglycemia and ketonemia in normal adult rats. Starvation decreases the glycogen reserve of the l i v e r but has no effeot on the vitamin A content. (b) The injeotion of gluoose intraperitoneally and the administra-tion orally both lead to a replenishment of the l i v e r glycogen supply, an elimination of the hypoglycemia and a slight re-duction i n the ketonemia. There appears to be no relationship between the administration of glucose and the vitamin A oontent of the l i v e r . (c) The values in Table 21 for the hepatic glyoogen content are open to some question i n view of the results shown in Table 22. Evidently there is some variation i n analyses of samples obtained from the same lobe and probably considerable variation between lobes. 76 5. This experiment was instigated to verify the hypoglycemia indioated by the deficient rats i n Experiment 1; and to follow the progressive development of this reduction i n blood sugar. Its second purpose was to correlate the work of Experiment 4 with previous investigations and determine i f the administration of glucose to vitamin A deficient rats had any effeot on blood glucose/ blood ketones or l i v e r glycogen. Fifteen weaner rats were divided into three groups, one male and two female. One of the female groups received the vitamin A deficient diet plus a vitamin A supplement; the two remaining groups, the deficient diet alone. The depleted rats began to show vi s i b l e signs of the deficiency after fi v e weeks on the diet. Throughout the duration of the experiment, individual blood samples were taken by heart punoture at weekly intervals for the determina-tion of blood glucose. Fooled samples of blood enabled group, determinations to be made for blood ketones and vitamin A. At the end of the experiment, the ten vitamin A defioient rats were subdivided and subjected to varying treatments. . Two males and two females were k i l l e d immediately in order t o determine the basal levels for blood and urine. Two males and two females were then each given five hundred mg gluoose per os; and one male and one female the same amount intraperitoneally. Blood samples were taken two hours after the injection. The five con-t r o l rats receiving the vitamin supplement were also k i l l e d to determine their blood values. Time of Experiment in Days 6th Day 12 th Day 19th Day 22nd Day 24th Day 29th Day Blood Blocd Liver Rat Group No. Gluo. Vit.A. IU/100 Gluo. Vit.A. iuAoo Gluo. Vit.A. Gluo. IU/100 r*n% Vit.A. IU/100 Gluo. Vit.A. IU/LOO Treatment Gluo-ose Ke-tones Gly-00 gen CD V 1*3./o 1 H ' 1 75.0 57.8 54.0 39.0 45.0 Control 66,8 3.4 2'l83 3 CD XXI 2 81.5 51.5 41.2 33,0 39.0 ti 41.0 7.-5 '4.19 M 3 90.8 56.5 39.0 36.5 36,5 83.3 1.6 0,15 an A- 4 94.2 61.0 52.5 41.5 39,0 # 70.5 0,0 1,-1- • 5 65.5 53.5 54.0 45,0 43.0 X 75.0 3.25 2.64 Avge 81.4 32.4 56.1 14.6 48.1 10.2 39.0 0.0 40.6 0.0 1 104.1 45.0 33.0 45.0 39.0 Control 35.4 4,1 4.2 O CD <l XXII 2 95.6 39.0 39.0 33.0 30.5 it 45.0 2,8 4.6- CD M F 3 78.1 54.0 45.0 39.0 40.5 92.2 2.2 2.26 O T J A- 4 85.1 51.5 41.5 42.8 45.0 # 83.3 1.6 1.77 3 CD 5 81.9 52.2 39.0 35.6 39.0 X 57.8 0.0 4.44 c i Avge 89.0 40.5 48.3 19.5 39.5 8.1 39.1 0.0 38.0 0.0 0 >-* 1 79.5 - 57.8 65.4 91.8 80,5 Control 62.3 1.51 a XXIII 2 84.7 69.4 81.5 83.6 50.3 .» 92.3 - 0^ ,75 O era F 3 93.4 71.8 57.8 65.0 60.9 a 66.8 mm 2,*75 tr* «< AT 4 89.8 83.5 105.5 90.0 85.4 n 63.0 - 3,48 O CD 5 98.7 81.4 95.8 87.8 76.7 ti 83.3 mm 3,37 B H » Avge 89.4 65.4 72.8 59.5 81.2 61.5 83.6 0.0 70.7 71.5 Average 73.5 - 2.37 SB. # — 500 mg. gluoose per os. X — 500 mg. gluoose intraperit. - 4 Observations (Table 23 and Figure 6): (a) Control values are a l l within the normal range. (b) The hypoglycemia in the WA W deficient rats developed almost immediately but did not reach i t s maximum u n t i l the twenty fourth day. At this time a slight ketonemia was i n evidence. (c) The administration of glucose peros raised the blood sugar level, caused a slight reduction in the ketonemia, and appeared to deplete the l i v e r glycogen reserve. (d) Gluoose injeoted intraperitoneally also increased the blood glucose, reduced the blood ketones, but spared the l i v e r glyoogen supply. Conclusions: (a) Vitamin A would appear to be neoessary for the conversion of gluoose to glycogen. In Experiment 4, the administration of gluoose to starved rats with normal vitamin A levels led to a building up of the hepatio glycogen supply. However, in Experiment 5, when glucose was given orally to vitamin A deficient rats, this build-up did not occur. By the intraperitoneal route, the rats were able to maintain their normal glycogen levels but no excess reserve was synthesized. (b) The time of depletion and the development of hypoglycemia were extremely short. For this reason, a further confirma-tory experiment would be desirable. 6. A further study of the effect of glucose administration on the blood gluoose and l i v e r glycogen levels of vitamin A deficient rats was carried out i n Experiment 6. Sixty-one weaner rats were placed on a vitamin A deficient diet for periods varying from four to six weeks. At the end of this depletion period, when sample hepati vitamin A determinations indicated that no vitamin A was present, the rats were subdivided into nine groups and subjected to varying pre-experimental treatments.1 Pour groups received five hundred I.TJ. of vitamin A orally per rat and then were subjected to a fi f t e e n hour fast. Four groups were fasted fifteen hours but received no vitamin 0 A. One group was not fasted and served as a control. The rats i n this last group received no vitamin A. At the end of the fast, two groups received glucose per os (five hundred mg per rat — allowing a one hundred percent increase over basal metabolic requirements) and four groups the same dose intraperitoneally, according to the plan indicated by Table 24. Blood samples were taken two hours after injection of gluoose and analyzed immediately. • The livers were extirpated as soon as the blood samples were drawn, placed in a salt-ice freezing mixture, and analyzed at once for l i v e r glyoogen content. Observations (Table 24): (a) A l l the animals in this experiment that received the depleted diet, showed visible signs of vitamin A deficiency after four to five weeks. Analyses of blood and l i v e r samples at this time indicated hypoglycemia, slight ketonemia and normal l i v e r glycogen. No."of „ Liver Glycogen % Blood Gluoose mg^ Group Rats Pre-Bxperimental, Treatment Range Avge. Range Avge. ( a y e N i l 3.01-3.94 3.29 42.0-53.9 47.01 00 10 I 5 h r . Fast 0.0 -0.42 0.19 39.0-62.25 49.27 10 500 IU Vit.A + 15 hr. Fast 0.0 -0.51 0.22 39.0-62.3 50.49 (d) 5 15 hr. Fast + 500 mg Glucose p.o. 0.48-0.51 0.49 45.0-52.25 49.36 to 5 500 IU Vit.A +- 15 hr. Fast -t-500 mg Gluoose p.o. 0.41-O.49 0.45 50.3-96.1 75.0 (f) 5 15 hr. Fast +• 500 mg Gluoose l.p. 0.42-0.48 0.44 59.0-81.25 68. 41 (6) 5 500 IU Vit.A 1-15 hr. Fast +-500 mg Gluoose l.p. 3.0 -3.42 3.45 65.0-121.5 98.4 (h> 8 15 hr. Fast -t- 500 mg Glucose l.p. 0.0 -2.54 1.31 53.9-76.0 63.37 (i) 8 500 IU Vit.A +- 15 hr. Fast +• 500 mg Glucose l.p. 0.94-3.04. 2.35 66.75-111.0 89.92 • Vitamin A content of Liver x n n u u 0.0 IU/lOOoo 31.3 " Blood Ketones 1.5 mg# In order to remove some of the dietary variation and diurnal fluctuation that might influence such an-experiment as this, the remainder of the animals were fasted fifteen hours to bring them to a common base l e v e l . The effeot of fasting (Group b - Table 24) was to reduce the glycogen oontent of the l i v e r . It .did not further reduce the blood sugar l e v e l . The administration of vitamin A to the deficient rats that were subsequently' fasted for f i f t e e n hours (Group e - Table 24) did not change the blood or l i v e r picture from that mentioned i n (b) above. If "A deficient" rats were fasted f i f t e e n hours and then given glucose per os, the hepatic glycogen oontent appeared to increase slightly though perhaps not significantly. The blood glucose level did not rise (Group d - Table 24). However i f these rats were given vitamin A previous to the above treatment, the blood glucose level returned to within the normal range (Group e - Table 24). The administration of gluoose intraperitoneally to rats that had been previously fed glucose, not only raised the blood gluoose level but also led to a return of the l i v e r glycogen to normal (Groups f to i - Table 24)• 82 Conclusions: (1) There does not appear to be any interference in the absorption of glucose intraperitoneally in the vitamin A deficient animals. There evidently is some blocking of the absorption per os. (2) In the vitamin A deficient rat, either: (a) the glucose-glycogen reaction is interfered with or (b) there is an interference somewhere i n the con-version of the oxidation, products of glucose to glyoogen. 7. The results obtained in Experiment 6 with weaner rats were quite significant and i t was f e l t that i t would be desirable to repeat this work using older rats. Twenty eight mature rats that had been on a vitamin A deficient diet (Experiment 3) for over twenty weeks were used for this experiment. Some d i f f i c u l t y had been experienced previously in depleting these rats on the diet fed. For this reason, six rats (three male and three female) were selected for li v e r vitamin A determinations. The results indicated that the rats had no reserve of vitamin A (Table 25), even though they were obviously obtaining the vitamin because their body weights were normal and they exhibited no vi s i b l e symptoms. However, i t was f e l t that these rats were bordering a vitamin A deficient state and rather than sacrifice them indiscriminately, they might be suitably adapted to this experiment. 83 TABLE 25 Rat No. Sex Body Wt. gm. Liver Vitamin A I.U./gm. Blood Glucose mg# # 1 M 240 21.87 103.5 2 M 295 0.0 99.75 3 M 288 0.0 83.25 # 4 P 186 19.3 78.75 5 P 167 0.0 66.75 6 P 180 0.0 75.00 # - 1000 I.U. vitamin A per os. The rats were divided into four groups. Group A was fasted f i f t e e n hours and fed eight hundred mg glucose per os. Group B received one thousand I.U. vitamin A, fasted fifteen ;hours,and then given gluoose per os. Group C was fasted fifteen hours and given eight hundred mg gluoose intraperitoneally. The rats i n Group D were each given one thousand units of vitamin A. They were then fasted fifteen hours and each injected with eight hundred mg glucose intra-peritoneally. The results of this experiment are presented in Table 26. It i s possible that the lack of response to gluoose administra-tion i n the vitamin A supplemented groups as reflected by the low l i v e r glycogen levels for groups C and D might be attributed to the fact that these rats were not completely "A deficient". In addition, Tables 25 and 26 indicate that the blood gluoose levels were within the normal range. No. of Liver Glycogen % Blood Glucose mg$ Group Rats Pre-Experimental Treatment Range Avge. Range Avge. A 7 15 hr. Fast +-800 mg Gluoose p.o. 0.59-0.72 0.66 99.75-119.25 110.3 B 6 1000 IU Vit.A + 15 hr. Fast + 800 mg Gluoose p.o. 0.54-0.74 0.65 75.5-114.7 97.75 C 7 15 hr. Fast + 800 mg Glucose l.p. 0.82-1.10 0.88 83.2-119.25 100.02 D 8 1000 IU Vit.A +-15 hr. Fast + 800 mg Glucose l.p. 0.85-1.14 0.94 75.0-104.16 91.0 85 8. In Experiment 6, blood and l i v e r samples were taken a r b i t r a r i l y at two hours post injection, of glucose. Analyses were made immediately following sampling. Experiment 8 was designed to find out how much time is required after injection to show optimum response to the glucose. It was also hoped that this experiment would verify the results of Experiment 6. Twenty five vitamin A deficient rats.of approximately the same stage of depletion as used i n previous experiments;were divided into two groups of ten. The five remaining rats were sacrificed.immediately. Analyses of their livers indicated that they were completely depleted of vitamin A. The average value for blood glucose was 64.8 mg$. The rats from Group A were fasted f i f t e e n hours and each was i n -jeoted with four hundred and fi f t e e n mg of gluoose intraperitoneally. Blood and l i v e r samples were taken from pairs of rats at time zero, fifteen, forty»five, ninety and one hundred and f i f t y minutes post injection. Group B received one thousand I.U. of vitamin A per rat, and was then treated similarly to Group A. 86 TABLE 27 Group A - 15 hour Fast plus .415 mg glucose Intraperitoneally (Time. 0) Time of Rat Sample i n Blood Glucose Liver Glycogen No. Min. mg# % 1 0 62.25 0.36 2 0 60.5 0,f28 3 15 55.6 0.48. 4 15 50.25 0^39 5 45 75.0 0.52 6 45 75.0 0,51 7 90 96,0 0.49 8 90 68.35 0.49 9 150 88,3 0,47 10 150 92.5 0.48 Group B - 1000 I.U. Vitamin A plus f i f t e e n hr. Fast plus 415 mg glucose Intraperitoneally (Time'O)  1 0 73.5 0.29 2 0 74.8 0.27 3 15 66.76 0,42 4 15 63.5 0,45 -5 45 72.25 0,43 6 45 73.0 0.42 7 90 88.5 0.42 8 90 88.25 0.42 9 150 83.9 0.50 10 150 80.25 0.52 Although the experimental conditions were similar to those of Experiment 6, the inorease i n l i v e r glycogen of the deficient rats that had been given vitamin A was not verified in this experiment. The reason for this lack of correlation was not immediately apparent, although the relatively high blood glucose values at time zero would suggest that these animals were not completely depleted of vitamin A. 87 Further experiments were planned to repeat this work under more closely controlled conditions i n an attempt to disolose the cause of the variation. 88 IV. SUMMARY A. Ketosis in Dairy Cattle 1. Ketosis i n dairy oattle is now universally recognized as a c l i n i c a l syndrome causing appreciable monetary IOBS to the dairy industry. It has been established as a definite pathological state within the area studied i n the present work. 2. No completely adequate speoific therapy has been suggested for the treatment of the various recognized forms of ketosis. . , 3. Present methods for the rapid detection of uncomplicated and complicated ketosis i n the f i e l d are not entirely satisfactory. However, sufficient data has been published to provide a reasonable picture of the blood and urine changes that occur i n ketosis; and the necessary laboratory procedures are available for i t s detection. The present work has added to this information and has afforded a f i r s t comparison of ketosis as reoognized here, with the con-dition observed elsewhere. Information was obtained from the Univer-sity herd of Ayrshire oattle permitting the establishment of normal levels for blood and urine constituents under winter and summer feeding conditions. It has been demonstrated that, under the feeding and management conditions prevailing i n the University herd, a sub-c l i n i c a l avitaminosis A exists not only in the winter, but probably in the summer months. 4. Prom the information published elsewhere, as well as from the present data, i t would appear that the heavy drain on the maternal energy reserve and on certain of the vitamins following 89 parturition, is sufficiently great to cause a physiological de-rangement of pathologioal significance. The possibility is suggested that vitamin A might play a c r i t i o a l role in the induotion of this derangement. It seems li k e l y that the fundamental metabolic disorder is concerned with the energy metabolism of the animal. It is not yet established whether this is solely within the carbohydrate oycle, the l i p i d cycle, or within both. Nor is i t established whether vitamin A is involved in this derangement. B. Ketosis i n Other Domestic Animals 1. In the present investigation, no experimental work has been carried out relative to ketosis in other domestic animals. 2. A review of the current literature concerning ketosis in other domestic animals has been made and the various conditions have been compared to the ketotic state in dairy cattle. 3. These observations confirm, in part, the tentative conclusion that ketosis is a disturbance of energy metabolism. C. The Etiology of Ketosis 1. The meohanisms involved in normal energy metabolism have been discussed i n relation to the energy picture in the ketotic state. A brief outline has been given of the various theories whioh have been suggested to acoount for the formation of ketone bodies. 2. The experimental work was planned on the assumption that the basic cause of ketosis is a deficiency of available carbo-hydrate and as a result the animal 1s energy requirements are not 90 satisfi e d . This deficiency may arise through a dietary inadequacy or through the inability of the animal .to metabolize carbohydrate arising from protein and l i p i d precursors. A working hypothesis was advanced proposing that the in a b i l i t y of an animal to make use of i t s energy resources through normal path-ways is due to a deficiency of vitamin A. 3. On the basis Of. this hypothesis, i t was assumed that animals suffering from avitaminosis A would be unable to metabolize administered carbohydrate in the normal manner. The experimental results obtained indicate that in young animals deprived of vitamin A1/ a hypoglycemia of cl i n i o a l l y significant proportions develops as the vitamin A deficiency progresses. The data suggests further that this hypoglycemia is one of the earliest symptoms of avitaminosis A and arises despite an adequate intake of dietary energy. 4. The development of a hypoglycemia on adequate energy intake followed by a characteristic loss i n body weight is offered as supplementary evidenoe that vitamin A may have a role in carbo-hydrate metabolism. Other experiments have been conducted that demonstrate the inab i l i t y of the vitamin 'A deficient animal to u t i l i z e gluoose administered per os. Fork is proceeding in order to confirm this finding. 5. In conclusion, results have been presented to suggest that vitamin A may possess, in addition to i t s other established funotions, a specific role i n carbohydrate "metabolism. The 91 experimental data is not yet sufficiently complete to assign the properties of a coenzyme to vitamin A* Moreover, the po s s i b i l i t y of vitamin A affecting the hormonal balance and thus indirectly, oarbo-' hydrate metabolism, cannot be precluded on the basis of the' present investigation. Pinal proof that vitamin A may possess this coenzyme activity must await-further studies. It is suggested that subsequent work might well exploit the advantages of " i n vitro" tissue s l i c e techniques. 6. Related experiments have been carried out and are appended for future reference. ' ' -V 92 APPENDICES Page I. Survey of the Incidence of Ketosis i n B r i t i s h Columbia. 9.3 II. Rothera's Qualitative Test for Urine Ketones. 95 III. Analytical Procedures. # 96 IV. Vitamin.A Injeotion by the Intramuscular Route.. 114 V. Colostrum.Data from University Herd. 118 VI. The L.D. 50 Dose Level in Acute• Acetone Poisoning. 119 VII. (a) Experiment 2 . 120 (b) Experiment 3 . 121 # - A l l chemicals used are of Merck's Reagent Grade except where otherwise noted. - Spectrophotometer used:- Coleman, Model 1 1 . Definition of Units: 1 . oubio centimeter es 0 . 9 9 9 9 7 m i l l i l i t e r 2 . mgfe as milligrams, percent » parts per 100 ,000 3 . 1 I.U. Vitamin A = 0 . 6 ug beta carotene APPENDIX I A SURVEY OF THE INCIDENCE OF KETOSIS IN BRITISH COLUMBIA - 1948. 1. Veterinarians oontacted: Dr. 6. Barton Chilliwack Dr. L. T. Clarkson Abbotsford Dr. G. L. Davis Ladner Dr. J. A. Fischer Duncan Dr. H. R. Hylton Mission Dr. G. H. Keown Victoria Dr. J. C. Lomas Hammond Dr. M. H. Milton Chilliwaok Dr. J. J . Murison Armstrong Dr. E. L. Nundal Langley Prairie Dr. B. W. Ray Abbotsford Dr. G. P. Talbot Kelowna Dr. K. H. Thompson Murr'ayville 2. Survey Procedure. A questionnaire was prepared asking the veterinarians i n eaoh d i s t r i c t to l i s t the cases as they occurred giving the treatment used and a brief history of each case. The forms were distributed to fourteen practising veterinarians in eleven rural oentres. Each veterinarian was contacted in person or by mail at least four times during the period of the survey. Replies were received from three practitioners as indicated i n the following table: RESULTS OF KETOSIS SURVEY FOR BRITISH COLUMBIA / Jan. to May, 1948. Veterinarian D i s t r i c t Case Date of Case Breed of Cow Age of Cow Treatment and Remarks Dr. G.H.K. Victoria Dr. L.C. Abbotsford Dr. H.R.H. Mission 1 12.12.47 Jersey 3 yrs. 'Dextrose — sub- 1 lb . porn syrup cutanebusly & 1 |- oz. chloral Intra-venously hydrate 2 3. 1.48 n 5 ii tt n 3 14. 1.48 » 2 ti II . « 4 31. 1.48 tt 4 II ii n 5 5. 2.48 n 5 tt tt it 6 10. 2.48 tt 3 n n n 1 25. 1.48 Guernsey . 3| it Dextrose and cobalt 2 26. 1.48 n tt it n it 3 28. 1.48 n Ik n Dextrose, oobalt and chloral hydrate 4 28. 1.48 it 4 tt Dextrose and molasses 5 13. 2.48 n 2 II « 6 18. 2.48 tt 2 n 1 l b . sugar per day 7 19. 2.48 ti 2 t i " ti • - . . . JI 1 21.11.47 Jersey 7 it Dextrose, Vitamin A, chloral hydrate and oalcium gluconate — milk fever 2 2. 2.48 II 4 tt chloral hydrate and sugar.(per os) 3 9. 2.48 tt 6 if Dextrose & chloral - high producer 4 12. 2.48 it 6 tt n " L i t t l e response 5 13. 2,48 n 5 ti « tt 6 14. 2.48 it 6 tt • fl & oobalt 7 15. 2.48 tt 8 it " ' " - paralysis 8 16. 2.48 « 4 <t M " & cobalt 9 16. 2.48 it 8 it n n « 10 22. 2.48 Holstein 6 it n it 11 23. 2.48 Jersey 5 it n M & cobalt 12 28. 2.48 ii 6 ti it it 13 6. 3.48 ii 9 n ii tt it 14 9. 3.48 n 5 n " ' M & HCL APPENDIX II ROTHERA'S QUALITATIVE TEST FOR URINE KETONES (95) Reagents: 1. A mixture of one part finely powdered sodium nitroprusside with one hundred parts pure ammonium sulphate. 2. Flaked sodium hydroxide. Procedure: One gram of the nitroprusside-sulphate mixture is dissolved in five oo. of the suspected urine, and then a small-flake of NAOH is added. A purple permanganata colour indioates a positive test. 96 APPENDIX III ANALYTICAL PROCEDURES (1) Somogyi's Mioro Modification of the Schaffer - Hartman Method for Blood Gluoose (94).  Reagents: 1. 0.0667 N Sulphuric aoid 2. 2.5$ Sodium Tungstate solution 3. Alkaline oopper reagent: 20 gm. anhydrous sodium carbonate ) — 25 gm.. sodium bicarbonate ) Dissolved in 600 25 gm. Rochelle salt ) oo. of water Dissolve 7 gm. orystalline copper sulphate i n 100 oc-of water. Introduce this into the carbonate-tartrate solution through a funnel touching the top of the solution to prevent oarbon dioxide absorption. Add ten gm. of potassium Iodide and 5 gm. potassium oxalate and 22 cc. alkaline 1 normal solution of potassium bi-iodate: (32.498 gm. pot. bi-iodate (83.3 oo. 1 N Sodium hydroxide (make up to one l i t r e — 0.715 (gm. pot. bi-iodate in 22 cc. (of approximately N NAOH Make up to 1000 cc. (5 cc. alkaline oopper reagent equals 22 00*0.005 N sodium thiosulphate) 4. I N sulphurio aoid 5. 0.005 N sodium thiosulphate solution (add a few drops of ten per oent sodium hydroxide per l i t r e ) . Prooedure: 2.0 co. 1/15 N sulphuric acid are placed into a centrifuge tube. Add 0.2cc. of oxalated blood and rinse the pipette in the solution. Add 0.8 cc. 2.5$ sodium tungstate. Mix the contents of the tube; oover; and centrifuge (can leave overnight). Pipette 2.0 cc. blood f i l t r a t e into a 25x 200 mm. test tube, add 2.0 cc.of the alkaline oopper reagent. Mix and oover with a funnel. Immerse in hot water for f i f t e e n minutes, then cool. 97 Add 2*0 oc. of N sulphurio aoid dropwise, shake to dissolve the cuprous oxide. Titrate after one or two minutes with 0.005 N sodium thiosulphate. When tit r a t i n g , add the thiosulphate u n t i l the colour of the mixture has beoome a pale green-blue, then add 1.0 c c o f a 1% starch solution. Treat 2.0 oc. of water i n the same manner as the blood f i l t r a t e . The difference between the two t i t r a t i o n figures-is referred to the conversion table and then multiplied by seven hundred and f i f t y to give the amount of gluoose in the blood (mg$). Conversion Table for Blood Gluoose Determination 0.005 N Sod. • Thiosulphate 0 1 2 3 4 5 6 7 8 9 0 - .036 .052 .067 .077 .089 .100 .111 .123 .133 1 .143 .153 .165 .176 .188 .199 .209 .221 .232 .243 2 .253 .264 .276 .287 .299 .308 .319 .331 .341 .352 3 .363 .373 .385 .396 .407 .417 .428 .440 .451 .461 98 Verification of Somogyi's Blood Gluoose Procedure - Sample of blood taken from colony rat. - Titration of water blanks:' 12 samples, avge. «J 8.55 ± 0.05 oo. sodium thiosulphate. 0.05 oc. «= 0.018 mg% glucose. Blood cc. sodium Sample # thiosulphate mg% glucose 1. 7.6 2. 7.5 3. 7.5 4. 7.5. 5. 7.5 avge. - 7.5 ss 111 mg$ 6. 7.55 7. 7.5 8. 7.5 9. x 15.7 10. x 15.7 -11. X 15.7 avge. B 15.7 ta 616.5 mg# 12. x 15.7 • -x 1.0 mg. dextrose added. (1.0 mg. to 0.2 oc} blood = 500 mg% ) 500 + 111 a 611 ng% % recovery = 99.02$ 99 (2) Blood Aoetone Bodies - Method of Behre and Benedict, (4). Procedure: Transfer a measured amount of oxalated blood to a flask having a capacity at least fifteen times that of the volume taken. For eaoh volume of blood taken (e.g. 2 cc) add slowly 7 x 2 c c of water and mix. Add 2 oc. of 10$ sodium tungstate solution and mix. Finall y add slowly and with shaking 2 cc. of 2/3 N sulphuric acid (35 gm. of con-centrated sulphuric and make up to one l i t r e ) . Stopper the flask and shake. Only a few bubbles should form i f a l l the proteins have been precipitated. Allow to stand for ten minutes. The oolour should change from a red to a dark brown. Pour the mixture on to a dry folded f i l t e r paper. Cover the funnel with a watch glass. Collect the f i l t r a t e in a olean dry container. From ten to one hundred o c are transfered to a two hundred and f i f t y oc. d i s t i l l i n g flask. Three or four drops of 1:1 sulphuric acid (500 oo. cone, sulphuric aoid into 500 oo. water) are added. The volume is made up to f i f t y to seventy five oc. with water and d i s t i l l off approximately one third of this volume. Five cc.of d i s t i l l a t e are transfered to a test tube and exactly five cc. of a 32$ sodium hydroxide solution and ten drops of s a l i c y l i c aldehyde are added. A water blank is prepared by adding five eo. of the NaOH and ten drops of the aldehyde to five co.of water* Shake the tubes and immerse in a boiling water bath for three to five minutes. Remove the tubes, cool, f i l t e r and read i n the spectro-photometer at five hundred and twenty mu. 100 Prepare a standard calibration curve from the following dilutions of stock 1 mg. per oc.acetone solution: G Concentration 520 0.01 mg 83 .02 66 .03 55 .04 44 .05 35 .06 28.5 .07 24 .08 18.5 .09 15 1.0 12.5 The amount of acetone i n mg. per 100 oc. blood (as-total acetone bodies) mg acetone i n Vol. of 100 =s 5 co. d i s t i l l a t e . X d i s t i l l a t e X (standard curve) 5 y o l # of blood Verification of Behre and Benedict's Blood Acetone Procedure 5 oo. blood samples taken from oolony rats. Sample $ Blood Ketones mg$. 1 . 0 . 0 2 . 0 . 5 3 . 0 . 0 4 . 0 . 0 avge. B 0 . 2 6 5. 0 , 8 6 . x 2 1 , 0 7 . x 1 9 , 4 8 . x 1 6 , 6 avge. a 19 ,2 9 . x 2 0 . 6 1 0 . x 1 8 . 5 x 1 mg. acetone added to 5 cc. sample of blood — 20 mg$ % recovery ss 9 6 . 0 $ 102 (3) Urine Glucose — Sohaffer - Hartman Method (85). Reagents: 1. Fehling's Solutions: I. 34.64 gm. copper sulphate dissolved i n water to make five hundred cc. II. Dissolve one hundred and seventy three gm. of Rochelle salt and f i f t y gm. sodium hydroxide in water to make five hundred cc. 2. Iodate - Iodide solution: Dissolve 5.4 gm. KIO and 60 gm.'KI in water to which a 3 small amount of a l k a l i has been added; and dilute to one l i t r e . Procedure: Five oc, of urine^ forty five cc.of water and twenty five cc, of Fehling's #1 and 25 cc. of Fehling's #11 solution are placed into a two hundred and f i f t y cc. flask. Cover with a beaker and heat. Bring to a boi l i n four minutes and boi l for two minutes. Cool for three or four minutes under running water. Add f i f t y oc. of the iodate - iodide solution plus f i f t e e n to seventeen co.5 K sulphuric acid (add quickly). Shake gently to dissolve the cupric oxide. Add twenty oc.of a saturated potassium oxalate solution. Titrate with 0.1 N sodium thiosulphate. Add starch as the indicator - just before the green colour disappears. Prepare a water blank as above using f i f t y cc. of water. (Blank Titration) - (Unknown) a cc.Iodine required 103 for the oxidation of the cuprous salt. Multiply this "by the copper faotor of the sulphate (1 cc. of 0.1 N thiosulphate ss 6.36 mg. copper). Find the amount of sugar equivalent to the copper from the Munsen and Walker Tables. (Methods of Analysis, Association of O f f i o i a l Agricultural Chemists Page 830)• 104 (4) Urine Acetone Bodies — Method of Behro and Benedict (4) . Five cc.of urine and ten oc. of water are placed in a two hundred and f i f t y oc. d i s t i l l i n g flask. Add three drops of 1:1 sulphuric acid. Add a few glass beads and connect immediately to a two hundred mm water-cooled condenser f i t t e d with a delivery tube drawn out to a fine t i p ( a l l glass apparatus or cork stoppers). Use a fifteen cc. graduated oentrifuge tube as a receiver for the d i s t i l l a t e , so arranged that the enlarged end of the delivery tube rests on the t rim of the centrifuge tube, aoting as a cover for i t ; and the fine tip of the delivery tube just reaohes to the bottom of the receiver. Place a minimal amount of water i n the reoeiver to oover the t i p outlet, heat slowly, d i s t i l l over one third of the original volume. Remove the receiver; rinse off the tip of the delivery tube with water; measure the total volume in the receiver or dilute with water to a definite volume and mix by inversion. For colour development transfer 0.1 cc. s a l i c y l i c aldehyde (Eastman's test grade) to a test tube graduated at five and ten oc. Add two 6c. of d i s t i l l a t e followed by 1.5 oc. of saturated potassium hydroxide. Mix the contents of the tube by several ohurning motions with a footed glass rod, leave the rod i n the tube, and aliow to stand for twenty minutes at room temperature. Final l y add either (a) water to the ten cc.mark or (b) seventy five peroent ethyl-alcohol to the five oo. mark. Rinse and remove the rod. Mix by tapping and inversion. For photometrio measurement determine the density in a photometer at five hundred and twenty mu within f i f t e e n 105 minutes after dilution by (a) or thirty minutes after dilution by (b) Set the photometer to zero density with a blank prepared by treating two oo of water by the same prooedure as used for unknown. Calculation: (total acetone bodies) mg. aoetone mg. aoetone in d i s t i l l a t e 100 per 100 c c ss 2 oo. of X volume X urine - d i s t i l l a t e # 2 urine # by reference to a calibration curve prepared previously from standard acetone solutions. volume To prepare a calibration curve for Procedure (A), two c c portions of standard acetone solution (1 mg. per cc.) containing from 0.0 to 0.16 mg. acetone give a satisfactory curve relating density and concentration at five hundred and twenty mu and 1 cm solution depth. For Prooedure (B) the corresponding range is 0.0 to 0.03 mg. acetone. Prooedure A: (wider range than above) . 6 Concentration 520 0.1 82. 0.2 69.5 0.3 58 0.4 48 0.5 40 0.6 33 0.7 27.5 0.8 23 0.9 20 1.0 16 106 (5) A Rapid Microdetermination of Glycogen i n Tissue Slices Van Wagtendonk method (105). Reagents: . . 1. 35 % KOE 2. 95 % ethyl alcohol 3. cone. HCL 4* Phenolphthalein: 1% solution in 50$ ethanol 5. Lugol's solution. 1 gm. iodine dissolved i n a solution containing 2*0 gm. EI. i n 20 oc. water. Keep i n a well-stoppered dark bottle. 6. Stock glyoogen solution. 25 mg. pure glycogen (Eastman Kodak Co., White Label) dissolved i n 25 cc. of a 35$ KOH solution. Store at room temperature. i Method: A tissue slioe weighing from f i f t y to seventy-five mg. is dropped into two cc. of thirty^five percent KOH contained in a six inoh pyrex test tube equipped with an a i r condenser. Place tube i n boiling water bath and reflux the contents for two hours. F i l t e r -the digest through starch free f i l t e r paper (Whatman #41) into a calibrated colorimeter tube i n which a crystal of potassium iodide is placed. Wash the residue on the f i l t e r paper with one co. of d i s t i l l e d water. The f i l t r a t e i s made up to ten cc. with ninety-five percent ethyl alcohol; one drop of indicator is added and the contents of the tube are mixed by lateral shaking. Neutralize with concentrated hydroohloric acid. Cool to room temperature. One drop of HCL is added i n excess to coagulate the glycogen. Centrifuge the tubes for five minutes at three thousand R.P.M. in an angle centrifuge. Decant the supernatant liquid and dissolve the precipitated glycogen in one to two co.of warm d i s t i l l e d water. Dilute to five oc.and exactly 0.05 co.of Lugols solution is added from a pipette. Mix the contents of the tube 107 well and read the colour i n the spectrophotometer at five hundred and forty mu. The blank reading is given by a solution of five cc* d i s t i l l e d water and 0*05 oo-of Lugol's. Preparation of the Calibration Curve. From the stock glycogen solution prepare standards of the following concentration in the potassium hydroxide solution: G Concentration 540 0.5 mg. 75 1.0 56 1.5 41.5 2.0 31 2.5 24 3.0 17.5 Calculation: Glyoogen in 100 ^ mg. glycogen from Liver ss sample wt. standard curve (wet) 108 Verification of Van Wagtendorik's Glycogen Procedure  - A l l samples taken from same lobe of l i v e r from a colony rat* Sample # 1. 2. 3. 4. x 5. x Sample Wt. 71.7 66.9 62.5 61.1 65.4 $ Glycogen 2.96 2,94 2.95 4,49 4.53 avge, 2.95 X 1 mg. glycogen added $ recovery 97.83$ total of 4.59$ 109 (6) Determination of Hepatic Vitamin A. Procedure: Grind the l i v e r with sufficient anhydrous sodium sulphate to remove water. Extract with chloroform. F i l t e r off the residue and note the volume of the chloroform extract. One oo. of this extract is anal«yzed for i t s vitamin A oontent by the Carr-Prioe reaction at six hundred and twenty mu using a spectrophotometer. Calibration Curve: Prepare a f i f t y I.TJ. per co. solution of vitamin A aoetate (TJ.S.P. Referenoe Standard) i n chloroform and set up the standard curve from the follovring dilutions: Dilution Concentration G 620 O.lcc 5.0 I.TJ. 86 0.2 10 75 0.3 15 64 0.4 20 55 0.5 25 46.5 0.6 30 40 0.7 35 34.5 0.8 40 30 0.9 45 26 1.0 50 22.5 Calculation: For a one gram sample of l i v e r , /total" \ I.TJ. of Vitamin A =[Vitamin A i n the) X I c c« o f J V loc. of extract/ \extract / Verification of Liver Vitamin A Procedure - colony rat Sample # Vitamin A* I.U./ gm. 1. 35,-5 avge. 36.15 2. 36,6 3. x 65,9 4. x 70,4 avge. = 68.2 5. x 68.2 x 45.4 I.U. vitamin A acetate added % reoovery = 95.1$ I l l (7) Determination of Vitamin A and Carotene i n Blood Plasma Method of Kimble (51). Reagents: 1. 95$ ethyl alcohol 2. Light petroleum ether 3. Chloroform 4. Antimony tri-chloride reagent: 25$ antimony tri-chloride i n chloroform Prooedure: 3.5 to 5.0 oo. of plasma from oxalated blood ( l drop of thirty peroent potassium oxalate to ten co. of blood) are pipetted into a narrow necked - glass stoppered twenty five oc.oentrifuge tube. Add an equal volume of ninety five percent ethyl alcohol and twelve cc. of petroleum ether. Stopper and mix contents by end over end inversion for ten minutes. Centrifuge for a short period of time. Ten oc of the supernatant ether extraot are pipetted into a c o l o r i -meter tube. The yellow oolour is read in a spectrophotometer at four hundred and forty mu with a petroleum ether blank. Evaporate the ether off in a current of warm dry a i r or COg. For petroleum ether boiling at 45° C the ten oo. oan be taken to dryness in about ten minutes, the tubes in a water bath at 40-43°C. The outsides of the tubes are rinsed and dried. The residue is dissolved i n 1 oc. of chloroform. Add one drop of aoetio anhydride. Determine the density in a spectrophotometer at six hundred and twenty mu using an antimony tri-ohloride reagent blank. Preparation of Standard Curves: 1. Carotene: & Concentration 620 0.025 mg# 86 0.05 73 • 0.10 54 0.15 49.5 0.20 29 0.25 21 multiply the carotene read from this standard ourve by 2.4 ( i f 5.0 oe. of blood plasma were used) and this — mg$ carotene i n the blood. 2. Vitamin A: The following dilutions of a 46.5 I.U. per cc. solution of vitamin A acetate in ohloroform were used to prepare the calibration curve: Dilution Concentration 6 620 O.lcc 4.65 I.U. . 85 .2 9.3 74 .3 13.95 64 .4 18.6 54 .5 23.25 47 .6 27.9 40 .7 . 32.55 34 .8 37.2 29.5 .9 41.85 25 1.0 46.5 22 Vitamin A I.U./1O0 oe. m 100 oc. of plasma" 10 I.U. Vitamin A from standard curve Verification of Kimble's Prooedure, for Blood Carotene and Vitamin A* - sample of venous blood taken from a dairy cow. (a) Carotene: Sample ffi Carotene mg$ 1 . 0 . 3 1 5 avge. ss 0 . 3 2 0 2 . . 3 2 5 5 . x . 5 2 0 avge. — 0 . 5 3 0 4 . x . 5 4 0 x 0 . 2 5 mg$ beta carotene added % recovery = 9 2 . 9 $ (b) Vitamin A: Sample # Vitamin A. I . U . / L O O C Q . 1 . 8 6 . 5 avge. ss 8 6 . 1 5 2 . 8 5 , 8 3 . x 1 2 6 . 6 ~ 4 . x 1 2 5 . 5 avge. = 1 2 5 . 9 6 5. x , • 1 2 5 . 8 x 4 5 . 0 I.TT./lOOoc added % reoovery = 9 6 . 0 4 APPENDIX 17 VITAMIN A INJECTION BY THE INTRAMUSCULAR ROUTE Large doses of vitamin A o i l were used in the treatment of a case of ketosis that developed in the University herd during nineteen hundred and forty-eight. It was f e l t at the time that a higher dose or a more prolonged course of treatment might have given more effective results.. The question was raised as to the efficiency of vitamin A absorption by the oral.route due to the time of passage through the gastro-intestinal tract and the possible deleterious effects of the ruminant f l o r a . It was deoided to investigate b r i e f l y the possi-b i l i t y of using the intramuscular route of injection as a means of delaying absorption arid maintaining the blood vitamin A le v e l . A method of approach to this problem was suggested by the pioneer work of Romansky (72) and others (100) with p e n i o i l l i n . "Two Ayrshire b u l l calves were available for this experiment. One calf was given a one oo. intramusoular injection of a high potency f i s h l i v e r o i l containing three hundred and twenty-three thousand I.U. vitamin A per gram. The other oalf was injeoted with the same amount of vitamin A mixed with an equal volume of aluminium mono stearate provided by Dr. Menotti of B r i s t o l Laboratories Inc./ Syracuse. New York. This compound is a two percent aluminium mono stearate in peanut o i l mixture, and is used as the repository for Procaine P e n i o i l l i n 6. In both animals, the injection was made into the 'biceps femoris' and 'reotus femoris' musoles of the right thigh. No swelling or exudate was i n evidence after injection. 115 Age Weight 23 days 120 lbs. 14 w 83 tt Blood samples were taken from the jugular vein just prior to injeotion and at one, four, seven, eleven, twenty four, thirty one, forty eight and seventy two hours after injeotion. The plasma from these samples was then analyzed for oarotene and vitamin A. Blood Plasma Conoentration Time in Carotene mg$ Vitamin A I.U. /lOOoo. hours Calf #1 Calf #2 Calf #1 Calf #2 0 0.057 55.8 39.94 1 .039 53.7 32.64 4 .036 141.0 82,94 7 .012 162,0 138,96 11 .011 139.5 40.4 24 .018 111.3 128.8 31 .04 88.5 107.2 48 .009 56.9 94.7 72 - - 36.4 It can beeseen from the appended graph that the aluminium mono stearate carrier does delay absorption of the vitamin A by a period of approximately twenty four hours. These, results are oomparable to those observed from the work on p e n i c i l l i n (100). Further investigations w i l l have to be carried out with vitamin Aj not only repeating the above experiment with a larger number and with different species of animals, but also varying the Age and Weight of Animals Calf No. 1 323,000 I.U. vitamin A Calf No. 2 323,000 I.U. vitamin A plus aluminium mono stearate 116 dosage and ratio of vitamin A to the mono stearate vehicle. Extensive analytical work is also required to determine exactly what happens to the injected vitamin A that is not recovered in the blood stream. Sufficient time was available to repeat the above experiment using a mature Angora rabbit obtained from the colony breeding unit. Sixty five thousand I.U. of vitamin A (same o i l as above) was i n -jected intramuscularly into this twelve pound doe. There was no inflammation or disturbance of any kind at the site of injection. A blood sample was taken from the ear vein just prior to injection and at six, twenty two, thirty, forty eight, seventy two, ninety six and one hundred and forty seven hours after. At this time, the animal was sacrificed to permit a l i v e r vitamin A determination. i Blood Vitamin A Content — Rabbit Time in hours Blood Vitamin A I.U./lOOoo 0 60,* 6 139.6 22 278.8 30 265.0 48 240.5 72 ' 223.2 96 92.7 147 64.3 The maximum plasma vitamin A concentration for the rabbit was higher than that observed^for the two calves. Also, i t would appear from the graph that there was a greater delay i n absorption. The vitamin A content of the l i v e r was found to be 157.5 I.U./gm. at the end of the experiment. This is within the 117 normal range and does not account for the exoess vitamin A that was administered. The fate of this surplus i s s t i l l * in doubt. It is suggested that the hepatic vitamin A might be determined when the blood level reaches i t s optimum concentration by analyzing a sample of l i v e r removed by partial hepatectomy. Lbs, of Colostrum per Milking after Parturition Name of Cow i * 2 3 4 5 6 7 8 9 io 11 12 Iona 9.3 11.4 15.0 10.2 15.2 13.3 15.0 13.0 17.0 11.9 15.4 13.0 Jacqueline 7.5 14.4 14.0 20.0 15.5 22.6 16.2 22.6 17.6 21.6 17.8 23.8 Janice 17.0 21.0 17.3 23.3 18.0 22.0 19.0 19.0 22.0 22.0 17.3 24.0 Lucerne 10.8 13.2 21.0 19.0 16.0 16.5 20.5 16.9 19.5 16.8 20.5 16.0 Lucy 24.5 16.3 15.0 19.9 16.5 21.7 17.0 23.8 17.4 25.2 18.8 25.7 Margaret 3.5 2.5 7.6 6.7 9.3 6.4 9.1 7.1 9.7 8.5 9.9 9.5 Midge . 23.0 18.8 19.0 21.8 19.3 26.0 17.5 23.3 18.0 26.8 19.8 27.8 Moira 19.0 14.5 13.0 20.2 19.3 20.3 19.0 26.7 21.2 25.5 20.5 27.5 Myra 24.2 8.8 16.4 13.9 21.4 16.7 21.1 17.2 23.3 17.0 24.0 18^ 5 Nora 19.3 12.4 17.7 16.0 21.4 17.2 23.7 18.5 23.4 18.5 25.6 18^ 0 Olive 12.0 7.5 10.2 14.9 11.5 14.5 10.7 17.0 14.5 17.8 17.0 18.0 O l i v i a 10.0 10.3 14.0 10.9 14.5 13.0 16.2 11.6 16.7 13.4 18.4 14.5 Ophelia 10.0 9.0 10.4 • 9.4 12.4 10.5 13.8 11.7 13.8 11.7 13.8 11.5 Orohid 9.0 10.2 15.2 12.1 13.2 15.2 13.0 16.2 14.1 17.4 14.7 17.0 I ca t-9 *1 3 3 00 119 APPENDIX VI i i The L.D. 50 Dose Level in Acute Acetone Poisoning via the Intraperitoneal Injection Route (Calculated by the Method of Reed and Muench (68) Mature white Wistar rats of weights ranging between two hundred and two hundred and f i f t y grams (four to five months old) were obtained from the colony breeding unit. These rats were sub-divided into groups and given intraperitoneal injections of C P . Acetone (82.04 % by analysis) at the following dose levels: Group Dose' levels of acetone A 0.3 oc B ' 0 . 5 C 0.7 D 0.9 E 1.1 -L.D. 50 for-Mature White Wistar Rats (5 mos.old) Dose of Alive at Dead at acetone gm/kg. 24 hours 24 hours Lived Died % Mortality 0.984 3 0 11 0 0 1.640 4 2 8 2 25 2.256 3 3 4 6 56 2.952 1 5 1 10 91 3.608 0 4 0 14 100 L.D. 50 = 2.15 gm A g - Body Weight 120 i APPENDIX VII (a) Experiment 2 i Twenty rats were selected at weaning and divided into four groups on the basis of weight and sex. Two of these groups were fed a synthetic ration which was vitamin A free. This ration was mixed in the Animal Nutrition Laboratory and was of the following com-position: 16$ vitamin free casein 4$ Salt mixture U.S.P. XII 8$ Irradiated yeast 65$ Starch 5$ Peanut o i l The two control groups received a vitamin A supplement at the rate of four I.U. per rat per day. In addition, a l l groups received a thiamine supplement (forty ;ug per rat per day) because i t was f e l t that the irradiation of the yeast might have destroyed the thiamine present. Table of Average Body Weight in grams No.& Group Sex Diet Age i n Weeks  4 5 6 7 8 9 10 11 12 13 A- 106 105 85 86 111 100 92 86 82 -78 A- 88 84 73 91 91 79 74 70 67 67 A* 87 89 75 91 105 111 112 131 172 182 A+ 80 84 73 83 94 86 91 100 115 164 The rats were sacrificed at the end of the ninth week (thirteen weeks of age). Two rats were placed i n a metabolism cage, and fed a 3X 5M X 5P XI 5M XII 5P 121 diet consisting solely of pork fat for twenty four hours. At the end of this period the urine was collected and a sample analyzed for total ketone body oontent whioh was found to be 9,0 mg $. This supplementary experiment was oonducted i n an attempt to find some means of aggravating the ketosis that was shown to develop s l i g h t l y i n vitamin A deficient rats (Experiment 1). • The possibility of such an approach to developing an acute ketosis would have to be explored more thoroughly with further investigations along this l i n e . APPENDIX VII (b) (b) Experiment 3 Composition of Diet (61): 70$ Wheat 15$ Sucrose 11$ Vitamin free casein 2$ Lard 1.4$ Calcium Carbonate 0.6$ Sodium'Chloride i Vitamin A supplement — 4 I.TJ. per rat per day (TJSP Vitamin A Referenoe Standard) , Eight groups of weaner rats were selected on the basis of sex and weight and fed the above diet. Pour similar groups received the same diet plus the vitamin A supplement. 122 (b) Experiment 3 (Cont'd) § 4 o u CJ h O P. w -p bZ •H O IS *» o m <o bo <a u CO C M O i CD in tO CO CM M rH CD OS •8 O 09 ss co o o os CM CO CM to CM CO in CM CM CM o t o CM I o CM t o OS CM Oi CM os p CO t-rH CM CM co CO CO CO CM CM CO CM CO O OS co CO rH 8 CM CM i H rH CM OS to CO tH P o CM |H 8 ; * to CO CM t o IO CD i H rH CM CM IO rH CM CO O CM CO OS OS CO CO to OS t o CO OS CO rH CM CO to CO <> rH C rH •H rH CM CM rH CO rH t o t o CM CO t o rH rH os t o tH CM b -rH rH rH O h O O <J H CO o CM 8 rH t o CM CM 8 CO t o rH t o CO CO CM t o OS t o OS rH t o rH t-O CM t o OS t o tH t o CM rH 3 rH o CM rH CO H O t o rH GO rH t o CM rH t> CM tH IO rH rH s H CM rH rH t> tH IO rH rH 8 rH CO rH rH OS s IO o rH 8 rH t o s co '8 rH s tH t o t o rH CO s CO CO t o CO CO a * t o rH rH rH CO OS CM tH rH CO OS t o t- t o CO os CO CM g t o t o 8 OS t o s I + + < < < I < I + + < < < H * a fe H H & M W H 123 Representative growth curves and graphs of feed consumption are illustrated for four of the groups. 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