M_______:_:_:__:___::__:_:_:_:__ mmm ' . a; flux u 0“ 5‘ L‘ ’ “-3 .‘ y, 0-159 This is to certify that the thesis entitled AN INVESTIGATION OI‘ VII‘ALfiIN bLZ SYNTHESIS IN A RUMINANI‘ presented by CLARA LODGE. RM DON has been accepted towards fulfillment of the requirements for M. Sc. degree in Biochemistry ' Major professor DateW f ._ ' "fr—- , - "____ _-' I o AN II‘TVESTIGATION OF VITAT’IN B SYI‘ITHESIS IN A RUTZI‘FAITT 12 By Clara Dodge Refson A T.ESIS Submitted to the School of Graduate Studies of Vichigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of TESTER CF SCIEKCE Department of Chemistry 1950 : itm‘... _ ._, ~. . ‘a'. . \~.L to... 2.. f“: (2‘ 1': A), AC KNOT-J'LEDG‘ CENT I wish to express my sincere appreciation to Dr. Keith B. ficCall and to Dr. Carl A. Hoppert whose constant encouragement and advice have been invaluable. I am sincerely grateful to Dr. Lester F. welterink who donated both material and technical advice for the tracer portion of this project. Thanks are also due to Dr. Joseph Neites, Dr. Carl F. Huffman, and Vr. Elbert 3. Churchill who kindly shared unavailable material. ********** ##ssssst ssssss **** ** 1.: 244557 TABLE OF CONTENTS IIJTPACDTICTIOII...0....0..00......0.0.0.0000...OOOCOOOOIOOOOOOOOO The Liver Factor......................................... The "Animal Protein Factor".............................. The Isolation............................................ Microbiological Assay.................................... Possible Yeohanism of Action............................. IISTOTRYOOOOOOOO0..OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO rips" *W‘T‘ A.“ 3.1..I‘LL‘iLALQOOQOOOOOO...COOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOC The Microbiological Assay................................ Sample Preparation....................................... Preparation of Extract................................... The Use of Radioactive Cobalt............................ Chromatography........................................... DISCUSSI 'I‘TOOOOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOOOOOOOIOOOOOOOOO CO}ICIJ[TSIOIIOO0.0.0....0.0.0.0...0.0000000000000000.0.0.0.000... BIBLIC)GI{AP£IYOOO0.00.00.00.000......OOOOOOOOOOOOOOOOOOOIOOOI... Page (DCDCJ'II\'3l--I 14 17 17 23 24 25 30 32 35 41 Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES Page 34 35 56 57 38 4O INTRODUCTION The Liver'Factor Vitamin 312 as we know it at the present time is yet a fraction of the form whose development is inevitable. Although its life as an independent vitamin has been a short one, the volume of research which has been published in the last three years is indeed impressive. The study actually began a century ago when pernicious anemia was recognized clinically.1 The specific importance of diet as an influenc- 2 announced in ing factor was not fully realized until Minot and Vurphy 1926 that at least partial alleviation of symptoms could be effected by treatment with whole liver. This discovery was paralled by the work of Koessler et al.3 and was preceded by that of Whipple et a1.4 and Elders5 who had also considered pernicious anemia to be a deficiency disease. Subsequent verification was again supplied by ”inot and T‘urphy6 in 1927 and by Castle and Bowie7 in 1929. As must follow, various groups of investigators now took up the task of liver fractionation with hopes of characterizing the unknown factor or factorS'which must be present. Virtually nothing was excluded in the never ending search as each of the major classifications of com- pounds was eliminated from each new fraction. By 1945, highly concen- trated preparations had been developed, and an extensive review of the painstaking fractionation studies was published by Subbarow, Hastings, and Elkin.8 The progress thus far attained appears all the more impres- sive when the only method at that time available for testing these -1... fractions is considered, - that of direct clinical therapy on perni- cious anemia patients. It was postulated early that the factor sought was not of singular 9'10’11 advanced the classic theory that an nature when Castle et a1. extrinsic factor, supplied by dietary constituents, must be combined with an intrinsic factor, supplied by certain glands of the stomach, to be nutritionally effective. Although this theory has been modified somewhat by later research, the initial postulation was a sound one and is still supported at the present time. Present evidence seems to indi- cate that the intrinsic factor of gastric juice functions primarily as an aid to absorption and hence more complete utilization of the orally administered vitamin.12’13’14 The "Animal Protein Factor" Gradually, investigators in other branches of nutrition took up the task, as the growth effects of an “animal protein factor" were realized. Parkhurst,15 as early as 1927, discovered that a protein of animal origin was necessary for normal hatchability of eggs. Two years later, a similar factor was reported in codliver'meal by McFarlane, Fulmer, and Jukes.16 Further studies were forthcoming, and in 1935 Van der Hoorn et a1.17 identified a strong growth promoting factor in casein. Nestler et al.18 postulated a "new factor" and indicated pork liver meal as a good source. Some essential nutrient(s), absent in vegetable protein, was obviously present and associated with animal protein. During 1940 - 1944, the beneficial effects of sardine meal and fish solubles toward growth and hatchability'were determined.19’20921,22 23 and co-workers noted that non-protein-nitrogen levels McGinnis in the blood of chicks fed a diet deficient in the "animal-protein factor" were increased when soy bean protein or vitamin-free casein was added to the basal diet. Also within this period came the discovery that cow manure was effective for growth under the same conditions in 24 Rubin, Bird, and co-worker325’26’27’ which sardine meal was effective. 28’29’30 further distinguished this factor from the known.vitamins and succeeded in concentrating it from cow manure by combined drying, pre- cipitative, and extractive methods. The new'factor gradually began to find its way into animal as well as poultry science. As early as 1932, flapson31 had recognized that rats maintained on a diet in which yeast and wheat germ were the only sources of B vitamins developed a deficiency which was corrected by feed- 32 ing liver. Cary and Hartman in 1946, using a purified diet containing alcohol-extracted casein and yeast, demonstrated an unidentified factor required for normal growth of the rat. An anti-pernicious anemia liver extract satisfied this requirement when added to the ration in milligram 33 34 quantities. Jaffe, Jaffe and Elvehjem, and Sporn, Reugamer, and Elvehjem;5 reported similar observations. Bowland, Esminger, and Cunha36 also emphasized the association of a growth factor with animal proteins. The rat growth factor was further shown to be present in such foods as milk, beef and pork liver, and egg yolk.37 The potency of the factor in egg yolk was observed to be effected by the diet of the hen. The yolk was apparently a storage depot, serving to transmit this nutrient to the chick. The research of poultry science nutritionists in the same year paralled that of Cary and.Hartman when it was demonstrated that the chick growth factor was likewise transmitted by way of the egg to the chick, under some conditions in quantity sufficient to support maximum growth to four weeks of age.38’39’40’41’42’43 The Beltsvilleworkers44 reported the chick growth factor to be present in hen feces. Evidences of bacterial origin were therefore suggested. This was partially verified by McGinnis etal.4‘5’46 and further in 1948 by workers at the Lederle Laboratories47 who succeeded in isolating a microorganism.which, by aerobic fermentation, formed the animal protein factor. This material, when refined and concentrated, proved therapeutically active against pernicious anemia in man by clini- cal test. Although chemical assay was impossible without further characteri- zation of this factor, Shorb,48 also of Beltsville, was attacking the situation by attempting to find a microorganism for which the rat growth factor was an essential nutrient. Lactobacillus lactis Borne: apparently required two constituents, a "TJ factor" found in tomato juice and casein, and an "LLD factor", active for rat growth, and found in highest concentrations in liver extracts. A linear relationship was observed between the potency of this latter factor and the anti-pernicious anemia -4- factor in liver concentrates.49 Further investigations identified the latter factor in other well known animal protein factor source mater- ials.50 At this point, the various lines of investigation began to converge. The Isolation In the spring of 1948, after several years of work, Rickes and co- workers51 announced the isolation of a pure, crystalline material from liver extracts, designated as vitamin B12. First aided by clinical test, then later by microbiological assay52’53 (using Lactobacillus lggti§_Dorner), the anti-anemia factor had been isolated. Clinical tests proved it to be effective not only in producing positive hemato- logical responses in pernicious anemia, but in the alleviation of neuro- logical symptoms as well.54’55’56’57’58’59’60’61’62 It was also effective in the treatment of tropical sprue and nutritional macrocytic C 1 6 anemia. Netzel 5 reported a statistical analysis of growth failure in school children as associated with vitamin B12 deficiency and the re- sponses to oral treatment. As would be expected by the use of Lacto- bacillus lactis Dorner in fractionation studies, the crystalline material exerted LLD growth factor activity.64 At the same time and independently of other investigators, Smith,65’66 at the Glaxo Laboratories in England announced the isolation of the anti-anemia factor. His fractionation studies were followed by means of clinical testing as well as the characteristic pink coloration. By means of partition chromatography, he had concentrated one gram of the product from four tons of ox liver. Reported attempts to determine the relationship between vitamin B12 and the animal protein factor were forthcoming. It had already been indicated that vitamin B12 and the LLD factor were probably identical. The crystalline material or vitamin B concentrate were observed to be 12 active in the promotion of both good growth and hatchability in chicks.67’68:69s70,71.72,73 Rat growth was stimulated as much as thirteen fold over control animals depending upon the protein concentration in the diet.74 Tissue distribution studies showed that vitamin B12 concentration increased in most organs and tissues when small amounts were added to the diet.75 The effectiveness of vitamin B12 for pig growth was also reported by several investigators and its efficiency observed to compare favor- 76,77,78,79,80 ably with that of the animal protein factor. The equiv- alence of vitamin B12 and anti-pernicious anemia liver extract could be demonstrated.81:82 Stokstad et al.,83 however, upon the feeding of more acutely defi- cient basal rations,found that maximum growth requirements of the chick were not completely satisfied by the addition of vitamin B12 to the B4 diet. Some indication of this had been noted by Hill a year earlier. The existence of other factors not replaceable by vitamin 31 which may 2 be present in the animal protein factor have been.suggested: (1) an additional liver rector85.86 and (2) a substance in Streptomyces -5- 87 cultures. Zucker and Zucker,88 however, did not obtain responses from the above factors in addition to those obtained from vitamin B12. Hartman, Dryden, and Cary89 have attempted to clarify some of the con- flicting evidence previously reported. They concluded that exceedingly high levels of riboflavin may play an important role in bringing about intestinal synthesis of vitamin B12 in the rat. Nichol et al.90 have found considerable variation in chick growth response to the same dosage of anti-pernicious anemia preparation in different experiments. The "multiple nature"91 of the animal protein factor is still under investi- gation, being at present inadequately defined. 92’93'94 who had also observed the syndrome pro- Zucker and Zucker, duced in rats by high protein diets, suggested the name "Zoopherin" for the missing dietary factor. The name was soon abandoned, however, since sources of microbia195’96 as well as animal origin were involved. Some relationship appears to exist between the vitamin and growth 97’98 Rats in a thyrotoxic condi- requirements in hyperthyroid animals. tion apparently require one or more factors not needed by normal animals. Purified liver extracts contained this factor, and crystalline vitamin B satisfactorily replaced the extracts. A similar condition in chicks 12 99 was counteracted by vitamin 312 and conversely, a vitamin 312 deficiency could be demonstrated in the chick much more quickly if a thyroxine- active substance was administered.100 The isolation of pure, crystalline vitamin B12 was followed imme- diately by further characterization of chemical properties and structure, 101,102,103,104,105,106 as would indeed be expected. Cobalt and phos- phorus were found to be present to the extent of one atom of each per molecule. A similarity in structure to riboflavin was evidenced when a 1,2-diamino - 4,5 - dimethyl benzene moiety was identified. Evidence of the multiple nature of this vitamin was shown when two other compounds, vitamin B 107 and vitamin B 108'109 12a 12b lized. These were later shown to be identical and to differ from.vita- 110,111 'were crystal- min 812 only by the absence of a cyano group. The isolation of crystalline 812 from.liver called forth a search for other more readily available sources. 'Workers at the Lederle and ”erck Laboratories were able to isolate the vitamin from cultures of 112 Streptomyces aureofaciens and from Streptomyces griseus113 respec- tively. The further testing of the bacterial production of the animal 114’115 not only resulted in protein factor and of B12 by other workers the development of numerous fermentation procedures, but served to fur- ther demonstrate the complex nature of the animal protein factor. Microbiological Assay Numerous procedures for the assay of vitamin B12 and the animal protein factor were developed during 1949. Vitamin B12 was found to elicit growth responses by other microorganisms in addition to Lacto- 116 bacillus lactis Dorner. These include Euglena gracilis, Lacto- bacillus leichmannii 313, ATCC 7aso,117:118'119 and Lactobacillus leich- mannii, ATCC 4797.120’121 A microbiological assay employing either -8- Lactobacillus lactis or Lactobacillus leichmannii based upon anti-biotic 122,123 124 type procedures was later published by Foster et al. A method involving paper chromatography in combination with microbiological pro- cedures was also published.125'1d6 127,128 Short-term rat assays also were developed. Non-specificity, especially in the case of the earlier broth culture assay procedures, sensitivity to oxygen tension, variation in oxidation- reduction potentials, and culture dissociation introduced troublesome 129,130,131,132,133,134,135 The fact that the growth factor 136,137,138 complications. can apparently be stored in the animal body as well as be transferred through the mother's milk139 further complicated animal assay procedures and deficiency experiments. Possible Mechanism of Action Two years prior to the discovery of vitamin B12, another factor, vitamin M or folic acid was studied and synthesized.l4‘0’141 With its discovery came the justified, although short-lived, hope that the vital factor of liver had at last been isolated. It was still apparent, however, that erythrocyte counts did increase somewhat with folic acid treatment. Also during 1948, the desoxyriboside of thymine was isolated from.liver. Shive et al.142 indicated a functional relationship between this factor and folic acid in that it was highly active in conteracting bacterial growth inhibition by methyl folic acid. Stokesl45 had previously demon- strated that thymine itself, if substituted in large quantities, could completely replace folic acid in the nutrition of lactic acid bacteria. It was, in fact, capable of replacing folic acid in the treatment of 144 macrocytic anemia if administered in the proper amount. Snell et 145 al. reported thymidine to be an essential growth factor for certain lactic acid organisms. Vitamin B12 was again brought into this series of events when 146 ”wright, Skeggs, and Huff interpreted their investigations of Lacto- bacilli to show replacement of vitamin 31 147 2 as a growth factor by thymidine. Shive, Raull, and Eaken also reported an interrelation- ship. Sinilar information was indicated by Kitay et al.148 in regard to other desoxyribosides as well as thymidine. The growth characteristics of certain other organisms (Streptococcus faecalis R,149 Leuconostoc citrovorum,150 and Lactobacillus bifiduslsl) has not indicated a specific thymidine - B12 relationship. Hoff- Jorgensen152 reported irregularities in regard to three other strains of lactic acid bacteria. Similarly, thymine will not replace thymidine for Lactobacillus leichmannii 313, Lactobacillus leichmannii 327, or Lactobacillus citrovorum.153 The possibility still exists, however, of a series of reactions in the animal body in which the above mentioned components are involved. In consideration of the apparent interrelation- ship of folic acid, thymine, thymidine (or nucleosides in general), and vitamin B12, Skeggs et al.154 have proffered a possible mechanisn1by which these substances may function: "The biochemical defect in perni- cious anemia may well be inability to synthesize certain nucleosides, -10... particularly thymidine, from parent purines and pyrimidines. The cura- tive effects observed in this disease with folic acid may arise from increased thymine snythesis, which, by mass action effects, yields more thymidine. The effectiveness of large amounts of thymine in pernicious anemia similarly may be explained." The possibility is further suggested that vitamin 312 may enter the above scheme as'a co-enzyme in the syn- thesis of thymidine from thymine.155’156 Shive et a1.157 presented further evidence of the interrelationship of purines and vitamin 812’ but did not preclude the possibility of a reverse situation in which purines or derivatives are involved in the biOSy-nthesis of the vitamin. Also in accord with the foregoing theory, Roberts et a1.158 employ- ing radioactive tracer techniques have demonstrated an increase in phosphorousuptake in the desoxyribonucleic acid fraction of Lactobacillus leichmannii cultures when supplemented with varying amounts of vitamin B although bacterial growth remained constant. 12 Vinute amounts of anti-pernicious anemia concentrate produce excel- lent growth of rats given folic acid and succinylsulfathiazole. Con- sideration of the quantity of concentrate needed indicates that vitamin B12 is the factor which is responsible for this increased growth as well as the fact that vitamin B12 may be necessary for the proper functioning of folic acid in the animal body. Jones at 211.159 have interpreted their data in part as followm: "The mechanism by which succinylsulfathiazole increased the need for the growth factor is unknown at the present time. -11.. The most logical explanation is that intestinal bacteria form small amounts of this factor, and that the bacteria are inhibited by the sulfa drug from doing so". A lipotropic effect in rats with dietary-induced liver injury was reported as another of the properties of liver extract.160 Endeavoring to reduce the number of possible factors to a minimum, repeat experiments concentrate continued to demonstrate a significant 161 using vitamin 812 lipotropic effect comparable to earlier results. Vitamin 812 is known to exert a sparing effect on the requirement for choline and meth- ionine in the prevention of hemorrhagic kidney. The addition of vitamin 812 to subprotective levels of choline produced significant weight in- creases although this was not the case when adequate choline levels were 162 maintained. Other investigations of a choline, vitamin 812 relation- ship served to substantiate this fact.163’164 Similarly, an increased requirenent of methyl group donors (choline and betaine) for chicks made partially deficient in the animal protein factor was observed.165 Cunha et al.166 reported that pigs on a corn, peanut meal ration showed growth response to either animal protein factor or methionine supplements, the latter eliciting the lesser response. Stokstad and Jukes167 found vitamin B12 to be involved in the methylation of homocystine to methionine. Homocystine promoted growth only if vitamin 812 was supplied. Vitamin Bl was found to exert a protective effect on hepatic injury 2 168 produced by carbon tetrachloride. The development of characteristic -12- a histologic changes such as fatty metamorphosis and depletion of ribose nucleic acid were prevented. Shaefer et al.169 have expressed the be- lief that vitamin B12 exerts its maximum effect in the presence of folacin. This was indicated a year earlier by Vichol et al.170 in re- gard to hemoglobin regeneration. Since it was known earlier that desoxyribonucleic acid synthesis was important in the multiplication of a type of bacteriophage (Tér), 171 Roberts and Sands investigated the possibility that vitamin B1 might 2 likewise be involved. The vitamin was found to be one of the rate limiting factors of virus synthesis in the presence of resting cells. The research already accomplished, although rather broad in scope, leaves many questions unanswered as does it call forth many new ones for future investigation. There can be little doubt that the initial problem has been solved, that of finding the active anti-pernicious anemia factor present in liver. 172 containing The recent biosynthesis of radioactive vitamin 812 isotopic cobalt, C060, has made available another technique for new and different types of research. -13.. HISTORY The importance of cobalt as a trace element in the nutrition of ruminants was late in receiving recognition. There is no definite evi- dence that cobalt is a dietary essential in the non-ruminant although its action in producing polyoythemia would indicate that some mechanism 173 for the utilization of this element does exist. Attempts to produce cobalt deficiency in laboratory animals (non-ruminants) have proven.un- 1,174,175,176 177 cobalt deficiency successfu and according to Harston, in horses does not occur. The syndrome characteristic of insufficient cobalt in the diet of ruminants, however, has been reported in many parts of the world under such titles as Denmark disease, coastal disease, enzootic marasmus, bush sickness, salt sickness, Nakuritis, and pining disease. A rather complete summary of the disease characteristics as well as areas of occurrence has been published by the National Research Council.178 The characteristic digestive system of the ruminant at once sug- gests the possibility that cobalt is involved in bacterial metabolism. 179 in 1944 suggested that cobalt may function in VcCance and'Widdowson this connection. An investigation of possible changes in the rumen flora of cobalt deficient sheep in 1949 showed that a marked alteration in the types and numbers of bacteria did occur.180 Although the retention of orally administered cobalt was shown to be negligible, it was not nutri- 1 tionally effective when injected.181’182’183’“84 The announcement that the vitamin B12 molecule contained an atom of cobalt at once revealed another possibility. The vitamin is readily -14.. metabolized by many microorganisms. It would therefore seem quite pos- sible that the influence of cObalt is exerted through microbial synthe- sis of vitamin B1 which is, in turn, utilized further to support 2 growth of rumen microflora. It was in support of this contention that the research reported in this paper was begun in the fall of 1949. If it could be definitely established that vitamin 812 was present in the ruminant stomach, fur- ther steps would be taken to demonstrate synthesis of the vitamin, provided that the metabolic pool was large enough at any one time to in- sure working quantities. In the event that results proved positive, it was hoped that this might be the starting point for future work, perhaps the isolation of the organisms involved. In Nevember of 1949, Burroughs et a1.185 and Hale et al.186 reported their investigations of this same problem derived from two entirely dif- ferent approaches to the situation. Burroughs attempted to duplicate in so far as possible the conditions existent in the rumen. The cellu- lose digestion of poor quality roughages was studied. Two of the most potent supplements effecting maximum cellulose digestion were: (1) a complex mineral mixture and (2) an autoclaved water extract of cow manure. Cow manure, as mentioned before, contained a factor which promoted growth and hatchability of chicks. Hale conducted direct chick growth assays of the rumen contents from sheep fed cobalt-deficient and cobalt-supple- mented diets. A significant growth difference was observed. Growth retardation could be completely overcome by adding vitamin B12 to the ration of the chicks fed the rumen contents from the cobalt deficient sheep. Additions of inorganic cobalt were ineffective. 157 were successful in culturing In.7hy'of 1950, Henlin and Ruger rumen isolants which synthesized LLD-active substances and demonstrated an increased synthesis with the addition of cobalt salts. -16.. E‘J‘IP-Ell" IEN‘TAL The study of cobalt metabolism in the ruminant at the time of initiation of this project was quite incomplete. Further, opinions con- cerning the involvement of vitamin 812 in this phase of animal nutrition ‘were many and varied. Several approaches to the problem.had been pub- lished, some of them more or less indirect. This project was planned as a direct investigation of the situation which would at least serve to elucidate and solve some of the minor issues involved. Direct isolation of the vitamin from ruman contents was beyond the scope of this project. Attention was turned to the preparation of suitable extracts whose potency could be determined by microbiological assay. The successful demonstration of a significant concentration of the factor active for Lactobacillus leichmannii would at least serve to establish the presence of an LLD active substance. By the use of radioactive tracer techniques involving 0060, the in— corporation of cobalt into the molecule of the active substance could perhaps be demonstrated. The Hicrobiological Assay It was apparent that the choice of a suitable assay procedure was one of the most important steps to be taken. The cup assay of Foster, Lally, and'Wbodrufle8 was finally selected since it appeared to be the most workable method. The rate of growth of either of the test organisms (Lactobacillus lactis or Lactobacillus leichmannii) has been reported to be affected -17.. by oxidation-reduction potential, by degree of aeration, and by the accumulation of peroxides in liquid media. The cup assay procedure permits better control of these factors and hence a greater degree of accuracy. Although animal assays have been developed, the diet has been known to affect markedly the weight gains produced by varying concen- trations of the vitamin. Until such a time as the importance of in- testinal synthesis has been determined and the multiple nature of the animal protein factor has been more clearly defined, the microbiological assay would appear to involve less question than other procedures. To this end, the situation was further complicated by the fact that the exact nature of impurities present in the extracts to be tested was un- known. The number and types of substances present in the rumen as a result of bacterial fermentation were without doubt many and varied. The assay organism used throughout the entire project was Lacto- bacillus leichmannii A.T.C.C. ,{é4797. The media of Foster et al.189 was used for the first few trial assay procedures, in so far as it could be duplicated. It was desirable to prepare fresh batches of media for each usage in 150 to 200 milliliter quantities but the proportions of the ingredients were such that this could be accomplished only by weighing aliquots from a mixture of the dry ingredients. Other methods were laborious and uniformity could be ascertained only by preparing the dry mixture in rather large quantities. For simplification, the choice of 'media was changed to the prepared vitamin Blz Agar Experimental of Difco -18... and Company, a medium essentially the same as that of Capps, Hobbs, and Fox.190 The stock cultures were prepared by stab inoculation of Bacto-Vicro Assay Culture Agar. Each new culture was incubated at 35°C for 48 hours and then stored in the refrigerator. Cultures stored in this manner were satisfactory if used within a period of thirty days. Slightly better results were obtained, however, by preparing fresh cultures immediately before each assay. Although good stab growth was observed during the first several trial procedures, the assays themselves were very unsuccessful, apparently due to culture dissociation. The incorporation of .01 - .02% of Tween 80 into the Micro—Assay Culture Agar was found to correct the difficulty after the first transfer. The inoculum for each assay was prepared by subculturing from.the stock culture into a tube containing ten milliliters of Bacto-Hicro Inoculum. Broth. This broth was supplemented with Tween 80 in the same concentration as employed in the preparation of the Micro-Assay Culture Agar. This was observed to produce an exceptionally large growth in- crease over that of the dissociated culture. The tubes were then incu- bated for 24 hours at 35°C. 'Following incubation, the broth was centrifuged at about 2500 r.p.m. for ten minutes. The cells were then resuspended in ten milliliters of sterile physiological saline and centrifuged as before. This was re- peated a second time. The procedure varied slightly at this point in that saline was added until a final volume of ten milliliters was reached. After the second washing, the volume was again made up to a ten milliliter level with saline. One milliliter of this suspension was used to inoculate each one hundred milliliters of melted vitamin B12 Agar, Experimental held at 45 - 50°C. Aseptic conditions were main- tained throughout. One to one and one-half percent of sodium chloride was added to the assay medium before plating. This had a tendency to diminish indistinct growth zones. The use of abnormal salt concentrations was also reported by Foster et al.191 to eliminate the diffuse growth response of the test organism to desoxy ribonucleic acid or its corresponding nucleosides. A sufficient volume of a sterile sodium chloride solution was added to the autoclaved assay medium to produce the desired salt concentration. This was added immediately before the inoculation and at a temperature of 45-500C. Occasionally a flocculent precipitate was observed to form after the media was autoclaved. This condition was improved to some degree by adding the sodium chloride as a sterile solution after auto— claving. Any flocculent material which formed after these precautions had been taken was easily resuspended by swirling the flask containing the mixture and this material apparently did not interfere with assay results. Twenty-five milliliters of the inoculated medium.was pipetted into each of four sterile petri dishes. Precautions were taken to be sure that the surface on which the plates were resting was perfectly level. -20- Since final results are dependent upon an even diffusion downward and outward from the assay cylinders, uniformity in depth of the agar was important. The agar was allowed to harden for ten minutes. Four to six stainless steel assay cylinders were then distributed evenly on the surface of the agar by dropping from a uniform height equidistant from the outer edge of the petri dish. The weight of the cylinder was sufficient to produce a tight seal between the lower edge of the cylinder and the agar surface. Heating of the cylinder before placing in position as advocated for antibiotic assay procedures was not found to be necessary, and a more uniform settling was obtained without it. The number and choice of standards was more or less arbitrary, de- pending upon the concentration of the unknown solution and the accuracy desired. Slightly better results were obtained when both the standards and the test sample were included on the same plate. This was not abso- lutely necessary, however, if care was taken to keep the procedure as uniform as possible. Standards usually ranged from 0.0 to 2.0 gamma of vitamin 812 per milliliter. Dilution of the test solution, when neces- sary, and use of the lower standards (.0. to 0.5 gamma per milliliter) gave greater accuracy. Standards were run with each new assay since growth sometimes varied from day to day. The cylinderS'were then filled'with the test solutions from sterile dropping pipettes. Air bubbles sometimes formed in.the bottom of the assay cups if care was not taken to prevent it. Bubbles inhibited proper diffusion of the liquid through the agar, and the volume of liquid which -21- could be placed in the cylinders was also diminished. Splattering from the dropper occurred occasionally. The resultant growth areas appear- ing after incubation of the plates were, however, easily distinguished from the true growth zones which were to be measured. After the first cylinder was once filled, the others were filled as rapidly as possible. Results were dependent upon the growth time of the organism.as well as the diffusion of the test solution. The more quickly the assay cylinders were filled, the less the difference in growing time of the organism for each test solution. To facilitate transfer from the working surface to the incubator, and to avoid any possible jarring, the plates were set on small racks prior to the positioning of the cylinders upon the agar. All the plates could thus be moved in a single operation. Glass petri dish covers were replaced by sterile porous covers to prevent subsequent condensation and dripping into the test solutions. After incubation at 35°C. for 24 to 48 hours, the diameters of the growth zones were measured as these values indicate the amount of vitamin B12 present when compared with standard solutions. Neasurements were best made over an ordinary plate counter or ruled scale such as is used for standard anti-biotic assay procedures. Good growth zones were white, distinct areas with smooth, regular edges. (Figure 1) This is not the case if culture dissociation is present. Tests for false positive reactions were made using both desoxy ribo- nucleic acid, ribo nucleic acid, and a mixture of the two plus vitamin 312. Nucleic acid concentrations as high as 4000 gamma per milliliter were used. The results are recorded in Figure 2. 'Despite the fact that measureable growth due to desoxy ribonucleic acid did occur, it was of an entirely different quality than that obtained from any concentration of vitamin 312 and could easily be distinguished from it. Growth was very slight, faint, and not easily measured. There was no significant additive effect as a result of combining the three compounds. Sample Preparation A rumen-fistula goat was selected as the experimental animal for this project. Four weeks were allowed to elapse after the rumenotomy before the first sample was withdrawn. From consideration of the phy- siology of the rumen, it was apparent that a definite time for removal of each sample must be set if results were to be consistent. It has been estimated192 that both the bacterial and protozoan populations in the rumen declined to their lowest numbers during a ten to twelve hour period after feeding. Also at this point, the volume of oxygen present in the rumen would have reached a maximum value due to the cessation df bacterial gas production. Whether or not the presence of aerobic organ- isms was of any importance was of course not known. However, the fact that the rate of disappearance of vitamin B12 due to bacterial metabolism would have reached a minimum value during this period was important. It was also possible that an excess of the vitamin was present at this time. Samples were therefore drawn directly from the rumen, eleven to twelve hours after feeding. The animal was fed twice daily. The diet consisted of alfalfa hay, water ad libitum, and a ground mash containing the following constituents: oats 400 pounds corn - 400 " linseed oil meal - 200 " salt - 23 An attempt was made to limit each feeding to a quantity such that it would be completely consumed by the animal within an hour's time. This, however, was not always possible. Preparation of Extract The fluid portion of each rumen sample was used in the preparation of extracts. In spite of this fact, samples were found to be very diffi- cult to handle. Attempts to filter the sample were unsuccessful. Centri- fugation resulted in the formation of several layers of material, and a clear solution could not be obtained. After numerous attempts, a success- ful extraction procedure was finally developed. Ruben, et a1.193 re- ported that the growth factor in cow manure could be precipitated by virtue of the association of the factor with protein, by adjusting the pH of a water extract to 3.0. Following this same suggestion, the rumen sample was acidified to pH 3.0 with 1N hydrochloric acid. Earlier use of .1N hydrochloric acid led to excessive dilution of the samples. Centrifugation at this point yielded two distinct layers, a brownish-gray precipitate and a cloudy supernatant. The supernatant was discarded, and the precipitate extracted seven to eight times with n-butanol. Warming to about 50°C hastened the separation of the butanol layer. The extraction was completed as quickly as possible, and the pH of the butanol extract adjusted again to 7.0 with .lN sodium hydroxide. The extract was then evaporated to dryness by vacuum distillation below 50°C. The dry residue was extracted with redistilled water, and this solution autoclaved at 121°C for 15 minutes followed by an assay for the vitamin 312 content. Attempts to obtain an accurate concentration value would have bean of little significance since the vitamin B12 concentration in the rumen, without doubt, varied from day to day. A vitamin B1 triturate* (95% 2 minimum purity) was used in the preparation of standards. All standards were prepared by serial dilution so that consistent results would be obtained. Assay results determined the vitamin 312 concentration to be approximately three gamma per milliliter of extract (Figures 3 and 4). Since one liter of rumen sample was used, and the total volume of extract was 55 milliliter, the concentration of vitamin 812 in the liquid portion of the raw rumen material was calculated to be .17 gamma per milliliter. The Use of Radioactive Cobalt Attempts to demonstrate bacterial synthesis of vitamin 312 by the use of tracer cobalt indicated early that some modification of the *‘ ferck & Co., Rahway, NeW'Jersey. -25... extraction procedure was necessary. Cobalt60 (as a water solution of cobalt sulfate) was added to a fresh rumen sample after removal from the rumen, and the material extracted in the usual manner. A much larger quantity of radio-cobalt was added to these samples than had been proposed for the actual tracer study. It was found that a large part of the in- organic radio-cobalt was dissolved in the butanol extract. This was assumed to be due to the partial miscibility of butanol and water. It was not necessary to make an accurate count of the extract to demonstrate the presence of inorganic cobalt. Ierely holding the flask containing the extract up to the G.H. tube of an ordinary laboratory monitor was sufficient. Radio-cobalt was added to a second rumen sample, and the pH again adjusted to 3.0. This material was then added to 500 milliliter centri- fuge bottles, centrifuged, and the supernatant discarded. The remaining precipitate was shell frozen and lyophilized. This operation was com- pleted as rapidly as possible. By the end of the lyophilization period, it was apparent that smaller samples must be used since only partial dry- ing had been accomplished. Nevertheless, the samples were extracted, and a large reduction in count was noted as compared to the previous sample. A third sample was prepared and treated in the same manner as the second sample, with only three exceptions. A 500 milliliter sample, before pH adjustment, was placed in a fixed position near the G.X. tube of the laboratory monitor. Radio-cobalt was added until the scale -26- registered 1000 counts per minute. This sample was to be treated as a blank for future work if necessary. Exactly 100 milliliter of sample was added to each of five centrifuge bottles. The supernatant was dis- carded after centrifugation as before, and the remaining precipitate lyophilized to complete dryness. This was then extracted with dry butanol. Dry butanol was prepared according to the following steps: 1. Fractionally distilled, and the fraction collected which boiled above 114°C. 2. Allowed to stand over potassium carbonate for forty-eight hours, with occasional shaking. 3. Redistilled from drying agent immediately before using. Fraction collected which boiled above 116.700. The extract was evaporated to dryness, extracted with 120 milliliter of water, and autoclaved, as previously described. This sample was then refrigerated until the actual tracer sample could be prepared. Although any attempt to calculate the quantity of cobalt which must be administered as a tracer dose would be rather futile, some sort of estimation was necessary since several factors were involved. There was little danger of introducing artifacts due to over exposure of the animal, since only a matter of days would elapse between the time of administra- tion of the tracer dose and the actual withdrawal of the test samples. 194 According to the data of Comar et a1., only 51.2% of a total cobalt dose remained twenty-four hours after rumen administration. Forty-eight .-4 7- hours after administration, 20.7% remained, and none was absorbed through the rumen walls. The ganma energies of Co60 were sufficiently high as to completely penetrate the animal, leaving only the beta energies to be considered. Since no attempt was made to eliminate co- balt from the diet, this fact alone introduced a sizeable dilution factor, and it was quite obvious that a large dose must be given. Once placed in the rumen, the dose would be further diluted by the mass of material which was present. In addition, the vitamin 312 concentration in the rumen would, at best, be small. A trial dose, calculated to give a maximum exposure of .l milli- roentgen per hour'at a distance of one inch, or approximately 7.1 x 10"9 curies of cobalt was placed directly into the rumen. The material was inserted by means of a hypodermic needle through the rubber stopper which closed the fistula opening. The needle was washed through into the rumen several times with distilled water after the cobalt injection. Twenty-four hours after adding the radio-cobalt, a sample of rumen contents was withdrawn and checked for radioactivity. The count on this raw sample was far too weak to produce an extract with a significant count. The second dose was calculated to give a maximum exposure of approxi- mately 1.6 milliroentgens per hour at one inch, or approximately 1.14 x 10'7 curies. This was about sixteen times as great as the first dose given, or about sixteen times the maximum exposure level at a distance of one inch. After a twenty-four hour period, another rumen sample was taken -28.. and measured (as was the blank) from a fixed position on the laboratony monitor. Fortunately, a 500 milliliter sample counted 1400 counts per minute on the monitor scale. This was very close to the count previously obtained for the sample which was to be treated as a blank. This sample was extracted in the same manner as was the blank, using the same quanti- ties of material for each step. The final extract volume in each case was 120 milliliters. When assayed for vitamin B12 content, the extract was found to con- tain approximately .03 gamma per milliliter of extract, or approximately .07 gamma per milliliter of liquid rumen sample. (Figures 5 and 6). Apparently, a sizeable quantity of vitamin B1 had been lost through the 2 added lyophilization step. It was found that the water extract of lyophilized samples had a tendency to gel after autoclaving. To prevent this, and to obtain a clear liquid sample which would diffuse readily, a larger quantity of water had to be used in the water extract than was previously intended. Even after this precaution, the extract had to be filtered, and the gelatinous material washed thoroughly with water. This step undoubtedly accounted for'the loss of an appreciable amount of the vitamin content. It was evident that any further work beyond that re- ported in this paper would necessitate the use of improved extraction procedures and the further purification of extracts. The two solutions (blank and sample) were plated on aluminum disks, by repeatedly applying one-half milliliter of solution at a time and dnying until a total of twelve milliliters of material had been dried on each. Each disk was then counted, and the data listed as shown in -29.. Figure 7. This data gives definite indication that some radioactive cobalt had been bound either in the form of vitamin 812, or some other organic compound which was extracted by the same treatment. Chromatography Further identification of the Lactobacillus leichmannii growth factor present in rumen digest was attempted. A type of filter paper chromatography similar to that of flinsten and Eigen195 was employed. A filter paper cylinder was used instead of filter paper strips, and Vitamin B12 Agar was used to replace the media of Winsten and Eigen. The solutions (pH 5.0) were spotted at the base of a filter paper cylinder (flatman:#l) and allowed to dry. The cylinder was then placed upright in a museum.jar_and wet n-butanol was slowly added. The top of the jar was then sealed tightly, and the chromatogram developed for approximately sixteen hours. The cylinder was then removed, allowed to dry in air for one hour at 30 - 35°C, and cut into strips. These strips were then laid on agar plates seeded with Lactobacillus leichmannii #4797. The paper strips were allowed to soak for six minutes on the moist agar surface in order to allow some of the test solutions to transfer'from the strip to the agar. After the strip was removed, its imprint could be plainly seen on the agar. The plates were then incubated for twenty- four hours at 35°C. The location of the vitamin 812 on the filter paper strip was identified by the corresponding zone of growth on the agar sur- face. Standard solutions of one gamma per milliliter of vitamin B12 developed perfectly, showing Rf values of .015 (based upon the distance -30.. traveled by the solvent). Growth resulting from the extracts tested was not found. Repeated tests of rumen extracts were completely void of growth, even when quantities as large as .04 milliliter were spotted on the chromatogram by repeatedly adding and drying .005 milliliter portions of the solution. This type of result was completely unexpected since good positive results had been obtained by microbiological assay on the same media. Apparently the vitamin was not released from the filter paper onto the agar when present in this crude extract. Four per cent solutions of ribonucleic acid, desoxyribonucleic acid, and a mixture of the two plus vitamin B 'were chromatogrammed in this 12 same manner and negative results again observed wherever vitamin B12 was absent. -31.. DISCUSSION The use of an agar cup assay method as a means of testing for the presence of vitamin 812 in rumen digest has revealed the presence of the vitamin in significant concentration. This is in accordance with the theory that inorganic cobalt may function in rumen metabolism as a part of the vitamin 812 molecule. Attempts to trace the dietary cobalt by administering radioactive 0060 indicated that the element was involved in a bacterial synthesis. An extraction procedure was developed by means of which vitamin B12 could be extracted from rumen samples and at the same time remove only a Very insignificant quantity of inorganic cobalt. It was evidenced, however, that further purification of these extracts was to be desired. This fact was demonstrated further when chromatography was attempted. The extraction of wet rumen samples with ordinary n-butanol resulted in a clear, liquid solution which could be easily tested by microbio- logical assay. Attempts to chromatogram these extracts were unsuccess- ful, although standard solutions of vitamin B12 gave clear, positive results. The extraction of lyophilized samples with dry n-butanol apparently removed more of the organic material present than did the wet extraction. Gelatinous material was observed to form in these solutions upon standing, but could be removed by filtration. Although this gelatinous material was extracted thoroughly by washing with water, assays proved the vitamin 812 concentration to be much lower under these conditions. \ C CI‘TC LETS I ON A microbiological assay for the determination of vitamin 812 was tested and modified. A procedure for the extraction of vitamin B from rumen samples 12 was developed. A tracer study was performed in an attempt to demonstrate bacterial synthesis of vitamin 812. A chromatographic study of rumen extracts proved unsuccessful and demonstrated the need for further improvement of extraction procedures. -33- H 8&3 Figure 2 Solution Tested Growth Diameters*(cm.) Description 1. Desoxyribonucleic acid (4,000 r/oc) Ribonucleic acid (4,000 n/cc) Vitamin B (.lr/cc) 12 Desoxyribonucleic acid (4,000 n/cc) + Ribonucleic acid (4,000 n/cc) + Vitamin B (.ln/cc) 12 2.4 negative 1.9** Growth very faint Clear, distinct growth Tore distinct growth than 1 * Average of two values ** Average of six values -35- 10 9-9 an m 6-0 5-0 o‘é 'T'. Solution Tested Growth Diameter (cm.) Plate 1 .5 rBlz/bc 1.0 rBlz/cc 5.0 rBlz/bc dumen extract Plate 2 .5 r31 /bc 1.0 rBlz/cc 3.0 rBlZ/cc Rumen extract Plate 3 .5 rBlg/bc 1.0 rBlz/bc 2.0 rB /cc l2 Rumen extract Plate 4.- 2/ .5 r51 cc .0 r31 g/cc 2.0 rB1 g/bc Rumen extract -37- v—v ' W 7 H W" 7’77V*W'7_77 7’ ' 7 'iii—ii" ' 7 STANDARD CROWN! CURVE an 7.0 6.0- 2.0 1.0. O. 0.8.- 0. 0.6.- 50. 0.4-- 0.3-- 0.2.- O. O-.. -.- Solution Tested Growth Diameter (cm.) Plate 1 .005 .l .5 1.0 Plate 2 .005 .l .5 1.0 Plate 5 Plate 5 .005 .1 .5 1.0 Rumen Plate 6 .005 .1 .5 1.0 Human rB Extgact rBlZ/cc rBln/cc “ 2 rnlz/bc r312 cc 5.312%: 5312 /cc rBIZ/CC rBlz/bc r8 2 cc rB /bc rBlz/bc rBlz/bc rB jkc rB cc extgact r3 /bc rBiz/bc rBlz/cc cc NNH 000 ‘31»th Hannah: 0 +401;me Figure 7 Sample Volume Counts/minute above Plated background 1 12.0 cc. 31.7 2 12.0 cc. 31.8 3 12.0 cc. 19.8 (Blank) 4 12.0 cc. 13.4 (Blank) 5 12.0 cc. 20.5 (Blank) Sample Average 31.8 Nos. 1 and 2 Blank Average 17.9 Tos. 3, 4 and 5 Average Count 13.9 Difference Specific activity (based upon vitamin B concentration as indicated by assay values) 6.43 countsysec/gamma. -40- 10. ll. BIBLIOGRAPHY Addison, T., "Anemia, disease of the supa-renal capsules", London ”8d. Caz. 438517 (1549). Vinot, G. R. and‘fi. P. Purphy, "Treatment of Pernicious Anemia by a special diet", J. Am. "ed. Assoc. 87:470 (1926). Koessler, K. K., S. ”auer, and R. Laughlin, "Relation of anemia, primary and secondary, to vitamin A deficiency", J. Am. "ed. Assoc. 87:476 (1920). Whipple, G. H. and F. S. Robscheit-Robbins, "Blood regeneration in severe anemia. I. Standard basal ration bread and experi- mental methods, Am. J. Physiol. 72:395 (1925). Elders, 0., "Tropical Sprue and pernicious anemia, etiology and treatment", Lancet 1:75 (1925). ffinot, G. R. d N. P. “urphy, "A diet rich in liver in the treatment of pernicious anemia", J. Am. Med. Assoc. 89:759 (1927). Castle, W. B. a H. A. Bowie, "Domestic liver extract for use in pernicious anemia; T""Tethod of preparation", J. Am. ”ed. Assoc. 92:1930, (1929). Subbarow, Y., A. B. Hastings, and V. Elkin, "Chemistry of anti- pernicious anemia substances of liver", Vitamins and Hormones 3:257 (1945). 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II. Studies on the nature and source of intrinsic factor." Proc. Cen. Soc. Clin. Research 22:18 (1949). Abstracted Annotated Bibliog. of Vit. 912, werck 3 Co., 17 (1950). Berk, L., W. B. Castle, A. D. thch, R. W. Heinle, R. Anker, & ’3 Epstein, "Observations on the etiologic relationship of achylia gastrica to pernicious anemia. X. Activity of vitamin Bl as food (extrinsic) factor", New Engl. J. Med. 239:911 (1948). Gardner, F. H., J. S. Harris, d‘X. B. Castle, "Erythropoietic activity of extrinsic factor on parenteral administration in pernicious anemia", Am. J. T“Ted. 7:421 (1949). Parkhurst, R. T., Univ. Idaho Agr. Exp. Sta. Bull. 149, (1927). "Borden's Rev. of Nutrition Research" X, Io. 1:5 (1949). VcFarlane, n. D., H. L. Fulmer, & T. H. Jukes, "Studies in embryonic mortality in the chick. I. The effect of diet on the nitrogen, amino nitrogen, tyrosine, tryptophane, cystine, and iron con— tent of the proteins and on the total copper of the hen's egg", Biochem. J. 24:1611 (1950). Van der Hoorn, B., H. . Branion, & W. R. Graham, "Studies in the nutrition of the chick. II. Effect of purification of casein in simplified diet." Poultry Sci. 143295 (1935). Nestler, R. B., T. C. Byerly, N. R. Ellis, & H. W. Titus, "A new factor, not vitamin G, necessary for hatchability", Poultry Sci. 15:67 (1936). Christiansen, J. B., H. J. Deobald, J. G. Halpin, & E. B. Hart, "Further studies on the nature of the effective supplements for soybean oil meal in chick rations", Poultry Sci. 18:481 (1939). Christiansen, J. B., H. J. Deobold, J. G. Halpin, a E. B. Hart, "Practical supplements for soybean oil meal in chick rations", Poultry Sci. 19.19 (1940). Hammond, J. C. and H. R. Bird, "Effects of nutrition on variability in the growth of chickens", Poultry Sci. 21:230 (1942). Hammond, J. C. & H. W. Titus, "The use of soybean meal in the diet of growing chicks", Poultry Sci. 23:49 (1944 . -42.. 23. 24. 25. 26. 27. 28. 29. 30. 31. 53. 34. 55. ”cGinnis, J., P. T. Hsu, and'W. D. 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Its stability and solubility", J. Biol. Chem. 174:611 (1948). Lillie, R. J., A. C. Dentow, & H. R. Bird, "Relationship of vitamin B 2 to the growth factor present in cow manure", J. Biol. Cgen. 176.1477 (1949). Uapson, L. W}, "Evidence of the existence of a dietary principle stimulating general growth and lactation", Biochem. J. 26:970 (1932). Cary, C. A., A. ”. Hartman, L. P. Dryden, and G. D. Likely, "An unidentified factor essential for rat growth", Federation Proc. 5:128 (1946). Jaffe,'W. G., "The growth stimulating effect of a heat-labile factor in liver extract on rats fed a natural diet", J. Biol. Chem. 165.387 (1946). Jaffe,'W. G. & C. A. Elvehjem, "Fractionation of growth stimulating factor in liver", J. Biol. Chem. 169:287 (1947). Sporn, E. W.,'W. R. Ruegamer, and C. A. Elveljem, "Growth and repro- duction in rats on synthetic rations", Proc. Soc. Exp. Biol. red. 65.5 (1947). -43- 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. Bowland, J. P., V. E. Ensminger, & T. J. Cunha, "Need for an uni- dentified factor or factors for growth, reproduction, and lactation of rats fed purified rations", Arch. Biochem. 16.257 (1948). Hartman, A. ". and C. A. Cary, "Occurrence in food of an unidenti- fied factor essential for rat growth", Federation Proc. 5.137 (1946). Bird, H. R., ”. Rubin, D. Whitson, & S. K. Haynes, "The effectiveness of dietary supplements in increasing hatchability of eggs and viability of progeny of hens fed a diet containing a high level of soybean oil meal", Poultry Sci. 25.265 (1946). Bethke, H. 7., P. R. Record, D. C. Kennard, e V. D. Chamberlin, "The relation of nutrition to production and hatchability of chicken eggs. I. Effect of protein supplements and alfalfa meal", Poultry Sci. 25.570 (1946). Bethke, R. T., J. T. Pensack, & D. C. Kennard, "The influence of hen's diet on growth of progeny", Poultry Sci. 26.128 (1947). Nichol, C. A., A. R. Robblee,'fl.'W. Cravens, & C. A. Elvehjem, "Distribution of an unidentified chick growth factor", Poultry Sci. 27.438 (1948). Cravens, W. W}, J. G. Falpin, & W. H. ”cGibbon, "The use of various vitamin supnlements in rations for laying and breeding hens", Poultry Sci. 25:99 (1946). ”cGinnis, J. & J. S. Garner, "The storage of an unidentified growth factor or factors in the egg, and its relation to chick growth and mortality", Poultry Sci. 26:457 (1947). Rubin, W., H. R. Bird, & I. Rothchild, "A growth promoting factor for chicks in the feces of hens", Poultry Sci. 25:526 (1946). 7' VcGinnis, J., J. ‘. Stevens, & K. Groves, "The in vitro synthesis of a chick growth promoting factor in hen.fEces”, Poultry Sci. 26.432 (1947). "cGinnis, J., J. 7. Stevens, & K. Groves, "Studies on an unidenti- fied factor required for chick growth and livability", Poultry Sci. 26.550 (1947). Stokstad, E. L. 3., A. Page, J. Pierce, A. L. Franklin, T. H. Jukes, R.'fi. Heinle, I. Epstein, & A. D. Helsch, "Activity of micro- bial animal protein factor concentrates in pernicious anemia", J. Lab. Clin. fied. 33.660 (1948). -44.. 48. 49. 50. 51. 53. 54. 55. 56. 57. 58. 60. Shorb, U. 3., "Unidentified essential growth factor for Lacto- bacillus lactis found in refined liver extracts and in certain natural materials", J. Bact. 53.669 (1947). Shorb, T. S., "Unidentified growth factors for Lactobacillus lactis in refined liver extracts", J. Biol. Chem. 7169:455 (1947). Shorb, I. 8., "Activity of vitamin B , for the growth of Lactobacil lus lactis", Science 107:397 (1948). *- Rickes, E. L., N. G. Brink, F. R. Koniuszy, T. R. Hood, & K. Foekers, "Crystalline vitamin B12", Science 107:396 (1948). Koditschek, L. K., D. Eenlin, E. B. hoodruff, "Investigations on the nutrition of Lactobacillus lactis Dorner", J. Biol. Chem. 179.1093 (1949). ' Caswell, V. C., L. K. Koditschek, & D. Lendlin, "The microbiologi- cal estimation of L. lactis Dorner activity with vitamin B12 as a standard", J. Biol. Chem. 180.125 (1949). ‘fiest, B., "Activity of vitamin B12 in addisonian pernicious anemia", Science 107.396 (1946). Smith, E. L., "Purification of anti-pernicious anemia factors from liver", Nature 161.636 (1946). Ungley, C. C., "Anti-anemic substances from liver", Lancet 1:771 (1946). Berk, L., D. Den r-E3rown, U. Finland, &”N. B. Castle§"3ffectiveness of vitamin Bl in combined system disease: rapid regression of neurologic manifestations and absence of allergic reactions in a patient sensitive to injectable liver extracts", YeW'Engl. J. fied. 239.326 (1946). Hall, B. E. and D. C. Campbell, "Effect of vitamin 31 on the hema- topoietic and nervous systems in addisonian pernicious anemia", J. Lab. Clin. med. 33.1646 (1946). Spies, T. D., R. ". Suarez, G. G. Lopez, P. ”ilanes, R. E. Stone, R. L. Toca, T. Aramburu, A Sam.Kartus, "Tentative appraisal of vitamin B as a therapeutic agent", J. Am. fled. Assoc. 139.521 (1949). Bethell, F. B., T. C. "eyers, & R. B. Heligh, "Vitamin B1 in perni- cious anemia and puerperal macrocytic anemia", J. Lab. Clin. Med. 33.1477 (1946). -45.. T1 61. Spies, T. D., R. 3. Stone, G. L. Garcia, :. "ilanes, R. T. Lopez, & T. Aranburu, "Thymidine, folic acid, and vitamin Bl in nutritional macrocytic anemia, tropical sprue, and pernicious anemia", Lancet 2.519 (1946). 62. Bethell, P., "Treatment of macrocytic anemias with vitamin 812"’ J. Am. Dietet. Assoc. 25.69 (1950). 63. wetzel, N. C., J. C. Fargo, I. H. Smith, 4 J. Helikson, "Growth failure in school children as associated with vitamin B19 deficiency - response to oral therapy", Science 110:651 (1949). 64. Ibid., 50. 65. Ibid., 55. 66. Smith, 3. L. A L. F. J. Parker, "Vitamin B Chem. Eng. Hews, 26.2216 (1946). 12 isolated in England", 67. Ott, IL 3., E. L. Rickes, & L. R. Wood, "Crystalline vitamin Bl activity for chick growth", J. Biol. Chem., 174.1047 (19483. 66. Lillie, R. J., C. A. Denton, & H. R. 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H .otll: 1 w no i . - L ;"s. ‘ A :hn‘ I“ "\F .- .C'f; .‘. . 1‘ . .33: In! 4), n":— t \ . . .5?! . r a w. v (.11. .rb\..r.r-l . 3.11:1 I r N 1 fr .1} C _‘l v . .4 ... ‘.\.-.o . n v I n I .- .\1|vJu.ovI ..v-. .. . . 54.94... , . 99.5,... 01.... ..a.. 7.3.2:? . . Q .grumhgun~.urnrn. mQ-lrr. , . \IHHIIHIW||||HIHHHlllllilllillHUIlllflllllllllllll 31293 02446 7080