’1‘ — —‘ v-u— '— ‘— H- IV?“ THE SENDING 0F VITAMlN 812 BY MiLK PROTEtNS {basis far the Degree of M. S. WCHiGAN STATE UNIVERSETY Young Suk Kim 1964 THESIS LIBRARY Michigan State University THE BINDING 0F VITAMIN B12 BY MILK PROTEINS BY Young Suk Kim A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1964 ACKNOWLEDGMENTS The author wishes to express her sincere appreciation to Dr. Bernard S. Schweigert, Professor and Chairman of the Department of Food Science, for his encouragement and motivating interest during the course of this study. The author also expresses her gratitude to Dr. J. Robert Brunner, Professor of Food Science, for his advice, efforts and counsel through this research. Grateful acknowledgment is due to the Department of Food Science, Michigan State University and the National Institute of Health through Research grant EF-284 for the funds and facilities which made this research possible. ii TABLE OF CONTENTS INERODUCTION . . . . . . . . . . . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . . . . History . . . . . . . . . . . . . . . . . . . . . . . . . . Source . . . . . . . . . . . . . . . . . . . . . . . . . Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . Stabilization of Vitamin B12 . . . . . . . . . . . . . . . Vitamin 312 binding factors . . . . . . . . . . . . . . . . Milk . . . . . . . . . . . . . . . . . . . . . . . . . . EXPERIMENTAL PROCEDURES . . . . . . . . . . . . . . . . . . . The microbiological assay . . . . . . . . . . . . . . . . . Assay organism . . . . . . . . . . . . . . . . . . . . . Media . . . . . . . . . . . . . . . . . . . . . . . . . Inoculum . . . . . . . . . . . . . . . . . . . . . . . . Method of determination of Vitamin B12 . . . . . . . . . Standard solution . . . . . . . . . . . . . . . . . . Sample solution . . . . . . . . . . . . . . . . . . . Procedure . . . . . . . . . . . . . . . . . . . . . Determination of total Vitamin . . Determination of free Vitamin 312 . . . . . Preparation of samples and analysis . . . . . . . . . . . . Page Experiment 1. Determination of total Vitamin 312 in pasteurized milk . . . . . . . . . . . . . . . . . . Experiment 2. Determination of total and free Vitamin B pasteurized milk . . . . . . . . . . . . Determination of free Vitamin B12 . . . . . . . . . . Method 1. Seitz filtration . . . . . . . . . . . Method 2. Ultrafiltration . . . . . . . . . . . . Determination of total Vitamin B12 . . . . . . . . . Method 3. Extraction with cyanide . . . . . . . . Method 4. Extraction with cyanide . . . . . . . . Method 5. Digestion with papain . . . . . . . . . Determination of bound Vitamin B12 . . . . . . . . . Experiment 3. Determination of total and free Vitamin B12 in raw skimmilk, raw whole milk, pasteurized skimmilk, and pasteurized whole milk . . . . . . . . . Preparation of raw skimmilk . . . . . . . . . . . Preparation of pasteurized whole and skimmilk . . . . . . . . . . in casein Experiment 4. Determination of total and free Vitamin B and whey samples prepared from pasteurize by ultracentrifugation, rennin coagulation, and electric precipitation methods . . . . . Preparation of casein and whey samples . . . . . . . a. Ultracentrifugal method . . . . . . . . . . . b. Rennin coagulation method . . . . . . . . . . c. Isoelectric precipflation method . . . . . . . Determination of total and free Vitamin 312 in casein Total Vitamin B12 . . . . . . . . . . . . . . . . Free Vitamin 312 . . . . . . . . . . . . . . . . . iii dzskimmilk iso- oooxbww 11 15 23 23 23 23 23 23 24 24 24 24 25 25 26 26 26 26 27 27 27 27 27 28 28 28 28 29 29 29 29 29 30 3O 30 Page Determination of total and free Vitamin 312 in Whey samples . . 3O Experiment-50ooooooooooooooo000000.000 Preparation of crude lactalbumin and lactoglobulin from raw m01e milk 0 O O O O O I O O O O O O O O O O O O O O I O O O O 31 Determination of total and free Vitamin B12 in crude lactal- . bumin and lactoglobulin . . . . . . . . . . . . . . . . . . . 32 Moisture determination . . . . . . . . . . . . . . . . . . . 32 Nitrogen and protein determination . . . . . . . . . . . . . 32 Electrophoretic studies of crude lactalbumin and lactoglobulin 32 Free boundary electrophoresis . . . . . . . . . . . . . . . 32 Zone electrophoreSiS o o o o o o o o o o o o o o o o o o o o 33 Experiment 6. O O I O O O O O O O O O O O O O O O O O O O O O O O 33 Preparation of skimmilk, whey, casein, crude lactalbumin and lactoglobulin from fresh whole raw milk . . . . . . . . . . . 33 Analytical methods 0 I O O O O O O O O O O O O O O O O O O O O 34 Experiment 7. O O O O O O O O O O O O O O O O O 0 0 O O O O O O O 34 Separation of a-lactalbumin and immuneglobulin fractions from crude IaCtogIObUIin C O O O O O O C O O O O O O O O O O O O O M Analyticalmethods......................34 EXPERIMNIAI‘ ESULTS O O O O O O O O O O O I O O O O O O O O O O O O O O 38 Experiment 1. I O O O O O O I O O O O C O O O O O O O O O O O O O O 38 Experiment 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Experiment 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Experiment 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Experiment 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 EXperiment 6. . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Experiment 7. . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 DISCUS SION . O O O C O C O O O O O O O C O O O O O O C O C O O O O O O O 60 CONCLUS IONS O O O O O O O C O O C O O C O O O O O O O O O O O O O O O O 6 5 mFERENwS CITED 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O 66 iV LIST OF TABLES Table Page 1. Vitamin B12 derivatives . . . . . . . . . . . . . . . . . . . . . . . 20 2. Naturally-occurring Vitamin B12 analogues . . . . . . . . . . . . . . 21 3. Vitamin 312 in the milk of different Species . . . . . . . . . . . . . 22 4. Total and free Vitamin B12 in pasteurized whole milk . . . . . . . . . 43 5. Total and free Vitamin B content of raw skimmilk, raw whole milk, pasteurized skimmilk and pasteurized whole milk . . . . . . . . . . . 44 6. Mug of total and free Vitamin B in skimmilk, casein and whey con- tained in 100 m1 skimmilk and percentage of total Vitamin 312 accounted for in fraCtiOnS I I I I I I I I I I I I I I I I I I I I I I I I I I I 45 7. Total Vitamin B calculated in pug per mg of casein, skimmilk protein and Whey Protei I I I I I I I I I I I I I I I I I I I I I I I I I I I 46 8. Electrophoretic mobilities of crude lactalbumin and lactoglobulin fractions of cows' milk in veronal buffer, pH 8.6, /-'/2 = 0.1, at 1° CI I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 47 9. Total and free Vitamin B 2 in pug per mg of protein for whey, crude lactalbmnin and lactog10%ulin I I I I I I I I I I I I I I I I I I I I 48 10. Percent protein in various milk fractions on dry basis . . . . . . . . 49 ll. Electrophoretic mobilities of crude lactalbumin and lactoglobulin of cows' milk in veronal buffer, pH 8.6, 7—7/2 = 0.1 at 1° C. . . . . . . 50 12. Total and free Vitamin 312 in protein fractions obtained from 100 m1 skimmilk and percentage accounted for in various fractions . . . . . . 51 13. Total and free Vitamin B12 in pug per mg of protein from skimmilk and protein fractions I I I I I I I I I I I I I I I I I I I I I I I I I I 52 14. Total Vitamin B12 in protein fractions obtained from 100 m1 skimmilk and percentage accounted for in various fractions . . . . . . . . . . S3 15. Percent protein in various milk fractions on dry basis . . . . . . . . S4 16. Total Vitamin 312 in pug per mg of protein from skimmilk and protein fraCtionS I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 55 17. Total Vitamin B12 in pug per mg of protein from skimmilk and protein fraCti-ons I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 56 LIST OF FIGURES Figure Page 1. Vitamin B12 (cyanocobalamin) . . . . . . . . . . . . . . . . . . . . 19 2. A schematic diagram showing the sample preparation for determination Of total and free Vitamin B12 I I I I I I I I I I I I I I I I I I I 35 3. A schematic diagram showing the fractionation of cows' milk with amoni‘m SUI fate I I I I I I I I I I I I I I I I I I I I I I I I I I 36 4. A schematic diagram showing the fractionation of cows' milk with amonim Su1 fate I I I I I I I I I I I I I I I I I I I I I I I I I I 37 5. Electrophoretic patterns of crude lactalbumin and lactoglobulin in Verona]. bUffer, pH 8.6, 7-7 l2 = 0.]- o o o o o o o o o o o o o o o o 57 6. Electrophoretic patterns of crude lactalbumin and lactoglobulin in veronal buffer, pH 8.6, /—’/Z = 0.1 . . . . . . . . . . . . . . . . 58 7. Electrophoretic patterns of crude lactoglobulin, immune globulin and (x-lactalbumin in veronal buffer, pH 8.6, /_'/2 = 0.1 . . . . . . . . 59 vi INTRODUCTION Vitamin B12 is required for the prevention of pernicious anemia in man. The relief of the symptoms of pernicious anemia, a disorder involv- ing impaired intestinal absorption of vitamin B12, by oral administration of vitamin 312 plus a source of intrinsic factor is evidence of its require— ment in the human diet. It is a known dietary requirement for the growth of variety of animals. For many years prior to the isolation of vitamin 312: liver extracts were used for the treatment of pernicious anemia. Later came the import- ant observations that the growth-factor potency of liver extracts for the microorganism Lgctobacillus lactis Dorner, correlated well with the cura- tive-potency when used in the treatment of pernicious anemia. Using this knowledge, rapid progress was made in the late 1940's in the isolation and identification of vitamin B12. Vitamin B is a cobalt-containing substance of high molecular weight 12 and complex polycyclic structure with a basic porphyrin configuration re- sembling that of hemoglobin and chlorophyll. Its metabolic function remains obscure, although various physiologi- cal roles at the cellular level, such as the synthesis of deoxyribosides, the metabolism of single carbon fragments, and the incorporation of amino acids into proteins, have been tentatively ascribed to vitamin 312. The occurrence of the vitamin is unique among vitamins in that it is absent in higher plants. Vitamin B12 is found exclusively in foods of animal origin (meats, fish, eggs, milk, etc.) and fermented products (cheese, silage, etc.) and most of the vitamin exists in the form of a protein complex. It originates either through synthesis by organisms within the animal's own digestive tract or from ingestion of foods derived -2- from animals. Extensive synthesis occurs within the forestomach of ruminants and these animals, therefore, do not show a dietary require— ment of the vitamin. Man and certain animals are dependent upon dietary B12 because the vitamin is either not formed in sufficient quantities, or not released in adequate amounts from the cells of synthesizing organisms in the region of the intestinal tract from which absorption occurs. The vitamin is carried by the blood-stream to all parts of the muscles and organs, and mainly is stored in the liver. Most of the vitamin is present as the bound form in the milk of different Species of animals. Available evidence indicates that the vitamin is bound to proteins and unavailable to test organisms without Special treatment of the sample. Free vitamin B12 is measured after Seitz filtration of samples. The value for bound vitamin B12 is obtained by subtracting the amount of free vitamin B12 from the amount of total vitamin B12, which is measured after releasing and converting bound vitamin B to the more stable cyanocobalamin by autoclaving samples at 12 pH 4.6 with a trace of cyanide added. The purpose of this study was to isolate and characterize vitamin B12 rich protein fractions from cows' milk and carry out preliminary studies on the nature of the binding and equilibrium in model systems with isolated milk proteins and added vitamin B12. As the first step of this study it was proposed to isolate and characterize vitamin 312 rich protein fractions from cows' milk. In cows' milk, approximately 95% of the total vitamin 312 was shown to be present as the bound form and the remainder of the vitamin was present as free vitamin B12. REVIEW OF LITERATURE History The study of Vitamin B12 actually began a century ago when pernicious anemia was recognized clinically (1). Pernicious anemia is a macrocytic anemia; the red blood cells are abnormally large, but relatively few in number, 1-3 million per cu. mm. instead of the normal 4.5-6 million. The bone marrow is megaloblastic; the blood-forming cells have become enlarged while still immature. In 1920 Whipple (90) found that feeding liver accelerated the regenera- tion of red cells in dogs made anemic by bleeding. Minot and Murphy (61) in 1926 demonstrated that patients with pernicious anemia could be main- tained in normal health by ingestion of liver. Subsequently it was dis- covered that the injection of liver extracts gave more reliable results with less inconvenience to the patients than oral feeding of liver prep- arations. Since that time, the use of liver extracts became routine prac- tice prior to isolation and identification of Vitamin B12 in the treatment of not only of Addisonian anemia, but also of pernicious anemia due to tapeworm, pernicious anemia of pregnancy, nutritional megaloblastic anemia, megaloblastic anemia of infancy and childhood and megaloblastic anemia accompanying steatorrhea. Cohn (l7) started extraction of the "factor" from liver in 1928. Isolation of the pure crystalline factor was announced by two independent teams, Merck and Co., Inc. in America and Glaxo Laboratory in.England, within a Space of a few weeks (66, 78) in 1948 and the factor was called Vitamin B12. Pernicious anemia is also characterized by achlorhydria, a failure -3- -4- to secrete hydrochloric acid and partial atrophy of the mucous membrane of the stomach. Castle (15), in 1928, thought that the atrophied stomach glands might be failing to secrete some essential substance present in normal gastric juice, which he later called "intrinsic factor". This factor was presumed to act upon something present in certain foods, such as liver, called "extrinsic factor" (8), which has now been identified as Vitamin B12, and the substance was given successfully by mouth along with gastric juice to treat patients with pernicious anemia (90). It has been postulated that intrinsic factor acts with extrinsic factor to yield the "Liver factor", the product of this reaction. Stokstad et a1. (81) suggested that an animal protein growth factor required by chicks and other animals and the antipernicious anemia factor (vitamin B12) are identical. Vitamins of the B group are growth factors for most bacteria, as well as for higher animals. Some bacteria synthesize all they need, but others rely upon an external supply of one or more vitamins. Lactobacillus laoggg Dorner would grow on a synthetic medium supplemented with tomato juice and liver extract (73). It was then found that the liver extract could be replaced by concentrates active against pernicious anemia and the "L.L.D. factor" required by the Lactobacillus has been shown to be identical with the antipernicious anemia factor (73). Source Vitamin B12 is unique among the vitamins in that it is synthesized by certain microorganisms but not by higher plants. Whenever it is found in nature, its origin can be traced back to bacteria or other microorganisms, growing in soil or water or in the rumen or intestine of some animals. The -5- activities of microorganisms in their natural habitats lead to the presence of Vitamin 312 at very low concentrations in soil, in pond-water and even in the sea. Some bacteria and actinomycetes however, produce much more than they need for their own growth, or at least they can be induced to do so under appropriate conditions of growth. Three out of twelve differ- ent proactinomycetes of the genus Nocardia produced the largest amounts of Vitamin B12; 3, erythropolis (12.2 mg/lOOml of medium) (24). Organisms selected from these groups are employed for the commercial production of Vitamin B12; among those recommended for this purpose are Streptomyces griseus, S, olivaceous, Bacillus megatherium and propionic bacterium and yields up to 3 pg of Vitamin 312 per ml of fermentation liquor have been claimed (53). The presence of Vitamin B12 in higher plants is still controversial. The absence of Vitamin B12 in these plants is confirmed in part by the fact that deficiency symptoms have been induced in several Species of animals and have also appeared in some human subjects on exclusively vege- table diets. The most recent claim, still unconfirmed, is that it is present in turnip greens (30). Vitamin 312 is found in animal tissues and animal food products, milk and milk products and eggs. It originates either from organisms within the animal's own digestive tract or from ingestion of foods of animal origin. Strictly noncarnivorous non-ruminant animals must pre- sumably acquire nearly all of their Vitamin 312 by absorption of what is synthesized within the gut. In man and some higher animals synthesis and absorption does not appear to occur to any significant degree, so that they are dependent upon dietary Vitamin 312° Liver is the main storage depot in most animal Species, although with some (e.g. the rat) kidney levels tend to be higher. ChemiStry Crystalline Vitamin B12 is a red, complex coordination compound con- taining a trivalent cobalt and a cyano group. Its empirical formula is C63H88014N12PC° and it has a molecular weight of 1355 which is considerably larger than that of any other known vitamin. Soon after Vitamin B12 was crystallized, considerable knowledge of its structure was obtained by degrad- ation studies, but the molecular structure was definitely established after the brilliant work of Hodgkin and her colleagues (40) using x-ray crystallo- graphy as shown in figure 1 (20). The cyanide group is attached to the cobalt atom which in turn is linked coordinately to a nitrogen of the 5,6-dimethylbenzimidazole group. Vitamin 312 contains a nucleotide component. However, the base present is 5,6-dimethylbenzimidazole rather than the various purine and pyrimidine bases of the nucleic acids and the sugar ribose has an a-glycosidic linkage unlike the B-linkage in the nucleic acids. The D-l-amino-Z-propanol moiety of the molecule is esterified to the nucleotide and joined in amide linkage to the porphyrin-like nucleus. The term "cobalamin" has been suggested for the entire B12 molecule except for the cyanide group, Cyanocobalamin (the cyano derivative) is used synonymously with Vitamin B12. Naturally occurring Vitamin B12 derivatives in which the CN group is substituted by other groups are summarized in table 1 (2,77). All the Vitamin 312 derivatives listed are converted into Vitamin B12 itself by cyanide treatment. Therefore all the compounds listed show biological activity towards microorganisms, animals and human patients with pernicious anemia. Table 2 (77) shows naturally occurring Vitamin 312 analogues in which the 5,6-dimethylbenzimidazole group is replaced by a purine or pyrimidine base or related derivatives. All of these Vitamin B12 analogues are in- active or less active than Vitamin 312’ as shown in the table, for various organisms. The role of Vitamin B12 has recently been brought into sharp focus on an enzyme level since the isolation of B12 derivatives acting as co- enzymes by Barker and his colleagues (6,7,49). Although the precise mechanism of this action is not known, Vitamin 312 participates in several types of reactions. These reactions are: (l) the isomerization of carboxy- lic acids, (2) metabolism of one carbon compound, (3) conversion of ribo- nucleic acid to deoxyribonucleic acid (82). Vitamin B12 is a neutral molecule, odorless and tasteless, and is soluble in water to the extent of 1.2% at 25° C. Vitamin B12 crystallizes as dark red needles or prisms. The crystals darken at 210-220°C but do not melt below 300°C. The crystalline cyanocobalamin is stable in the solid state, even for several hours at 100°C. Aqueous solutions are most stable between pH 4 and 6; within this range solutions can be sterilized by autoclaving at 121°C with the loss of only a few per cent of activity. Aquocobalamin is less stable, eSpecially in alkaline solution, but both are about 90% inactivated by one hour at 100°C at pH 8 (27). On exposure to light, cyanide is slowly Split off, leading to hydroxocobalamin, but this change is reversed on keeping the solution in the dark (87). Pro- longed exposure to sunlight, however, causes irreversible destruction. The absorption Spectrum of Vitamin 312 shows three characteristic maxima at 278, 361 and 550 mu which are relatively independent of pH. The -g- .. 1% extinction co-efficients (E cm) at the above wavelengths are 115, 207 and 64 resPectively (4). Assay Numerous procedures for the assay of Vitamin B12 have been developed. Very commonly the vitamin occurs in such minute concentration that at present the microbiological assays are the best methods. A description of the assay methods follows: 1. Chemical assays. a. Spectrophotometric. As little as 25 ug of B12 per ml can be determined. This method is rapid and accurate but many of the Vitamin 312 analogues also have absorption maxima at 361 mu, and therefore the usefulness of the assay is limited for the most part to pure samples of Vitamin B12. b. Colorimetric. Cyanide is liberated by reduction or by photo- lySims and is measured by a sensitive colorimetric procedure. An alternate mettuad has been proposed which is based on the difference in the Spectrum of cyanocobalamin and its purple dicyanide complex (71). Other colori- metrix:nm¢hods, less widely used, are based on the presence of 5,6-dimethyl- benzinddazole (12) and on the hydrolysis products resulting from treatment of Vitamin B with strong hydrochloric acid (22). 12 c. Isotope dilution. This technique involves the addition of a known amount of radioactive Co-labeled Vitamin B12 to a crude sample. This method is highly Specific but sufficient amounts of the sample must be taken to permit isolating a measurable quantity of the pure vitamin. (Ihemical methods although very accurate are less sensitive than micrc”biological methods and may be time consuming and tedious, and therefore -9- are not amenable to routine assays of many food samples. 2. 'Microbiological assays. Microbiological assays are based on the fact that certain organisms grow slowly or not at all in the absence of Vitamin B12. A few kinds of microorganisms are more commonly used as test organisms. Limitations on the use of these methods are similar to those with other microbiological assays for B vitamins and include lack of Specificity of the method and problems in releasing bound forms of the vitamin prior to assay. Vitamin B12 has been shown to be bound to other constituents in different degrees (70, 30). The bound form cannot be assayed unless the vitamin is released in the free form. The bound form can be released by simple boiling of the sample in some cases such as for blood serum and liver, however, muscle tissues, human's and sows' milk have been reported to require treatment with trypsin and papain (72). a. Lectobacilli. Vitamin B12 was first measured microbiologi- cally by Shorb (74), with a strain of Lactobacillus lactis. In subsequent work reasonable satisfactory tube and cup-plate methods were developed for this organism, but it has since been almost entirely di8placed by more reliable lactobacilli. Skegg et al. (75) have recommended L, leichmannii ATCC 4797, Hoffman et al. (42) independently pioneered the use of the strain.ATCC 313, which has also been adopted by other workers (57). The method official in the U.S. Pharmacopoeia uses L, leichmannii ATCC 7830 in the titrimetric assays (84). The L, leichmannii tube assays are sensitive enough for the assay of 312 levels in blood serum (28). A complication with all the lactobacilli is that they respond not only to Vitamin 312 but to deoxyribonucleosides. The ratio of interfering substances, permissible compared to Vitamin B12 to avoid interference is 106 (DNA): 1 (Vitamin 312), and permissible amount of desoxyribotides compared to -10- Vitamin B12 is 2 x 104:1 (39). In general, except when the test extracts are rich in nuclear material such as liver, interference in the assays is largely eliminated simply by dilution. L, leichmannii strains also res- pond in varying degrees to the range of Vitamin B analogues containing 12 different nucleotides in place of the benzimidazole moiety (23) and this can be a serious complication when assaying samples of microbiological origin (Table 3). These analogues do not have significant Vitamin B12 activity for man and other animals and are synthesized by certain micro- organisms. b. E. coli mutant. One of the E, ggli_mutants, strain 113-3 has come into popular use as a test organism (55). It is somewhat less sensi- tive than the lactobacilli. This organism does not respond to deoxyribo- nucleosides, but it can use methionine as a substitute for Vitamin B12. The levels of methionine required for interfering with the Vitamin B12 assay is 5 X 104 parts of methionine to 1 part of Vitamin 312° This mutant responds to Vitamin 312 analogues, such as factor B and for this reaSon it has been widely used in the assay of Vitamin B12 analogues. c. Euglena gracilis. This photosynthetic algae (44) was intro- duced for assay of the vitamin. ‘Its advantages over other microorganisms are extreme sensitivity and the fact that it does not respond to deoxyribo- sides or other compounds unrelated to Vitamin 312' Its disadvantages are slow growth, and the fact that fairly intense illumination must be provided during the growth period. d. Other organisms. Ochromonas malhamensis (30) and Poterio- chromonas stipitata have higher Specificity towards true Vitamin B12 than any other organisms previously used. 9. malhamensis (26) has an animal- like Specificity for the vitamin and can be used to measure the vitamin -11- even when related compounds are present, and has been recommended for the assay of the vitamin in animal feeds. 3. Biological assays. Chicks (16) are most widely used, because it is relatively easy to rear depleted birds since little Vitamin B12 is synthesized in the gut. Rats are also used. There are, however, some difficulties; first young animals bred from normal parents may be endowed by the mother with an adequate reserve of the vitamin. Secondly, since the vitamin is a product of fermentation, it frequently results from bacterial activity in the gut and may be absorbed by the animal either directly through the gut wall or by intake of its own feces. Assays made in higher animals are somewhat more difficult and time consuming than microbiological assays. Large amounts of samples and approximately 2-4 weeks are needed for results. 4. Clinical assay. Before Vitamin B12 was isolated a great many assays of liver extracts were carried out with only semiquantitative precision in human subjects with pernicious anemia. Potency was deter- mined by the magnitude of the increase in red blood cell counts, hemo- globin, and the rise in reticulocyte percentage. We considered that among all these methods, the microbiological method using LP leichmannii was the most adequate one for this study because of its simplicity, the very small amount of the vitamin required, negligible interference from other substances in milk, and the relatively short time of the assay. Stabilization of Vitamin B11. Many workers (79, 72, 65) have tested stabilizers of Vitamin B12 during autoclaving of the samples. Reducing agents Show someWhat unpredict- -12- able effects. Thiol compounds at low concentrations are alleged to pro- tect the vitamin from destruction but in larger amounts they can cause destruction (50). Sulphite has also been recommended esPecially for the stabilization of hydroxocobalamine (Vitamin Ble)' The addition of ascorbic acid destroys Vitamin Ble quite rapidly, but shows no effect on cyanoco- balamin itself (27). A combination of thiamine and nicotinamide, or nicotinic acid alone has been shown to result in slow destruction of Vitamin B12 in solution although either alone shows no effect (11). Iron has been reported to protect the vitamin from destruction by this combina- tion (27). Lees et al. (56) reported that the growth resPonse of L, leichmannii to Vitamin B12 is related to the oxygen content of many media. A larger surface area and smaller depth of medium increased the rate of diffusion of oxygen into the medium and caused a decrease in growth of the organism. Ford (25) studied the factors influencing the destruction of the vitamin by heat in milk. He concluded that destruction of the vitamin was caused mainly by oxygen dissolved in milk and this destruction could be reduced by deaeration of the milk before heating. Vitamin B12 binding factors. Several of the B vitamins occur naturally bound to peptides or proteins. As these conjugates differ in physical and biological proper- ties from the free vitamin they may be more or less effective for any specific biological system. As early as 1928, Castle showed that pernicious anemia could be treated with intrinsic factor in the normal human gastric juice or from dried hog stomach, along with the so-called "extrinsic factor" present in meat and other foods. Crude intrinsic -13- factor preparations were shown to combine with Vitamin B12 ifl.!i££2.<83)- The resulting "bound Vitamin B12" could be distinguished from the free vitamin in various ways. It was non-dialysable; it was not available to the microorganism used for Vitamin B12 assay (10); it was not appreciably taken up from dilute aqueous solutions by bacterial cells, e.g. the wild strain of E, 221; (41). On the other hand it was much more effective than the free vitamin in the oral treatment of pernicious anemia. Vitamin B12 binding has been measured by microbial growth inhibition (83), electro- phoresis (46, 54), absorption on charcoal (59), ultrafiltration (35), and dialysis binding (67). These are methods of separation of the free and bound form of the vitamin. With the use of radioactive Vitamin 812’ work on the isolation of intrinsic factor proceeded in America, Britain, Holland and Scandinavia. Latner, Merrills and Raine (52) extracted intrinsic factor from hog mucosa by fractionation at pH 4.5 with ammonium sulfate and removal of salts by ultrafiltration. The molecular weight was between 15,000 and 20,000 with 6.5% hexosamine, 2.2% fucose, 10% N, 13% reducing sugar expressed as glucose, 10.4% tryptophan and 19.6% tyrosine and it was clinically active at l to 4 mg per day. Williams et a1. (91) also applied ammonium sulfate precipitation to mucosal extract, but they introduced a new step of diges- tion with trypsin. The product was clinically active at l-2 mg daily; however, it had a molecular weight of only about 5,000, contained 5.2% glucosamine and 11.8% N, and showed negligible binding capacity for Vitamin B12. Latner suggested that the trypsin had degraded the mucro- protein molecule to a still active mucopeptide. Holdsworth (43) purified hog stomach extract by repeated treatment with DEAE—cellulose. The ultra- centrifugally homogeneous product bound 15 ug of Vitamin B12 per mg and -14- the molecular weight was higher than Latner's preparation showed. GrHsbeck (29) obtained intrinsic factor which had a molecular weight of 70,000 by using electrophoresis on starch. O'Brien et al. (62) claimed to obtain a homogeneous product with a molecular weight of 40,000 from human stomach. Smith (77) suggested that the "native" substance as secreted has a high molecular weight, but it is degraded by relatively mild treatment to sub-units of several sizes, all retain- ing clinical activity. GrHsbeck (29) concluded from his experiment that binding of B was a prerequisite for intrinsic factor activity and that, 12 in addition, another part of the intrinsic factor molecule was necessary for physiological activity. In 1961, Bromer and Davisson (13) presented a preliminary report on the preparation of a Vitamin BIZ-intrinsic factor complex active at less than 50 pg per day. The complex contained 25 ug of Vitamin B12 per mg and had a molecular weight of 53,000. Intrinsic factor is by no means the only natural substance that binds Vitamin B 2. Many substances without intrinsic factor also bind Vitamin 312: bile constituents, hemoglobin, heparin, milk, saliva, and tear fluid (29). Rosenthal described multiple cyanocobalmin binding sites in serum from different Species of animals (68). Normal serum contains about 300 pug of Vitamin B12 per ml (63, 76) attached to proteins that are either identical with, or electrophoretically indistinguished from theglobulin fraction of human serum was assumed to be a kind of glycoprotein. These glycoproteins represent small fractions of the total proteins of the source material. Though similar in chemical and physical properties they are all different immunologically, even when derived from different organs of the same Species (e.g. hog pylorus and sows' milk: chick serum and chick proventriculi). A few attempts have been made to ascertain the point of attachment of the peptide chain on the vitamin molecule. Hydroxocobalamin can easily combine to yield compounds of the "cobalichrome" type (47) when a suitable terminal amino acid, such as histidine, is present that can link directly to the cobalt atom. The binding of cyano- and hydorxo- cobalamin by normal serum proteins was studied by Meyer (60). Blocking of cyanocobalamin binding by analogues with substituted amide groups in the pyrrol ring side chains was also investigated. The results obtained suggested that some of these amide groups was involved in binding. More hydroxo- than cyanocobalamin is bound, suggesting that the cobalt atom is also involved in binding. Milk At the present time the most accurate and rapid method of measuring Vitamin 312 in milk is by microbiological assays. Lactobacillus strains -l6- have been mostly widely used. Certain proteins and peptides have the properties of combining with cyanocobalamin and inactivating it for microorganisms (36, 34). Gregory found that treatment of samples with cyanide increased the microbiological value for Vitamin B12 in milk sample extracts.. This increase is probably due to action of cyanide in releasing the vitamin from bound forms, and to converting cobalamin derivatives to the more Stable cyanocobalamin. The presence of cyanide in the extracts is therefore a necessary precaution, and in some instances treatment with proteolytic enzymes is also essential for releasing bound Vitamin B12. Preliminary treatment of milk for re- leasing bound Vitamin B is necessary before the vitamin could be measured 12 quantitatively by the test organism. Table 3 (31) gives some published values of Vitamin 312 in the milk of different Species of animals (32). The term Vitamin 312 refers to the total Vitamin B12 activity measured by using Lactobacillus leichmannii. A difference in the Vitamin B12 content of milk between morning and afternoon milking is controversial. Some reports (86) claimed the after- noon milk contain more Vitamin B12 than morning milk, but some other reports (3) were the other way. Other workers (48) studied the Vitamin 312 content of cows' milk for different seasons, and found the values to be the highest in the autmn and lowest in Spring. On the contrary, Collins et a1. (19) observed that the cobalamin content of cows' milk did not vary Significantly with season or between the Jersey, Guernsey, Ayrshire, Friesian and Brown Swiss breeds. Collins et al. (18) described that when cows were fed a trace- mineralized salt containing 0.02% cobalt, their colostrum contained sig- nificantly higher cobalamin than when iodized salt was given. Contrarily -17- other workers (38) observed that the milk of cows fed a cobalt supplement did not contain significantly more Vitamin 312 as measured by rat growth assay. Koetsveld (86) found that Vitamin B12 activity of milk increased when the cows were put on to pasture after being indoors. Gregory (32) and Lichtenstein (57) measured the comparative Vitamin 312 content in foods and milk using different microorganisms. The latter showed that higher Vitamin B contents were obtained by using Ochromonas 12 malhamensis than using Lactobacillus leichmannii but the former group of workers showed approximately the same value of B12 content using each micro- organism. The results of microbiological assays showed that 79-86% of added Vitamin B12 in milk was destroyed by heat treatment at 115° C for two hours, but heating at 116° C for 15 minutes Showed no marked effect on the Vitamin B content (85). Almost all the Vitamin B12 was lost during 12 the Sterilization of evaporated milk or in—bottle sterilized milk. This loss was reduced by deaeration of the milk before heating (80). When raw milk was subjected to 0.125-l.0 Mrad y-radiation either when no-gassed, air-gassed or NZ-gassed, no losses were observed for Vitamin B12. By the use of an ultrafiltration technique, Gregory (32) has shown that Vitamin B12 occurs in a bound form in the milk of the cow, goat, pig, rat, sheep and human. The bound Vitamin 812 in cows', goats', rats', and ewes' milk was available to the test organisms after treatment with cyanide _but that the bound Vitamin B12 in sows' and human's milk was more firmly held and not released by autoclaving with cyanide, although digestion with a papaiIl rendered it available. Gregory and Holdsworth (36) Studied further -13- the occurrence of Vitamin BIZ-binding proteins in milk. They reported that pseudo-Vitamin 312’ factor A and factor B were bound by intrinsic factor and by sows' whey concentrates to the same extent as cyanocobalamin. The binding capacity of both concentrates was much greater before than after heating. In 1954 (33), the cobalamin-binding protein of sows' milk was isolated, by continuous electrophoresis on paper, in the form of its complex with cyanocobalamin. The complex contained 16.1% of N and 23.6 ug of cyanocobalamin in 1 mg of the complex. They assumed that with equimole- cular combination the molecular weight of the protein would be 55,000. It contained 17.6% of tyrosine and 7% of hexosamine. The cyanocobalamin- protein bond did not involve the -CN of the vitamin or the -SH of the protein. -19... C H20 H1O ONM2 Fig. 1. Vitamin B12 (cyanocobalamin) menn0neo «engages AnnoumenanuaonnsESSEne-o-mv-o no anamnmnooon0neo oanmnoo oaonsonamnoo -Amanenumnev-Ansnoumansnuamnnseumane-o”mv-n- manenumnm manenumnm A.ouov opnnoHLo oEonsonHmnoo oanmnoooanEm afl%aouman8nnconnksuoancuonmvunv mmz mnaoaad opnamnoooumammoonzu AnnnoumaneHusmnnnrumane-o-mv-o no oumcmmoonrn munEmnoo AamaoumsnEnuconamenoEnpuonmvunv ansmamnoooumcmmoonnH uzum ounEmnooounnune AnnnowmannuamnnseSmane-onmv-o no ounnnna unamflmnooonunz o - ounEmnoo AamaouocHEHNcmnH%£uoanpuoumvua no enamfimnooonnnnnz nozo Nam anamun> MW A.ouo «onmsmfism noV ownnoaso AdonnDHOm n - ounfimnooosvm AaxaoumanEHNemnaknuoEnpuonmvuxv anamamnooosv< pnomv omm Nam :nEmuH> munampoooxonmzn AconnSHOm m AnnnoumananucmnnseSSEne-o-mv-o unsansnouoxonenm mannmxnmv-mo mnm anemon> opnamnooocmzo Annnonanannamnnseumane-e-no-5 no opncmxo «unannoo AH%HoumannNconH%£noancumumvud anamnmnooocmho uzo Nam anamun> mam: mam: ponmdnpnOIOU mama onnmEoummm onumEonmmmnnaow oHsooHoa no SOH Hmanwnno Annv mo>num>nnmo Nnm enamun> n unamn -21- m>Huom nHuemHHm - .uoa .m m>Huo< - .uoa amnaowmue< - .us< more amonHm .uu< ammo oz -<-nouumm .uod omnemso Awe nouommvlu o:n:owmonamo HOV HOOO HOsO HOmO HOOHO -nmaHnruozTN Han nouoamv.. . woennsm onmmn .uo< %nmnunou csocxcb In O n 2:33. anon-03H oHoumenEnnaon AHHH nouommumev .uoa m 8.23 Om mm on OmHOOH Axe-PEA H non-own mansuamxom%£ O OO-mH OS OON -eroozwm m nouumm O OOH-O~ OOH managemxoenm O nonomm AsocHSOOmonmmo N Om OOHOOH Amalgam-2.3 H. .H non-own O O O .uo< n m nouowm .Hca O O O O O n O nouomn O OH SN 82 22:55: O n83...- O O ...--< O O O OH OONOOH wen-832- 02 m non-use 32.. .m H v H v O OO OS Om OOH «enamOmHseumfm < .832 O O 35H O OOH Om OH OOH SHE-3H NHO flash-H?) oaoumanEnNCon OOH OOH OOH OOH OOH OOH OOH OOH -erumann-oum «Hm unsoun> mammm conuooncn nusoe umou umou umou umou noon opnuooHosz oamz Eu .3 .3 was... one» was... mean 339 no ommm unanHo mmcoa mconsm nndmfi nHoo HHoo seams xuneo -onnuo -emHmH .H .m .m .AOOH no NHm anamun> on eonumHon an oommonmxo monnn>nuo wennnsoooumHHmnSumz .N oHan -22- Table 3. Vitamin B12 in the milk of different Species (31). Average content Range Assay organism mug/m1 mus/m1 COWS"MILK -- 1.0-6.0 L. leichmannii -- 2.0-24.0 L. leichmannii 6.6 3.2-12.4 L. leichmannii -- 2.7-9.0 L. lactis 3.0 L. leichmannii -- l.3-1l.5 L. leichmannii 3.9 3.2-4.8 L. leichmannii COWS' COLOSTRUM -- 3.0-78.0 L. leichmannii -- 5.8-38.0 L. leichmannii -- 4.0-30.0 L. leichmannii EWES' MILK 14.0 8.0-20.0 L. lactis 1 4 1.0-2.0 L. leichmannii -- l.0-6.0 L. leichmannii 7.0 -- L. leichmannii GOATS' MILK 0.9 0.3-1.4 L. lactis 0.1 0.1-0.2 L. leichmannii 0.7 -- L. leichmannii GOATS' COLOSTRUM 4.2 1.3-8.5 L. leichmannii 5.0 -- L. leichmannii HUMAN MILK. 0.4 0.1-1.5 L. leichmannii -- 0.06-0.16 L. lactis 0.3 -- L. leichmannii 3rd-8th day of lactation 0.9 0.3-2.4 L. leichmannii lst-8th month of 1actation0.3 0-0.8 L. leichmannii HUMAN COLOSTRUM 0.2 RATS"MILK -- 11.0-95.0 L. leichmannii -- 4.5-139.0 L. leichmannii 12.0 -- L. leichmannii SOWS"MILK 1.1 0.03-2.7 L. leichmannii 2.0 -- L. leichmannii -- 0.5-7.6 L. leichmannii -22- Table 3. Vitamin B12 in the milk of different Species (31). Average Vitamin B ‘content Range Assay organism mug/m1 mug/m1 COWS' MILK -- 1.0-6.0 L. leichmannii -- 2.0-24.0 L. leichmannii 6.6 3.2-12.4 L. leichmannii -- 2.7-9.0 L. lactis 3.0 L. leichmannii -- 1.3-11. L. leichmannii 3.9 3.2-4.8 L. leichmannii COWS' COLOSTRUM -- 3.0-78.0 L. leichmannii -- 5.8-38.0 L. leichmannii -- 4.0-30.0 L. leichmannii EWES' MILK, 14.0 8.0-20.0 L. lactis 1.4 1.0-2.0 L. leichmannii -- 1.0-6.0 L. leichmannii 7.0 -- L. leichmannii GOATS' MILK 0.9 0.3-1.4 L. lactis 0.1 0.1-0.2 L. leichmannii 0.7 -- L. leichmannii GOATS' COLOSTRUM 4.2 1.3-8.5 L. leichmannii 5.0 -- L. leichmannii HUMAN MILK 0.4 0.1-1.5 L. leichmannii -- 0.06-0.16 L. lactis 0.3 -- L. leichmannii 3rd-8th day of lactation 0.9 0.3-2.4 L. leichmannii lst-8th month of 1actation0.3 0-0.8 L. leichmannii HUMAN COLOSTRUM 0.2 RATS' MILK -- 11.0-95.0 L. leichmannii -- 4.5-139.0 L. leichmannii 12.0 -- L. leichmannii SOWS"MILK 1.1 0.03-2.7 L. leichmannii 2.0 -- L. leichmannii -- 0.5-7.6 L. leichmannii -22- Table 3. Vitamin B12 in the milk of different Species (31). Average Vitamin B content Range Assay organism mug/m1 mug/m1 COWS' MILK -- 1.0-6.0 L. leichmannii -- 2.0-24.0 L. leichmannii 6.6 3.2-12.4 L. leichmannii -- 2.7-9.0 L. lactis 3.0 L. leichmannii -- l.3-11.5 L. leichmannii 3.9 3.2-4.8 L. leichmannii COWS' COLOSTRUM -- 3.0-78.0 L. leichmannii -- 5.8-38.0 L. leichmannii -- 4.0-30.0 L. leichmannii EWES' MILK 14.0 8.0-20.0 L. lactis 1.4 1.0-2.0 L. leichmannii -- 1.0-6.0 L. leichmannii 7.0 -- L. leichmannii GOATS' MILK 0.9 0.3-1.4 L. lactis 0.1 0.1-0.2 L. leichmannii 0.7 -- L. leichmannii GOATS' COLOSTRUM 4.2 1.3-8.5 L. leichmannii 5.0 -- L. leichmannii HUMAN MILK 0.4 0.1-1.5 L. leichmannii -- 0.06-0.16 L. lactis 0.3 -- L. leichmannii 3rd-8th day of lactation 0.9 0.3-2.4 L. leichmannii lst-8th month of lactation0.3 0-0.8 L. leichmannii HUMAN COLOSTRUM 0.2 RATS' MILK -- 11.0-95.0 L. leichmannii -- 4.5-139.0 L. leichmannii 12.0 -- L. leichmannii SOWS"MILK 1.1 0.03-2.7 L. leichmannii 2.0 -- L. leichmannii -- 0.5-7.6 L. leichmannii EXPERIMENTAL PROCEDURE The experiments in this study were undertaken primarily to determine which protein fractions of cows' milk contain the largest amount of bound Vitamin 312° It is hoped that this study would provide some information on the relationship between the Vitamin B12 content of blood proteins as compared to milk proteins. Throughout this study, a modified Gregory's method (32) was applied along with the method described in U.S.P. XVI (84) and A.O.A.C. 9th edition (5) for the determination of Vitamin B12. The Microbiological Assay Assay organism Lactobacillus leichmannii ATCC 7830 was used. Media Standard official media (Bacto-B culture agar, Bacto-B 2 inoculum 12 1 broth, and Bacto-Blz assay medium U.S.P. were used. Inoculum One loop of cells from the stock culture in inoculum broth was trans- ferred to a tube of the inoculum broth. The culture was incubated for 16- 24 hours at 37° C j;0.5° C. This transferring had been done daily for three days before performing the Vitamin B assay. The culture was washed 12 three times by means of centrifuging and decantation and was then suSpended in 50 m1 of saline solution. One drop of suSpended cells was inoculated into each tube for assay of the vitamin. Method of determination of Vitamin B12 All procedures were carried out under minimum exposure of the samples -23- -24- to light, and amber colored volumetric flasks were used. Standard solution One mg of Vitamin 312 (cyanocobalamin) from an ampule standard solution was diluted to contain 50 ppg per m1 of working standard Solution. Sample solution Samples were treated as outlined below and diluted to contain about 20-50 ppg of Vitamin 312 per m1 of assay solution and kept in a refrigera- tor after addition of a small amount of toluene. Excee‘lter. Standard curve. Predetermined quantities of standard solution were added to the standard size test tubes, in triplicate, at 0.5 m1 interval and made to 10 m1 volume with 5 ml medium and sufficient water. These tubes were covered and autoclaved at 121° C for five minutes. After cooling to room temperature one drop of cell suSpension was added to each tube. Following incubation at 37° C for 72 hours, the lactic acid produced was titrated with the use of an automatic titrimeter or by using bromthymol-blue solution as an indicator. A standard curve was prepared by plotting titration values, expressed in m1 of alkali solution for each level of standard Vitamin 312 solution used, against the quantity of reference standard. Determination of total Vitamin 312° To the same size of test tubes, in duplicate, 1.0 ml to 5.0 ml of sample solution was added at 1 m1 interval. A procedure was followed -25- similar to that used for preparing the standard curve. The quantity of Vitamin B12 for each level of assay solution was determined by interpolation of the standard curve. The amount of Vitamin B in each original sample 12 was obtained by multiplying by the dilution factor. Determination of free Vitamin Bifi Each suitably diluted sample was passed through a Sietz filter or ultra- filtered through a regenerated cellulose tubing under vacuum, aseptically, and 0.0 ml to 5.0 m1 of Sietz filtered or ultrafiltered solution was added aseptically to the tubes for assay. The concentration of free Vitamin 312 was obtained by interpolation of the standard curve. Preparation of Samples and Analysis A series of experiments were carried out in seven Steps to isolate and characterize Vitamin B12 rich fractions from cows' milk. 1) Experiment 1 -- Determination of the total Vitamin 312 in pasteurized milk. 2) Experiment 2 -- Determination of total and free Vitamin B12 in pasteurized milk. 3) Experiment 3 -- Determination of total and free Vitamin B12 in raw skimmilk, raw whole milk, pasteurized skimmilk and pasteurized whole milk. 4) EXperiment 4 -- Determination of total and free Vitamin B12 in casein samples and whey samples which were prepared from pasteuri- zed skimmilk by three different methods (ultracentrifugally sedimented, rennin coagulation and isoelectric precipitation). 5) Experiment 5 -- (a) Preparation of crude lactalbumin and lacto- globulin from raw whole milk. (b) Determination of total and free -26- Vitamin B12 in the fraction of proteins on a dry basis. (c) Electrophoretic studies of the fractions. 6) Experiment 6 -- (a) Preparation of skimmilk, whey, casein, crude lactalbumin and lactoglobulin from raw whole milk. (b) Determin- ation of total and free Vitamin B12 in the fractions of proteins on a dry basis. (c) Electrophoretic studies of the whey, crude lactalbumin and lactoglobulin. 7) EXperiment 7 -- (a) Separation of monm pom Hmuou mo conumananouop now connmnmmonm mHeEmm man menacnm amnmmnw onumaosom < .N onswnm Hm mwnm NHm Hmuon -uonuHHH unnmm o>memwsm «Hm owns NHO Hmuon NHm mane NHm Hanan NHm «one «Hm Hauon _ _ _ _ _ onmnanwiunnom m>meons< oumnanmrmunom opmfiwons< oumnanmnunnom o>oHofimn4 _ 5 A. NHm monm NHm Hanoa _ _ NHm omnm «Hm Hmuoa oumnanm-unnom o>oHotws< _ _ # onmnanm_Ww«om o>mmm%u3< enomoo mangoes anommu wcnmmms anomwv wannmmz ONe euHs ON: runs one euHs wannmos wcnswoz mangoes Snowmo mosszHo< anommu hmsz possum anommw moms oosuozneonumunonownm nanom unnuomaoowH vogue: conumstmoo anamom wanna: Hmw9mnnuwoomnuas P _ - anaanxm pmanSoummm .NHm anamnn> menu was Honon mo connmcnanOHOO now connmnmmonm oHaamm man menacnm amnwmnp onnmaoaom < .N onswnm Hm owns «Hm Hmuon wumnuHHm — unnom oponmwsm NHm monm NHm Honoa «Hm monm NHm Honoa NHm oonm «Hm Honey _ _ _ _ _ oumnanwmunom o>meonS¢ oumnanmrmunom oemfiwonam onmnnanm-uunom o>mHomws< «.3 .q «Hm omnm Nam Honoa H _ _ N m monm «Hm Houoa muwnuHHH-Nunmm o>aHomm=< _ _ — onmnuHHMHuunom m>mmmwus< anommw wanmmmz anomwo maniacs anommo wansmms Owe runs ON: nuns Omm HHHS wanemuz wanemmz. menses: Snowmo mosziwno< cnommu . moss noeaom enommm hows Oofioz eoHumuH-Hnomn-H nanom onnnonoOOH vogue: eonanswooo cannon vogue: HowsmnnHWoomnnHD L _ xHHaame OmNHnSSSmmm 'I -36- Whole Raw Milk ljieparate 3X at 40° C Skimmilk Acidify with 0.1 N HC1 to pH 4.6 Filter Casie in WTi'ey (Discard) Adjust pH to 6.5 with 0.1 N NaOH Add (NH4)2$O4 to 0.5 saturation Centrifuge at 2000XG, 15 min. Phecipitate (Fraction A) Shpernatant (Fraction B) Redissolve at 3% protein conc. Dialyze against water Adjust pH to 4.6. Adjust to pH 4.6. Add (NH ) $0 to 0.25 saturation Filter Centrifuge at 25000XG, 15 min. , 1 fl PP]: Sjupernat ant Precipitate (Fraction C) Supernatant (Discard) (Fraction F) (Discard) Filter through glass wool Adjust to pH 6.5 Adjust to pH 6.0 Add (NH4)2SO4 to 0.: Add (NH4)2804 to 0.4 saturation saturation Centrifuge at 25000XG, 15 min. Centrifuge 2000XG. 15 min I . '1 l i1. Precipitate (Fraction D) Supernatant Supernatant PreCipitate Redissolve at 3% protein (Discard) (Fraction.G) (Discard) conc. Dialyzed against Adjust to pH 4.5 water Filter off insoluble residue Lyophilize Precipitate Shpernatant (Fraction.E) (Discard) Dialyze against water Lyophilize Crude Lactalbumin Crude Lactoglobulin Figure 3. A schematic diagram showing the fractionation of cows' milk with ammonium sulfate (88). -37.. Whole Raw Milk l Separate 3X at 40° C H.. Skimmilk Acidify with 0.1 N HC1 to 4.6 Filter I _1 Casein Whey Adjust pH to 6.5 Add (NH4)ZSO4 to 0.5 saturation Centrifuge at 2000XG for 15 min. | 1 Precipitate (Fraction A) S ernatant (Fraction B) Redissolve at 3% protein conc. Dialyze against water Adjust pH to 4.6 Lyophilize Centrifuge at 25000XC, 15 min * ‘fi Crude Lactalbumin l Precipitate S ernatant (Discard) Dialyze against water Lyophilize Crude Lactoglobulin Figure 4. A schematic diagram showing the fractionation of cows' milk with ammonium sulfate (88). EXPERIMENTAL RESULTS Experiment 1 Four different samples of pasteurized whole milk were found to contain 212, 234, 338 and 531 mpg of total Vitamin B12 per 100 m1 of milk. The average value obtained was 329 mpg per 100 ml of pasteurized whole milk. Experiment 2 The content of total and free Vitamin B12 in pasteurized cows' whole milk is shown in Table 4. Values for the total Viatmin B12 content were found to be similar for the three methods used for the release of the vitamin (extraction with cyanide and HC1, extraction with cyanide and NaAc, and digestion with papain). The extraction method with cyanide and hydrochloric acid was used in all subsequent experiments. Seitz filtration was considered to be the method of choice for preparation of samples for Vitamin 312, because of the small variation in the values obtained by this method and its simplicity as compared to the ultrafiltration method in maintaining aseptic condition. Experiment 3 The amounts of total and free Vitamin B12 in raw skimmilk, raw whole milk, pasteurized skimmilk and pasteurized whole milk are shown in Table 5. The different samples gave similar results. However, to assure the greatest similarity in the history of milk samples, raw -38- -39- milk obtained immediately after milking was used in all future experiments. The content of free Vitamin B12 was approximately five per cent of the total Vitamin 312. Experiment 4 The results for the total and free Vitamin B12 content of casein and whey samples which were prepared from pasteurized skimmilk by three differ- ent methods--ultracentrifuga1 method, rennin coagulation method, and iSo- electric precipitation method-- are shown in Tables 6 and 7. The ultracentri- fugal method gave the lowest content of Vitamin 312 in casein. The Vitamin B12 content of casein and whey prepared by the isoelectric precipitation of casein was in good agreement with the Vitamin B12 content of the skimmilk from which they were prepared. Casein contains slightly larger amounts of Vitamin 312 than the whey remaining after the precipitation of the casein. For further study the isoelectric precipitation method was used for prepar- ing casein samples, since this was the simplest method. Table 7 shows that whey protein contains more Vitamin B12 than casein protein when expressed in ppg/mg of protein. Whey samples were used in future experiments, since the B12 content in whey proteins was higher than that in casein for all samples tested on the basis of B concentration per mg of protein. 12 Experiment 5 In the first preparation of crude lactalbumin and lactoglobulin from raw whole milk, 3.8 grams of crude lactalbumin and 0.5 grams of lactoglobulin were obtained from 3,400 m1 of whey. In the second preparation, 4.0 grams of crude lactalbumin and 0.7 grams of crude lactoglobulin were obtained from 3,400 m1 of whey. -40- The moisture contents were 8.3% and 15% in crude lactalbumin and lactoglobulin, reSpectively, for the first preparation, and 11.1% and 11.1% in crude lactalbumin and lactoglobulin, reSpectively, for the second preparation. Protein contents, obtained by multiplying the nitrogen content by 6.38 of the crude proteins were 99.5% and 90.0% in crude lactalbumin and lactoglobulin, reSpectively, for the first preparation, and 94.4% and 85.5% in crude lactalbumin and lactoglobulin, respectively, for the second preparation. The free boundary electrophoretic mobilities of these proteins are given in Table 8, and mobility patterns are shown in Figure 5. The proteins present in each of the fractions were identi- fied from the descending electrophoretic patterns (14). Four bands were obtained from crude lactalbumin in the first and second preparations by zone electrophoretic study. They were tentatively identified as proteose- peptone, blood serum albumin, mixture of B-lactoglobulin A and B and as lactalbumin. When the crude lactoglobulin fractions from the first and the second preparation were examined, the bands were spread out and no clear zones were obtained. The total and free Vitamin 312 contents of eadh fraction are shown in Table 9. It will be noted that crude lactalbumin was found to contain more Vitamin 312 per mg than crude lactoglobulin in this experiment. EXperiment 6 By ammonium sulfate fractionation, 2,990 m1 of whey, 352.4 grams of moist-crude casein, 19.0 grams of crude lactalbumin, and 4.7 grams of crude lactoglobulin were obtained from 3,500 m1 of skimmilk. The protein content for each fraction is shown in Table 10. Free boundary electrophoresis showed proteose-peptone and a-lactalbumin in the fraction of crude lactal- -41- bumin. Immune globulin or proteose-peptone or a mixture of these and a-lactalbumin were identified in the lactoglobulin fraction. Absolute identification is difficult since the descending mobilities of these proteins are very similar. The mobilities are shown in Table 11, and the mobility patterns are shown in Figure 6. By zone electropharesis, five bands were observed in both fractions. The content of total and free Vitamin 312 in the skimmilk, casein, whey, crude lactalbumin, and lacto- globulin is tabulated in Tables 12 and 13. The total Vitamin 312 in casein and whey accounted for 95% of the total Vitamin B 2 present in 1 skimmilk. Total Vitamin B12 in the crude lactalbumin plus that in the crude lactoglobulin fraction accounted for 90% of the total Vitamin B12 found in the whey. Whey proteins again were shown to contain more Vitamin B12 per unit of protein than casein. Total Vitamin B12, un- corrected to unit protein concentrations, in the crude lactalbumin fraction is twice as high as that found in crude lactoglobulin. A very high con- centration of free Vitamin B12 was shown in casein. The values for free Vitamin B12 are very low in most cases, and precise values are difficult to obtain (Table 13). Data for total and free Vitamin B12 are shown in Table 14 and data for total 312 were also calculated in ppg B12 per mg on a dry protein basis in each fraction (Table 16). As is shown in the table, whey protein contains twice as much total Vitamin B12 per mg as casein; crude lactoglobulin contains more total Vitamin B12 per mg than crude lactalbumin. The highest total Vitamin 312 content per mg was found in crude lactoglobulin; next highest, in crude lactalbumin; next, in Whey; then skimmilk; and the smallest: total Vitamin B12 content was observed in -42- the casein (all values were compared on the basis of Vitamin B12 content per mg of protein). Both total and free Vitamin B12 contents are high in the whey and crude lactoglobulin fraction. Experiment 7 From the ammonium sulfate fractionation procedure less than 5 m1 of immune globulin and a-lactalbumin solution in veronal buffer were obtained. The protein content in the immune globulin and a-lactalbumin solutions was 0.8 mg/ml and 1.26 mg/ml, reSpectively. The mobility patterns of crude lactoglobulin, immune globulins, and a-lactalbumin are shown in Figure 7. The total Vitamin B12 content in each fraction is shown in Table 17. As indicated in the table the immune globulin fraction contains approximately five times more Vitamin B12 than a-lactalbumin. .mvsum mnnu an pom: OOHmEmm Hospn>nvan mo noses: onm momosuaonmm man an mnonasz . m . Hmv HH-S Hmv mn-w HOV «Om-mm~ HOV «SN-OOH HnO OOO-OH~ amuse «H OH new mmN mnm owmnm>< .cHOO o HH menu can HmHOH .¢ maan -44- Table 5. Total and free Vitamin B content of raw skimmilk, raw whole milk, pasteurized s immilk, and pasteurized whole milk. Raw Pasteurized B12 mpg/100 m1 Skimmilk whole milk Skimmilk whole milk Total 514 504 550 515 Free 35 21 23 22 OH om mH O NH O ON cmoz wH u OH m NH O ON N OH on HH NH oH N HN H omnm mm mm HO OO mm 0O ooH mcoHuomnm an now Ownssooom NHm unsouns HmuoH no N OHM SH SH mm OHH NHL... a H 5% .Em NNH NwH me HmH omH NmH HOm Ono :moz .r nOH NwH OOH OOH mmH OOH 0mm O 4. NNH ONH HNH nOH u mNH Nmm m HmH NHN NoH OmH u OmH 0mm N moH mOH mmH OmH oNH wNH Nmm H Hmuoy monz cHommu koez :Hommu mon3 :Hommu nooEHnoexo mo nonesz noumuHeHoonm poanswmoo OomSOHnucoomnqu xHHEame .m.m.H :Hcaom .mcoHuomnm on now Ooucsooom NHm :HamnH> Hmuou mo owmncmonom Odo xHHaEme Ha OOH OH 833:8 his new 533 .xHHE-Em OH «HO ans-3; use Ea. H33 H0 was .O SHOE. -46- Table 7. Total Vitamin B12 calculated in ppg per mg of casein, Skimmilk protein and whey protein. Rennin I.E.P. Skimmilk Ultracentrifuged Coagulated Precipitated Casein Whey Casein Whey Casein Whey B ppgfmg 87 47 139 51 147 61 135 -47- :HESOHmuomHud aHHsnonoo:0Osomm MN.Ou Om.Nu mo onsuxna no eHHsnonoODOOQ MH.Nu om.Nu aHHseonouomH HHHHHHHHDHN ESHOm @OOHm Nomi Odom: m Odo < mo onSanE no .O no a anHOOononomH-O «.m- HH.O- anaanmuomH-nv o.Ou mN.mu cHaanmuomH connmnomonm OcN N.O u CHEDOHO Esnom OooHn aHESHHmuomHnB m.H u cHHsnonso aHHsnonSOuOOSOOQ o.N u cHHsnonOOsomm mo onsnxna no GHHnnonovsomm o.Nu HN.N- cHHDOonouomH N.O - Hm Ono < OOxHaO m GHHDOonouomHum m new < mo enuana no m.m - < OHHSOononomH-O .m no a OHHsnononomH-O as.m- Om.m- N.O . GHESOHmuomHuo GHESOHmuomH-B om.m- NH.On cHEDOHmuomH conumnmoonm umH HOan HHnHHHOoa An-Oan Hm-OHxV connomnn msHm> wcHanoOOO Eonm OoumHsonov wcHanomon waHpcoom< on5umnonHH poemonm cHouonm anHHnozonuonosmonuoon .U oH no «H.o n N\-lb «O.w mm «nommsn Hmconm> CH xHHE .m300 mo mcoHuomnm CHHseononomH Ono cHeseHmuomH epono mo OOHuHHHOos onuonoseonHUOHm .m oHOmH -48- Table 9. Total and free Vitamin B in ppg per mg of protein for whey, crude lactalbumin and 1a6foglobulin. Prep. Total . 2 Free No. whey lactalbumin lactoglobulin lactalbumin lactoglobulin 1 272 233 157 9 20 2 272 263 205 10 18 _49— Table 10. Percent protein in various milk fractions on dry basis. Skimmilk Whey Solids Casein Solids Lactalbumin Lactoglobulin 35.9 90.0 11.9 77.9 84.5 -50- Table 11. Electrophoretic mobilities of crude lactalbumin and lactoglobulin of cows' milk in veronal buffer, pH 8.6, F/2=o.1, at 1° c Electrophoretic mobility Protein present Descend§ng determined from Fraction (x10- ) descending mobility lactalbumin -3.0 proteose-peptone (-3.0) -4.1 (x-lactalbumin (-4.2) lactoglobulin -2.6 pseudoglobulin (-2.0) euglobulin (-1.8) proteose-peptone (-3.0) -4.5 (x-lactalbumin (-4.2) Data in parenthesis are from literature (14). -51 - m H OH HO .OH Ha OOH\O18 NHO monm O.OO meannomnn an n m.Hm O.Om OOH amen Hanan on N H.ms O.mO muonnomnn an N m.mH O.ON O.OO m.OO OOH anesnxm Hmnon on n mmN me mcoHuomnw mH HmuOH SO OOH HON mON mqm Ha OOH\w15 NHm HmUOH aHHsnonouomH GHEDOHmuomH hon: aHommo xHHaEme moonuomnh mGOHuomnm msonnm> on now moussooom owmuaoonoe Oam xHHastm Ha OOH aonn Omenmnno muonnomnn nnmnona OH NHO anewnn> mmnn Ocm Hanon .NH OHOmn -52- Table 13. Total and free Vitamin B12 in ppg per mg of protein from skimmilk and protein fractions Assayed Skimmilk Casein Whey Lactalbumin Lactoglobulin Total B12 ppg/mg 160 105 404 461 550 Free B12 ppg/mg 3 3 35 3 26 -53- O.Om OcOHuomnm :H N N.ON N.OO OOH Omsk Hmnon on O N.OO 0.00 Ononnomnm an N N.OH O.mm O.Nm m.Om OOH xHHeeHxO Hmnon on N OO OOH HON OOH OOO Ha OOH\Wfla m cHHsnonouomH cHaanmuooH . mozz :Hommo xHHEEme OnoHnomnm mcoHuomnm OsoHnm> cH now noucsooom owmnemonme cam xHHEEHxO HE ooH Eonm OmchnOo meoHuomnm cHononm :H NHm aHamuH> HouOH .OH OHOOH -54- Table 15. _Percent protein in various milk fractions on dry basis Skimmilk Whey solids" casein " Solids "' Lactalbumin 'LaCtbglObulin 38.0 _ 101.5 _12.3 7 , 74.2 . .2, 85.2; -55- Table 16. Total Vitamin B1 in ppg per mg of protein from skimmilk and protein fracfions Assayed Skimmilk Casein Whey Lactalbumin Lactoglobulin B12 ppg/mg 143 65 333 370 430 -56- om Nam omm HOO OOO moH OOH wE\Wfl1 m cHaanmnomH cHHsnon GHHseonouomH cHaanmuomH hogs Gnommu xHHEEme Oozmmm< no oosaeH mpsno oODnU meHnomnm :Houone new xHHEEme aonm anemone mo ma non win cH NHm cHamnH> Hmu0H .NH oHan -57- ASCENDING DESCENDING First Preparation: Crude lactalbumin Fraction: M N 4900 sec 11.0 volt cm. Crude lactoglobulin Fraction: adh- A 4900 sec. 11.0 volt cm. Second Preparation: Crude lactalbumin Fraction. HA IN.— 4800 see 11. 2 VOlt cm.- Crude lactoglobulin Fraction: m W 6500 sec. 11.2 volt cm. Figure 5. Electrophoretic patterns of crude lactal- bumin and lactoglobulin in veronal buffer, pH 8. 6, F72: O. l ~58- ASCENDING DESCENDING Crude lactalbumin Fraction: W W 5500 sec. 11.0 volt cm Crude lactoglobulin Fraction: .46- Jan: 4400 sec. 11.0 volt cm. Figure 6. Electrophoretic patterns of crude lactal- bumin and lactoglobulin in veronal buffer, pH 806' [72 = 0.1 -59 -. ASCENDING DESCENDING Crude lactoglobulin Fraction: 5 hours 11.0 volt cm."1 Immune globulin Fraction: _ ~ 3350 sec. 11.0 volt cm.- 1 OK - lactalbumin Fraction: w 3620 sec. W 11.0 volt cm.-1 Fi re 7. Electrophoretic patterns of crude lacto- g'u globulin, immune globulin and til-lactalbu- min in veronal buffer, pH 8.6, FVZ = 0.1 DISCUSSION The Vitamin B12 content of milk varies from Species to Species and from animal to animal. The vitamin content of milk from the same animal apparently varies with the season, the lactation period, the time of milk- ing, and feeding regimen. The Vitamin B12 content in milk was reported to be significantly increased by adding cobalt in the feed. In Spite of these factors, the values for total Vitamin 312: 255-550 mpg per 100 m1 of whole milk or skimmilk, from our experiments (Tables 4, 5, 6, 12, 14) were in good agreement with other worker's results (Table 3). Samples of milk proteins were autoclaved after the addition of cyanide and HC1, in order to release the bound Vitamin B12 and convert any hydroxocobalamin into the more stable cyanocobalamin. Extraction by autoclaving with cyanide and HC1, or with cyanide and NaAc, and digestion with activated papain (Table 4) were satisfactory methods for this purpose. Recoveries of cyanocobalamin added before extraction by autoclaving with cyanide and HC1, the method selected for our experiment, were between 95-105% of the original amount added. The free Vitamin B12 content in Experiment 2 was approximately 5% of the total Vitamin B12 content (Tables 5 and 6). Both methods, Seitz filtration and ultrafiltration, gave 60-80% recoveries of standard cyano- cobalamin added to milk extracted by the cyanide and HCl autoclaving method. Gregory (32) described that when cows’ milk was extracted by cyanide or papain before ultrafiltration, 50 to 75% of the total Vitamin B12 present in the milk could be ultrafiltered. An explanation for this -50- -61- low recovery could be that some substances present in regenerated cellu- lose tubing combine with free Vitamin 312° The same phenomenon could have occurred in the pad of the Seitz filtrater when free Vitamin B12 is pass- ing through the pad of the Seitz filter. Table 5 shows slightly different total Vitamin B12 contents between raw and pasteurized whole milk and between raw and pasteurized skimmilk. The Vitamin 812 content in raw and pasteurized skimmilk is slightly higher than in raw and pasteurized whole milk. This difference could be accounted for on the basis of dilution by the fat phase. The data in Table 6 Show that three different methods of whey prep- aration gave slight but probably nonsignificant differences in Vitamin 312 content, ranging from 102 to 171 mpg of total Vitamin 312 in the whey prepared from 100 m1 of skimmilk. Casein contained more Vitamin B12 than whey when prepared by rennin coagulation or isoelectric preparation. The data in the table show that casein and whey contain approximately the same amount of Vitamin B12 when these fractions were prepared by centri- fugation. The free Vitamin 312 in isoelectrically prepared casein showed an extremely high value. No rationale is apparent to explain this obser- vation. Whey samples showed higher free Vitamin 312 contents than the casein preparations except for the casein prepared by isoelectric precipi- tation. As shown in Table 7, whey protein contains two to three times more Vitamin B12 than casein from all preparations when the amount of the Vitamin per mg of protein is considered. -62- From the data in Tables 12 and 14 it could be concluded that Vitamin B12 is distributed among all the milk proteins, or that the Specific protein-B12 adsorption complex was a contaminant in all prep- arations, since the Vitamin B 2 content in skimmilk is randomly dis- 1 tributed approximately half and half between casein and whey. When the Vitamin B12 content was calculated per mg of milk protein, it is two to three times higher in whey protein than in casein as shown in Tables 7, 13, 14 and 17, and lactalbumin contains two to three times more Vitamin B12 than lactoglobulin in skimmilk. Lactalbumin contains more Vitamin B than lactoglobulin on a dry basis (Table 9), but, 12 contrarily, a higher Vitamin 312 content was observed on the second ex- periment for lactoglobulin than lactalbumin on a dry basis (Tables 13, 16 and 17). It is accepted that Vitamin B12 is synthesized in the digestive tract by rumen microorganisms, and it is carried to the mammary gland through the blood stream. The proteins (58) present in milk have been considered not only to arise from the synthetic activitiescf the mammary gland but also to include some preformed which enter the gland from the blood. The isolation and recognition in recent years of 97% of the protein of cows' skimmilk as Specific chemical and biological entities have made it possible now to determine which of these proteins are syn- thesized in the mammary gland. Larson et al. (51) described from their radioisotope C14 experiment that the levels of C14 incorporated by ab casein, B-casein, a-lactalbumin and B-lactoglobulin suggest that these milk proteins are synthesized in the mammary gland from a common amino -63- acid pool. The levels of C14 incorporated by y-casein, the immune globulins, and milk serum albumin, and similarity with the levels present in the blood proteins, suggest these proteins enter the milk preformed from the blood stream. Vitamin B12 is possibly carried by the blood proteins and interchanged between the blood proteins and milk proteins synthesized by the mammary gland. In one experiment lactalbumin was found to contain more Vitamin B12 than lactoglobulin (Table 9), while in other experiments lactoglobulin was found to contain more Vitamin 312 than lactalbumin (Tables 13 and 16). Whey proteins contained two to three times more Vitamin B12 than casein (Tables 7 and 13) and five times more Vitamin B in another experiment 12 (Table 16). Probably no equilibrium exists between the Vitamin B bound 12 with different milk proteins. Vitamin B12 is being randomly distributed among the binding sites, or, also, possibly the binding state of Vitamin B12 and different milk proteins is changed continuously with some biochemi- cal mechanism. Distribution of Vitamin B12 between different proteins of the same biological systems has been found in serum. Cresseri (21) described that six different fractions with microbiologically available Vitamin B12 exist in the serum of the normal rabbit, and Rosenthal (69) reported that more than one major binding site exists in the serum of man, dog, rabbit, frog and chick. These reports are consistent with the concept that the cyanocobalamin- binding phenomena in sera is associated with multiple binding sites that may be different for various animal Species and that Special orientation -64- between the protein and cyanocobalamin moieties is necessary for binding to occur. It appears from the results of this study that Vitamin B12 is dis- tributed among more than one milk protein and that equilibrium does not exist between Vitamin B12 bound with different milk proteins, since there is no regularity in the distribution of the vitamin between differ- ent milk proteins. The evidence suggests that Vitamin B12 is carried into milk via blood proteins (immune globulin, serum albumin, and possibly a blood protein in the casein complex) in the mammary gland. For further study preparation of more highly purified milk protein is very necessary. Purified protein will give more information of the occurrence of B12 in milk protein and relationships between milk and blood proteins. Sampling of milk at certain intervals obtained from the same cow may also be of value to determine changes in Vitamin B12 binding between different milk proteins. If blood and milk are sampled at the same time from the same cow, important relationships in Vitamin B12 bind- ing by blood and milk proteins may be obtained. 1) 2) 3) 4) 5) 6) CONCLUSIONS Extraction by autoclaving with cyanide and HC1 was a satisfactory method for the release and conversion of the bound form of Vitamin B12 to cyanocobalamin in cows' milk prior to microbiological assay. The Vitamin B12 content of whole milk or skimmilk was found to range from 255 to 550 mpg per 100 ml. The free vitamin accounted for 5% of the total Vitamin B12 content in whole milk or skimmilk. Slightly higher Vitamin 312 contents in raw skimmilk and pasteurized skimmilk than in raw whole milk and pasteurized whole milk were found. Higher Vitamin B contents in casein than in whey were obtained from 12 a given amount of skimmilk by rennin coagulation or iSoelectric precipi- tation than by ultracentrifugation. Vitamin B12 content in whey is two to three times higher than in casein when the results are expressed per unit weight of protein. The Vitamin B12 content in crude lactalbumin is higher than in lacto- globulin from a given amount of whey. Immune globulin contains more Vitamin 312 than