VITAMINS 



374 



VITAMINS 



L-amino acids, L-hydroxy acids, alde- 

 hydes and purines. In Warburg's "old 

 yellow enzyme", the respiratory en- 

 zyme, riboflavin exists as the mono- 

 nucleotide. The j^ellowish-green fluo- 

 rescence of riboflavin has been used for 

 its detection in tissues by Ellinger, 

 P., and Koschara, W., von Euler, H., 

 et al., Hert, A. and Wimmer, K. and 

 Metcalf, R. L. and Patton, R. L. See 

 Glick, D., Techniques of Histo- and 

 Cyto-Chemistry, New York, Inter- 

 science Publishers, Inc., 1950, pp. 531 

 Metcalf, R. L. and Patton, R. L. claim 

 that another form of riboflavin exists 

 which gives a yellow-orange fluores- 

 cence. 



B3. Pantothenic acid, first designated 

 B3. filtrate factor, factor II, anti- 

 grey-hair-factor. This vitamin is a 

 component of coenzyme A involved 

 in the acetylation of aromatic amines 

 and choline, and in the metabolism of 

 fats and carbohydrates. Pantothenic 

 acid is determined microbiologically 

 using Lactobacillus arabinosus 17-5 

 (Johnson, 1949, pp. 79-80). 



Be. Group (Pyridoxal, Pyridoxine, and 

 Pyridoxamine). Pyridoxamine is basi- 

 cally as important as pyridoxine, 

 and pyridoxal is even more so. Pyri- 

 doxal phosphate is the coenzyme of 

 this vitamin group and is involved in 

 the decarboxylation of amino acids and 

 in transamination. Pyridoxine can be 

 determined chemically by techniques 

 based upon the indophenol test (Gy- 

 orgy, 1950, p. 239). This method, 

 however, is only suitable for pyridoxine 

 and not for the other two forms so 

 that it is not adaptable for tissue analy- 

 sis. Saccharomyces Carlsbergensis and 

 Neurospora sitophila are employed for 

 the microbiological determination of 

 the Be group of vitamins. Pyridoxal, 

 pyridoxamine and pyridoxine have ap- 

 proximately the same activity in stim- 

 ulating growth (Gyorgy, 1950, pp. 406- 

 414). 



B12. Aniipernicious anemia factor (ani- 

 mal protein factor). This vitamin con- 

 tains 4 per cent of cobalt. It produces 

 a positive hematological response in 

 pernicious anemia in quantities as low 

 as 3 micrograms. This vitamin also 

 improves the hatchability of hen's eggs, 

 the growth and survival of chicks, and 

 the growth of rats. Vitamin B12 

 has been implicated in the following 

 processes: (1) the synthesis of purines 

 and pyrimidines and their derivatives, 

 (2) the synthesis of methionine and 

 folic acid, and (3) the utilization of 

 p-aminobenzoic acid and folic acid. 

 It is related metabolically to folic acid 

 (see The Nutritional and Chemical 



Significance of Folic Acid, Lederle, 

 American Cyanamid Co. 1950, 121 pp. 



Choline, a constituent of phospho- 

 lipids, lecithins and sphingomyelins, 

 acts as a donator of labile methyl 

 groups and prevents hemorrhagic de- 

 generation of kidneys of j^oung rats 

 deficient in choline. Choline can be 

 determined chemically or microbio- 

 logically (Gyorgy, 1950, pp. 243 and 

 464). 

 C. Antiscorbutic vitamin (Cebione, Re- 

 doxon). Bourne, G., Anat. Rec, 1936, 

 66, 369-385, has made a critical study 

 of cytological methods for the deter- 

 mination of vitamin C. The technique 

 reconomended is based on the assump- 

 tion that the only substance, other 

 than vitamin C, capable of reducing 

 an acid silver nitrate solution in the 

 dark is hydrogen sulphide "which is 

 not by any means a common constituent 

 of living tissue, if it occurs at all." 



To demonstrate reduced vitamin C, 

 frozen sections of fresh tissue are 

 treated with 5% aq. silver nitrate, to 

 which 5 cc. acetic acid is added for 

 each 100 cc, for a few minutes. The 

 vitamin C granules blacken. After 

 washing in aq. dest. fat may be stained 

 by a solution of Sudan III or Scharlach 

 R in 90% ale. and the section cleared 

 and mounted in glycerin. 



To reveal both reduced and oxidized 

 vitamin C is more difficult. Bourne 

 advised: Subject fresh tissue to glacial 

 acetic acid vapor for several minutes. 

 Cut into very thin slices and put in 

 atmosphere of hydrogen sulphide for 

 15 min. All vitamin C is thereby con- 

 verted to reduced form. Remove hy- 

 drogen sulphide by keeping in partial 

 vacuum for 10 to 30 min., follow by 

 exposure to a strong stream of nitrogen 

 gas for 15 min. Treat with acid silver 

 nitrate solution as described. 



If there is reason to believe that 

 glutathione inhibits the reaction, 

 Bourne suggests, after the hydrogen 

 sulphide treatment, to momentarily 

 wash the section, then to plunge into 

 mercuric acetate solution for a few 

 minutes, wash and apply acid silver 

 nitrate solution. See Barnett, S. A. 

 and Bourne, G., J. Anat., 1940-41, 75, 

 251-264 for methods of demonstration 

 of vitamin C in chick embryos. 



Modification of Giroud and Leblanc 

 silver method (Tonutti, E., Proto- 

 plasma, 1938, 1, 151-158). Briefly wash 

 tissue in 5.4% aq. levulose and 10% aq. 

 AgNOs plus 2 drops glacial acetic per 

 cc, up to 30 min. Rinse in aq. dest. 

 30 15-min. and in 3% aq. NajSoOj, 

 15-30 min. Rinse in aq. dest. 15-30, 

 min. All this is to be done in the dark 



