VITAMINS 



263 



VITAMINS 



formalin and examination of frozen 

 sections with Fluorescence Microscope 



wittiin 24 hrs. The green fluorescence 

 fades during irradiation especially in 

 the Kupffer cells. The same fluo- 

 rescence is found in epithelial cells of 

 fascicular and glomerular zone of 

 adrenals (but it is absent in adrenals of 

 newborn infant), in the cells of the 

 corpus luteum, interstitial cells of the 

 testis and several others. Greenberg, 

 R. and Popper, H., J. Cell. & Comp. 

 Physiol., 1941, 18, 269-272 report that 

 vitamin A, gives "striking green and 

 quickly fading" fluorescence and A2 

 "faint yellow-brown and slowly fading." 



B. Complex contains many factors. 



Nicotinic acid and nicotin amide. 

 An excellent chemical method for 

 quantitative determination of nicotinic 

 acid has been advocated by Dam, W. J. 

 and Handler, P., J. Biol. Chem., 1941, 

 140, _201-213_, 755-762. As to tissue 

 localization it is reported by the same 

 authors that nicotinic acid exists as 

 part of nucleotide molecules (see 

 Pentose Nucleotides) but in muscle 

 or renal cortex most of it occurs in some 

 other form. The status of the fluo- 

 rescent substances present in the urine 

 of pellagrins is not clear (Najjar, V. A. 

 and Cleckley, H. M., Proc. Soc. Exp. 

 Biol. & Med., 1941, 48, 413-414). 



Pantothenic acid. Filtrate factor. 

 Factor W., Anti-grey hair factor. 

 Localization as between blood plasma 

 and cells apparently is possible (Pear- 

 son, P. B., J. Biol. Chem., 1941, 140, 

 423-426. 



Choline. No histochemical method. 



Bi. Thiamine hydrochloride; thiamin 

 chloride, anti-BeriBeri or antineuritic 

 vitamin; Aneurine, Betabion, Beta- 

 toxin, Oryzaniu, Torulin. C12H18, 

 ON4SCI2, mol. wt. 337.26. There is no 

 microchemical test for thiamin but 

 tissue analysis reveals the curious fact 

 that the amount in the adrenal cortex 

 of the bull is more than 7 times that in 

 the medulla while in the cow the medulla 

 contains 1.9 times as much as the cortex 

 (Wright, L. D., et al., Univ. Texas 

 Publ., 1941, 4137, 38-60). 



B2. Vitamin G, Riboflavin; Lactoflavin. 

 C17H20O6N4, mol. wt. 376.19. No micro- 

 scopic methods are available but a 

 microbiological technique for riboflavin 

 has been described by Snell, E. E. and 

 Strong, F. M., Univ. Texas Pub., 1941, 

 4137, 11-13 which Gyorgy says results 

 in satisfactory agreement with those 

 secured in other ways. Riboflavin can 

 now be determined by polarographic 

 analysis (Lingane, J. J., and Davis, 

 O. L., J. Biol. Chem., 1941, 137, 567- 

 574. Sherman (p. 373) states that the 



respiratory enzyme (Warburg's yellow 

 enzyme) is a combination of riboflavin 

 phosphate and protein (see Cyto- 

 chrome). 



B3, 4, 6. Data insufficient. See Sher- 

 man (p. 390). 



Be. Pyridoxin, adermin, antidermatitis 

 vitamin. CgHuNOs, mol. wt. 169.18. 

 This essential nutrilite apparently oc- 

 curs in tissues to a large extent in bound 

 form. The trouble with microbiological 

 methods of analysis is that it may be 

 only incompletely extracted as noted 

 by Gyorgy. 



C. Antiscorbutic vitamin, Cebione, Re- 

 doxon. CeHgOe, mol. wt. 176.06. 

 Bourne (Anat. Rec, 1936, G6, 369-385) 

 has made a critical study of cytological 

 methods for the detection of vitamin C. 

 The technique recommended is based 

 on the assumption that the only sub- 

 stance other than vitamin C capable 

 of reducing an acid silver nitrate solu- 

 tion 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. sol. of silver nitrate 

 to which 5 cc. acetic acid is added for 

 each 100 cc. for a few minutes. The 

 vitamin C granules blacken. After 

 w'ashing in aq. dest. fat may be stained 

 in 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 

 advises: Fresh tissue is subjected to 

 glacial acetic acid vapor for several 

 minutes. Cut into ver}^ thin slices 

 and put in atmosphere of hydrogen 

 sulphide for 15 min. All vitamin C 

 is thereby converted to reduced form. 

 Remove hydrogen sulphide by keeping 

 in partial vacuum for 10 to 30 min. 

 followed by 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 hydrogen sulphide treat- 

 ment, to momentarily wash the section, 

 then 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--U, 75, 251-264 for methods of 

 demonstrating vitamin C in chick 

 embryos. 



Modification of Giroud and Leblanc 

 silver method (Tonutti, E., Proto- 

 plasma, 1938, 31 (1), 151-158). Briefly 

 wash tissue in 5.4% aq. levulose. 10% 

 aq. AgNOj -f- 2 drops glacial acetic per 



