Osborne and Wakeman28 added alcohol gradually to a 0.01 per cent N. acetic acid extract of yeast vitamin and tested the different fractions for growth-promoting qualities. That obtained from 52 per cent alcohol (by weight) was almost inert, while that soluble in 52 per cent, but precipitated by 79 per cent contained most of the active substance. That precipitated from 90 per cent alcohol was almost as active per unit as that from 79 per cent, but the amount obtained was small, only 10 per cent of the total extract, while the second fraction contained 37 per cent of the total. The portion soluble in 90 per cent alcohol also contained the vitamin, but not in so great a concentration as the preceding.

19a Palmer and Kennedy, J. Biol. Chem. 46, 550, 1021.

20 Chamberlain and Vedder, Phil. J. Sci., B6, 305, 1911; McCollum and Davis, J. Biol. Chem. 33, 181, 1015.

21 Cooper, J. Hyg. za, 436.

22 Cooper, 1. c.

23Suguira, J. Biol. Chem. 36, 101.

24 Stepp, Techr. Zt. f. Biol. 57, 135; Eijkman, Arch. f. path Anat, 148, 523, 149, 107; Fraser and Stanton, Lancet, March 12, 1910, 733; Chamberlain and Vedder, Phil. J. Sci. 6B, 305; Tervvchi, Y., Saiking akuashi, Tokio, 1010, No. 170; Zeits. Bioch. Biophys. 11, 710; Punk, Trans. Soe. Trop. Med.

5,86.

25 Osborne and Mendel, J. Biol. Chem. 31, 140.

26 Drummond, Bioch. J. zz, 255, 1017.

27 Osborne, Wakeman and Perry, J. Biol. Chem. 39, 35, 1010.

28Osborne and Wakeman, J. Biol. Ch. 40, 383,1010.

On the other hand, Funk 29 and Fraser and Stanton30 have successfully used absolute alcohol to extract the antineuritic substance from rice polishings and Cooper31 has made use of it for extraction of various foodstuffs.

Vedder and Williams 32 found the vitamin from rice only slightly soluble in cold 95 per cent alcohol. McCollum and Simmonds33 state that it is very difficult to extract it completely from cooked beans. They believe that the failure of alcoholic extracts of cooked beans to induce growth is due to the physical changes in the bean during cooking whereby the vitamin is protected from contact with the solvent, not because of destruction of the active substance. Even with raw beans extraction with hot alcohol in a Soxhlet was not complete in 18 hours. Eddy 34 found it possible to extract the vitamin from sheep's pancreas with cold 95 per cent alcohol containing 0.8 per cent HC1. Methyl alcohol dissolves the vitamin from yeast35 and from some vegetables.36

In general the antineuritic vitamin appears to be insoluble in ether,37 although Cooper39 finds that it can be extracted from egg-yolk, and to a lesser degree from meat, by this solvent.

It is soluble to some degree in acetone, as noted by Stepp89 and confirmed by McCollum and Davis,40 who state, however, that it is a poorer solvent than water or alcohol. McCollum and Simmonds 41 found that B is not extracted directly from beans, wheat germ, or pig kidney by ether, benzene, or acetone, but is readily extracted in great part by alcohol. After being removed by alcohol it is soluble in benzene, but very slightly in acetone.

29 Funk, J. Physiol. 43, 385, 1911-12.

30 Fraser and Stanton, Lancet, 1910, ii, 1755.

31 Cooper, J. Hyg. la, 436, 1913.

32 Vedder and Williams, Phil. J. Sci. SB, 175.

33 McCollum and Simmonds, J. Biol. Chem. 33, 55, 1918.

34 Eddy, J. Biol. Chem. 37, 113, 1916.

35 Myers and Voegtlin, J. Biol. Chem. 4a, 199, 1920.

36 Brill and Alencastre, Phil. J. Sci. 12A, 127, 1917.

37 Stepp, Zeitschr. f. Biol. 57, 135; Chamberlain, Vedder and Williams, Phil. J. Sci. 7B, 39, 1912; Cooper, Bioch. J. 7, 268, 1913; 8, 347, 1914.

38 Cooper, J. Hyg. 12, 436.

39 Stepp, Zeitschr. f. Biol. 6a, 405, 1913.

40 McCollum and Davis, J. Biol. Chem. 33, 192, 1915.

41 McCollum and Simmonds, J. Biol. Chem. 33, 55, 1918.

McCollum and Kennedy 42 report that the vitamin from fat-free wheat embryo can be extracted by acetone, benzene, and ethyl acetate, but McCollum and Simmonds43 noted that the scheme of applying successively to raw navy beans the solvents ether, benzene, and 95 per cent alcohol, in order named, and of feeding the extract and residue in separate experiments, showed that benzene does not remove B from ether-extracted beans while hot alcohol does. When material which was dissolved from beans by hot alcohol was deposited on dextrin and the latter then extracted with hot benzene, the benzene-soluble material thus obtained was very effective in inducing growth and in causing prompt recovery of polyneuritic rats. A benzene extract prepared directly from ether-extracted beans does not contain an appreciable amount of B. Benzene therefore does not remove B from beans directly, but once this substance is extracted by alcohol it is soluble in benzene.

Steenbock 44 states that he was able to cure polyneuritis in pigeons by means of intraperitoneal injections of a water-acetone soluble substance from egg-yolk.

Cooper 45 prepared a curative substance from horse-flesh which was moderately soluble in water, and insoluble in alcohol, benzene, chloroform, ether, and ethyl acetate, and states that the brain contains an antineuritic substance which can be extracted with alcohol and precipitated by a mixture of acetone and ether.

Myers and Voegtlin 46 found that olive oil and oleic acid remove the vitamin from autolyzed yeast filtrate, thus showing that in the form of the crude extract it is fat-soluble as well as water-soluble.

Water-soluble B is not colloidal, since it can be dialyzed through parchment47 and collodion48 membranes.

It is readily absorbed by charcoal,49 kaolin,50 mastic,51 fuller's earth,52 Lloyd's reagent (colloidal hydrous aluminum silicate,53 colloidal ferric hydroxide,54 and, according to Drummond, by flocculent precipitates of all kinds, a property which doubtless accounts for some, at least, of the losses observed in attempting to purify this factor, although Funk55 regards this loss as unimportant.

42 McCollum and Kennedy, J. Biol. Ch. 24, 491.

43 McCollum and Simmonds, J. Biol. Chem. 33, 65, 1918.

44 Steenbock, Proc. Am. Physiol. 80c Am. J. Physiol. 4a, 610.

45 Cooper, Bioch. J. 7, 268, 1913; 8, 347, 1914.

46Myers and Voegtlin, J. Biol. Chem. 42, 199, 1920.

47 Eijkman, 1. 0.; Chamberlain and Vedder, Phil. J. Sci. 6B, 395, 1911; Drummond, J. C. Bioch. J. 11, 255, 1917.

48 Sugnira, J. Biol. Chem. 1918, 36, 191.

49Chamberlain and Vedder, Phil. J. Sei. 6B, 251, 395; Voegtlin, J. Wash. Acad. 8ci. Oct. 4, 1916.

50Voegtlin, J. Wash. Acad. Sci. Oct 4, 1916.

51Voegtlin, J. Wash. Acad Sci. Oct. 4. 1916.

52 Seidell, U. S. Patent 1, 173, 317; Emmctt and McKim, J. Biol. Chem. 3a, 403, 1917; Public Health Reports Feb. 18, 1916, p. 364. Reprint No. 325; Harden and Zilva, Bioch. J. 12, 93, 1918.

53 Seidell, U. S. Patent 1, 173, 317; Emmett and McKim, J. Biol. Chem. 32, 409, 1917; Public Health Reports Feb. 18, 1916, p. 364. Reprint No. 326; Eddy and Roper, Proc. Soc. Exp. Biol. Med. 14, 52, 1916.

54 Harden and Zilva, Bioch. J. za, 93,1918.

55 Punk, Biochem. Bull. 5, 1, 1916.