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    (From the Chemical Laboratory of the New York Agricultural Experiment Station, Geneva, N. Y.)

    (Received for publication, April 2, 1912.)

    In continuation of the physiological investigation concerning the metabolism of the organic-phosphorus compound known as phytin, which has been and is being carried out at this institution by Dr. Jordan, a closer study of the chemical properties of this substance, phytin, became necessary. Much work has already been done and reported on this subject by various investigators. Definite information, however, concerning different kinds of salts formed by the free phytic acid or inosite phosphoric acid is seldom met with in the literature. Frequently impure salts have been analyzed.

    Posternak, who first successfully prepared phytin in pure form,l also studied its chemicalproperties. Among the salts mentioned2 is one, calcium-magnesium, as well as one crystalline, calcium- sodium, double salt, for which he gives the formula, 2C2H4Pz09Na4 +C2H4PzCaz + 8H20. Winterstein describes a calcium-magne-

    sium compound which, after removing the calcium with oxalic acid and precipitating with alcohol, contained 42.24 per cent PZOs and 12.97 per cent MgO. Patten and Hart,4 working in this laboratory, isolated from wheat bran an impure magnesium- calcium-potassium compound. Levene5 describes a semi-crystal- line barium salt which corresponds to a tetra-barium phytate. Vorbrodt! mentions a crystalline barium salt obtained by partially

    1 Rev. gkn. de bot., xii, p. 5; Compt. rend. acad. des xi., cxxxvii, p. 202. 2 Compt. rend. acad. des xi., cxxxvii, pp. 337 and 439. 8 Ber. d. d. them. Gesellsch., xxx, p. 2299. 4 Amer. Chem. Journ., xxxi, p. 566. 6 Biochem. Zeitschr., xvi, p. 399. 6 Anzeiger Akad. Wiss. Krakau, 1910, Series A, p. 414.


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  • 472 Phytin and Esters of Inosite

    neutralizing phytic acid with barium hydroxide and evaporating in vacuum, to which he assigns the formula, C12H26046Ba7Pll. Although crystalline, this compound was undoubtedly impure. By neutralizing the mother-liquor from the above with barium hydroxide he obtained an amorphous precipitate of the composi- tion C, 5.75; H, 0.77; Ba, 52.97; P, 11.60 per cent. This corre- sponds approximately with a hexa-barium phytate.

    Of the several salts mentioned in this paper some were obtained from commerbial phytin and from an organic-phosphorus-mag- nesium compound by precipitating with barium chloride and barium hydroxide; others were prepared from previously purified phytic acid. These products will be more fully described in the experimental part.

    The tri-barium phytate, CsH1209[(P0~H)z Ba18, is obtained pure as an amorphous white powder by repeatedly precipitating barium phytate in 0.5 per cent hydrochloric acid with a like volume of alcohol. It may also be obtained in crystalline form by dissolving the amorphous salt in a 10 per cent solution of phytic acid in which it is very soluble and from which it again slowly crystallizes out on standing at ordinary temperature.

    A penta-barium phytate, C6H14027PsBai, is obtained when a solution of the tri-barium phytate in 0.5 per cent hydrochloric acid is neutralized with barium hydroxide and then made faintly acid with acetic acid.

    The penta-barium ammonium phytate, CsH12027P6Ba5(NH&, is obtained when the above mentioned amorphous tri-barium salt is digested with dilute ammonia.

    Thepenta-magnesiumammoniumphytate,CBH,2027PsMgS(NH1)2, is thrown down as a white amorphous precipitate when excess of magnesia mixture is added to an aqueous solution of phytic acid, or when ammonium phytate is precipitated with magnesia mixture.

    A tetra-cupric di-calcium phytate, CsHlzOzrPsCu&az, in nearly pure form is obtained when a slightly acid solution of calcium ammonium phytate is precipitated with excess of copper acetate. If the magnesium ammonium phytate is precipitated under the same conditions an impure compound is obtained which contains about 1 per cent Mg, 0.6 per cent N, 34 per cent Cu and 15.6 per cent P. No effort was made to obtain these salts pure. It was

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  • R. J. Anderson 473

    only desired to find out to what extent other bases were removed when precipitating with copper acetate.

    Starkenstein claims that commercial phytin always contains free inosite together with inorganic phosphates and that merely drying the substance at 100C. causes nearly complete decomposi- tion into inorganic phosphate and free inosite.

    That phytin is so easily decomposed seemed very improbable as several months work on the substance has shown that it is relatively stable when pure and when no miheral acids are present, Moreover Contardi8 reports that when phytin is heated in an auto- clave with pure water for several hours to a temperature of 200C. only very small quantities of inosite could be isolated.

    In order to determine if inosite is present in determinable quantity 100 grams of commercial phytin in the form of the acid calcium salt, imported from Europe and which had been kept, in the laboratory for several years, was shaken up with 1 liter of water, filtered at once and washed with water. The filtrate was precipitated with barium hydroxide, again filtered and the excess of barium precipitated with carbon dioxide and the filtrate from the latter evaporated on the water-bath. In the very slight residue which remained, consisting mostly of barium carbonate with a trace of barium chloride, no trace of inosite could be detected by the most painstaking method of isolation. Of the same phytin, lQ0 grams were dried to constant weight at 115C. and was then treated in the same manner. Even here no trace of inosite could be obtained. Subjecting to the same treatment 50 grams of the same phytin, after previously mixing with 0.5 gram ihosite, resulted in the recovery of 0.4 gram inosite.

    This proves that phytin is by no means so easily split as Starken- stein claims. The results in his case may have been due to other causes besides mere drying at 100C.

    The same author (Zoc. cit.) also states that when phytic acid is precipitated with ammoniacal magnesia mixture it is not the mag- nesium ammonium compound which is formed but only the diffi- cultly soluble magnesium phytate. This is an error. Under these conditions the previously mentioned penta-magnesium ammonium phytate, CsH12027PaMg,(NH&, is formed.

    For the free phytic acid Pbsternakg proposed the empirical for- mula, CzHs09Pz, which he considered to have the following con- stitution:

    7 Biochem. Zeitschr.. xxx, p. 59. 8 Atti R. Accad. dei Lincei Roma (5), xviii, i, p. 64. 9 Compt. rend. acad. des sci., cxxxvii, p. 439.

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  • 474 Phytin and Esters of Inosite

    H I OjCHO. po (OHI

    $THO. PO (OH)3

    and which finds expression in the name anhydro-oxymethylen di-phosphoric acid.

    As is well known the free acid, as well as its salts, is easily split under the influence of dilute mineral acids into inosite and ortho- phosphoric acid. This fact and the discovery by NeuberglO that both inosite and phytin yield furfurol when distilled with phos- phorus pentoxide and phosphoric acid, respectively, lead him to believe that the inosite ring exists already formed in phytin. In accordance with this view he proposed the following structural formula fop the acid:

    /O\ / \ (OH)aP $(OHJ3


    (OH>3 1 1 (OH)3 P- 0-CH CH-O-P

    O< II > (OH), P-0-CH-CH-O-P (OH)3

    This is just treble the molecular weight of the anhydro-oxymeth- ylen di-phosphoric acid of Posternak.

    Suzuki and Yoshim;rall considered that phytic acid was the hexa-phosphoric acid of inosite.

    Starkenstein12 believes that phytin represents a cqmplex pyro- phosphoric acid compound with inosite and he proposes the follow- ing constitutional formula:

    lo Biochem. Zeitschr., ix, pp. 551 and 557. I1 Bull. Coil. of Agric. Tokyo, vii, p. 495. I2 Biochem. Zeitschr., xxx, p. 56.

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  • R. J. Anderson 475

    (OH)2 (OHIs P=O.HO.HC-CH.OH.O=P

    I 10 /

    (0% P=O.HO.HC CH.OH.O=P

    I I


    (OH)2Pq0.H0.HC-CH.OH.O=P (OH)2






    Vorbrodt (Zoc. tit.) proposes still another formula. It is impossible at the present time to decide definitely beiweeri

    any of the above constitutional formulas, as the substance has not, yet been synthesized in the laboratory.

    As represented by the empirical formula, CsH2402,Pe, phytic acid corresponds to a hexa-phosphoric acid ester of inosite plus 3H20, CsHsOti PO(OH)zle, + 3H20.

    At present it is impossible to say whether the compound repre- sents a pyrophosphate or if the water is linked in some other way. That the acid contains twelve acid (OH) groups as expressed in the formula of Starkenstein, which wouldalso be the case if it were a hexa-phosphoric acid ester of inosite, and not eighteen (OH) groups as in the formula of Neuberg, seems certain, for in no case have we been able to prepare any salt in which more than twelve H- valences were replaced by bases.

    As observed by Starkenstein only one-half of the twelve (OH) groups are particularly reactive. This finds expression in the fact that the barium salt obtained from .acid solutions contains only 3Ba to 6P. As