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NEW York STATE COLLEGES
OF
AGRICULTURE AND HoME EcoNoMICS

CORNELL UNIVERSITY

898.P6A54
organic phosph

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The Occurrence of Organic
Phosphorus .Compounds in Plants

A THESIS

Presented to the Faculty of the Graduate School
of Cornell University for the Degree of
Doctor of Philosophy

By
RUDOLPH J. ANDERSON

May, 1919

The Occurrence of Organic

Phosphorus Compounds in Plants

A THESIS

Presented to the Faculty of the Graduate School
of Cornell University for the Degree! 'of
Doctor of Philosophy

By
RUDOLPH J. ANDERSON

CORNELL
UNIVE! SELY
May, ,1919
r

Table of Contents

PAGE
ITE FOG MCELON > .xeorecn aa ahs wanes nord caermcysh acted ath hese omcre nt omone Rate ON 5
Inosite phosphoric acid’ or phytin: << cacacccaasiusew otaes ohh sn aesieaiadanowes 7
Distribution of inosite phosphoric acid in the plant world................... 8
Nomenclature and constitution of inosite phosphoric acid.................... 9
Properties of inosite phosphoric acid.............c0c cece cece eee eee eeeeees 14
Properties of salts of inosite hexaphosphoric acid.................0.eseeeae 16
Synthetic esters of inosite and phosphoric acid............. 00.0 ccc eee ee eee 18
Methods for estimating inosite phosphoric acid............0.....ccceseeaeeee 19
Other phosphoric acid esters occuring in plant material.................0.006 20
Effect of enzymes upon inosite phosphoric acid.............0 000. cece eee eeee 23
Methods for isolating inosite phosphoric acids from plant material............ 24-32
Preparation of crystalline tribarium inosite hexaphosphate.................. 28
Preparation of the free inosite hexpahosphoric acid....................00008 29
Method of analysis of barium inosite hexaphosphate...............0...00005 29
Composition of crystalline barium inosite hexaphosphates................... 31
Composition of free inosite hexaphosphoric acid...............00. cee ee ee eee 31
Preparation of inosite monophosphoric acid..............0...00 ecu eeeeeee 34-37-47
Properties of inosite monophosphoric acid...............c cece cece ee eeeees an, 235
Cleavage of inosite monophosphoric acid..............0000cc cece eee btanitiadiiceas 36
Isolation of inosite triphosphoric acid............00 000s ccc eee eee cee eee eae 37
Preparation of strychnine and barium salts of inosite triphosphoric acid..... 38-39
Preparation of the free inosite triphosphoric acid................e0eceee eee 41
Properties of inosite triphosphoric acid............e cece ccc e cece eee encase 42
Cleavage of inosite triphosphoric acid.......... 0... ccc cece ccc eee eee 42
Hydrolysis of phytin by the enzyme phytase...........0.0....000. cece eee 43
Tsolation4Of ANOSIt@h ses coenais aise cleemnanueuid wiu'e ae gee eine aga A ved oe ecen ne 36-42-47
Hydrolysis of the organic phosphorus compound of wheat bran by the enzyme
SIDES OP sissy ah ay Mca de cohub ash Gs ursnaiva seems atuipad peat an diley eA oes apeaagmaabaaannaela Ue nde & gue hs Mater 48
Destruction of the enzyme phytase.......... 0... 0c cece eee e ete n ee eee ens 51
Concerning some chemical properties of inosite hexaphosphoric acid......... 52
Is inosite formed in the spontaneous decomposition of inosite hexaphosphoric
BUTE cg oe cay cesta vt by Seuenavee Akh hse easienny Hsu Pate OR SEAHES Sota leans lee thcwate oee aykk ai TaN 55
Decomposition products of inosite hexaphosphoric acid after drying at 105°.. 57
Methyl ester of inosite hexaphosphoric acid............. 00. cc cece cece veces 61
Summary .cungeadnds sive ereer eed cee wa ted Aen awe es ea eS ees 62

Bibliography: ceiteesciaaeia2 each s Madnmiaa ere aed ed oGncoeebadad oduct invades 64

The Occurrence of Organic Phosphorus

Compounds in Plants*

INTRODUCTION

It has been recognized for a long time that only a small part of
the total phosphorus contained in plant material is present as inor-
ganic phosphate because only very small quantities of phosphoric acid
could be precipitated from solutions obtained by digesting powdered
plant material, rich in phosphorus, in water or dilute acids (Hart and
Andrews 1903). In order to determine total phosphorus it was neces-
sary either to ash the material or subject it to complete hydrolysis with
mineral acids.

It is evident, therefore, that the greater percentage of the phos-
phorus existing in plant cells is combined in some way with organic
compounds. The knowledge of the various groups of organic phos-
phorus compounds existing in plants has been gradually developed
through the efforts of numerous investigators. Although much work
has already been done on this subject, the field is not exhausted; future
investigations will probably reveal a number of new phosphorus com-
pounds elaborated in plant cells.

About the middle of the last century it was found that a small
percentage of the phosphorus in vegetable material, particularly in
seeds, could be extracted with ether or other solvents for fats (Knop
1860 and Tépler 1861). This class of ether-soluble phosphorus com-
‘pounds was shown to be closely related to lecithin and the phospha-
tides, if not identical with similar substances isolated from animal
tissues (Hoppe-Seyler 1867; Jacobson 1889; Schulze and Likiernik
1891). The wide distribution of lecithin in the plant world was shown
‘by Heckel and Schlagdenhauffen (1886). The plant phosphatides have
been extensively investigated in Schulze’s laboratory. A large num-
ber of important papers dealing with methods of estimation, com-
position and properties have been published by Schulze and Winter- .
stein and their students.

* The experimental work reported in this thesis was done in the Chemical
Laboratory of the New York Agricultural Experiment Station, Geneva, N. Y.,
in the Chemical Laboratory of the University of Berlin and in the Ludwig Mond
Biochemical Research Laboratory of the Institute of Physiology, University
College, London; and the series of papers dealing with this subject, as given in
-the bibliography, was originally published in the “Journal of Biological Chemistry.”

A monograph on Iccithin in which the above subject is reviewed,
with a complete list of references, by Maclean (1918) has recently been
published.

The first plant nucleic acid was discovered by Altmann (1889)
as a constituent of yeast cells. This substance was similar to nucleic
acids isolated from animal tissues, but it showed certain differences in
composition, particularly in cleavage products (Kossel 1891, 1893,
1894). Osborne and Harris (1902) described the triticonucleic acid
obtained from wheat embryo. This substance was found to be similar
to yeast nucleic acid and the identity of the two acids was established
by the investigations of Levene and La Forge (1910). It appears,
therefore, that there is only one plant nucleic acid. This substance
has been very extensively investigated by Levene, Levene and Jacobs
and more recently by Jones and Read (1917). The structure of plant
or yeast nucleic acid has not yet been fully established. It has been
shown, however, by Levene and Levene and Jacobs that the molecule
of yeast nucleic acid contains four nucleotides which are made up of
the following constituents:

Phosphoric acid — d — ribose — guanine
Phosphoric acid — d — ribose — cytosine
Phosphoric acid — d — ribose — uracil
Phosphoric acid — d — ribose — adenine

This subject is extensively reviewed in the monograph by Jones
(1914), “Nucleic Acids: Their Chemical Properties and Physiological
Conduct.”

The observation that fermentation is accelerated by the addition
of phosphates was first made by Wroblewski (1901) and later by
Buchner and Hahn (1903). Studies made since that time have shown
that during fermentation inorganic phosphate disappears and that an
organic phosphoric acid is produced. Phosphates are apparently essen-
tial in the process of fermentation and in the absence of phosphates no
fermentation can take place (Harden 1914). The nature of the organic
phosphoric acid and the conditions under which it is formed have been
studied by Harden and Young (1905, 1908, 1909, 1911), by Iwanoff
(1905, 1909), Young (1909, 1911), Lebedeff (1909, 1910. 1911, 1912,
1913) and by Euler (1912) and his students (1911, 1912).

There is no complete agreement among these investigators as to
the constitution of this organic phosphoric acid. Iwanoff regards it
as a triosemonophosphoric acid, C,H,O, (PO,H,), while Harden and
Young believe that it is a hexosediphosphoric acid, C,H,,O, (PO,H,)>.
Euler believes that a mixture of these two acids is formed during fer-
mentation. For further particulars regarding this interesting phos-
phoric acid, reference is made to the monograph by Harden (1914).

Another interesting substance, the phosphoric acid of starch, has

recently been investigated by Northrup and Nelson (1916). It has
been known for some time that starch contained a small percentage of
phosphorus which could not be completely removed by washing,
dialysis or by repeated precipitations (Thomas 1914). Samec (1914)
believed that the phosphoric acid in starch was present in an ester com-
bination and he called it amylophosphoric acid. Northrup and Nelson
succeeded in isolating a new organic phosphoric acid from partially
hydrolyzed starch, corresponding to the formula C,,H,,0,,P. The acid
is optically active, reduces Fehling’s solution and on hydrolysis yields
glucose and phosphoric acid.

The phosphorus compounds occurring in plant or vegetable
material may be divided, on the basis of our present knowledge, into
the following groups:

1. Inorganic phosphates.

2. Nucleic acid.

3. Lecithin or phosphatides.

4. Hexosephosphoric acid.

5. Starch phosphoric acid and other combinations of organic
radicals with phosphoric acid.

6. Inosite phosphoric acids or phytin.

The inorganic phosphates will not be discussed in this paper.

According to Osborne (1918), there is no evidence of the occur-
rence of phosphoproteins in plants although references are frequently
found in the literature to plant phosphoprotein.

The inosite phosphoric acid, or phytin, is quantitatively the most
important phosphorus compound found in plants. The chemistry and
properties of this substance, as well as closely related inosite phos-
phoric acids, will be particularly considered in this paper.

A large number of investigations have been made regarding the
metabolism and utilization of organic phosphorus compounds in the
animal organism. This subject has been exhaustively reviewed by
Forbes and Keith (1914), to which reference is made.

INOSITE PHOSPHORIC ACID OR PHYTIN

In the excellent resumé of the literature on the subject of inosite
phosphoric acid by Rose (1912) will be found an interesting histori-
cal sketch of the investigations which led up to the discovery of inosite
phosphoric acid. It may be stated briefly here that the first chemical
examination of the substance was made by Brandau under Pfefters
direction (1872), the object being to determine the constituents of the
aleuron grains which had been described and named earlier by Hartig
(1855). This investigation revealed the fact that the aleuron grains
from seeds, named “globoid” by Pfeffer, contained phosphorus, cal-
cium and magnesium.

The next contributions to this subject were made from Schulze s
laboratory. Palladin, Schulze and Winterstein, Winterstein and later
Posternak studied the chemical properties and composition of the sub-
stance. ‘Palladin (1894+) discovered that a substance containing phos-
phorus, calcium and magnesium, insoluble in hot but soluble in cold
water, could be obtained from the seed of senapis niger. The work of
Palladin was later confirmed and amplified by Schulze and Winterstein
(1896). These authors believed that the substance in question was
identical with the globoid of Pfeffer. \Winterstein (1897) described
the substance more closely and also studied its cleavage products.
This author was able to show that it gave inosite and phosphoric acid
when hydrolyzed in a sealed tube and, therefore, suggested the name
inosite phosphoric acid. Somewhat later Posternak took up the study
of this subject (1900, 1903, 1904, 1905). ‘This author not only studied
the chemical nature of the product, preparing and analyzing many salts
as well as the free acid, but he also elaborated plans and methods for
the production of phytin on a commercial scale.

During the course of a study of the effect upon the metabolism of
ash constituents, particularly phosphorus, inaugurated by Jordan
(1906), Patten and Hart (1904) showed that phytin was present in
wheat bran. The occurrence of the same substance in rice bran and
other plant material was subsequently demonstrated by Suzuki, Yoshi-
mura and Takaishi (1907). They also discovered that rice bran and
wheat bran contained an enzyme capable of hydrolyzing phytin into
inosite and phosphoric acid. This enzyme they named “phytase.”

Notable contributions to the chemistry and distribution of phytin
were made later by Vorbrodt (1910) and particularly by Starkenstein
(1908, 1910, 1911). Levene (1909) also studied a preparation obtained
from hemp seed. Hart and Tottingham (1909) carried out extensive
investigations regarding the nature of the organic phosphoric acid
occurring in a number of grains, tubers and feeding material. They
concluded that phytin was present in all of them except dried alfalfa
and rutabagas. Rising (1909) reported the occurrence of the same sub-
stance in barley and other grains and in flour.

More recent investigations of phytin or inosite phosphoric acid
preparations have been reported by Anderson (1912, 1914, 1915),
Plimmer and Page (1913), Plimmer (1913), Thompson (1915), Rather
(1913, 1917, 1918), Jegorow (1913, 1914, 1915), Clarke (1914, 1915),
Heubner (1914), Robinson and Mueller (1915) and Boutwell (1917).
Special reference to these and other investigations will be made below.

DISTRIBUTION OF INOSITE PHOSPHORIC ACID IN THE
PLANT WORLD

It is evident from the result of investigations mentioned above

that salts of inosite phosphoric.acid exist in all seeds. In the cereal
grains the outer layers or bran are particularly rich in organic phos-
phorus which is largely composed of inosite phosphoric acid. The
substance is also present in various roots and tubers, such as potatoes,
radishes, turnips and dahlias, also in clover leaves and in flowering
millet. This list will undoubtedly be extended through further investi-
‘gations, but it shows that inosite phosphoric acid is very extensively
distributed in the vegetable kingdom and that it is present in practi-
cally all of the material so far examined.

AS TO THE NOMENCLATURE AND CONSTITUTION OF
INOSITE PHOSPHORIC ACID

Winterstein, as mentioned above, found that inosite and phos-
phoric acid were produced on hydrolyzing the substance in a sealed
tube. Without determining the actual relation between carbon and
phosphorus, he named the acid inosite phosphoric acid, but suggested
no constitutional formula. Posternak, as a result of his extensive in-
vestigations, concluded that the empirical formula of the acid was
C,H,O,P,; i. e., the relation of C : P was as 2 :2 and he believed it to
have the following constitution:

H
| OH
C.H.O-P=0
fp OH
O
X& OH
C.H.O—P=0
a OH
H

‘This relation found expression in the name “anhydro-oxy-methylen
diphosphoric acid.” On hydrolysis the “anhydrooxymethylen,” accord-
ing to this author, combined, producing inosite by synthesis.

The same author gave the name “phytin” to the calcium salt of
this acid, which was placed on the market as a pharmaceutical prepar-
ation. Since that time the organic phosphorus compounds isolated
from various plant material have been described under the name of
phytin, phytates or phytic acid, as well as inosite phosphoric acid. Phytin
is a trade name for a commercial product and the use of this name for
various organic phosphorus compounds isolated from plants which fre-
quently differ in composition is likely to lead to confusion. The use of
the terms inosite, hexa-, penta-, etc., phosphoric acid in the scientific
literature to designate-such substances would probably be more
desirable.

Missing Page

and rice polish. All of these preparations are said to be identical and
ail correspond to an acid having the following composition: C,,H,,O,,P,
_or else inosite pentaphosphoric acid, C,H,,O,,P;.

It is very evident from this resumé that there is still great con-
fusion in the minds of the investigators who are working on this sub-
ject as to the actual composition of the inosite phosphoric acid.
Nearly every one who embarks upon an investigation in this field
reports entirely different results from any predecessor. This condition
merely emphasizes the difficulty encountered in purifying the prepara-
tions which are isolated. Further complications that arise are due to
the fact that the substance originally present in the plant material may
undergo changes of various kinds during the process of isolation. It
may suffer partial hydrolysis through the activity of the enzyme
(phytase) contained in the material itself or such partial hydrolysis
may be caused by the reagents or processes employed in the purifica-
tion ; the final product which is obtained may represent, therefore, not
the substance as it existed in the plant, but a mixture of such frag-
ments of the molecules of the inosite phosphoric acid as escaped either
the action of the enzymes or the more or less drastic action of the
reagents. The fact that the same investigator has produced appar-
ently identical results from a number of different materials is not
necessarily a sign of the purity of the preparations; being isolated by
identical methods, the same errors are carried over. As an example of
this kind of error we need only mention the fact that we (Anderson
1912, (3) 1914 (5) ) were unable to obtain any inosite phosphoric acid
having the composition of inosite hexaphosphoric acid from a 0.2 per
cent. hydrochloric acid extract of wheat bran. Instead of inosite hexa-
phosphoric acid we obtained consistently an amorphous barium salt
corresponding to the formula, C,,H,,0,,P,Ba,;, which on hydrolysis
gave inosite and phosphoric acid. This compound was obtained re-
peatedly and we were entirely unable to isolate a substance having any
other composition. Continued experiments with this compound
showed, however, that it was not homogeneous but that it could be
separated into various fractions and finally a substance was obtained
from this compound which corresponded in composition to inosite tri-
phosphoric acid, C,H,,O,,P,. The original substance was therefore
undoubtedly a mixture of inosite di- and tri-phosphoric acids with pos-

. sibly some other admixtures. The extracts obtained on digesting
ground seeds, bran or other plant material in dilute acid contain a
large number of soluble constituents in addition to the phosphorus
compounds. It contains among others, also, soluble carbohydrates,
such as pentoses which are carried down in the precipitates of the
inosite phosphoric acids. Levene (1909), for instance, noted a pentose
in the preparation which he analyzed. In the barium salt prepared

from wheat bran we (Anderson 1912 (3) ) obtained evidence of the
presence of pentose and Rather (1913) also showed that such prepara-
tions contain varying amounts of pentose. Small quantities of these
soluble carbohydrates adhere to the inosite phosphoric acid prepara-
tions with great tenacity. We have noticed that barium salts of
inosite phosphoric acid isolated from various plant material gener-
ally show too high a percentage of carbon in the early stages of puri-
fication.

In order to obtain salts having constant composition it is neces-
sary to recrystallize them a great number of times. We have found
that it is possible to obtain preparations having uniform composition
after repeated, careful recrystallization of the products. After this
degree of purity has been attained further crystallization does not alter
the composition. All of the crystalline barium salts which we have
thus obtained correspond in composition to salts of inosite hexaphos-
phoric acid; i. e., the relation of C: P is as 6:6. We believe for this
reason that the inosite phosphoric acid exists in plants or in the
majority of plant material at least as salts of inosite hexaphosphoric
acid. In this connection it should be notéd that it is very much easier
to obtain preparations from plant extracts which differ in composition
from those just mentioned. In order to obtain preparations corre-
sponding in composition to salts of inosite hexaphosphoric acid the very
greatest care is necessary in the manipulations connected with the isolation
and purification. In isolating the material from wheat bran, for instance,
the bran must not be digested in 0.2 per cent. hydrochloric acid, but in
an acid solution of such strength as to inhibit the activity of the
enzyme phytase. The same applies when isolating the preparation
from rice bran. Unfortunately, several investigators appear to have
overlooked this important fact. It might be argued that repeated re-
crystallizations might cause decomposition or partial hydrolysis of
the product, for these purifications naturally require considerable time.
Of course, that danger always exists. We have always employed as
mild methods as possible, avoiding contact with strong acid and care-
fully avoiding heat. The various operations have been performed as
rapidly as possible. But even when working with the greatest pos-
sible rapidity it is sometimes not possible to complete the isolation
and crystallization of a particular preparation in less time than a week
or ten days. Ifa notable amount of hydrolysis occurred during this
time when the substances during crystallizations are in contact with
dilute acid for varying periods, the resulting product should show a
steady increase in carbon content and a decrease in the percentage of
phosphorus because the hydrolysis is progressive, the molecules of
phosphoric acid splitting off one by one. This is not the case, how-
ever, for, as mentioned above, such carefully recrystallized barium

salts contain carbon and phosphorus in the ratio of 6 :6, whereas in
less carefully purified products the percentage of carbon is generally
higher.

The contradictory results in ultimate composition obtained by vari-
ous investigators of the inosite phosphoric acids from seeds or other
plant material can be explained, it seems to us, only as being due to
any one of the following three causes; viz:

1. Insufficient purification. The preparations which have been
analyzed have either contained unknown quantities of inorganic im-
purities or else organic complexes, such as pentoses or other carbohy-
drates, have been carried down by absorption with the salts of inosite
phosphoric acid.

2. Partial hydrolysis during isolation due to enzyme action or
partial hydrolysis during the process of purification due to repeated
boiling of the solutions of salts of the inosite phosphoric acid or the
repeated evaporation of aqueous or alcoholic solutions of the acid
itself.

3. The existence in plant material of different inosite phosphoric
acids.

This last cause might be quite important in connection with cer-
tain seed cakes, stich as cottoonseed meal. Varying degrees of heat are
applied to the crushed seed during the expression of the oil. This
process might affect the composition of the resulting residue or the
meal might be stored under conditions of heat and moisture which
may favor enzymatic processes. In one report Rather (1917 (1) ) states
that he obtained an inosite phosphoric acid from cottonseed meal
which correspond in composition to inosite triphosphoric acid, but
he was never able to isolate this acid from any other sample of the
material.

PROPERTIES OF INOSITE PHOSPHORIC ACID

The inosite hexaphosphoric acid, when prepared from pure salts,
is obtained as a colorless syrup, the consistency, of course, depending
upon the amount of water which is present. When the acid has been
dried in a desiccator over sulfuric acid until it is practically water
free it forms a solid, glassy, brittle mass which is very hygroscopic.
The acid, which is colorless when freshly prepared, assumes gradually
at room temperature a light yellow, yellowish, straw and finally, after
many months, a very dark color. The color change, which is very
gradual at room temperature, is very much hastened by heat. After
drying at 100° for a few hours to constant weight the acid is practi-
cally black in color. Drying at the temperature of boiling chloroform
or alcohol gives a product which is light brown or brown in color.
This change in color is associated with some decomposition and sep-

aration of inorganic phosphoric acid. Concentrated solutions of the
acid undergo gradual spontaneous decomposition at room temperature,
but much more rapidly when heated. The decomposition is only par-
tial; i. e., there is formed inorganic phosphoric acid but no inosite.
After standing for 18 months a sample of the acid contained con-
siderable free phosphoric acid but no inosite. Heating this same acid
for 48 hours to 100° caused some of the acid to decompose completely
because inosite was found as one of the products of decomposition, but
only to a small extent. Dilute solutions of the freshly prepared acid
give no precipitate with ammonium molybdate either in the cold or
on heating for half an hour. at 60°. Continued heating at 60°, however,
causes a gradual separation of a small amount of yellow precipitate
due to hydrolysis of the acid with formation of phosphoric acid. In
concentrated solutions ammonium molybdate produces a _ white
crystalline precipitate. The acid is soluble in all proportions in
alcohol, but insoluble in other organic solvents. The addition of
ether to an alcoholic solution of the acid causes a cloudiness and the
acid gradually separates out in minute oily drops. The acid coagu-
lates egg albumin immediately, even in dilute solutions. The alkali
salts are very soluble in water. Posternak states that the acid alkali
salts are also soluble in alcohol and water. Aqueous solutions of the
acid are not precipitated by the chlorides of the alkaline earths, but
the acetates and hydroxides of these metals give white amorphous pre-
cipitates. Magnesium acetate, however, precipitates the acid only
from neutral solutions. Alcohol produces in solutions containing the
acid and the above chlorides white amorphous precipitates, but the
salts of magnesium and calcium are less completely precipitated than
those of strontium and barium. While barium chloride and the acid
give no precipitate, yet on allowing such a solution to stand a few
hours, needle-shaped crystals of the barium salt separate. Silver nitrate
in excess produces a heavy, white, amorphous precipitate. Precipitates
are also produced with other heavy metals. Copper acetate in excess
produces green colored precipitates, even in the presence of dilute
acetic, hydrochloric of sulfuric acids. Lead acetate gives white pre-
cipitates in solutions of the free acid or in solutions of its salts in
acetic acid. The copper and lead salts of inosite phosphoric acid are
particularly suitable for isolating the acid because thesé ‘metals can.
be easily removed by hydrogen sulfide. All of the salts of inosite
hexaphosphoric acid except the iron salt are more or less easily soluble
in dilute mineral acids. The iron salt is very sparingly soluble, even
in strong hydrochloric acid. The great insolubility of this salt in
dilute acids has led to the development of a volumetric method by
Heubner and Stadler (1914) for determining _ iflosite phosphoric acid

by titrating with a standardizéd solution of if

ifon chloride, using ammo-

nium thiocyanate as indicator. The barium salt can be obtained im
the form of spherical masses consisting of microscopic needles; other:
salts with inorganic bases crystallize much less readily or not at all.
Posternak describes, however, several double salts which were crystal-
line. The only organic base found so far that forms readily crystalline
salts with inosite hexaphosphoric acid is strychnine, as was first shown
by Clarke (1914).

PROPERTIES OF THE SALTS OF INOSITE HEXA-
PHOSPHORIC ACID

The alkali salts of the acid have not been obtained in crystalline
form. The magnesium and calcium salts have no definite crystalline
structure, but are obtained under certain conditions as small spherules
or globules of uniform size. Posternak obtained double salts of the
acid with alkali and alkaline éarths which crystallized in small needles.
Of the salts with inorganic bases the acid barium salts crystallize most.
readily. If the barium salt dissolved in dilute hydrochloric acid is pre-
cipitated with alcohol an amorphous precipitate is formed which, how-
ever, on standing in contact with the aqueous alcoholic mother-liquor,,.
usually assumes the crystalline form after a few hours. These crystals.
are minute rosettes consisting of microscopic needles. The same
shaped crystals may be obtained by carefully adding alcohol to the
dilute hydrochloric acid solution of the barium salt until a slight
cloudiness appears. The solution is now filtered and allowed to stand,
when the salt separates out in the above-mentioned crystalline form.
These salts are crystalline, but they do not contain a constant quantity
of barium unless an excess of barium chloride is added to the solution.
before crystallization begins. It is also necessary to provide as nearly
identical conditions as possible as to concentration of the salt and
acid ; slight variation in the amount of free hydrochloric acid present
will cause’a difference of 1.5 to 2 per cent. in the amount uf barium in
the crystalline salt. Similar crystalline barium salts are obtained
when solutions of the barium salt in dilute hydrochloric acid are heated’
nearly to boiling, only in this case the salt contains about 4 per cent..
more barium than when separated from the acid alcohol mixture men-
tioned above. A salt of similar crystal form and composition is also:
formed when the dilute hydrochloric acid solution of the salt is nearly
neutralized with dilute barium hydrate, filtered and allowed to stand...
The first salts mentioned above correspond to tribarium inosite hexa-
phosphate, C,H,,0,,P,Ba,, and the last mentioned salts correspond
to heptabarium inosite hexaphosphates, (C,H,,0,,P,). Ba,, or else
to equimolecular mixtures of tribarium and tetrabarium inosite hexa-
phosphates, C,H,,0,,P,Ba, + C,H,,O0,,P,Ba,. The tribarium ino--

site hexaphosphate contains water of crystallization corresponding to
8 molecules H,O and the water of crystallization in the heptabarium
salt corresponds to 14 molecules H,O. These crystalline barium salts
are free from inorganic phosphoric acid. However, after standing for
several months at room temperature in a well closed bottle a slight
reaction for inorganic phosphoric acid will be obtained with ammo-
nium molybdate. Drying these salts at 100° likewise causes a slight
liberation of free phosphoric acid.

As intimated above, inosite phosphoric acid generally forms acid
salts; in fact, it is very difficult to obtain any salt of this acid, even
from neutral solutions, which does not show an acid reaction on moist
litmus paper. The silver salt, for instance, obtained by precipitating
a solution of the acid neutralized with ammonia contains only about
8 or 7 equivalents of silver and has a strong acid reaction.

It has been mentioned previously that only one-half of the acid
hydroxyls enter readily into reaction with bases. Salts with binary
bases, such as barium, contain therefore only 3 atoms of the base when
separated from slightly acid solutions. Starkenstein (1911) also men-
tions this fact. In titrating the acid with standard uranium acetate
he observed that he obtained only one-half the value for phosphorus.
These reactive acid hydroxyls are probably adjacent, but linked to dif-
ferent phosphoric acid residue, giving salts with binary bases of the
following type:

The salts of inosite phosphoric acid are more soluble in cold than
in hot water. This fact was first observed by Palladin (1894) and led
to the discovery of the substance in an aqueous extract of senapis
niger. Advantage has frequently been taken of this property in iso-
lating crude salts of the acid from acidulated aqueous extracts of plant
material. The dilute acid extract is heated to boiling. This coagu-
lates the proteins and the alkaline earth salts of inosite phosphoric
acid are also precipitated, but they redissolve on cooling the liquid.
After cooling, the permanent precipitate, consisting of proteins, etc.,
is filtered off. The filtrate is again heated to boiling and the precipi-
tate which then forms represents the crude phytin preparation mixed
with some inorganic phosphate. ;

Pure salts of inosite phosphoric acid are generally white or color-

less. The copper salt is bluish green and the iron salt is yellowish
brown in color. Various salts of inosite phosphoric acid which we
have prepared and analyzed will be described in the experimental part.

SYNTHETIC ESTERS OF INOSITE AND PHOSPHORIC ACID

Several unsuccessful attempts have been made to synthesize phy-
tic acid or the inosite phosphoric acid. Neuberg and Kretschmer
(1911) report obtaining a polyphosphoric acid ester of inosite by
their method of preparing phosphoric acid esters of carbohydrates
and glycerin; that is, by the action of phosphorus oxychloride. They
could not obtain any pure product, however, as it was found impos-
sible to separate the new compound from inorganic phosphates.

Contardi (1910) claims to have succeeded in preparing inosite
hexaphosphoric acid by heating inosite with an excess of phosphoric
acid at a temperature of 160 to 165° in an atmosphere of carbon dioxide.
The product was purified as the barium salt and the free ester whicn
was finally obtained he described as identical with “phytic acid” which
was prepared from rice bran. The same author claims to have pre-
pared polyphosphoric acid esters of mannite quercite and glucose by
the same methods.

Carré (1911) was unable to substantiate the results of Contardi
and stated that the reaction mixtures in question consisted only of
free phophoric acid and the poly-hydric alcohols, together with some
decomposition products.

Contardi (1912) answered this criticism by repeating his work and
claimed to show that his original conclusions were correct.

The results of our experiments showed that inosite and phosphoric
acid react when heated to 140 to 160° in vacuum and that water is
given off. The reaction product after purifying as a barium salt and
removing inorganic phosphoric acid gave results on analysis corre-
spending to inosite tetraphosphoric acid, C,H, (OH), (PO,H,),,
(Anderson 1912 (1) ). 1t was also found that inosite and pyrophos-~
phoric acid react at a temperature of about 200° with the production
of esters (Anderson 1912 (2) ).

Jegorow (1914 (1) ) has more recently reported some experiments
along the same line. Inosite and phosphoric acid were heated in a
flask in an atmosphere of hydrogen or nitrogen. He noted that the
final reaction product contained 22.6 per cent of organic phosphorus.

There is no doubt but that esters of phosphoric acid are produced
when inosite and phosphoric acid are heated together, but we do not
believe that inosite hexaphosphoric acid or phytic acid can ever be
prepared in this way. Contardi, for the reasons mentioned below,
must have been in error when he claimed to have synthesized the sub-
stance by this method. Numerous experiments have shown that phytic

acid can not be dried even at a temperature under 100° in vacuum
over phosphorus pentoxide without undergoing a very considerable
decomposition with liberation of free phosphoric acid. Attempts to
synthesize the substance by subjecting mixtures of inosite and phos-
phoric acid to a high temperature would appear, therefore, to be
doomed to failure.

METHODS FOR ESTIMATING INOSITE PHOSPHORIC ACID

Inosite phosphoric acid or its salts can not be extracted from plant
material and quantitatively determined by any direct method. Indi-
rect methods of estimation, therefore, were developed and used. More
recently efforts have been made to perfect direct volumetric methods
for the determination of the acid. The earlier methods consisted in
extracting the plant material with dilute hydrochloric or acetic acids
and determining the inorganic phosphoric acid and the total soluble
phosphorus in the resulting extract. The difference between the two
values thus obtained was regarded as phytin phosphorus. Ammonium
molybdate precipitates the inorganic phosphoric acid but not organic
phosphoric acids, hence it was possible to effect an approximately
accurate separation of inorganic phosphate in the presence of inosite
phosphoric acid. The total soluble phosphorus in the extract was
determined after destroying organic matter by the Neumann method.

Hart and Andrews (1903) worked out the first method along the
line mentioned above for determining organic and inorganic phos-
phorus in plant material. They employed a practically neutral solu-
tion of ammonium molybdate. These authors believed that it was
necessary to restrict as much as possible the amount of free acid in
the precipitating mixture in order to prevent hydrolysis of the organic
phosphorus compound by the nitric acid. They examined a large
number of plant products and found that practically all of the phos-
phorus was present in organic combination. The method which they
employed has been criticised by several authors, but their results have
in general been confirmed by all other investigators. Various modi-
fications have been proposed for the indirect estimation of organic or
phytin phosphorus in plant material by several investigators, among
whom may be mentioned Schulze and Castoro (1904), Stutzer (1908),
Vorbrodt (1910) and Rising (1911) in Europe and particularly Forbes
and his co-workers (1914, 1915, 1916) in this country. It was shown
by Suzuki and Yoshimura (1907) that the ordinary nitric acid solu-
tion of ammonium molybdate could be used for precipitating. inor-
ganic phosphate in the presence of organic or phytin phosphorus without
effecting a notable cleavage of the organic phosphorus compound. Plim-
mer (1913) (2) has also shown_that it is necessary to heat phytin
dissolved in N/1 or 2N/1 nitric acid for several hours to 75° or higher

19 ‘

before appreciable quantities of inorganic phosphoric acid are split off.
In view of these facts, it appears to be unnecessary to determine inorganic
phosphate in plant material in the presence of a minimum quantity of acid.
lf the usual quantity of nitric acid is present the precipitation of the phos-
phomolybdate is more rapid and complete. Many determinations of inor-
ganic phosphate in plant material have been made by using the ordinary
ammonium molybdate solution and there appears to be no reason to believe
that these results are very far from the truth.

Starkenstein (1911) studied the direct determination of inosite
phosphoric acid by titrating with uranylacetate, using cochineal or
ferrocyanide as indicator. The most promising method for the direct
determination of inosite phosphoric acid is undoubtedly that proposed
by Heubner and Stadler (1914). This method is based upon the fact
that the iron salt of inosite phosphoric acid is very sparingly soluble
in dilute acid. It is possible, therefore, to determine the amount of
inosite phosphoric acid in the presence of phosphoric acid or other
phosphoric acid esters whose salts are soluble in dilute hydrochloric
acid by titrating with ferric chloride, using ammonium thiocyanate as
indicator. The method has recently been used by Rather (1917) in
determining the percentage of inosite phosphoric acid present in a
number of feeding materials.

OTHER PHOSPHORIC ACID ESTERS OF INOSITE
OCCURRING IN PLANT MATERIAL

Evidence of the existence of various phosphoric acid esters of
inosite formed by hydrolysis from phytic acid or inosite hexaphos-
phoric acid was obtained in our study of the properties of phytic acid
(Anderson 1914 (4) ). At that time, however, no definitely homo-
geneous compound could be isolated. Clarke (1914) reported that he
had obtained from wild Indian mustard compounds which corre-
sponded to inosite tetra- and di-phosphoric acid.

Probably the most interesting substance of this kind that has been
obtained from plant material is the inosite monophosphoric acid which
was isolated from wheat bran (Anderson 1914 (6) ). The properties of
this acid differ from those of all other inosite phosphoric acids so far
described. When pure it crystallizes very easily and may be purified
by recrystallization from dilute alcohol or water. All other inosite
phosphoric acids have been obtained only as non-crystallizable syrups.
Inosite monophosphoric acid separates in beautiful, colorless crystals
which melt under decomposition at 190°. All of the salts of this acid,
with the exception of the lead salt, are easily soluable in water, whereas
other inosite phosphoric acids are precipitated by the alkaline earth
and heavy metals. In addition to obtaining this acid from wheat bran

we have isolated the same substance from a mixture of phytin after
this has been acted upon by the enzyme phytase from wheat bran
(Anderson 1915 (2) ). A good yield has been obtained on the partial
hydrolysis of phytin with dilute sulfuric acid in an autoclave. At first
we believed that inosite monophosphoric acid occurred naturally in
the wheat bran. But since the substance is produced through the
action of phytase on phytin and also by the partial hydrolysis of this
substance with sulfuric acid, it seems more reasonable to believe that
it is produced during the extraction by the action of the phytase upon
inosite hexaphosphoric acid which is present in the wheat bran.

As a result of the action of phytase upon phytin certain com-
pounds were also produced which appeared to consist of a mixture of
inosite tri- and di-phosphoric acid. These substances, however, could
not be separated into definitely homogeneous salts (Anderson 1915
(2) ).

We have separated from the mixture of inosite phosphoric acids
obtained from wheat bran on digesting this material in 0.2 per cent.
hydrochloric acid a substance which on analysis gave results corre-
sponding to the composition of inosite triphosphoric acid (Anderson
1915 (1) ). This compound had been purified by means of the crystal-
line strychnine salt and then converted into the barium salt. The
barium salt was amorphous and the free acid was a non-crystallizable
syrup. The oniy claim to homogeneity for this substance, therefore,
is based upon a crystalline strychnine salt and, in view of this fact,
we would not emphasize this claim too strongly. The molecular
weight of such strychnine salt is so high that the acid part of the mole-
cule is very small in comparison with the strychnine and it is quite
impossible to determine accurately from the analysis of these salts
the composition of the acid.

The reactions of the free inosite triphosphoric acid prepared from
the barium salt differed in several particulars from the reactions given
by inosite hexaphosphoric acid. The concentrated aqueous solution
gave no precipitate with ammonium molybdate, while inosite hexa-
phosphoric acid gives a white, crystalline precipitate. Silver nitrate
in excess gave no reaction, while this reagent causes a white precipi-
tate with inosite hexaphosphoric acid. The aqueous solution of the
acid gave only a slight turbidity when added to egg albumin and only
on longer standing was a white precipitate produced.

Rather (1917 (1) ) described a preparation obtained from cotton-
seed meal which corresponded in composition to inosite triphosphoric
acid. This substance had also been separated as a crystalline strych-
nine salt. This author was never able to duplicate this result with any
other sample of cottonseed meal.

Boutwell (1917), in working on the inosite phosphoric acids in

wheat bran, believed that a part of it existed as inosite tetraphosphoric
acid, but no definite compound of this composition was isolated.

As previously mentioned, Rather (1918) in an extensive series of
investigations disputes the existence of inosite hexaphosphoric acid
in any plant material. All of the preparations which he obtained were
regarded as salts of inosite pentaphosphoric acid, C,H, (OH)
(PO,H,);, or of a hypothetical acid having the formula C,,H,,O,,Py.

As a result of our studies we believe that the whole series of pos-
sible esters of inosite and phosphoric acid exists; viz., inosite hexa-,
penta-, tetra-, tri-, di- and monophosphoric acid. \We believe, however,
that the lower esters are formed by hydrolysis through enzymes, acids
or through spontaeous decomposition of a mother substance which is
inosite hexaphosphoric acid. Judging by our experience, the lower
esters are always obtained as mixtures and it is extremely difficult to
separate them. The amorphous barium salts of such lower esters as
the inosite di- and triphosphoric acids are comparatively soluble in
cold water and may, therefore, be removed from the higher inosite
tetra-, penta- or hexaphosphoric acids with a fair degree of complete-
ness by repeating the operation of extraction with cold water several
times. In like manner, it is possible to effect a partial separation of
inosite di- and triphosphoric acids from a mixture of these two com-
pounds, but it is practically impossible to separate completely the two
substances by this method.

When these esters occur in conjunction with inosite hexaphos-
phoric acid, as they may frequently do when isolating the latter sub-
stance from plant material in which the enzyme phytase exists, it is very
difficult to remove the lower esters completely and obtain compounds
which give results on analysis corresponding to salts of inosite hexa-
phosphoric acid.

While we do not doubt the existence of all of these esters, as stated
above, we do not believe that experimental evidence has been presented
sufficient to prove the existence of more than the following:

Inosite hexaphosphoric acid.

Inosite tetraphosphoric acid (synthetic).

Inosite triphosphoric acid.

Inosite monophosphoric acid.

As has already been mentioned, it has been observed that very
carefully purified preparations of inosite phosphoric acid from different
sources, either the crystalline barium salt or the free acid, undergo
spontaneous decomposition with the gradual liberation of inorganic phos-
phoric acid and the production of lower phosphoric acid esters of
inosite. This process is very much hastened by the enzyme phytase in
aqueous solutions under proper conditions of temperature and acidity.
Evidently the same reactions do not occur in the dry plant material.

Apparently the cleavage of the inosite phosphoric acid in the plant occurs
only during germination or when the material is digested in aqueous or
faintly acid solutions favorable to enzyme activity.

THE EFFECT OF ENZYMES UPON INOSITE
PHOSPHORIC ACID

Suzuki, Yoshimura and Takaishi (1907) observed that the amount
of inorganic phosphoric acid increased when wheat or rice bran was
allowed to stand in contact with water. These investigators were also
able to separate a preparation from rice bran which, when added to a
solution of phytin, caused a rapid formation of inorganic phosphoric
acid and led finally to a complete hydrolysis of the phytin molecule into
inosite and phosphoric acid. This enzyme was termed phytase. The
existence of phytase has been demonstrated in other plant material and
in animal tissues by several investigators. McCollum and Hart (1908)
showed that enzymes extracted from various animal tissues were capable
of liberating inorganic phosphoric acid from phytin. Vorbrodt (1910)
showed that it was present in wheat, rye, barley, etc. Investigations by
Dox and Golden (1911) and by Jegorow (1913) demonstrated that cer-
tain fungi cause a cleavage of the phytin molecule with production of
inorganic phosphoric acid. Plimmer (1913) also found that certain
enzymes of animal tissue liberated inorgan'c phosphoric acid from phytin
but the most active phytase was contained in wheat bran.

We have studied quantitatively the activity of the wheat bran phy-
tase by determining the amounts of inorganic phosphoric acid formed
on digesting wheat bran in water or dilute acids (Anderson 1915 (3)).
The enzyme is very active in aqueous solutions and also in 0.1 per cent.
or 0.2 per cent. hydrochloric or acetic’ acid. The hydrolysis of the
inosite phosphoric acid begins at once and proceeds with great rapidity.
After digesting wheat bran in water for thirty minutes about 45 per
cent. of the soluble phosphorus is inorganic and after 24 hours about 88
per cent. of the total soluble phosphorus is present as inorganic phos-
phoric acid. The enzyme is very sensitive to certain concentrations of
hydrochloric acid. Greater amounts of free phosphoric acid are lib-
erated when the bran is digested in 0.1 or 0.2 per cent. hydrochloric acid
than when distilled water is used, but when the bran is digested in 0.3
per cent. hydrochloric acid the activity of the enzyme is almost entirely
inhibited. With increasing strength of hydrochloric acid the amount of
inorganic phosphoric acid in the extract decreases, the minimum amount
being obtained when 1.0 per cent. hyrochloric acid is used. The activity
of the enzyme is likewise inhibited or destroyed by 0.25 per cent. am-
monia or by the action of boiling water, as well as by exposing the bran
for a short time to the action of 0.5 per cent. hydrochloric acid. -

Boutwell (1917) has also studied the activity of the wheat bran

phytase and his results confirm in the main those mentioned above.
This author found that heating the dry bran to temperatures much above
boiling water apparently did not destroy the enzyme.

It is evident that certain plant materials contain an enzyme, phytase,
which under proper conditions of moisture, temperature and acidity
rapidly hydrolyzes inosite phosphoric acid into inorganic phosphoric
acid and inosite. This hydrolysis occurs in stages with the intermediate
formation of certain lower phosphoric acid esters of inosite (Anderson
1915 (2) ). These lower esters must consist of inosite penta-, tetra-,
tri-, di- and monophosphoric acids. Since there is a rapid formation of
inorganic phosphoric acid when certain plant material is digested in
water or dilute acids, it is self-evident that it is necessary to choose such
solvents as will inhibit the activity of the phytase while the inosite phos-
phoric acid is extracted from the plant substance. In the case of wheat
bran 1.0 per cent. hydochloric acid may be safely used for this purpose.
It is likely that the same strength acid would also destroy or inhibit the
activity of the phytase during the extraction of the organic phosphorus
compound from other seeds or plant material containing this enzyme.

METHODS FOR ISOLATING AND PURIFYING INOSITE
HENAPHOSPHORIC ACID AS BARIUM SALT

The barium salt of inosite hexaphosphoric acid has been isolated
from cottonseed meal by the following method:

The cottonseed meal, 25 pounds, was digested over night in 0.2 per
cent. hydrochloric acid in porcelain percolators covered on the inside
with a double layer of cheesecloth. It was then percolated using 0.2
per cent. hydrochloric acid until about 20 liters of extract were obtained.
The extract was of a dirty, dark color and contained some suspended
particles from which it was freed as much as possible by centrifugalizing
the solution. A concentrated solution of 300 grams of barium chloride
was then added and the precipitate allowed to settle. The precipitate
was centrifugalized and finally brought upon a Buchner funnel and freed
as far as possible from the mother-liquor. It was then digested in sev-
eral liters of about 5 per cent. hydrochloric acid until no further solution
took place. The insoluble residue was removed by centrifugalizing and
the still very dirty colored solution precipitated by adding barium hydrox-
ide until the free acid was neutralized. The barium hydroxide was
added slowly, with constant shaking, when the precipitate separated in
crystalline form. It was then filtered and washed thoroughly in water
and again dissolved in dilute hydrochloric acid, filtered and reprecipitated
with barium hydroxide. These operations were repeated three times.
The hydrochloric acid solution was then precipitated by gradually adding
an equal volume of alcohol when the substance again separated in crystal-
line form consisting of globular masses of microscopic needles. It was

then precipitated a fourth time with barium hydroxide and after that
two more times with alcohol. It was then filtered, washed free of
chlorides with dilute alcohol and then in alcohol and ether and dried in
vacuum over sulfuric acid. The product was then a nearly white, cry-
stalline powder and it weighed 94 grams.

The dry substance was shaken up with about 1.5 liters of cold water,
allowed to stand for several hours and then filtered and washed in
water. The aqueous solution contained very little substance precipitable
with alcohol and it was therefore discarded.

The washed precipitate was dissolved in dilute hydrochloric acid
and precipitated a fifth time by the very gradual addition of barium
hydroxide; after filtering and washing, this operation was repeated a
sixth time. After again dissolving in dilute hydrochloric acid, nearly
neutralizing the free acid with barium hydroxide and filtering, the sub-
stance was brought to crystallization by the gradual addition of an equal
volume of alcohol. After standing for several hours the substance was
filtered and washed in dilute alcohol, alcohol and ether and dried in
vacuum over sulfuric acid. It was then a voluminous snow-white cry-
stalline powder.

The dry substance was again dissolved in dilute hydrochloric acid,
the free acid nearly neutralized with barium hydroxide and the solution
filtered and allowed to stand over night. The substance soon began to
crystallize. Under the microscope it appeared perfectly homogeneous
and consisted as before of globular masses of microscopic needles. The
substance was filtered, washed free of chlorides with water and then in
alcohol and ether and dried in vacuum over sulfuric acid. The dry,
snow-white, crystalline powder weighed 69 grams.

Qualitative analysis failed to reveal any heavy metals other than
barium and from 0.5 gram of the salt no weighable residue of alkali was
obtained. It gave no reaction with ammonium molybdate in nitric acid
solution. It was free from sulfur and nitrogen.

It was analyzed after drying at 105° in vacuum over phosphorus
pentoxide.

0.4641 gram substance gave 0.0556 gram H,O and 0.1125 gram CO,,.

0.1982 gram substance gave 0.1333 gram BaSO, and 0.1203 gram
Mg,P,O,.

Found: C=6.61; H=1.34; P=16.91; Ba = 39.57 per cent

For tribarium inosite hexaphosphate :

C,H,,0.,4P,Ba, = 1066.

Calculated: C=6.75; H=1.12; P=17.44; Ba=38.65 per cent.

A portion of this salt was re-crystallized as follows: 5 grams were
dissolved in a small quantity of 5 per cent. hydrochloric acid and the free
acid nearly neutralized with barium hydroxide, the solution was then
filtered and 2 grams of barium chloride dissolved in a little water added

and the solution allowed to stand. The substance separated slowly im
the usual crystal form. After two days it was filtered, washed free of
chlorides with water, again dissolved in the dilute hydrochloric acid, the
solution filtered and alcohol added gradually until a slight cloudiness re-
mained. After standing for 24 hours at room temperature the substance
had crystallized in the usual form. It was filtered, washed free of
chlorides in dilute alcohol and then in alcohol and ether, and dried in
vacuum over sulfuric acid. The dilute nitric acid solution of the sub-
stance gave no reaction with ammonium molybdate. The snow-white
crystalline powder was analyzed after drying at 105° in vacuum over
phosphorus pentoxide.

0.3588 gram substance gave 0.0464 gram H,() and 0.0867 gram CO,,.

0.1726 gram substance 0.1138 gram BaSQ, and 0.1058 gram
Mg,P,0O,.

Found: C=6.59; H=1.44; P=17.08; Ba = 38.79 per cent.

Ancther portion of the substance was re-crystallized as follows:
2 grams were d'ssolved in a small amount of the dilute hydrochloric acid,
barium hydroxide added, with constant shaking, until a faint permanent
precipitate remained, and the solution filtered. The filtrate was then
heated to boiling and allowed to stand for a few minutes. As the
temperature rose the solution began to turn cloudy and finally a heavy
precipitate separated which appeared to be amorphous at first but it
soon changed into the same crystal form as previously described. This
was filtered and washed free of chlorides in boiling water and then in
alcohol and ether and allowed to dry in the air. The dry substance
weighed 1.6 grams. The snow-whité, crystalline powder was free from
inorganic phosphate. It was analyzed after drying at 105° in vacuum
over phosphorus pentoxide.

0.4695 gram substance lost 0.0443 gram H,O.

0.4252 gram substance gave 0.0423 gram H,O and 0.0981 gram CO,,.

0.1238 gram substance gave 0.0885 gram BaSO, and 0.0735 gram
Mg,P,O,.
Found: C=629; H=1.11; P=16.54; Ba=42.06; H,O 9.43
per cent. ‘

For heptabarium inosite hexaphosphate (C,H,,O,,P,),Ba, = 2267

Calculated: C=6.35; H=0.97; P=16.40; Ba=42.39 per cent.

For 14 H,0O calculated, 10.00 per cent.

Still another portion of the substance was re-crystallized in the fol-
lowing manner: 2 grams were dissolved in the dilute hydrochloric acid
and then nearly neutralized with barium hydroxide as befote. The
solution was filtered and 10 cc. N/1 barium chloride added and allowed
to stand over night. The substance had then separated as a heavy cry-
stalline powder of the same form as before except that’ the individual

crystals were much larger. The crystals were filtered, washed free of
chlorides with water and finally in alcohol and ether and allowed to dry
in the air. It was analyzed after drying as above.

0.6430 gram substance lost 0.0745 gram H,O.

0.5685 gram substance gave 0.0603 gram H,O and 0.1258 gram CO,,.

0.2208 gram substance gave 0.1608 gram BaSO, and 0.1252 gram
Mg,P,O,.

Found: C=6.03; H=1.18; P=15.80; Ba= 42.85; H,O = 11.58
per cent.

For heptabarium inosite hexaphosphate (C,H,,0,,P,),Ba,; — 2267.

Calculated: C=6.35; H=0.97; P=16.40; Ba = 42.39 per cent.

For 16 H,O calculated: 11.27 per cent.

With very slight modifications in the method of isolation identical
preparations were obtained from oats, corn and from commercial phytin
(Anderson 1914),

In preparing the substance from wheat bran the following method
was used: .

The bran, 700 grams, was digested in 5 liters of 1 per cent. hydro-
chloric acid for 5 hours. It was then strained through cheesecloth and
the liquid filtered. Barium hydroxide (Kahlbaum) was added to the
filtrate until the reaction was alkaline. The precipitate was filtered and
washed with water and then dissolved in about 3 per cent. hydrochloric
acid. The opalescent solution was filtered through charcoal and the
filtrate precipitated by adding about an equal volume of alcohol. After
standing over night the precipitate was filtered, washed in dilute alcohol
and again dissolved in 3 per cent. hydrochloric acid and filtered through
charcoal. A dilute solution of barium hydroxide was gradually added
to the filtrate until a precipitate began to form. After standing over
night the substance had separated out in semi-crystalline form. It was
filtered and washed in water, dissolved in 3 per cent, hydrochloric
acid and precipitated by alcohol. After filtering and washing with dilute
alcohol it was again dissolved in the dilute hydrochloric acid and pre-
cipitated by barium hydroxide.

The precipitate was still dark colored and it contained some im-
purities, not completely soluble in the dilute hydrochloric acid, apparently
of colloidal nature, which could not be removed by filtration. In order
to eliminate these impurities the barium precipitate was suspended in
water and the barium removed with a slight excess of dilute sulfuric acid.
The barium sulfate was filtered off and the filtrate precipitated with
excess of copper acetate. The copper precipitate was filtered and washea
free of sulfates with water. It was then suspended in water and decom-
posed with hydrogen sulfide. After filtering off the copper sulfide a clear
and colorless solution of the free acid was obtained. By these various
operations the oxalic acid had also been removed, as, after nearly neu-

tralizing with barium hydroxide and adding barium chloride, no precipi-
tate or turbidity occurrred. The solution was precipitated with barium
hydroxide, filtered and washed in water. The substance was again twice
precipitated with barium hydroxide from 3 per cent. hydrochloric acid
and finally twice precipitated with alcohol from the same strength hydro-
chloric acid. After finally filtering, the substance was washed free of
chlorides with dilute alcohol, alcohol and ether, and dried in vacuum
‘ over sulfuric acid. It was a snow-white, semi-crystalline powder which
weighed 11 grams. It was free from chlorides and inorganic phosphate
and did not contain any bases except barium.

For analysis it was re-crystallized as follows: 2 grams of the sub-
stance were dissolved in the minimum quantity of 2 per cent. hydro-
chloric acid and the free acid nearly neutralized with barium hydroxide
and the solution filtered. The clear filtrate was heated to boiling when
the substance separated as a heavy crystalline powder. This was fil-
tered and washed in boiling hot water and finally in alcohol and ether
and dried in the air. Yield, 1.5 grams. It was re-crystallized a second
time in the same manner except that 20cc. of N/1 barium chloride were
added to the solution before boiling. After filtering, washing and drying,
as before, about 1.3 grams substance were obtained. It consisted of
fine, microscopic, needle-shaped crystals. It was free from chlorides
and the nitric acid solution gave no precipitate with ammonium molybdate,
showing that inorganic phosphate was absent.

It was analyzed after drying at 105° in vacuum over phosphorus
pentoxide.

0.3771 gram substance lost 0.0385 gram H,O.

0.1917 gram substance lost 0.0196 gram H,O.

0.3386 gram substance gave 0.0361 gram H,O and 0.0794 gram CO,.

0.2571 gram substance gave 0.0269 gram H,O and 0.0617 gram CO,.

0.1715 gram substance gave 0.1218 gram BaSO, and 0.1026 gram
Mg,P.O,.

Found: C=639; H=1.19; P= 16.67; Ba= 41.79 per cent.

C=6.54; H=1.17.
H,O, 10.20 and 10.22 per cent.

For heptabarium inosite hexaphosphate (C,H,,O,,P,)»Ba, = 2267.

Calculated: C=635; H=0.97; P=16.40; Ba = 42.39 per cent.

For 14 H,0 calculated, 10.00 per cent.

PREPARATION OF THE CRYSTALLINE TRIBARIUM
INOSITE HEXAPHOSPHATE
This was prepared by dissolving 5 grams of the original substance

in 2 per cent. hydrochloric acid, nearly neutralizing with dilute barium
hydroxide, filtering and adding alcohol gradually until the solution turned

28 ae ee

cloudy. It was then allowed to stand for two days at room temperature.
The substance separated slowly in the form of globular masses or rosettes
of microscopic needles. The crystal form was identical with that pre-
viously described for the tribarium inosite hexaphosphate. The substance
was filtered and washed in dilute alcohol, alcohol and ether, and dried
in the air. It was re-crystallized a second time by dissolving in the
minimum quantity of 2 per cent. hydrochloric acid, filtering, and adding
alcohol until a slight permanent cloudiness remained. After standing
for two days the substance had separated in the same form as before.
It was filtered, washed free of chlorides with dilute alcohol, and then in
alcohol and ether and dried in the air. It was obtained as a snow-white,
crystalline powder. It was free from chlorides and inorganic phosphates.

For analysis it was dried at 105° in vacuum over phosphorus pen-
toxide.

0.2922 gram substance lost 0.0334 gram H,O.

0.2588 gram substance gave 0.0291 gram H,O and 0.0648 gram COQ,.

0.1421 gram substance gave 0.0944 gram BaSO, and 0.0867 gram
Mg,P.O,.

Found: C=6.82; H=1.25; P=17.00; Ba=39.09 per cent

H,O = 11.43 per cent.

For tribarium inosite hexaphosphate, C,H,,O.,P,Ba, = 1066.

Calculated: C=6.75; H=1.12; P=17.44; Ba=38.65 per cent.

For ,H.O calculated 11.90 per cent.

PREPARATION OF THE FREE INOSITE
HEXAPHOSPHORIC ACID

Inosite hexaphosphoric acid is easily prepared from the purified
barium salt by the method of Patten and Hart (1904). The barium salt
is suspended in water and decomposed with a slight excess of sulfuric
acid. After removing the barium sulfate, the filtrate is precipitated with
a hot, concentrated solution of copper acetate. The precipitate is filtered
on a Buchner funnel and washed thoroughly in water. It is then sus-
pended in water and decomposed with hydrogen sulfide. The copper
sulfide is filtered off and the filtrate is concentrated in vacuum at a
temperature of about 45° until a thick syrup remains. This is trans-
ferred to an evaporating dish and dried in a vacuum desiccator over
sulfuric acid.

METHOD OF ANALYSIS OF THE BARIUM
INOSITE HEXAPHOSPHATE

Most authors working with organic phosphorus compounds report
much difficulty in obtaining proper values for the carbon. This difficulty
is particularly great in burning compounds such as those reported above

acid and, consequently, an increase in weight. The actual composition
can therefore never be determined by analysis of the free acid itself.

The inosite phosphoric acid preparations which have been isolated
and analyzed recently by Rather (1918) were obtained by the following
method:

“Digest the ground material in 0.2 per cent. hydrochloric acid for
3 hours with frequent stirring. Pass the digestion mixture through a
double cloth and filter the resulting extract. Add copper acetate in excess
immediately, filter and wash the precipitate with water on a Buchner
funnel. Suspend the washed copper precipitate in water and decompose
it with hydrogen sulfide. Filter off the copper sulfide and wash with
water. Evaporate the filtrate to small volume at a temperature of 55° to
60° under a strong current of air. Take up the residue in six volumes
of 95 per cent. alcohol. Filter off any insoluble matter. Dissolve the
insoluble matter, consisting of acid salts, in dilute acid and precipitate
this solution with copper acetate. Decompose the copper precipitate with
hydrogen sulfide and evaporate the filtrate to small volume as before and
take up in alcohol. If any alcohol-insoluble matter results repeat the
copper acetate precipitation as before. Evaporate the combined alcoholic
solutions to a syrup at 50° under a strong current of air, take up the
residue again in alcohol, filter if necessary, and evaporate as before.
This process removes all inorganic bases. The inosite phosphoric acid
. is then separated as follows: Weigh the crude acid and dissolve it in
about 3 liters of water, filter off the insoluble matter, if any, and add to
the warm solution an amount of reprecipitated strychnine equal in weight
to the acid syrup. It is necessary to use this ratio of strychnine to acid
in order to get a salt of the inosite phosphoric acid which will not give
equivocal results on analysis. All of the strychnine should go into solu-
tion. Filter if necessary and concentrate at a temperature of 55° to 60°
under a strong current of air until crystals begin to form. Then allow
to cool. Filter off the crystals and concentrate the mother-liquor as
above until crystals begin to appear, cool and filter off the crystals. Con-
centrate the mother-liquor again for another crop of crystals. Purify
the fractions by re-crystallization in the usual manner. This process
separates the inosite phosphoric acid from orthophosphoric acid, oxalic
acid and other acids... .

“The silver salts are prepared from the strychnine salts as follows:
Three grams of the purified strychnine salt are dissolved in warm water
and the strychnine precipitated by excess of ammonia. The mixture is
cooled rapidly and the strychnine is filtered off. The filtrate is washed
with ether to remove any strychnine which may remain in solution. The
filtrate is then boiled to expell the excess of ammonia. A solution of 3
grams of silver nitrate is then added to the hot solution of the ammonium
salt and the resulting silver salt is filtered off and washed with water and

where the percentage of carbon is so low. Under ordinary conditions it
is impossible to obtain a complete combustion—the ash is usually more
or less dark colored. Some authors recommend mixing the substance
intimately with fine copper oxide. This procedure is very serviceable
when burning salts of these organic phosphoric acids with organic bases,
like the strychnine salts which we have previously reported, but with
barium salts we have not found copper oxide to be of much use. In the
analyses reported above we have used the following method, for the
suggestion of which we are indebted to Prof. E. Fischer of Berlin.

The substance is first burned in the usual way in a current of
oxygen, the combustion lasting about an hour. The calcium chloride tube
and the potash bulb are then weighed. The increase in weight of the
calcium chloride tube is taken as the correct weight of the water. The
residue in the boat, which is dark colored from particles of unburned
carbon, is powdered in an agate mortar with some recently fused potas-
sium bichromate and again placed in the boat, the mortar being rinsed
out with some more powdered bichromate. The whole is again burned
in the usual way. The potassium bichromate fuses and oxidizes all tlie
carbon in the residue. The increase in weight in the potash bulb is
added to the first, giving the total carbon dioxide.

The analytical data of these various crystalline barium salts, as well
as of the free acids from them, is reproduced below in tabular form.
These compounds represent the purest preparations that we have been
able to produce.

The relation of carbon to phosphorus in all of these salts is very
closely that of 1 :1 or 6 :6, and the composition agrees fairly closely with
calculated percentages for salts of inosite hexaphosphoric acid. We
were forced to the conclusion, therefore, that phytin or inosite phos-
phoric acid existing in the natural plant material which we have examined
is represented by the formula C,H,,O,,P, or inosite hexaphosphoric acid.
In the beginning of our study we assumed that the old formula of Post-
ernak, C,H,O,P., or as formulated by Neuberg, C,H.,0.,P,, was correct.
The analysis of amorphous and less carefully purified material gives
results in close agreement with this formula. In the carefully purified
re-crystallized barium salts, which we have since prepared and which
have been described above, the composition is slightly different and the
results agree more closely with the phytic acid formula of Neuberg
minus 3 molecules of water; viz., C,H.,O0.,P,—3H,O = C,H,,0.,P,. In
the analysis of the free acid which we have made we have always found
that the hydrogen is too high and the phosphorus too low, in some cases
one per cent, too low. The free acid is not suitable for analytical de-
terminations for, as we have shown, it can not be dried for analysis
without undergoing considerable hydrolysis. With the hydrolysis is
associated the absorption of water and production of free phosphoric

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dried in vacuo over phosphorus pentoxide. When the silver salt is
protected from light it shows only a slight change in color.”

Using this method of isolation and purification, Rather has analyzed
the strychnine and silver salts of the inosite phosphoric acid obtained
from a large number of feeding materials. He believes, as previously
mentioned, that all of his preparations represent salts of inosite pen-
taphosphoric ac‘d and that this is the only inosite phosphoric acid which
exists in natural plant material.

Clarke, (1914, 1915), in his examination of the inosite phosphoric
acid existing in wild Indian mustard and in wheat bran, isolated the
acid from 0.2 per cent. hydrochloric acid extracts of the material by a
method similar to that first used by Palladin. The crude phytin thus
obtained was purified by means of the crystalline strychnine salts.
Judging by the analytical results reported, these salts were evidently
mixtures of various inosite phosphoric acids.

Boutwell (1917), who has recently reported an investigation of
the inosite phosphoric acid of wheat bran, also followed in principal
the method used by Clarke. The different steps in his process were
as follows: The bran was macerated in 2.0 per cent. hydrochloric
acid at a temperature of about 20° for five hours. The mixture was
then pressed through cheesecloth and allowed to stand over nigh.
The clear extract was then heated to boiling to precipitate the protein
and allowed to cool. After filtering off the insoluble matter the clear
filtrate was heated to boiling and neutralized with ammonia. The
sticky, brown precipitate which formed was filtered from the hot solu-
tion and washed with boiling water. The precipitate was then ex-
tracted with 8.0 per cent. acetic acid, filtered and the filtrate heated
to boiling and, after cooling, the insoluble matter was filtered off. The
clear filtrate was diluted with an equal volume of water, heated to
boiling and made alkaline with ammonia. The white precipitate
which formed was filtered off from the hot mother liquor and washed
in boiling water. This precipitate was extracted with 0.8 per cent.
acetic acid and the insoluble matter filtered off. The clear solution
was heated to boiling, when the phytin separated out as a heavy, pow-
dery precipitate which was filtered off, washed with boiling water and
dried.

The product thus obtained consisted of spheroidal crystals which
had the following composition:

C=7.99; H=2.49; P=20.42; Ca=3.65; Mg=—10.81 per cent. From
this material crystalline barium salts were prepared which, however,
did not correspond closely in compostion to salts of inosite hexaphos-
phoric acid. This author expresses the opinion that the inosite phos-
phoric acid existing in bran is probably inosite hexaphosphoric acid
and that his preparation may have undergone some change during the
repeated heating of the solutions in the purification process.

PREPARATION OF INOSITE MONOPHOSPHORIC ACID.

Wheat bran is digested in 0.2 per cent. hydrochloric acid over night.
Strain through cheesecloth and filter the extract. Add about 4 volumes of
95 per cent. alcohol, allow the precipitate to settle and then filter on a
Buchner funnel and wash with dilute alcohol. Dissolve the precipitate
in 0.2 per cent. hydrochloric acid, filter through a heavy layer of paper
pulp on a Buchner funnel and precipitate with 4 volumes of alcohol.
These operations are repeated six times or until a nearly white product
is obtained which dissolves in the dilute acid, giving a perfectly clear
solution. The substance is then dried in vacuum over sulfuric acid.
Dissolve it in water with the aid of a few drops of hydrochloric acid
and precipitate the solution with a concentrated solution of pure barium
hydroxide. Filter off the precipitate and wash with cold water. Evap-
orate the filtrate to about one-fourth its volume, remove the excess of
barium hydroxide with carbon dioxide, filter and evaporate the filtrate
to a thin syrup on the water bath. Pour the syrupy residue into a large
volume of 95 per cent. alcohol. Filter off the precipitate and dry it in
vacuum over sulfuric acid. Dissolve the dry substance in water and re-
precipitate with a-large volume of alcohol. Filter, wash with alcohol
and dry as before. There is then obtained a nearly white amorphous
powder. One preparation obtained in this way had the following com-
position :

C= 39.22; H=5.43; N=14.26; Amino N (by Van Slyke method)
= 0.99; organic Phosphorus = 0.87; Ba =10.43 per cent.

The crude nitrogen-containing substance was dissolved in water and
a concentrated solution of lead acetate added in excess. The resulting
precipitate was filtered, washed thoroughly in cold water and then sus-
pended in hot water and decomposed with hydrogen sulfide. The lead
sulfide was filtered off and the filtrate boiled to expel hydrogen sulfide.
The solution was then strongly acid to litmus and it had a sharp acid
taste. It was again precipitated as above three times with lead acetate.
The pure white colored lead precipitate which was finally obtained was
decomposed with hydrogen sulfide. The filtrate was concentrated in
vacuum at a temperature of 40 to 45° and then dried in vacuum over
sulfuric acid until a thick, practically colorless syrup remained. On
scratching with a glass rod this immediately began to crystallize, form-
ing a white, solid mass. It was very soluble in water, but insoluble in
alcohol. It was extracted several times with 95 per cent. alcohol, fil-
tered and washed in absolute alcohol and ether and allowed to dry in the
air. For recrystallization it was dissolved in a small quantity of water
and absolute alcohol added until the solution turned slightly cloudy. On
scratching, the substance began to crystallize. After standing in the
ice chest over night it had separated in large, colorless plates or prisms
arranged in star-shaped aggregates. It was recrystallized a second time
in the same manner.

The substance was free from bases and also free from nitrogen and
sulfur, but it contained organically bound phosphorus. The aqueous so-
lution gave no precipitate with ammonium molybdate on being kept at a
temperature of 65° C. for some time but after decomposing by the
Neumann method it gave an immediate precipitate of ammonium phos-
phomolybdate with this reagent. ;

The substance has no sharp melting point. When rapidly heated in
a capillary tube it softens at 200° C. and decomposes under effervescence
at 201 to 202° ; when slowly heated it begins to soften at 188° and melts
under decomposition at 190° to 191° (uncorrected).

It is optically inactive. A 10 per cent. solution in a 1 dem. tube
shows no rotation.

For analysis it was dried at 100° in vacuum over phosphorus pen-
toxide but it did not lose in weight.

0.1550 gram substance gave 0.0749 gram H,O and 0.1566 gram CO,.

0.0766 gram substance gave 0.0325 gram Mg,P,O,.

Found: C = 27.55; H= 5.40; P=11.82 per cent.

For inosite monophosphate, C,H,,0,P = 260.

Calculated: C= 27.69; H=5.00; P=11.92 per cent.

Titrated against barium hydroxide, using phenolphthalein as indi-
cator, it forms a neutral barium salt, C,H,,O,P Ba.

0.1985 gram substance required 7.6 cc., N/5 Ba (OH),.

For C,H,,O,P Ba, calculated: 7.6 cc., N/5 Ba(OH),.

PROPERTIES OF INOSITE MONOPHOSPHORIC ACID.

The acid is very soluble in water. The acqueous solution shows a
strong acid reaction to litmus and it has a sharp, somewhat astringent,
acid taste. It is insoluble in alcohol, ether and the other usual organic
solvents.

Its aqueous solution gives no precipitate with barium hydroxide or
with calcium or barium chloride; ammonia produces no precipitate in
these solutions but the addition of alcohol causes white amorphous pre-
cipitates. Silver nitrate produces no precipitate even in a solution neu-
tralized with ammonia. When alcohol is added to the solution contain-
ing silver nitrate a white amorphous precipitate is produced which dis-
solves on warming; on cooling the silver salt separates in small, round
crystal aggregates. It gives no precipitate with ferric chloride or mer-
curic chloride nor with copper sulfate. In the cold no precipitate is
produced with copper acetate, but on warming this solution a bluish-white
precipitate separates which again dissolves completely on cooling.

With excess of lead acetate a white, heavy amorphous precipitate
is formed which is but slightly soluble in dilute acetic acid, but readily
soluble in dilute hydrochloric or nitric acid. Ammonium molybdate pro-
duces no precipitate in either dilute or concentrated aqueous solutions.

The acid crystallizes without water of crystallization from either
water or dilute alcchol.

The aqueous solution of inosite monophosphate does not precipitate
egg albumin, differing in this respect from phytic acid.

CLEAVAGE OF INOSITE MONOPHOSPHORIC ACID INTO
INOSITE AND PHOSPHORIC ACID

I. steid Hydrolysis. °

The acid, 0.35 gram, was heated in a sealed tube with 15 cc. of 3
per cent. sulfuric acid to 120—125" for about 3 1/2 hours. After cool-
ing, the liquid was of a pale straw color. The sulfuric and phosphoric
acids were precipitated with barium hydroxide and the excess of barium
hydroxide removed with carbon dioxide. The filtrate was evaporated
to dryness on the water-bath. The residue gave no precipitate with
ammonium molybdate, but after decomposing by the Neumann method,
a heavy. precipitate of ammonium phosphomolybdate was obtained, show-
ing that only a portion of the acid had been hydrolyzed under the above
conditions. The residue, however, contained some inosite which was
isolated as follows: The substance was taken up in a few cubic centimeters
of hot water, a little more than an equal volume of alcohol was added
which caused a voluminous, white amorphous precipitate consisting of the
barium salt of the unchanged inosite monophosphate. After filtering, the
precipitate was again treated with water, again precipitated with alcohol
and filtered. The filtrates were evaporated on the water-bath, taken up in
a little water and the inosite brought to crystallization by the addition of
alcohol and ether. It crystallized in the usual needle-shaped crystals.
After standing several hours in the ice chest the crystals were filtered,
washed in alcohol and ether and dried in the air. Yield, 0.06 gram. It
gave the reaction of Scherer and melted at 224° C. (uncorrected).

II. Alkaline Hydrolysis.

Another portion of the acid, 0.4 gram, was heated in a sealed tube
with 10 cc. of 10 per cent. ammonia for six hours to 120°. The solution
then contained some free phosphoric acid as it gave a precipitate with
ammonium molybdate but the greater portion of the acid remained un-
changed. It was found impossible to isolate any inosite from this re-
action mixture.

The residue was therefore again heated in a sealed tube with 10 per
cent. ammonia for about 4% hours to 150°. In this case complete hy-
drolysis had taken place, and after isolating the inosite in the usual way
0.15 gram was obtained. This was recrystallized three times from
dilute alcohol with addition of ether and was then obtained in colorless
needles free from water of crystallization. It then melted at 224° C.

|
(uncorrected), and it gave the reaction of Scherer. The identity of the
substance was further confirmed by the analysis.

0.1206 gram substance gave 0.755 gm. H,O and 0.1761 gm. CO,,.

Found: C= 39.82; H =6.97 per cent.

For C,H,,0, = 180.

Calculated: C= 40.00; H = 6.66 per cent.

The yield of inosite monophosphoric acid by this method of prepara-
tion is naturally very small and its isolation is a very tedious and expen-
sive operation. The substance can be obtained more readily and in larger
quantity on partially hydrolyzing phytin in an autoclave. From 500
grams of phytin after heating with 25 per cent. sulfuric acid in an auto-
clave to about 150° for 45 minutes we obtained nearly 10 grams of inosite
monophosphoric acid in beautiful crystalline form. The reaction mix-
ture is diluted with water, precipitated with barium hydroxide and fil-
tered. The filtrate which contains inosite and the barium inosite mono-
phosphate is evaporated to small bulk on the water-bath. About 2 vol-
umes of alcohol are added to the concentrated solution. The precipitate
which forms consists of barium inosite monophosphate. This is filtered
off and the inosite monophosphoric acid is isolated by means of the lead
salt and finally crystallized as described above.

PREPARATION OF INOSITE TRIPHOSPHORIC ACID

The wheat bran was digested over night in 0.2 per cent. hydrochloric
acid. It was then strained through cheesecloth and filtered. The free
acid in the extract was nearly neutralized by adding a dilute solution of
barium hydroxide until a slight permanent precipitate remained. A con-
centrated solution of barium chloride was added and the precipitate al-
lowed to settle over night. The supernatant liquid was syphoned off
and the residue centrifugalized. The precipitate was finally brought
upon a Buchner funnel and freed as much as possible from the mother-
liquor and then washed in 30 per cent. alcohol. For further purification
the substance was precipitated alternately six times from about 1 per
cent. hydrochloric acid with barium hydroxide (Kahlbaum, alkali free)
and six times with about an equal volume of alcohol.

After this treatment the substance was obtained as a snow-white,
amorphous powder. It was free from inorganic phosphate, and bases
other than barium could not be detected. But it still contained oxalates.
The oxalates were removed as follows: The barium salt was dissolved in
the least possible amount of 0.5 per cent. hydrochloric acid; 5 grams of
barium chloride dissolved in a little water was added and the solution
allowed to stand for 24 hours. A heavy, white crystalline powder, con-
sisting principally of barium oxalate, separated slowly. This was fil-
tered off and the solution precipitated with alcohol, filtered, washed with
alcohol and ether and dried in vacuum over sulfuric acid. The above

operation was repeated a second time when a further slight precipitation
occurred, but on repeating a third time the solution remained perfectly
clear. The solution was precipitated with alcohol, washed free of chlor-
ides with dilute alcohol, alcohol and ether and dried in vacuum
over sulfuric acid. The substance was finally precipitated from about
1 per cent. hydrochloric acid with alcohol, filtered and washed free of
chlorides with dilute alcohol and then in alcohol and ether and dried in
vacuum over sulfuric acid. The dry preparation weighed about 66
grams.

This crude acid salt was rubbed up in a mortar with about 500 cc.
of cold water, allowed to stand a few hours and then filtered and washed
with water, alcohol, and ether and dried in vacuum over sulfuric acid.
The water-insoluble residue weighed about 30 grams.

The water-soluble portion contained in the filtrate was precipitated
with barium hydroxide and reserved for a future investigation.

PREPARATION OF THE STRYCHNINE SALT

The water-insoluble portion of the barium salt mentioned above
was suspended in water and the barium precipitated with slight excess
of dilute sulfuric acid. The barium sulfate was removed and the fil-
trate precipitated with excess of copper acetate. The copper precipi-
tate was filtered and washed with water until the washings gave no
reaction with barium chloride. It was then suspended in water and
the copper precipitated with hydrogen sulfide and the copper sulfide
filtered off.

The solution containing the free organic phosphoric acid was
diluted to about 2 liters with water and heated on the water-bath and
44 grams of powdered strychnine added. After heating for a few
minutes the strychnine was dissolved. The solution was filtered and
concentrated in vacuum at a temperature of 45 to 50° to about one-half
of the volume. The strychnine salt soon began to separate in long
needle-shaped crystals. After standing in the ice chest over night the
crystals were filtered off and washed in ice cold water and finally in
absolute alcohol and ether and dried in the air. Yield 45.8 grams.

For further purification the substance was twice recrystallized
from hot water. It was then obtained in pure white, needle-shaped
crystals which looked perfectly homogeneous.

From more concentrated solutions the substance sometimes sep-
arates in the form of colorless plates which differ from the needle-
shaped crystals in that they contain about 4 per cent. more of water
of crystallization.

The strychnine salt has no sharp or definite melting point. Heated
in a capillary tube it softens at about 200° C. but it does not melt com-
pletely even at a much higher temperature. On moist litmus paper it

shows an acid reaction. The substance does not change in color on
drying at 105° C.

‘For combustion, the substance was mixed with fine copper oxide.
After drying at 105° C. in vacuum over phosphorus pentoxide it was
analyzed.

The needle-shaped crystals gave the following:

0.1534 gm. substance gave 0.0901 gm. H,O and 0.3329 gm. CO,,.

0.2944 gm. substance gave 0.0702 gm. Mg.,P,0,;.

0.1175 gm. substance gave 6.75 cc. nitrogen at 22° C. and 763 mm.

0.1688 gm. substance lost 0.0154 gm. H,O on drying.

0.1982 gm. substance lost 0.0183 gm. H,O on drying.

Found: C=59.18; H=6.57 ; P=6.64; N=6.56; H,O=9.12 and 9.23
per cent.

The plate-shaped crystals gave the following:

0.1786 gm. substance gave 0.0993 gm. H,O and 0.3909 gm. CO,.

0.4972 gm. substance gave 0.1089 gm. Mg,P,O,.

0.1955 gm. substance lost 0.0256 gm. H,O on drying.

0.5997 gm. substance lost 0.0783 gm. H,O on drying.

Found: C=59.60; H=6.22; P=6.10; H,O~13.09 and 13.05 per
cent.

The compounds apparently do not represent any definite strych-
nine salt of inosite triphosphate but it would seem as if they were mix-
tures of the tri- and tetrastrychnine salts.

For tri-strychnine inosite triphosphate, C,H,,;O0,,P,(C,,H,,N.O»)s
=1422.

Calculated: C=58.23 ; H=5.70; P=6.54 ; N=5.90 per cent.

For tetra-strychnine inosite triphosphate, C,H,,O,,P,(C.,H.,.N.O.),
=1756.

Calculated: C=61.50; H=5.86; P=5.30; N=6.37 per cent.

PREPARATION OF THE BARIUM SALT FROM THE
STRYCHNINE SALT

The recrystallized strychnine salt, 27 grams, was dissolved in
about 750 cc. of hot water and the solution rendered alkaline with
ammonia. After standing in ice water for some time the strychnine
was filtered off. The filtrate was shaken with several portions of
chloroform to remove the last trace of strychnine.

The solution, which contained the ammonium salt of the organic
phosphoric acid, was precipitated by adding a solution of barium
chloride in excess. After settling over night, the precipitate was fil-
tered and washed several times with water. It was then dissolved in
1 per cent. hydrochloric acid, filtered and precipitated with barium
hydroxide in excess. The precipitate was filtered and washed with

water until free from chlorides. It was again dissolved in 1 per cent.
hydrochloric acid, filtered, and the solution rendered neutral to litmus
with barium hydroxide. The precipitate was filtered and washed free
of chlorides with water and then in alcohol and ether and dried in
vacuum over sulfuric acid. The substance was a pure white, amor-
phous powder. On moist litmus paper it showed a very faint acid
reaction. It was free from nitrogen.

For analysis it was dried at 100° in vacuum over phosphorus
pentoxide.

1.2323 gm. substance gave 0.0370 gm. H,O and 0.0758 gm. CO).

0.2085 gm. substance gave 0.1668 gm. BaSO, and 0.0868 gm.
Mg.P,O,.

Found: C=8.89; H=1.78; P=11.60; Ba=47.07 per cent.

The substance was again dissolved in 1 per cent. hydrochloric acid
and the solution neutralized to litmus with barium hydroxide. It was
filtered, washed free of chlorides with water and then in alcohol and ether
and dried in vacuum over sulfuric acid.

It was analyzed after drying as before and the following results
obtained:

0.3168 gm. substance gave 0.0481 gm. H,O and 0.0972 gm. CO,.

0.2366 gm. substance gave 0.1968 gm. BaSO, and 0.0960 gm.
Me,P.O,.

Found: C=8.36; H=1.69; P=11.31; Ba=48.94 per cent.

For the neutral barium salt of inosite triphosphate,
C,H,O,;P,Ba,=826.

Calculated: C=8.71; H=1.08; P=11.25; Ba=49.88 per cent.

In the two analyses reported above the carbon is somewhat low.
It must be noted, however, that these barium salts burned with ex-
treme difficulty. Traces of carbon remained after prolonged heating
ia a current of oxygen. The residues were mixed with fine copper
oxide and re-burned when one or two milligrams of carbon dioxide
were obtained, but we believe that the combustion even under these
conditions was incomplete.

PREPARATION OF THE ACID BARIUM SALT

The above neutral barium salt was dissolved in the minimum
quantity of 1 per cent. hydrochloric acid, the solution was filtered, and
precipitated by adding about an equal volume of alcohol. The result-
ing precipitate was filtered, washed free of chlorides with dilute alco-
hol and then in absolute alcohol and ether and dried in vacuum over
sulfuric acid. The substance was then a pure white, amorphous
powder which showed a strong acid reaction on moist litmus paper.

It was analyzed after drying at 105° in vacuum over phosphorus
pentoxide. - .

0.2588 gm. substance gave 0.0538 gm. H,O and 0.1046 gm. CO,.

0.1691 gm. substance gave 0.1027 gm. BaSO, and 0.0890 gm.
Mg,P.O,.

Found: C=11.02; H=2.32; P=14.67 ; Ba=35.74 per cent.

In the combustion of this substance a practically white ash was
obtained.

This compound is evidently a complex acid salt of inosite tri- ‘phos-
phate and agrees with the following formula:

C,,H,;0,;P,Ba,== 1937.

(Calculated: C=11.15; H=1.80; P= 14.40: Ba=35.46 per cent.)
which may be graphically represented as follows:

C,H,,0,,;P;=Ra
a

a
CH ©. Ba
“a

a
C,H,,0,;P,=Ba

That is, three molecules of mono-barium inosite triphosphate
joined by two atoms of barium. Whether it is a compound as repre-
sented above or a mixture of various acid salts of inosite triphosphate
can hardly be determined.

PREPARATION OF THE FREE ACID

The barium salt from above was suspended in water and the
barium precipitated with a slight excess of dilute sulfuric acid.

The barium sulfate was filtered off and the filtrate precipitated
with excess of copper acetate. The copper precipitate was filtered
and washed with water until it gave no reaction with barium chloride.
It was then suspended in water and decomposed with hydrogen sulfide.
The copper sulfide was filtered off and the filtrate evaporated in
vacuum at a temperature of 40 to 45° to small bulk and then dried in
vacuum over sulfuric acid. The substance was then obtained as a prac-
tically colorless syrup. After continued drying it forms a hard, sticky
hygroscopic mass. It is extremely soluble in water and also readily
soluble in alcohol and in absolute alcohol. Much time was consumed
in endeavoring to obtain it in crystalline form but without success.
The syrupy substance was therefore analyzed after drying first for
several days in vacuum over sulfuric acid at room temperature and
finally at 100° C. in vacuum over phosphorus pentoxide. On drying at
this temperature it turned quite dark in color.

0.1685 gm. substance gave 0.0557 gm. H,O and 0.1054 gm. CO,.

0.1693 gm. substance gave 0.1363 gm. Mg,P,O,.

Found: C=17,06; H=3.69; P=22.44 per cent.

For inosite triphosphoric acid C,H,,O,,;P,—420.

Calculated: C=17.14; H=3.57; P=22.14 per cent.

In the above combustion a slight residue of unburned carbon re-
mained enclosed in the fused metaphosphoric acid. It was mixed with
some fine copper oxide and again burned when a few additional milli-
grams of carbon dioxide was obtained.

PROPERTIES OF INOSITE TRIPHOSPHORIC ACID

The reactions of this acid differ in several particulars from phytic
acid or inosite hexaphosphoric acid.

The concentrated aqueous solution gives no precipitate with
ammonium molybdate either in the cold or on warming. The cold
aqueous solution of inosite hexaphosphoric acid gives a white crystal-
line precipitate with ammonium molybdate.

The aqueous solution of the acid is not precipitated with silver
nitrate. Inosite hexaphosphoric acid gives a white amorphous precipi-
tate with silver nitrate in excess. However, a solution of inosite
triphosphate neutralized with ammonia gives a white amorphous preci-
pitate with silver nitrate.

An aqueous solution of inosite triphosphoric acid when added toa
solution of egg albumin causes only a slight turbidity ; on longer stand-
ing a white precipitate separates slowly. Inosite hexaphosphoric acid
precipitates egg albumin immediately.

The acid is very soluble in water and readily soluble in alcohol
and absolute alcohol; from the latter solution it is precipitated by
ether in small, oily drops.

The acid is not precipitated by barium or calcium chlorides but
alcohol produces in these solutions white amorphous precipitates;
these salts are likewise precipitated with ammonia.

CLEAVAGE OF INOSITE TRIPHOSPHORIC ACID INTO
INOSITE AND PHOSPHORIC ACID

One gram of the acid, dissolved in a little water, was heated with
10 ce. of S5N/1 sulfuric acid in a sealed tube for three hours to 150 to
155° The contents of the tube were then slightly yellowish brown in
color. The sulfuric and phosphoric acids were precipitated with
barium hydroxide and the inosite isolated in the usual way. Unfor-
tunately a portion of the solution was lost but from what remained 0.15
gram inosite was obtained. After twice recrystallizing from dilute
alcohol with addition of ether, 0.12 gram of inosite in the characteristic

needle-shaped crystals was obtained. It gave the reaction of Scherer
and melted at 222° C. (uncorrected). After mixing with some previ-
ously isolated and analyzed inosite the melting point did not change.
The substance was therefore undoubtedly inosite and the analysis was
omitted.

THE HYDROLYSIS OF PHYTIN BY THE ENZYME PHYTASE
CONTAINED IN WHEAT BRAN

It has been shown by the investigations of Suzuki, Yoshimura and
Takaishi that rice bran contains an enzyme which rapidly hydrolyzes
phytin with formation of inosite and inorganic phosphoric acid. These
authors concluded that wheat bran likewise contained a similar enzyme
because the inorganic phosphorus increased in wheat bran extracts on
standing.

Plimmer examined a large number of extracts prepared from the
intestines, liver, pancreas, castor beans, etc., as to their action on
organic phosphorus compounds. While some of these showed a slight
cleavage action on phytin, none could be compared in activity to an
aqueous extract of wheat bran. The hydrolytic action of these extracts
was determined by estimating from time to time the amount of inor-
ganic phosphorus split off from phytin solutions of known concen-
tration.

Since the above experiments clearly demonstrated that large quan-
tities of inorganic phosphate were liberated from phytin by wheat
bran extracts, it appeared of interest to determine what products, in
addition to inosite and inorganic phosphoric acid, were formed under
these conditions. For this purpose wheat bran extract was allowed to
act upon a dilute solution of phytin at a temperature of 37°. Inorganic
phosphoric acid was determined in the solution from time to time.

It was found that about two-thirds of the total phosphorus was
split off during the first 16 days. Afterwards there was no appreciable
change even on standing for about two years.

The solution had been prepared and the original determinations
made by Dr. Plimmer. At his suggestion the writer undertook to
examine the final reaction mixture for such products as had been
formed.

These products were separated into two portions by precipitating
the original solution with barium hydroxide. The precipitate con-
tained inorganic barium phosphate, and also those barium salts of
organic phosphoric acids that were insoluble in the dilute alkaline
solution. The filtrate, on the other hand, was found to contain inosite
monophosphoric acid and free inosite.

The inorganic phosphate and other impurities were removed from
the crude barium hydroxide precipitate, as will be described in the

experimental part. The organic phosphoric acids which remained were
obtained as amorphous barium salts. It was impossible to isolate
any unchanged barium phytate. It is evident, then, that all of the
phytin had been partially hydrolyzed.

The above amorphous substance appeared to consist mainly of
barium inosite triphosphate, but probably mixed with some barium
inosite diphosphate. Owing to the difficulty by separating these com-
pounds, their isolation was not attempted.

Among the soluble substances which had been formed we were
able to isolate and identify inosite monophosphoric acid, a substance
which we have previously isolated from wheat bran. In addition to
this, the solution also contained some free inosite which was isolated
by means of its lead compound.

The action of this enzyme phytase upon phytin appears to proceed
in several stages. Only a portion of the phytin is completely decom-
posed into inosite and phosphoric acid, but all of the phytin is par-
tially hydrolyzed with formation of certain lower phosphoric acid
esters of inosite; viz., inosite tri-, di- and monophosphoric acid and
inorganic phosphoric acid. The formation of these intermediate pro-
ducts is possible only through the destruction or inhibition of the
enzyme before the hydrolysis is complete. The reason for this inhibi-
tion is not clear but it may be due to the excess of phosphoric acid
which is liberated.

It is interesting to note, and we call particular attention to the
fact, that the organic phosphoric acids which remain as intermediate
products of the action of the enzyme upon phytin; -viz., inosite tri-
phosphoric acid and inosite monophosphoric acid, are identical with
the substances which we have isolated previously from wheat bran
after it has been digested in 0.2 per cent. hydrochloric acid.

EXPERIMENTAL PART

Commercial phytin, 100 grams, was dissolved in 500 c.c. of water
and filtered from the insoluble matter which weighed 3.5 grams when
dried at 100°. The pale yellow solution was treated with 38 grams of
oxalic acid dissolved in about 250 cc. of water. The calcium oxalate
was filtered off, washed and dried. It weighed 48 grams. The solution
was diluted to 6000 cc. with water and was then found to contain 40
grams of phosphorus pentoxide. To it were added 800 cc. of an
aqueous extract of wheat bran which contained 2.2 grams of P,O,.
The solution was kept under toluol at a temperature of 37°. No hydroly-
sis occurred in a week. This was evidently due to the strong acid reaction
of the solution: It was nearly neutralized with ammonia and 735 cc. of
bran extract containing 1.53 grams of P,O; were added. It was again
kept at a temperature of 37° under toluol. In 9 days one-half of the total

phosphorus was hydrolyzed; in 16 days two-thirds was hydrolyzed. In
35 days the amount of hydrolysis had not altered and after about two
years it was again the same. The total and inorganic phosphorus was
determined as described by Plimmer and Page.

The dark-colored solution was filtered and barium hydroxide
(Kahlbaum) added in slight excess. After standing over night the
precipitate was filtered and washed in water. The filtrate and wash-
ings were evaporated on the water-bath and the residue was examined
as will be described later.

The barium precipitate was dissolved in about 2.5 per cent. hydro-
chloric acid, filtered, and precipitated by adding about an equal volume
of alcohol. The precipitate was filtered and washed in dilute alcohol.
The substance was again precipitated four times in the same way. It
was then precipitated by barium hydroxide three times from about 2
per cent, hydrochloric acid and finally two times more with alcohol
from the same strength hydrochloric acid. After the final filtering it
was washed free of chlorides in dilute alcohol and then in alcohol and
ether and dried in vacuum over sulfuric acid. The substance was then
a snow-white, amorphous powder. It weighed 28.4 grams. It was free
from chlorides and inorganic phosphate, and bases other than barium
could not be detected.

The substance was then rubbed up in a mortar with 300 cc. of
cold water and allowed to stand with occasional shaking for a few
hours. It was then filtered and washed in water, alcohol and ether,
and dried in vacuum over sulfuric acid. The dry water-insoluble
portion weighed 5.4 grams.

The filtrate from above was neutralized to litmus with barium
hydroxide. The precipitate was filtered and washed in water, alcohol
and ether, and dried in vacuum over sulfuric acid. It weighed 23.6
grams.

These precipitates were analyzed after drying at 105° in vacuum
over phosphorus pentoxide.

The first, water-insoluble portion gave the following result:

Found: C=11.46; H=1.93 ; P=11.59 ; Ba=39.94 per cent.

This substance is apparently largely composed of the di-barium
inosite triphosphate; calculated for this, C,H,,O,;P;,Ba,—=690.

C=10.43; H=1.59; P=13.47; Ba=39.71 per cent. It is, however,
not pure but apparently contains some barium inosite di-phosphate
because the carbon is high and the phosphorus is low.

The water-soluble substance which was precipitated with barium
hydroxide gave the following:

Found: C=9.63; H=1.63; P=10.91; Ba=47.41 per cent.

This substance also appears to consist largely of the neutral
barium salt of inosite triphosphate. Calculated for the latter

C,H,O,;P,Bas—826. C=8.71; H=1.08; P=11.25; Ba=49.88 per cent.
The carbon, however, is high and the phosphorus as well as barium
are low, which points to the presence of barium inosite di-phosphate.

In the hope of approximately separating these barium inosite tri-
and diphosphates, the substance, 23.6 grams, was digested in dilute
acetic acid for several hours with occasional shaking. It was then
filtered and washed in water and the insoluble portion dried in vacuum
over sulfuric acid. It weighed 10 grams.

The filtrate and washings containing the soluble portion of the
substance was precipitated by adding lead acetate in excess. After
standing over night the white, amorphous precipitate was filtered and
washed in water. It was suspended in water and decomposed by
hydrogen sulfide, filtered, and the excess of hydrogen sulfide boiled
off. It was again precipitated in the same manner with. lead acetate
and decomposed with hydrogen sulfide. The solution still contained
a considerable quantity of barium. The barium was therefore re-
moved with a slight excess of dilute sulfuric acid. After filtering off
the barium sulfate the solution was precipitated by adding copper
acetate in excess. The copper precipitate was filtered, washed and sus-
pended in water and decomposed with hydrogen sulfide. After remov-
ing the copper sulfide, the filtrate was evaporated in vacuum to small
bulk and finally dried in vacuum over sulfuric acid. There remained
a thick, nearly colorless syrup. It was readily soluble in alcohol. The
addition of chloroform to this solution caused the substance to separ-
ate in small oily drops; the addition of ether produced a cloudiness
and, on standing, a flocculent, amorphous precipitate separated. These
solutions could not be brought to crystallize. The acid preparation
itself was kept for several weeks in the desiccator over sulfuric acid.
It became a hard but sticky mass but showed absolutely no tendency to
crystallize. Kept in this manner the color of the preparation gradu-
ally darkened.

Since the acid would not crystallize, the syrupy substance was
analyzed after drying at 105° in vacuum over phosphorus pentoxide.

Found: C=18.58; H=3.82; P=20.38 per cent.

For inosite triphosphoric acid, C,H,,O,,;P,=420.

Calculated: C=17.14; H=3.57; P=22.14 per cent.

For inosite diphosphoric acid, C,H,,O,,P,—340.

Calculated: C=21.17; H=4.11; P=18.23 per cent.

This acid preparation is evidently also a mixture of the inosite
tri- and diphosphoric acids.

EXAMINATION OF THE FILTRATE AFTER THE WATER-
INSOLUBLE BARIUM SALTS HAD BEEN PRECIPITATED

The filtrate was evaporated, as mentioned above, and the residue

taken up in hot water. It was decolorized with animal charcoal. The
solution was neutral in reaction. It strongly reduced Fehling’s solu-
tion on boiling, possibly due to sugars introduced with the bran ex-
tract. The solution was found to contain barium and also phosphorus
in organic combination, evidently inosite monophosphate. The aque-
ous solution was precipitated by adding about an equal volume of
alcohol and the white, amorphous precipitate filtered off, the filtrate
being reserved for further examination.

ISOLATION OF INOSITE MONOPHOSPHORIC ACID

The above precipitate, which formed on the addition of alcohol,
was dissolved in water, slightly acidified with acetic acid, and then
precipitated with lead acetate in excess. After settling, this was
filtered, washed in water and suspended in hot water and decomposed
with hydrogen sulfide. It was then filtered and the filtrate boiled to
expel excess of hydrogen sulfide. It was re-precipitated several times
with lead acetate in the same manner until a white lead precipitate
was obtained. This was finally decomposed with hydrogen sulfide,
filtered, and evaporated to small bulk in vacuum and then dried in
vacuum over sulfuric acid until a thick syrup remained. On scratch-
ing with a glass rod, this crystallized to a white, solid mass. It was
digested in alcohol and filtered, washed in alcohol and ether, and dried
in the air. It weighed 1.6 grams. It had all the properties of inosite
monophosphoric acid. [Tor further purification it was dissolved in a
few cubic centimeters of water and filtered. Alcohol was then added
until the solution turned cloudy; it was heated until it cleared up and
more alcohol was added until a faint permanent cloudiness remained.
It was allowed to stand for about 48 hours at room temperature, when
the substance had separated:in massive, practically colorless crystals.
After filtering, washing in alcohol and ether, and drying in the air,
one gram. substance was obtained. When heated in a capillary tube
it began to soften at 188 to 189° and melted under decomposition and
effervescence at 190° (uncorrected). The appearance and properties of
the substance corresponded exactly with those described for inosite
monophosphoric acid and the analysis was therefore omitted.

Isolation of Inosite.

The filtrate, after precipitating the above barium salt of inosite
monophosphate with alcohol, was evaporated on the water-bath until
the alcohol was removed. It still contained barium, chlorides, etc.
The barium was quantitatively precipitated with dilute sulfuric acid
and the solution again concentrated on the water-bath.

The addition of lead acetate caused no precipitate. Basic lead
acetate was then added so long as any precipitate formed. This pre-

cipitate was filtered off and discarded. The solution was then heated
to boiling and more basic lead acetate added and the solution was
finally made strongly alkaline with ammonia and allowed to stand over
night. This precipitate was filtered and washed in water and then
decomposed in aqueous suspension with hydrogen sulfide. The filtrate
was concentrated on the water-bath and the inosite brought to crystal-
lization by the addition of alcohol. After recrystallizing several times
€.5 gram of pure inosite was obtained in the characteristic needle-
shaped crystals. It gave the reaction of Scherer and melted at 218° C.
(uncorrected).

THE HYDROLYSIS OF THE ORGANIC PHOSPHORUS
COMPOUND OF WHEAT BRAN
BY THE ENZYME PHYTASE

Total and inorganic phosphorus were determined in extracts pre-
pared from wheat bran. The extracts were prepared by digesting
wheat bran in water or dilute acid for varying lengths of time, the
particulars and details being given at the beginning of each table.
Total phosphorus was determined after decomposing by the Neumann
method. Inorganic phosphorus was determined as follows: The ex-
tract was diluted with some 50 cc. of water, 15 grams of ammonium
nitrate were added and then warmed to 65° on the water-bath. It
was then strongly acidified with nitric acid and the phosphorus pre-
cipitated with ammonium molybdate and the solution kept at the above
temperature for half an hour. Under these conditions there is no
danger of eleavage of the organic phosphorus, at least weighable quan-
tities of phosphorus are not precipitated during this time from prepar-
ations which are free from inorganic phosphate. Plimmer has also
shown that it is necessary to heat phytin solutions with N/1 or 2N/1

TABLE 2

CHANGES IN CONDITION OF PHOSPHORUS IN WHEAT BRAN
BY WATER DIGESTION

Total Inorganic Part of Total

Time Phosphorus Phosphorts Phorphorus in

in Extract in Extract Inorganic Form
% % %
Vp NOUtitse on wiees ae hued dos ah eteses 0.362 0.165 45.77
DO alent tame aie Sin tae inten! 0.420 0.239 56.95
SAMMOUCS 046-49 08 verbo oe Sees 0.508 0.383 75.34
DO: SE" care cictine se ioe tine tea eas alae 0.655 0.576 88.00
Dae EE so Maison dis Gide WAN Nas Ghat 0.747 0.660 88.43

nitric acid for several hours to a temperature of 75° or higher before
appreciable quantities of inorganic phosphorus are split off.

The phosphomolybdate precipitate was then filtered off and the
phosphorus determined as magnesium pyrophosphate in the usual way.

The digestions were made throughout these experiments at room
temperature, about 16° C.

Ten grams of the bran were digested in 100 cc. of water for the
time mentioned and then filtered and 20 cc. used for each determina-
tion. The same quantities were used throughout.

It will be noticed that the hydrolysis is greatest during the first
four hours. The percentage of phosphorus represents only that amount
which was soluble in the water.

After digesting in water for 24 hours and then acidifying with
hydrochloric acid and shaking for half an hour the following results
were obtained:

TABLE 3

CHANGES IN CONDITION OF PHOSPHORUS IN WHEAT BRAN
AFTER WATER DIGESTION AND ACIDIFYING

Total Inorganic Part of Total
Time Phosphorus Phosphorus Phosphorus in
in Extract in Extract Inorganic Form
% % %
DA NOUS ee 65 .c4h cetk e8 ey dec saris Aa 1.06 0.649 60.85

After digesting the bran for 48 hours in water and then adding
100 cc. of 2 per cent. hydrochloric acid and allowing to stand for an-
other 24 hours, the following results were obtained:

TABLE 4

CHANGES IN CONDITION OF PHOSPHORUS IN WHEAT BRAN
AFTER WATER DIGESTION, ACIDIFYING AND STANDING

Total Inorganic Part of Total
Time Phosphorus Phosphorus Phosphorus in
in Extract in Extract Inorganic Form
Hours % % %
48 in water; 24 in weak acid.... . 1.27 0.803 62.79
48 in water; 24 in weak acid..... 1.28 0.796 62.13

The addition of toluol to the water appears to diminish the amount
of phosphorus dissolved without affecting the degree of hydrolysis.

TABLE 5

EFFECT OF TOLUOL ON HYDROLYSIS OF PHOSPHORUS
COMPOUNDS IN WHEAT BRAN

Total Inorganic Part of Total
Time Phosphorus Phosphorus Phosphorus in
With Toluol in Extract in Extract Inorganic Form
% 7 %
24 DOUES iit acer emda aod wet dein danas 0.518 0.462 89 25

Using hydrochloric acid as the extracting medium, the strength
varying from 0.2 to 2.0 per cent., the following results were obtained :
10 grams of bran were digsted in 100 cc. of the acid for the length
of time mentioned in the table. It was then filtered and 20 cc. of the
filtrate used for each determination.

TABLE 6

EFFECT OF CONCENTRATION OF ACID ON HYDROLYSIS OF PHOSPHORIC
ACID COMPOUND IN WHEAT BRAN

Total Inorganic Part of Total
Solvent Time, Phosphorus Phosphorus Phosphorus in
HC1 Hours in Extract in Extract Inorganic Form
% % Oo Go
0.2 3 1 130 0.415 36.52
0.2 24 1.200 0.925 76.87
0.3 24 0.999 0.203 20.22
0.4. 24 0.939 0.146 15.61
0.5 20 0.922 0.124 13.44
1.0 5 0.894 0.081 9.13
2A) 30 1.080 0.117 10.83

The presence of toluol did not materially alter these results.
Hydrochloric acid, 0.5 per cent. after 20 hours, with toluol gave the
following:

TABLE 7

EFFECT OF TOLUOL ON HYDROLYSIS OF PHOSPHORUS
COMPOUNDS IN WHEAT BRAN

Time Total Inorganic Part of Total
Phosphorus Phosphorus Phosphorus in
With HC1 and Toluol in Extract in Extract Inorganic Form
% % C0
Pitre 2G Sake el ee 0.890 0.117 13.14

When the bran is digested in dilute acetic acid the hydrolysis is
greater than with the same strength hydrochloric acid, as shown by
the following results :

TABLE 8
EFFECT OF ACETIC ACID ON HYDROLYSIS OF PHOSPHORIC ACID COM-
POUND IN WHEAT BRAN

Solvent, Total Inorganic Part of Total
Acetic Acid, Time Phosphorus _| Phosphorus Phosphorus in
% Hours in Extract in Extract Inorganic Form
% % %
0.2 3 1.21 0.695 57.03
0.2 24 1.23 1.050 85.54

The maximum activity of the enzyme, as shown by the greatest
hydrolysis, was obtained by digesting bran for 48 hours in water and
then adding 0.1 per cent. hydrochloric acid and allowing to stand for
another 24 hours. Ten grams of bran were digested in 100 cc. of
water for 48 hours and then 100 cc. of 0.2 per cent. hydrochloric acid
added. After standing for 24 hours more it was filtered and 20 cc. of
the filtrate used for each determination.

TABLE 9

MAXIMUM HYDROLYSIS OF PHOSPHORIC ACID COMPOUND
IN WHEAT BRAN

Total _ Inorganic Part of Total
Time Phosphorus Phosphorus Phosphorus in
in Extract in Extract Inorganic Form
% % %
72 Molise: is need Ge dee 1.24 1.14 92.37

DESTRUCTION OF THE ENZYME “PHYTASE” BY HEAT

As is shown by the following experiments, the hydrolytic action
of the enzyme is completely destroyed when bran is exposed to the
action of boiling water or boiling dilute hydrochloric acid for a short
time. Ten grams of bran were placed in an Erlenmeyer flask and
100 cc. of boiling water poured over it. It was then heated for a few
minutes until the water boiled. It was allowed to digest for 24 hours
at room temperature. Another lot of bran was treated in the same
way but using 0.2 per cent. hydrochloric acid and allowed to stand a
few minutes to settle. Lots of 20 cc. of the liquid were then taken
for each determination.

TABLE 10

EFFECT OF BOILING WATER OR BOILING ACID ON HYDROLYSIS OF
PHOSPHORIC ACID COMPOUND IN WHEAT BRAN

Total Inorganic Part of Total
Time, Phosphorus Phosphorus Phosphorus in
Solvent Hours in Extract in Extract Inorganic Form
% To %
Boiling water....... 24 0.886 0.108 12.66
Boiling 0.2% HC1.. 24 1.140 0.105 9.22

The hydrolytic action of the enzyme is likewise destroyed by ex-
posing bran for a short time to 0.5 per cent. hydrochloric acid or by
digesting bran in dilute ammonia. Ten grams of bran were digested
in 100 cc. of 0.5 per cent. hydrochloric acid for 114 hours. The acid
was then nearly neutralized with ammonia, leaving the solution
faintly acid, and then standing for 24 hours. Ten grams of bran were
digested in 100 cc. of 0.25 per cent. ammonia for 24 hours. It was
then acidified with dilute hydrochloric acid.

TABLE 11

EFFECT OF STRONG ACID AND OF AMMONIA ON HYDROLYSIS OF
PHOSPHORIC ACID COMPOUND IN WHEAT BRAN

Total Inorganic Part of Total
Solvent Time Phosphorus Phosphorus Phosphorus in
Hours in Extract in Extract Inorganic Form
% % %
05% Helse. occas 24 0.661 0.115 17.46
0.25% NHsz........ 24 0.922 0.087 9.51

CONCERNING SOME CHEMICAL PROPERTIES OF INOSITE
HEXAPHOSPHORIC ACID

Spontaneous Liberation of Inorganic Phosphoric Acid at Ordinary
Temperature and On Drying

The freshly prepared acid is a practically colorless syrup, especi-
ally when, in the concentration of its aqueous solution, the tempera
ture is not allowed to rise above 50°. When the acid has been pre-
pared from a pure salt, free from inorganic phosphate, the free acid
does not give any reaction with ammonium molybdate for inorganic
phosphoric acid. Whenever such colorless specimens of the acid are
preserved for any length of time the color always darkens. The

change in color is more rapid when the concentrated aqueous solution
is allowed to stand exposed to the air or preserved in a well stoppered
bottle, than when the acid is kept in the desiccator; but even under
the latter condition the color gradually deepens to light yellow, deep
yellow, light brown and finally, after several months, to dark brown
or black. When the acid is dried for analysis either in vacuum or in
an air bath the color darkens very materially in a short time, especi-
ally when dried at 100° or higher. When dried at a temperature of
60 or 70° in vacuum the color darkens somewhat, but very slightly in
comparison with that produced at higher temperatures.

Patten and Hart asserted that the acid turned dark in color on
drying at 110° without undergoing any decomposition. As mentioned
by Vorbrodt the grounds for this statement are not quite clear. A
striking change in color, such as inosite hexaphosphoric acid suffers
in drying or on mere keeping either in the desiccator or under ordi-
nary conditions, would very likely indicate a more or less serious
decomposition.

In order to determine to what extent decomposition occurs it
was decided to make a series of inorganic phosphoric acid determina-
tions by the usual molybdate method on inosite hexaphosphoric acid
preparations before and after drying. While absolute accuracy could
hardly be expected or claimed for this method, at least comparable
results would be obtained when the precipitations were done under
similar conditions.

One portion of the acid was dried at 105° in vacuum over phos-
phorus pentoxide to constant weight. It was then dissolved in water,
neutralized with ammonia, acidified with nitric acid, ammonium ni-
trate added and heated to 65°. Ammonium molybdate was then added
and kept at above temperature for one hour. The precipitate was
then determined as magnesium pyrophosphate in the usual way.

Another portion was treated in the same manner without dry-
ing, the amount of moisture found on drying as above being deducted
from the weight taken.

The fresh acid preparation, dried in vacuum over sulfuric acid
as described, contained about 15 per cent. of water and it gave no
reaction with ammonium molybdate. It was allowed to stand in the
laboratory at summer temperature (about 80 or 90° F.) in a loosely
covered dish for three or four weeks. The color had then changed
to light brown. On drying at 105° in vacuum over phosphorus pen-
toxide for about 24 hours to constant weight it lost about 22 per cent.
of its weight, showing that it had absorbed about 7 per cent. of water
during this time. The acid contained 27.31 per cent. of phosphorus.
The dried preparation gave the following as inorganic phosphate:

0.2508 gram dry substance gave 0.0000 gram Mg,P,O,, equiva-
lent to 7.73 per cent. phosphorus or 28.30 per cent. of the total phos-
phorus was precipitated as inorganic phosphoric acid.

Before drying:

0.1889 gram (dry substance calculated) gave 0.0039 gram
Meg.,P,O,, equivalent to 0.57 per cent. of phosphorus or 2.08 per cent.
of total phosphorus. ,

As will be noticed from the above figures, 26.2 per cent. of the
total phosphorus had been hydrolyzed by drying at 105° for about
24 hours.

An old sample of inosite hexaphosphoric acid, which had been
kept in the laboratory for about 18 months, was examined in the
same manner. It was practically black in color. It lost about 22 per
cent. of its weight on drying as above for about 20 hours. After
decomposing by the Neumann method it was found to contain 27.08
per cent. of phosphorus.

The dry preparation gave the following:

0.2348 gram dry substance gave 0.0733 gram Mg,P.O,, equiva-
lent to 8.70 per cent. of phosphorus, or 31.43 per cent. of the total
phosphorus was present as inorganic phosphoric acid.

Before drying:

0.2651 gram (dry substance calculated) gave 0.0295 gram Mg,P.O,,
equivalent to 3.10 per cent. of phosphorus or 11.19 per cent. of the
total phosphorus had been hydrolyzed in about 18 months under ordi-
nary room conditions.

In the above case about 20.2 per cent. of the total phosphorus
had been hydrolyzed on drying at 105° for about 20 hours.

A sample of the pure recrystallized barium inosite hexaphos-
phate was examined for inorganic phosphoric acid in the same way.
After standing in the laboratory for five or six weeks the following
results were obtained:

After drying at 105° in vacuum over phosphorus pentoxide 0.2108
gram substance gave 0.0104 gram Mg.P,O,, equivalent to 1.37 per
cent. of phosphorus.

Before drying:

0.2060 gram (dry substance calculated) gave 0.0030 gram Mg.P.O.,
equivalent to 0.40 per cent. of phosphorus.

By drying at 105° the inorganic phosphorus increased about 214
times.

A portion of the inorganic phosphoric acid found in the above
determinations was probably due to cleavage of the inosite hexaphos-
phoric acid by the dilute nitric acid. Such cleavage appears to take
place slowly and uniformly, as shown by the following experiment:

Another portion (0.1876 gram substance) of the same barium inosite
hexaphosphate without previous drying gave 0.0022 gram Mg,P.O,
after heating one hour with the ammonium molybdate, or 0.32 per
cent. in organic phosphorus; after heating the solution one-half hour
more 0.0010 gram Mg,P.,O, was obtained ; further heating for one hour
gave 0.0022 gram Mg,P.O, and a fourth hour’s heating gave 0.0040
gram Mg,P,O,.

The total inorganic phosphorus obtained after heating 314 hours
as above was 1.39 per cent. The results indicate that the cleavage
under these conditions is slow and that it proceeds at a very uniform
rate.

EXPERIMENT TO DETERMINE \VWHETHER INOSITE IS
FORMED IN THE SPONTANEOUS DECOMPOSITION
OF INOSITE HEXAPHOSPHORIC ACID

The sample of old inosite hexaphosphoric acid previously referred
to was used. As shown by the analysis, the preparation contained 3.10
per cent. inorganic phosphorus. Of this acid, 12.8 grams (correspond-
ing to 10 grams of the dry substance) were dissolved in about 500 cc.
of water and barium hydroxide (Kahlbaum, alkali free) added to
slight alkaline reaction. The precipitate was filtered and washed sev-
eral times in water. The barium precipitate was reserved for special
cxamination.

The filtrate was examined for inosite as follows: The excess of
barium hydroxide was precipitated with carbon dioxide filtered and
evaporated on the water-bath nearly to dryness. The residue was
taken up in a few cc. of hot water, filtered from a small amount of
barium carbonate and the filtrate mixed with alcohol and ether and
allowed to stand for several days in the ice chest. A trace of a white,
amorphous precipitate had separated, but absolutely no inosite crystals
appeared.

In case the organic part of the inosite hexaphosphoric acid mole-
cule, corresponding to the inorganic phosphoric acid present, had sep-
arated as inosite, the above quantity, 10 grams, should have contained
about 0.3 grams of inosite and sucha quantity could not have escaped
detection. Since no inosite could be isolated, it seems fair to assume
that under the above conditions of spontaneous decomposition inosite
hexaphosphoric acid does not decompose into inosite and phosphoric
acid, but phosphoric acid plus some unknown substance.

EXAMINATION OF THE ABOVE BARIUM PRECIPITATE

In the hope of throwing some light upon the nature of this un-
known substance, the barium precipitate obtained on the addition of

barium hydroxide was examined as follows: It was rubbed up with
about 400 cc. of 0.5 per cent. hydrochloric acid and brought into solu-
tion by the careful addition of dilute hydrochloric acid. After filter-
ing it was precipitated by adding an cqual volume of alcohol. The
precipitate was filtered, washed in dilute alcohol, dissolved in 0.5 per
cent, hydrochloric acid and reprecipitated by barium hydroxide. The
substance was then precipitated twice from 0.5 per cent. hydrochloric
acid with alcohol, finally filtered, washed in dilute alcohol, alcohol
and ether and dried in vacuum over sulfuric acid. A white, amor-
phous powder was obtained which weighed 14 grams. It was free
from chlorides and inorganic phosphate. After drying at 105° in
vacuum over phosphorus pentoxide the following results were ob-
tained:

Found: C=7.83; H=1.46; P=16.72; Ba = 36.96 per cent.

The carbon found is much too high for a pure barium inosite
hexaphosphate.

PREPARATION OF CRYSTALLIZED BARIUM INOSITE HEXA-
PHOSPHATE FROM THE ABOVE AMORPHOUS
BARIUM SALT

The substance was rubbed up in a mortar with about 150 cc. of
cold water and allowed to stand for several hours. The insoluble por-
tion was changed slowly into a semi-crystalline precipitate. It was
filtered and washed in water and then recrystallized as follows: It
was dissolved in a small quantity of about 3 per cent. hydrochloric
acid, the free acid nearly neutralized with barium hydroxide; a con-
centrated solution of 10 grams of barium chloride was added, the solu-
tion filtered and alcohol added gradually with constant shaking until
a slight permanent cloudiness was produced. On standing the sub-
stance crystallized slowly in the usual crystal form; 1. e., in globular
masses of microscopic needles. After two days the crystals were fil-
tered off, washed free of chlorides in water and then in alcohol and
ether and dried in the air. Yield, 4.5 grams. The substance gave no
reaction with ammonium molybdate.

A further quantity of the same-shaped crystals was obtained
from the aqueous solution containing the water-soluble portion of the
amorphous salt by adding to it 2.5 grams barium chloride and allowing
to stand over night. The balance of the water-soluble portion of the
substance was recovered by precipitating with an equal volume of
alcohol. The resulting precipitate was filtered, washed free of chlorides
with dilute alcohol, alcohol and ether and dried in vacuum over sul-
furic acid. Yield, 4.1 grams.

These substances were analyzed after drying at 105° in vacuum
over phosphorus pentoxide.

The recrystallized salt gave the following results:

Found: C= 6.28; H = 1.28; P= 15.93; Ba = 42.18; H,O = 11.81
per cent.

The crystalline salt which separated from the aqueous solution
gave:

C—647; H=1.23; P=15.95; Ba= 42.77; H.O — 12.62 per
cent.

These substances are therefore nearly pure heptabarium inosite
hexaphosphate.

Calculated for (C,H,,0,,P,),Ba, = 2267.

C=635; H=0.97; P=16.40; Ba = 42.39 per cent.

The water-soluble substance precipitated with alcohol gave the
following :

Found: C=858; H=1.62; P=15.86; Ba = 38.28 per cent.

This substance was again treated with about 100 cc. of cold water,
the insoluble portion filtered off and the filtrate, after adding 1 gram
of barium chloride, precipitated with alcohol. After washing in dilute
alcohol, alcohol and ether and drying in vacuum over sulfuric acid 1.4
grams of a white, amorphous substance was obtained. For analysis
it was dried at 105° in vacuum over phosphorous pentoxide.

Found: C=8.08; H=1.68; P=15.64; Ba=39.75 per cent.

This water-soluble substance apparently represents a mixture of
the barium salts of penta- and tetraphosphoric acid esters of inosite.

CONCERNING THE DECOMPOSITION PRODUCTS OF
INOSITE HEXAPHOSPHORIC ACID AFTER DRYING
AT 105° UNDER REDUCED PRESSURE

The specimen of old inosite hexaphosphoric acid previously exam-
ined was used; 12.8 grams (corresponding to 10 grams dry acid) was
dried at 105° for about 48 hours over sulfuric acid under slightly re-
duced pressure. It was then dissolved in about 200 cc. of cold water.
The solution was practically black in color and contained particles
of carbonized material. It was decolorized by shaking with animal
charcoal. The clear, colorless solution was then precipitated with
barium hydroxide to slight alkaline reaction, the precipitated filtered
and washed in water and reserved for examination. The filtrate and
washings were freed from barium with carbon dioxide and evaporated
on the water-bath to dryness. The residue was taken up in a small
amount of hot water and filtered. On adding a little alcohol a heavy,
voluminous, white, amorphous precipitate was produced. This was

removed from the solution by adding about 3 volumes of alcohol. The
precipitate settled, leaving a clear supernatant liquid; adding more
alcohol produced no further precipitate. It was then filtered and
washed in alcohol and the filtrate reserved.

After drying, the above precipitate was obtained as a heavy, white,
amorphous powder. It was free from inorganic phosphorus, but con-
tained barium and after combustion the ash gave a heavy yellow pre-
cipitate with ammonium molybdate. This substance was purified, as
will be described later.

The filtrate from the above precipitate was again evaporated on
the water-bath nearly to dryness, taken up in hot water, filtered and
mixed with alcohol and ether, On scratching with a glass rod a sub-
stance began to crystallize in needles. It was allowed to stand in
the ice chest over night. The crystals were then filtered, washed in
alcohol and ether and dried in the air. Yield, 0.25 gram. The sub-
stance was recrystallized four times in the same manner and was
finally obtained in colorless needles free from-water of crystallization.
It gave the reaction of Scherer and melted at 222° (uncorrected). It
was, therefore, no doubt pure inosite. This was further confirmed by
the analysis:

0.1215 gram substance gave 0.0737 gram H,O and 0.1780 gm. CO,.

Found: C= 39.95; H=6.78 per cent.

For C,H,,0, = 180.

Calculated: C= 40.00; H = 6.66 per cent.

PURIFICATION OF THE BARIUM AND PHOSPHORUS CON-
TAINING PRECIPITATE REMOVED FROM THE
INOSITE SOLUTION WITH ALCOHOL

The substance mentioned above, precipitated with alcohol, was
apparently the barium salt of an organic phosphoric acid. It was
very soluble in water and was not precipitated from the aqueous solu-
tion by barium hydroxide. The dry substance weighed 1.2 grams. It
was dissolved in a small quantity of water, a few drops of dilute hydro-
chloric acid added and 10 ce. of N/1 barium chloride. The solution
was heated to boiling and alcohol added until a slight cloudiness was
produced. On standing in the cold over night a small amount of a
hard crust had separated on the bottom of the flask. This was removed
and the solution again heated and more alcohol added when a further
quantity separated in the same way. The substance was finally fil-
tered and washed thoroughly in 80 per cent. alcohol, alcohol and ether,
and dried in the air. Without further purification the substance was.
analyzed after drying at 105° in vacuum over phosphorus pentoxide.

Found: C= 20.03; H=3.58; P=853; Ba=25.55 per cent.

The small quantity precluded recrystallization and we are there-
fore unable to state whether the substance was pure. The analytical
result indicates that it was a barium salt of inosite monophosphate
and agrees approximately with this formula:

C,,H;,0,,P,Ba, = 1051.

Calculated: C=20.55; H=3.33; P=8.84; Ba= 26.16 per cent.

Such a salt could be represented by the following formula:

a2
C,H,,O,—P=O
o)

re
C,H,,O,—P=0
“~OH

EXAMINATION OF THE PRECIPITATE PRODUCED WITH
BARIUM HYDROXIDE AFTER DRYING THE
ABOVE ACID

A portion of the barium precipitate was dried in vacuum over sul-
furic acid and then examined for total and inorganic phosphorus in
the same way as before:

Found: Total phosphorus (by Neumann method) 9.98 per cent.

Found: Inorganic phosphorus 7.46 per cent.

As will be noticed from these figures, 74.76 per cent of the phos-
phorus was present as inorganic phosphoric acid.

The substance was freed from inorganic phosphate by precipi-
tating four times with alcohol from 0.5 per cent. hydrochloric acid.
After finally drying in vacuum over sulfuric acid 3.2 grams of a snow-
white, amorphous powder were obtained. The substance was free
from chlorides and inorganic phosphate.

It was shaken up with about 75 cc. of cold water in which the
greater portion dissolved; 10 cc. of N/1 barium chloride was added
and allowed to stand for several hours; the insoluble portion was then

filtered off, washed free of chlorides with water and then in alcohol
and ether and dried in vacuum over sulfuric acid. It weighed 0.65
gram.

The filtrate from above containing the water-soluble portion of
the substance was acidified with a few drops of dilute hydrochloric
acid, heated to boiling and alcohol added until a slight, permament
cloudiness remained. On standing over night a portion had sep-
arated in the form of a heavy, granular powder. Under the micro-
scope no definite crystal form could be observed, but it appeared to
consist of transparent globules. It was filtered off, washed free of
chlorides in 30 per cent. alcohol, alcohol and ether, and dried in the
air. Yield, 0.67 gram. It was free from inorganic phosphate.

The mother-liquor from above was precipitated with alcohol.
After settling, the precipitate was filtered, washed with dilute alcohol,
alcohol and ether, and dried in vacuum over sulfuric acid. Yield, 1.55
grams. The substance was a snow-white, amorphous powder. It was
free from chlorides and inorganic phosphate.

These three different portions were analyzed after drying at 105°
in vacuum over phosphorus pentoxide.

The water-insoluble portion gave:

C=9.40; H=1.65; P=13.76; Ba = 39.56 per cent.

Judging by the analysis this substance consists mainly of barium
inosite tetraphosphate.

The granular powder which separated from the hot, dilute hydro-
chloric acid solution and alcohol on cooling gave the following result:

C=-12.70; H=240; P=13.84; Ba = 32.29; H,O=12.77 per
cent.

This substance appears to be mainly barium inosite triphosphate,
although not pure. It was mixed probably with some barium inosite
diphosphate.

The water-soluble portion precipitated with alcohol gave:

C=1407; H=231; P=12.92; Ba = 33.04 per cent.

Deducting the barium found, allowing for hydrogen and water,
and calculating to the free acid these results became:

C= 20.88; H = 4.11; P= 19.16 per cent.

This is approximately the composition of inosite diphosphoric
acid, C,H,,0,,P, = 340.

Calculated: C=21.17; H=4.11; P=18.23 per cent.

That these substances, separated from the partially decomposed
inosite hexaphosphoric acid, are inosite esters of phosphoric acid and
not condensation or other decomposition products, is evident from the
fact that on complete cleavage inosite is obtained. Unfortunately, the
amount of each of the above substances was too small to permit of

examination in this direction except the last one; viz., the water-
soluble product precipitated by alcohol and which analyzed for inosite
diphosphate. The remainder (0.94 grams dry substance) was hydro-
lyzed with dilute sulfuric acid in a sealed tube at 150 to 160° for about
21% hours and the inosite isolated in the usual way. The amount of
inosite obtained was 0.28 gram or about 88 per cent. of the theory.
The substance gave the reaction of Scherer and melted at 222° (uncor-
rected) which leaves no doubt that it was pure inosite.

It is evident that all of the barium precipitates described above
are mixtures. It could hardly be expected that a complete separation
into pure chemical compounds of the salts of these inosite esters could
be effected by the method used. The analytical results, however, show
that it is possible to isolate from partially decomposed inosite hexa-
phosphoric acid certain substances approximating in composition vari-
ous phosphoric acid esters of inosite which on complete cleavage
yielded inosite, just as does inosite hexaphosphoric acid itself. This
fact, we believe, supports the view previously expressed that inosite
hexaphosphoric acid suffers a gradual and partial decomposition; i. e.,
molecules of phosphoric acid are eliminated one by one. We believe
also that these facts taken in connection with the formation of inosite
from the acid on mere drying at 105° must be considered as a strong
support of the theory that it is inosite hexaphosphoric acid and not
some complex compound, as previously held. '

ATTEMPT TO PREPARE A METHYL ESTER OF INOSITE
HEXAPHOSPHORIC ACID

The silver salt previously described as hepta-silver phytate (An-
derson 1912 (2) ) was used. Of this salt, 5.4 grams were suspended in
100 ce. of absolute methyl alcohol and 4 grams of methyl iodide (a
little over the required amount) were added and the mixture shaken
for several hours, the flask being protected from the light. At the
end of this time the white silver phytate had changed into the yellow
silver iodide. The precipitate was filtered off and washed several
times in absolute methyl! alcohol and the filtrate several times evap-
orated in vacuum to dryness under addition of methyl alcohol for the
removal of the excess of methyl iodide. The residue was dissolved in
methyl alcohol and evaporated to dryness in vacuum over sulfuric
acid. The substance was then obtained as a light-yellow-colored, thick
syrup of faint, aromatic odor. It was strongly acid in reaction and
of sharp acid taste. For analysis it was dried in vacuum at 105° over
phosphorus pentoxide. It then turned very dark in color.

0.1985 gram substance gave 0.0608 gm. H,O and 0.1016 gm. CO,.

Found: C= 13.95; H = 3.42 per cent.

This agrees with a dimethyl ester of inosite hexaphosphoric acid.
For C,H,,0.,P,(CH;), = 688.
Calculated: C=13.95; H=3.19 per cent.

THE WATER-INSOLUBLE SUBSTANCE \WHICH SEPARATES
FROM INOSITE HEXAPHOSPHORIC ACID AFTER
DRYING

As has been mentioned earlier, inosite hexaphosphoric acid, which
has been dried to constant weight in vacuum over phosphorus pen-
toxide, is not completely soluble in, water. We have observed this
insoluble substance in many instances after drying the acid at 60°, at
78° and at 105°. It always separates, on adding water to the dry sub-
stance, in thin gelatinous plates. It appears to be practically insolu-
ble in hot or cold water. Continued boiling in acidulated water is
necessary to dissolve it. It is also insoluble in alcohol and ether.

In order to obtain some knowledge of the composition of this
insoluble substance 2.7 grams of the acid, containing about 12 per
cent. of moisture, were dried to constant weight at 105° in vacuum
ever phosphorus pentoxide. After treating with water the insoluble
portion was filtered, washed thoroughly in water and finally in alcohol
and ether and dried in vacuum over sulfuric acid. It was then obtained
as a dirty, gray powder which weighed 0.23 gram. Jt was non-hygro-
scopic. For analysis it was dried at 105° in vacuum over phosphorus
pentoxide at which no change in color was noticeable. The substance
was burned with copper oxide and the phosphorus determined in the
ash.

0.2118 gram dry substance gave 0.0569 gram H,O and 0.1357 gm.
CO, and 0.1822 gm. Mg,P,O,.

Found: C=17.47; H=3.00; P= 23.98 per cent.

The quantity of the substance obtained was so small that it was
only sufficient for one analysis. Of course, we are unable to state
whether it was homogeneous or not, but the analytical results agree
approximately with inosite triphosphoric acid minus one molecule of
water. The substance may therefore be a partial pyrophosphoric acid
ester of inosite or it may represent some complex decomposition pro-
duct of inosite hexaphosphoric acid.

SUMMARY

The evidence from experimental work shows that the organic
phosphorus compound known as phytin or inosite phosphoric acid

corresponds in composition to inosite hexaphosphoric acid, C,H,,O,,P,
or C,H,O,. (PO(OH), ),.

Crystalline barium salts have been prepared and analyzed which
correspond to the following formulas, C,H,,O0,,P,Ba, and (C,H,,0,4P,).
Ba,.

Salts of other metals are generally obtained as amorphous pre-
cipitates.

The acid yields readily crystalline strychnine salts.

The free acid may be obtained as a colorless, non-crystallizable
‘syrup.

The acid decomposes on complete hydrolysis, yielding inosite and
phosphoric acid.

On partial hydrolysis of the substance phosphoric acid and various
dower inosite tetra-, tri-, di- or monophosphoric acids are produced.

When inosite and dry phosphoric acid are heated in vacuum to a
temperature of 140 to 160° a reaction occurs and inosite tetraphos-
phoric acid is formed.

Inosite monophosphoric acid is formed from inosite hexaphos-
phoric acid on partial hydrolysis with mineral acids or by the action
of the enzyme phytase in dilute acid solutions.

Wheat bran contains an enzyme phytase which in faintly acid
‘solution rapidly hydrolyzes the inosite phosphoric acid with the pro-
‘duction of inorganic phosphoric acid and various lower inosite phos-
Phoric acids and some free inosite.

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