opening of the peritoneal cavity. With the carcass lying on its back, the muscles and fascia to the right of the median line of the abdomen were thoroughly cooked with a soldering iron. An incision was made, with a sterile scalpel, through the cooked tissues into the peritoneal cavity. The liver was removed piecemeal, with sterile scissors and forceps, and placed in a sterile mortar with sterile broken glass, and then ground to a pulp. No effort was made to weigh the liver-substance used, all endeavors aiming at transference to sterile flasks as soon as possible, to avoid contamination. Approximately 10 gm. of the liver pulp and glass were placed in one Erlenmeyer flask (a) and about 20 gm. in another (b), while the third flask was a control (c) :

Liver pulp

A, 10 gm.

B, 20 gm. C, none
15 c.c 5 c.c.
4
4

B-Imidazolylethylamin in saline sol., 1-1000 (c.c.)... IO C.C.
Toluene (c.c.)

4

All flasks were incubated at 37° C., for 24 hr. Inoculations from all flasks were then made on agar and in bouillon to test the sterility, which showed no growth in 48 hr. The contents of the flasks were filtered and the filtrates used for injections into guinea-pigs. Dale (2), as well as the writer (3), has shown that 0.5 mg. of ẞ-imidazolylethylamin, injected into the blood stream, kills a 300 gm. guineapig in 6 min., from spasm of the bronchioles and suffocation. Injected into guinea-pigs on a basis of 0.5 mg. per 300 gm. of weight, there was no effect for the solutions that were incubated with liver, but for the control solution there were the usual fatal symptoms.

That this action is due to some enzyme seems probable, for heating to boiling inhibits the detoxicating action. Further study will no doubt elucidate this problem but the scarcity of material has, for the present, required postponement of the experiments. It is also of interest to note that the power of causing urticarial lesions, possessed by this amin, to which the writer called attention last year (4), is also destroyed by heat.

Full protocols of these experiments will be published as soon as a sufficient quantity of the amin can be obtained for final tests, but justification for this preliminary note is found in the hope that some one more fortunate than the writer may have sufficient of the amin to be able to complete the study; or that these results may lead to further experimental work on the several amins of intestinal origin, with a view to extending our knowledge on the detoxicating function of the liver.

BIBLIOGRAPHY

1. EWINS and LAIDLAW: Jour. of Physiol., 1910, xli, p. 78.

2. DALE and LAIDLAW: Ibid., p. 318.

3. EUSTIS: Amer. Jour. Med. Sciences, 1912, cxliii, p. 862.

4. EUSTIS: New Orleans Med. Surg. Jour., 1914, lxvi, p. 730.

:

THE ORGANIC PHOSPHORUS COMPOUNDS OF
WHEAT-BRAN

CHARLES J. ROBINSON AND J. HOWARD MUELLER (Laboratory of Physiological Chemistry, University of Louisville, Ky.)

Introduction. The organic-phosphorus materials, or phytins, obtained by alcoholic precipitation of aqueous or dilute acid extracts from various sources, are not identical, but ultimate analyses show a fair degree of similarity. Thus, the phosphorus content varies between 14 and 17 percent, and there are varying proportions of magnesium, potassium and calcium. There has been prepared, also, from the phytins from many sources, the free phytic acid, corresponding to the formula C2H3P2O, (anhydro-oxymethylene phosphoric acid, Posternak),1 or C6H24P6O27 (Neuberg, Starkenstein3).

2

In the case of the material extracted from wheat bran, however, there has been difference of opinion regarding its identity with phytin and its ability to yield phytic acid. Patten and Hart claimed to have obtained an acid containing 10.63 percent of carbon, 3.38 percent of hydrogen, and 25.98 percent of phosphorus, figures agreeing very well with the formula C8H24O27P6. They therefore called their product phytic acid. Anderson, on the other hand, was unable to obtain such a compound, and ascribed Patten and Hart's supposed error to contamination with inorganic phosphates and phosphoric acid. Anderson obtained his material by a method of procedure different from that used by Patten and Hart, a fact that may explain the divergent results.

It was with a view to clearing up this matter that the work described in this paper was undertaken. Wheat-bran contains a much larger percentage of organic-phosphorus extractives than most other materials so far examined, and probably is the best source of phytin and phytic acid for further investigations.

We have repeated Patten and Hart's work. Their so-called tri-barium phytate has been prepared from wheat bran, with care to insure the absence of inorganic phosphates by means of the method recommended by Anderson, viz., repeated solution of the salt in dilute hydrochloric acid sol. and reprecipitation with alcohol. With Anderson's new method, we have been able to prepare this barium salt in crystalline form and identical in properties with that obtained by him from cotton-seed meal, oats and corn, but corresponding more closely in composition with the formula, C6H18024 PeBa3, than with Anderson's formula C6H12O24P6Ba3. Our data leave no question as to the presence of substances in wheat-bran which yield, by the usual treatment to be described in the experimental part, a substance very similar to phytic acid, but apparently having the composition represented by the formula C6H24024P6. From his crystalline tri-barium salt from cotton-seed meal, oats and corn, Anderson obtained an acid to which he ascribed the formula, C6H18O24P6. Hence, both in the case of the barium salt and the free acid, our compounds appear to contain six more hydrogen atoms to the molecule; while in carbon, barium and phosphorus contents, they agree very well with Anderson's compounds.

In comparing the results of the analyses, the method used in combustion must be taken into consideration. It is a well known fact that, in the combustion of organic compounds containing phosphorus, the phosphorus is converted into metaphosphoric acid, HPO3, which remains as a glossy coating in the boat, and may occlude more or less carbon. Anderson states that in decomposing his crystalline barium salts, it was necessary to burn a second time with chromic acid, in order to insure combustion of all the carbon, but that this was unnecessary with the amorphous barium salts. Since he does not say that he burned the free acid with chromic acid, we presume he did not do so. It is inevitable, if this is true, that his hydrogen analyses gave low results for phytic acid. It is a noteworthy fact that his formula shows six atoms less in the Anderson: Jour. Biol. Chem., 1914, xvii, p. 160.

molecule than ours; and, since the molecule contains six atoms of phosphorus, the formation of the metaphosphoric acid residue would account for the discrepancy, if our substance is identical with his. In the case of the barium salt, the explanation is less evident, for, of course, barium phosphate or metaphosphate would be formed, together with some barium carbonate and metaphosphoric acid, although a reaction between the latter two substances might take place, liberating both the hydrogen and carbon.

In each of our combustions, we burned the material a second time: in the case of the free acid and the brucine salt to be described, with well dried, powdered lead chromate; in the case of the barium salts, with a mixture of lead chromate and potassium dichromate. There was always an increase in weight in both the potash bulbs and the calcium chloride tube after the second burning. It is probable, therefore, that our compounds from wheatbran are identical with those obtained by Anderson from various other sources.

We believe, however, that in addition to the phytic acid derivative in our extracts of wheat-bran, there were at least two other organic-phosphorus compounds, which we have been prevented from investigating completely by lack of time. It was one of these substances which Anderson investigated, and found to yield an acid, to which he ascribed the formula, C20H55049P9, combined with the elements of a pentose. In regard to this substance, we wish to point out that his analytical results show rather wide departures from the calculated formula, and that none of the barium salts were obtained crystalline; hence may not have been pure. It is also noteworthy that the analytic data for the crystalline brucine salt [to which he ascribed the formula C20H55O49P9 (C23H26O4N2) 10], accorded better (except in the case of carbon which is low) with brucine phosphate, (C23H26O4N2) 3. (H3PO4)2 than with his calculated formula. Anderson himself has shown that phytic acid is broken down into phosphoric acid and other substances by drying at 100° C., and even to some extent by drying at ordinary temperatures. The new acid prepared by him from wheat-bran