THE NATURE OF THE ACID SOLUBLE PHOSPHORUS COM- POUNDS OF SOME IMPORTANT FEEDING MATER1ALS.l BYE. B. HART AND W. E. TOTTINGHAM. (From the Agricultural Chemical Laboratory of the University of Wisconsin.) (Received for publication, July 13, 1909.) In rgoo Posternak published his first paper2 on the occurrence of phytin in vegetable tissue. This was followed by subsequent papers3 in which he described the separation of this body from the seeds of the red fir, pumpkin, pea, bean, white and yellow lupine and the potatoe. In 1904 Patten and Hart* isolated phytic acid from wheat bran, and in Igo7 Suzuki5 succeeded in separating the same body from rice bran. The indications are that phytic acid as a complex salt of potas- sium, magnesium and calcium, is widely distributed in nature, particularly in the seeds as a reserve material. In our early work6 on this subject it was found that the natural grains, such as corn, oats and wheat, yielded to an extraction with dilute hydrochloric acid, from 30 to 50 per cent of their total phos- phorus. This, interpreted in the light of available methods, meant that practically the entire hydrochloric acid-soluble phos- phorus was organic in form. The investigation incorporated in this paper is confined to a continuation of our study on the nature of the phosphorus-bear- ing bodies contained in this dilute hydrochloric acid extract ot a few of our natural feeding materials. Representatives of different botanical families have been chosen. Although the work of Palladin7 and Posternak had 1 Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station. 2 Rev. gin. de botanique, xii, pp. 5 and 65, 1900. 3 Conspt. rend. de l’acad. des xi., no. 3, no. 5, no. 8, cxxxvii, 1903. 4 Amer. Cheun. Journ., xxxi, no. 5. 5 Bulletin of College of Agriculture, Tokio Imperial University, vii, no. 4. B Hart and Andrews: Amer. Chem. Journ., xxx, no. 6. 7 Zeitschr. f. Biol., 1894, p. 199. 431 by guest on February 2, 2020 http://www.jbc.org/ Downloaded from
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THE NATURE OF THE ACID SOLUBLE PHOSPHORUS COM- POUNDS OF SOME IMPORTANT FEEDING MATER1ALS.l
BYE. B. HART AND W. E. TOTTINGHAM.
(From the Agricultural Chemical Laboratory of the University of Wisconsin.)
(Received for publication, July 13, 1909.)
In rgoo Posternak published his first paper2 on the occurrence of phytin in vegetable tissue. This was followed by subsequent papers3 in which he described the separation of this body from the seeds of the red fir, pumpkin, pea, bean, white and yellow lupine and the potatoe. In 1904 Patten and Hart* isolated phytic acid from wheat bran, and in Igo7 Suzuki5 succeeded in separating the same body from rice bran.
The indications are that phytic acid as a complex salt of potas- sium, magnesium and calcium, is widely distributed in nature, particularly in the seeds as a reserve material. In our early work6 on this subject it was found that the natural grains, such as corn, oats and wheat, yielded to an extraction with dilute hydrochloric acid, from 30 to 50 per cent of their total phos- phorus. This, interpreted in the light of available methods, meant that practically the entire hydrochloric acid-soluble phos- phorus was organic in form.
The investigation incorporated in this paper is confined to a continuation of our study on the nature of the phosphorus-bear- ing bodies contained in this dilute hydrochloric acid extract ot a few of our natural feeding materials.
Representatives of different botanical families have been chosen. Although the work of Palladin7 and Posternak had
1 Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station.
2 Rev. gin. de botanique, xii, pp. 5 and 65, 1900. 3 Conspt. rend. de l’acad. des xi., no. 3, no. 5, no. 8, cxxxvii, 1903. 4 Amer. Cheun. Journ., xxxi, no. 5. 5 Bulletin of College of Agriculture, Tokio Imperial University, vii, no. 4. B Hart and Andrews: Amer. Chem. Journ., xxx, no. 6. 7 Zeitschr. f. Biol., 1894, p. 199.
already revealed the presence of phytin in the potatoe, a rep- resentative of the Solanacecz; in the seeds of the pea and bean- Legumivtos~, and in the black mustard seeds-Crucifercz, never- theless it was thought not impossible that a chemical difference might obtain in the phosphorus complexes in other species of these families of plants. In addition, studies of parts of the plant other than the seeds were undertaken. With this end in view, representatives of the GraMinece, Cruciferca and Legwninos~ were chosen.
In our choice of materials we have always selected those parts of the plant that are used preeminently for direct animal feed- ing. It is not to be doubted that the different parts of a plant will vary, not only in the proportion but also in the nature of its phosphorus bearing bodies. We would expect to find bodies of greater mobility and simpler structure in the stems of plants than in the seeds. This is true of nitrogenous structures, as exempli- fied in our hays, where the simple amino acids predominate, as compared with a much lower content of these forms in the seeds. For example, in our previous work1 it was found that while 70 per cent of the phosphorus in alfalfa hay was soluble in dilute hydrochloric acid, 52 per cent of this acid soluble phos- phorus was inorganic in form. This is very different from the condition prevailing in some of our seeds, as oats, corn and wheat, where 30 to 50 per cent of the total phosphorus was acid soluble, but practically all in organic form.
Isolation of Phytic Acid from the Corn Grain (Zea mays).
Ten kilos of finely ground corn meal were extracted with 0.2
per cent hydrochloric acid, strained through cheesecloth and filtered through paper. The clear brown solution was treated with copper acetate in dilute acetic acid solution to facilitate the removal of any bases existing in combination with phytic acid. After thoroughly washing, the copper precipitate was suspended in water and decomposed by hydrogen sulphide. The filtrate from the copper sulphide was made alkaline with sodium hydrox- ide and precipitated with barium chloride. The barium salt was washed free from alkali, suspended in water and dilute sul-
phuric acid added in sufficient quantity to decompose the salt and throw down the barium as a sulphate. After removal of the barium sulphate by filtration, the filtrate was again precipi- tated in alkaline solution with barium chloride and treated as before. This process was repeated three or four times and after final removal of the barium, copper acetate was addedin excess. The copper precipitate was filtered, thoroughly washed with water, and finally suspended in water and decomposed by hydrogen sulphide. The copper sulphide was removed by filtra- tion and the filtrate evaporated on the water bath to a syrupy consistency.
The residue was dark in color and sharply acid to indicators, Analysis of the acid dried at 100’ C. gave the following results:
o. I I 3 gram of substance gave o .0426 gram CO, = I o .3 per cent C. o. 200 gram substance gave o. 187 gram Mg,P,O, = 26.08 per cent P.
Calculated for C~H~P,OQ: Found:
C . . . . . . . . . . . . . . . . . . . . . . I o .08 per cent IO. 30 per cent P . . . . . . . . . . . . . . . . . . . . . 26.07 u 26.08 “
Decomposition of the Cowzpound into Inosite and Phosphoric Acid. In this investigation an unweighed portion of the acid was heated in a sealed tube with 50 cc. of 30 per cent sulphuric acid at a temperature of 155 to 160~ C. for five hours. After cooling, the tube was opened, the sulphuric and phosphoric acids removed by barium hydroxide and the excess of barium with carbon dioxide. The filtrate was evaporated nearly to dryness, taken up with hot water and filtered from the remaining barium carbonate. This filtrate was treated with absolute alcohol and ether until a cloudiness was produced. It was then surrounded by ice and allowed to stand. A crystalline precipitate separated which after recrystallization was identified as inosite. It gave the reactions of Scherer and Gallois and melted at 220’ C. (uncor- rected). Inosite melts at 218’ C. (uncorrected). The substance dried at I IO’ C. gave the following results:
o. TOO gram of substance gave 0.148 gram CO, and 0.062 gram H,O.
CdC~&td,”
C.....................40.00percent. H .t................... 6.66 “
The isolation of inosite and the data on the ultimate analysis identify the body separated from corn as phytic acid.
Preparation of Phytin.
Two kilos of corn meal were extracted with water for several hours with frequent agitation. The extract was strained through cloth and finally filtered through paper to a clear solution. The filtrate was treated with a large volume of 95 per cent alcohol, which threw down a voluminous flocculent precipitate. The precipitate was allowed to settle to the bottom, the supernatant liquid siphoned off and the precipitate washed with alcohol by decantation. It was then redissolved in a small volume of water, filtered from an insoluble residue, reprecipitated with alcohol and washed as before by decantation. The precipitate was finally brought upon the filter, washed with alcohol and ether and dried at 100' C. The product was a yellow amorphous powder, veryrich in phosphorus, and but slightly soluble in water. Apparently some substance,very probably of a protein nature- as the substance still gave color reactions for protein-was con- taminating the material and modifying the solubilities of the en- tire mass. The material manifestly does not represent a pure product. Nevertheless a partial analysis is appended below, which indicates that we had an impure salt of phytic acid and that the same bases that were found in a preparation of phytin from wheat bran’ were to be found here.
Per cent Ca.................................................. 0.48 K.................................................. 5.60 Mg................................................ 6.70 P...................................................14.1,
Distribution of Phytin in the Seed.
In the wheat kernel phytic acid as a salt exists largely in the outer aleurone layers and consequently is found in very large pro- portion in wheat bran. In order to determine whether similar distribution obtained in the corn kernel this seed was mechan- ically divided into three parts-the outer layer or corn bran
(pericarp), the germ and the starch and gluten cells (endosperm). The following table shows the distribution of total and dilute acid soluble phosphorus. These determinations were made on the air-dried material.
It is clear from these data that in the maize grain, phytin is not localized in the outer layers. It is probably very nearly accurate to consider that the phosphorus of cereal grains sol- uble in dilute hydrochloric acid largely exists as phytin. In this instance there appears to be none in the outer skin or seed coats, while there is, on the contrary, more or less uniform distribution throughout the entire seed.
Isolation of Phytic Acid jrovn the Oat Kernel (Avena sativa).
Eight to ten kilos of finely ground seed were extracted with 0.2
per cent hydrochloric acid, strained through cheese cloth and filtered through paper. The details of the preparation and puri- fication of the acid were in every particular similar to those given under the separation of this product from corn, and consequently it will be unnecessary to repeat them.
The final preparation was black in color, of a syrupy consist- ency and a sharp acid taste. It was dried at roo” C. and gave the following results on analysis :
o I 28 gram of substance gave o. 048 gram of CO, = 10.22 percentc. 0.29 I gram of substance gave 0.27 I gram of Mg,P,O, = 25.97 per cent P.
After removing the sulphuric and phosphoric acids, the residual solution was evaporated nearly to dryness, treated with alcohol, and ether, iced and allowed to stand. The crystalline precipitate which separated was again recrystallized, washed with ether and dried at 100~ C.
This body melted at 217.5’ C. (uncorrected) and gave the reactions of Scherer and Gallois. Inosite melts at 2 18' C. (uncor- rected). These reactions and the melting point fully identified the body as inosite and established the presence of the phytic acid radical in the oat grain.
Preparation of Phytin from Oats.
Tbo to three kilos of the finely ground grain were extracted with water and the extract finally obtained clear by frequent filtration through paper. The further separation and purifi- cation of the preparation was conducted in exactly the same manner as has been previously detailed in the case of the prep- aration from the corn grain. The color reactions for protein persisted even after a third resolution and reprecipitation of the substance. The final preparation was a white friable powder and but partially soluble in water. Estimation of the bases and phos- phorus gave the following figures :
Per cmt Ca.................................................. 8.6 Mg.,................................................ 4.7 K................................................... 1.5 P...................................................16.7
Distribution of Phytin in the Seed.
The seed was mechanically separated into the hull or bran layers (pericarp) and kernel. The former is fibrous and forms a considerable portion of the grain. The latter consists of the aleurone layer and starch cells (endosperm) and the embryo, No further divisions were made. The analyses are reported on the air-dried material.
It is apparent from the data that while the seed coats carry a total amount of phosphorus comparable with the other parts of the grain, the proportion of phytin in the outer layers is rela- tively small. On the other hand it constitutes 50 per cent of the total phosphorus bearing bodies in the remaining parts of the seed.
Isolation of Phytic Acid from Barley (Hordeuun sativunz) .
Five kilos of finely ground barley were extracted with 0.2 per cent hydrochloric acid. The extract was filtered clear and pre- cipitated with copper acetate. This precipitate was decomposed with hydrogen sulphide, the copper sulphide removed and further manipulation continued as has already been described. The product was a dark sticky mass and less fluid when perfectly dry than the preparations so far studied from the other grains. It was dried at IIO’ C. and gave the following results:
o. 154 gram of substance gave o. 058 gram of CO, = 10.5 percentc. o. 485 gram of substance gave 0.450 gram of Mg,P,O, = 25.8X per cent P.
An unweighed portion was decomposed under pressure with 30 per cent sulphuric acid heated to I 5 5’ C. for five hours. After removing the sulphuric and phosphoric acids, the clear solution was evaporated to 25 cc., cooled and treated with alcohol and ether. After icing and allowing to stand a few hours, a crystal- line precipitate separated which after recrystallization was dried at 110~ C. This substance had a melting point of 219~ (uncor- rected). It gave the Scherer reaction and a carbon determina- tion gave the following results :
o I 20 gram of substance gave o . I 74 gram of CO, - 39.5 per cent C.
These results fully identified the body as inosite and furnished conclusive evidence that the preparation from barley was phytic acid.
Preparation of Phytin.
Five kilos of ground barley were extracted with water for 36 hours under frequent stirring and in the presence of chloroform and toluol to inhibit bacterial fermentations. The liquid, both supernatant and expressed, was filtered through paper, boiled to coagulate the proteins, filtered, concentrated and precipitated with 95 per cent alcohol. This precipitate was filtered off, digested with water and again filtered from an insoluble residue. The clear solution was treated with alcohol and the precipitate filtered out. This was redigested with water and the process as above described repeated a third time. The preparation was difficultly but completely soluble in water and after drying at 100' C. for five hours showed the following proportion of bases and phosphorus :
Per Cent Ca................................................... 0.00
No calcium was present in this preparation from barley.
Distribution of Phytin in the Seed.
The separation of the entire seed into the outer fibrous coats (pericarp), here designated as bran, and into the aleurone layer, starch cells and embryo (kernel) was carried out mechanically. The separate portions of the seeds gave the following results. The determinations were made on air-dried materials :
proportion of the total phosphorus existing there. Phytin appears to be distributed throughout the entire seed.
Study of the Dilute Hydrochloric Acid Soluble Phosphorus in Rutabagas (Bras&a rutabaga).
Posternak has already isolated phytic acid from the potatoe. The fact that roots and tubers are agriculturally valuable be- cause of the store of nutrients which they deposit in their sub- terranean branches or roots lead us to select for investigation another representative of this class of materials.
Twenty-five to thirty kilos of rutabagas were thinly sliced and dried. After grinding, the material was extracted with 0.2 per cent hydrochloric acid and the clear extract filtered and precipitated with copper acetate. The further treatment and manipulation was exactly comparable to that followed in the preparation of phytic acid from the grains. The product finally obtained, dried down to a gummy, dark-colored mass. It gave no protein reactions and was free from nitrogen, but had partly lost its original solubility in water. It reduced Fehling’s solu- tion, an important distinction from phytic acid, which does not have this reducing property. An ultimate analysis of this prep- aration, dried at 100~ C., gave the following results:
Per cent P . . . . . . ..*....*.......*......*.I........*.l...... 16.92 c................................................ 17.81
This first preparation was again treated with water, filtered from an insoluble residue, and the insoluble barium salt again prepared in alkaline solution. After its resolution in dilute sul- phuric acid and a repeated precipitation and solution a second and third time, the material was separated as a copper salt, de- composed with hydrogen sulphide, evaporated and dried at 100~ C. A phosphorus determination on this preparation showed 25.98 per cent. Too small an amount, however, of the second preparation was available for further analytical work. It did not directly reduce Fehling’s solution, indicating that the further process of purification had removed a contaminating and reduc- ing substance from preparation I.
An unweighed quantity of preparation I was heated for five hours with 30 per cent sulphuric acid in a sealed tube at 155' to
I 60’ C. After removing the sulphuric and phosphoric acids with barium hydroxide the solution was concentrated and treated with 95 per cent alcohol and ether. The mixture was iced and allowed to stand several days. A trace of a white amorphous substance separated which was finally collected on a filter for further examination. It did not give the reactions of Scherer and Gallois. It reduced Fehling’s solution in contradistinction to the behavior of inosite with this solution. These tests exclude the possibility of this body being inosite. Further, it makes it very improbable that phytic acid exists in rutabagas.
A quantitative extraction of the dried rutabagas with dilute o. 2 per cent hydrochloric acid showed that 88 per cent of the total phosphorus was soluble in this menstruum. Application of the Hart-Andrews method’ for inorganic phosphorus to the hydrochloric acid extract, showed that 73 per cent of the acid soluble phosphorus was inorganic in form. However, in this estimation on rutabagas somewhat more nitric acid than the method specified was used to effect precipitation by the neutral molybdate. The rapidity with which the separation of the molybdate took place when just sufficient nitric acid was present indicates the suppression of some retarding influence on the molyb- date separation, rather than a progressive hydrolysis of organic forms of phosphorus. It appears from the data that there was no phytin in the rutabagas examined and that the greater part of the hydrochloric acid-soluble phosphorus exists in inorganic forms. It is, however, probable that in addition to the inorganic forms, there was in small amounts some complex of phosphoric acid paired with an unknown organic radical. The amount of material obtained was too small for a thorough study of these preparations and this work is only preliminary to their further investigation.
Examitiation of the Dilute Hydrochloric Acid Extract of Cured Alfalfa Hay (Medicago sativa).
The material used was clean, field-cured alfalfa, cut in early bloom. Six to eight kilos of the air-dried material were finely chopped, ground and extracted with 0.2 per cent hydrochloric acid. The details for the separation of phytic acid were strictly followed. The final preparation dried at 100’ C. was a dark, limpid liquid, differing in this physical respect from any of the grain or rutabaga preparations, these usually being either thick and syrupy in character, or occasionally even gummy. It gave none of the protein color reactions and was free from nitrogen. It strongly reduced Fehling’s solution. An ultimate analysis gave the following results:
Per Cent P................................................ 16.91 C................................................ 9.19 H................................................ 3.50
This analysis differs from that of a pure phytic acid, the per cent of phosphorus being materially lower.
Hydrolysis with Strong Acids.
A portion of the preparation was hydrolyzed with 30 per cent sulphuric acid in a sealed tube at 155’ C. for five hours. After removal of all the sulphuric and phosphoric acids with barium hydroxide and the excess of barium as a carbonate, the clear solu- tion was evaporated to a small bulk; it was then treated with alcohol and ether, iced and allowed to stand several days, as in the usual procedure for the separation of inosite. Finally a small amount of an amorphous material separated. This substance was collected and tested for inosite. It gave none of the charac- teristic color reactions for that body and reduced Fehling’s solution. These facts exclude the possibility of the existence of phytic acid in alfalfa at that stage of its development.
Hydrolysis with Dilute Acids.
2.2 5 grams of the material were treated with IOO cc. of a 2.5
per cent solution of sulphuric acid for two hours at the tempera-
ture of the water bath and under a reflux condenser. After re- moving th sulphuric and phosphoric acids a quantitative esti- mation of the reducing power of the filtrate was made. This was equivalent to 0.035 gram calculated as dextrose. The material in 50 cc. rotated + I .o” in a 400 mm. tube, Soleil-Ventzke in- strument, 0.03 5 gram of dextrose under similar conditions, should have given a rotation of approximately + .7O. As the material on hand was too small for further study, it is at present impos- sible to definitely decide whether this simple hexose was present. The readings are within experimental error for small amounts of dextrose.
Distillation of a small sample of the original material with strong hydrochloric acid gave no positive test for furfurol, ex- cluding the pentose sugars in this instance as possible paired bodies with phosphoric acid.
Studies on this subject are being continued. There is strong evidence for the supposition that a class of organic phosphorus bodies, which are not phytins, exists in the transition stages of growth of the alfalfa plant.
It is, however, necessary to observe that a large part of the dilute soluble phosphorus in the material at this stage of growth was inorganic in form. Eighty per cent of the total phosphorus was soluble in 0.2 per cent hydrochloric acid, while by the Hart- Andrews method, 79 per cent of this soluble phosphorus was in- organic in form.
In the following table is displayed a summary of the analytical data covering the estimations on the several materials of total dilute acid soluble and inorganic phosphorus. The determina- tions were made on air-dried materials :
If we assume that the phosphorus in the dilute acid extract of the grains is practically all as phytin, and this assumption is probably very near the truth, then the amount of this form, as shown in the table under column four, can be approximately derived. Column five shows that over 60 per cent of the total phosphorus in the rutabaga and alfalfa hay was inorganic, while of that soluble in dilute hydrochloric acid the amount of inorganic phosphorus was 79 and 73 per cent respectively.
SUMMARY.
(I) Phytin is present in the seeds of the corn, oat and barley plants. It has already been isolated from the wheat kernel.’
(2) Phytin could not beisolated from rutabagas or alfalfa hay. (3) It appears to be distributed throughout the entire
seed of the cereal grains reported in this investigation, differing in this respect from wheat, in which it is largely localized in the outer layers.
(4) The proportion of phytin phosphorus as approximately estimated was 38 to 48 per cent of the total phosphorus in these seeds.
(5) The principal form of phosphorus in the rutabaga was inorganic, 64 per cent of the total existed as such; there was in addition some unknown organic phosphorus complex, soluble in dilute acids, nitrogen free and yielding reducing substances on hydrolysis, the phosphorus of which approximated 24 per cent of the total phosphorus of this root.
(6) In alfalfa, cut when just coming into bloom, and repre- senting that stage of growth recognized as most suitable for for- age, the inorganic phosphorus found was about 63 per cent of the total phosphorus.
(7) There was present in the dilute acid extract of alfalfa hay an organic phosphorus complex, separated by the modified Posternak method, which was nitrogen free, yielded reducing bodies on hydrolysis with acids, but did not produce inosite; it consequently was not phytic acid. The phosphorus of this body constituted about 17 per cent of the total phosphorus of the plant.
1 Patten and Hart: Amer. Chem. Journ., xxxi, no. 5.
(8) It is possible that the reducing substance, separated by dilute acids from the alfalfa hay preparation, was dextrose. Further work is necessary to fully decide this point. No pentose sugar could be separated from the same preparation.
