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PETROLEUM ETHER- AND ETHER-SOLUBLE CONSTIT- UENTS OF CRANBERRY POMACE* BY K. S. MARKLEY AND CHARLES E. SAND0 (From the Division of Fruit and Vegetable Crops and Diseases, Bureau of Plant Industry, and the Food Research Division, Bureau of Chemistry and Soils, United States Department of Agriculture, Washington) (Received for publication, April 20, 1934) The annual production of cranberries in the United States amounts to approximately 500,000 barrels, valued at 5 to 6 million dollars. Despite the economic importance of the American cranberry, Oxycoccusmacrocarpus (Vaccinium moxrocarpon), it has received considerably less attention from a chemical view-point than has been accorded the cowberry, Vaccinium vitis id~cz,which serves in the northern European dietary much as the cranberry does in America. Wehmer (1) cites numerous instances of the identification of citric, malic, and benzoic acids in cranberries. Quinic acid, which was presumed by Blatherwick and Long (2) to be the precursor of hippuric acid secreted in the urine following ingestion of cranberries, was isolated from fresh berries by Kohman and Sanborn (3). The red pigment of the cranberry has been identified as 3+glucosidyl peonidin chloride by Grove and Robin- son (4). The mineral constituents of the ash have been deter- mined- by Morse (5), who also made comparative proximate analyses of a large number of cranberry varieties (6). He reported as wax the carbon tetrachloride extract of cranberries, and showed that this fraction increased during growth; for example, during the month of September from 0.40 to 0.47 per cent (fresh weight basis) and from 0.28 to 0.35 per cent for Early Black and Howes varieties, respectively. In view of the fact that most of the residue extracted from cran- berry pomace with petroleum ether and ether originates from the surface of cranberries and because some of the substances might * Food Research Division Contribution No. 225. 643 by guest on April 6, 2018 http://www.jbc.org/ Downloaded from

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PETROLEUM ETHER- AND ETHER-SOLUBLE CONSTIT- UENTS OF CRANBERRY POMACE*

BY K. S. MARKLEY AND CHARLES E. SAND0

(From the Division of Fruit and Vegetable Crops and Diseases, Bureau of Plant Industry, and the Food Research Division, Bureau of Chemistry

and Soils, United States Department of Agriculture, Washington)

(Received for publication, April 20, 1934)

The annual production of cranberries in the United States amounts to approximately 500,000 barrels, valued at 5 to 6 million dollars. Despite the economic importance of the American cranberry, Oxycoccus macrocarpus (Vaccinium moxrocarpon), it has received considerably less attention from a chemical view-point than has been accorded the cowberry, Vaccinium vitis id~cz, which serves in the northern European dietary much as the cranberry does in America. Wehmer (1) cites numerous instances of the identification of citric, malic, and benzoic acids in cranberries. Quinic acid, which was presumed by Blatherwick and Long (2) to be the precursor of hippuric acid secreted in the urine following ingestion of cranberries, was isolated from fresh berries by Kohman and Sanborn (3). The red pigment of the cranberry has been identified as 3+glucosidyl peonidin chloride by Grove and Robin- son (4). The mineral constituents of the ash have been deter- mined- by Morse (5), who also made comparative proximate analyses of a large number of cranberry varieties (6). He reported as wax the carbon tetrachloride extract of cranberries, and showed that this fraction increased during growth; for example, during the month of September from 0.40 to 0.47 per cent (fresh weight basis) and from 0.28 to 0.35 per cent for Early Black and Howes varieties, respectively.

In view of the fact that most of the residue extracted from cran- berry pomace with petroleum ether and ether originates from the surface of cranberries and because some of the substances might

* Food Research Division Contribution No. 225. 643

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644 Constituents of Cranberry Pomace

be similar or related to the constituents previously identified from the surface of apples, it seemed desirable to undertake an investiga- tion of the cranberry extracts. It was also thought that the results of such work might suggest possible commercial uses for the constantly accumulating cranberry waste which is produced in the processing of the berries for canned sauce and jelly.

EXPERIMENTAL

The material used in this investigation was air-dried cranberry pomace consisting of cuticle with some adhering cell tissue and seeds, the latter comprising about 25 per cent of the weight of the residue. This material was furnished through the courtesy of Cranberry Canners, Inc., South Hanson, Massachusetts.

After being ground to pass a 2 mm. sieve the material was extracted first with petroleum ether (skellysolve, b.p. 30-60’) and then with ethyl ether. 4 kilos of ground cranberry waste gave 9.6 per cent petroleum ether-soluble extract and 10.6 per cent ether- soluble extract by the hot extraction method, and 10 kilos gave 9.8 per cent and 10.2 per cent respectively by the cold percolation method. Altogether, 1.40 kilos of petroleum ether-soluble extract and 1.49 kilos of ether-soluble extract were obtained.

Petroleum Ether Extract

The petroleum ether extract of the cranberry waste, which was a yellowish green semisolid mass at room temperature, became increasingly pasty with rising temperature until at about 55-60” it was transformed into a clear yellowish green liquid. That the crude extract contained considerable free fatty acid, calculated as oleic acid, was indicated by the fact that it required for neutrali- zation 1.52 ml. of N p’otassium hydroxide per gm., equivalent to 43 per cent of free acid, or approximately 72 per cent of the total fatty acids subsequently isolated.

A portion (192 gm.) of the extract was saponified for 10 hours with 4 per cent alcoholic potassium hydroxide, the alcohol partly removed by evaporation, and the residue poured into water. The mixture was then extracted with ether and the latter evaporated to dryness. The residue representing the unsaponifiable fraction was resaponified and again extracted with ether, which was then evaporated. After recovery of the acids from the dissolved soaps

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K. S. Markley and C. E. Sando

of the combined aqueous layers, four fractions were obtained as follows: aqueous filtrate, fatty acids (115 gm.), unsaponifiable fraction (67 gm.), and insoluble interfacial layer (15 gm.). The fraction mentioned last was discarded after repeated attempts had been made to crystallize it without success.

GEyceroZ-After recovery of the acids from the soluble soaps, the aqueous filtrate and washings were brought to neutrality by the addition of alkali and concentrated on the steam bath until most of the sodium salts separated from the concentrated solution. These were removed and the liquid was evaporated almost to dryness, The residue was extracted with 95 per cent alcohol containing a small amount of ether. The alcohol was evaporated off, the residue taken up in hot water, and clarified with lead acetate. The deleaded filtrate was colorless and on evaporation yielded a small amount of a viscous liquid, which on heating with potassium bisulfate gave off acrolein, as was evident from its acrid odor and the fact that it strongly reduced ammoniacal silver &rate and Fehling’s solution. When dissolved in 1 per cent copper sulfate, it gave a clear deep blue solution on the addition of concentrated sodium hydroxide. The above tests indicate that the aqueous filtrate contained glycerol, but the amount obtained was insufficient to account for the total fatty acids being present as glycerides. This conclusion is in keeping with the high titration value of the crude petroleum ether extract.

Liquid Fatty Acids

The fatty acid fraction (115 gm.) was dissolved in 95 per cent alcohol, treated with charcoal (darco), and allowed to stand at low temperature for several weeks, whereupon a crystalline mass slowly separated. After filtering and washing there was obtained a light yellow powder weighing 7.5 gm., which appeared to be solid fatty acids. The bulk of the acids which remained soluble in the alcohol was submitted to the lead salt-ether method of Varrentrapp in order to separate the saturated from the unsaturated acids. The insoluble lead salts gave an additional 3 gm. of solid acids. The soluble lead salts, after decomposition and removal of the lead, gave 102 gm. of liquid fatty acids.

Linolenic Acid-The unsaturated acids were dissolved in a mix- ture of glacial acetic acid and ether, which was cooled to - 5” and

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brominated dropwise with an ice-cold solution of bromine in glacial acetic acid. Crystals began separating immediately, but the brominated solution was permitted to stand several days in the refrigerator before filtering. 20 gm. of grayish white bromides were collected and crystallized several times from benzene, where- upon there were obtained 9 gm. of hexabromolinolenic acid melting at 182-183’. Two bromine determinations’ with the micro-Carius method gave 63.23 and 63.50 per cent. Theory, 63.28 per cent.

Linoleic Acid-After separation of most of the impure hexa- bromides, the filtrate was heated on the steam bath under reduced pressure to remove the ether, and the remaining solution was poured into water. After standing in the refrigerator for 24 hours, the gummy precipitate was separated by filtration, dissolved in ether, and the ethereal solution dried over anhydrous sodium sulfate. It was then evaporated to dryness and the residue re- peatedly extracted with hot petroleum ether.

On standing at about 10’ for some days, the petroleum ether solution slowly deposited crystals. The liquid was separated from the precipitate (Crop 1) by decantation and the solution returned to the refrigerator. After several days an additional crystalline precipitate (Crop 2) separated and was removed as before. Fur- ther separation of solids did not occur after 2 weeks. The solution was therefore concentrated to a thick sirup, from which crystals slowly formed at low temperature. The crystals (Crop 3) were separated from the sirup by careful solution of the latter in cold petroleum ether. Altogether the three crops of impure tetra- bromides amounted to 25 gm., and the sirupy dibromide fraction, obtained after the final removal of the petroleum ether, weighed 124 gm.

The impure tetrabromides comprising Crop 1 were extracted with petroleum ether, in which they were found to be only partly soluble. The insoluble portion was recrystallized from benzene, which gave 3 gm. of hexabromolinolenic acid melting at 180-181’. The soluble portion was combined with Crop 2, and the combined material crystallized from petroleum ether. The tetrabromides

1 For the combustion and halogen determinations reported throughout this paper, the writers are greatly indebted to R. T. Milner and Mildred S. Sherman of the microchemical laboratory, Fertilizer Investigations, Bureau of Chemistry and Soils.

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obtained on recrystallization weighed 19 gm. and were still yellow- colored. They were therefore dissolved in ether, treated with charcoal, and fractionally precipitated by the addition of petroleum ether. After recrystallization from petroleum ether, two fractions of approximately the same weight (10 gm.) were obtained, one melting quite sharply at 107.5-108” and the other considerably below 100’. The latter fraction contained a low melting solid fatty acid and is therefore described later under solid fatty acids. The fraction melting at 107.5-108” was repeatedly fractionated from ether by the addition of petroleum ether, until finally there were obtained 5.7 gm. of tetrabromides melting at 109.5-110’ and having a bromine content of 54.65 per cent. The tetrabromides comprising Crop 3 were also submitted to fractional separation from ether by the addition of petroleum ether, yielding 3.5 gm. of nearly pure tetrabromolinoleic acid melting at 113-113.5” and having a bromine content of 53.68 per cent. Pure tetrabromo- linoleic acid contains 53.28 per cent of bromine and melts at 113-114”.

Oleic Acid-The sirupy dibromide fraction, weighing 124 gm., obtained by evaporation of the petroleum ether solution after separation of the impure tetrabromides as described under linoleic acid, was taken up in 90 per cent alcohol, in which medium it was debrominated with the aid of metallic zinc. The oleic acid was recovered by the usual saponification procedure, after removal of excess zinc and decomposition of the zinc sal%s. 62 gm. of oleic acid were obtained having an iodine number (Hanus) of 95.4. Theory, 89.93.

Solid Fatty Acids

The solid fatty acids came from three sources; namely, (a) alcoholic solution of the original fatty acids fraction (7.5 gm.), (5) ether-insoluble lead salts (3 gm.), and (c) Crop 2 under linoleic acid (10 gm.). Acids from (a) were crystallized once from ethyl acetate-alcohol solution, submitted to the lead salt-ether method of Varrentrapp to remove further traces of liquid fatty acids, and the regenerated solid fatty acids fractionally crystallized from ethyl acetate. Final crystallization was from a mixture of acetone and glacial acetic acid. The acids from (b) were fractionally crystallized from ethyl acetate-alcohol solution, while those from (c) were recrystallized from petroleum ether, ether, and benzene.

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The approximate weights, melting points, and neutralization values of the various fractions were determined and are recorded in Table I.

The first three fractions recorded in Table I were combined, converted into the corresponding barium salts, and exhaustively extracted with ether. The regenerated acids were then crystal- lized twice from dilute alcohol; 4.5 gm. of fine needles were ob- tained, having a melting point of 53.5-54.5° and a neutralization value of 181. The last two fractions mentioned in Table I were submitted Lo x-ray examination.” The value of the roughly determined spacing (67.6 f 1.0 A.) and the melting points and neutralization values indicated that the fractions were mixtures.

TABLE I

Aplfroximate Weights, Melting Points, and Neutralization Values of Various Fractions of Solid Fatty Acids Obtained from Petroleum Ether-

Soluble Fraction of Cranberry Pomace

Source mgd. fraction

a-4 b-l b-2 a-3 a-l c-l a-2

Weight

gm. “C.

4.0 52.3-53.5 1.7 54.0 0.9 55.s56.0 0.8 68.5-68.8 1.5 72.0-73.0 1.0 78.5-79.0 1.2 79.5-80.0

M.P. Neutralization due

198 200 208 177 158 142 144

It is obvious, therefore, that the solid fatty acids consisted of the usual mixture of plant acids of the series CDJ to CL,, which, in view of the small amount originally available, cannot be separated into definite chemical entities, as has been demonstrated by Francis, Piper, and Malkin (7).

Parafin Hydrocarbons

The unsaponifiable fraction (67 gm.) was heated for 7 hours under a reflux condenser with a large excess of acetic anhydride. After standing overnight the insoluble solid was separated by

* Measurements of the crystal spacings were kindly made by Sterling B. Hendricks of Fertilizer Investigations, Bureau of Chemistry and Soils.

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filtration and crystallized from 95 per cent alcohol. The light yellow solid (28 gm.) was again treated with acetic anhydride and recovered. From the acetic anhydride filtrates 45 gm. of an oily material were obtained, which after saponification and frac- tional distillation at 3 mm. pressure yielded unidentifisble liquid oily and solid resin fractions and a small amount of microcrystalline substances of neutral reaction, melting at 56-57”.

Nonacosane and Hentriacontane-The acetic anhydride-insoluble portion was crystallized first from alcohol containing a small pro- portion of ethyl acetate and then twice from a petroleum ether- acetqne mixture (1 :,l), whereupon 5 gm. of hydrocarbon were obtained melting at 63.2-65’. Four main fractions melting be- tween 63.2-64.5” were obtained from this material by repeated fractionation from petroleum ether-acetone mixture, saponification with sodium ethoxide, and treatment with acetic anhydride. Although the crystal spacings (41.2 to 42.0 A., all f0.3) indicated that the material consisted of a mixture of hydrocarbons, never- theless the results of combustion (C, 84.20 to 84.28; H, 14.46 to 14.76) showed the presence of oxygenated impurities. The vari- ous fractions were therefore combined, and the resulting sample repeatedly treated with concentrated sulfuric acid at 104’ for periods of 14 to 20 hours. The recovered material was finally crystallized several times from a mixture of petroleum ether and acetone, after which it melted sharply at 64.2” and set solid at 63.4-63.0”. On heating, the sample showed a transition at 56.0- 56.5” and on cooling a very sharp transition at 55.0-54.8”. With Cr K, radiation a pressed specimen gave an x-ray spacing of 39.20 f 0.20 A. with eight orders of reflection showing on the original photograph. Analysis gave C 85.24, 85.11; H 14.60, 14.56. Nonacosane, C&sH,, requires C, 85.19; H, 14.81; and hentriacontane, C&Hc4, requires C, 85.22; H, 14.78.

Considering only the melting point and combustion results, one might conclude offhand that the cranberry hydrocarbon fraction consisted of slightly impure triacontane. However, the presence of this hydrocarbon in plants, although repeatedly reported in the literature, has been seriously questioned by Collison and Smedley- MacLean (8). In all the cases where triacontane has actually been reported it seems more than probable that it was either non- acosane or a mixture of hydrocarbons. It was pointed out by

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Cwstituents of Cranberry Pomace

Piper et al. (9) that if a hydrocarbon gives the correct melting and transition points its identity must be further confirmed by x-ray analysis.

In view of the uncertainty relative to the presence of triacontane in plants, we consider our fraction a mixture of hydrocarbons and, from a comparison of our data with similar data reported by Piper et al. for pure synthetic hydrocarbons and their mixtures, it is concluded that the final hydrocarbon fraction obtained from cran- berry pomace probably consisted of a mixture of nonacosane and hentriacontane, the former predominating to the extent of 80 to 90 *per cent.

Ether Extract

The ether-soluble fraction consisted of a light cream-colored, high melting resinous powder, insoluble in water but partly soluble in hot aqueous alkali. A quantity (113 gm.) of the crude extract was digested several times for 3 hours with 1.5 liters of water containing 25 ml. of 40 per cent sodium hydroxide solution. The insoluble material was filtered off with suction, washed, and dried, whereupon there were obtained 70 gm. of an aqueous alkali- insoluble sodium salt.

The aqueous filtrates contained in solution a considerable quan- tity of a second resinous acid in the form of its sodium salt. The free acid was precipitated by the addition of dilute hydrochloric acid, filtered off, washed, and dried. Repeated attempts to purify this acid or to obtain any of its derivatives in crystalline form were unsuccessful.

Ursolic Acid, HO. C29H46. COOH-The crude water-insoluble sodium salt obtained as described above was dissolved in hot 95 per cent alcohol containing a slight excess of sodium hydroxide. The solution was filtered from a small quantity of gummy sub- stances, and an equal volume of boiling water added to it. Upon evaporation of most of the alcohol a copious precipitate of white crystalline sodium salt occurred. After cooling, the precipitate was collected on a Buchner funnel and recrystallized several times by repetition of the process. There were thus obtained 48 gm. of nearly pure sodium ursolate.

The sodium salt was dissolved in the smallest possible amount of hot 95 per cent alcohol and the free acid regenerated by the addi-

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tion of 4 volumes of hot dilute hydrochloric acid. After cooling, the free acid was filtered off with the aid of suction, well washed with hot water, and dried. The recovered acid (38 gm.) was fractionally crystallized from 75 per cent alcohol, fractions melting below 275” being rejected. The remaining portions, amounting to 30.5 gm., were combined and crystallized from 95 per cent alcohol, giving 24 gm. of pure ursolic acid in the form of long slender white needles or blades. The substance melted sharply at 283-283.2’ and, after drying for 6 hours at 140”, gave on analysis C, 79.18, 78.92; H, 10.69, 10.53. Ursolic acid, CS0H4803, requires C, 78.88; H, 10.60.

Methylursolate, HO. C&Y4s. COO(CH3)-5 gm. of ursolic acid were dissolved in absolute alcohol and boiled under a reflux condenser with an excess of both sodium ethoxide and methyl iodide. After 3 hours, the alcohol was partially removed by evaporation and the solution poured into water to separate the methyl ester. After two crystallizations from 70 per cent alcohol and several crystalliza- tions from 50 per cent alcohol, a fairly pure derivative was obtained. Placed in the bath at room temperature, the substance melted indefinitely between 110-115”, resolidified, and finally melted at 168.0-168.4”. After drying at 130’ for 20 hours, the methyl ester gave on analysis C, 78.84, 78.89; H, 10.23, 10.62. Methyl- ursolate, CS1HL003, requires C, 79.08; H, 10.71.

Acetylmethylursolate, CH&‘O. 0. C29H46. COO(CH3)-4 gm. of the methyl ester were refluxed for 3 hours with 40 cc. of acetic anhydride and the solution was allowed to stand for 2 days, whereupon the acetylmethylursolate separated in the form of elongated plates. These were filtered from the acetic anhydride and recrystallized from absolute alcohol. The melting point was found to be 246“. After drying at 125’, analysis gave C, 77.19; H, 10.25. Acetylmethylursolate, C33H5204, requires C, 77.28; H, 10.23.

DISCUSSION

The investigation reported above indicates that the wax-like coating of the cranberry is quite as unique chemically as the whole epidermis is anatomically. Anatomically the cranberry is unusual, having no stomata in the epicarp but many non-functional stomata in the endocarp, according to the observations of Winton (10) and

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Bergman (11). The outer wall of the epidermis is reported by Stevens (12) to be comparatively thick, varying from 8.7 to 13.7 p. Despite the thickness of the epidermis, the absence of stomata, and the imperviousness of the waxy coating as evidenced by the extreme difficulty experienced in dehydrating the uncut fruit, the cranberry nevertheless respires at an appreciable rate, approxi- mately 5 mg. of carbon dioxide per kilo per hour at 10” (13).

With these facts in mind, it is therefore interesting to note that the petroleum ether extract of cranberry pomace is approxi- mately 2 times, and the ether extract 3 times as large as that ob- tained from grape pomace extracted under the same conditions. The total extract amounts to more than 20 per cent for the cran- berry pomace as compared to about 7.5 per cent for the grape. How- ever, it is not alone the amount but particularly the composition of the extract which commands attention. Previous work from this and other laboratories has established the fact that the petroleum ether extract of apple pomace and cuticle is composed principally of the higher hydrocarbons, primary and secondary alcohols, and that it cont,ains only very small amounts of solid fa,tty acids. The cranberry extract, on the other hand, cont,ains no recognizable alcohol other than a small amount of glycerol, apparently derived from a true fat, and only a small quantity of hydrocarbon, but large amounts of free fatty acids, principally oleic acid. The ether extracts of cranberry and apple pomace were found to be similar, both consisting of free ursolic acid and another unidentified resin acid.

The large amount of free liquid fatty acids occurring in or on the cranberry cuticle no doubt partly explains the imperviousness to water and also the unusual stickiness observed in berries which have been frozen.

SUMMARY

Cranberry pomace obtained in the commercial canning of cran- berry sauce has been examined with respect to the constituents present in the petroleum ether and ether extracts. The petroleum ether extract was found to consist of the hydrocarbons, nonacosane, CZ9HG0, and hentriacontane, CS~HM; free solid fatty acids of the series C16 to C&e; the liquid acids, linolenic, linoleic, and oleic, the last predominating; a small amount of glycerol probably originally

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combined with acid in the form of a fat; and small amounts of unidentified solid and liquid substances. The ether extract (following extraction with petroleum ether) was found to consist of free ursolic acid together with an unidentified resin acid,

BIBLIOGRAPHY

1. Wehmer, C., Die Pflanzenstoffe, Jena, 2nd edition, 2, 917 (1931). 2. Blatherwick, N. R., and Long, M. L., J. Bid. Chem., 67, 815 (1923). 3. Kohman, E. F., and Sanborn, N. H., Ind. and Eng. Chem., 23,126 (1931). 4. Grove, K. E., and Robinson, R., Biochem. J., 26, 1706 (1931). 5. Morse, F. W., J. Biol. Chem., 81,77 (1929); 79,409 (1928). 6. Morse, F. W., Massachusetts Agric. Exp. Stat., Bull. 86’6 (1930). 7. Francis, F., Piper, S. H., and Malkin, T., Proc. Roy. Sot. London,Series

A, 128, 214 (1930). 8. Collison, D. L., and Smedley-MacLean, I., Biochem. J., 26, 606 (1931). 9. Piper, S. H., Chibnall, A. C., Hopkins, S. J., Pollard, A., Smith, J. A.

B., and Williams, E. F., Biochem. J., 26,2072 (1931). 10. Winton, A. L., Connecticut Agric. Exp. Stat., d&h Ann. Rep., 288 (1902). 11. Bergman, H. F., Bull. Torrey Bot. Club, 47, 213 (1920). 12. Stevens, N. E., Am. J. Bot., 19,432 (1932). 13. Morse, F. W., and Jones, C. P., Massachusetts Agric. Exp. Stat., Bull.

1.98 (1920).

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K. S. Markley and Charles E. SandoCRANBERRY POMACE

ETHER-SOLUBLE CONSTITUENTS OF PETROLEUM ETHER- AND

1934, 105:643-653.J. Biol. Chem. 

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