654
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
Acknowledgment
TABLE 11. COLORIMETRIC ESTIMATION OF TUNQSTEN No. 1 2 3 4 5 7.
Mineral Scheelite Scheelite Scheelite Scheelite Scheelite Scheelite Psilomelane
Location Alaska California Alaska Alaska Alaska Alaska Arisona
8 9 10 11 12 13
Scheelite Scheelite Scheelite Coronadite Scheelite Psilomelane
Alaska Alaska Alaska New Mexico Alaska New Mexico
14
Molybdenite
Idaho
6
~~~
Per Cent of W OProcedure 1 Procedure 2 colorimetrii colorimetri; Gravimetrio
...
0.08 0.15 0.11 0.18 0.31 0.48 0.55
0.04 0.10 0.11 0.19 0.26 0.48 0.52
0.60 0.71 0.73 2.0 1.1 1.2
0.83
... ... ... ...
0.60 0.88 0.75 11 .. 81 52 1.29
...
None
None
0.14
... ... 0.31
0.45 0.54
0.74
c
Results on Tungsten Ores The tungsten or- were first analyzed gravimetrically, using 4gram portions easentially by the procedure outlined by Scott (7). Colorimetric estimations using procedures 1 and 2 are given in these are seen to be good in the I1 for range up to 1 per cent tungsten trioxide.
Vol. 15, No. 10
Notes
... ~ontainkb:AS Contained As
...
Total M n 4 8 % BaO 14% ’ ”
The authors wish to thank M. Fleischer for the analyses of samples 7 and 13 and C. Milton for No. 3; also William Schlecht and R. C. Wells for kindly reviewing the manuscript.
Literature Cited
(1) Feigl, F., and Krumholz, P., 2.angew. ... Chem., 45, 674 (1932). (2) FernjanEiE, S., Z. anal. Chem., 97, 332 Pb30%”‘ (1934). Total Mn 50% (3) FernjanEiE, S., Zapodakuya Lab., 6, 289 BaO SrO 15% (1937). MoSzand 1.86% (4) Grimaldi, F. S., and Wells, R. C., IND. ENO.CHEY.,ANAL.ED.,15,315 (1943). (5) Poluektov, N. S., Zavodakaua Lab., 10, 92 (1941). (6) Popov, K. M., and Dorokhova, M. N., ZM., 9,1315 (1940). (7l . , Scott. W. W.. “Standard Methods of Chemical Analvsis”. 5th ed.; Vol. 1, p. 1005,New York, D.Van Nostrand Cia, 1939. (8) Shakov, A. s., Zaoodskuya Lab., 470 (lggl). (9) Voznesenskii, A. T.,ZM., 9, 25 (1940).
...
PaleemNTEn before the Division of Analytical and Micro Chemistry at the 108th Meeting of the AMERICAN CEEMICAL SOCIETY. Pittsburgh, Penna. Published by permiasion of the Director, U. 8. ~eOlOgiC81Survey
Microbiological Determination of Pantothenic Acid Further Studies A. L. NEAL AND F. M. STRONG, College of Agriculture, Udveroity of Wisconsin, Madison, Wis.
The procedure for microbiologiixtl determination of pantothenic acid has been modified by additions to the balal medium and improvements in the method of growing inoculum. The modified method is capable of measuring smaller amounts of pantothenic acid and gives concordant results at increasing levels of sample.
The present paper is concerned (a) with a study of methods for liberating “bound” pantothenic acid in the sample, (b) with procedures designed to remove interfering fat-soluble substances from the test solution, and (c) with efforts to modify the b a d medium in such a way as to avoid the effect of water-soluble, stimulating factors and to prevent “drift”. Except for the modifications detailed below, the general procedure previously described (19) has been followed in the present work for setting up assays and calculating results.
Cultures and Inoculum
A
LTHOUGH published methods for the micrpbiological assay of pantothenic acid (8, 13) have been widely Used
and have been made the basis for various conclusions regarding the physiological and nutritional functions of this vitamin, certain discrepancies raise serious questions as to the reliability of the results. Among these difficulties may be listed lack of agreement with animal tests, the large “drift” frequently observed, and the fact that minor variations in the method of preparing the sample may cause wide fluctuations in the final assay values. Furthermore ( l a ) ,anomalous results were obtained in attempting t o carry out recovery experiments on hydrolyzed samples of fresh liver and kidney. Some of the reasoas underlying these troubles have recently come to light. The effect of fatty acids on the growth of Lactcrbacillwr casei has been discovered (d, I d ) , and methods for avoiding their interference in the riboflavin assay have been worked out ( I , 1.2). It has also become apparent that certain watcrsoluble substances of an unknown nature, and apparently prcsent mainly in brans, are capable of stimulating the bacterial response (5, 7 , 1 5 ) .
Evidence has accumulated in carrying out a large number of assays that the best rcsponse is obtained when the inoculum is not carried through several transfers in the liquid medium as formerly recomrnendcd, but is grown directly from a stab culture. It also appears advisable to transfer the stab cultures weekly instead of monthly. To grow inoculum, cells from a suitable stab are transfcrred into a tube containing 10 cc. of the basal medium (see below) plus 1 microgram of calcium pantothenate. After 24 hourdincubation at 37’ ( * 1”) C. the cells are centrifuged out and resuspended in 10 cc. of saline, and the resulting cell suspension is used as inoculum. Thus there is no “subculture” made between the stock stab culture and the inoculum and the cell suspension is not so extensively diluted with saline as before (IS). One should return to a stab each time inoculum is to be grown. One stab culture may bc used several times if proper precautions are taken to avoid bacterial contamination.
Basal Medium The previous basal medium haa been modified by adding glutamio acid and a supplement prepared from Vitab, by increasing
ANALYTICAL EDITION
October 15, 1943
the sodium acetate concentration to 2 per cent, and by using a more concentrated yeast supplement. The remaining ingredients of the medium were prepared as previously described (18).
This was prepared as before (19) exYEASTSUPPLEMENT. cept that Darco G-60 was used for the adsorption, and the adsorption was carried out in a volume of 240 ml. After the second charcoal treatment the filtrate was neutralized with solid sodium hydroxide and diluted to 250 ml. The supplement was thus four times as concentrated as previously described, since each milliliter waa equivalent to 100 mg. of whole autolyzed yeast. VITAESUPPLEMENT.Seventy-five grams of Vitab (National Oil Products Co.) were dissolved in 500 ml. of water, the pH was adjusted to 1.2 to 1.5 with concentrated hydrochloric acid, and the mixture was stirred with 50 grams of activated charcoale. g., Darco G-60-for 20 minutes at room temperature. The charcoal was filtered off, the pH readjusted to 1.2 to 1.5, and the adsorption repeated. The filtrate was neutralized to pH 6.8 with concentrated sodium hydroxide, filtered again, and diluted to IO00 ml. with distilled water. One milliliter of the final solution therefore contained the equivalent of 75 mg. of Vitab. GLWPAMIC ACID. One gram of 2-glutamic acid hydrochloride was dissolved in 80 to 90 ml. of water by warming on the steam bath and stirrin until solution was effected. The solution was then cooled and %luted to 100 ml.
655
pantothenic acid added as the volume is bein made up to 100 ml. The amount of pantothenic acid added siould be approximately equal to the quantity of the vitamin in the sample. In either case the mixture is autoclaved for 15 to 20 minutes at 1 kg. per sq. cm. (15 pounds per square inch) pressure. After cooling, 2 ml. of 2.5 M sodium acetate are added, and the mixture is brought to H 4.8 (green to bromocresol green outside indicator) with N hyirochloric acid. The solution is then diluted to 200 ml. and is filtered through a fluted quantitative paper-. g., Whatman No. 40. If the filtrate is not erfectly clear it is refiltered with the aid of a small amount orfine Filter-Gel. Fifty milliliters of the filtrate are neutralized to pH 6.8 with N sodium hydroxide, and diluted to 100 ml. for assay. If a sample very high in fat is to be assayed, or if the sample as above prepared shows a drift, it is necessary to extract the 50 ml. of filtrate with ether after neutralizing but before diluting to volume. The filtrate is shaken out with about 30 ml. of ether. The extraction is twice repeated, and the aqueous phase is then diluted to 100 ml. without removal of the dissolved ether. IO
,
,
,
,
,
,
,
,
,
,
,
.
,
,
-8. -J
a
-
The amounts of these materials used in setting up an assay, and the composition of the modified medium are shown in Table I. This medium will be referred to henceforth as the basal medium. To set up a 100-tube assay, the amounts of the various reagents and solutions indicated in Table I are mixed, adjusted to pH 6.6 to 6.8, and diluted to 500 ml. with distilled water. This gives a mixture having twice the concentration of the basal medium. The rest of the procedure is carried out as before (19).
o TABLE I. COMPOS~ION OF BASALMEDIUM AND AMOUNTS REQUIRED FOR ONEHUNDRED TUBES Ingredient
Amount for 100 Tubes
Amount in Medium
100
0.5
Grams
Anhydrous glucose Sodium acetate
10 14
1 .o
2.0
MI. Cystine Asparagine Glutamic acid Riboflavin Yeast supplement
100
Vitab supplement
100
Salts solution A Salte solution B
25 25
2 50
5 5
FIGURE 1. STANDARD CURVES Curve 1, present basal medium. Curve 2, original medium. 72 hours' incubation.
%
M1. NaOH-treated peptone
0.02 0.04 0.06 0.08 0.10 0.12 a14 MICROGRAMS d-CALCIUM P A N T O T H E N A T E
0.01
0.025 0.025 0 . 1 p p. m.
Equivalent t o 50 mg. of autolyzed yeast per tube Equivalent to 75 mg. of Vitab ertube (5 ml. oteach per liter of medium)
Standard Curve Since the linear portion of the standard curve extends to 0.08 to 0.10 mi( rogram of calcium pantothenate, rather than to approximately 0.15 microgram as was formerly tho case, it has seemed desirable to work with a more dilute standard solution. The standard solution now used is.prepared as previously described, but is diluted to 0.02 microgram per ml. The standard curve is constructed from the titration values of tubes containing 0.00, 0.00, 0.5, 0.5, 1.0, 1.0, 2.0, 2.0, 3.0, 3.0, 4.0, 4.0, 5.0, and 5.0 ml. of the standard solution. A typical standard curve, given in Figure 1 (curve l ) , shows that the useful assay range is about 0.01 to 0.08 microgram of calcium pantothenate per tube.
Preparation of Materials Tested Dry samples are finely ground. Fresh tissues are homo enised with a small amount of water in a Waring Blendor or %otterElvehjem homogenizer (9). An amount of the material containing approximately 10 micro rams of pantothenic acid is then suspended in 100 ml. of disthed water and adjusted to pH 6.8. If recovery experiments are to be carried out, the amount of sample taken is reduced by one half, and a known amount of
ENZYME TREATMENT. An aliquot of the homogenized sample estimated to contain 10 micro rams of pantothenic acid after digestion, is suspended in 25 mf. of distilled water plus 2 ml. of 2.5 M sodium acetate. An amount of clarase or pa ain powder equal to twice the dry weight of the sample is adled and the mixture brought to pH 4.8 with N hydrochloric acid. The volume is made to 50 ml. with water and the mixture incubated at 37" C. under toluene for 48 hours. The suspension is then prepared for assay as described above. A portion of the enzyme powder is carried through the same procedure and assayed to ascertain its pantothenic acid content. Digestion with pancreatin was carried out in a similar manner, except that 50 ml. of 0.05 M phosphate buffer, pH 7.2, replaced the sodium acetate. ETHER-SOLUBLE FRACTION FROM YEAST. A pancreatindigested yeast suspension was adjusted to pH 6.8 and subjected to continuous ether extraction for 3 hours. The ether was removed from the extract, the residue taken up in 5 ml. of alcohol, and the solution diluted to the same volume as that of the aqueous phase extracted. OLEICACID. The oleic acid was prepared by saponifyin a pure sample of ethyl oleate and was tested in the form of a f n e suspension obtained by diluting a methanol solution with water. The small amount of methanol used had no detectable effect on the test organism.
Results and Discussion Although satisfactory agreement was reported (IS) for animal and microbiological pantothenic acid determinations on several samples, a further comparison has revealed that in the case of yeast serious discrepancies exist. Thus the data in Table I1 show about fivefold higher values by chick assay of a number of yeast samples than by the L. casei method as originally published. It has been reported (IS)that such discrepancies are attributable to a "bound form" of pantothenic acid not measured by the bacteria, and efforts have been made to release it by enzymatic digestion (4,10,14,16).
656
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Vol. 15, No. 10
(5, 7, 15), a rice-bran concentrate, Vitab, was selected as the OF ANIMALAND MICROBIOLOGICAL starting material. It was successfully freed of pantothenic acid TABLE 11. COMPARISON ASSAYOF PANTOTHENIC ACIDIN YEAST by charcoal treatment. Pantothenic Acid, Fou,nd-When the basal medium was modified by the addition of this Dry Yeast. Microbiological Assay b Sample Chick ClaraseVitab supplement together with extra sodium acetate (8, 11), a No. assaya Direct digested steeper standard curve was obtained (Figure l),and the drift was Micrograms per gram-almost entirely eliminated. The data in Table V show excellent 106 200 47 134 107 220 45 187 agreement over a wide range of increasing levels of sample, even 108 190 38 123 though widely d ~ e r e ntypes t of natural materials were analyzed. 109 510 131 298 110 250 52 132 I t is important in carrying out these analyses to employ wide a Chick assays carried out by Jukes by method previously described (6) ranges in sample levels; otherwise a moderate amount of drift Each value is average of 2 or 3 concordant individual assays. b Carried out according to original procedure ( 1 9 ) . may easily be overlooked. I n the present work the criterion c A fortified yeast sample. has been set up that in order for an assay to be regarded as reliable, a t least three sets of duplicate tubes must fall in the assay range-i. e., contain amounts of pantothenic acid corresponding to the linear portion of the standard curve-and that the results In the early part of this work a study was made of the effectivecalculated from any one of these tubes must not deviate by more ness of clarase, pancreatin, and papain for the liberation of than * 10 per cent from the average. Furthermore, the amount bound pantothenic acid in yeast. Systematic variations in the of sample in those tubes representing the highest level should amount and type of enzyme and in the conditions of time, tembe a t least four times that a t the lowest level. perature, and pH under which the digestions were carried out With this improved assay procedure available it has been posshowed that the values obtained could be increased to a maximum sible to re-examine with more confidence the question of enzyme of about 60 to 80 per cent of those found by chick assay. Some of digestion of the sample. The data in Table VI show clearly that these results are given in Table 11. it is often neressary to include an enzyme treatment in order to However, many of the assays run in connection with this study showed considerable drift-i. e., a regular decrease in apparent pantothenic acid content was noted as larger amounts of sample were tested. The amount of drift was usually increased TABLE 111. EFFECTOF METHODOF PREPARATION OF SAMPLE following the enzyme treatment and i t was difficult to obtain ON APPARENTPANTOTHENIC ACIDCONTENT OF YEAST" concordant duplicate results. Typical data are shown in Table ,-Treatment of Sample Test Suspended in Digested with Clarase 111. Since the authors' experience in general has been that asSolution water and Suspension Extracted Added per assayed assayed with says showing drift are unreliable, it was questionable whether Tube directly directly Filtered ether the increase found after enzyme treatment actually represented M1. Micrograms per gram pantothenic acid. Inasmuch as methods of avoiding such drifts 57 165 ... ... 161 58 ... in the riboflavin assay had in the meanwhile been worked out ... 121 49 114 114 ( I B ) , it seemed desirable to apply similar procedures to the di117 53 118 118 42 105 102 102 gested yeast samples. When this was done, lower values for the 109 41 102 102 apparent pantothenic acid content were obtained. As shown ... . ... 86 90 . . . 86 90 in Table 111, filtration or ether extraction reduced the value Av. 50 130 101 103 found for yeast 110 after clarase treatment by about 20 per cent, a Ex eriments done on yeast 110, and assays carried out aocording to originarprocedure (13). but the result was still twice that obtained by assay of the original sample. When the same yeast was digested with pancreatin, TABLEIV. EFFECTOF FATTYMATERIALS ON RECOVERY OF and then assa>ed,a value of about 210 micrograms per gram was PANTOTHENIC ACID found. This value was reduced to 47 after filtration of the digest Pantothenic and to 41 after ether extraction. Similar treatment after papain -Fatty Material AddedAcid Pantothenic digestion gave a value of 42 micrograms per gram. Control exper Tube Added per Acid Type Amount Tube Recovered periments showed that these procedures did not remove pantoP9 * Microgram % thenic acid. It was evident, therefore, that a large part of the in28.2 0,025 Oleic acid 116 28.2 0.050 100 crease observed after enzyme treatment was attributable to the 28.2 0.075 94 liberation of interfering substances, rather than pantothenic acid, 28.2 0.100 92 and it seemed likely that the interfering substances were fat-sol56.4 0.025 176 uble in nature. 56.4 0.050 156 This suspicion was strengthened by testing the effect of oleic 56.4 0.075 118 56.4 0.100 97 acid and an ether-soluble fraction from yeast. The data pre112.8 0.025 84 112.8 0.050 sented in Table IV summarize the results of this work. It is 183 112.8 0.075 132 evident that as in the riboflavin method (IB), oleic acid exerts a 112.8 0.100 100 large influence on t t e bacterial response, and that the magnitude 169.2 0.025 128 of the stimulation or inhibition observed is dependent not only 169.2 0.050 192 on the amount of fatty acid used but also on the level of panto169.2 0.075 138 169.2 0.100 105 thenic acid being tested. The ether extract of yeast was likewise Mi. stimulatory. Ether extract 1 0.100 96 I n further experiments it became obvious, however, that even of yeasta 2 0.100 94 after fat-soluble interfering substances were removed, an ob3 0.100 94 jectionable amount of drift still persisted (Table 111). It was 1 0.050 153 assumed, therefore, that water-soluble materials of a stimulatory 2 0.050 136 3 0.050 127 nature were being introduced with the sample, and efforts were 4 0.050 131 made to prepare a supplement to the medium which would pro3 0.025 159 vide such stimulants in excess. Since it has been reported that a 1 ml. contains extractable substances from 1 mg. of dried yeast obtained as described in text. water extracts of various brans contain materials of an unknown nature which stimulate growth and acid production by L. casei 7 -
-
-
October 15, 1943
ANALYTICAL EDITION
achieve maximum values. It has not yet been possible to carry out by the improved assay procedure an extended comparison of the effectiveness of various types of enzyme treatment of different samples. Whether the clarase digestion described represents the optimum method for liberating combined pantothenic acid must be left an open question a t present.
657
TABLE VII. RECOVERY OF ADDEDPANTOTHENIC ACID Sample
Treatment
Dried yeast 110 Dried human feces Oil-packed sardines
Clarase Direct Direct Clarase Direct Clarase Direct Clarase Clarase
Canned beets Canned Deas AT INCREASING LEVELSOF SAMPLE TABLE V. AGREEMENT
Test Solution Added per Tube MI. 0.5 1 .o 1.5 2.0 2.5 3.0 3.5 4.0
Pantothenio Acid Found Hydrolyeed Canned hog tqmato Canned Oil-packed hog liver" juice peas sardines liver Microproma per pram 0.90 3.2 15 1.3 0.49 2.8 0.45 0.90 17 1.6 0.43 2.7 1.5 17 0.43 2.9 15 i.08 1.6 0.42 3.0 17 ... 1.5 3.0 0.42 0.96 1.5 16 2.9 1.4 15 0.84 ... 3.2 1.4 16 16 3.0 Av. 1.5 0.44 0.96 a Suspended in 6.7 volumes of 2 N sodium hydroxide autoclaved 1.5 hours at 15 pounds per sq. inch pressure, filtered at pH 4.8,'and neutralized. 7
Canned pineapple Clarase
...
Recovery
% 102 99
103 90 98 100
92 101 100
99
Fresh
-
of pantothenic acid as they do on Medium C of Light and Clarke (7). These calculations apply only to the recommended assay range in each case-i. e., to conditions under which pantothenic acid supposedly is the only factor limiting acid production. It is obvious that a large discrepancy still exists between the ... ... pantothenic acid content of the yeast samples in Table I1 aa determined by the present microbiological method and by the chick assay. What the cause of this discrepancy may be, and TREATMENT ON DETERMINATION whether it will persist when a large variety of other natural TABLE VI. EFFECTOF ENZYME OF PANTOTHENIC ACIDIN FOODS materials are assayed by improved animal methods, must be left -Pantothenic Acid Found--an open question at present. Sample Direct Clarase-treated0 Micrograms per gram Sardines 3.0 6.5 0.8 0.56 Pineapple 0.94 Beets 0.89 1.8 Peas 0.98 S inach 0.42 0.55 east no. 110 52 98 54 130 Yeast no. 107 Tuna fish 1.5 2.4 Corrected for pantothenic acid content of enayme preparation used.
Summary
9
a
That the enzyme does not destroy pantothenic acid, and that no appreciable amount of vitamin is lost by the filtration of the test suspension, are indicated by the recoveries given in Table VII. The recovery on clarase was made by treating 5 micrograms of calcium pantothenate with 2 grams of clarase. The results in Table V relating to alkali-hydrolyzed fresh liver are noteworthy. It was possible to carry out an entirely satisfactory analysis on this material, and recovery of added calcium pantothenate was good. This is in sharp contrast to previous experience (13). The slight residual activity is not destroyed by further treatment with alkali under conditions which result in complete inactivation of pantothenic acid. No effort has been made in the present work to extend the range of the standard curve to titration levels appreciably above 10 ml., although it is well known that if both the glucose and pantothenic acid are increased much larger amounts of lactic acid will be formed. Extending the titration range above 10 or 11 ml. is considered inadvisable, since the large amount of acid formed may unfavorably influence the growth and vigor of the test organism. Furthermore, no one has yet succeeded in devising a basal medium which will permit a continuation of the linear response to still higher titration values. By linear response is meant a constant ratio between added increments of the vitamin to be determined and the extra acidity thereby produced. It is important that this ratio be as high as possible in order for the assay to be sensitive, accurate, and specific, For example, the titration values for the standard curve (Figure 1, curve 1) go from 2.0 to 8.7 ml. as the amount of calcium pantothenate is increased from 0.01 to 0.08 microgram. This corresponds to an average increased titration in this region of the curve of (8.7 2.0) + (0.08 0.01) X 100 = 0.956 ml. of 0.1 N acid for each additional 0.01 microgram of calcium pantothenate. This figure is much higher than corresponding values which may be calculated in the same manner from the data given in previous papers-viz., 0.56 (IS), 0.49 (8),and 0.0715 (7) ml. per 0.01microgram. Thus, for example, in the present method the bacteria produce about 14 times as much lactic acid for a given increment
-
-
The procedure for carrying out the microbiological determination of pantothenic acid has been modified by additions to the basal medium and improvements in the method of growing inoculum. The modified method is capable of measuring smaller amounts of pantothenic acid and gives perfectly concordant results a t increasing levels of sample. The apparent increase in pantothenic acid following enayme digestion as measured by the original method is shown to be largely due to the liberation of fat-soluble and water-soluble stimulants. Methods of avoiding these effects have been worked out, and an enzyme treatment which results in an actual increase of free pantothenic acid in many samples is described.
Acknowledgment The authors wish to express their appreciation to F. W. Quackenbush for a sample of pure ethyl oleate, to T. H. Jukes for carrying out the chick assays reported in this paper, and to the National Oil Products Co. for the gift of the Vitab.
Literature Cited Andrews, J. S., Boyd, H. M., and Terry, D. E., IND. ENQ.CHEM., ANAL. ED.,14,271(1942). Bauernfeind, J.. C., Sortier, A. L., and Boruff, C. S., Ibid., 14, 666 (1942).
Black; A,, personal communication. Cheldelin, V. H., Eppright, M. A., Snell, E. E., and Guirard B. M., University of Texas, Publ.4237,p. 23 (1942). Clarke, M. F., Lechyoka, M., and Light, A. E., J . B i d . Chem., 142,957(1942).
Jukes, T. H., J. Nutrition,21,193 (1941). Light, A. E., and Clarke, M. F., J . B i d . Chem., 147,739(1943). Pennington, D.E., Snell, E. E., and Williams, R. J., Ibid., 135, 213 (1940).
Potter, V. R., and Eivehjem, C. A., Ibid., 114,495(1936). Rohrrnann, E., Burget, G. E., and Williams, R. J., Proc. Soo. Ezptl. Biol. Med., 32,473(1934). (11) Stokes, J. L., and IMartin, B. B., J. Bid. Chem., 147,483 (1943). (12) Strong, F. M.,and Carpenter, L. E., IND.ENQ.CKEM..ANAL. ED.,14,909(1942). (13) Strong, F. M.,Feeney, R. E., and Earle, A,, Ibid.. 13, 566 (1941). (14) Waisman, H. A., Henderson, LaVelle M., McIntire, J. M., and Elvehjem, C. A., J. Nutrition,23, 239 (1942). (15) Wegner, M. I., Kemrnerer, A. R., and Fraps, G. S., J . Bwl. Chem., 144,731 (1942). (16) Willerton, E., and Crornwell, H. W., IND.ENQ.CHEM.,ANAL. ED.,14,603(1942). P R S S ~ N T Ebefore D the Division of Agricultural and Food Chemistry, Joint Program on Vitamins, at the 105th Meeting of the AMERICAN CHEMICAL SOCIETY, Detroit, Mich.
