Microbiological Assay for Pantothenic Acid F. M. STRONG, R. E. FEEh'EY, AND ANN EARLE University of Wisconsin, Madison, Wis.
A convenient method of determining the pantothenic acid content of biological materials has been developed on the basis of the essential nature of this vitamin for a lactic acid bacterium. Crude suspensions of the sample are fermented by the organism, and the acid produced, as determined by direct titration of the entire culture, is a
measure of the pantothenic acid in the sample. The method gives results which agree well with values determined by chick assay, and is applicable to a wide range of natural materials. No expensive or unusual apparatus is required. One worker can assay approximately 15 samples per day.
T
time of this monthly transfer several extra stock cultures are repared (Nos. la, lb, 2a, 2b, etc.) and are used to grow inocufum for assay tubes as required during the month. To grow inoculum a stab from a stock culture-e. g., 1ais made into 10 cc. of the basal medium to which is added-0.5 microgram of calcium pantothenate. This culture, "subculture" in Figure 1, is incubated 24 hours a t 37" C., and again transferre: into the same medium. After 24 to 40 hours' incubation a t 37 (but not longer) the cells are centrifuged out aseptically and resuspended in 10 cc. of sterile 0.9 per cent sodium chloride solution. One cubic centimeter of this suspension is then further diluted with 10 cc. of the sterile saline, and 1 drop of the h a 1 cell suspension is used to inoculate each assay tube. Thus ' A s
HE bacterium Lactobacillus casei
E, which is used as the test organism in a microbiological assay procedure for riboflavin (IO), also requires pantothenic acid' (11). A medium has been devised which is essentially free from pantothenic acid but appears to be otherwise adequate for L. casei E. It has been found that on this medium the addition of graded amounts of synthetic calcium pantothenate results in a proportionate increase in lactic acid production. This relationship has been made the basis of a rapid assay method for pantothenic acid.
Cultures and Inoculum -one Original culture-
The method of carrying cultures and preparing inoculum is indicated schematically in Figure 1.
month-tone Stock
month+ Stock culture 2+
etc.
cdturel-/ Stock cultures Stock cultures 2a, 2b, 2c4etc. la, lb, IC etc.
The test organism, which may be obtained under the serial number 7469 from The American Type Culture Collection, Georgetown University Medical School, Washington, D. C., is carried as stab cultures in yeast water-glucose agar. This medium is prepared by dissolving'0.5 per cent glucose and 1.5 $er cent agar in yeast water, which in turn is made by steaming resh, starch-free baker's yeast for 2 houn with 10 times its weight of water, autoclaving 45 minutes at 1 kg. per sq. cm. (15 pounds per sq. inch) pressure, allowing the cells to settle, and decanting the supernatant liquid. A l to 2 per cent solution of Difco yeast extract may be substituted for the yeast water if desired. Sterilized tubes containing 10 cc. of this medium are inoculated with L. casei E, incubated a t 37" C. for 24 hours, and then stored in the refrigerator until needed. One such culture (Nos. 1, 2, etc.) is ke t at all times as a reserve stock culture, which is never disturbej except to transfer at monthly intervals. At the
,
c___*__-
.1
Subculture
-
J. Inoculum a J. Inoculum b J. etc.
Inoculum for assays during one month
1 I n order t o conform t o recognized nomenclature (i), t h k organism will henceforth be termed Lactobacillus helteticus in publicationa from this laboratory.
Subculture
J. J. Inoculum b J. etc. Inoculum a
c -
-
Inoculum for assays during a second month, etc.
FIGURE 1. METHODOF CARRYING CULTURESAND PFZEPARINO INOCULUM 566
ANALYTICAL EDITION
August 15, 1941
from the agar medium pass throu h a t least one subculture in the liquid medium before being used as inoculum. If assays are to be made on each of several successive days, it is not necessary to grow inoculum from a different stock culture each day, but a drop from a tube of inoculum-e. g., a in Figure 1-may be transferred to a similar tube, b, which is incubated for use the next day. One should return to a stock culture about every 5 days, however, to minimize chances of contamination and bacterial variation.
Basal Medium The basal medium contains sodium hydroxide-treated peptone 0.5 per cent, sodium acetate 0.6 per cent, glucose 1 per cent, cystine 0.01 per cent, asparagine 0.025 per cent, riboflavin 1.0 microgram per 10 cc. of medium, 0.05 cc. each of inorganic salt solutions A and B per 10 cc. of medium, and yeast supplement equivalent t o 50 mg. of autolyzed yeast per 10 cc. of medium. SODIUMHYDROXIDE-TREATED PEPTONE. To a solution of 40 grams of peptone (Difco Bacto) in 250 cc. of water are added 20 rams of sodium hydroxide also dissolved in 250 cc. of water. he mixture is allowed to stand a t room temperature for 24 hours, the alkali is neutralized with glacial acetic acid (ca. 28 cc.), 7 grams of anhydrous sodium acetate are added, and the volume is made to 800 cc. with water. The final solution, which contains 5 per cent of peptone and 6 per cent of sodium acetate, is stored
567
centimeters are pipetted into each of the 50 tubes and a suitable aliquot of the sample is added. The contents 01 each tube are then diluted to a total volume of 10 cc. Thus, as much as 5 cc. of liquid may be added with the sample. The volumes indicated should be measured with an accuracy of *0.1 cc. The tubes are plugged with cotton, sterilized in the autoclave a t 1kg. per sq. cm. (15 pounds per sq. inch) ressure for 20 minutes, allowed to cool, inoculated, and incubate$ a t 37” C. (* 1 C.) for 72 to 84 hours. The contents of the tubes are transferred to 125-cc. Erlenmeyer flasks, and titrated with 0.1 N sodium hydroxide to a definite pH of about 6.8 or 7.0. Bromothymol blue is a suitable indicator. A standard flask is used for color comparison. O
Standard Curve The range of the assay is roughly between 0.03 and 0.12 microgram of calcium pantothenate per 10 cc. of culture medium. Duplicate tubes should be set up at about the following concentrations of pure &calcium pantothenate: 0.00, 0.025, 0.05, 0.075, 0.10, 0.125, and 0.15 microgram per 10 cc. of medium.
%
under toluene in the refrigerator. YEASTSUPPLEMENT.A solution of 25 grams of whole, autolyzed baker’s yeast, obtainable from Difco Laboratories, Inc., Detroit, Mich., in 500 cc. of water is adjusted to H 1.5 with concentrated hydrochloric acid, 25 grams of Norit are added, the mixture is stirred 20 minutes at room tem erature, and the charcoal is removed by either centrifuging or kterin with suction. In the latter case a 0.5-cm. layer of moistenedFilter Cel (Johns-Manville, standard) is used on the paper. In either case the Korit is not washed, and a small amount of charcoal coming into the filtrate or centrifugate is disregarded. The pH of the liquid is readjusted to 1.5, and the adsorption repeated as before. The solution is then brought to neutrality with sodium hydroxide, filtered to remove any charcoal present, and diluted to 1000 cc. One cubic centimeter of the final solution is thus equivalent to 25 mg. of autolyzed yeast. It is desirable that the preparation of the yeast supplement be carried through as rapidly as convenient, so that the solution is not kept strongly acid for longer than 1 to 2 hours. This preparation is stored under toluene in the refrigerator. ISORGAXIC SALTS. Solution A consists of 25 grams of dipotassium hydrogen phosphate and 25 grams of potassium dihydrogen hosphate dissolved in 250 cc. of water. Solution B consists o r 10 grams of magnesium sulfate heptahydrate, 0.5 gram of sodium chloride, 0.5 gram of ferrous sulfate heptahydrate, and 0.5 gram of manganese sulfate tetrahydrate dissolved in 250 cc. of water. GLUCOSE. Only c. P. anhydrous glucose is used, and is added dry as needed. CYSTINE. 2-Cystine is dissolved in the minimum amount of hydrochloric acid, and the solution is diluted with water to a concentration of 0.1 per cent cystine. The solution is stored under toluene at room temperature. ASPARAGINE.A 1 per cent aqueous solution of I-asparagine monohydrate is prepared, and is stored under toluene. RIBOFLAVIS. Merck’s synthetic riboflavin is dissolved in 0.02 N acetic acid (dim light), and diluted with 0.02 N acetic acid to a concentration of 50 micrograms per cc. The solution is stored in the refrigerator under toluene and is carefully protected from light a t all times.
Procedure The assay cultures are grown in ordinary test tubes (16 X 150 mm. to 20 X 150 mm.) containing the basal medium plus the sample to be assayed in a total volume of 10 cc. A metal rack
which holds each tube separately and upright and which can be autoclaved is very convenient but not necessary if the tubes are properly marked. If 50 assay tubes are to be set up from stock solutions of the above concentrations, 50 cc. of sodium hydroxidetreated peptone solution, 50 cc. of cystine solution, 12.5 cc. of asparagine solution, 5.0 grams of lucose, 1.0 cc. of riboflavin solution, 2.5 cc. each of inorganic sayt solutions A and B, and 100 cc. of yeast supplement solution are mixed, adjusted to pH 6.8 to 7.0 with sodium hydroxide, and diluted to 250 cc. Five cubic
I
l
l
I
I
1
1
1
1
1
I
I
l
I
I
I
1
0.10 0.15 0.05 0 MICROGRAMS d-CALCIUM PANTOTHENATE
FIGURE 2. STANDARD CURVEOF RESPONSETO PANTOTHENIC ACID 1. 2.
I n presence of aotive yeast supplement In absence of yeast supplement and asparagine
Acid production is plotted against calcium pantothenate concentration to obtain the standard curve (curve 1, Figure 2). This curve should be linear from the blank (1.0-cc. titration or less) very nearly to the maximum titration (8.5 t o 9.5 cc.). Failure of the curve to meet these specifications is usually due to contamination or weakening of the test organism or to improper preparation of the yeast supplement. For comparison a typical “standard” curve as obtained on the basal medium without any asparagine or yeast supplement is included in Figure 2 (curve 2). A standard curve is run with each individual set of assays. The pantothenic acid content of the unknown is determined in terms of calcium pantothenate b y interpolation on the standard curve. Preferably, three levels which fall within the range 0.03 t o 0.12 microgram should be run on each sample under test, with duplicate tubes at each level. The pantothenic acid content of the sample calculated from the various levels must agree within a t least 20 per cent; otherwise the
INDUSTRIAL AND ENGINEERING CHEMISTRY
568
results should not be trusted. The values a t the three levels are averaged for the h a 1 results. The complete data for a typical analysis are given in Table I. For the above purpose two stock solutions of pure d-calcium pantothenate (Merck, synthetic) in 0.05 M phosphate b d e r (pH 6.8 to 7.0) are prepared. One is diluted t o a concentration of 50 micrograms per cc., and the other to 5 micrograms per cc. The former is used as required t o replenish the latter, which in turn is used from day to day in setting up assays. Aliquots are diluted accurately (with water) for setting up the standard curve. Both solutions are preserved under toluene in the refrigerator.
TABLE
11. RECOVERY O F PANTOTHENIC ACID ADDED TO BIOLOGICAL MATERIALS
Sample
Dried yeast Whole milk Yellow corn Wheat middlings Rat kidney 1
Preparation of Sample for Analysis Solids are finely ground. Fresh tissues are mechanically homogenized in water. In either case a suspension is then prepared in approximately 50 volumes of water, adjusted to pH 6.5 to 7.5, autoclaved 20 minutes at 1kg. per sq. cm. (15 pounds per s inch) pressure, and neutralized t o bromothymol blue (pH 6.3, and water is added to give a convenient dilution for assay. Aliquots of the final suspension are pipetted directly into the assay tubes. Blood is diluted fivefold with water, and assayed directly without autoclaving. Other liquids such as milk or urine, and materials which are easily and completely soluble in water are simply diluted t o a convenient volume, neutralized if necessary, and assayed directly without autoclaving.
TABLEI. SAMPLE CALCULATION FOR PANTOTHENIC ACID ASSAY
-
S u spen sion Added Titer, per 0.1 S Tube Base cc , Cc. 0 . 2 0 4 gram of dry 2.0 4.5 yeast 250 ce. 2.0 4.4 2.5 5.0 of water, autoclaved, diluted to 2.5 4.9 3.0 6.0 500 cc. 3.0 5.9 Preparation of Sample
+
Pantothenic Acid Content Per cc. Per gram Per tube suspension sample Microgram Microgram Micrograms 0.067 0.34 83.4 0.064 0.32 78.4 0.078 0.31 76.0 0.30 73.6 0.076 0.100 0.33 80.9 0,097 0.32 78.4 Av. 7 8 . 4
Reliability of the Method The reliability of the present method as a measure of pantothenic acid has been investigated, (1) by means of recovery experiments both on intact materials and on other materials from which the pantothenic acid was removed by autoclaving with strong acid or alkali, ( 2 ) by testing the effect of other known growth-promoting substances, and (3) by comparison with results secured on the same samples by chick assay. Data for typical recovery experiments are collected in Table 11. The yeast extract (Difco) was hydrolyzed by autoclaving in N sodium hydroxide solution for 1 hour a t 1 kg. per sq. cm. (15 pounds per sq. inch) pressure. The liver extract was autoclaved in N sulfuric acid solution for 1.5 hours a t the same pressure. Each sample was neutralized before assaying. The recoveries were satisfactory in all cases. Attempts to carry out similar recovery experiments on whole tissues which had been hydrolyzed were in most cases unsuccessful. When beef liver or kidney was autoclaved for varying lengths of time up to 6 hours with N hydrochloric acid or N sodium hydroxide, and the neutralized product assayed, it was found that the bacteria did not grow normally in the assay tubes. Apparently some pantothenic acid remained intact in nearly every case, because a slight response was noted in the assay tubes containing the lowest level of the hydrolyzed samples. However, growth-inhibitory substances also seemed to be present, since in attempting to carry out the assay at several different levels in the usual manner, the tubes containing the larger amounts of sample actually showed less response than those containing smaller amounts. It was, therefore, impossible in such cases to de-
Vol. 13, No. 8
Rat kidney 2 Human urine 1 Human urine 2 Beef liver
Hydrolyzed yeast extract Hydrolyzed liver extract
Pantothenic Acid Added Found MMicrograntsl .Micrograms/ gram gram None 22 20 43 None 4.0 5 8.4 None 9.0 8.6 17.2 Sone 23 39 62 None 16 28.8 47.3 None 47 139 181 None 2.25 1.67 3.93 None 6.2 5.0 11.6 None 61.5 35.7 97.1 71.5 129 None None 125 137 None 9.0 12.5 21.5
Recovery
70 105 88 95 100 115 96
101
108 100 94
110
100
termine how much pantothenic acid remained undestroyed, and reliable recovery experiments could not be carried out. The effect of known growth-promoting substances was d e termined by assaying pure pantothenic acid in the presence of the following mixture of compounds (figures are micrograms in each culture tube) : biotin 0.004, cozymase 0.8, indoleacetic acid 40, asparagine 800, pimelic acid 200, choline hydrochloride 400, uracil 200, i-inositol 200, adenylic acid 40, cholic acid 200, taurocholic acid 40, cocarboxylase 4, vitamin Be 20, vitamin B1 100, nicotinic acid 1000, and Norit eluate factor (9) 40 (a preparation of which 1 microgram was sufficient for maximum growth). The recovery of pure pantothenic acid in the presence of the above mixture was 106 per cent. As a further check on the method a number of samples of which the pantothenic acid content had been determined by the chick assay were also assayed by the bacterial method. The results are collected in Table 111, from which it will be seen that the agreement was good in all cases. The data in this table also illustrate the reproducibility of results obtained by the bacterial method.
Pantothenic Acid Content of Biological Materials The pantothenic acid content of a variety of biological materials as determined by the present method is given in Table IV, together with pertinent literature values.
Discussion When attempts were made to use the basal medium of Snell, Strong, and Peterson ( l a ) for the determination of pantothenic acid in biological materials in general, it was soon found that the bacteria were responding to substances other than pantothenic acid, or, in other words, that the medium lacked certain essential or a t least stimulatory factors in addition to pantothenic acid. This inadequacy is apparent from a comparison of the curves in Figure 2. I n the absence of asparagine and yeast supplement the response to pure calcium pantothenate is not linear, and the titration for 0.15 microgram is below 7 cc. Much larger amounts of the vitamin-e. g., 1 to 3 micrograms per tube-often gave titrations of 8 to 9 cc., but the response was very irregular and entirely unsuited for assay purposes. It was therefore necessary to add a supplement to the medium which would contain an excess of the stimulating
August 15, 1941
substances but would be free from pantothenic acid. A suitable supplement was found in autolyzed yeast from which the pantothenic acid had been removed by adsorption on Norit. The complete removal of pantothenic acid from autolyzed yeast solutions proved to be difficult, and an extended series of preparations revealed that the separation could be satisfactorily accomplished only in strongly acid solution and by repeated treatment with an active Norit. Under these conditions a considerable portion of the desired stimulating substances appeared to be lost, probably both by adsorption and destruction by the acid. As a result of these difficulties the preparation of satisfactory yeast supplements, which would permit low blank and high maximum titrations, was somewhat uncertain. The recent discovery that l-asparagine is one of the stimulatory substances in question (14) made it possible to include this chemical in the medium. With this addition and the addition of a yeast supplement prepared as above described, consistently satisfactory results have been obtained. Most of the assays reported in this paper have been carried out on a medium containing an active yeast supplement but no asparagine. Repeated tests have shown that identical results are secured whether or not asparagine is present, so long as the medium contains an adequate amount of active yeast supplement (14). Throughout the present work a 3-day incubation period followed by titration of the lactic acid produced has been uniformly used to evaluate the bacterial response in preference to the quicker turbidimetric method (6, 10). The reasons for this choice are twofold. First, the effect of the abovementioned stimulatory substances is greater during the first 12 to 24 hours than after the lapse of 72 to 84 hours (14). Although the present medium has been made as complete as feasible with respect to such stimulatory substances, the possible existence of others has not been excluded, and it is felt that the likelihood of error from this source is materially reduced by using the 3-day assay period. Secondly, when the cultures are titrated i t is unnecessary to prepare a clear extract which contains all the pantothenic acid of the sample, as it is when the turbidimetric method is used. The difficulties attendant on such an extraction are especially great in the case of pantothenic acid, which appears to be largely combined in nondialyzable form in intact tissues (8),and which cannot be extracted with either acid or alkaline solutions because of the danger of hydrolytic destruction (1.2, 16). The primary standard adopted for the present assay is synthetic d-calcium pantothenate (IS). Before this material became available an arbitrary standard had been employed, which consisted of a concentrate prepared from liver extract, and the pantothenic acid content of which had been determined to be 10.8 mg. per cc. by comparison with a preparation from R. J. TTilliams. Tj7hen the older standard was assayed against the synthetic material it was found to
TABLE111.
COMPAR4TIVE PANTOTHENIC --Pantothenic
Sample Yeast 1 Yeast 2 Sweet potato Louisiana cane molasses Cuban cane molasses Cow rumen contents Dried skim milk Green s lit peas Wheat {ran 1 Wheat bran 2 0
569
ANALYTICAL EDITION
Per gram dry weight.
Chick assay Micrograms 50 31 80 la t erial Egg yolk
Beef muscle Beef kidney Dog blood Human urine Whole wheat Wheat bran Polished rice Rice polishings Yeast extract (Difco) Lettuce Rat muscle Rat muscle, autolyzed Rat liver Rat liver autolyzed Dried buttermilk Dried whey
Microbiological Literature Assay Values Micrograms per gram 72 5W 10 9.8 40
0.22 2 to 6 8.3 27.2 8.8 128 240 0.82 4.41
.. . . . 24 .....
27 43
o:ibb
.....
11.2 25.2 4.2
..... ..... .....
2.72 11.76 2.88 36.0
.....
56
Method of Away Referenoe Chick Chick
lo
.......... ..810 Yeast growth ,
.........
Chick Chick Chick
..9 9
3 .......... *. ...... .... .. . . . . .. . .. . .4. Yeast growth
Yeast growth Yeast growth Yeast growth
.. ... . .... Chick
4
4 4
.,
9
Calculated from chick assay results by multiplying “filtrate factor value” by 14 (8). b Calculated from yeast growth results by multiplying reported values by 80 [(since Williams’ “milligram unit” equals 0.08 microgram of pantothenic acid (6), and “yeast growth unit” is 1000 times “milligram unit” (16,17)l. 0
materials assayed. There is frequently a slight drift in these values, the direction being from higher apparent pantothenic acid content at low levels to lower results at high levels of sample per assay tube. However, the magnitude of the drift very seldom exceeds 20 per cent, and it is not observed consistently on any one type of sample. Secondly, pantothenic acid added to various materials was satisfactorily recovered, and a series of biologically active compounds had no significant effect on the assay of pure pantothenic acid. Thirdly, there is substantial agree ment between the results of chick assays and values sesured by the present method (Tables I11 and N).The question of possible inhibition of the growth of the organism by various materials has been previously studied (IO), and the information obtained presumably also applies to the present assay. Furthermore, it has been shown that a high degree of structural specificity is required for pantothenic acid activity toward bacteria (4,6,7,16). An inspection of the data in Table IV reveals a great discrepancy between the pantothenic acid content of certain materials, such as rat liver and rat muscle, as determined by the present method, and by Williams’ yeast growth method (8), the former values being higher. However, the yeast growth results were secured on a water extract of the tissues, and presumably represent only the free pantothenic
570
INDUSTRIAL AND ENGINEERING CHEMISTRY
acid present. After autolysis the values rise to the same order of magnitude as those found by the chick and bacterial assay methods. I n view of these comparisons it seems very likely that the bacterial method measures not only the free but also the combined pantothenic acid of intact tissues. [Since this manuscript was submitted for publication it has been found, however, t h a t certain materials, notably yeasts and animal tissues, contain a combined form of pantothenic acid which is not available to the bacteria. Recent chick assay results on such materials have been much higher than values secured by the microbiological method as described in this paper. In order t o determine the total pantothenic acid content of meats and yeast samples it is necessary first t o subject them to autolysis, or t o an enzyme digestion. Although the optimum conditions for the enzyme treatment are still under investigation, preliminary work indicates that 48 hours’ incubation of a slightly acid (pH 5) a ueous suspension of one part of the sample plus two parts of Cyarase powder (obtainable from the Takamine Laboratories, 193 Arlington Ave., Passaic, N. J.) at 37” C. ives satisfactory results. Fruits, vegetables, cereals, eggs, and fairy products do not show any significant increase in apparent pantothenic acid content when subjected to this treatment. The values for yeasts and animal tissues reported in this pa er must be regarded as a measure of the free antothenic aciapresent at the time the samples were assayecf The actual figures found in such cases obviously depend on the amount of autolysis which the sample has undergone before the assay is carried out.] I
Although too few samples have been assayed to warrant an extended discussion of the distribution of pantothenic acid in biological materials, a few interesting points regarding the data in Tables 11, 111, and IV may be noted. Liver, yeast, kidney, egg yolk, dried whey, dried buttermilk, sweet potato, and the bran of grains are all good sources. Whole grains are much lower. Molasses may be very high or fairly low in pantothenic acid. The high value found for rumen contents probably indicates bacterial synthesis. I n general pantothenic acid seems to be more abundant than riboflavin in foods. The values reported for human urine are based on analysis of samples from 15 normal adult subjects of both sexes. The 24-hour urinary excretion of pantothenic acid by 10 of these subjects fell between 3 and 5 mg. with the exception of one which was 1.95mg. The usefulness of the assay method here reported is substantially similar to that for riboflavin (IO). Crude suspensions of the sample are suitable for assay, so that no extraction of the pantothenic acid is necessary. Approximately 15 samples can be run by one person per day. The range of the assay is from about 0.03 to 0.12 microgram of pantothenic acid, so that very small amounts of material are needed. No unusual or expensive apparatus is required. Finally, since the identical organism and same general setup are used for the present assay as for the determination of riboflavin (IO), it is now possible to assay samples for both vitamins with relatively little additional effort.
Vol. 13, No. 8
Summary A rapid biological assay for pantothenic acid has been developed, which is based on the effect of this substance on acid production by Lactobacillus casei E . The reliability of the method is supported by agreement of the assay results a t different levels, recovery of added pantothenic acid, absence of any disturbing effect from other growth-promoting substances, comparison with results of chick assay on the same samples, and specificity of structure required for activity.
Acknowledgment The authors wish to thank C. A. Elvehjem for making available the chick assay results on the yeast samples listed in Table 111, and T. H. Jukes for similar data on the remainder of the samples in Table 111. The sample of Norit eluate factor was kindly supplied by Brian L. Hutchings. The pantothenic acid concentrate from liver extract was prepared by David Klein of The Wilson Laboratories, Chicago, to whom the authors wish to express their thanks.
Literature Cited (1) Bergey, “Manual of Determinative Bacteriology”, 5th ed., p. 135. Baltimore. Williams & Wilkins Co.. 1939. (la) Jukes; T. H., J. Biol. Chem., 117, 11 (1937). (2) Ibid., 129, 225 (1939). (3) Jukes, T. H., and Lepkovsky, S., Ibid., 114, 117 (1936). (4) Mitchell, H. K., Snell, E. E., and Williams, R. J., J . Am. Chem. SOC.,62, 1791 (1940). (5) Mitchell, H. K., Weinstock, H. H., Jr., Snell, E. E., Stanbery, S. R., and Williams, R. J., Ibid., 62, 1776 (1940). (6) Pennington, D., Snell, E. E., and Williams, R. J., J . Biol. Chem., 135, 213 (1940). (7) Reichstein, T., and Griissner, A,, Helv. Chim. Acta., 23, 650 (1940). (8) Rohrmann, E., Burget, G . E., and Filliams, R. J., Proc. SOC. Exptl. Biol. Med., 32,473 (1934). (9) Snell, E. E., and Peterson, W. H., J . Bact., 39, 273 (1940). (10) Snell, E. E., and Strong, F. M., IND. ENQ.CHEM., Anal. Ed., 11, 346 (1939).
(11) Snell. E. E., Strong, F. M., and Peterson, W. H., J . Am. Chem. . SOC.,60, 2825 (1938). (12) Snell, E. E , Strong, F. M., and Peterson, W. H., J. Bact., 38, 293 (1939). (13) Stiller, E. T., Harris, S. A., Finkelstein, J., Keresztesy, J. C., and Folkers, K., J. Am. Chem. SOC.,62, 1785 (1940). (14) Strong, F. M., and Feeney, R. E., unpublished work. (15) Weinstock, H. H., Jr., May, E. L., Arnold, A., and Price, D., J . Biol. Chem., 135, 343 (1940). (16) Weinstock, H. H., Jr., Mitchell, H. K., Pratt, E. F., and Williams, R. J., J . Am. Chem. SOC.,61, 1421 (1939). (17) Williams, R. J., Truesdail, J. H., Weinstock, H. H., Jr., Rohrmann, E., Lyman, C. M.,and McBurney, C. H., Ibid., 60, 2719 (1938). PRWENTED before t h e Division of Biological Chemistry at the 100th Meeting of the American Chemical Society, Detroit, Mich. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from the Wisconsin Alumni Research Foundation.
