Microbiologic Assay of Natural Pantothenic Acid in Yeast and Liver

Influence of Clarase Digestion

ELEANOR WILLERTON AND HOBART W. CROMWELL Bacteriological Research Department, Abbott Laboratories, North Chicago, 111.

ICROBIOLOGIC assays of natural pantothenic acid have often given much lower values than chick assays,

because of the existence of part of the vitamin in combined form, in which i t is unavailable to the test organism. That this combined form of pantothenic acid could be freed by enzyme digestion was noted in 1934 by Rohrmann, Burget, and Williams (2) . Using yeast as the test organism, they obtained much higher assays with extracts from autolyzed tissue, especially liver, than from fresh tissue. Pennington, Snell, and Williams (1) advised autolysis of samples, wherever suitable, previous to microbiologic assay with Lactobacillus casei. Strong, Feeney, and Earle (3) also using L. casei, found that clarase digestion of yeast and animal tissues satis- factorily increased the pantothenic acid values obtained.

[Since the submission of this manuscript for publication a report by Waisman, Henderson, McIntire, and Elvehjem (4) has appeared, comparing the use of pepsin, clarase, and pancreatin in preparation of meats for such an assay. Pan- creatin was found more effective than clarase in treatment of muscle tissue, and gave about the same results with softer tissue such as liver and kidney. Both were more effective than pepsin.]

Since accounts of the use of clarase have been rather lack- ing in detail, i t seems worth while to record here experiences with this enzyme in digestion of yeast and liver preparations used in pharmaceutical manufacturing. These materials have consistently given the authors much lower values for pantothenic acid by the microbiological method than by the chick method. Sfter adequate clarase digestion, however, the values obtained by the microbiologic method are higher, and compare favorably with those obtained by the chick method. In their assays the authors have followed the method outlined by Strong, Feeney, and Earle (5).

Test materials were digested at pH 5.0 and 37" C. under toluene. To 0.5 gram of the test sample were added 1.0 gram of the clarase powder and water to 10-cc. volume. If necessary, coarse particles, and particularly yeast cells, were broken up in a small glass colloid mill before incubation of the mixture. After digestion the toluene was removed, and appropriate dilutions of the mixture were made for testing.

Clarase is available as a dry powder in crude and con- centrated forms, and from the standpoint of expense, i t was of considerable interest to know whether or not the crude mate- rial would be satisfactory. In Table I are shown comparative results with the two forms. Only 10 per cent mixtures of the concentrated clarase are considered since, in the authors' experience, lower concentrations were inadequate with 48 hours' digestion time. In no instance was tile recovery of pantothenic acid as great after digestion with crude clarase as with the concentrated enzyme, even though the crude material was used in as much as 20 per cent concentration. Possibly longer digestion would release more of the vitamin but, for control of manufacturing, 48 hours is about the limit of convenience, and some of the results suggest a possibility of loss of pantothenic potency with prolonged incubation.

I n the light of this information, a number of different lots of yeast powder, yeast extract, and liver extract were tested, and one lot of Solvamin (Commercial Solvents Company). Assays were made without digestion, and after digestion of the samples with 10 per cent concentrated clarase for 48 hours a t 37" C. Independently, assays on the same samples by the chick method were made by Mrs. F. Peirce Dann of the company's bioassay laboratories. Table I1 shows the results. The assays for pantothenic acid were much higher after digestion than before, and generally approximated the values reported by the animal method.

The clarase itself contains an appreciable amount of panto- thenic acid which must be considered in evaluation of the results. Since the different lots contain varying amounts, a sample of each lot should be assayed after self-digestion when it is first used and occasionally thereafter.

The digestion of these materials, especially yeast, appar- ently produces, or releases, a substance into the digest that interferes with the metabolism of the test organism. The evidence of this is the progressively lower values obtained a t

TABLE I. COMPARISON OF CRUDE AND CONCENTRATED CLARASE IN PAXTOTHENIC ACID A s s . 4 ~

(Clarase digestion, 48 hours) 7- Crude , Concentrated

Test Material 10% 15% 207, 10% .Microgram8 p e r y m m

Yeast powder 154 107 170 Yeast extract 346 . . . . . 358 Liver paste S o . 1 324 323 . . . 170 Liver paste S o . 2 . . . 178 213 Liver paste KO. 3 . . 170 565 Liver powder 30. 1 . . . . 130 198 Liver powder KO. 2 . . . 388 548 Liver powder No. 3h . . . . . . 338

. . . . . . . . . 335

. . . . . . . . . 331 . . . . . . . . 327

a Assay values shown as inicrogranis of pantothenic acid per gram of test

b Values of 338, 335, 331, and 327 were obtained after digestion 48 hours, sample.

72 hours, 4 days, and 6 days, respectively.

TABLE 11. PANTOTHESIC ACID VALUES OBTAINED BY MICRO- BIOLOGIC AND BY CHICK METHODS

(Expressed in micrograms per gram of test material)

S o t Microbiologic

Test Material digested Digesteda Chick Method 55 137 Slightly over 165 Yeast powder No. 1

About 200 Yeast powder S o . 2 55 170 Yeast extract KO. 1 223 321 360 Liver extract paste Nu. 1 357 370 Slightly over 300 Liver extract paste No. 2 165 213 S o t over 300 Liver extract paste No. 3 466 565 600 Liver extract paste N o . 4 288 448 450 Liver extract powder No. 1 219 33s 360 Liver extract powder No. 2 204 357 Liver extract powder No. 3 124 198 300 or more Liver extract powder KO. 4 377 54s 600 or more Solvamin 426 1240 1200 Clarase (crude) 4 . 8 . . . . . Clarnse (concentrated), Lot 1 1 1 . 5 . . . . . Clarase (concentrated), Lot 2 1 0 . 0 1 6 . 0 . . . Clarase (concentrated), Lot 3 . . . 2 7 . 0

a Digested 48 hours with concentrated clarase 10%

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604 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y Vol..14, No. 7

TABLE 111. CALCULATIONS AT VARIOUS TEST LEVELS OF PANTOTHENIC ACID IN A YEAST POWDER AFTER CLARASE

DIQESTION Test Solution

Added per Culture

CC . 0.5 0.75 1.0 1.5 2.0

Digestion with Concentrated Ciarase 10%

188 173 177 153 133

the higher levels tested (Table 111). More difficulty has been encountered with yeast than with liver preparations, perhaps because the lower potency does not permit sufficient dilution of the test sample beyond the range of activity of the inhibitor. With such a progressive decrease in values for pantothenic acid as the test level is increased, it is sometimes difficult to decide which, if any, of the figures are correct. Usually an average of calculations from the two or three lowest test levels has approached fairly closely to the potency obtained by the chick assay. It is probable, of course, that materials of lower potency would present a real difficulty be- cause of the stronger dilutions required for testing.

The assays of the clarase itself also show the presence of an inhibiting substance, with some lots more than with others, but in none has this been enough to account for the effect in the digested samples of yeast or liver, which are tested in much weaker dilutions than the clarase alone.

Obviously, if this inhibition phenomenon could be over- come, less difficulty would be encountered in making calcula-

tions and the dependability of the method would be increased. The authors have not studied this problem except to t ry extraction of the digested material with hexane and with ether before testing. The interfering substance was not removed.

Summary and Conclusions Digestion of 0.5-gram samples of yeast and of liver prepar-

ations with 1.0 gram of concentrated clarase in a total volume of 10 cc., for 48 hours at 37" C., has proved adequate for re- lease of combined pantothenic acid. After such treatment the assay values by the microbiologic method compared favor- ably with those obtained by the chick method, although averaging somewhat lower.

Digestion with double this amount of crude clarase powder was inadequate.

Digestion of these materials with clarase apparently pro- duces or releases a substance which interferes with the me- tabolism of the test organism, L. casei. This does not interfere seriously in testing materials of higher potency where test levels are out of range of the inhibiting factor. If this effect can be overcome, the dependability and usefulness of the method will be increased.

Literature Cited (1) Pennington, D., Snell, E. E., and Williams, R. J., J. Biol. Chem.,

(2) Rohrmann. E., Burget, G. E., and Williams, R. J., Proc. SOC. 135, 213 (1940).

Ezptl. Bo!. Med., 32,473 (1934). (3) Strong, F. M., Feeney, R. E., and Earle, Ann, IND. ENQ. CHEM.,

(4) Waisman, H. A., Henderson, L. M., McIntire, J. M., and Elveh- ANAL. ED., 13,566 (1941).

jem, C. A., J. Nutrition, 23, 239 (1942).

Direct Determination of Sulfur G. L. MACK AND J. M. HAMILTON

New York State Agricultural Experiment Station, Geneva, N. Y.

An ammoniacal cuprous sulfate reagent is prepared by reducing a solution of cupric ammonium sulfate with hydroxylamine hydrochloride. The reaction of this re- agent upon elemental sulfur in acetone solution to form colloidal cuprous sulfide has been made the basis of a turbidimetric method for the direct determination of sulfur.

RAPID method of analysis for sulfur in spray residues A is much needed in the practical evaluation of commer- cia1 fungicides.

The usual procedure (3, 11) has been to oxidize the elemental sulfur to sulfate, which is then precipitated and weighed as ba- rium sulfate. The gravimetric method is so time-consuming that various volumetric (6, 8, 18, 14) and photometric (16) procedures for the estimation of sulfate ion have been proposed. However, for work u7ith spray residues, the oxidation itself requires a con- siderable amount of time, no matter whether the sulfur is first extracted from the plant material (1 1) or the whole sample is oxi- dized in an alkaline fusion (1). The same objection would apply to methods based upon preliminary reduction of sulfur to sulfide

Thus a direct determination of sulfur in the solvent used for extraction from the plant material would be the most desirable procedure. A direct iodometric method has already been pro-

(6).

The reagent was found to be capable of detecting 1 microgram of sulfur in 10 ml. of solution. The determination is fairly specific and accurate. The rapidity with which a large number of samples can be analyzed makes it especially useful for routine work. The method has been ap- plied particularly to the analysis of sulfur in spray residues.

posed by Fleck and Ward (4 ) , and an alkalimetric method first suggested by Kuhl (7) has been adapted to the analysis of spray residues by Small (ZO), but large errors inherent in these methods make them of little use for accurate work.

In 1929 Pierce (9) found that von Nagy Ilosva's reagent (1.2) could be used as an extremely sensitive test for carbon bisulfide in adulterated olive oil and noted that elemental sulfur also gave a test with the reagent.

After considerable experimentation the authors have modi- fied the ammoniacal cuprous sulfate reagent so that it reacts quantitatively with elemental sulfur in solution to give cu- prous sulfide which can be determined turbidimetrically.

Reagents STOCK SOLUTION A. Cupric sulfate pentohydrate, 4.0 rams;

ammonium hydroxide (28.3 per cent ammonia by weighty, 48.0 ml.; distilled water to make 100 ml.