Three Sources of Amino Acids for Niacin Assay A. R. KEMMERER AND FAY SHAPIRO D e p a r t m e n t of Nutrition, University of Arizona, Tucson, A r k .
produced for any given level of niacin with trypsin-hydrolyzed casein than with either acid-hydrolyzed casein or charcoaltreated peptone. The titration values for the blank tubes with the trypsin-hydrolyzed casein are higher than with either of the other two preparations. Although the increase in acid production with the trypsin-hydrolyzed casein was slight, it was consistently obtained in five different experiments. The addition of tryptophane and cystine was found necessary in media containing charcoal-treated peptone, and of cystine but not tryptophane in media containing trypsin-hydrolyzed casein. Determination of Niacin. Media containing the three prepara-
HE Snell-Wright (5) microbiological assay for niacin as Tmodified by Krehl, Strong, and Elvehjem (9)has been widely employed in nutritional research. In this method casein hydrolyzed with hydrochloric acid has been med as a source of amino acids for the assay microorganism, Lactobacillus arabinosus. Recent investigations have shown that tryptic digests of casein and other proteins contain one or more unidentified substances which stimulate the growth of certain microorganisms ( 4 , 6, 7 , 10). Strepogenin (7), which may also stimulate growth in mice (9) and rats (8), was the name tentatively given one of these factors. Since the acid-hydrolyzed casein used in the Krehl, Strong, and Elvehjem (2) method is laborious to prepare and expensive to buy, and may lack certain necessary factors, nutritionists have suggested other sources of amino acids for microbiological assays. Isbell (1) reports that charcoal-treated peptone replaces the acidhydrolyzed casein satisfactorily in media used for the assay of biotin, niacin, or pantothenic acid. Roberts and Snell (5) have recently found that a pancreatic digest of casein gives superior results in media for Lactobacillus casei and heavy growth iri media for Lactobacillus arabinosus. The work presented in this paper attempts to determine which of these sources of amino acids is the most satisfactory for the niacin assay.
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EXPERIMENTAL
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Procedures. Trypsin-hydrolyzed casein was prepared by suspending 50 grams of purified casein (GBI vitamin test) in 500 ml. of water, adding 1 gram of potassium monohydrogen phosphate, and agitating the suspension continuously by means of a motor stirrer while concentrated sodium hydroxide solution was added until the casein was dissolved. The pH was maintained between 8.0 and 9.0, during the entire procedure. After the casein was dissolved, the pH was adjusted to 8.0 to 8.4, 2.5 grams of trypsin were added, and the solution was covered with ~f thin layer of toluene and allowed to stand overnight a t 37" C The pH was readjusted to 8.0 to 8.4 and the solution alIowed to stand 24 hours a t 37" C. The toluene was removed from the casein solution and the pH was adjusted to 4.0 with dilute hydrochloric acid. Twenty grams of activated charcoal (Sorite A) were added, and the mixture was stirred for 1 hour a t room temperature and then filtered by gravity. The amount of filtrate was measured and its equivalent in casein per milliliter recorded. It was covered with a thin layer of toluene and stored in the refrigerator. A small amount of tyrosine, which crystallized after storage for several days, was easily brought back into solution by heating. Acid-hydrolyzed casein was prepared by the procedure of Krehl, Strong, and Elvehjem (9)and the charcoal-treated peptone (Bacto-Peptone) by the procedure of Isbell (1). The basal media, stock cultures, inoculum, and assay procedure were similar to those of Krehl, Strong, and Elvehjem ( 2 ) with these exceptions: When the trypsin-hydrolyzed casein was used no tryptophane was added to the media and when charcoaltreated peptone was used, it was placed in the media a t twice the concentration of the hydrolyzed caseins. Response of Lactobacillus arabinoszis to Pure Niacin in Different Media. The amount of acid produced by the microorganism when the three different amino acid sources were used in the media are compared in Figure 1. There was slightly more acid
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04 08 08 MICROGRAMS NIACIN
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Figure 1. Response to Pure Niacin I. Trypsin-hydrolyzed casein
11. Acid-hydrolyzed catasin 111. Charcoal-treated peptone
Table I. Comparison of Niacin Contents with Three Sources of Amino Acids Sample Dried skim niilk Corn meal Oat meal Broccoli (dried; Cottonseed meal Barley Bran Grain sorghum Sweet corn (dehydrated) Wheat
358
TrypsinHydrolyzed Casein .//B. 7.1 12.0 7.0 63.8 35.4 49.5 240.0 72.3 66.3 75.8
AcidHydrolyzed Casein
CharcoalTreated Peptone
Y/O
Y/Q.
7.1 13.0 7.6 69 6 30.4 47.1 244.0 78.6 72.6 76.7
8.2 12.7 8.4 66.9 39.6 59.5 254.0 70.3 72.0 81.3
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V O L U M E 19, NO. 5, M A Y 1 9 4 7 tions were used for the assay of ten samples (Table I!. Each sample w&s assayed a t the same time with each of the three media. The amount of niacin found in the samples varied only slightly among the three preparations. Better agreement between assay levels seemed to result with the trypsin-hydrolyzed casein for the single assays reported.
The increased response of Lactobacillus arabinosus with the trypsin-hydrolyzed casein indicates (4, 6, '?',IO) that this preparation contains still unidentified factors not present in acid-hydrolyzed casein or charcoal-treated peptone, which slightly stimulate Lactobacillus arabinosus. LITERATURE CITED
DISCUSSION
The above results show that any of the three sources of amino acids will give reliable results in the niacin assay. Trypsinhydrolyzed casein is considerably less tedious to prepare than the acid-hydrolyzed casein and media in which it is used did not require the addition of tryptophane. Charcoal-treated peptone is simple to prepare, but like acid-hydrolyzed casein required the addition of tryptophane, and it had to be used at double the concentration of the other two. From the standpoint of expense these are important factors. Cystine was required with all three preparations.
Isbell, H., Science, 102, 671 (1945). Krehl, UT.A., Strong, F. M.,and Elvehjem, C. A , , IND.ENQ. C H E M . , . ~ S AED., L . 15, 471 (1943). Roberts, E. C., and Snell, E. E., J . BioZ. Chem., 163, 499 (1946). Snell, E. E.,I b i d . , 158,497 (1945). Snell, E. E., and Wright, L. D., Ibid., 139, 675 (1941). Sprince, H., and Woolley, D. R., J . Am. Chem. SOC.,67, 1734 (1845).
Sprince, H., and Woolley, D. IT.,J . E s p t . M e d . , 80, 213 (1944). Womack, M., and Rose, W. C., J . B i d . Chem., 162, 735 (1946). Woolley, D. W., Ibid., 159, 753 (1945). Wright, L. D.. and Skeggs,H. R.. J . Bact., 48, 117 (1944).
Determination of Saponification Value of Natural Waxes B. H. KNIGHT 50 East 41st S t . , New York 17, N . 1..
HE ordinary procedure, using alcoholic potassium hydroxide Tand varying reflux periods, gave unsatisfactory end points in an effort to establish a clean-cut difference b e k e e n t,he saponification values of two wax samples, one of which was a processed portion of the other. A search of the literature showed that the standard saponification methods of the U. S. Pharmacopoeia, the Association of Official Agricultural Chemists, the American Oil Chemists' Society, and the American Society for Testing Materials made no special provision for wax. Alcoholic potassium hydroxide was the only saponification reagent mentioned, with one exception, but when used alone i t does not give dependablc results. Consequently, a method was sought which would, without special apparatus or manipulation, give consistent check results in the hands of the same analyst and also in different laboratories. Carbitolized potassium hydroxide offered promise because of its high boiling point (about 200" C.), but when used alone its marked tendency to develop color after completion of the refluxing period made the end point on the back-titration uncertain. Excellent check results were obtained by adding an equal volume of alcohol to t,he carbitolized potassium hydroxide and refluxing very gently for one hour. In a series of determinations, figures on refined carnauba and candelilla samples were within the limits tabulated by Lewkowitsch. .-1 simplified method, in which an equal volume of Carbitol was added to the measured alcoholic potassium hydroxide, gave figures for carnauba, candelilla, and ouricury samples that checked excellently with original figures on the same samples. Twenty duplicate determinations on the same candelilla sample had an over-all variation of only 0.05 ml. in alkali consumption. Results by this method were markedly more consistent than those obtained by several other procedures tested, and it, has worked equally well in the author's hands on both crude and refined samples of the hard waxes such as candelilla, carnauba, and ouricury. A complete determination can be made in less than 2 hours. Glassware can be much more easily cleaned-a point which the wax analyst will appreciate, as the residue on the flasks remaining after ordinary procedures is very difficult t,o remove. REAGENTS AND EQUIPMENT
Carbitol solvent (Carbide and Carbon Chemicals Corp.), practical grade. Alcoholic potassium hydroxide, 0.5 M . Aqueous
hydrochloric acid, 0.5 .V. Phenolphthalein indicator. Ethyl alcohol, 95%. Regular 200-ml. Erlenmeyer flasks, and condensers, preferably of Pyrex, connected by rubber stoppers. PROCEDURE
Prepare hard waxes for analysis by selecting small lumps from the entire shipment, grinding them in a loosely set laboratory plate mill and screen, and regrinding them until the entire sample will pass a 20-mesh screen. Roll the screened sample on paper t o ensure uniformity and weigh 0.5 gram directly from the rolling paper. Transfer the sample to a 200-ml. Erlenmeyer flask and pipet in 20 ml. of 0.5 N alcoholic potassium hydroxide, followed by 20 ml. of Carbitol from another pipet. Set up a blank with exactly the same volume of both reagents, and fit the flasks to the condensers. Heat with a low flame, adjusted so that they boil very gently,. and reflux for one hour. Titrate the blank first with aqueous hydrochloric acid and phenolphthalein. Before titrating the sample, bring it to the boiling point; all the precipitate disappears, but reappears soon after addition of acid, thus requiring considerable rotation of the flask during titration to absence of indicator color. Bring the solution to the boiling point and disappearance of precipitate and again titrate to absence of indicator color. Finally reheat and titrate further if indicator color develops. Generally the color is all discharged after the second titration. Subtract the total determination reading from the blank reading and calculate the saponification figure. The end point IS vrry easily distinguished, as not even crude samples of carnauba or ouricury developed a troublesome color when this method was used. This combination of reagents, while an excellent hot solvent for the wax saponification end products, does not generate sufficient heat to produce a marked end color. I n rare instances a lot of Carbitol will develop color when heated with alcoholic potassium hydroxide; it is therefore necessary to test each delivery of Carbitol by refluxing a small volume with an equal volume of 0.5 S alcoholic potassium hydroxide, rejecting any which devclops color. ACKNOWLEDGMENT
The author extends his thanks to the Mamaroneck Chemical Corporation and to its vice president and chemical director, C. S. Treacy, for their cooperation and assistance in making this investigation possible.
