ANALYTICAL EDITION
January 15, 1943
TABLE I. DETERMINATION O F ARSEKICI N SODIUM CACODYLATE AND CACODYLIC ACID
-
Arsenic Found SemimicroDistillation method methoda
7 -
Compound Sodium cacodylate
Cacodylic acid
70
R
46.63 46.85 46.65 Av. 46.64 54.30 54.38
46.71 46.55 46.85 46.70
Arsenic Calculated
%
77
grams of potassium acid sulfate and 0.5 ml. of concentrated sulfuric acid, attach the condenser, and heat gently until all the potassium acid sulfate is melted. Continue heating vigorously until no further signs of carbon remain, allow to cool, and wash the condenser and walls of the flask with water. Dilute to a volume of approximately 20 ml., insert the glass stop er, and shake until the fused mass is dissolved. Add 3 grams orsodium hypophosphite (NaH2P02.HZO) and follom the original procedure
(4). 46.83
.... .. ...
.kv. 54.34 54.31 a Sample was decomposed in distillation flask with acid sulfate a n d sulfuric acid a n d analysis completed according t o procedure of Association of Official Agricultural Chemists ( I ) .
pounds is suitable for the analysis of cacodylic acid and sodium cacodylate. Weigh the dried sample (of size to yield approximately 15 mg. of arsenic) on a piece of cigaret paper or in other convenient manner and transfer to the flask. Introduce into the flask 5 to 6
The results in Table I show the method to be satisfactory. .4n investigation is being carried out in applying the method to the determination of arsenic in arsenic-containing insecticides, such as calcium, magnesium, lead, and copper arsenates.
Literature Cited (1) Assoc. Official Agr. Chem., Official and Tentative Methods of Analysis, 5th ed., p. 615 (1940). (2) Low, W e i n i g , and Schoder, "Technical Methods of Ore Analysis", 11th ed., pp. 31, 32, 89, New York, John Wiley & Sons, 1939. (3) Sloviter, McNabb, and Wagner, ISD. ENO.CHEM.,~ N A L .ED., 13,890 (1941). (4) Ibid., 14,516 (1943).
Microbiological and Chemical Assay of Nicotinic Acid in B Complex Products R . D. GREENE AND ARCHIE BLACK, E. R. Squibb a n d Sons, New Brunswick, N. J., AND F. 0. HOWLAND, E. R . Squibb a n d Sons, Brooklyn, K. Y.
T
HE important role of nicotinic acid (niacin) in human nutrition has stimulated the search for a method of assay which would possess the specificity of the biological assay with dogs and yet afford greater accuracy, speed, and economy. A number of chemical procedures based on the Konig cyanogen bromide reaction and several microbiological methods have been reported t o meet these requirements. The dog method has not been used extensively for quantitative tests, so that comparative data are limited. The recovery experiments and duplication studies in connection with the various rapid methods are subject to limitations &s criteria of the reliability of these procedures. It is believed that further comparative assay data from procedures as widely different as the chemical and microbiological tests will provide useful evidence as to their applicability. In the present studies tests have been made on various natural sources of the B complex. The chemical method as modified by Jones (4) and the Snell and Wright (8) microbiological method were used. Microbiological assays were made by R. D. Greene and A. Black, and chemical assays by F. 0. Howland.
In supplying the biotin requirement of the organism they have used the supplement prepared from egg yolk and later pure biotin with equal results. In their experience the free pure biotin a t a level of 0.001 microgram per 10 ml. of medium was
TABLEI. RECOVERY OF PURENICOTIXIC ACID IN ESCE OF WHEATGERMEXTRACT Added
Micrograms
Recovered
nicotinic acid p e r 10 ml. of mediuma 0.026
___
n- . nnti
0.100 0.130 0.200
0,203 0,264 0.324
0.078 0,107 0.168 0.228
PRES-
Recovery
%b 104 1 1. 2_ .
104 107 112 114
a Medium according t o Snell and Wright +1.4 mg. of wheat germ extract per 10 ml.
TABLE11. EFFECT OF ACIDIC TREATMEKT OF SAMPLESOK MICROBIOLOGICAL ASSAY Sample No.
Methods The procedure is essentially that of Snell MICROBIOLOGICAL. and Wright (8). However, in the preparation of the hydrolyzed casein supplement, the optional charcoal treatment has been used as a regular procedure. In performing this step the authors treat a solution of hydrolyzed casein of pH 3.5, containing about 25 mg. of solids per ml., with 2.5 mg. of Darco G-60 per ml. Recently Isbell (3) has reported poor growth with charcoaltreated casein hydrolyzate, due to p-aminobenzoic acid deficiency. The authors' hydrolyzate, treated at greater dilution with charcoal, supported optimum growth. Although p aminobenzoic acid was not a limiting factor in their tests, its routine addition seems a desirable safeguard against variations in the casein supplement. For the preparation of inoculum they have used a dilution of 1 to 25 rather than 1 to 10. For incubation of cultures they have used temperatures of 30" C. and later 37" C. (9) with equal success.
Found of
THE
2463 2488 2514 2522 2640 2560 2661 2294 2541 2342 2287 2426 2485 2523 2459
a
Description Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast extract Yeast extract Liver extract Rice bran concentrate Rice bran concentrate Rice bran concentrate Rice bran concentrate Wheat germ extract Flour p a t e n t Flour' whole wheat Yellok corn, ground Rolled oats Whole milk powder
Nicotinic Acid Aqueous Acidic"
MOJO.
MOJO.
0.214 0.603 0.218 0.342 0.560 0,506 0.194 1.80 3.04 0.508 1.64 1.1s 0.97 1.02 0.049 0,0069 0.0293 0.0156 0,0051 0.0049
0.231 0.599 0,229 0.371 0.488 0.512 0.216 1.85 2.83 0.462 2.15 1.53 1.33 1.42 0,069 0.0095 0.0362 0,0194 0.0057 0.0051
Autoclaving for 15 minutes a t 15 pounds with 25 volumes of 2 N HCI or
2 N H2S04.
INDUSTRIAL AND ENGINEERING CHEMISTRY
78
TABLE 111. MICROBIOLOGICAL AND CHEMICAL ASSAYSFOR NICOTINIC ACID Microbiological 5 Sample No. of No. testsb
Chemical
Range
Av.
iMg./g.
Mg./g.
Rice 1.69-1.74 1.41-1.42 1.47-1.47 1.78-1.79 1.63-1.69 1.67-1.72 1.65-1.73 2.08-2.21 1.88-1.91
No. of testsb
Bran Sirup 1.71 1 1.41 1 1.47 1 1.78 2 1.66 1 1.69 1 1.69 1 2.14 1 1.89 1
15,037 13,917 12,727 9,279 7,922 7,594 7,280 6,093 5,538
2 2 2 2 2 2 2 2 2
13,917 12,009 11,322 10,811 9,925 6,532 12,874 11,724 11,725 12,875
2 4 4 2 2 4 4 2 4
0.23-0.23 0.21-0.25 0.34-0.43 0.43-0.46 0.34-0.36 0.54-0.55 0.52-0.65 0.54-0.62 0.54-0.58 0.58-0.62
14,236 7,633 6,347 6,000 5,514 5,248 4,871
2 5 4 2 2 2 2
1.63-1.75 1.79-1.96 1.80-2.22 2.00-2.23 1.91-1.95 1.98-2.15 1.91-2.05
7,991 7,646 11,716
2 2 2
Wheat Germ 0.067-0.071 0.069 2 0.071-0.071 0.071 2 0.074-0.079 0.076 3
9,414 4,902 98,750 1,610 15,351
4 4 2 2 2
Malted Wheat Germ 0.096-0.111 0.102 0.087-0.112 0,100 0.114-0.115 0.114 0.100-0.108 0,104 0.103-0.104 0.103
6A
2 2
Cane Molasses 0.0047-0.006 0.0053 1 2 0.0098-0.01 0,0099
6
Dried Yeast 0.23 0.23 0.38 0.44 0.35 0.54 0.59 0.57 0.56
1 3 2 2 2 1
2 4 1
0.60
3
Yeast Extract 1.69 1.87 1.98 2.12 1.93 2.06 1.98
2 2 1 1 2 1
1
Range
Av.
MO./O.
nrg. 10.
....... ....... ....... 1.78-1.82 ....... ....... ....... ....... .......
1.61 1.24 1.24 1.80 2.10 1.50 1.90 2.20 1.80
.......
0.36 0.20 0.33 0.38 0.39 0.44 0.65
0.17-0.24 0.32-0.35 0.36-0.40 0.37-0.42
.......
0.54-0.76 0.54-0.74
.......
0.42-0.57
.......
.......
2.0 2.1 2.06 1.8 1.8 2.2 1.8
0.066-0.071 0.060-0.070 0.064-0.089
0.068 0.065 0.078
1.7-2.5 2.0-2.1
.......
....... 2.1-2.3
Extract 1 ....... 3 0.07-0.13 3 0.08-0.15 a
0.60 0.39 0.50
....... .......
0.068 0.093 0.123
...
...
Vol. 15, No. 1
dependent to a marked degree on how drastic were the conditions of extraction. Recently other reports (1, 9, 10) have gone into this matter more extensively, showing that cereal products, as distinguished from yeast or animal products, contain a water-soluble precursor of nicotinic acid which yields maximum values in the microbiological assay only after treatment with alkali or strong acid. The question has been raised as to whether this microbiologically inactive precursor of nicotinic acid is active for animals, and the consequent issue as to the available nicotinic acid of cereal foods and feedstuffs must await clarification by further research. The authors’ experiences with a variety of products of cereal origin, ranging from original grains to highly potent concentrates, have paralleled those of other investigators. I n order to determine the conditions necessary for the preparation of samples for test, they have made comparative microbiological assays of a variety of products subjected to treatments ranging from aqueous to 4 N acid. As a result of these experiments, summarized in Table 11, they have adopted the use of strong acid as a standard preparatory treatment for cereals, whereas other materials have been prepared by aqueous extraction.
Microbiological and Chemical Assays The results of parallel microbiological and chemical assays of various cereal and yeast products are presented in Table 111. For the most part the results from the two methods are in agreement and indicate that both these rapid and relatively inexpensive tests are reliable measures of nicotinic acid, although somewhat greater individual variations were observed in the chemical tests of materials of lower nicotinic acid content.
Summary
more than sufficient for Lactobacillus arabinosus 17-5, whereas the methyl ester was almost inactive. Shull, Hutchings, and Peterson ( 7 ) have reported that the methyl ester is not utilized by Lactobacillus casei e. CHEMICAL.The chemical assays of nicotinic acid were carried out by a modified cyanogen bromide method which has been fully described by Jones (4).
I n agreement with recent reports it has been shown that preliminary acid treatment of cereals, as distinguished from other products, leads to higher microbiological assay values for nicotinic acid. Comparative assays of various pharmaceutical B complex products for nicotinic acid by the Snell and Wright microbiological method and by the cyanogen bromide method a.s adapted by Jones have shown substantial agreement between the results from these widely different procedures and tend to establish the validity of both as measures of this factor. In the testing of the materials of lower nicotinic acid content in this series the microbiological niethod appeared to be somewhat more suitable.
Factors Affecting Microbiological Assay
Acknowledgment
SPECIF~CITY OF RESPONSE.I n order to test the sufficiency of the basal medium the authors added extracts of rice bran, yeast, and liver in amounts judged by assay to be equivalent to the levels of nicotinic acid in the standard series, up to optimum. The titration curves produced from the addition of these supplements to the basal medium followed the standard curve very closely and indicate that results based on the usual assay range involve negligible stimulation due to substances other than nicotinic acid. This question has been approached in a different way by determining the recovery of varying increments of pure nicotinic acid in the presence of a constant quantity of a wheat germ extract, a product relatively low in nicotinic acid. The recovery data from this experiment, shown in Table I, confirm the results obtained from the addition of the other B complex supplements. PREPARATION OF SAMPLES FOR ASSAY. Several of the earlier reports on both chemical (5, 6) and microbiological (8, 11) assays of cereals indicated that the results were
I n connection with the microbiological data the authors are indebted to Miss Edna Heacock for supervision of stock cultures and to Miss Frieda Bamman and Miss Lois Herbert for technical assistance.
8A 0
b
.......
None
None-0.04
All other than yeast products prepared by acid treatment as in Table 11. Independent tests.
Literature Cited (1) Andrew, J . S., Boyd, H. M . , and Gortner, W. A., IND. ENO. CHEM.,ANAL.ED., 14, 663 (1942). (2) Cheldelin, V. H., and Williams, R. R., Zbid., 14,671 (1942). J. B i d . Chem., 144,567 (1942). (3) Isbell, H., (4) Jones, W.S., J . Am. Pharm. Assoc., Sci. Ed., 30,272 (1941). (5) Kodicek, E.,Biochem. J., 34, 712, 722 (1940). (6) Melnick, D.,Oser, B. L., and Siegel, L., IND. ENG.CHEM., ANAL.ED., 13, 879 (1941). (7) Shull, G. M., Hutchings, B. L.,and Peterson, W. H., J. BioE Chem., 142, 913 (1942). (8) Snell. E. E . , and Wright, L. D., Ibid., 139,675 (1941). (9) Teply, L. J., Strong, F. M., and Elvehjem, C. A., J . Nutrition, 23, 417 (1942). (10) Ibid., 24, 167 (1942). (11) Waisman, H.A.,and Elvehjem, C. A., IND.ENQ.CHEM.,ANAL. ED., 13, 221 (1941).
