ANALYTICAL EDITION
August 15, 1942
The use of clarified aqueous extracts. The preliminary extraction of the dried sample with lipoid solvent, after which an autoclaved aqueous suspension of the product may be used for assay. The inclusion of photolyzed extracts of the product to be assayed in the riboflavin assay medium, or the inclusion of an alkaline or acid-treated extract of the product to be assayed in the pantothenic acid assay medium, as has been previously suggested by other workers.
Summarv Growth stimulants have been shown to exist in certain foodstuffs for the growth of L. casei in the microbiological assavs for riboflavin and Dantothenic acid when suboptimum amounts Of the are present in the assay medium* and +‘ttention i’ directed to the presence Of the treatments are discussed to avoid them.
67 1
Literature Cited (1) Eckhardt, R. E., Gyorgy, P., and Johnson, L. V., Proc. SOC. Ezptl. Bid. Med., 46, 405 (1941). (2) Feeney, R. E., and Strong, F. M . , J . B i d . Chem., Proc. xxxviii (1941). (3) Hodson, A. Z., and Norris, L. C., J . Bid. Chem., 131,621 (1939). (4) Ishell, H., Wooley, J. G., and Fraser, H. F., U. S. Pub. Health Service, Pub. Health Rept., 56, 282 (1941). (5) Pennington, D., Snell, E. E., and Williams, R. J., J . BbZ. Chem., 135.213 (1940). (6) Snel1,’E.E., and Strong, F. M . , IND. & ENG.CHEM.,ASAL. ED., 11, 346 (1939). (7) Strong, F. M., Feeney, R. E., and Earle, Ann, Ibid., 13, 566 (1941). PRESENTED before t h e Divisions of Agricultural and Food Chemistry, Biological Chemistry, and Medicinal chemistry, program on Vitamins, a t t h e 102nd Meeting of t h e AMERICANCHEMICAL SOCIETY, Atlantic City, h-.J .
Extraction and Assay of Nicotinic Acid J
from Animal and Plant Tissues Comparison of Methods VERNON H. CHELDELINI
AND
ROBERT R. WILLIAMS, 297 Summit .4ve., Summit, N. J.
I S C E the discovery of the vitamin properties of nicotinic acid in 1937 (6) considerable interest has been focused upon the estimation of this substance. I t s widespread importance to clinicians and students of nutrition has made obvious the desirability of having standardized procedures for its quantitative determination wherever it may occur. The practical use of such methods for the control and the manufacture of enriched bread and enriched flour is especially timely a t present. At the present time some 25 methods or modifications of methods for the determination of nicotinic acid have appeared in the literature] and several points of controversy exist as t o what experimental steps are t o be preferred. The present paper is a report of progress with various aspects of this problem as applied to a number of natural substances.
After digestion the samples were steamed 30 minutes t o inactivate the enzymes and remove the benzene. Each sample was filtered through a very thin layer of kieselguhr on a Hirsch filter and the residue was washed with a volume of water equal to about twice that of the filtrate. The combined washings and filtrates were diluted, usually to a concentration of 1 ml. per 25 mg. of fresh tissue. The extracts were placed in tubes or flasks stoppered with cotton plugs, steamed 5 to 10 minutes, and stored in the refrigerator until used. Materials which were received in a homogeneous state, such as milk powder, flour, and other cereals, were not ground or mixed thoroughly prior to the preparation of extracts. The samples of whole wheat flour and bread, white flour, enriched white bread, and dry yeast were obtained as “collaborative samples” from the Research Corporation Committee on Assay Methods. The other materials were obtained in nearby stores and markets.
Enzyme Digestion
Table I shows that in general maximum amounts of nicotinic acid are extracted from tissues by digesting with takadiastase and papain, either separately or in combination. (Since this combination has been found effective in releasing other vitamins from tissues, 2, the authors have employed it routinely in the preparation of extracts for nicotinic acid assays.) Other means, such as autolysis or brief hot water extraction] are somewhat less effective. Extraction with hot water has been deemed insufficient for chemical analysis, because of incomplete hydrolysis of nicotinamide or nicotinuric acid (7, 12, 14). This objection does not hold for the microbiological assays by the Snell and Wright method, since both of the above compounds as well as cozymase are as effective as nicotinic acid in promoting bacterial growth ( I Q ) , but it would seem, then, that nicotinic acid may exist in non-growth-promoting combinations from which it cannot be freed completely by hot water. The utility of nicotinuric acid as a pellagra preventive is at present uncertain, owing to the contradictory findings of Woolley and eo-workers (22) and of Dann and Handler (5) regarding the antiblacktongue potency of this compound. Since its presence in measurable quantities seems to be con-
S
For the liberation of nicotinic acid from possible complexes, autolysis or digestion with added enzymes seemed an attractive procedure. Accordingly, a number of materials were treated with enzymes and the extracts were assayed according to the Snell and Wright microbiological method (19). Seven enzymes were employed: takadiastase, malt diastase, pancreatic amylase, papain, pepsin, trypsin, and pancreatin.
Procedure Samples of fresh tissue were ground several times in a ment chopper, mixed thoroughly, and weighed into sterile tubes or flasks. Each portion was then suspended in ten times its weight of 0.1 per cent buffer solution, the pH of which was chosen to coincide xyith the supposed “optimal pH” for activity of the enzyme. A weighed amount of enzyme equal to 2 per cent, of the wei ht of the tissue sample was added to each flask, a few drops of benzene were added, and the samples were allowed to digest under specified conditions (see Table I). 1 Present address, Austin, Texas.
Department of
Chemistry, University of
Texas,
Results of Enzyme Digestion
,
672
INDUSTRIAL AND ENGINEERING CHEMISTRY
fined to urine, this point is of small iniportance to assays of plant and animal tissues.
Other Methods of Preparation of Extracts In order to convert the nicotinic acid present in natural materials into a form available for assay, two other methods have been used generally-namely, hot extraction with sulfuric acid or sodium hydroxide, Although animal tissues and milk powder show essentially the same apparent nicotinic acid content when determined microbiologically after treatment by the three methods discussed (Table 11),acid or alkaline extraction of cereals or cereal-containing diets has in the authors' hands given generally higher results than those obtained by treatment with enzymes. The observations with regard to alkali treatment are in agreement with data obtained by other workers (8, 16, 19). The increases due to treatment with alkali, although in some cases small, are often appreciable. A possible source of these increases seemed to be trigonelline. llelnick, Robinson, and Field (14) noted that alkaline hydrolysis of trigonelline in the presence of urine produced a substance in yields varying up to 30 per cent which gave the color reactions for nicotinic acid in their test. Sarett, Perlzweig, and Levy (18) found further that these yields could be increased to 70 per cent in the presence of 6 N alkali and a high concentration of ammonium salts or urea. That the conversions are genuine is indicated by similar results with the microbiological technique. The conversion of samples of trigonelline to nicotinic acid (or its equivalent) was studied under varying conditions b>microbiological estimations. The results are listed in Table 111. I t is apparent from Table 111 that, although trigonelline alone is unaffected by autoclaving 30 minutes with 2 A; sodium hydroxide, it may under sufficiently drastic treatment viith alkali be converted to nicotinic acid in rather high percentage yields. However, under the conditions of hydrolysis ordinarily employed only a slight error is to be expected. This may sometimes assume greater significance in the presence of high concentrations of substances ,which promote the reaction, such as asparagine, which are probably present, in the framework of plant proteins in fairly large amounts. The authors are of the opinion that the higher conversions obtained in the presence of asparagine or beta-alanine may be due to slow production of ammonia (or -SH2 groups) in the reaction mixture. The increases due to acid hydrolysis parallel in most cases the increases with alkali hydrolysis within the limits of experimental error. However, they cannot be explained by conversion of trigonelline, as may be seen from the experiments xith whole wheat bread listed in Table IV. Large amounts of trigonelline failed to affect the apparent nicotinic acid content when heated with hydrochloric or sulfuric acid. The addition of reducing agents, cystine and hydrogen sulfide, also failed to produce significant increases. If the assay values obtained after acid extraction can be assumed to represent no converted trigonelline, the method of Sarett, Perlzweig, and Levy (18) can then be used to indicate the amount of trigonelline present. This has been done for whole wheat bread.
Vol. 14, No. 8
T.4BLE I. EFFECTOF ENZYME TRE.4TMENT ON YIELDS FROM VARIOUS TISSUES
OF
NICOTINIC ACID
(Micrograms per gram of fresh tissue)
Material Hog heart
Beef leg muscle
Beef brain
Green peas
Alilk powder
Treatment
pH
Autolyzed 24 hours a t 37'. natural p H Digested 24 hours a t 37' with malt diastase With pancreatic amylase With papain With pepsin With trypsin With ancreatin D i g e s t e f w i t h takadiastase 3 hours, then with apain 5 hours a t 45-47' Steamez 30 minutes with 0.1 .V H O h c Autoclaved 30 minutes with steam a t 1.5pound pressure Autolyzed 24 hours a t 37', natural p H Digested 24 hours a t 37' with takadiastase With malt diastase With pancreatic amylase With pepsin With trypsin With pancreatin Digested with takadiastase 3 h o y , then with papain 5 hours a t 45-47 Autoclaved 30 minutes with steam a t 13pound pressure Autolyzed 24 hours a t 37O, natural H Digested 24 hours a t 37' with taka&astase W k h malt diastase With pancreatic amylase With papain With pepsin With trypsin With ancreatin Digestefwith takadiastase 3 hours, then with papain 5 hours at.45-47' Autoclaved 30 minutes with steam a t 15pound pressure Autolyzed 24 hours a t 37', natural p H Digested 24 hours a t 37' with takadiastase With malt diastase With pancreatic amylase With papa,in With pepsin Wit,h txvnsin .."~..~ With ancreatin Digestefwith takadiastase 3 ho:rs, then with papain 5 hours a t ,45-47 Autoclaved 30 minutes with steam a t 15pound pressure Autolyzed 24 hours a t 37O, natural p H Digested 24 hours a t 37' with takadiastase ( T a b l e I continued t o p OJ' next p a i r )
Relative Yield, Maximum 100%
Nicotinic Acid Content
, . .
85
98
4 3
77 77 82 77 87 74
89 89 94 89 100 8.5
4.7
...
81 62
93 71
...
79 41
91 89
44
8.4
46 46 46 46 44
96 100 100 100 100 96
4.7
42
91
...
40 30
8i
7 0 5.0 2.1 8.4
8.4
...
3.0 4.5
7.0 1.8 8.4
...
30 29 35 35 34 30 3.5
4.5 4 3
!,8
2.1 8.4 8.4 4.7
4 5 4.5 7.0 5 0 2.1 8.4
86
.
8fi
35
100
29 20
91
83
8.4
20 20 22 20 22 16 21
4.7
35
91
20 7.6
91 100
7 .5
99
4 .5
91 91 100
91 100 73 96
WITH SULFURIC .ACID OR SODIUM HYT A B L E 11. EFFECTO F EXTRACTIOX DROXIDE ON ~\lICROBIOLOGICAL ASSAYS FOR -\JICOTINIC d C I D IN V.4RIOUS
SUBSTANCES
Sicotinic Acid Conteut
RIaterial Beei muscle Beef brains Milk powder Green Deas "Avera'ge American diet" Batch 31 35 .. 36
Digested with takadiastase 3 hours Autoclaved Autoclaied then with 30 minutes 30 minutes papain with 1 N with 2 .V 5 hours a t NaOH 45-470 HzSOi Macrograms p e r g r a m of fresh t w u e 36 35 .. 37 38 38 7.5 7.8 8.2 22 22 22
-3 3 4 0
..
13.8a 16.0a 16.44 15,Ob (10.9)c 21 (12.5): 16.8 (11.8)' 13.8 (11.2)~18.2 (11.2) 15.8 (14.8)e 6.4 9.4 8.4
16" 40 32 47
19a 12 15 31
Increase Due t o Treatment with HzSOr NaOH
70
% 3 9
0
Corn m e i i Whole wheat 43 47 42 62 bread 48 36 60 Whole wheat flour 44 . . . . 21b Takadiastase '9 .. 19b Papain .. 40 i8.4 13.2 Blackeyed peas Germinated lima 33 11.5 8.5 beans 0 Samples assayed after 2 months' storage a t 0-5'. b Autoclaved 30 minutes with steam a t 15-lb. pressure. c Values in parentheses represent assays on same samples after 8 months' storage ac 0-5'.
..
..
ANALYTICAL EDITION
August 15, 1942 ~
TABLEI. (Coqtinued) (Micrograms per gram of fresh tissue)
Material 31i1k powder (Contd.)
COJn meal
\$-hole wheat bread
Treatment Digebted 24 hours a t 37' with papain Autoclaved 30 minutes with steam ar 15 t o 20 pound pressure Autoclaved, t h e n digested with takadi. astase 24 hours a t 37' Autoclaved, $en digested with papain 24 hours a t 37 .4utolvzed 24 hours a t 37'. natural DH Digesied with takadiastase 3 hours,-then with papain 3 hours a t 3 i c Digested with takadiastas; and papain together, 24 hours a t 37 Autoclaved 30 minutes with steam a t 15pound pressure Autoclaved, then digested with takadiastase 12 hours, then with papain 12 hours a t 37' .4utoclaved, then digested,with tak,adiastase 3 hours, then with papain 5 hours a t 45' Butoclaved with steam 30 minutes a t 15pound pressure Autoclaved, then digested with takadiastase 3 hours; then with papain 5 hours a t 45-47 Autoclaved, then digested with takadiastase 12 hours, then with papain 12 hours a t 37' Digested 24 hours takadiastase$t 37' Digested 24 hours clarase a t 37 Digested 24 hours papain a t 37' Digested 24 hours pepsin a t 37'0 Digested 24 hours trypsin a t 37'" Digested 3 hours takaiiastase, then papain 5 hours a t 45-4; .4utolyzed 24 hours a t 37 Autolyzed 24 hours a t 3 i 0 Autoclaved, then digested with takadiastase 3 hours, then with papain 5 hours a t 45-47' Autolyzed 24 hours a t 37' hutoclaved 30 minutes with steam a t 15pound pressure rlutoclaved then digested with takadiastase 3 'hours, then with papain 5 hours a t 45-47'
pH 5.0
+ ++ + ++
K L i t e flour
Brewer's yeast, diro --i
a
Sicotinic Acid Content 7.1
Relative Yield, Maximum 100%
93
6.2
6.2
82
4.5
7.2
95
5 0 , . .
7.2 5.6
95 85
4.i
4.5
68
4.7
5.7
66
...
5.9
89
4.7
6.1
92
4.7
6.6
100
Satutal
24
57
4.7
42
100
4.i
42 37 39 37 35 37
100
4 5 4 5 5 0 1 5 6.5 4 7 Natural Satural 4.7 Natural
93 88 83 88
43 23 4 3
51
8.5
100
490
100
490
100
490
100
. I .
4.7
88
p H adjusted t o approximately 5.5 before steaming.
OF TRIGONELLINE TO NICOTINIC ACIDCNDER TABLE111. CONVERSIOX VARYINGCONDITIONS WITH SODIEM HYDROXIDE
Weight
-
Con c e n tration of NaOH
07
Treatment
Other Reagent
Autoclaved 30 minutes a t 15-pound pressure
.MQ. Sone (xH4)2S04, 100
s 2
Total Volume
.W. 40 25
5 21 12.5 12.5 12.5 12.5 12.5 12.5 -'71) -,
Autoclaved 30 minutes in stoppered flask
3
ti
Heated 2 hours a t 75' Heated 2 hours a t 75'
Heated 45 minutes a t 7 5 O in stoppered flask
("4)&06, 50 Urea, 50 Urea, 100 Asparagine, 50 Glutamine, 50 @-Alanine,50 Urea, 50 (KHdnSOa, 50 Urea, 50, Asparagine, 50 a-Alanine, 50 ,%Alanine, 50 (NH32SO4, 300 b r e a , 300 Asparagine, 300 @-Alanine,300 Acetamide, 300 Glutamipe, 300 Urotropine, 300
12.5 12.5 12.5 12.5 12.5 12.5 2.5 2,5 2.5 2.5 2.5 2.5 2.5
Trigonelline MicroQrams 4000 1000 500
Theoretical Conversion
Per Cent 0.11 2.4 3.4
200
4.0
500 500 500
2.8 2 5 3.1 6.4 1.2 18
500 500
500
500 500 500 500 500 500
200 200 200 200 200 200 200
0.6 8.3 5.3 34 4.4 33 42 52 52 69 51 10
2 Y/ Q.
rqutvalent of nicotinic acid
2
Autoclaved 30 minutes at 15-pound pressure None
Asparagine, 100 @-Alanine,50 Asparagine @-Alanine
10 10
..
0 0 0 0
