VITAMIN ASSAY
Microbiological and Chick Assay of Vitamin B,, Activity in Feed Supplements and Other Natural Products
WILLIAM 1. WILLIAMS, ARTHUR V. STIFFEY, and T. H. JUKES Nutrition and Physiology Section, Research Division, American Cyanamid Co., Pearl River, N. Y.
Recent modifications and improvements of the Ochromonas and E. coli methods, as well as the details of a chick growth method for determining vitamin Bn, are presented. On the basis of comparative determinations of vitomin BI2 activity in 40 samples, it is concluded that pseudocobalamins-i.e., substances which promote the growth of certain microorganisms but not the growth of animals-do not occur commonly to a large extent in vitamin Bla feed supplements. The USP L. leichmannii method will give reliable results with most vitamin Bl2 feed supplements, but it is advisable to make at least one test with both the Ochromonas and chick methods. Certain fermentation materials contain substances which are inhibitory to chick growth, so that the chick assay method cannot be used in such cases.
T
HE
ISOLATION
OF A CRYSTALLINE
of the anti-pernicious anemia factor from liver extracts (25, 28) led to the proposal (25) that the name “vitamin B12)’ be given to the substance which had been crystallized and which, like concentrated liver extract (3, 7), bad “animalprotein factor” or “factor X” activity for chicks and rats (22). T h e substance also promoted the growth of suitable microbiological test organisms such as L. laclis Dorner (26), L. leichmannzi (16, 37), and Euglena gracilis (78). The simplification brought to the field by the new nomenclaturewas short-lived, for other compounds with equal bialogical activity were shown to be present in liver extracts and other natural materials (24). A fresh attempt to rationalize the terminology was made with the proposal that the compounds be termed “cobalamins,” and it was suggested that the original “vitamin BIZ’’ he named “cyanocobalamin” in recognition of the presence of the coordinate cyano group which characterized its molecule (2). New complexities were soon to appear; a number of other cobalt-containing pigments were shown to be present in natural materials and to differ from the cobalamins in lacking the 5,Gdimethylbenzimidazole group. These other pigments, which may be termed “pseudacobalamins,” have successfully eluded a uniform system of classified namenclature. T h e first to be partially characterized was “pseudovitamin BlS” (8, 23), which contained adenine instead of 5,6dimethylbenzimidazole. The pseudocobalamins are characteristically inactive FORM
364
for animals and exhibit varying degrees of biological activity in microbiological assays. Coates and others (4-5) have reported that vitamin Blz activities of gut contents and feces as determined by E. coli and Euglena gracilis were higher than those found by L. leichmannii, while assays with chicks gave lower results than any of the microbiological test: Ford, Kon, and Porter ( 7 7 ) showed the existence of two pseudacobalamins which they named “factors A and B.” Factor B is evidently the pigment which may be obtained by removing the rihatide portionof the vitamin B,s molecule by acid hydrolysis (73). Ford and Porter in furtherchromatographic studies found and named factor C (72). All three cornpounds possessed full activity for E . coli and various
Figure 1.
AGRICULTURAL AND FOOD CHEMISTRY
degrees of activity far L. Ieichmannii and Euglena gracilis, but were inactive for chick growth. Sjostrom (27) has reported that half the vitamin BISactivity in anaerobically digested municipal sewage sludge is due to factor A, pseudovitamin Bu, factor B, facio? C, and factor CP. The need for a n assay method specific for animal activity is obvious, in view of the larg-e amounts (15) of vitamin B I ac~ tivity currently used for various nutritional purposes. Hamilton, Hutner, and Provasoli (74) have shown that certain chrysomonads require vitamin BIZ. O n e culture, Poteriochrornonos stipitalo, was studied as an assay organism by Barber and others ( I ) , who found that fermentation materials showed much lower vitamin BU content by this method than
Pad p l a t e assay of vitamin 812using E. with a fexv exceptions, fair agreement was found between the U S P a n d the Ochromonas methods. T h e sewage sludge concen.4 typical standard curve is shown in trates, although below the claimed Figure 2. potency, showed similar vitamin BIZ conT h e chick assay was based on the tent by the U S P and the Ochromonas method described by Jukes a n d Williams methods. This differs from Sjostrom's (79). T h e breeding flock a t 6 months of (27) observation that about half the age \vas placed on basal diet I (Table vitamin BIZ activity of certain se\vage 11) a n d a t least 2 weeks elapsed before sludges is due to pseudocobalamins. eggs were used for hatching. Chicks Lillie and others (27) obtained vitamin hatched from these eggs were placed in BIZ values for t\vo sewage sludge prepelectrically heated battery brooders with arations ivhich \cere similar by chick s; 4-inch mesh \?ire floors in a n air-condiand microbiological assay. a n d higher tioned room a t 28' C. a n d fed diet I1 microbiological values with three other (Table 11) supplemented tvith the varisludge preparations apparently due to ous levels of cyanocobalamin, Duplicate their pseudocobalamin content. T h e groups of 12 chicks were used for each difference may reflect the widely varied level of assay. T h e assay period was 28 microbial action which takes place in the days and the average weights a t each processing of this material. level were used in constructing a standard T h e values for samples 36 through 40 response curve to cyanocobalamin, as ilare the 1954 .40AC collaborative study lustrated in Figure 3. values obtained in this laboratory. Sample 36 contained 0.5 y of pseudoResults and Discussion vitamin B I ? per ml. and 0.5 y of cyanocobalamin per ml. T h e USP and the E. T h e vitamin BI:! values obtained by the coli methods gave values very close to 1 y four methods are shown in Table 111. per ml., Lvhile the Ochromonas method deT h e values are averages of t\vo to four termined only the cobalamin. determinations, which agreed within T h e values obtained by the chick 10Tc for the microbiological methods growth method in general agree \vith a n d \yithin 30% for the chick method. those obtained with the Ochromonas T h e chick assay values were in general method. Samples 14, 15, 16. and 33 the lo\vest a n d the E. coli pad plate values were inhibitory to chick groivth. The \yere the highest. I n samples 1 through vitamin B,? content given in the table for these four samFigure 2. Typical standard curve obtained in Ochromonas ples \vas calculated method for vitamin BIZ Response of Ochromonor molhomenrir to vitamin B m determined with Coleman Model 1 1 A spectrophotometer
from the lowest amount of the sample which promoted growth. T h e nature of the response made it impossible to assess the biological value accurately. Higher amounts of these samples added to the chick diet gave less growth than this lowest level of sample. If antibiotics \\;ere omitted from the chick assay diet. samples containing antibiotics gave a higher apparent vitamin B1? content. T h e addition of a n antibiotic to the chick assay diet appeared to eliminate this effect. Penicillin was used in preference to chlortetracycline, because the latter antibiotic is prepared from a fermentation which contains vitamin BIZ. Lillie and others (27) compared vitamin B19 assay values obtained by the U S P L. leichmannii and chick groivth methods. Their chick growth method gave vitamin BI? values for commercial feed supplements. liver products, and fish solubles Ivhich ivere higher than the microbiological values. However? the basal chick diet \vas not supplemented \vith B vitamins a n d a n antibiotic, as was the case in the present investigation.
Litercrture Cited (1) Barber, F. LV., Baile, D. L., Troescher, C. B., Huhtanen. C. N.. Ann. AV, I-. dcad. Sci. 56, 863 (1953). ( 2 ) Brink, N. G., Kuehl, F. A , , Jr.. Folkers, K.! Science 112, 354 ( 1950). ( 3 ) Cary, C. A.: Hartman, A. M.. U. S. Dept. -4gr. Yearbook Agr. 1943-1947, p. 783. (4) Coates, h l . E.: Ford, J. E., Harrison, G. F., Kon, S. K., Porter. J. \V. G., Biochem. J . 52, vi ( 1952). (5) Coates, M. E.? Ford, J. E., Harrison, G. F., Kon, s. K., Porter, J. M:. G.. Brit. J . ,Yutrition 7 , 319 (1953);. (6) Coates, M. E., Ford, J. E., Harrison, G. F.: Kon, s. K.: Porter,
Absorbance
Figure 3. Average weights of groups of 24 chicks at 28 days in response to four amounts of vitamin 812
225
0.0
366
0.05
0.io
0.20 my/tube
0.40
I
0.8
AGRICULTURAL A N D F O O D CHEMISTRY
y Crys. B,r/Kg. Diet
Table 111.
Comparison of Vitamin BLz Assay Values by Four Different Methods Assay Resulfs, M g . Vifamin Bir/lb.
Sampfe No.
1
Type o f Product
Bacterial fermentation Bacterial fermentation
2 3 4 3
6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Bacterial fermentation Bacterial fermentation Bacterial fermentation Bacterial fermentation Bacterial fermentation Bacterial fermentation Bacterial fermentation Bacterial fernlentation Bacterial fermentation Bacterial fermentation Pseudovitamin BI? fermentation Streptomyces fermentation Streptomyces fcrrnentation Str@ptomyces fcrmentation Sfreptomyer fermentation Streptoniyes fcrrnentation Streptomyes fcrrnentation
Streptomyes fcrrnentation Sfreptomyes ftrmentation Streptomjces fermentation SfrefltomycPs fermentation Streptomyes fermentation Streptonyes fermentation Streptomyces fermentation Streptomyes fermentation Streptomyces fermentation Streptomyces fermentation StreptomyPs fermentation Streptomyces fermentation Concentrates from sludge Concentrates from sludge Concentrates from sludge Concentrates from sludge
Stated Content
60 60 70 70 60 80 120 80 399 454 50 3.33a 100 20 19 16 16 12 12
12 12 0.45 0.45 0.45 0.45
...
19 19 16 17 y
36 37 38 39 40 a
Vitamin Blz ,and pseudovitamin B12 solution Vitamin Bl? concentrate Vitamin B1z feed supplement Fish solubles Dried liver
USP 1 . feichmannii
29
...
45 41 32 54 96 110 226 499 43.2 2 .?5a 45 14 12 14 14 12 10
12 10 0.27 0.36 0.52 0.56 17 13 18 12 14
E. coli pad plafe
Chick mefhod
Ochromonas
81
45
31
99
88 59
50
45 43 82 95 86 238 544
44
82 76 73 98 155 140
... ... ... ..
64 126 ... ...
40
2.13~ 0
78 23 23 18 17 12 12 30 8 8 1.7 7 12 13 ...
. .
23 12 14
6 (toxic) 0 .5. (toxic)
4 (toxic)
10
13 13 17 4
, . .
... , . .
... 21 13 22
8 9 5
5 10
13 10
12 12
0.10 0.31 0.57 0.47 17 15
16 23 3 (toxic)
15 15
15
18
15
Vitamin BIZper Gram or 141. 1
450 20 0.2 0.9
0.80 450 18.1 0.20 1.3
0.90 454,5 458 0.29 2.6
0.56 335 15.6 0.14 1.1
0 20 190 6 6 0 20
1.3
Mg. of vitamin Bl? per ml.
(7) (8)
(9) (10) (11) (12) (13) (13) (15) (16)
J. W.G.! Cuthbertson. W.F. G., Pegler. H. F.? Biorhem. J . 49, lxviii (1951). Cravens: \Y. \ V , > LlcGibbons, W. H., Halpin, J. G., Poultry Sei. 24, 304 (19451). Dion, H. W., Calkins: D. G.? Pfiffner. J. J., J. A m . Chem. Sac. 74, 1108 (1952). Ford, J. E.: Brit.J . *Vutrition 7 , 299 (1953). Ford, J. E.?Holdsworth, E. S., Kon, S. K.: Biochem. J’. 59, 86 (1955). Ford, J. E., Kon. S. K., Porter, J. W. G.. Ibid., 50, ix (1951). Ibid., 52, viii (1952). Gant, D. E., Smith: E. L., Parker, L. F. J., Ibid., 56, xxxiv (1 954). Hamilton, L. D., Hutner, S. H., Provasoli, L., .halyst 77, 618 (1 952). Hester, A. S., Ward, G. E., Ind. Eng. Chem. 46, 235 (1954). Hoffman. C . E.. Stokstad, E. L. R., Franklin, A . L.. Jukes, T . H., .I. Biol.Chem. 176, 1465 (1948).
(17) Hutner, S. H . ? personal communication, 1954. (18) Hutner, S. H., Provasoli, L., Stokstad, E . L. R., Hoffman, C. E., Brlt. M., Franklin. A. L., Jukes, T. H., Proc. SOC.Expti. Biol. &3 Med. 70, 118 (1949). (19) Jukes, T. H., Williams, W. L., “ T h e Vitamins.” W . H. Sebrell, Jr., and R. S. Harris, eds., vol. I, p. 452, Academic Press, New York. 1954. (20) Krieger, C. H., J . Assoc. Oj%. Agr. Chemists, 35, 726 (1 952). (21) Lillie, R. J., Bird, H. R., Sizemore, J. R., Kellogg. W.L.. Denton. C. A,; Poultr)-&‘ci. 33, 686 (1955): (22) Ott, iY.H., Rickes: E. L., Wood, T. R., J . Biol. Chem. 174, 1047 (1948). (23) Pfiffner, J. J., Calkins: D. G., Peterson, R. C., Bird, 0. D.? McGlohen. V., Stipek, R. \V., Abstracts of Papers, 120th Meeting, ACS, p. 22C, 1951. (24) Pierce, J . V.: Page, A. C.. Jr., Stokstad, E . L. R.: Jukes, T. H.,
(25)
(26)
J . -4m. Cheni. So:. 71, 2952 (1949). Rickes, E. L., Brink, S. G., Koniuszy, F. R.. Wood. T. R.. Folkers, K., Science 107, 396 (1948). Shorb, A t . S., J . Bacteriol. 53, 669 (1947). Sjostrom, ,4. G. 51.. Seujahr, H. Y., Lundin, H.! Acta Chem. Scand. 7, 1036 (1953). Smith, E. L.? A\7uture 161, 638 (1948). U. S. Pharmacopoeia SIV Revision, 4th Supplement. p. 10, Mack Publishing Co.. Easton, Pa., 1952. ivilliarns, W. L.. Esposito. R. G.. Pierce, J. V., Federation Proc. 11, 458 (1952). Wright, L. D., Skeggs. H. R., Huff, J. W., J. Biol. Chem. 175, 475 (1948). %
(27) (28) (29)
(30) (31)
I
Received for reLiew July Id, 7955. .4cceptrd Noaember 4, 7955.
V O L . 4, NO. 4, A P R I L 1 9 5 6
367
