Stability ascorbic

of vitamin acid1
Jacob M.D.

B12 in the

presence

of

Harold L. Newmark, M.S., and Mukund Prabhudesai,

Schemer,

M.S.,

Martin

Marcus,

M.S.,

ABSTRACT own and levels present these involved, and contrary meal Jacob of preparations (J. Am. of daily added studies meals 3) there were

Experiments which Med. food L-ascorbic performed in general values published obtained no deleterious and were 2) the was to their Assoc. intake acid

were were 230:

performed exactly 241, similar 1974), of modest C)

in

two order

independent to check increasing official calculated than acid 29: those showed their vitamin methods. that from on the 645-649,

laboratories, to the meals report that content

each described with

using

their meals

in composition
in

by Herbert increasing B12. The determined of foods Jacob, the and

incubating

(portions

by man) (vitamin with agreement were effect results.

or high and

B3

produced The results higher ascorbic C/in. J.

destruction Vitamin 1) the the vitamin 1976.

of vitamin B12 was for vitamin literature by Herbert B12 content

standardized method. with manyfold of added Am.

Downloaded from ajcn.nutrition.org by guest on March 26, 2014

microbiologically

by radioassay

B12 contents

values

reported

of meals,

Nutr.

Recently, Herbert and Jacob (1) reported that increasing levels of ascorbic acid, added to homogenized meals prior to incubation at 37 C for 30 mm as a laboratory mimic of the gastric environment in man, produced increasing destruction of vitamin B12. This was determined by radioassay against a series of intrinsic factor concentrate standards. From these results they inferred that high doses of ascorbic acid, popularly used as a home remedy against the common cold, destroy substantial amounts of vitamin B12 when ingested with food. If this is true, the timing of supplementary ascorbic acid ingestion would have to be adjusted to a minimum exposure to vitamin B12 in the digestive tract. Review of the Herbert and Jacob paper revealed that the reported vitamin B12 assay values of the meals that were used were considerably lower for the foods involved than were calculated values taken from previously published food composition tables (2-5), which in turn led to concern about the analytical methodology employed in the investigation. In view of these observations which could have influenced the results of Herbert and Jacob, it was decided to reexamine the effect of supplementary ascorbic acid on vitamin B12 in meals with standardized and official extraction procedures, using both microbiological and radioassay methods.
The American Journal of Clinical Nutrition 29: JUNE

Experimental
Microbiological Four (Table sets each 2) vitamin

methods
assays

of the modest (Table I ) and high B12 meals of the composition and preparation previously described (1) were prepared in the Product Development Laboratories, H offm an n- La Roche Inc., and incubated at 37 C for 30 mm with 0, 0.1, 0.25, or 0.5 g L-ascorbic acid (vitamin C). Immediately after this incubation the meals frozen until assayed. The diets directly, 2) after extractions were frozen and kept were assayed either I) by the Association of

Official Analytical Chemists (AOAC) procedure (6), or 3) by the procedure published by the British Analytical Methods Committee (7). The following quantities of AOAC extractant were added to 20 g of the homogenized meals: modest B12, 25 ml; high B12 meal, 300 ml. The meals were then extracted for 10 mm at 15 psi steam pressure (121 C), brought to appropriate volume, centrifuged, the pH of aliquot was adjusted to 6.0 and diluted to assay range. For the British method, 4 ml of a I mg/mI KCN solution and 16 ml water were added to 20 g of homogenized meal; the pH was adjusted to 4.6 to 5.0. After standing for 30 mm with occasional stirring, the pH was readjusted to 4.6 to 5.0 as necessary. The containers were placed in a boiling water bath and were left in the bath for 30 mm after the internal temperature reached 90 C. The meal samples were cooled and brought to a volume of 100 ml. A portion of the samples was centrifuged and an aliquot was adjusted to pH 6.0 and diluted to assay range. All assays for vitamin B13 were run by the AOAC microbiological procedure (6), using Lactobacillus leichmannii. From the Product Development Department, mann-La Roche Inc., Nutley, New Jersey 07110, Department of Pathology, Fordham Hospital, cordia-Fordham Affiliation, Bronx, New York Hoffand the Miseri10458.

1976,

pp.

645-649.

Printed

in U.S.A.

645

646 Radioassays

NEWMARK

ET AL. For the modest B12 content meal, with and without added ascorbic acid, microbiological assays indicate that the AOAC extraction procedure gives somewhat higher results than the British procedure, while the results without extraction are about /2 to / of the total B12 (P < 0.01; Table 4). It is noteworthy that the various levels of added ascorbic acid have significant effect neither on the free vitamin B12 (no extraction after homogenization) content of the meal nor on total vitamin B12 after extraction.
TABLE 2 Composition

radioassay, separate preparations similar to those for the microbiological assays of both the modest B12 content meal and a high B12 content meal were prepared in a Fordham Hospital laboratory. Proportional amounts of freshly prepared ascorbic acid solution were added to 20 g portions of these homogenized meals equivalent to 0. 1, 0.25, and 0.5 g per meal. These preparations, as well as control meal aliquots without ascorbic acid, were incubated for 30 mm at 37 C. After incubation, KCN was added to one set and the pH was adjusted to 4.8. This set was placed in a boiling water bath and timed for 30 mm after the temperature reached 90 C. The pH ofthe other set was adjusted to 4.8 and the samples were kept at room temperature. Both sets were subdiluted with deionized water so that expected values would lie within the linear portion of the standard curve. Four ml of a solution containing 0.6 g of glutamic acid and 2 mg of KCN per 100 ml adjusted to pH 3.3 were added to 1.0-mI sample aliquots. Preliminary assays revealed the presence of high nonspecific binding which was eliminated by heating the sample to 90 C for 2 1 hr. Cyanocobalamin standards that were similarly treated were unaffected by this procedure. Aliquots of the final dilution were used for the saturation analysis which followed. With the exception of extended heating time lander the (8). method was essentially that of Wide and Kil-

For

the used

of high Component

vitamin

B12 meal Amount

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Chicken noodle soup Crackers Grilled beef liver Catsup Whole kernel corn Egg salad Apple Bread Butter Milk

177 ml 6 g 90 g 15 ml 90 g 45 g l75g 25g 5 g 237 ml

Results The major sources of vitamin B12 in the modest meal (Table 1) are cottage cheese and milk, each of which supply about 1 g of vitamin B12 to the meal. In the high B12 meal (Table 2), the major source of the vitamin is liver, with milk adding a small fraction of the total content. A comparison of calculated values and the various assay values of the two meals is given in Table 3. Clearly the results of the microbiological assays and radioassays by standardized extraction procedures employed in this study are in rough agreement with the values calculated from the literature. On the other hand, the results of Herbert and Jacob (1) are only a small fraction of the expected values.
TABLE Composition I of modest Component Cream Cottage Lettuce Canned Crackers Bread Butter Milk of potato cheese peaches soup vitamin B12 meal Amount 177 ml 100 g lOg 360g 6g 25g 5g 237 ml data

TABLE
Calculated

3
and high assay vitamin values for B12 meals modest vitamin

B12 and

Meal

Calculated

Microbiological assay extraction procedure

AOAC

#{149}

Radioassay

British sg/meal

Herbert and Jacob (1)

Modest B12 High B12 Based on

ca 2.0 ca73 values in

2.7 96 Home

2.0 78

1.6 46

0.37 8.9 Research

Economics

Report 36, United States Department 1969, Washington, D.C. Assuming had B12 content similar to 90 g fresh

of Agriculture, 90 g grilled liver liver.

TABLE
Effect vitamin
Ascorbic

4 of extraction B12 content

acid

procedure of modest

on apparent B12 meal

added to meal prior to incubation at 37 C

g

Microbiological

assay

extraction None AOAC

g/meaI

procedure British

Herbert

and Jacob (I)

0
0.1 0.25 0.5 Average in paper. of

0.98
0.90 0.92 0.99 duplicate

2.7 3.1 3.0 3.5 assays.

2.0 2.1 2.2 2.0 Calculated

0.37 0.21 O.07b 0.02 from

STABILITY

OF VITAMIN

B12 IN THE

PRESENCE Discussion

OF ASCORBIC

ACID

647

For the high B12 content meal with and without added ascorbic acid, the results obtamed by microbiological assays follow a similar pattern (Table 5). The data indicate once again that neither the free vitamin B12 levels nor the total B12 levels by AOAC extraction show significant changes after incubation at 37 C for 30 mm with graded levels of added ascorbic acid. The results obtained by the British extraction and microbiological assay indicate that the results for all three levels of ascorbic acid addition do not differ significantly, considering the normal variability of this method of assay. As a further check on the validity of the assay results, extracts of both the high B12 and modest B12 diets prepared by the AOAC procedure were hydrolyzed at pH 12 for 30 mm at 121 C. This procedure is known to destroy vitamin B12 (9, 10) and any microbiological activity remaining after such treatment could be considered as microbiological growth factors for the assay organism that are not cobalamins. Assays of these treated extracts indicated that at best only a small fraction (e.g., less than 20%) of the vitamin B12 activity as measured by L. leichmannii in the diets might be due to nonspecific stimulation of the test organism. Therefore, the microbiological activity reported is essentially vitamin , activity. The radioassays indicate that lower results are obtained in the absence of initial heat and cyanide treatment than when they are used (P < 0.2; Table 6). Hence by another analytical approach, neither set of results for the modest B12 meal or the high B12 meal indicates any loss of vitamin B12 activity in the presence of the added levels of ascorbic acid. TABLE
Effect vitamin

A review of the literature indicated that the results obtained with L. leichmannii for the high B12 and modest B12 meals were in agreement with assessments of the vitamin B12 content as assayed by a variety of methods. The vitamin B12 of the high B12 meal came almost entirely from liver. In this context it had been shown by Kamikubo and Oguni (3) and by Lichtenstein et al. (4) that for beef liver the results of L. leichmannii assays were essentially similar to the results of Ochromonas malhamensis assays, the latter organism also being more specific for cyanocobalamin and hydroxycobalamin than for pseudo cobalamins (1 1). The vitamin B12 of the modest B12 diet came from milk and cottage cheese. In this case Gregory (5) and Lichtenstein et al. (4) had shown that similar results were obtained for milk and cheese, respectively, by both of the above assay organisms. When the vitamin B12 content of the meals as determined by Herbert and Jacob (1) was compared to the amount calculated from literature values of the meal components (2), it was evident that the values reported by these investigators were considerably lower. Thus, the modest meal was one-fifth and the high meal one-eighth of the calculated values. These discrepancies in vitamin B12 content suggested the possibility of inadequate methodology. It is now accepted that a common error in the early years of vitamin B12 research into TABLE 6
on modest incubation B12 and at 37 C high B12

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Radioassays meals after

5
of extraction B12 content procedure of high B1, on apparent meal

Ascorbic acid added g

No added cyanide pg/meal Modest 0.84 1.1 1.4 0.9 High B12

Added cyanide

Herbert and Jacob (I)

Ascorbic acid added to meal prior to incubation at 37 C

Microbiological assay extraction procedure None AOAC pg/meal British

B12 1.6 2.0 1.7 1.9 0.37 0.21#{176} 0.07#{176} 0.02#{176}

Herbe and Jacob (I)

0 0.1 0.25 0.5

g 0 0.1 0.25 0.5

#{176}

37 35 34 37 of duplicate

96 89 83 96 assays.

I
I

78 68 61 60 Calculated

8.9 ca8.5b 6.7 ca5.l from

#{176}

0
0.1 0.25 0.5 Calculated from

32
36 31 31 data in paper.

46 49 62 47

8.9 ca8.5#{176} 6.7#{176} ca5.I#{176}

data

Average in paper.

648

NEWMARK

ET

AL.

the cobalamin content of food was inadequate extraction from the tightly-bound cobalamin forms. Several extensive extraction procedures have become universally accepted for food assays for cobalamin content over the last two decades. However, Herbert and Jacob (1) used a method which was developed for assays of vitamin B12 in serum, where mild extraction appeared to be adequate by the references cited. On the other hand, consulting the now extensive literature reveals that a more extended extraction procedure is required to release the more tightly-bound cobalamins present in many foods (7, 9, 12). In addition, according to Skeggs (9), many discrepancies in assay values determined by radioactive methods are the result of failure to extract the total cobalamin present in the sample and convert it to cyanocobalamin against which it is to be compared. The short extraction time apparently used by Herbert and Jacob (1) was considerably less than that found necessary by Rosenblum (12) for total cobalamin in liver, the recommended extraction time being boiling for 1 hr at pH 4.0 in the presence of sodium nitrite and potassium cyanide. In the present study the vitamin B12 values obtained by standardized extraction and assay methods of both meals were in rough agreement with those calculated from literature values and manyfold higher than those reported by Herbert and Jacob. No significant deleterious effect of supplementary ascorbic acid on the vitamin B12 content of the meals was found, contrary to that indicated by Herbert and Jacob. It is interesting to note that the microbiological assay medium for vitamin B,2 used by both the AOAC (6) and the United States Pharmacopeia (13) contains ascorbic acid added at the level of 4 g/liter. This is about 106 times the level of vitamin B12 at the lowest level of standard used in the microbiological assay. The vitamin B12 and vitamin C are autoclaved together in this media for 5 mm at 121 C as part of the normal test procedure without destruction of vitamin B12. It has long been known that low levels of iron in solution in various ionic or complexed forms will markedly enhance vitamin B12 stability in the presence of ascorbic acid. Thus, Shenoy and Ramasarma (10) found

that the iron content was the stabilizer of vitamin B12 in liver extracts. Commercial pharmaceutical liquid formulations have used low levels of iron salts or complexes to stabilize vitamin B12 against degradation by high concentrations of ascorbic acid for long periods of months or years. Levels of iron as low as 17 ppm in the solution (14) or 12 ppm (15) have been used successfully in such systems. The United States Food and Drug Administration has recognized this use of iron as a vitamin B12 stabilizer in aqueous multivitamin solutions in a Food Additive Regulation (16). Many foods have iron levels in this range. The vitamin B12 in foods, however, unlike that in pharmaceutical preparations, is usually tightly bound. For example, the major part of the cobalamin present in liver is in the form of a coenzyme bound to a liver protein. This binding serves to enhance further the stability of vitamin B12 activity, presumably by reducing accessibility of vitamin B12 binding sites to prolonged chemical attack.

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Summary Herbert and Jacob have reported vitamin B12 values for two specific test meals that are only a small portion (one-fifth to one-eighth) of the levels calculated from literature values of the meal components. Their low results are apparently due to inadequate extraction of the vitamin B12 in the samples during preparation for assay. The vitamin B12 values obtained by officially recognized methods of extraction and assayed by microbiological or radioassay were manyfold higher than those of Herbert and Jacob and were in rough agreement with those calculated from literature values. When performed with standardized and official extraction methods and assayed either microbiologically or by radioassay, there is no significant deleterious effect of added ascorbic acid on vitamin B12 stability in foods when tested in meals and under conditions reported by Herbert and Jacob.
The authors wish to thank Miss Ann Sami Wassef for helping in performing Drs. Myron Brin and Jack Bauernfeind suggestions. Dowell and Mr. these assays and for their helpful

STABILITY References
I.
HERBERT,

OF VITAMIN

B1, IN THE

PRESENCE
8.
WIDE,

OF ASCORBIC

ACID

649

V.,

AND

E.

JACOB.

B1, 2.

by ascorbic

acid.

J. Am.

Destruction Med. Assoc.

of vitamin 230: 241,

1974.
Acid, Vitamin B, and Vitamin B1, in Economics Research Report No. 36, Agricultural Research Service, U.S. Dept. of Agriculture, Washington, D.C., 1969. 3. KAMIKUBO, T., AND Y. OGUNI. Microbiological determination of vitamin B1, with Ochromonas malhamensis. J. Vitaminol. 5: 51, 1959. 4. LICHTENSTEIN, H., A. BELOIAN AND H. REYNOLDS. Comparative vitamin B1, assay of foods of animal origin by Lactobadillus leichmannii and Ochromonas maihamensis. J. Agr. Food Chem. 7: 771, 1959. 5. GREGORY, M. E. The microbiological assay of vitamin B12 in the milk of different animal species. Pantothenic

Foods

Home

L. AND A. KILLANDER. A radiosorbent techfor the assay of serum vitamin B12 Scand. J. Lab. Invest. 27: 151, 1971. 9. SKEGGS, H. R. The Vitamins (2nd ed.) New York: Academic Press, 1967, vol. 7, pp. 277-293. 10. SHENOY, K. G. AND G. B. RAMASARMA. Iron as a stabilizer of vitamin B1, activity in liver extracts and
nique Clin. the nature Biochem. of so-called alkali stable Biophys. 55: 293, 1955. factor. Arch.

11.

BRIGGS,

G. M.
part

AND

F. S.
acid). Ann.

DAFT.

vitamins,

I (Vitamin

B13, folic

Water soluble Acid, choline and Biochem. 24:

para-aminobenzoic 339, 1955. 12. 13.

Rev.

6.

Brit. J. Nutr. 8: 340, 1954. Official Methods of Analysis (12th ed). Washington, D.C.: Association of Official Analytical Chemists, 1975, p. 842. 7. Analytical Methods Committee: The estimation of vitamin B12. Analyst 81: 132, 1956.

14. 15.

ROSENBLUM, C. Analytical applications of radioactive vitamin B12. Talanta II: 255, 1964. The United States Pharmacopeia, 19th Rev. United States Pharmacopeial Convention, Inc., Rockville, Md. 1975, p. 613. NEWMARK, H. L. U.S. Patent 2,823, 167-February II, 1958, Stable vitamin B12-containing solutions. FREEDMAN, EIGEN.

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L., M. US.

BLITZ,

D. B.

SABINE

AND

16.

Patent 2,939,82 I-June vitamin B1, solutions. Food Additives. Federal Register

7, 27:

1960,

E. Stable

883,

1962.

Erratum
In the article Stability of vitamin B12 in the presence Newmark, Jacob Schemer, Martin Marcus, and Mukund 29: 645, 1976, line 8, second paragraph, page 646 should 90C for2hr. of ascorbic acid by Harold L. Prabhudesai, Am. J. Clin. Nutr. read 90 C for I hr instead of

932

The American

Journal

ofClinical

Nutrition

29: AUGUST

1976,

p. 932.

Printed

in U.S.A.