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Agriculture Handbook No. 97

UNITED STATES DEPARTMENT OF AGRICULTURE
ACKNOWLEDGMENTS

Thanks are due Mary E. Wigmore and Victoria

M. Chappell, formerly of the Human Nutrition

Research Branch, Agricultural Research Service,

United States Department of Agriculture, for their

contributions to the standardization and application of the laboratory procedures.

Deep appreciation is expressed to Madelyn Womack, Human Nutrition Research Branch, for the

rat bioassay on yeast, and to L. R. Richardson,

Texas Agricultural Experiment Station, for the rat

bioassays on 5 foods and the microbiological assays

on the 5 foods and yeast.

CONTENTS

Page

Summary 1

Background 1

Part I. Development of microbiological procedure. 2

Stock solutions for medium 2

Assembling the medium 3

Other reagents 3

Reagents for enzyme preparation 4

Enzyme preparation 4

Test organism—its care and inoculum preparation 4

Standard pantothenic acid solutions and standard sample 5

Extraction of pantothenic acid from foods 5

Page

Parti. Development of microbiological procedure—

Continued

Microbiological procedure 6

Investigations to establish the method 7

Results with the established procedure 11

Part II. Quantitative distribution of pantothenic

acid in typical foods 14

Food samples 14

Moisture 15

Fat 15

Detailed data obtained in the study of 237 food

items 16

Literature cited 17

TABLES

Page

1. Composition of basal medium used for assay of

pantothenic acid with L. plantarum 3

2. Pantothenic acid values, reported on a dry, fatfree basis, found in ethyl ether extracted and

nonextracted food samples with and without

double-enzyme treatment 9

3. Influence of pH on the destruction of pantothenic acid during sample extraction 10

4. Recovery of pantothenic acid added to samples

of air-dried ground beef round 10

5. Pantothenic acid values obtained by 2 laboratories by assay with L. plantarum in 5 foods

and yeast, with and without the doubleenzyme treatment 11

Pantothenic acid values obtained by rat bioassay

and by assay with L. plantarum using the

double-enzyme treatment

Pantothenic acid values reported in the literature,

determined by microbiological assay using

the double-enzyme system, and by bioassay

with rats and chicks in comparison with values

found in this study

Pantothenic acid in edible portion of foods, as

determined by microbiological assay with L.

plantarum with and without double-enzyme

extraction

Page

11

13

18

U. S. GOVERNVENT PRINTING OFFICEi 19B6

Issued September 1956

For sale by the Superintendent of Documents, Ü. S. Government Printing Office

Washington 25, D. C. - Price 20 cents
PANTOTHENIC ACI

in foods

By ELIZABETH GATES

H

SUMMARY

A detailed laboratory proceduiT employing the

double-enzyme system of intestinal phosphatase

and pigeon-liver extract for release of pantothenic

acid from foods and subsequent microbiological

assay with Lactobacillus plantarum (formerly Lactobacillus arahinosus) is given along with results

of its appHcation. In a 2-year period this laboratory procedure, applied 104 times to a dried

yeast sample, gave an average value of 111.0 ¡ig.

pantothenic acid/gm. with a standard deviation

of 10.7 and a coefficient of variation of 9.7. Recoveries of pantothenic acid added to samples of

dried beef, taken through the entire assay procedure,

averaged 98.8 percent of the amount added and 99.9

percent of the total amount present. Reliability

was also shown by good agreement between values

obtained by this double-enz3nne microbiological

procedure and those obtained by rat bioassay on

5 widely different foods and yeast.

The results of the laboratory analyses of 208

food samples, representing 161 différent foods

using this method, are summarized (see table 8).

Analyses were always made on edible portions

prepared from samples purchased in the retail

market. In a few instances, processed foods were

analyzed, but usually the assays were made only

on raw products.

Lean muscle meat and poultry generally tended

to be higher in pantothenic acid content than

fruits and vegetables on a fresh-weight, edibleportion basis, although individual items in each

class were found to overlap. Shell eggs, especially

the yolks, were higher in pantothenic acid than

meat. Most ready-to-eat cereals, whole-grain

breads, and nuts (including peanuts) had about

the same pantothenic acid content as meat.

Fluid milk had about half the pantothenic acid

found in meat.

BACKGROUND

Pantothenic acid, one of the B-vitamins, is of

known nutritional importance because of its role

in biochemical reactions as a part of coenzyme A,

a mechanism only recently understood. Pantothenic acid has been demonstrated as a dietary

ZooK, MARILYN J. MACARTHUR, AND EDWARD W. TOEPFER

uman Nutrition Research Branch, Agricultural Research Service

requh-ement for growth of a number of animals

and different micro-organisms.

In 1933, R. J. Williams and coworkers {2Sy

reported an acid-yeast-growth factor and named

the factor pantothenic acid. Seven years later

the vitamin had been isolated, the chemical

structure had been confhmed, and pantothenic

acid was S3nithesized. Pantothenic acid is 2,4-

dihydroxy-3,3-dimethylbutyryl-beta-alanide, and

this dipeptide structure accounts for its instability

to acid and alkali. Pantothenic acid has from the

earliest studies been known to occur to some

extent in all tissues or foods tested.

In early studies it was found that bioassays for

pantothenic acid in natural materials employing

rats or chicks generally gave higher results than

microbiological assays on extracts of the materials

treated with mylase-P, Takadiastase or papain

(4, 5, 6, 10, 12, 18, 22), Microbiological assays

using different test organisms also had been reported to give divergent results. These differences

were subsequently found to be due to the utilization by the animals or microorganisms of a bound

form or forms of the vitamin. Three bound forms

of pantothenic acid have been found to occur

naturally. While different forms of pantothenic

acid serve as growth factors for various organisms,

only coenzyme A serves as the biochemically

active form of pantothenic acid in acetylation

reactions.

In 1947 Lipmann and coworkers {11) reported

that coenzyme A contained pantothenic acid, since

established as 11 percent of the total coenzyme

weight. Coenzyine A acts as an acetyl group

transfer agent in tissue metabolism. This function

helps to explain the mechanism whereby 2-carbon

fragments resulting from fatty acid breakdown

enter the cycle in which carbohydrate is oxidized to

carbon dioxide and water, by way of pyruvic acid.

Coenzyme A, through an active sulfhydryl group

( —SH), unites with acetate and is the thiol ester

shown to be ^^active acetate" which reacts with

oxalacetate to form citric acid. The sulfhydryl

group is part of the thioethylamine attached by

peptide linkage to the alanine of pantothenic acid.

Not only does coenzyme A take part in many acet1 Italic numbers in parentheses refer to Literature

Cited, p. 17.

1
ylatioiis believed to be essential to the oxidative

metabolism of carbohydrate, fatty acids, and amino

îiads, but also it has a role in steroid synthesis,

through coenzyme A, pantothenic acid becomes

one of the essential activators for the reactions

concerned with cellular oxidation of food materials.

Since the pantothenic acid fraction of coenzyme A

apparently cannot be manufactured by the human

body but must be supplied in food, it is recognized

as an essential nutrient.

Pantothenic acid occurs in foods in both free and

bound forms. Release of the pantothenic acid

molecule in coenzyme A was found to require enzymes other than those that had been used for

liberatmg many vitamins from tissues. Novelli

and coworkers {Uj) found pantothenic acid to be

released quantitatively from coenzyme A only by

a double-enz}Tne system involving intestinal phosphatase and pigeon- or chicken-liver extract.

Another bound form of pantothenic acid—the

pantothenic acid conjugate (PAC) reported by

King and coworkers (5, 9) as active for the growth

of Acetobacter suboxydans—was found to contain

phosphorus and glutamic acid. PAC apparently is

not as easily dial3^zable as coenzyme A, indicating

a greater molecular weight than coenzyme A; and

PAC apparently contains more glutamic acid on a

molecular basis than coenzyme A. In the study,

it was found that intestinal phosphatase and avianliver enz3Tnes liberate pantothenic acid quantitatively from PAC also.

Another form of bound pantothenic acid was reported by W. L. Williams and coworkers (24-) to be

required for the growth of certain strains of Laciobacillus bulgaricus. L. bulgaricus factor (LBF) was

probably formed by the action of phosphatase on

coenzyme A. It was similar to and possibly identical with the compound synthesized by Snell and

coworkers (¿*0), which was pantothenic acid joined

by a peptide linkage to thioethylamine to yield

pantetheine. The double-enzyme system also

splits this peptide linkage to give free pantothenic

acid. LBF was an important compound in that it

furnished a basis for establishing part of the probable structure of coenzyme A.

Colorimetric procedures (2, 16, 17, 21) for the

determination of pantothenic acid in foods depend

on reaction with a hydrolysis product of the vitamin, either beta-alanine {2, 16,21) or pantolactone

{17). They are restricted to relatively pure preparations of pantothenic acid in a fair concentration

and have only limited application to extracts of

foods and other natural materials {16).

Application of the double-enzyme system of

Novelli and coworkers {1^) to foods was made

by Neilands and Strong {13). Thus, for the first

time, microbiological assays on foods gave results

equivalent to bioassay values. NoveUi and

Schmetz {15) improved the method, permitting

the determination of pantothenic acid in lowpotency material. With modification and standardization of the above procedures in the Human

Nutrition Research Branch laboratories. Agricultural Research Service, United States Department of Agriculture, it became possible to obtain

reliable data on the pantothenic acid content of a

large number of foods.

The results reported here were on studies to

standardize the extraction and microbiological

procedures, to apply them systematically in the

assay of common foods, and to report the quantitative distribution of total pantothenic acid

among many typical foods which make up the

American diet. Foods were also analyzed for

their free pantothenic acid content, so that a

comparison could be made with data found in

literatiu-e reporting results obtained by older

extraction methods which usually residted in

values only slightly higher than the free pantothenic acid content.

PART I. DEVELOPMENT OF MICROBIOLOGICAL PROCEDURE

Studies on the method for the microbiological

assay for pantothenic acid and its comparison

with rat bioassay have been reported earlier

{22). The microbiological assay method is being

given in suflBcient detail in this handbook for

direct use by the average laboratory worker.

The conditions and times of storage of the following reagents, enzymes, and test organisms

have been found to give reproducible standard

curves and media free of contamination. These

concentrations and amounts have been found

convenient in this laboratory where the number of foods being assayed was large. Smaller

amounts of stock solutions would probably be

more practical in a laboratory where only a few

analyses were contemplated.

The mention in this publication of a commercial company or of any commercial products

does not imply endorsement by the United States

Department of Agriculture over other companies

or products not named.

STOCK SOLUTIONS FOR MEDIUM

Acid-hydrolyzed casein.—100 gm. of vitamin-free

casein were mixed with 500 ml. 5 N HCl and refluxed 8 hours. The HCl was removed from the

mixture by distillation under reduced pressure until a thick paste remained. The paste was dissolved in distilled water and concentrated again in

the same manner. The resulting paste was redissolved in distilled water, adjusted to pH 3

(indicator paper) with concentrated NaOH, and

sufficient water added to bring the volume to approximately 600 ml. ; 40 gm. of Darco G-60 were

added to the solution which was stirred 2 to 4

hours, and then filtered with suction through a

pad of filter-eel.

The treatment was repeated using 10 gm. Darco

for 1 hoiu* if the filtrate was not colorless. Sufficient distilled water was added to bring the volume

to 1 liter and the solution was stored under toluene

at 5° C.
1 ml.OlOO mg. of hydrolyzed casein.

Tryptophan solution.—5 gm. of DL tryptophan

were dissolved in 25 to 30 ml. of 1 N HCl, and

sufficient water was added to make 250 ml. Solutions were stored up to 1 week under toluene at

5°C.

1 ml.OlO mg. of L tryptophan.

Adenine^ guanine, uracil solution.—200 mg. each

of adenine sulfate, guanine hydrochloride, and uracil were dissolved with the aid of heat in 10 ml. of

20 percent HCl, and sufficient distilled water was

added to make 1 liter. The solution was stored

under toluene at 5° C.

1 ml.O200 Mg. each of adenine sulfate, guanme

hydrochloride, and uracil.

Xanthine solution.—400 mg. of xanthine were

dissolved in 20 ml. of concentrated NH4OH with

heat, and distilled water was added to make 1 liter.

The solution was stored under toluene at 5° C.

1 ml.=O=400 iJLg. of xanthine.

Vitamin-mixture solution.—40 mg. of riboflavin

were suspended in 500 ml. of 0.02 M CH3COOH,

and the solution was protected from the light.

40 mg. of thiamine hydrochloride, 40 mg. of nicotinic acid, and 80 mg. of p^^idoxine Iwdrochloride

were dissolved in about 200 ml. of distilled water

in a liter volumetric flask. 10 mg. of p-aminobenzoic acid were dissolved in distilled water

and made to 100-ml. volimie. 40 ml. of this pamino-benzoic acid solution were pipetted into the

liter volumetric flask; biotin to furnish 100 ng. and

the riboflavin solution were added quantitatively.

Distilled water was added to volume, and the mLxture was stored in a dark glass-stoppered bottle

under toluene at 5° C.

'40 /xg. each of riboflavin, thiamine

hydrochloride, and nicotinic acid;

1 ml.OsSO Mg- of pyridoxine hydrochloride;

4 Mg. of ^-amino-benzoic acid;

.0.1 Mg. of biotin.

Salts solution A.—10 gm. of KH2PO4 and 10 gni.

of K2HPO4 were dissolved in distilled water, diluted to 100 ml., and stored under toluene at room

temperature. This solution was made fresh

weekly.

1 ml.OlOO mg. each of KH2PO4 and K2HPO4.

Salts solution B.—20 gm. of }.igS04.7H20, 1

gm. of NaCl, 1 gm. of FeS04.7H20, and 750 mg.

of MnS04.H20 were dissolved in distiUed water;

1 ml. of concentrated HCl was added; the whole

was diluted to 500 ml., and stored under toluene

at room temperature.

(40 mg. of MgS04.7H20;

1 ml.O 2 mg. each of NaCl and FeS04.7H20;

(1.5 mg. of MnS04.H20.

''Tween 80'' solution.—2.5 gm. of polyoxyethylene sorbitan monooleate ^ were dissolved in warm

2 "Tween 80," Atlas Powder Co., Wilmington, Delaware.

distilled water (45° C), diluted to 500 ml, and

stored under toluene at 5° C.

1 ml.05 mg. of polyoxyethylene sorbitan

monooleate.

Table 1 lists the ingredients used in the basal

medium and gives the concentration in 2 ways—

the amount of each ingredient per tube, and the

amount of each solution or weight of solid for 1

hter of double-strength medium, as prepared and

used in these investigations.

ASSEMBLING THE MEDIUM

Distilled water to approximately half of the

expected volume was placed in the container for

the medium, and the liquids were added without

warming, in the order shown (table 1). The solids

were then added and dissolved with a minimum

of stirring. The pH was adjusted to 6.8 with

HCl or XaOH, with the use of a pH meter. The

medium was made to volume, layered with toluene,

and returned to the refrigerator until time to put

it into the assay tubes.

TABLE 1.—Composition of basal medium used for

assay oj pantothenic acid with L. plantarum

Reagent

Acid - hydrolyzed

casein.

DL tryptophan

A d e n i n e, g u a n i n e,

uracil.

Xanthine

Vitamin mixture

Salts A

Salts B

''Tween 80"

Dextrose, anhydrous.

Sodium acetate-3H20

L cj^stine ^

Concentration of

solid per assay

tube

50 mg

2mg

50 /xg. each adenine,

guanine, and uracil.

100 Mg

20 Mg. pyridoxine;

10 Mg. each thiamine,

riboflavin, niacin;

1 Mg. 7>aba;

0.025 Mg. biotin.

5 mg. each KH2PO4

and K2HPO4.

2 mg. MgS04-7H20;

100 Mg. each NaCl

andFeS04-7H20;

75 Mg. MnS04-H20.

500 Mg

200 mg

83 mg

1 mg

Amount

for 1 liter

of doublestrength

medium

100 ml.

20 ml.

50 ml.

50 ml.

50 ml.

10 ml.

10 ml.

20 ml.

40 gm.

16.6 gm.

200 mg.

1 Dissolve in about 5 ml. of 10 percent HCl before

adding to the medium.

OTHER REAGENTS

The following reagents are needed for either the

microbiological-assay procedure or the extraction

of the pantothenic acid from the foods.

Physiological salt solution.—9 gm. of NaCl were

dissolved in water and diluted to 1 liter. Sterile

salt tubes were prepared by placing 10 ml. portions

in test tubes, plugging with absorbent cotton, and

autoclaving for 15 minutes at 15 pounds of steam

pressure.
Bromthymol blue indicator saturated solution.—

1 gm. of bromthymol blue was suspended in 16.5

ml. of 0.1 N NaOH, and the volume was brought to

250 ml. with distilled water. This was discarded

after standing if the solution was not still green.

Solubilized liver extract.—5 gm. of Bacto liver

were suspended in 100 ml. of distilled water. The

mixture was held at 50° C. for 1 hour and at 80° C.

for 5 minutes, filtered, and the filtrate stored under

toluene in a glass-stoppered bottle at 5° C.

''Tris'' hufer—1 M, pH8.3.—24.2 gm. 2-amino2 - (hydroxymethyl) -1,3 -propanediol [(CH20H)3-

CNH2]^ were dissolved in distilled water and diluted to 200 ml., the pH adjusted with a pH meter

to 8.3 with 30 percent HCl, the solution filtered

through Whatman No. 40 filter paper into a glassstoppered bottle, and stored at room temperature.

This solution was discarded when more than 2

weeks old.

^;rm" hufer—1 M, pH 11.3.—24.2 gm. 2-

amino-2 - (hydroxymethyl) -1,3 - propanediol were

dissolved in distilled water, diluted to 200 ml.,

filtered, and stored in a glass-stoppered bottle at

room temperature.

Sodium bicarbonate bußer—0.1 M, pH 8.5.—850

mg. NaHCOa were dissolved in distilled water and

made to 100-ml. volume. This solution was prepared fresh daily.

REAGENTS FOR ENZYME PREPARATION

The following reagents were used in the preparation of enz}Tnes for this determination.

Potassium bicarbonate—0.02 AI.—1 gm. KHCO3

was dissolved in ice-cold distilled water, diluted to

500 ml., and stored in the refrigerator until used.

This solution was prepared fresh each time it was

used.

Activated Dowex.—100 gm. of Dowex-1 (200-

400 mesh) were stirred by means of a glass stirrer

with suflficient power to keep the Dowex completely suspended, in a liter of 1 N HCl for 10

minutes, then iiltefed through Whatman No. 50

filter paper on a Büchner funnel by means of suction. The residue was treated in the same manner

with a second liter of N HCl for another 10 minutes, and then filtered. The residue was washed

with a liter of distilled water, being stirred and

filtered under identical conditions. The washings

were repeated 10 times. The acid-treated washed

Dowex was wetted suflBciently with water to mLx

well, and '^tris" buffer pH 8.3 was added drop by

drop until the pH was 8.0. The slurry was stored

in a refrigerator for use within about 2 days.

ENZYME PREPARATION

Intestinal-phosphxitase solution—2 percent.—A

water extract of the alkaline intestinal-phosphatase preparation (Armour and Company) was

prepared fresh daily, 5 ml. being allowed for

each 4 ml. to be used.

3 P-4833—Eastman Chemical Products, Inc.—tris (hydroxymethyl) aminomethane.

Pigeon-liver extract—10 percent.—All equipment

used, including centrifuge tubes and cups, mortar

and pestle, and pipettes, were cooled overnight in

the freezing compartment of a home type refrigerator. As the acetone-dried pigeon liver received

from Armour had a higli percentage of feathers

and connective tissue, it was sifted through a

50-mesh Monel-metal sieve to concentrate the

liver. After being sifted, the liver was weighed

into 10-gm. lots, placed in small bottles, and

returned to tlio freezer. For the extraction, 10

gm. were rubbed to a paste with about half of the

100 ml. ice-cold 0.02 M KHCO3 in a mortar and

pestle held in an ice-salt bath. The suspension

was transferred as quantitatively as possible to

ice-cold centrifuge tubes with the assistance of the

remainder of the KHCO3.

The contents of each centrifuge tube were

thoroughly stirred with a small glass rod, placed

in balanced centrifuge cups, and chilled in the

freezer compartment for 10 minutes. Centrifugation at 3,000 revolutions per minute (r. p. m.) for

5 minutes was followed by a cooling period of 10

minutes in the freezer. The supernatant fluid was

transferred to a cold 125-ml. Erlenmeyer flask,

about half the 100 grams of activated Dowex

were poured in, and the flask was shaken for 5

minutes in the ice-salt bath.

The mixture was then transferred to balanced

cold centrifuge tubes, centrifuged for 5 minutes

at 3,000 r. p. m., and returned to the freezer for

a 10-minute chilling.

The supernatant fluid was treated again in the

same manner with the remainder of the activated

Dowex; and after being chilled, it was poured into

cold centrifuge tubes and centrifuged for 5 minutes,

then cooled for 10 minutes. The supernatant

fluid was then transferred to balanced cold centrifuge tubes, centrifuged at 3,000 r. p. m. for 5

minutes, returned to the freezer for a 10-minute

chill, and then pipetted into small, sterile, cold

tubes in amounts ranging from 2 to 5 ml., depending upon the expected need. The small tubes

were then frozen in the refrigerator and thawed

just prior to use.

TEST ORGANISM—ITS CARE AND INOC

ULUM PREPARATION

The following reagents were used in maintaining

the culture.

Test organism..—Lactobacillus plantarum (formerly Lactobacillus arabinosus 17-5), culture

No. 8014, obtained from the American Type

Culture Collection, 2029 M Street NW., Washington 6, D. C, was used for these assays.

Agar culture medium.—5 gm. Bacto yeast

extract, 5 gm. dextrose, and 7.5 gm. agar, were

made up to 500 ml. with distilled water. The

agar was dissolved by steaming for 10 minutes.

The hot solution was tubed in 10-ml. amounts;

the tubes were then plugged with cotton and

autoclaved 15 minutes at 15 pounds pressure.
If refrigerated, the sterile-agar tubes were usable

for about 6 months; if kept at room temperature,

about 2 months.

Liquid culture medium.—Broth-culture-medium

tubes were prepared by placing 5 ml. basal

medium (see table 1), 5 ml. distilled water, and 1

drop of solubilized liver extract into lipless test

tubes. These tubes were plugged with cotton

and autoclaved 15 minutes at 15 pounds pressure.

The sterile-broth tubes were kept at room temperature, and fresh tubes were prepared every 2 to

3 weeks.

Stock culture of L. plantarum.—The pure culture

of L. plantarum was maintained by monthly

transfers from agar through broth tubes for 24

hours and back to agar. The broth tubes were

incubated in a water bath held at 34° C. for 24

hours, and agar tubes inoculated from these

rapidly growing cultures were incubated for 18

to 24 hours, and stored in the refrigerator. At

biweeldy intervals the inoculum was started from

these agar tubes.

Broth inoculum.—Cells of L. plantarum to be

used for inoculum were maintained by daily transfer from broth to broth. Cells were never used for

inoculum unless they had been transferred twice

through broth. For use as inoculum, the cells were

separated from the broth by centrifuging for 15

minutes at 2,500 r. p. m.; the liquid was then decanted, and the cells were resuspended in sterile

saline and centrifuged for 10 minutes; the wash was

repeated once, and the cells were resuspended in a

third portion of sterile saline.

STANDARD PANTOTHENIC ACID SOLUTIONS AND STANDARD SAMPLE

Stock A.—^Approximately 45 mg. of referencestandard calcium pantothenate,* dried under

vacuum over P2O5, were weighed on a microbalance, transferred to a liter volumetric flask, and

dissolved in about 500 ml. distilled water. 100 ml.

0.2 N sodium acetate and 10 ml. 0.2 N acetic acid

were added, and the whole was brought to volume

with distilled water. Using the factor—

1.087 fig. calciimoL pantothenate= 1.000 fig.

pantothenic acid,

the concentration of pantothenic acid per ml. was

determined. This solution was stored in the refrigerator, and was prepared fresh each year.

Stock B.—4-fig. pantothenic acid/ml.—A portion

of stock A pantothenic acid solution was taken to

give 1 liter of a solution containing exactly 4.0 fxg.

pantothenic acid per ml. To this portion, 90 nil.

0.2 N sodium acetate and 9 ml. 0.2 N acetic acid

were added, and the whole was diluted to volume

with distilled water. This was prepared every S

months and was also kept refrigerated. Working

standards were prepared from this stock.

* Obtained from U. S. P. Reference Standards, 46 Park

Ave., New York 16, N. Y.

Working standards.—The 3 working-standard dilutions were prepared fresh for each assay. For

the 0.1-/xg-per-ml. pantothenic acid standard, 5 ml.

stock B (4.0-/xg. pantothenic acid per ml.) was diluted to 200 ml. with distilled water. For the 0.02-

/ig-per-ml. pantothenic acid standard, a second 5-

ml. portion of stock B pantothenic acid standard

was diluted to 1,000 ml. with distilled water. The

0.01-Mg. pantothenic acid per ml. standard was

prepared by diluting 25 ml. of the 0.1 /xg. standard

to 250 ml. with distilled water.

Standard reference sample.—A quantity of moist

brewer's yeast ^ was prepared for a standard sample in the laboratories of the Human Nutrition

Research Branch. The wet yeast was separated

by filtration with suction on a large Büchner funnel, dried in a vacuum desiccator over CaCl2,

ground through a 60-mesh screen with a Wiley

mill, and stored in a glass bottle in the refrigerator.

EXTRACTION OF PANTOTHENIC ACID

FROM FOODS

Sample extractions.—The edible portion of most

food samples was divided into 6 representative

parts, sealed in No. 2 sanitary enameled cans, and

stored until used at —40° F. For analysis, the

cans of meats, poultry, nuts, cheese, fresh fruits,

and vegetables, and also the dried fruits, were

opened as soon as they were removed from the

freezer. The entire contents of each can of frozen

food was put through a home electric food chopper

with a plate having openings of )Í6-inch diameter,

or ^á2-inch if impossible to use the finer plate.

From 4 to 100 gm. of the finely ground, unthawed

food was weighed immediately on a small torsion

balance, and transferred quantitatively to a

Waring Blendor cup containing 10 ml. ''tris"

buffer pH 8.3. Sufficient distilled water was

added to obtain a good suspension while the total

volume was kept below 150 ml. About 1.5 ml.

of caprylic alcohol was added, to prevent foaming

in the Blendor and bumping in the autoclave.

The whole was blended for 3 to 5 mmutes and

then transferred quantitatively, with the aid of a

funnel and an 8-inch swab stick, to a 200-ml.

volumetric flask, the volume bemg kept below 190

ml. The flasks were plugged with cotton, autoclaved at 15 pounds pressure for 15 minutes, and

cooled to room temperature. The samples were

then brought to volume and shaken vigorously.

For many samples, a second Blendor treatment

lasting about 1 minute was necessary at this

time, to obtam a suspension that could be pipetted

accurately. . ,

An enth-e subsample of cereals, grains, wholewheat flour, or dried vegetables, was put thro^^gû

a 60-mesh screen in an intermediate model Wüey

mill. Amounts of 10 to 25 gm. of this finely

divided sample were then treated m exactly the

same way as the moist samples.

5 Obtained from Christian Henrich Brewing Co., Washington, D. C.
Breads and certain dried foods had to l)e

handled by a shc^ht modification of tlie above

procedure. For these foods, the finely ground

samples were put directly into 500-ml. Eilenmeyer

flasks marked at the '200-ml. volume. ^Tris'^

buftVr pH 8.3 and caprylic alcohol were added,

and the whole was brought to volume. The

flasks were shaken as thoroughly as possible by

hand, plugged with cotton, and autoclaved a*^s

usual. After being cooled to room tempeiature,

the volume was adjusted if necessary, and the

entire contents of the flask were transferred to a

Waring Blendor and blended from 3 to 5 minutes.

Free pantothrjiic acid.—To determine the free

pantothenic acid value, 3 different-sized aliquots

of the food extracts were treated (omitting

enz^ones) under conditions identical with those

described in the following paragraphs. Because

the free pantothenic acid was always less than

the total, the aliquots taken for determination

were at least twice those used for enzyme treatment and, in some instances, considerably more.

Total j>aiitothfmc acid.—To determine the total

pantothenic acid content, a double-enzyme treatment was given to 3 different-sized aliquots of each

food. Test tubes measuring 16 mm. in diameter

were used for these extractions and the total

volume in each tube treated with enzymes was

kept at 2.1 ml. A 0.1 ml. of 0.1 M sodium

bicarbonate buffer, together with sufficient distilled water to bring the total volume to 2.1 ml.

(allowing for the sample aliquot and the 0.6 ml.

of the enzyme system), were pipetted into each

tube.

Sample weights taken for enzyme digestion

varied from 8 to 700 mg. of the fresh foods, whose

moisture content varied from 65 to 96 percent.

For the dry foods, whose moistures ranged from 2

to 12 percent, the samples used for the enzyme

treatment were from 20 to 175 mg.

Aliquots of the food extracts were pipetted with

care to keep the sample from the sides of the tube,

and the tubes for total pantothenic acid determination were placed in an ice bath. To each tube,

0.4 ml. 2 percent intestinal phosphatase and 0.2

ml. twice-Dowex-treated 10 percent pigeon-liver

extract were added. Each tube was thoroughly

mLxed by hand shaking—care being used to keep

the sample off the sides of the tube—then layered

with toluene, plugged with cotton, and incubated

overnight (or 4 hours if more convenient) in a

water bath held at 37"^ C.

Standard sample and blank.—A standard sample

of brewer's yeast—aliquoted at 0.8, 1.2, and 1.6

ml. for free pantothenic acid content, and at 0.4,

0.6, and 0.8 ml. for total pantothenic acid content—was run with every assay. A blank determination was also run with each assay ; and because

the pigeon liver is responsible for most of the pantothenic acid found in the blank, it was added in 1,

1%, and 2 times the amoimt used with the samples.

Dilution and filtration.—After incubation, the

contents of the tubes were transferred quantitatively to 100-ml. volumetric flasks, made to volume, and flltered through dry fluted Whatman No.

40 filter paper. With certain foods low in pantothenic acid, where increased sample weight could

not be handled, it was necessary to limit the final

volume to 50 ml. Refiltering through the same

filter paper helped to clarify most samples and was

routinely done.

MICROBIOLOGICAL PROCEDURE

Assay procedure.—Pyrex 16-mm. tubes with

plastic screwcaps were used for the microbiological

assay. The filtrates were used dir-ectly in amounts

of exactly 1, 2, 3, 4, and 5 ml. in triplicate assay

tubes. A standard curve, also in triplicate, was

run for each of 15 levels at 0.0, 0.0, 0.005, 0.010,

0.015, 0.020, 0.025, 0.030, 0.040, 0.050, 0.060,

0.080, 0.100, 0.100, and 0.200 Mg- pantothenic acid

per tube, respectively. The first 0.0 tubes were

not inoculated and were used as a check on sterility

of the medium and tubes. A different working

standard was used in the second set of 0.100-/¿g.

tubes, thus giving a check on the dilution of the

standard.

Routinely in advance of assay, the tubes were

numbered, and water was added in sufficient

amounts to give a total volume of 5 ml. per tube

after the standard or sample was added. These

watered tubes were then stored in a walk-ÍQ

refrigerator held at 32° F. The laboratory

temperature was kept around 70° F. in the winter

and was air-conditioned at 80° F. in the summer.

The filtrates and standards were kept at room

temperature until used unless 3 hours or more

were expected to elapse. Under these conditions,

the extracts and standards were refrigerated

until a short time before use. The assay tubes

were refrigerated at 5° C. after the samples or

standards were added.

When all extracts, blanks, and other solutions

had been pipetted, each tube received 5 ml. of the

cold double-strength basal medium (see table 1)

by means of an automatic pipetting machine.

Each tube was capped, and the entire set was

autoclaved for 10 minutes at 15 pounds pressure.

(If a pipetting machine is not used, the contents

of the tubes must be shaken before they are

autoclaved.) After being cooled to room temperature, all tubes except the fiLrst triplicate 0.0

standard tubes were inoculated aseptically with

1 drop of the washed L. plantarum cells, by use

of the apparatus shown (fig. 1). After inoculation, the tubes were incubated in water baths

held at 34° C. for 72 hours. At the end of the

incubation period, the contents of each tube were

titrated with 0.1 N NaOH, 2 drops of the bromth^Tnol blue indicator being used per tube.

Titration.—Tubes were titrated with a titration

apparatus (fig. 2) designed in a laboratory of the

Human Nutrition Research Branch. Essentially,

the apparatus is a titration stand with a buüt-in

fluorescent light to which 2 burettes with 2-way

stopcocks are mounted so that they drain into 2
r~\

A L

A B \jk=^

FIGURE 1.—Apparatus used for aseptic inoculation of

microbiological-assay tubes: A^ Transferring inoculum

to sterile syringe; B^ tube in position for drop of inoculum; a, straight test tube (16- by 150-mm.) in which

inoculum was grown and washed; &, 22-gage needle

embedded in rubber stopper; c, 5-ml. syringe with

spring holder; á, glass guard (22- by 70-mm.); e, screwcap

assay tube in position for drop of inoculum.

pyrex separatory funnels (used as titration vessels)

connected to a filter pump (water-operated) for

emptying the titrated samples and the rinse water

down the drain. The level of alkali in the burettes

is automatically maintained by another airejector

(filter pump). The samples are added to the

titration flasks via tubing connected to a funnel

which also has a built-in rinsing system. Compressed air is used to agitate the samples during

the titrations. The stopcocks contain no grease

and, with practice, 1,000 tubes could be titrated in

an 8-hour working day.

Calculation.—'V\i^ concentrations in us;, of standard pantothemc acid per tube versus the average

ml of standard alkah used in titrations were plotted on J log-log- paper. The type of standard

curve obtamed with these data is shown (fig. 3).

By using the standard curve of the assay^period

and the titrations and dilutions of the sample the

tree and total pantothenic acid for each food were

calculated, taking into account the blank determination obtained with the enzymes for the total

pantothenic acid calculation. For every food 3

subsamples were analyzed in at least 2, and in most

cases 3 assay periods. Fresh extracts from replicate subsamples were prepared when repeat assavs

lor any food were needed.

INVESTIGATIONS TO ESTABLISH THE

METHOD

The first experiments performed to develop,

standardize, and evaluate the microbiological procedure just described for determining free and

total pantothenic acid in foods are reported by

Toepfer and coworkers (22). The results of these

mvestigations established: (1) The amounts of intestinal phosphatase and pigeon-liver enz}mie preparation used in the extraction procedure; (2) the

fact that mylase-P released only 9 to 60 percent of

the bound pantothenic acid released by intestinal

phosphatase-liver enzymes in the 10 foods studied;

(3) reproducibility with a 5-fold variation in sample

size for extraction and a 10-fold change in samplealiquot size for enzyme incubation; and (4) the

fact that bioassays with rats were in satisfactory

agreement with the microbiological assays. Further experimental work undertaken during the

course of the study, and the results obtained with

the microbiological assay in another laboratory, as

well as in this study, are given later in this

publication.

Basal medium.—In the beginning of the study,

several media and organisms were tested for

response to increments of pantothenic acid. The

optimum response was obtained with the Skeggs

and Wright (19) medium with L. arabinosus (now

L. plantarum). Further experimentation was

limited to this medium with this organism. In

checkmg additions to the medium, a growth

response was obtained with an increase of xanthine; the amount of xanthine was therefore

doubled over the original concentration.

Considerable attention was given to obtaining

a basal medium not stimulated by fat, since fat

has-been reported (6, 19) and also observed in this

laboratory to stimulate L. plantarum (formerly

L. arabinosus) growth. The addition of 100

mg. ^Tween 80'^ per liter of medium was found

satisfactory for samples to which known fatty

acids were added. To check further on the

adequacy of this medium in the assay of foods,

6 foods varying from 1.7 to 51.4 percent fat and

from 1.4 to 76.6 percent moisture were chosen,
A.—Stand holding 2 funnels, each used for introduction of sample to its titration flask.

B.—Distilled-water-rinse system used to rinse contents of tubes into titration flasks and also to rinse

titration flasks after titration.

C.—Pyrex separatory flasks used as titration vessels,

connected to airejector (filter pump) for emptying

titrated samples and rinses.

D.—Titration stand with fluorescent light, holding

two 50-ml. burettes with 2-way stopcocks and a rack

for holding titration vessels.

E.—Glass tubing with a fine tip connected by a

T-tube and clamps to either a second airejector, for

automatic zeroing of the standard alkali in the burettes,

or to the distilled-water reservoir for rinsing the burettes

after use.

F.—Distilled-water reservoir with 3-hole rubber

stopper.

G.—Distilled-water inlet.

H.—Overflow tube leading to the sink.

7.—Standard alkali reservoir with 3-hole rubber

stopper.

/.—Connection to burettes with a clamp shutoff.

K.—Glass tubing with a clamp shutoff connected to

the airejector for vacuum to use in filling the alkali

reservoir.

L.—Connection to alkali reservoir, used for filling.

M.—Glass tubing with a capillary ending connected

to a compressed-air source for agitation of the samples

during titration.

FIGURE 2.—Apparatus used for semiautomatic titration of microbiological-assay tubes.
20.0

£ 10.0

g 8.0

^ 6.0

O

o

Z

4.0

2.0

1.0

Logarithmic

coordinates

X

y

/

y

/

/

I 11

0.005 0.01 0.02 0.04 0.06 0.08 0.10 0 20

CONCENTRATION OF PANTOTHENIC ACID (MICROGRAMS)

FIGURE 3.—Acid production of L. -planiarum in response to increments of pantothenic acid.

and pantothenic acid assayed on both the residue

after eth}^ ether extraction and the fresh food.

The finely ground fresh foods were packed tightly

into small Whatman extraction thimbles, placed in

a Soxhlet apparatus, and subjected to continuous

ethyl ether extraction for 24 to 30 hoiu-s. The

samples were then transferred to beakers, broken

up with a spatula, and dried in a desiccator over

CaCl2 until used. For assay, a weighed quantity

of this fat-free dry residue was treated exactly

as a regular food sample.

The data thus obtained are presented (table 2).

In general, the agreement between the adjusted

values obtained from the moisture, fat, and

pantothenic acid determinations and the values

obtained on the ether-extracted sample are good.

TABLE 2.—Pantothenic acid values, reported on a dry, jat-jree basis, found in ethyl ether extracted and

nonextracted food samples with and without double-enzyme treatment

Fat (ether

extract) Moisture

Free pantothenic acid Total pantothenic acid

Food Ethyl

ether

extracted

Adjusted

from fresh

weight

Ethyl

ether

extracted

Adjusted

from fresh

weight

Beef, eround round

Percent

12.8

34.5

1. 7

3. 7

24 5

51.4

Percent

65.9

33.3

76.2

76. 6

60.9

1. 4

fig./gm.

13.68

6. 17

27. 61

31.87

29.44

32.52

ßg.lgm.

13. 80

7.55

30.81

42. 54

32.88

34 68

fig.lgm.

24 30

12.23

39.06

50.35

46. 10

43.80

fig.lgm.

30.80

Cheese, cheddar 12.58

Chicken, breast

Chicken, legs _ _

37.92

59.80

Chicken, skin 47.40

Peanuts - -

51. 14

37895ft—56
pH of the extracting solution.—''Tvis'' buffer first

reported by Gomori (S) had been used by Novelli and

Schmetz (15) with excellent success in the enzyme

treatment of samples for microbiological assay.

Since its buffering capacity was known to be effective for large amounts of HCl, it was chosen for the

food extractions. With fresh fruits such as lemons

and limes, where the pantothenic acid content was

low, the food was very acid, and large samples had

to be taken (100-gm.). There were indications

that the 10-ml. ''tris'' buffer used in the standard

procedure were insufhcient to neutralize the food

sample. An experiment with limejuice of pH 2.3

was run, in which various amounts of ''tris" buffer

pH 11.3 were used. The lime aliquots incubated

for this experiment were 1.2, 1.5, and 1.75 gm. per

tube without enzyme; and 300, 400, and 500 mg.

per tube with enzyme. This ÍOO-gm. limejuice

sample required 89 ml. of the "tris" buffer pH 11.3

to bring the pH to 6.6. The results of this experiment and of a series of standards run over the

same pH range (table 3) show considerable pantothenic acid destruction below pH 6.0. The destruction was greater in the limejuice sample.

Because of these results, 14 of the more acid foods

were checked. Lemons, limes, cranberries, rhubarb, and sour red cherries were too acid for the

standard procedure. These were run by the modified procedure to give values for the table of pantothenic acid content of foods.

TABLE 3.—Influence of pH on the destruction of

pantothenic acid during sample extraction

Pantothenic acid found

pH 0.0140 Mg./

ml. standard

solution

Limejuice

Free Total

2.3

ßg.lml. ng./gm.

0.36

fig./gm.

0. 19

2.5 0.0111 . 28

3.5 . 34

4.5 .._ .0124

.0129

. 0133

. 0137

. 0137

. 91

5.5 - - 1. 29

6 0

6.6 _ . 65 1. 45

7.0

7 2 1. 41

7.5 - _ .0135

. 0137

.0102

80

11 0

Recovery of added pantothenic acid.—Three levels

of standard pantothenic acid were added directly

to duplicate 6-gram samples of dried ground beef

round before extraction, so that the recovery figures would be representative of the entire procedure. The data (table 4) show the recoveries

calculated in 2 ways: First, as total recovery,

which ranged from 94.5 to 104.7 percent, with an

overall average of 99.9 percent; and second, as

added pantothenic acid recovery, which ranged

from 85.7 to 108.5 percent, with an average of 98.8

percent.

TABLE 4.—Recovery of pantothenic acid added to

samples of air-dried ground beef round ^

Standard pantoCalculatedtotal

pantothenic

acid

Pantothenic

acid

found

Pantothenic acid

recovered

thenic acid added

Total Added

13.83/xg./gm

20.66 Mg./gm

27.67 Mg./gm

fig./gm.

36. 03

42.86

49.87

fig./gm.

/ 35. 07

I 34. 05

/ 42. 22

1 44. 45

/ 50. 18

1 52. 22

Percent

97.3

94.5

98.5

103. 7

100. 6

104.7

Percent

93.1

85.7

96.9

107.7

101. 1

108.5

Average _ 99.9 98 8

1 Pantothenic acid content of samples was 22.20 tig./gm.

Reproducibility of assays.—To check further the

reproducibility and reliability of the microbiological-assay procedure, 5 foods and yeast were

also assayed by the microbiological procedure at

the Texas Agricultural Experiment Station. The

data obtained in both laboratories, together with

the 95-percent confidence limits calcidated from

the standard error of the average, are given

(table 5).

Rat-bioassay comparison.—To be certain that

the enz3^me system chosen was adequate, rat

bioassa^^s were also conducted on the same 5 foods

and 3^east. As a further check on the medium

using high-fat foods, 2 high-fat foods (peanuts

and dried whole egg) were included. The foods

chosen and the results of the rat bioassays and of

the microbiological assays, together with the 95-

percent confidence limits, are given (table 6).

These are the bioassays reported by Toepfer and

coworkers (22) along with the detailed procedures

used in conducting the bioassays. The results by

microbiological assay agreed with the results of

the rat bioassay with the exception of carrots.

The results of the microbiological assays on the 2

high-fat foods—peanuts with 50.8 percent fat, and

dried whole egg with 41.4 percent fat—fall well

within the 95-percent limits of the bioassays of

those foods.

Taking agreement with rat bioassays as the

criterion, the microbiological method used for this

study gave very satisfactory values for total

pantothenic acid as utilized by mammals.

10
TABLE 5.—Pantothenic acid values obtained by 2 laboratories by assay with L. plantarimi in 6 foods and

yeasty with and without the double-enzyme treatment

Free pantothenic acid

_.

Total pantothenic acid

Food Laboratory No. 1 Laboratory No. 2 Laboratory No. 1 Laboratory No. 2

Average 95-percent

limits Average 95-percent

limits Average 95-percent

limits Average 95-percent

limits

Carrots

fxg.lgm.

14.2

50.7

41. 4

12.4

96.0

46.3

ßg./gm.

14. 6-13. 8

55. 0-46. 4

44. 0-38. 8

13. 3-lL 5

98. 9-93. 1

47. 4-45. 2

¡ig.lgm.

17.5

57. 6

44. 1

12. 0

85.7

54.8

fxg./gm.

19. 9-15. 0

61. 7-53. 5

45. 8-42. 4

12. 9-11. 1

101. 6-69. 7

64. 2-45. 3

¡igjgm.

20.7

76.2

64.9

23.8

203. 0

117. 6

txg./gm,

21. 9- 19. 5

80. 1- 72. 3

68. 4- 61. 4

25. 5- 22. 1

213. 7-192. 3

122. 1-113. 1

fjig./gm.

23. 1

66.4

59.4

17.7

15L6

117.7

ßg.Igm.

Dried whole egg

Kale

26. 6- 19. 5

72. 7- 60. 0

66. 6- 52. 2

21. 4- 14. 0

166. 2-137. 0

140. 4- 94. 9

Peanuts _ _

Liver, pork_ _

Yeast, brewer's_

TABLE 6.—Pantothenic acid values obtained by rat bioassay and by assay with L. plantarum using the

double-enzyme treatment

Food

Bioassay (rat)

Number of

animals Average 95-percent

limits

Microbiological assay

Average 95-percent

limits

Carrots

Dried whole egg

Kale

Peanuts

Liver, pork

Yeast, brewer's.

60

60

2 48

60

60

48

ng.lgm.

35.7

6L5

56.2

25.4

176.7

138.0

ßg./gm.

51. 4- 24. 9

83. 0- 45. 6

81. 7- 38. 6

33. 6- 19. 3

244. 7-127. 5

169. 0-112. 7

ßg.Igm.

120.7

76.2

64.9

23.8

203.0

117.6

fig./gm.

21. 9- 19. 5

80. 1- 72. 3

68. 4- 6L 4

25. 5- 22. 1

213. 7-192. 3

122. 1-113. 1

1 Significant difference from bioassay mean.

2 High-level supplement not eaten; therefore only low-level-supplement animals used.

RESULTS WITH THE ESTABLISHED PROCEDURE

Using this standardized procedure, the free and

total pantothenic acid contents were determined

on meats, poultry, eggs, nuts, fresh and dried

fruits and vegetables, breads, breakfast cereals,

rice, flour, milk and cheese.

Sample titrations.—To illustrate the agreement

among aliquots in response to increasing amounts

of sample, the titrations obtained for each tube in

4 samples for both free and total pantothenic acid

are plotted (fig. 4). To make it possible to follow

the points for each sample more easily, a smoothed

curve was drawn for each, but in actual calculation the pantothenic acid content of each aliquot

of a food was determined individually. All the

titration values for the 3 different ahquots of each

food are given.

These 4 foods, taken as examples of laboratory

data from 4 different assay periods, illustrate

directly the range of sample sizes used and indirectly the pantothenic acid content found during

the assay of foods.

As an example of liver, one of the foods high in

pantothenic acid, rabbit liver averaging 45.03

;ug./gm. was the first food presented. The original

laboratory sample taken for analyses weighed 5

gm., and the 3 aliquots used for enzyme treatment

represented, respectively, 15, 20, and 25 mg. of

liver.

As an example of lean-muscle meat, a good

source of pantothenic acid, leg of lamb averaging

8.86 Atg./gm. was the second food illustrated. The

laboratory sample taken for analysis weighed 32

gm., and the 3 aliquots taken for enz5ane treatment represented, respectively, 96, 120, and 160

mg. of the lean meat.

As representative of fresh vegetables, carrots

assaying 2.72 fjLg./gm. was the third food chosen.

The laboratory sample weighed 60 gm., and aliquots taken for enzynie treatment were 180, 240,

and 300 mg., respectively.

The fourth food, green seedless grapes with

0.40 fjLg./gm., was the lowest in pantothenic acid

content of the common foods analyzed. The

laboratory sample weighed 100 gm., and the 3

aliquots taken for enzyme treatment were 500,

n
15.0

I

O

o

Z

0.10 0.20 0.40 0.60 0.80 1.0 2.0 4.0 6.0 8.0 10.0 20.0 40.0 60.0 80.0,100

CONCENTRATION OF SAMPLE (MILLIGRAMS)

200 400

FIGURE 4.—Titrations obtained in different foods with and without double-enzyme treatment, as measured by L.

vlantarum.

600, and 700 mg., respectively. The grape aliquots were diluted after incubation to 50 ml., in

order to be in a good range on the standard curve.

The agreement between the 3 aliquots, as

judged by the titrations shown, was excellent. In

all examples, at least 3 mcrements of sample in

each aliquot were in agreement; in many, all 5.

In the medium used, L. plantarum also responded

to increments of samples, showing excellent

agreement mth the response obtained to standard

pantothenic acid.

Effectiveness of the Doivex treatment of pigeon

liver.—During tlie 2-year period of this study,

the average amount of pantothenic acid remaining

in the liver extract after 2 Dowex treatments was

0.0023 Atg. Since the standard curve is best from

0.01 to 0.100 Mg., a blank of this magnitude could

be handled.

At one point in the study, a metal stirrer

(stainless-steel) was substituted for the glass

stirrer in the Dowex activation. It was found

that the pigeon liver treated with this Dowex

lost almost 20 percent of its activity, as judged

by the release of pantothenic acid from the

standard brewer's yeast. Differences between

values obtained in different laboratories are

probably due to differences in the enzyme

preparation.

Standard reference sample.—The brewer's yeast

was analyzed 104 times during this study, exclusive of the experimental runs. The average

value for the free pantothenic acid was 44.04

/zg./gm., with 95-percent and 99-percent confidence limits of 44.73 to 43.35 and 44.96 to 43.12,

respectively. For total pantothenic acid content,

the average value was 110.6 jug./gm., with 95- and

99-percent confidence limits of 113.05 to 108.87

and 113.73 to 108.19, respectively.

Comparison with literature values.—Since Novelli

and COworkers published the results of their

research in 1949 (14), 49 of the foods reported in

this handbook were analyzed in other laboratories

by the double-enzyme S3^stem. These data have

been assembled (table 7) along with 3 rat and 24

chick bioassay values, as well as the microbiological-assay data obtamed in the Human

Nutrition Research Branch laboratories on these

foods. If several values were given in the published findings, an average value was used on the

table, and the number of reported values used to

obtain the average are indicated. Of the 4

microbiological-assay values obtained with the

double-enz}Tne system before Dowex treatment

of the pigeon liver for removal of the pantothenic

acid in the liver (Í5), 3 are considerably higher

than an}^ obtained in the present study, and are

in line with the results obtained in this laboratory

with untreated pigeon liver.

The 3 rat-bioassay values were reported from

2 different laboratories; and 2 of these foods—

walnuts and wheat bran—have chick-bioassay

values in addition. The walnut values obtained

by rat- and chick-bioassay were in fair agreement,

and the microbiological-assay value feU between

them. The rat bioassay value for wheat bran

was double the chick-bioassay value. The microbiological-assay value in this study checked a

value found in literature, and both feU nearer the

chick-bioassay value. All the chick bioassays

were conducted in 1 laboratory and represent

several years of work, including certain assays

conducted before crystalline pantothenic acid was

synthesized. With 5 foods, the chick-bioassay

values were higher than the microbiological-assay

values obtained in this study.

Since this study was initiated, Schweigert and

Guthneck (18) have published a method for the

determination of pantothenic acid in meats, in

which Dowex-treated hog-kidney extract is used

with excellent results instead of the pigeon-liver

extract. These values are included in table 7.

12
TABLE 7.—Pantothenic acid values reported in the literature, determined by microbiological assay usina the

double-enzyme system, and by bioassay with rats and chicks in comparison with values found in this

study

Food

MEATS

Beef:

Kidney

Liver

Rib

Dried

Lamb:

lüdney

Liver

Leg

Pork:

Liver

Ham, precookedRabbit:

Liver

Muscle

Free pantothenic

acid

Microbiological

assay in

Literature

(ig.lgm.

7.9

WhitesYolk—

WholeEGGS

NUTS

Almonds _-

Cashews- -

Coconuts: Meat--.

Milk—_

Walnuts--

VEGETABLES, FRESH

Beans, green snap_

Broccoli

Cabbage

Carrots

Corn, yellow

Eggplant

Kale

Lettuce

Onion

Peppers, green

Potatoes, white

Spinach

Squash, zucchini. _

Sweetpotatoes

Tomatoes

VEGETABLES, DRIED

Beans:

Cowpeas

Kidney.

Lima

Navy

Lentils

Split peas

See footnotes at end of table.

This study ^

L2

5. 1

10. 2

7.5

ßg./gm.

26.57

60. 57

2.02

8. 70

36. 41

44.08

2. 89

30.01

3.82

28.75

5.20

.79(4)

36.08(4)

13. 14(4)

2.81

5.50

.84

.35

5.91

.38

7.74

1. 56(3)

1.50

3.06

.90

7.25

L 19

.52

.71

2. 87(3)

1.36

1.67

8. 16

.69

7.68

2.53

6.01

4. 15

9.80

12. 79(2)

Total pantothenic acid

Microbiological assay in

Literature ^

ng./gm.

34. 10

68.38

4. 1

2 47.5

43.3

81. 4

5.9

66.98

5. 5

75

9.9

2.87

31.21

252.6

4.09

14.88

(2)

(4)

(5)

(2)

2.07

.0052

2. 14

1.63

3. 15

2.29

L36

1.27

2.29

2. 67

2 25. 7

3.06

5.33

.92

9.22

5. 12

7.98

4.98

n. 44

(2)

(3)

(4)

(4)

(4)

(2)

This studv

fig./g7n.

40. 64

93. 36

5.42

23. 40

47.43

70.99

8.86

70.08

6.94

45.03

7.90

1. 44(4)

42. 35(4)

15. 76(4)

5.78

11.62

L93

.50

9.70

2.02

12.87

2. 80(3)

2.72

8.89

2.05

12,88

3.63

1.68

2.37

4. 05(3)

3. 12

3.40

9.35

3. 14

12.43

6.50

12.99

12.08

14.99

2L 16(2)

Bioassay in literature

Rat 1

fxg./gm.

Chick 1

ßg./gm.

10.9

10

63 (4)

27 (37)

13 (2)

2

8 (3)

3 (2)

"i.'2(2)'

6.5(2)

L2(2)

3

11 (2)

1 (2)

18 (2)

21 (2)

Literature cited

(18)

{18)

(18), (7)

(13)

(18)

(18)

(18)

(18)

(18)

(14)

(14)

(1)

(1)A7)

(13), (7)

(1)

(1)

(1)

(1)

(5), (7)

(1)

(7)

(1)

(1)A7)

(7)

(1)

(7)

(1)

'" Sí!

U),<7)

(IS), (7)

(1),(7)

U),'7)

a),(7)

(Í)

^^} (1)

(7)

13
TABLE 7.—Pantothenic acid values reported in the literature, determined by microbiological assay using the

double-enzyme system, and by bioassay with rats and chicks in comparison with values found in this

study—Continued

Free pantothenic

acid Total pantothenic acid

Food Microbiological

assay in Microbiological assay in Bioassay in literature Literature cited

Literature This study i Literature ^ This study i Rati Chick 1

FRUITS

Avocados

ßgjgm- ixg.lgm.

9.27

L84

2.91

1.89

. 65

1. 26(2)

.75

5.03

2. 58(2)

4,46

6. 34(2)

19.40

26. 04(2)

2. 78(2)

2.34(6)

L 15

2. 09 (3)

fjLg./gm.

11.35

3.06

4.03

2.65

L38

2.21(2)

L75

14. 13

5. 93(2)

6.40

10. 95(2)

29.03

99. 24(2)

3. 75r2)

3.23(6)

2. 80

fj^gjgm. ng./gm.

0)

U)A7)

U)

il)

(1)

U)A7)

(i)

U)A7)

(1)

Bananas .7(2)

Figs, dried 7.0

Lemons. __ _ _ _ _ 1.08

3.14

1. 89 (2)

1.47

10.35

4.31 (2)

5. 21 (2)

12.06 (16)

2 30.0

124. 56 (2)

Lime, juice

Oranges __ .7(2)

Pineapple _ __

CEREALS, OTHER GRAIN

PRODUCTS

Oats, rolled 11

Cornmeal

Rice, white _ 4 (2)

11 (2)

24 (4)

(1)A7)

iie)A7)

{is)Aio)A7)

U)

(7)

a)A7)

U)

Wheat

Wheat bran_ _ 23.0 48 (2)

Yeast, brewers . _

MILK, CHEESE

Mük:

Skim 3. 6(9)

Whole 3.43 2. 8(4)

Cheese, cottage __ ___ L98

1 Number in parentheses is number of microbiological or animal assays averaged to obtain figure.

2 Data obtained before Dowex treatment of pigeon-liver enzyme.

PART II. QUANTITATIVE DISTRIBUTION OF PANTOTHENIC ACID IN

TYPICAL FOODS

FOOD SAMPLES

Meat.—Meats were purchased during the winter

months, to avoid transportation and preparation

during Washington, D. C, summers. Hamburger,

ground beef, stew meat, Hver, ham shoes, and other

samples were purchased from at least 3 retail

markets in the city, and pooled. For pork loins,

veal legs, and similar cuts, care was taken to make

certain that the cut was from at least 3 different

animals and, usually, that more than 1 market was

represented. For packaged meats, such as frankfurters, sausage, and liverwurst, 1 package of each

brand found in the stores on the day of purchase

was obtained and combined.

Laboratory-sample preparation involved obtaining the edible yield from each item and dividing

the lean from the separable fat. The laboratory

sample used for analysis was prepared by combining the edible lean from each item on an equalweight basis, the weight of the smallest item being

used as the combining unit. On the packaged

meats, all brands were cut to the smallest unit of

weight, and the whole combined for analysis. Six

subsamples were made of each of the combined

samples of food purchased; these subsamples were

sealed in No. 2 sanitary enameled tin cans and

stored at —40° F.

Poultry,—For poultry, legs and breasts were

purchased separately for the chicken samples, and

the right halves of 3 birds were procured for the

turkey samples. All the edible meat was used for

analysis; the dark and white muscle meat, organs,

and skin were analyzed separately. Subsamples

were prepared and stored, as for meats.

Eggs,—Four lots of eggs, each lot consisting of

2 dozen assorted-sized brown and white eggs, were

obtaiaed from Animal and Poultry Husbandry

Research Branch, Agricultural Research Service,

14
m June, July, and October. The analyses were

started immediately. No samples were stored, and

yolks and whites were analyzed separately.

Nuts.—Nuts, with the exception of cashews,

were purchased in the shell. The peanuts were

purchased roasted, the others as they are sold for

home consuniption. The nuts were shelled in the

laboratory, yields obtained, and subsamples stored,

as were meats.

Fresh vegetables andfruits.—Fresh vegetables and

fruits were obtained on the Washington, D. C., market at the peak season whenever feasible. State

of origin and variety, if known, were also recorded.

Each market lot purchased was analyzed separately, and usually only one lot was obtained.

Lots of foods arrived in the laboratory in the late

afternoon and were stored overnight in a large

walk-in refrigerator held near 35*^ F. The foods

were washed in distilled water, if necessary, and

dried back to the original weight by blotting with

cheesecloth. Edible portions were prepared and

yields calculated for all vegetables and fruits. If

more than one part of a vegetable or fruit was

edible and the structures were very different, the

parts were analyzed separately. Six 200-gm. subsamples of each vegetable and fruit were prepared

and stored, as for meats. Potatoes and apples were

not frozen ; a preliminary assay was run as soon as

the food was received in the laboratory; the remainder of the sample was stored in the walk-in

refrigerator; and final anal3^ses were run as soon as

the results of the first analysis were completed.

Analyses were rim separately on the peeled apples

and potatoes and also on the peel.

Dried vegetables andfruits.—One package of each

brand of a single type of bean, pea, or fruit easily

available on the Washington, D. C., market was

purchased. The various brands of each item were

combined in equal weights, the edible weight of the

smallest unit being used as the combining weight.

Subsamples of approximately 50 gm. of dried vegetables and 100 gm. of dried fruits were canned

and stored at —40'' F.

Breads.—One loaf of sliced bread of each type

baked by the main commercial bakeries of the

Washington, D. C, area was purchased on the

open market. Breads from 1 to 9 bakeries were

included in each of the 8 types of bread analyzed.

For laboratory samples, each loaf of each type of

bread was cubed; an equal weight of cubes from

each bakery was combined; the whole was thoroughly mixed; and subsamples were withdrawn,

packed in cans, and stored at —40° F.

Breakfast cereals.—One package of each breakfast cereal usually found in the large markets of

the Washington, I). C, area was purchased. These

packages were then grouped first according to kind

of grain, and second according to preparation of

the grain. Products from 1 to 5 food processors

were included in each of the 13 cereal composites

analyzed. Equal weights from each manufacturer

were combined for the laboratory samples. SLx

50-gm. subsamples were stored at —40° F. for the

laboratory analyses.

Flour.—With the exception of white enriched

all-purpose flour which came from three milling

companies, the flours were a single brand obtained

on the Washington, D. C, market.

Grains.—OwQ sample of the 1952 spring-wheat

crop was obtained from the Grain Division of the

Agricultural Marketing Service. The other grains

were purchased on the Washington, D. C, market.

Each sample analyzed was a composite of products

obtained from 1 to 5 processors. The composites

were made on an equal-weight basis from each

processor; and 6 subsamples, each 50-gm., were

canned and stored at —40° F. until analyzed.

Milk.—The buttermilk and the evaporated milk

were obtained on the Washington, D. C., market

from 4 dairies and 3 milk-processing companies,

respectively. The nonfat dry-milk sohds were

from 2 market purchases, 1 in each year of the

study. The first 3^ear's purchase was from the 1952

process, and the laboratory sample included the

product of 3 food processors. The second purchase

was from the 1953 process, and the laboratory sample included the product of 4 food processors.

Two samples of raw whole milk and one each of

heavy cream, pasteurized whole milk, skim milk,

and homogenized milk were obtained from the

Dairy Husbandry Research Branch of the Agricultural Research Service. One sample each of pasteurized whole, skim, and homogenized milk were

obtained from one dairy in the Washington area.

Each of these fluid milk samples was separately

anal^^zed immediately upon receipt at the laboratory. Evaporated-milk samples were stored in a

home t^^pe refrigerator and were opened and composited at the time of analysis. Dried milk composites were stored at —40° F. until analyzed.

Cheese.—All the cheese samples were obtained

from the Washington, D. C, market. The cheddar was from the 1951 process from both New

York and Wisconsin. The cottage cheese was a

composite of 5 brands carried in the large markets

of the Washington area. The processed-cheese

sample was American type cheese, composited from

products made by 7 processing companies. AU

laboratory composites were made from equal

weights of each brand, and the subsamples were

canned and stored at —40° F. untu analyzed.

MO/srü/?f

Moisture was determined on weighed 1- to 10-

gm. samples of food, depending on the expected

moisture content. Samples were dried to constant

weight in a vacuum oven at 70° C, in aluminumfoil moisture dishes.

FAT

Fat was extracted from weighed, dried samples

under continuous extraction with ethyl ether m a

Soxhlet apparatus for 18 hours. After the ether

was evaporated, the fat was dried for 5 days m a

vacuum desiccator over fresh calcium chloride and

then weighed.

15
DETAILED DATA OBTAINED IN THE STUDY

OF 237 FOOD ITEMS

A large summary of free and total pantothenic

acid in the edible portion of foods, containing data

on 237 food items, is given (see table 8). All the

foods were analyzed between November 1952 and

December 1954. A total of 1,139 assays in 109 assay periods, consisting of 509 subsamples for the

free and 630 subsamples for the total pantothenic

acid data, form the basis of the report. Of these

items, 29 are values calculated to include the fat

trim of meats as well as the separable lean, which

was analyzed. For certain vegetables and fruits,

the yield data on different portions, such as roots

and leaves, were used to calculate a composite

value for the total edible material. The pantothenic acid content of whole fresh eggs was obtained from the proportions of wliite and yolk

which were analyzed separately.

With most foods, the free pantothenic acid was

about half the total pantothenic acid. For each

food item, the yield figure is shown which represents the portion used for the laboratory sample

and from which the subsamples were prepared.

The reported moisture figure and percentage of fat,

or ether extract, are averages of duplicates on each

subsample of food tested.

Analyses were made of 25 meat items. The

separable lean of beef averaged 0.661 mg./lOO

gm.; that of pork, lamb, and rabbit, 0.788 mg./lOO

gm.; and that of veal averaged the highest, with

1.063 mg./lOO gm. fresh weight. It is interesting

to note that the muscle of the young, rapidly

growing animal was the highest in pantothenic

acid content. The overall separable lean muscle

from all species analyzed was 0.883 mg./lOO gm.

fresh weight and 2.632 mg./lOO gm. dry weight.

As expected, organ meats of all animals were high

in pantothenic acid content. The fresh kidney

of large animals (beef, veal, and lamb) averaged

4.273 mg., with a range of 4.011 to 4.743 mg./lOO

gm. In general, liver had the highest pantothenic

acid content of the fresh foods analyzed, with

beef, lamb, pork, veal, and rabbit averaging

7.531 mg./lOO gm. Beef and veal were the

highest of the livers with an average of 9.522

mg./lOO gm., while pork, lamb, and chicken liver

averaged 7.090 mg., with a range of only 7.008 to

7.163 mg./lOO gm., and rabbit liver was lowest

with 4.503 mg./lOO gm. fresh weight.

In both chicken and turkey, the dark meat was

higher in pantothenic acid than the white. The

dark meat of both averaged 1.153 mg./lOO gm.

fresh weight, while the white meat averaged 0.715

mg./lOO gm. fresh weight. Generally the chicken

meat contained more pantothenic acid than the

turkey.

On a fresh-weight basis, the 4 lots of egg 3^olks

from the Agricultural Research Center, with an average of 4.235 mg./lOOgm., contained 30 times the

amount found in the egg whites, which averaged

0.144 mg./lOO gm. On a dry-weight basis, however, the yolks with 8.241 mg./lOO gm. contamed

7 times the amount in whites, with 1.229 mg./lOO

gm. Whole eggs were a good source of the

vitamin, averaging 1.576 mg./lOO gm. fresh

weight. Over 80 percent of the pantothenic acid

found in eggs was in the free form.

Eight varieties of tree nuts were analyzed. On

a fresh-weight basis, coconuts were the lowest in

pantothenic acid, with 0.193 mg./lOO gm., and

pecans were the highest with 1.707 mg./lOO gm.

On a dry-weight basis, Brazil nuts were lowest,

with 0.241 mg./lOO gm., while pecans were still

highest, with 1.751 mg./lOO gm. The average

of all the common tree nuts was 0.808 mg./lOO

gm. fresh weight.

Three market lots of roasted peanuts were

anal3^zed and an average pantothenic acid content

of 2.137 mg./lOO gm., with a range of 1.651 to

2.414 mg., was found.

The 5 varieties of dried beans averaged 1.009

mg./lOO gm. of pantothenic acid; lentils contained

1.499 mg./lOO gm.; and dried peas contained

2.116 mg./lOO gm. Full-fat soy flour, which was

the only form of soybeans analyzed, contained

1.681 mg./lOO gm. Corn cereal with soya added

contained considerably more pantothenic acid

than plain-corn cereals.

No relationship could be found between pantothenic acid content and protein content of the tree

nuts, peanuts, and dried legumes. In these foods,

the protein content varied from 36.54 to 2.34 gm./

100 gm., while the pantothenic acid content of the

same foods varied from 2.414 to 0.193 mg./lOO gm.

in a highly irregular fashion. In 4 of these foods,

the protein content varied only from 24.11 to

24.33 gm. while the pantothenic acid varied from

0.650 to 2.182 mg./lOO gm. m no defijiite order.

Fresh vegetables are not a particularly concentrated source of pantothenic acid. However,

in the amounts usually consumed, they would

contribute a considerable amount of this vitamin

to the diet. Mushrooms were the highest source

of the vitamin among vegetables, having 2.713

mg./lOO gm. Other important fresh-vegetable

sources were broccoli, cauliflower, kale, and sweetpotatoes. Other fresh vegetables having more

than average amounts of pantothenic acid were

sweet corn, peas, brussels sprouts, parsnips, and

asparagus. Green onion bulbs, with 0.168 mg./lOO

gm., had the lowest pantothenic acid content of

any common vegetable analyzed. On a freshweight basis, peeled potatoes of 3 varieties averaged 0.404 mg./lOO gm., while the whole potatoes

averaged 0.380 mg. It is interesting to note that

the pantothenic acid content of the paring (0.230

mg./lOO gm.) is about half that of the potato.

Of the foods analyzed, fresh fruits were the

lowest in pantothenic acid. Avocado, with 1.135

mg./lOO gm., was the fruit analyzed with highest

content; and green seedless grapes, with 0.040

mg./lOO gm., the lowest. Fresh fruits with about

0.3 mg./lOO gm. were bananas, watermelon, strawberries, and apricots.

16
Raisins, like grapes, were very low in pantothenic

acid. However, the other commercially dried

fruits analyzed averaged 0.575 mg./lOO gm. Dates

contained the greatest amount, 0.780 mg./lOO gm.

Grain products followed a pattern, the highest

in pantothenic acid being the least refined.

Whole-wheat bread with 0.789 mg./lOO gm. was

highest, and vienna bread with 0.378 mg./lOO gm.

was lowest. Breakfast cereals averaged 0.863 mg./

100 gm. Wheat bran assayed 2.903 mg./lOO gm.,

oatmeal 1.413 mg./lOO gm., and corn cereals 0.239

mg./lOO gm. Buckwheat and whole-wheat flour

ranked with breakfast cereals as sources of the

vitamin.

Bearing out the observation on refinement, the

amount of pantothenic acid found in enriched allpurpose flour was approximately 50 percent of the

amount found in whole-wheat flour. White rice

contained only 62 percent of the vitamin found in

brown rice. Precooked rice was still lower, mth

less than 50 percent of the vitamin content of the

white rice on a dry-weight basis. These findings

indicate that the cooking and drying processes

leached or destroyed some of the pantothenic acid.

Fluid whole milk averaged 0.323 mg./lOO gm.

Fresh fluid skim milk and reconstituted dry skim

milk averaged 0.399 mg./lOO gm. with a range of

0.362 to 0.426 mg./lOO gm. The fresh skim milk

tested was a little lower than the reconstituted

nonfat solids, indicating little or no loss in processing.

Of the cheese samples analyzed, cottage was

lowest in pantothenic acid content with 0.280 mg./

100 gm.; Cheddar contained 0.405 nig./lOO gm.;

and processed American type was highest, with

0.482 mg./lOO gm. When cheese on a dry-weight,

fat-free basis is compared with milk on the sanae

basis, only 40 percent of the pantothenic acid is

accounted for in the cheese, indicating a loss during

processing.

LITERATURE CITED

(1) AsENJo, C. F., and MUñIZ, A. I.

1955. PANTOTHENIC ACID CONTENT OF TROPICAL

FOODS. Food Res. 20: 47-54.

(2) CROKAERT, R.

1949. DU DOSAGE CHIMIQUE DE L'ACIDE PANTOTHÉNiQUE. Soc. de Chim. Biol. Bul.

• 31:903-907. Illus.

(3) GOMORI, G. XT A r; ^r. Q fi

1946. BUFFERS IN THE RANGE OF PH 6.5 TO 9.0.

Soc. Expt. Biol. and Med. Proc. 62: 33-34.

(4) HALL, A. P., MORGAN, A. F., and WHEELER, P.

1953. THE AMOUNTS OF SIX B-VITAMINS IN FRESH

AND DRIED FIGS. Food Res. 18: 206-216.

(5) WHEELER, P., THIELEN, A., and MORGAN,

A. F.

1953 A NEW B VITAMIN STUDY OF WALNUTS.

Food Res. 18: 574-577.

(6) HoAG, E. H., SARETT, H. P., and CHELDELIN, V. H.

1945. USE OF LACTOBACILLUS ARABINOSUS 17-5

FOR MICROASSAY OF PANTOTHENIC ACID.

Indus, and Engin. Chem., Analyt. Ed.,

17: 60-62. lUus.

(7) JUKES, T. H.

1941. THE DISTRIBUTION OF PANTOTHENIC ACID IN

CERTAIN PRODUCTS OF NATURAL ORIGIN.

Jour. Nutr. 21: 193-200.

(8) KING, T. E., FELLS, I. G., and CHELDELIN, V. H.

1949. PANTOTHENIC ACID STUDIES. VI. A BIOLOGICALLY ACTIVE CONJUGATE OF PANTOTHENIC ACID. Amer. Chem. Soc. Jour.

71: 131-135. Illiis.

(9) — LOCHER, L. M., and CHELDELIN, V. H.

1948. PANTOTHENIC ACID STUDIES. III. A PANTOTHENIC ACID CONJUGATE ACTIVE FOR

ACETOBACTER SUBOXYDANS. Arch. Biochem. 17: 483-485.

(10) LiH, H., KING, T. E., HIGGINS, H., and others.

1951. GROWTH-PROMOTING ACTIVITY OF BOUND

PANTOTHENIC ACID IN THE RAT. Jour.

Nutr. 44: 361-370.

(11) LIPMANN, F., KAPLAN, N.O., NOVELLI, G. D., and

others.

1947. COENZYME FOR ACETYLATION, A PANTOTHENIC ACID DERIVATIVE. JOUF. Biol.

Chem. 167: 869-870.

(12) NEAL, A. L., and STRONG, F. M.

1943. MICROBIOLOGICAL DETERMINATION OF PANTOTHENIC ACID. Indus, and Engin.

Chem., Analyt. Ed., 15: 654-657. Illus.

(13) NEILANDS, J. B., and STRONG, F. M.

1948. THE ENZYMATIC LIBERATION OF PANTOTHENIC

ACID. Arch. Biochem. 19: 287-291.

(14) NOVELLI, G. D., KAPLAN, N. 0., and LIPMANN, F.

1949. THE LIBERATION OF PANTOTHENIC ACID FROM

COENZYME A. Joup. Biol. Chem. 177:

97-107. Illus.

(15) and SCHMETZ, F. J., Jr.

1951. AN IMPROVED METHOD FOR THE DETERMINATION OF PANTOTHENIC ACID IN TISSUES.

Jour. Biol. Chem. 192: 181-185.

(16) REFAI, F. Y., and MILLER, B. S.

1952. THE DETERMINATION OF PANTOTHENIC ACID

IN WHEAT. Cereal Chem. 29: 469-475.

Illus.

(17) ROBINSON, F. A.

1951. THE VITAMINS PROGRESS IN 1950. Food

Manufacture 26: 143-148. Illus.

(18) ScHWEiGERT, B. S., and GUTHNECK, B. T.

1953. LIBERATION AND MEASUREMENT OF PANTOTHENIC ACID IN ANIMAL TISSUES. JOUF.

Nutr. 51: 283-293.

(19) SKEGGS, H. R., and WRIGHT, L. D.

1944. THE USE OF LACTOBACILLUS ARABINOSUS IN

THE MICROBIOLOGICAL DETERMINATION OF

PANTOTHENIC ACID. JouF. Biol. Chem.

156: 21-26. lUus.

(20) SNELL, E. E., BROWN, G. M., PETERS, V. P., and

others.

1950. CHEMICAL NATURE AND SYNTHESIS OF THE

LACTOBACILLUS BULGARICUS FACTOR.

Amer. Chem. Soc. Jour. 72: 5349-5350.

(21) SzALKowsKi, C. R., MADER, W. J., and FREDIANI,

H. A.

1951. CHEMICAL DETERMINATION OF CALCIUM

PANTOTHENATE. Cereal Chem. 28: 218-

225. Illus.

(22) ToEPFER, E. W., ZooK, E. G., and RICHARDSON, L. R.

1954. MICROBIOLOGICAL PROCEDURE FOR THE ASSAY

OF PANTOTHENIC ACID IN FOODS: RESULTS

COMPARED WITH THOSE »^ BIOASSAY.

Assoc. Off. Agr. Chem. Jour. 37: 182-190.

(23) WILLIAMS, R. J., LYMAN, C. M., GOODYEAR, G. H.,

and others. ,, ^^^„

1933. ''PANTOTHENIC ACID" A GROWTH DETERMINANT OF UNIVERSAL BIOLOGICAL OCCURRENCE. Amer. Chem. Soc. Jour. 55:

2912-2927.

(24) WILLIAMS, W. L., HOFF-J0RGENSEN, E., and SNELL,

F E

1949. DETERMINATION AND PROPERTIES OF AN

UNIDENTIFIED GROWTH FACTOR REQUIRED

BY LACTOBACILLUS BULGARICUS. JOUr.

Biol. Chem. 177: 933-940. lUus.

17
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