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MICROCOPY RESOLUTION TEST CHART
MICROCOPY RESOLUTION TEST CHART
NATIONAL BUREAU OF STANDARDS-1963-A
NATIONAL BUREAU OF STANDARDS-1963-A'
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Teclanicnl Bulletin No. 1023
•
January
11TACKS
Cpnlparison of the 2,6-Dichlorophenolindo­
phenol and 2,4-Dinitrophenylhydrazine
Methods with the Cranlptoll Bioassay for
Detenuining Vitanlin C Values in :Foods 12
ELIZABETH M, HtW'STON, 1;"1.'/11;111., MUIIIIAY ~'ISIIER, biologist, a'tld ELSA
ORENT-KEII.ES;' /Ullrilill'l/ I;/II'II,isl, 8/0'1'(/'/1. of Hilma'll. Nutrition and Home
By
ECOIIO'II,.ics, AgricuUu'I"n[, Rt'HCIU'ch Adm'inist.Tution
CONTENTS
Jlagl:
Introduction ~""""""".
,Experimental dll'ocedure
,,,,' ,
Chemical pI1lScedure . """"
Biological Pi:ocedure ' ,
SUltS and dllkussion
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1 J Appendix A, summary of data
4 ' from the bioassay
. " , ... ,
5 Appendix B. interference of glu7
('oreductone in the RO and
10 1 RMOD 2,4-dinitrophenylhydra­
18; zine methods , ... ,.,'
19
Page
22
26
.INTRODUCTION ~ Th6l5.Rppli'tf\tion of chemical methods to the measurement of
8scor6Tc acicFin animal and vegetable tissues is often complicated
by the presEfuce of interfering substances. The most troublesome
of these are~he ascorbic-acid-Iike compounds which react in many
respects Iik&ltrue m,corbic acid with the two most commonly used,
reagents, 2,6-dichlorophenolindophenol and 2,4-dinitrophenylhy­
drazine. Since these substances are entirely inactive in the animal
body as antisc(ll'butic agents, however, (8, H)" they give rise to
estimates of vitamin C potency which are falsely high when values
are calculated from chemical amllYHefl, The reductones (9, 10),
which are derivatives of the carbohydrates and which may be
present in plants and animals under natural conditions (29, 44),
unquestionably reduce indophenol and may also react with phenyl­
1 SUbmitted for pUblication May 31, 1950.
, This research was donc as part of 1\ project Rupported by an allotment
made by the Secretary of Agriculture from Special Research Funds
(Bankhead-Jones Act of June 29, 1935),
'The authors are indehted to James F, Conch, Bureau of Agricultural
and Industrial Chemist.ry, for his generosity in supplying the rutin used in
this investigation,
• Italic numbers in parentheses refer to Literature Cited, p, 19.
2
TECHNICAl. IlULLI<:'1'IN No. 102:1. lJ. 1:;. DEI'T. (W AGIUCUlJl'UHE
hydrazine (:J.1). The othcr intcl"fcrence which ii' encountered most
frequently is caused by 2,3-diketogulonic add, a biologically inactive oxidation pr.oduct of ascorbic acid, This substance combines
with phenylhydrazine to give the same characteriRtic red color by
which ascorbic acid is measured in this reaction, but is entirely
inactive with indophenol (31, :Ii).
Both reductones and diketogulonic acid are generally thought
to be absent from fresh fruits and vegetables; however, their
presence has been demonstrated even in fresh products by several
lIlvestigators (12, 44). Animal tissues-Hver, blood, and adrenals
-have been shown to contain diketogulonic acid (92). It is also
found in small quantities in normal urine, a.id may be present in
conRiderable amounts in both blood and urine following injectionR
of either dehydroascorbic acid or diketogulonic acid (.'12). Reduc­
tones have been found in the adrenalR (2.9).
1'he products which contain the largest amounts of interfering
substances, howevet:, and in which the greateRt analytical prob­
lems are encountered, are fruitR and vegetable~~ that either have
undergone some form of processing that employs heat, or have
been stored for a considerable period of time. Reductones have
been identified in many such foods (2.1,22, 2fi, 3/i, 4;1. 44). Diketo­
gulonic acid has recently hf'en identified in a number of both fresh
and stored foods by Goldblith and Harris (.12), and discrepancies
in aSRay results have been attributed, probably rightly, to diketo­
gulonic acid by other investigators (.1 fI, .'14) .
Mapson (22) was able to correct successfully the ascorbic acid
values determined by the indophenol method for the presence of
reductones by treating the food extracts with formaldehyde, which
condenses with the ascorbic acid.
In an attempt to eliminate the effect of interfering substances
other thu'_ reductoneR (glutathione, cystein, cystine, etc.) from
the ascorbic acid analyseR of urine, blood, and other animal tissues,
and to diRpense with the URe of H:!S, the 2,4-dinitrophenylhy­
drazine method was introduced by Roe and his coworkers
(20. 36). By this met.hod, all of the ascOi bie acid is oxidized to
dehydroascorbic acid which is then treat.ed with the phenylhy­
drazine reagent.; the reRtllting precipitate is dissolved with 85
percent H:!SO.I and the color measured photocolorimetrically.
Norit wa!'; the first oxidizing agent used b~' Roe and 0thers; later
bromine water and finally bromine vapor (.17) were substituted
for norit. By employing a modified pt'ocedure for extraction of
the t.issue, Roe and Oesterling (i/8) adapted the method to the
measurement of the vitamin C content of foods. These investi­
gators decided that it was unlikely that sufficient reductones
occurred in processed foods to have an effect of any appreciable
magnitude upon the ascorbic acid values obtained by their method,
Snow and Zilva (41,42), however, had demonstrnted the pres­
ence of reductones in considerable amounts in processed foods.
Furthermore, Penney and Zilva (33) prepared in the laboratory
pure glucoreductone and mixtures of reductones, which reacted
with phenylhydrazine under the conditions employed in the Roe­
•
•
VITAMIN C-COMPARISON 0.' CHEMICAL AND BIOLOGICAL ASSA1:
3
Kuether (86) method. Penney and Zilva had found also that
diketogulonic acid interfered even more markedly than reductones
in the reactions. III fact, they had published a method (~rl) for
the quantitative measurement of 2,3-diketogulonic acid whi'ch used
dinitrophenylhydrazine as the reagent and had employed it in
a study of the chemical beh£vior of dehydroascorbic acid in vivo
and in vitro (82).
In the light of these experiments, Roe, Mills, CeRterling, and
Damron (37) further modified the Roe-Oesterling procedure so
as to differentiate between ascorbk, dehydroascorbic, and diketo­
gulonic acid8 present in the same tissue extract. No cognizance
waR taken of the possible presence of l'eductones, whieh could
interfere with the measurement of any or all of these llscorbic­
acid-like substances.
During a study of the effect of home cooking procedures on the
vitamin and mineral content of a series of foods (17), retentionR
of 102-113 percent ascorbic acid were obtained in french-fried
and country-fried potatoes, when the indophenol method of Bessey
and King (1) was employed. Ascorbic acid retentions were re­
duced to 57 and 67 percent, respectively, when two of the same
lots of potatoes were analyzed, using the Mapson formaldehyde
modification (22) to correct for reductoneR. This indicated the
presence of interfering substances, probably biologically inactive
reductones, equal to about 45 percent of the apparent ascorbic acid
content.
Other investigators have reported anomalous asc;orbic acid
values for canned fruit" and vegetables that had been kept at
elevated temperaturel'l 1.)1' varying periods of tim€l. In 1945,
Guerrant, Vavich, and Dutcher (13) observed losses of aRcorbic
acid in samples of fruit juices held at 85° F. for 270 days, but
increases in canned yellow corn and lima beans that had been
expmled to 11 or' for the same length of time. The authors
nttributed these increases "to the formation of nonascorbic acid
compounds which reacted with the dye during the course of the
assay." They were unsuccessful in their attempt to discover either
the nature of the interfering substances or their probable concen­
tration by means of additional analyses by the method of Roe
and Oestl1rlin~.
Feaster, Tompkins, and Pearce (11) summariz(!d the available
data on retention of ascorbic acid in canned fruits, vegetables, and
:iuices; storage at 21 0 C. resulted in "small sacrifk~es of ascorbic
acid" and even higher temperatures for a longer time, 27° for
] year, resulted in los!;es of only 5 to 15 percent. From their own
experiments they reported 90-percent or bette I' retentions of
ascorbic acid in canned tomato juice stored at temperatures of 4°,
10°, 16°, and 21°. It seemed logical to que~tion whether or not all
figures for ascorbic acid retentions of food~ after prolonged
storage might not. in reality. be partially attributable to the
inadvertent inclusion of "interfering substance!';'" ir. the ascorbic
acid measurements, although it was only when the interfering
substances were present in amounts sufficient to give apparent
4
TECHNICAL BULLETIN No. 1028, U. S. DEPT. 0.' AGRICULTURE
J'
retentions of 100 percent or more that their presence became
evident. Only a bioassay of vitamin C value would prove whether
or not the conclusions drawn from chemical analyses were true.
A comparison of the indophenol tib'ation with the bioassay as
measurements of vitamin C value of a number of fruits and vege­
tables was made by Harris and Olliver (15). They found good
agreement between the results of the two methods and concluded
that interfering substances were not found in any fresh fruits
and vegetables, or various processed materials. No attempt was
made by these investigators to show the presence of reductones
01' other interfering substances by chemical procedures.
No data have been published making comparisons among the
vitamin C values obtained by the indophenol method, the phenyl­
hydrazine method, and the bioassay when substances that inter­
fere with both chemieal methods are known to be present.
•
EXI'EUlMENTAL l'HOCEDUHE
Interfering subAances are present in largest amounts in foods
which have undergone either sume heat treatment or prolonged
storage. Since canned foods are processed products likely to have
uniform composition, especially If all cans are from the same lot,
and are convenitmt for handling and storage, they were selected
for expel'imentation. Preliminary chemical tests carried out on
nine different fruits and vegetables canned in tin showed that
peaches and sauerkraut were too low in ascorbic acid content for
convenient analysis by bioassay, pineapple in heavy sirup dark­
ened excessively with the phenylhydraZIne reagent, and the red
pigment in raspberries made photocolorimetric readings difficult.
Tomato juice (two brands), "dextrose-sweetened" on~~!!e juice,
"sugar-sweetened" orange-grapefruit juice (two brands), grape­
fruit segments in heavy sirup, and spinach proved to be suitable
for the study.
Suflkient amounts of these canned foods for the entire investi­
gation were purchased at one time on the Washington, D. C.
market. Four cans of each product wel'e opened upon arrival at
the laboratory. Two of these were analyzed individually and two
were combined and analyzed for ascorbic acid, dehydroascorbic
acid, and reductones, and these results were averaged. Since
chemical tests showed that reductones were entirely absent from
the jukes and a small quantity only was present in spinach, as
received from the market, 24 to 30 unopened cans of each product
were stored at several elevated temperatures for different lengths
of time in order to produce the interfering suhstances it was
designed to study. Tomato juice was stored at room temperature
(22 9 -35° C.) for 1,19, and 30 weeks; all the foods were stored in
constant-temperature oven8 held at 45°,60°, or 73° C. for intervals
varying from 3 days to 6 weeks, depending upon the particular
experiment. To equalize the effect of temperature gradients within
the own, the positions of the cans were interchanged at weekly
.­
•
VITAMIN C-CUMI'AIUSON 0'" CHEMICAl. AN)) 1II0V)GICAL ASSAY
•
5
intervals during the long storage pel'iods and daily during the
short periods.
In order to hold the samples unchanged for bioassay or chemical
analysis, an equal number of cans of each food was quiek-frozen
as purchased and then held in a freezer at _40 0 C. until needed.
The cans that had been stored at elevated temperatures were
similarly frozen and held for assay after the required time in
storage had elapsed.
All five of the canned products were analyzed by the indophenol
method of Bessey and King (1), including H~S reduction to
measure both ascorbic acid and dehydroascorbic acid (15), and
with the addition of the Mapson formaldehyde procedure (22).
The Roe-Kuether phenylhydrazine method (dU), in which norit
is an oxidizing and adsorbing agent, was applied in preliminary
studiei;, but wai; replaced by the Roe-Oesterling (RO) (:18) proce­
dure, which s\lbstttutes bromine for 110rit and thm; eliminates
incomplete elution of ascorbic acid from norit ai; a source of error.
Parallel samples of stored and uni;tored orange-grapefruit juice,
tomato juice, and spinach were a!'iHayed biologically for vitamin C
value, for comparison with the chemical analyses. In addition,
these three products were analyzed by the Roe-Mills-Oesterling­
Damron (37) modification (RMOD) of the phenylhydrazine
method which differentiates between the ascorbic, dehydro­
ascorbic, and diketogulonic acids present in animal or plant tissues
and which ,vas published while this study was in progress.
CIIEMICAL PIIOCEIIl!ItE
EXTRACTION.-The contents of the cans of juices to be analyzed
were thoroughly mixed in a beaker under nitrogen. A sample was
removed and extracted under CO.,;' in a Waring blendor with
200 ml. of a mixture of 5 percent -HPO;: and 10 percent glacial
acetic acid at pH 1.8. The quantity of juice taken was in the
proportion of one part of :i uice to five or seven parts of acid,
depending lIpon the ai-icorbic acid content of the food. A weighed
portion of the slurry was filtered through glass wool into a
volumetric flask, and diluted to volume with the acid mixture.
The amounts of food and degree of dilution were calculated so
that 1 ml. extract contained approximately 6 to 50 Itg. (micro­
grams) of ascorbic acid; the final ratio of acid to food varied
from 7 to 40 parts to 1. Aliquots of this extract were used for
analysis by both the indophenol and RO phenylh~rdrazine methods.
For analysis by the RMOD method, extracts were prepared by
thoroughly stirring' in a beaker ('qual pal·ts. by weight. of the juice
or slurry and a lO-percent solution of Snel:! in 5 percent HPO;;,
filtering the mixture through glass wool, and adding Rufficient
5 percent HPO:1 to give approximately a 0.5 percent concentration
of SnCI:!, and diluting to a convenient volume for analysis.
'CO:! was preferred 1.0 nitro~en for its cooling effect, which helped offset
hesting cllused by hlclldor.
6
TECHNICAL nULLETIN No. 1023, U. S. DEPT. OF AGRICUI.1'URE
It was necessary to sample the grapefruit sections and spinach
in a different manner, The juice was first thoroughly drained
from the solid and both l)ortions were weighed; samples of the
solid and juice were ana YZt1d separately, Spinach was analyzed
also by combining aliquots of the juice and leaves in a Waring
blendor in a ratio corresponding to their original weights, Cen­
trifuging was substituted for filtering in separating the extract
from the solid material.
2,(;-DICHLOIWPHENOLINDOPJl"~NOL METIIOD.-Ascorbic acid was
measured by the method of Bessey and King (.l) and dehydro­
ascol'bic acid by reduction with H::S, as described by Harris and
Ol1ivel' (.15). The Mapsoll procedure (:!2) as modified by Hewston
and coworkers (17) was uHed to correct theHe values for the
presence of reductolles. 'I'he chief modification consisted in the
replacement of H:!SO'I with HPO;; for al'l'esting the formaldehyde
reaction.
Experiments of MapRon (22) had shown that iodine would
oxidize cCl'tain intel'fel'ing substance:; that tended to increase the
indophenol value and that were not amenable to the fOJ'maldehyde
correction. J<'ollowing this suggestion, an additional procedure was
introduced in these analyses by which bromine vapor, the oxidant
employed by Roe and otherH, was substituted for iodine as an
oxidant and the extract Willi then reduced with H.,S before
titrating- with indophenol. This is, in effect, the principle-employed
in the RMOD procedure, in which the final step, however, is
color production with phenylhydrazine,
The dye solution waH freshly prepared euch week and stored in
the dark at 30 C. Standardization of the dye with sodium thio­
SUlphate (4, i, ;!S) instead of ascorbic acid proved to be accurate
and more economical of both time and matel"ial.
2.4-DI NITIWPH ENYLHYIJRAZI NE l\fETHODS.-The Roe~Oesterling
method (8) waH employed without modification,
.
The Roe-MiIlR-Oestcrling-Damron method (7) wafol used for
diffel'entiating between diketogulonic acid, dehydroascorbic acid,
and ascorbic acid. These mlthors recommend a 6-hour incubation
of the extracts with the phenylhydl'azine reagent. Either a shorter
or longer period was more convenient in this laboratory, and pre­
liminary tests after 3 and 23 hoUl"s' incuiJation showed that the
23-hoUJ' pel'iod was satifolfactory, ThE.'refore, 23 hours were sub­
stitut£!d for the 6 hours of the originHI method.
The Evelyn photoelectric colorimeter, with the 540 m", filter, was
used for most of the spectJ'ophotometric readings: later, when a
Beckman spectrophotometCl", Model DU, became available, it wus
employed. Compal'ison of the two instruments, with identical
samples, showed that they could be used interchangeably with
no error.U
• Typical rl'sult!! with identkal sample!! read with both Evelyn colorimeter,
and Reckrnan spectrophotollleter: OrangL'-grapefruit jUice as purchased:
E reuding, :18.2 mg.; R rending, 37.7 mg. Stored orange-grapefruit jlJiee:
.E reuding, 10.0 mg.; n reading, 9.5 mg. Stored tomato juice: E l'eading,
!1.4 mg.; B n:uding, ,10.2 mg.
....
...
•
•
VITAMIN G- ('OMI'AIUSON OF ,'lIt:MICAI. ANI) 1lI01.0GlCAI. ASSAY
•
7
The Crampton bioassay procedure (6) which relates odonto­
blast growth to vitamin C intake, was used.
MANAGEMENT OF ANIMALS.-Weanling male guinea pigs weigh­
ing 200 to 250 gm. were purchased from one breeder as required.
They were given a basal scorbutogenic diet supplemented with
cabbage and kale until they reached 300-310 gm. in weight. T
During this period, which varied in length from 1 to 3 weeks,
de~ending upon how readily the animals accepted the new ration,
dally weighings were made. When the required weight had been
attained, the assay period was begun. At this time assay groups
of six to eight animals were given a modified basal diet, with
measured supplements of either the food under investigation or
pure ascorbic acid instead of cabbage and kale.
DIETS.-The basal ration used was a commercial pellet rat dietg
found to be free of ascorbic acid by chemical tests, supplemented
with 21 mg. alpha-tocopherol in cottonseed oil,1! and percomorph
oil to supply 2,770 IV (International Units) vitamin A and 350
IV vitamin D per week.
This basal diet, although not identical with the one used by
Crampton, was very similar to it. Yellow corn meal, which was
absent from the Crampton diet, was present in this one, and beet
pulp and fish meal, present in his, were absent from this diet.
Crampton used "dried, long-stored grass clippings" as roughage,
which he regarded as essential. Filter paper was used as rough­
age (5) in these experiments; one 2- by 3-inch strip per day per
animal was eaten readily, and produced the desired effect upon
fecal consistency. Guinea pigs fed this diet with a daily supple­
ment of either 100 gm. fresh cabbage and 50 gm. kale or 5 mg.
ascorbic acid grew steadily for periods up to 6 weeks and on
autopsy showed no signs of scurvy, such as joint hemorrhages,
softness of bone, rough hair, or lack of muscle tone.
Two conditions, which have been troublesome to other investi­
gators who have attempted to feed dry experimental diets to
guinea pigs (.7, 5), were also encountered in the pre~~ent study:
Refusal to eat, and dialThea of irregular occurrence, when dietary
ascorbic acid was not present in optimum amounts. 'Therefore, it
was found nece8Rary to furnish additional concentrates with the
basal diet, in order to stimulate appetite, and control diarrhea in
T Crampton prefers to place guinea pigs on the ~corhutogenic diet at 28 ± :l
days of age; however, sinc(' the animlll~ lIsed in this study were not raised
in this Ilihorarory, ;iOO-:310 gm. weight appeared to be II more constant
criterion for beginning the assay.
• Percent composition of commercial rat diet: Whole ground yeIJow corn,
20; whole pulverized oats, 10.25; whole ground wheat, 20.5; linseed oil meal,
5; soybean melt!. 5: dehydrnh~d beef meal, 5; beef liver meal, 2; powdered
American cheese, 4; skim milk powder, 7; dried cheese whe~', 3; alfalfa leaf
meal (U. S. No. L sun-cured, carotene content not less than 60 pg. per
gm.), 10; irl'ltdiated yeast. 0.25; iodized NaCI, 0.5; CaCO:1, 1; vacuum­
processed hone meal. 0.5; manganous sulphate, 36.8 gm. percent per ton of
reed; and cold-pl'esBed wheat germ oil, 8 oz. per ton of .feed.
• Alpha tocopherol acetate (Merck) in We!l!lon oil.
8
TECHNIC."I. IIULLE'!'IN No.
IO~3,
U. !:i. UEI''!', OF AGWGUlll'UH!o;
the assay animals, 'I'he following f;upplements were given b~'
(1) Approximah'ly 0.4 gm, brewer's yeast'" as an
aqueous f;uf;pcmlion, daily; (2) 0.45 ml. liver extract" twice
a week; (3) 100 mg. rutin'~ weekly tc animals under 400 gm,
in weight and twicc thifi amount to the heavit'r animals, The rutin
appeared to be particularly effective in prcventing diarrhea,
s~Tingc:
•
PREPARATION AND FEEDING 01-' ASSAY SUPPLEMtJNTS.-During
the assay the vitamin C supplements wel'c fed to the animals
thl'ice wcekly (.~5). As a standard of refercnce with each assay,
ascorbic acid was fed at three levels-3.5, 7,0, and 14.0 mg, per
week. It was freshly prepal'ed af; an aqueous solution beforc each
feeding period; feeding was complett'(\ within 15 minutes of the
time the solution W,:\·'! made, to avoid oxidative destruetion of
the vitamin.
The foods uncleI' aSf;ay were al';o fed at thrce levels, calculated
to supply vitamin C valuc equivalent to thc three ascorbic acid
le\"els. Six to C'ight guinE'a pigs for each af;say were fed the
scorbutogenic diet only, as negative controls,
The juices prf'senl:l.'d no unusual problems of sampling and
feeding. t;sualiy one or two cans, depending upon the number of
animal::-; to be fed, were opened under nit.. o~en every othe.. feeding
day; once a wcek samples we ..e withd ..awn for chemical anal~'sis
by both the indophenol and phenylhyd ..azine methods, and the
juicE' for the r1('xt feeding was st:QI'ed in hottles under nit..ogen
at ~)' C. The amounts of juice to be fed were calculated from the
total ascorbic add content of the food, as measured by the .indo­
phenol I·eactioll, co .... ected 1'0" reduct0lw::,;.
Canned spinach presented certain problem::.; in the bi()a::.;sa~·.
It was planned originally to w('igh the ::.;pinach leaf directly into
[('('ei CLIpS and allow the guinea pi~s to ('at it \'oluntaril~·. Howevel',
they had no liking for spinach, and frequently did not eat the
sUPj)lement for s(>\'eral hOllrs. Experiments showed that ascorbic
aCl( was lost from the leaf rapidly under these conditions. There­
fore, feeding by syrin~e became a necessity.
A ::-;Iurr,v consisting of a weighl'd amount of the leaf and a
proportional amount: of drained liquid, which had been made
3 percent acid with I-IPO:;. was prepared in the Waring blendor,
Sinec further dilution was necessary in order that the slurry
would flo\\' freel,\' in the (e('ding syringe, a quantity of 5 per'cent
mC'taphosphoric-l0 percent acetic- acid mixture equivalent in
weight to the acid juice in the sluI'ry was added. An aliquot of this
l:Hlspension was weighed, adjusted to pH4 with 2 N XaOH and
diluted with 0.05 N pH4 citrate to a volume such that 25 ml. of the
suspension was (·quivalent to .15 !,'1l1. of the diluted slurry. This
pI'ocedureresulted in a supplement of relatively low vitamin C
content, of which it was necessary to feed a large volume and
1. A suspension of 10 gm. brewer's type 200-B dry yeast, in 25 ml. dis­
tilled \\'ater. and. 1 ml. 95 per<;'t'nt ethyl alcohol,
II Lederle's concentrated liver extrad (injectable), ;) U.S.P, units per ml.
U Solution of 8 gm. rutin in 40 ml. propylene glycol, and 20 drops 50
peret:nt sucrose Bolution.
•
•
VITAMIN C-C.OMI'AJUSON .W CJU;MIGAL AN)) BIOLOGICAL ASSAY
•
9
which contained HPO a in appreciable amounts. In order to reduce
the volume of a sJingle feeding, one-half of the ascorbic acid value
required by the assay was given as pure ascorbic acid and the
other half as spinach slurry. The supplements were fed five times
a week instead of three, ns in the other assays.
In order to validate this procedure, the ascorbic acid controls
for the spinach assay nlso were fed five times weekly, and six
guinea pigs w()re fed a quantity of the slurry sufficient to assure
that they would get the equivalent of 1 mg. of ascorbic acid per
day as their sole source of vitamin C. This was the largest amount
of the acid slurry received by any of the animals on the spinach
assay. The addition of a small amount of sucrose to the feeding
solution increased the acceptability of spinach to the guinea pig.
The same amount of sucl'ose was fed to both the ascorbic acid
reference animals and to the negative controls.
Also, eight; guinea pigs were given 7 mg. ascorbic acid per pig
per week fol' (j weeks, with the addition of pure glucOl'eductone,
equivalent to 7 mg. of ascorbic acid. 13 The glucoredllctone was
prepared in the labol'atol~Y accOI'ding to the method of Euler
and MartiuH (!J).
HISTOLOG'/CAL PROCEDUKE.-At the end of 6 weeks, the experi­
mental klnimnls wel'e killed by chloroform, autopsied, examined for
gross signs of scurvy, and the lower incisor teeth removed for
histological examinatiOIl. The teeth were fixed, stained, and sec­
tioned as r1i!commended by Crampton (U). The final measurement
of odontoblast length was facilitated by projecting 11 brilliant
image of the section, enlarged approximately 1,600 times, to a
high-glaze! white paper. This was done by mounting a rig-ht-angle
prism to the 10 X ocular of the compound microscope, using the
4-mm. objective. One of the newer powerful microscope spot
lamps" was used directly beneath the condenser, The inner and
outer ma'l"gins of the odontoblast layer in the critical region of
mature odontoblasts (u) were tmced by hand on the paper; the
height of this layer was then measured with a centimeter ruler
to 0.05 mm. at five locations.
The places selected for measurement were chosen at random,
except that when the cells differed markedly in height, care was
tak~~n to include both long and short cells in those measured. The
aye rage odontoblast length of each section was finally converted
to microns by means of a fador obtained by projecting a stage
micrometer scale to the same magnification as the sections. It was
possible to measure the actual height of the odontoblast layer
to 1 micron.
Thl,'! projection method proved supeJ"ior to the filar micrometer
usually employed. and res lilted in a great saving of time and
eyest;rain. with no sacrifice of accuracy.
II On thc bllsis of til(' indophenol tit.ni:ion value, 0.79 mg. giucul"cductOjlC ill
equivalent t() 1 1lI~. aseorhk add.
.
.
I'
"FrCijl1cl hiJfh-inlcnsit}'
Spot-lit,·, dl,;'gk
1110dt'l, 10
TECHNICAl. BULLETIN No.
lO~:J.
U. S. ))EI'T.
AGHICULTUHE
(JF
Four sections of each of the two lower incil;or teeth of each
animal were examined and the results averaged, in evaluating
responses to the vitamin C supplements.
•
HESULTS AND DISCUSSION
Data from the ehemical analyses of the foods before and after
SUbjection to a variety of stomge conditions are summarized in
tables 1 and 2 and compared with the biological evaluation,r.
in table 3.
CANNED 'FOODS, AS PUlWIIASED.-Both dehydroascorbic and
ascol:bic acids were present in the canned foods amllyzed soon
after plII'chafie, with the exception of spinach, which showed no
evidence of dehyci"oaHcorbic acid. The dehyd"oasco"bic acid of
canned fruit!;; and juices rCI)I'esclltcd a somewhat larger fraction
of the total ascorbic acid than is usually reported for fresh
foods UfO).
TAI:IL"~
acid
1.·-E'Jj'cct of
stO/'n{le
t'<llllCS (1I1f!. pCI' .100
at, severol fCmpCl'(lt'iwe.'l1lpon (L.';corln:c
(J1l/.) (l'Iul 'illte1'fcn:lI(J sub.<;t(lllcCS in ,Ii
1UccuiII/'cd b!J the 2.u-dich!orophellolindo­
phcuol alld ;!,.Hli/l'itl'o]J/u'lIy'hy(//'(/zinc ·method.,;
('(oUH'd pl'Odllcf.,;.
(1.'1
-------_._,---_.­
I Ph,·n"l·
f
l'rodtlt"t nJld
J'llOfU~t·
t
i1udilinl1H
Itt':;ti!-Iy-Kill~
,I
II~S
,
Canned gl'apefl'uit (in "sirup"):
Segments; At purchase
8101'l.·d .10 days at !iO"C.
!
n04c.
Stored 5 days at 'i::J"C.
---,
22,li
14.7
2:3.0
14.!l
!l.5
27.8
17.n
(23.5 )
-"
;).~)
(U.5)
15.lj
(ll.!)
(i,a
(1:3.:1)
Canned orange juice ("swN·lel1('d wilh
dextrose") ;
Sal11plc I; At purehase
Slor'ed 21 days at '15"C.
Stored 5 days at 'iaoc.
Sample 2 : At purchllse
Slor'('d :I days ai 73°C.
If:!!"; tn'nl~
HrH'_
OI~Hif!~linK
nH.~nt
!
5.5
«(i.8)
Drained liquid; At pun·hast·
StoJ'('d 1.0 days at
Afu.j'
II!S
---­
hyd~nzine
AI'tt·r Orq
nl1tl"
1\"fOI'''
"
27.1
2:.1.8
(29.ft)
8.0
(11.3)
25.7
(::2.5)
2.:3
(15.9)
•
24.3 23.9 8.2
26.6 25.1 12.1
28.2
2a.7
(27.2)
10.1
( 12.2)
;W.:3
ao.o
:l5.ft
2!l.5
27.9 24.2 15.4
(Hi,6)
]5.6
(20.4)
11.3
:H.fl
18.1
(25.2)
:38.2
24.9
(:31.5 )
34.6
21.6
(30.0)
38.8 21.7 '\;;'8;;(;' Apj'H:ndix A for di:;tW;sion of dduils of bioussay and calculaliollil of
yit;alllill C pot.('l1cy.
•
11
VITAMIN (",.. cnMI'MHf;ON OF ('In;~fI(·'''. ANIi mOJ.O(;JCAI. AilSA:\'
I.-Effect nf .qtomoc at .q(',l)(wal tnnpcmt1l.1'c.'J u.pon (t,scorbic
acid va.lue.'1 (m,!/. lW1' .100 om.) u,lId 'i1It(l1'f(~1'ino substances in 5
canned products. (IS wW(I.';IIl1'ed b11 the 2,6-dichlorophmwlindo­
phenol and 2,~-di1l'it)'Ophenylh1ld1'Uzine methods-Continued
TABLE
Phenyl­
hydrnzine
I ndUllhenol '
Produet and a10rnlte c:ondit ionR
nt!K:ll'y-I{in~ Act~r
IIr.. Hud
Canned ornnge-grapefrult juil'e ("l!\\'I,·('t.
ened with sugar") :
Sample 1: At purchalle Stored 7 days at HOoO. Stored 10 day;; at, (iO°C. Stored 7 days at 7:.1°0. Sampl;J'2: At purchase
,
Stored 7 days at (iOoO. Stored 10 days at GOoO. Oanned tomato juice:
Sample 1:
At pUJ'chase
Stored 4G days at room tel11pera­
turc (22°_35°0.)
Stored 1:1:.\ days at J'()0111 tPI11Jlera­
ture (22°-:35·0.)
Stored 210 day!> at l'OO))) t.CI11Jll'I·H'
ture (22°_::5·0.)
•
Storcd 14
day~
at: 45"('.
Stol'ed 28 days lit 45"C. Stm'cd 5 days lit 7:1·0. Samplc 2; AtpUJ'chase Stored 5 days at 73°0. Canned spinach (drained leaves):
At purchase
Storcd 21 days at 45°0.
Stored ,l2 days at .t1l"0.
,Jtored 10 day!; at (iOoO.
Stored 7 days at 73°0.
-
Ro.....
II~!-; tr'(!nt- O~~fltl!rlinll'
Il,!forc
Aftlll'
IbS
II~:;
mcnt
;,12.0
28.0
3,1.8
36.3
34.2
33.1
10.2
(18.3 )
3S.8
2S.8
30.7
33.G
33.G
24.:1
8.9
(10.8)
:n.:1
;19.5
42.8
2G.4
2:l.5
aa.5
31.5
30.S
37.3
30.9
30.0
,15.4
W.O
17,4
lS.0
18.7
17.9
19.7
18.5
(22.2)
13.2
(15.4 )
15.6
(22.0)
17.0
8.1
33.3
1(i.O
Hi, 0
11.1l
( 1:1.8)
14.1
(i.0 (7.!i ) .17.(i
.13.1
1(i,8
14.9
( 18.4)
Hi.5
13.0
( 14.9)
11.4
(.14.6 )
20.0
22.0
17.6
20.0
17.8
11l.8
(lS.i!)
16.9
13.2
7.2
17.7 15.1
(I n.O)
J9.8
22.5
14.7
(1M)
20.5 18.a
18.2
19.0
20.(i
17.1
22.6
(::!-i.(l)
(:2(;.9)
lli.2
(17.2) 18.4
(20.3)
22.9
(23.5)
12.1l
10.5
(15.5)
(22.6)
(Hi.7)
18.4
I All values determined by indophenol titration
have hcen corrected for
interfering substances by the l\fapson fOJ'mllldehyde condensation: when only
1. figure is given, the cOlTection is zero, indicating that no reductones were
present; when a subtraction has been made for the presence of reductones,
the uncorrected "apparent aSCOI ~ic acid" figure is in parentheses.
~
~
2.-Effect of tempc1'(lillre alld storage on total a.sco·rbic aci{l, ascQ/'bie acid, dehydroascorbic aeid and. ;J
reductone content in 5 canned foods as memmred by the 2,6_(/-ichlorophenoii11dophenol-jonnaldehydc 'method :x;Q
Z
(8essey-King-Mupson procedure)
(3
TABLE
>
Total ascorbic acid
Tot.al
Product and
storat:e oonditions
l)f:'T
100
~m.
Chang-e
durinl:
acid
J/:;.
~tonlg'p
Content
Pen'CHt
My./
-3(;
-80
22.6
14.7
5.5
14.9
6.5
15.6
6.3
l"}r )00
,;tn.
-._--
j Pcrcolt
100 gill. :
27.S
17.9
5.5
All{larcnL I
Aftc:
, correctIon
DchydrO:lscorbic acid
Ascorbic acid
-,,--
Grapefruit:
Segments:
At purchase
Stored 10 days at 60 o e.
Stored 5 days at 73°e.
Drained liquid:
At purchase
Stored 10 days at 60 o e .....
Stored 5 days at 73°e.....
Orange juice:
Sample 1:
At purchase ................
Stored 21 days at 45"e.....
Stored 5 days at 73°e.....
Sample 2: At purchase ..............
Stored 3 days at 73°e.....
a~corbic
I
Contlt!nt
I
I
I
81
82
100
5.2
:t2
0
19
18
0
.7
0
-58
8.1
6.3
5.3
22
21
34
-35
:14.9
18.1
91
73
3.3
6.8
9
27
38.2
24.9
eo
Percent or
apparent.
aRcorbic
acid
I
I
38.2
31.5
Z
?
o
!= P<:rccnt.
:r. 27.8
17.9
5.5
15.6
6.3
l'7.9
13.3
36.3
'27.2
16.6
~
Z
,....,
j..'III.
JIg.
27.8
23.5
11.5
4
0
78
79
66
-17
Jig.
Percent
Mg';
100 gm.
28.2
23.7
10.1
36.3
30.0
15.4
I
Cont.ent
I..,r 100
=
c:
,
-~----
..
96
100
t"
Reductone interference
Distribution of total us(orLic add
I
0
24
52
13
53
e
to!
~
~:.­
::e
is
C'l
c:
:.;
36.3
23.7
15.4
0
13
7
38.2
24.9
21
0
c:
::e
l':
•
•
Orange-grapefruit juice:
Sample 1:
At purchase
Stored 7 days at 60 C.
Stored 10 days at 60 0 C.
Stored 7 days at 73 0 C.
Sample 2:
At purchase
Stored 7 days at 60 C.
Stored 10 days at 60 c C.
Tomato juice:
Sample 1:
At purchase .....
Stored 14 days at 45 0 C.
Stored 28 days at 45°C.
Stored 5 days at 73 0 C.
Sample 2:
At purchase
Stored 5 days at 73 C.
Spinach:
Drained leaves:
At purchase ............ .
Stored 21 days at 45 DC.
Stored 42 days at 45 0 C.
Stored 10 days at 60 0 C.
Stored 7 days at 73 0 C.
0
0
0
t
a4.8
aa.13
:.11.3
8.1
42.8
:33.5
:n.5
16.0
13.2
16.8
13.0
20.0
15.8
3
---10
-~,77
-22
- -26
az.o
~8.0
24.3
8.9
39.5
26.4
23.5
92
83
78
100
fl2
79
75
.)
'f
~.~
7.1
8.0
o
8
21
25
84
46
4
13
16
54
88
85
2.4
!!.4
12
15
fli5
87
·19
21
17.6
13.4
o
o
34.8
33.6
31.3
'10.8 _
8
17
22
.6
1.7
2.7
7.0
15A
11.5
14.1
6.0
18
2.8
5.6
7.0
42.8l
a3.5
:31.5
!
I
34.8
33.6
31.3
8.9
42.8 ;
a3.5j·,
31.5
16.0
15.4
16.8
14.9
16.0
13.2
16.8
13.0
20.0
.18.5
20.0
15.8
o
o
o
18
o
o
o
o
o
14
13
o
~
<
:;
>
ll: Z
~
""
e-
ll: ~
~
~
i
:::. "'! 15 ~
~
ll:
15.1
18.3
2D.6
18.4
12.5
+21
-~36
-~
22
:-17
17.7
14.7
20.5
16.2
80
100
88
3.6
.1
2.2
23
o
12
19.0
'16.0
24.6
20.3
22.6
15.1
14.7
20.6
18.4 I
12.5
20
8
16
9
45
Reductones in this product were destroyed by II;!S treatment; this value represents reductones present before H;!S treatment.
>
~
>
z
-':::
g
g
to
>
'£
1'1>
>
0(
~
w
··7~
.
3.-Comparisun of the 2,6-dichiol'uphellolin<iophenol ami. 2,J,-dillit'rophe'tylhydl'azine methods ·/cith the
Crampton bioassay in the 'measurement of vitamin C (mg. per 100 gm,.) in J canned foods, before mul after
storage
TABLE
~
~
..:
tr.
-
----,----~---.-..
D.~t"(,'I'if1tion
(If sample
f;r
----.~---
hio:H;!-\ay
Orange-grapefruit juice:
Sample 2:
Held at -40· C. fo), I8!) days hefon' ;.;tart of
assay.
Befnt°t.'
H~
I
I
;\ I
I
After storage at -no C. for 5 days, held at
__ 40° C. 189 days before start of aSiiay.
0
-----. --_.-
- ..
~
_----
l'l
:3
z
30.5
30.0
29.5
32.61
29.4 .
30.7
31.6 •
(33.6) .
30.4
30.5
30.4
32.2 1:13.0 (89-162 percent)
(332) ,
17.6
13.0
(13.1)1'
11.8
(12.8)
15.5
(16.3) ;
36.0
:l3.1 I
30.3 ' = =
I
6.6
(9.4)
7.5
(12.7) ,
17.9 I
(27.5) j
11.5 I
(21.7)
13.2 I
(24.2) ,
11.6 I
(24.4)
10.7 i
(23.6)
9.5
(23.3)
8.3
14.2 ;
10.6 ;
I
23.5
18.3
3
1
Tomato juice:
Sample 2:
Held at -40 C. for 1 day before start of assay.
c
I
.
---~---
30.8 I
10.7
Average
Ro...·":
1
hioas,,~s
31.7
1
4
~
~
Cramllton;;
Roe-Millo,Ot>sterlinJ;-"
treatment j Oest.;>dm!-!"l Damron
After"
H~
41~
Average
Aft"r
Br:,!-H::S
...-.._-_.-
------
I'!
----~1
BeHHey·Ki n~
\\·f"'\,k
c::
Phenylhydl'Hzille:
J ntlophenol 1
I
1
18.0
2
17.1
22.3
4
17.1
19.4
i
I
(23.6)
19.8
I
I
15.6
(20.3) I
17.4
16.6
17.2
33.1
(35.1)
35.5
(34.5)
28.0
(30.0)
r­
~
I
21.8
~
-;
c·
-.;
I
::­
:>
13.4 f !l.6 (75-136 percent)
(14.1)\
S
~-= ~
22.4
20.1
c
;r.
~
!
(21.1) j
15.0 I
19.9
(15.9) .
•
•
ti
8
i
21.7
1!I.7
19.7 '
15..l :
20.2
18.3
20.1
21.4
18.3 r---ZO-:8"I'-16.2 : 19.4 (75-180 percent)
( 17.1)
15.9
----
Average
t
16.7 I
After storage at 73" C. for 5 days, held at
_40" C. for 1 day before 5ta,·t of assay.
1
2
4
![
I
I
8.6/
9.0
I
!J..J ;
I
I
!
8 I
6
A.verage
....... ............. .
Average
-----]:(8
5
14.:l
(17.5) ;
1:3.7
14.2
J--t
I
]3.0
(16.8)
13.0
t ~-I
14.6
( 18.8)
16.2
--~I
13.2'
(17.9).
J 4.4 I
13.4
(20.0)
14.0
8.6
(12.6)
9.7
(13.9)
1:3.5
~---
5
'-.
10.9
(14.2)
9.4
(13.5)
12.1
(]3.9)
---.--1 1
Average
Aftel sturage at 45" C. for 42 days, held at '
40' C. for 51l days before start of assay.
!L5
9.2
,.
Spinach:
Leaves and liquid:
Held at 40" C. for 5\; days befOl'C start of assay.
9.-J
(21.'i )
1.4.8
I
13.2
I
I 13.0
I
j
,12.1
12.4
(18.9)
16.0
13.1
]8.6·~
19.{j
8i!:
9.9
(10.5)
10.2
(10.9) J
8.5
(9.4)
"CI
>
I
]1.1
!
f
'''.5,
12.9
~
z
(14.0)
13.9/- - 18.6 I
(18.4)
I
17.2
r
14.6
<:
10.7,
12.7
L
15.9 '
( 17.0)
14.2 ;
(14.2 )
i!!
y:
o
z
c·
"!
10.51 U.(i (92 -170 pcn·t:nt) C toJ
==
(11.2) ::C='---.. c - - : - = ::
..
18.1
;;( 19.2)
r~-
>
]8.1 122.1 (fl6-221 percent)
o
20.6
(22.7)
1~6- I-Zo.6
~
~
IIG.l (82-187
PH~~nt) r­~
1 All values determined by indophenol titration have been corrected for interfering substances by the Mapson formaldehyde con­
densation: when one figure is given, this correction is zero, indicating that no reductones were present; when a subtraction has
been made for the presence of reductones, the uncorrected "apparent ascorbic acid" figure is in parentheses.
: The figures in parentheses represent ascorbic-dehydroascorbic acid before the correction for diketogulonic acid has been made.
'Figures in parentheses indicate exact fiducial limits for 95 percent of the cases.
>
~
~
.
.....
01
16
,'ECHNICAL HUl.LETIN No, 1023, U, S, DEPT. 01-' AGIUCULTURE
The juices and fruit gave no evidence of reductones by the
Mapson formaldehyde correction. Spinach, on the contrary, con­
tained small amounts of reductones following treatment with
H!,!S. Smythe and King (Sf)), and Miller (2U) have shown that
reducing substances may actually be formed in plant or aTlimal
m~terial by reaction of H~S with many carbonyl compounds,
including intermediates of carbohydrate metabolism, and a num­
ber of qui nones. Not all compounds of this nature are differen­
tiated from ascorbic acid by the Mapson procedure.
Only traces of diketogulonic acid were indicated (table 3) in
the freshly purchased canned foods that were analyzed by the
RMOD method (27). The vitamin C value of the unstored juices
measured by bioassay agreed well with the chemical measure­
ments, and the chemical methods agreed among themselves. It
appears from these experiments that the original Bessf;y-King
method, omitting even reduction with H~S, is the simplest and
most practical chemical procedure to employ in routine analyses
of foods, and at the same time accurately represents their vitamin
C value, provided it is first ascertained that the proportion of
dehydroascorbic acid is relatively small, and that no interfering
reducing substances are present.
The vitamin C value of the canned spinach, measured by bio­
assay, was higher than the figures obtained after taking into
account the reductones and diketogulonic acid present. The reason
for this is not clear, and none of the data explain this discrepancy.
CANNED FOODS, STORED AT ELEVATED TEMPERATURES.-Changes
in both ascorbic acid and a:.;corhic acid-like substances occurred
in these canned foods during storage at elevated temperature:.;
for short periods of time. The higher the temperature, the more
marked a.nd extensive were these alterations. These data are
presented in table 1 and fmmmarizetl in table 2.
Tomato juice was the most Htable of the products studied.
Spinach was unique in that there appeared to be actual increases
in the ascorbic acid during holding at a moderately high tempera­
ture (tables 1 and 2) ; it was only when held at 73° C. for 7 days
that a loss of 17 percent occurred. Table 2 also demonstrates
that falsely high ascorbic acid retentions would have been re­
ported for approximately half of the stored samples if no correc­
tion had been made forreductoneR, and emphasizes the necessity
for measuring reductones in foods which have been subjected to
heat or storag-e.
The proportion of dehydroascorbic acid in the total ascorbic
acid increased at elevated temperatures except in the grapefruit,
and orange-grapefruit juice held at 73°. In these instances,
dehydroascorbic acid was ausent, preHumably because destruction
of ascorbic acid was proceeding so rapidly (table 2) that dehydro­
ascorbic acid was disappearing as soon as formed.
Although the results of the several chemical methods agreed
reasonably well with one another and with the bioassay when
the unstored foods were analyzed, this uniformity did not occur
•
•
•
VITAMIN C-COMPARISON 0.' CHEMICAL AND DlOLOGJCAL ASSAY
•
•
17
in the stored foods, except tomato juice, in which agreement
among all the methods was good throughout.
'
The Roe-Oesterling procedure gave somewhat high values, espe­
cially for the grapefruit segments, orange-grapefruit juice, and
spinach: in some instances the RO values agreed more clostlly
with the uncorrected than with the formaldehyde-corrected
vaiues. From this it is concluded that certain reductones for which
the Mapson method makes correction are not completely removed
by bromine oxidation. In other instances the RO value was lower
than the corrected Bessey-King figure. The most obvious explana­
tion for these reactions is that under some circumstances, reduc­
ing substances are formed in foods for which correction is made
more adequately by bromine oxidation than by formaidehyde
condensation, However, the fact that "reductone:;" differ among
themselves quantitatively in the intensity of color produced with
the phenylhydrazine reagent, makes interpretation of these figures
ditlicult.
Penney and Zilva (31) have published results comparing the
chromogenic activity of the compounds formed with phenylhydra­
zine by a number of such SUbstances, including glucoreductone,
reductic acid, and ascorbic acid, Further, these investigators
showed that there were also differences among these reducing
substances in their reactivity toward indophenol. In the present
study, comparison has been made between ascorbic acid and
glucoreductone in their reactions with both phenylhydrazine and
indophenol (see Appendix B) .
In geneml, the results obtained using the Br2-H:?S treatment
agreed better with the indophenol than with the phenylhydrazine
values; however, this was not invariably true. The vitamin C
activity of the stored orange-grapefruit juice indicated by bio­
assay was lower than the ascorbic acid values obtained by any
of the conventional chemical methods, but agreed well with those
given by the Br~-H~S procedure, Stored tomato juice presented
.fewer problems in chemical analysis than did orange~grapefruit
JUice, possibly because of ibl lower sugar content. Likewise, the
bioassay agreed well with all of the chemical methods. Spinach,
when tested biologically, showed a potency higher than that given
by the two indophenol methods, and lower than the two phenyl­
hydrazine procedures.
The reductones present in the canned foods had no vitamin C
activity. Bioailsay of pure glucoreductone served as further evi­
dence of this fact (table 4). This is in agreement with the obser­
vations made byE~ller and Klussmann (8) and Cruickshank as
cited by Harris and Mapson (H) that "reductones" do not pre­
vent scurvy in guinea pigs.
The problems presented by the vitamin C analysis of processed
or stored vegetables and fruits are complex. Neither 2,6-dichloro­
phenolindophenol nor 2,4-dinitrophenylhydrazine is specific for
ascorbic acid. 'rhere are many reducing compounds other than
ascorbic acid which are formed in foods during preparation for
eating, and which read with the two substances more or less
18
TECHNICAL BULLETIN No.
IO~3.
U. S. DEPT. OF ,\GIUCULTURE
readily, depending upon the conditions under which the analyses
are performed. Some of these "reductones" are artifacts produced
by the reagents used in the analysis.
There is no single chemical procedure which can be employed
routinely for the ascorbic acid analysis of fruits and vegetables
in which reducing substanees are present which behave like
ascorbic acid chemically but not biologically. The bioassay, par­
ticularly the Crampton odontoblast-growth method, is specific for
vitamin C activity and although less precise than chemical tests,
remains the only sound method for the evaluation of vitamin C
potency of such foods.
•
SUMMARY
1'he mcmmrcment of biologically active ascorbic acid in foods
by chemical means is often made di/licult by the presence of
biologically inactive substances which react with 2,6-dichloro­
phenolindophenol and 2.4-t\initl'ophenylhydrazine, the two com­
pounds commonly ufo;ed in this meafo;urcment. 1'hese interfering
reducing substances are most often encountered in veg'~tables or
fruits which have been processed or stored and may give rise
to crr'oncow;I~' high values when vitamin C is measured by
chemical methodi'\.
I!1 the cxperi!11cnt:-; rep?rted here. five canned pr?,ducts-g~ape­
frUIt segmenti; In heavy sIrup. "dextrose-sweetened orange JUIce,
"sugar-sweetened" orange-grapefruit juice, tomato juice, and
spinach-wcI'e analyzed for a!;corbic acid, dehydroascorbic acid,
and interl'cl'ing substances behaving like a:-;corbic acid by methods
which employed both indophenol and phenylhydrazine. lnter­
ference was removed by either formaldehyde condensation, bro­
mine oxidation. 01: both. The canned foods were analyzed as
received from the market, and after storage at room temperature
and at elcvatC!d tempemturcs for relatively short periods of time.
Parallel bioassays by the Crampton odontoblast-growth method
were performed with three of the foods-canned orange-grape­
fruit juice, tomato juice, and spinach-before and after storage.
The studies indicated that these canned foods, as received from
the market, contained the largest proportion of their ascorbic acid
value as ascol'bic acid, although more was present in the dehydro­
form than is usual in the fresh products. Interferences, rnmovable
by either MapRon's formaldehyde condensation procedure or
bromine oxidation, were absent. The results of all the chemical
methods, as well as the bioassay, were in good agreement. Canned
spinach Was exceptional in that it contained no dehydroa!'!corbic
acid and gave evidence of redl/ctones by the formaldehyde reac­
tion. It is concluded that lise of the relatively simple Bessey-King
indophenol method i!-! ju!-!tified as a routine procedure in the
analysis of fruits and vegetables for biologically active ascorbic
acid, if pr:eJiminary test:.; have first eliminated the presence of
other reducing substances as a source of error. Even reduction
with H ~S may he omitted as part of the routine. provided it is
•
•
•
first ascertained that the proportion of dehydroascorhic acid is
negligible.
Storage of the canned foods at elevated temperatures "cl'ulted
in losses of al;corbic acid and inc..eases in the dehydroascorbic
acid fraction, except when dest..uction of asco..bic acid was accel­
erated. Relatively la ..ge amounts of "reductones" were also pro­
duced. If no co ....ection had been made fo .. the biologically inactive
"reductones," the vitamin C ..etentions would have appeared con­
siderably higher than those shown by bioassay. However, the
Mapson formaldehyde condensation, 01' b..omine oxidation, or a
combination of the two, g-ave chemical estimates of vitamin C
value which agreed well with the bioassay in two of the foods
that were analyzed by all of the procedu ..es.
Orange-grapefruit juice, which was relatively high in sug-a..
content, decreased more in vitamin C value and gave evidence
of the production of a mOI'e complex a ..ray of inte..fering· reducing
compounds than did tomato juice. Canned spinach behaved in
an anomalous manner, the reaSons for which are not apparent.
The bioassay value of the canned product, as pu ..chased, was higher
than even the unco....eded chemical measu..ements; sto..age at
high tempe..ature decreased only slightly both ascorbic acid
(chemical) and vitamin C value (biological).
These stUdies indicate that when extraneous reducing sub­
stances are present, no single chemical pl'ocelllll'e as ~ret devised
can be applied to all types of foods to obtain an acc:u ..atc measure
of vitamin C potency, Neither the .2,(i-dichlorophenolindophenol
nor the 2,4-dinit..ophenylhydrazine reagent is specific for aHcorbic:
acid, but each reacts with a vHI'iety of reducing compounds,
under the conditions of the tCflb,. No sint\'le method or combination
of methods used in these studies eliminated all intel'ference under
all circumstances,
Under some conditions, the oxidizing ~lI1d reducing reagents
themselves reacted to form additional interfering compounds.
A further complication is introduced in that the various reducing
substances react quantitatively to different degrees both in the
titration of indophenol and the production of color with phenyl­
hydrazine.
Bioassay remains the only specific measure of vitamin C in the
presence of interfering redlldn~ Huhstances, since no existing
chemic:al procedure is entirely adequate JOI' this purpose.
1.ITER'\TLIRE CITED
•
(1 ) BESSEY. O. A" and KING. C. G.
193:3.· TilE IH!':TIURUTION O~' VITAMIN C IN PLANT AN.D ANatAL TISSUES,
ANI) ITS 11En~IIMINATION. Jour. BioI. Chem. 103: 687-698.
(2) BLISS. C. I., and MAIIKS. H. P.
1939. Tilt: RIOJ.(I(;II~AI, ,\SS.H OF INSULIN. II. TilE ESTIMATION OF DRUG
POTENCY FROM A I:RAIlEJl RESPONSE. Quart. Jour. Pharm. and
Pharmacol. 12: 182-205, ill us.
(3) BOOTII, A. N .. ELVEIIJEM. C. A., lind HAIIT, E. B.
1949. TilE IMPORTANCE (W RIJLK IN TilE Nl!TIIITIf):>; (W THE GUI:>;EA PIG •
Jour. Nutr. 37: 21;:1-274.
20
'I'tx;tfNICA1. JlUJ,{.fo..'·IN No. 1023. U. S. VEI''I'. (II' MiIUCUL'I'Ullt:
(-I) HUCK. R. E., and RITCIIIE, W.
(5)
(6)
(7)
(8)
(9)
(10)
(1I)
(12)
( 13)
(14)
(15)
( 16)
(17)
( 18)
S.
1!I:iS. A NEW METHOD f'OR Til.: $TANJlAIIJlI1.ATIO;'I: O~· Tilt: I.n: USEII FuR Til.: DETERMINATIIlN "" rF.VITAMIC ACIJl (VITAMI!\: c).
(Ab­ stract) Indus. lind Eng-in. Chern., Analyt. I-~d. 10: 2fi. COllEN, H., lind MENIIEL, L. B. 1918. EXPERIMENTAL SCURVY (IF THE GUISEA PH: IS ItELATIOS TO THE DIET. Jour. BioI. Chern. 35: 425~453. CRAMPTON, E. W. 1947. TilE IiR(lWTH (It. TilE ODONTOBLASTS OF Til.: INCISOIt ToOTH AS A CIUTERION tit· THE VITAMIN C I!\:TAKE Of' TilE IiUINEA PIG. Jour. Nutr. ;1:.1: 491.504, ill us. DICK, H. .\!I:.I1. Disscrtlltion (Pl'illlkJurt) quoted 1.9:1:1 in Handbuch del' Pflan­ zenlinalY!le. cd. G. Klein. Viennll. EULER, H .., WiN. and KLUSSl\lANN, E. Ifl33. /'11 YSIOLOmSCIi t: VEItSU<;IIE \)Bf:1t VITAMIN f' (ASCOItHINS'AURE) UN!)
RE!)UKTON
(ENOL-TAItTI""N AUIEHYII).
Hoppe-Seylers Ztschr..f. Physic.!. Ch(!rn. !.! 17 : Hi7-17(;. - - - and MARTIUS. C.
\!I;!:'l. ·(JHf:lt. £1 N HOCIiItEIIUCIEItEN()ES Zl.ICI..:ItIl.:ItIV AT (II.:IIUKTON).
Svcnsk. Kern. Tidskr. 45: 7:.1 74.
- - - - and MAIITIUS, C.
19:13. UDEll REIIUKTON (EN(lI.-TAItTIlONALm:HYIJ) UNIJ ASCC/KHL"'SAURE. Justus Liebigs Ann. del' Chern. 505: ia-87. FEASTEIt, J. F .• TOMPKINS, M. D., and P.:ARO:, W.E. 1949. E.·.·ECT (It' STORAGE ON VITAMINS AND QUALITY IN CA:-'-NIW t'VOIIS. Food Res. 14: 25· :.19, iIIus. GOLUBLlTIf, S. A .• and HARItIS, 'R. S. 1948. ESTIMATlllN OF ASCORBIC ACID IN FOOD PREPARATIONS. Analyt. Chern. 20: 649-651. GUERRANT, N. B., VAVIGII. M. G., and DUTCllf:It, R. A. 1945. NUTRITIVE VALUE OF CANNEU H'(lJlS. INFLUENO: OF n:MPERA­ TUII.: AN!) Tun: Of' STOItAI1E ON VITAMIN CONTENTS. Indus. and Eng-in. Chern., Indus. I-:d. :37: 1240-124:1, ill us. HARRIS. L. J., und MAPSON, L. W.
1947. DETERMINATWN ot' ASCOIIIIIC ACIII IN PRESf:NCE OF INTERf'EIUNf: SUBSTANCES BY TilE 'CONTINUOUS-.'I.(,W' METHOD. Brit. Jour. NutI'. 1: 7-.:18, ill us. - - - and OLLIVER, M.
.1942. VITAMIN METHODS. 3. THE RELIABILITY OF TilE METlW[) FOR ES­
TIMATING VITAMIN C BY TITRATION AGAINST 2 :6-OICHWRO­
PHENOLINDOPHENOL. 1. CONTROL TESTS WITH PLANT TISSUES.
Biochem. Jour. as: 155-182, ilIus.
HARTZLER, E.
1948. FALSE HWU VALUES f'OR ASCORBIC Aem IN GUAVA JIJICE. A NOTE
ON TilE USE OF THE COLOIUMETRIC METHOD WITH 2,4-OINITRO­
PHE:-'-YLlfynRA1.INE. Jour. NutI'. :15: 419--424.
HEWSTON, E. M., DAWSON, E. H., ALEXANDER, L. M., and
ORENT-KEILES. E.
1948. VITAMIN AND MINERAL CONTENT OF CERTAIN FOODS AS AFFECTED
BY 1I0ME PREPARATION. U. S. Dept. Agr. Misc. Pub. 628, 76
pp., ilIus.
IRWIN, J. O.
1943. ON THE CALCULATION OF THE ERROR OF BIOWGICAL ASSAYS. Jour.
H3'1. [.London I 43: 121-128, ill us.
•
•
(19)
(20)
1944. A STATISTICAL EXAMINATION OF THE ACCURACY OF VITAMIN A
ASSAYS. Jour. Hyg. I, London 4a: 291-314.
KUETHER. C. A.. TELFORD, I. R., and ROE. J. H.
1944. THE RELATION ()f'THE Bwon LEVEl, 0 .. ASCORBIC ACID TO THE TIS­
SUE CONCENTRATIONS OF THIS VITAMIN AND TO THE HIS­
TOWGY OF THE INCISOR TEETH IN THE GUINEA PIG. Jour. NutI'•
28: 347-368, iIIus.
J
•
VITAMIN ('
('OMI'AIU!;ON tit' tJllf:MICAI. ANI) 1II01",'GII:AI. .ARRAY
21
(21) LUGG, J, W. H,
.1942, TilE USt: OF ~'nIlMAI.mmYllt; ANII 2.6-HICIiLOIlOl'IIF.NOLINDII­
Pllt:NOL I N 'I'll t: Ef.iTI M ATION (I~' ARCOIUtl('ACm ANIl 11.;11 YDRtI­
ASCfllUIJC "CII), Allstral. .JOIII·. I~xpt. BioI. and 1\f('d, Sci. 20:
27:1.285, illus.
(22) MAPSON. L, W,
l!)4;1.\'IT";\IIN ~1t:Tllons. \'1. Tilt: t;8'rIMATION (W .ASCflIUlIC ACllJ IN THE
PRt:St:NCt: m' 1It:IIIJCToNt;S ANII AI,l.IED SURSTAN(,ES. Soc.
Chem. Indus, Jour. Tl'llns. and COll1l11un. (;2: 22:'1~2:12. ilIus.
(2:!) MEN,\Kt;R, .M. H., and GUEIlRANT, N. B.
I!I:.I8.
(24)
STANI!AlUlI1.ATION Of' 2,(;-111('11 1.0I101'lIt:NOLl !IO[lOPIU:Ntll.. AN 11'01­
1'ltOYEil ;\I t:T 11 (II), Indlls. and Engin, Chcm., Anal~'t. Ed. 10:
25 2(;, illlls.
.
l\fll.um, E. S.
19:1.. , AI'I'LICATION (I~' (IU"NTlT,\TI\'~J Sl't:CTII,\L ,\NAI,Y"t;S 1'0 RINARY
l\IIXTUItES OF TIIt~ ('0l\1l\10N (' AIIOn:N{lIDS. 1'111111; Physiol. 9:
681-78".
(25)
W:.IS, 1'1I0TOELfXTIIIC SI'fX:TlWI'W.TOMETIIY AI'I'Llt:1)
1'(' TII~J (IUANTITA1'IV': AN,\I.YS':S (lP CMIOTENom AND ('IILOIIOI'IIn.t. 1'H:;\n:NTS 1:-':
n:II:o.:'AIIY,\NIl QI1,\n:IINAIIY sysn:;\Is. C('J'(~al Ch(!II1. Iii: aJO··
aI0,
HillS.
(2(;) )IIL"':II, M. C,
In·II, IIt:IIU("fONf: INn;IIFEllt;Nn: IN t;STIM,\TIOS
(27) MILLS,
•
(28)
OF \'11''\.\1/:0.:' ('.Food
Res. 12: :1"~la5!), illus.
M. 11., n,\;\IIWN, C. M., lind ROt" .1. Ii,
1!l4!l. Mi('OUIlIC M'II), nEIIY(lUOAH(,OIUlIt' .,\CW, ANti IllKt:TOGULONIC ACIII
IN FUESII "",(l I'IWC~;S;;EIl "'(lr.n~;. Analyt. Chcm. 21: 707-709,
Hlus,
n nd ){Ot:, ,J. H.
.\947, A ('I(('I'I('AI, STUD" (W l'II\II'OSf,l) ~lOnn'I('ATJ(l:-';S o I" TilE ItOE ANI)
KU~:TIlt:U~";l'IJ()1) FOil 1'lIf: m;n:U;\IlNATION (W,\SCOItBIC "cm,
WI'I'II FUUTIIEII COI'TIUIIUTIONS TO Tilt; (,IIt:MISTII'" OF' TillS
l'IWn:II!.llIf:. ;I(lur. BioI. Chclll. 170: 15!1 )(;1, ill us,
(29) OI'I·t'NIIEL\It:U, G.. 8n:IIN, K, G., and ROMAN, W., <:Ollllb.
I n:l!l. IIJ(lLl.I(ac ,\I, ox illATION. ~{I i pp. The Hl\g'lI~·.
(:10 ) OSt:u, B. L. I!150. I~ood. Anlliyt.. Chcll1: 22: 221 ~227. (31 ) Pt;NSt;Y, ;1. R., lind ZII,VA, S, S.
Hi4;3, Tilt: 1.1.:,..;11;\11 NATION (It' 2 :;{-lllln:To-I-!;\JLONIC J\('IJ). Ri(lchem .
•Iour. ;1/: ;.I!l~44, illlls.
_..." - .. anei ZII,\'A, S. S.
(:12)
I f14:1. 'I'lIt: ('IH:~lIe AI, liE II " ywun Ot' nt:IIYHltO-I-ASC(lRBIC ACIO IN VITIlI)
ASH II' \,IVO.
Bi{,lchcm, .IOllr. :"17: "O~I "17, illus.
(:1:1) anei Z 11,\',\ , S, S.
19·15. IN'I'EHFEItING SI1I1STA,,'('t:S IN Tilt; IWt: '\:0.:'1) KUt:TII.:1t METlH.[l t'.,1t
TII~; Ot:n:ItMIN,\TlnS III' MWOItIlI(: ,\CIIl.
Rif,('hclll. ;I(.ur. 39:
an2
(:l,U PIJO,\N,
(15)
(:lIj)
•
.~197.
~I., and Gt;IIJO\'ICII,
H ••1.
19·1(i. Tilt; USt] or 2,4-IIlNITnol'lIt:Nn,IIY()IIA1.I="E Fnlt Tllf: nt;TEItMINA­
TIM; Of' Mi('(.ItIIIG ACIIl. Science (n.s.) ]03: 202-20:1.
POLl,AIIIl. A .• KIESEI(, M. ~-:., and STF:t:n:\IAN, .J.
19-14. Tilt: M;(,OIUlIC ,\CIll ('ONn:="T 0.' SOME FItUIT SYltllPS ANII OTIIEII
l'II01Hl(:TS,
SII<:. ChclIl. Indll;;, .Jollr. 'I'mn5. lind Crolllllllln. 6:{:
215218, illll:l,
ROE, .J. H .. IlndKIJf:TIIEIt. C. A.
194:.1. TilE UEn:UI\II=".ATION ot' .ASCOIIBIC ACII) IN ,,"II0LE IILOOI) ANI)
URINE TIlItOI]GII TIfE 2.4-J)l:o.:'ITIIOPIfENYLHYIlRAZINE IlEItIVATIVE
OF DEIIYDIlOASCORBIC ACIil. Jou!:. Riol. Chem, 147: 399-407,
lHus,
MII,I.!;. M. H., OESTEIII.IN(;, M. J., and DAMIl"N, C. M.
(It'
IIJKt."(I-I-(WL(lNIC Acm. DEHYDRf,-I­
M1CflIlRI(' A(cm, ANI) I-ASCORBIC Acm IN TilE SAME TISSUE EX­
TRACT BY TilE 2,4-IIINITROPHENYLIlYDRAZINE METHOD. Jour.
BioI. Chem. 174: 201-208, illus.
C:18) - - - and OESTEItLING. M. J. 1944. TilE UETERMINATION (It. UEIIYDROASCORBIC ACID AND ASCORBIC ACUI IN PLANT TISSUES BY TilE 2,4-nJNITROPIlENYLHYDRAZINE METW)J). Jour. BioI. Chem. 152: 511-517. (39) SMYTHt:, C. V., and KING.• C. G . .1942. A STUDY ot' ASCORBIC ACJJ) SYNTHESIS BY ANIMAL TISSUE IN VITIIO. Jour. BioI. Chem. 142: 529-541. (4(1) Sl':t:ot:COIt, G. W. 1 !140. STATISTiCAL METIJODS AI'PLIED 1'1; EXPEfUMENTS IN AGRICULTUIlE
,\Nri BI(H.O(;\'. F.d. a, 422 pp., illus. Ames, Iowa.
(41) SNOW, G. A., and ZILVA, S. S.
H14a. A (:IIITICAL t:XAMINATION Ot' LUGG'S METIIOD .·OR THE DETERMI­
NATION OP I-ASCOIUIIC M:JI). Biochem. Jour. 37: (i30-li40. illu~.
(42) - - - lind ZIL\'A, S. S, IH44. A ClIITICAt, EXAMINATION (W I.UGI:'S IIU:TIIOI) .'Olt TilE UETERMINA­
THIN Ot· I-AS('OIlRIC Aem. 2. Biochem. Jour. 38: 458-467,
ill us. (-Ia) WOKES, F.. OnGAN, J. G., DUNCAN, J., and JACODY. F. C. 19·':3, AI'I'AnF.NT VITAMIN C IN .'(IOI)S. Biochem. Jour. 37: 695-702, (:17)
J !HFI,
,.11.: ...:n:IlMINATIfIN
illu~.
OIlI:A:-I,
(-1-1) -
•
.1. G.. and JACOBY, F. C. 1!).1:1. TilE .:STIMATJ(\N 01' Al'l'AnENT VITAMIN-C IN Foons.
Soc. Chem. I nllus. Jour. Trans. and Commun, 62: 2a2-2a6, illus. (-15) ZU,\' A, S. S. IS·U.
INf"I,UENCt: Of" INTEIIMITTENT CONSUMPTION OF VITAMIN C ON Tilt: IIEVELUI'MENT 0.' SCUIIVY. Biochem. Jour. 35: 1240­
12-15, ill us. TlU:
.APPENDIX A
SI'~I!\IAIl\' OF DATA FilUM TilE BIOASSAY
The expel'imental data .from whkh the vitamin C hioa~say valu!'s were
dedvc:d 111'(' SUIlIIlHII'ized in table 4. Analysis of variance (~O) demonstrated
that in t.he individual assay groups, variation from linear regression of
(,dontohlast len~th on log-dose of ascorbic acid was im;ignificant and that the
F values for: variation (:aused by differences in dose were above the 1 percent
levd of signitkance in all groups. When the data for all of the guinea pigs
IIsed in the as!<ay of a single food were combined and tested for variations
all10ng vitamin C doses and sources. and for variations .from linear regres­
sion. the differen(:es hetween doscs were highly signific.ant for every .food
as wl~ll as the reference standard: differences between vitamin sources were
not significllnt. Therefore, the odontohlast method can be used in the bioassay
as a measure of differences in vitamin C intake; reductones which were
present in tlw stored .foods did not affect their vitamin C biological activity.
When fresh cahbage and kale were fed in addition to the hasal diet plus
the rutin, yeast, lind liver extruct supplements, odontoblast growth averaged
sa ± 7 microns, which was greater than the response to a 2-mg. intake of
ascorbic acid. This demonstrates that the highest assay level of vitamin C
induced a le;;s than optimal response.
The negative control animals showed either a complete lack of the odonto­
blast layer or a layer that averaged only .14 to 19 microns in length. Plate 1
illustrates the typical microscopic appearance of odontoblasts from guinea
pigs on the lowes!. Ilnd highest levels 1)( ascorbic acid and orange-grapefruit
juice. and n negative control. The precision attained in these assays was
comparahle to that reported by Crampton for the odontoblast method, when
fruit juices were analyzed (6).
To calculate the vitamin C potency of the foods, it was necessary to use
the formula of. Irwin (18, 19) in!ltend of the.faetorial analysis designed by
•
•
• .$if'!11I\I
2
•
•
c
L,;,
PLATE 1
Technical Bulietin 1023. U. S. De,Jllrlmrnt of Agri(uh"re
j. ..' ('
,,:
'rypknl 'wi(lnllO,(I(lJ1U' UPtlP:1I1HW(' -t,C d,lont .. bl;h ... frum ~HiI1N' pigs nn Ulfl lnwt'!'t IUld hight·... '
k\l'b of U't'lltim' lind nud ornnI!{'-l!rnpdntlJ jl1lt'l' !'-lIpph'HW.IHS~ Ilwl !loUt tl )Wg-:,lll\p·('lIlltro)
pUU1l'tI II!g~ (;uuwn 1111-: lin: .1. {L!i lUg. ttM'PI'Iiic+ lIf'h!.. 1J.. '! 1I111g, ll~l'orht(' ntHI; ('~ 0'111lt!I'"
J!ntJitllflllt JUIl'(' p'lllt\nh'ul to n!"1 JUl!, !h('ul'hl~l ~lt'J.[' J), tl"llIlJ!'··J!nlpi (run Jtth'!' N.ltll\tll('tll to
211111!!' H"'-turlUt und. r-:,. nOH!'o['orhH Hli,I~t1Jtlllf,-nHUl (IH:gntl\l'('Wnruh, X 1·lu.
•
TABLE
•
•
4.-Statisti(·al compari.'5on oj (I'l.'('/"{l!/e lel/fJth of o(i(i)/toblasts oj g/liw;a piUl) jed /J levels 01 vitamin C as
pure ascorbic acid, canned t(l11l(lto juice, orcmge-gnlpe!i'uit juice, and spinach
......
--_._-------------­
food
_......
R'.'''llOtl~ to
Rf'!"IKmlM! to r~ft!T~nc~ fttundnrcl. wtcorLk ndlt
A"("orbl(;
,
acid
j
or
equi "'n I~nt
f~d
I
,j ,..,-.,.-~--~ --P-~~~h~~-
"'---.- .--~ ---"'-"~'-' VarIation
Num},.:l'
OtlonioblnHt.
Jc-l1brth
of
Jo:uint,.n
Ili5.:M
from
lint}ur
I}Qfl.c
rl..·J;n!~~lon
,,1 _ _ _ _ _._" ___.-.,;f_·_,'~:hlf!
. . ;,unl~r
of
Odonto­
~lIiJl·):t
hla:!t
Icni;1h
1)1.;.8
__....__...
I)(.~~ ~,~_ •...,.,'"'_
I
I Jf ie/'Oll:'
Mg.
from
~lIinp:t
Jin{lur
rt:a:ression
l.iJ:li
As Jlurchased
1.0
2.0 28 ± 6'
28 .~
1}·16.54 ••
40::: 1 a
4.81
j
.5 1.0
2.0 3
5
8
I
24 -:1.~ ;3
35 ::: 3 } 36.93"
38 ± 4 ~': :J ± 6 }14.91*'" I 6
6
2·1
1.0
2.0
G
~li ~
.5
:~1
4
Odonto­
blaKt
I,·n.,nh
J~
from
linear
reM'~8iOD
5
-1
26 .;.. 5
2'.l .L 6
4
41 :!: 7
I
\.
f
8.(j3
0.88
LLt
26
.09
3.06
!
!}
(j
(j
(j
As purchased ~-----
-
--.~
25 .;.. 4 ;n ::': (j
'I~~~~
-!-'
3
41::': 6
11.83
1.62
--S-t-or-e-d--G-\-~'e-e-k-s-a-t-45-a-C-.---
I
3
6
35 ~;:
1 }
5 , 11.59 H
I
6
40 ±
5
I·
'>4 +
!
-
I
_._--_.. _------------------_.
1.73
The average length of odontoblasts of 8 guinea pigs fed 1.0 mg. of ascorbic acid plus 0.79 mg. glucoreductone was 34 ± 2' microns.
Standnrd de\'intion.
2 Two a&teriHk!i indicate sh:nlficancf! at the I lh.'f'Ct"'nl h.·\"d (1.· nlJO,'e. 'Vitamin C fed. hulf U!4 Ill.rt! :l::;~'~~ruic acid und half as "pjnhl'h. • Vitnmin C f~ u!t Rilinltch 01-1)'.
c
:z
c
OJ:
____________~ _________O_r_a_n~ge-gr~_ef_r_u_it~J~·u~i-ce--~~------~~_
,_ _ _ As purchased
Stored 5 days at 73° C.
2.2 .;.. 2 t------,
6
I 25 ± 4
4
0.31
a5 ~ 4 (55.0
0.80 i
6
:35"~ 7:
9.08"
G
__ .. ~,._.__1
(j
. 38 ± 4 i
44 :" 3 '
5
Spinach'
.,
('"J
~
± 3 '}
5 , 35 ± 4 • 14.4S"
G
44 ± 25 I
I
?
e>
I
<-
6.89
Z
a:
I MicroJls
1
~a:
"II
Stored 5 days at 73° C.
r;ificro713 4
6
6
f.~ " alue or
Tomato juice perc/au
0.5
.
l-=-T:puriRtion
_ N"um~r
vari,.ttion r
('"J
=
a:
tIJ
i5
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24
n:QIINICAL BULLETIN No, 1023. U, S,
n~:p'I',
OF AGrUCUI.TURE
Bliss and Marks (t!), alld applied by Crampton (6) in his studies, because
the guinea pigs in the vllrious gl'Oups to be compared completed lhe 6-week
assay period in unequal nurnber[i, This formula relates logarithms of the
mean doses of the standard and t.he unknown to theil' mean odontoblast
responses lind to the average slope of thcrc3(>onse curve, The following
('(Iuation is used:
1\1 .;;;
X
.1.
•
11., 'y.
•• --b-,­
1\1 is the logarithm of pcd:cncy of the food; X. and X" the mean log dosl's for the stlllldard and unknown; Jf. lind iT., the mean responses for standal'd and unknown; I" the IIvcragc slope of the CUl'Ve of rcsponsc, The eXlict fiducilll limits of each asslIY were also caltulatcd by the method
of Irwin (/8).
Only one of the neg-alivc COli trois survivt)d the (i-week assay period. and
all showed gross si,,6ls of scurvy Oil autop.,y. There were many fewcr
hemorrhag-es lind no ('xh ihition of lhe classic "faee-lItht)" position after rutin
and yeaRl' WI'I'C int.rodutNI into the dict. A few animals thllt were l'I!cciving
ascorbic add, t'specially lit tht· 0.5 mg. per day lcvel. di('d during- the assay
period; a small propol'tion of lhese showed sign:; of scurvy. The!'t) we!'e /I
few dcat.hs alllOIl~ lhe :iupplcnlC'lIt()d animals which wel'e attl'ibulable to
respiratory infection, or refuSli1 to cat, rather lhan scurvy.
COIllJ\lIrison of the weight gains of the alii mal:; at. different levels of
ascorbic acid intake l'cv('aled 110 significant. difren'nees. Some of the animals
that r('c:eh'ed 0.5 IIlg, (Jf aSCOl'bie acid ~~I'ew a~ well as those 011 the highcr
levels. TI](I 11(ll1SPl'C'itkil~r of growth a~ II Illea"lll'e of vitamin C deficiency
WIIS in mal'ked cont.rllst. to lll\' hig-h Spt·citic.:iLy of the odontoblast re:;;ponse,
Figures I, ~, allli :l show g-llillea pigs that l'cc'dved the assay diet alone, the
aSSily diet with cabbug-c and kale supplements, and the assay diet supple­
Il1cnt('d with a low level of ascorbic ucid, respectively.
•
J ,-ExpcrinWlIlal guinea pig al end of a,;say period: Neglltivc cont.l'ol, rcteivcd assay diet only.
F1GU"tJ
•
VI'I'AMIN
I:
t'tlMI'AWSON Of.' CIU~MH.!AI. AND 1II1)I.OGlC:\I. ASSAY
25
8
2,-J~:xpel'illlcntal 1{1Iinl':1 pig' at cnd of as;:;ay pcriod: Received assay
diet with f:uhhul{c unci knit' slIpprcmcnts,
FIGUUE
•
3,-J<Jxp~l'imental guinea pig lit end of assay pel'iod: Received assay
diet supplemlmted with O,n Illg', of aSt:OI'hic acid,
FIGURE
•
26
'n:GIINHlAI. IIUI,U;'I'IN N", IlI2:I, U, f;, IIEP'I', (II' A(:ttlCUlil'UIlE
APPENOlX .B
nn: RO AND RI\IOD
2,4.J)I~ITROPln:NYI.I'YORAZI~.: METIIODS
I~TERFER.:NCE I,.. GI,UemU:ulJCTONE IN
!n order to secure more complete information abOUL the RO reaction when
applied to a product in which reductones were known to be present, complete
absorption curves for this reaction with orange.grapefruit juice were
measured between wave lengths ,!50 and 550 mil. Comparison of the curve for
the juice with that of a solution of pure ascorbic acid which had also been
subjected to bromine oxidation is shown in figure 4, The two differl·d con·
siderably; the c;U1've for omnge.gl'apefruit juice absorbed markedly in the
region of 450 to ,170 mil, which, according to data published by Mills and Roe
(:!8) chal'llctt'rizes the phenylhydrazine del'ivutivc of "reductone." Simillir
complete absorption curves wcre made for the diketol~ulonic, dehydroascorbic,
ascorbic acid (DKA.DHA.AsA) aliquot of unstored orange.grapefruit juice
alid the diketogulonic acid (DKA) aliquot of the stored product in the RMOD
I'eaction (fig, 4, B), These curves demonstrate that reductones ruther than
diketogulonic acid were prohably responsihle for much of the color produced
in the diketogulonic acid (DKA) aliquot of the stored juice,
Penlley and Zilva (:I.V) showed that not only dehydroascorbic acid, but also
reductic acid, reductone, glucoreductone, and diketogulonic acid react with
phenylhydrazllle reagent. as ulled in the Roe·Kuether method (36) for measur·
ing a~corbic acid. Reductone dcveloped 50 percent, and reductic acid 92 pel'·
cent of its final color after only 15 minuu.s at 25" C, For this reasor it was
considc.red desh'ahle to detel'mine whether or not "reductones" were also
likely to interfere under the conditions of the Roe.Mills·Oesterling·Damron
modification (.17), whieh allows corn'ction to be made for diketoguloni('
acid, hut not for the other interfering substances mentioned,
The action of H~S and Br~1 I'eagen!.); used in the RMOD method, on gluco­
reduci.one had not been thoroughly investigated, although Mapson U~)
interpreted the results of his experiment.s with "reductones," produced by the
action of alkali on either glucose 01' <:ane sugar, as indicating the pres('nce of
two types of rcducton{', hoth of which were oxidized by iodine. Suhsequent
reduction with H~S completely regenerated one of the compounds in 75 min· utes, the seeond only after G hours exposure, Snow and Zilva (.U) reported that the Pl'opOl'tion of each form of reductone in solutions prepared from glucose and alkali, varied with the concentration of alkali and the heating ttme ('mployed, •
•
:\tATEIII,\LS, METllflOS, PRflCEOlJRI-:S
Crystalline j!lucol'eductnne was prepared by the method of Euler and
Martius (f), /0), omitting final recrystallization from ethyl acetate which
gave the purest compound, but from which yields Were too small to be
practicable for pl'cparing the IImounts of material necessary for u bioassay.
The crudel' fl'llction, recrystallized from petroleum ether" and acetone, was
used instead.
The IInalytical techni'lues (If Roe, Mills, Oesterling, and Damron (37) for
measuring ascorbic, dehrdroascnrhic, and diketogulonic acids in plant and
animul tissues were applied to solutions of known amounts of the pure
j!lucol'eductone, and 1I!;(:orbic acid; a and 2:1 hours of coupling were sub·
stituted Jor the (i hours employed hy Roc and associates in order to observe
both early and late changes taking place in t.he reaction mixtures; other
minor modifrcation!; were introduced only to serve the purpo!;;es of the
experinumts.
Glucoreductone (solution I) and ascorbic acid (solution II) were dissolved
in 5 percent HPO:I containing 0.5 percent SnCl~ in concentrations ranging
from 5 to 200 Itg. pCI' ml. Three aliquots were taken from each solution and
subjected to t.he analytical treatment employed in the RMOD method to
measure t.he three ascorbic acid fractions, i.e, (A) diketogulonic and dehydl'D­
,., .Puriticd Skcll~'solve, fraction F., h,p, 30°_60° C.
•
27
VITAMIN C--COMPARISON OJ<' CHEMICAL AND BIOLOGICAL ASSAY
ascorbic acid (DKA-DHA): (B) diketogulonic acid (DKA): (0) diketo­
gulonic, dehydroascorbic, and ascorbic acids (DKA-DHA-AsA). From these
values the "true" ascorbic acid and dehydroascorbic. acid, as well as diketo­
culonic acid present in the original tissue or food extract are calculated.
100r---~~
_________________________________
A
\
90
~
\
\
80
70
'\-60,..9. ascorbic acid
x
60
,
"x
/
',,---A....
I
I
I
x
.....x--­x/
/
' - Orange-grapefruit juice,
stored 73·C.,5 days
•
z
Q
en
!!! 90
~
en
B
z80
ct
a::
~
70
60
~,/
Orange-vrapefruit juice,
as purchased (OKA +
OHA+AsA aliquot).
/
'®,
50
00.....00 ­
40
-oo-oo-®"
~
/ci
30
• Orange-grapefruit JUice,
,-stored at 73°C.,5days
(OKA aliquot>.
20
0
450
•
470
490
510
530
WAVE LENGTH -(mil)
550
570
4.-Ahsorption curves: A. for Roe-Oesterling reaction with stored
orange-grapefruit juice and with a solution of pure ascorbic acid; B. fer
Roe-Mills-Oesterling-DamrG!l reaction with aliquots of unstored and stored
orange-grapefruit juice.
FIGURE
28
'I'E(JHNICAI. IIULLN'I'IN Nu. Itl~;j. U. S. IJEP'I'. OF' AGRIOULTURE
AFTER 3HRS. COUPLING
0.7
BC
IBtl
0.6
A
•
BA ·0 BS
0.5
0.4
0.3
0.2
0.1
0.0
::L E
0
v
10
O.B
~
>­ 0.7
~
enz
0.6
0
0.5
AFTER 23 HRS. COUPLING
BC
B
taU
JIa-o
•
UJ
0.4
0.3
0.2
0.1
®--®Ascorbic acid,JIA @}---@Glucoreductone. (DKA aliquot)
IA (DKA aliquot) II---K Glucoreductone,
I C (DKA +DHA+AsA
aliquot)
....--.Ascorbic acid, JIC (OKA +OHA + As A aliquot) 5.-Roe·Mills·Uestcl'ling-Da/lll'Un reaction: Calibration curves for
aliquots of glucol'cductone solution Bnd ascorbic acid solution. A. after
3 hours coupling with 2,4·dinitrophenylhydrazine; B, after 23 hours
coupling with 2,4.dinitl:Ophenylhydrazinc.
FlGUlUiJ
I
Color produced varied unpredicb\bly from time to time.
•
VITAIIUN
•
•
~COMPAlUSON OF CHEMICAL AND BIOLOGICAL ASSAY
29
The percent transmission of the final solution contair.ing the phenylhy­
drazine chromogen was mea!:.ured with the Beckman spectrophotometer,
model DU, at wave length 540 mIl. This is the wave length usually employed
in ascorbic acid calibration curves. In addition, complete curves between wave
lengths 450 and 580 mIl were read for at least one concentration of each
aliquot of solutions I ancl II and their respective blanks.
The same substance is responsible for the color in all of the ascorbic acid
aliquots; it is apparently I:n osazone-type compound formed by the coupling
of diketogulonic acid wit.\ 2,4-dinitrophenylhydrazine in the presence of
H:!SO~. The preliminary trehtment to which each of the aliquots is subjected
in the RMOD method determines which of the reducing compounds originally
present will take pal·t in the final coupling reaction.
A bioassay of the pure solutions was made also, in which eight guinea pigll
were fed the ascorbic-acid-fl'ee diet supplemented with 1 mg. ascorbic acid
and 0.79 mg. glucoreductone (equivalent to 1 mg. ascurbic acid, by the
indophenol method) pel' day.
In figure 5, A and H, are plotted the absorptions at 540 mIl for aliquots
A, B, and C of solutions I and U in the concentl'ation range of 20 to 80 "g.
per 4 m!. of reaction mixture, after coupling with phenylhydrazine for a and
23 hours, respectively. These are similar to the calibration I!UrVes usually
prepared with a pUl'e ascorbic acid standard when measuring ascorbic acid
by either the RO or RMOD reaction.
The curves in figure 5 indicatc that glucol'eductone, if present in the
reaction mixtul'c of aliquots A 01' C, would interfere appreciably with a
reading made at 540 mIl after either a or 23 houl's' coupling; if present in
aliquot B, glucoreductone would give no interference at 3 hours, and only
u small amount, which would val'y unpredictably from time to time in these
experiments, after 23 hours. It is noteworthy that glucoreductone subjected
to the treatment given aliquot C intel·fered considerahly more than the same
amount when present in aliquut A. Apparently dehydl'oreductones, analogous
to dehydroallcorbic acid, which arc formed by the oxidation of reductones
according to Snow and Zilva (.41), do not I'eact with phenylhydrazine pre­
cisely like glucoreductune. This circumstance further complicates calculation
of the various IIsCUI'bic acid del'ivatives in the pl'esenCIl of glucoreductone, and
gives rise to uncel'tllinty abuut thc accul'llcy of the final values obtained.
The ascorbic acid present in eithel: aliquot A or B interfel'es but little,
even after 2a hours' coupling; this justifics one of thc fundamental assump­
tions of thc RMOD mcthod, i.e., that ascorbic acid participates in the I'eactioll
with phcnylhydrazine only in aliquot C.
Ahsol'ption curves betwccn wave iengths 450 and 570 m.... for the three
aliquots of the RMOD reactioll as aPJllied to the purc glucol'eductone solution
and aliquots A and C of the aSl!ol'bic acid solution after coupling for a and
2:1 hours are shown in figure (j.
In aliquot A, only slight intcl'icl'cncc frulI! ascol'hic acid is indicatcd cven
aftcr 23 puurs, with as much as 240 ....g. ascorbic acid per 4 m!. reaction mix­
tUI'c. Glucoredut;tonc, whcn Hli:dyzcd as the sanlc ailljuot, gavc cUl've;.; after
both a and 2:{ hours whi!'h suggcst slight intcrfercncc of questiunable quanti­
tative significance at 540 Ill".
Both solutions I and II in aliquot B reactcd slightly with phenylhydrazine
after 2:.: hours; however, absorption at 540 mIl was affected but little. It may
be significant that SnCb is the stabilizer employed in aliquot B, and thiourea
in aliquots A and C.Figure 6, A, includes an absorption curve for glueo­
reductone in aliquot B, with thioul'ca substituted fOI' SnCI~. Under these
conditions, which arc identical with those of the RO reaction, glucoreductone
gives a characteristic absorption curve in the 45~70 mIl region of the
spectrum, and absorption at 540 m.... is not negligible. This observation suggests
that SnCI~ Illlppresses interference from glucoreductone and that the possible
substitution of SnC!:! fOI' thiourea in aliquots A and C especially when
glucOl'eductone is 11I'cllent, merits investigation.
The absorption curves for solutions I and II following treatment as for
aliquot C (DKA-DHA-AsA) are the most significant. After 3 hours' coupling,
ascorbic acid shows the characteristic diketogulonic acid derivative curve;
glucoredllctone also gives a characteristic curve whose maxima and minima
ao
TECHNICAL BULLETIN No. 1023, U. S. nUT. 010' AGRICULTURE
differ from those of ascorbic acid, although both derivatives absorb strongly
in the same region of the spectrum. These observations suggest the possibility
of correcting quantitatively for substances like reductones whose phenyl­
hydrazine del'ivativc,; giv(' chul'acteristic reproducible absorption curves. This
could he done hy lIIeasuring the specific absorptions of reductic acid. and
reductone:; derived frolll other tYlles of lIuglirs. liS well liS tho!!e from
glucose, and upplying the IlldhodfrelJuently used til calculate individual
components of a mixture of carotenoid pigments (!!.4. !!:».
•
A
100 AFTER 3HRS. COUPLING
90
80
I
I
70
i= 60
~
~ 50
W
.....'.
I
Ix
v
I
.....L&.-
~40
I
X
J.
Z
'.
Q
30
CJ) lIA}
Ie CJ) ~ 20 CJ)
DC--'
~
UB·O
·0
~ 10
a:
.... 0 I.....I--'-.....~-"--I.-l--L.............'"'--I.....I~ 450
4)0
530
IBJI
®--® Ascorbic acid -IlA,
~g./4ml. l{--K Dehydroascorbic
•
570
450
490 WAVE LENGTH (m~) <!r--@ Glucoreductone -lA,
60
I
240
acid­
~g./4mi.
00---<> Glucoreductone - I Bt ,
( I" thiourea) ,80 ala./4ml.
IC,40 ~g./4ml.
___ G lucoreductone-IC,
60
~g.
14ml.
(,i.-Roe-Mills-Ot.·Slcrling-lJalllroll rcm:tioll: Abso/'pt'nn ("urVl'" be­
t.wecn \\'aVl' lengths 450 and 570 ml' for aliquots of glucorl'd"ctone solution
and ascorhiC' acid solution. A, afte/' ;-l hours coupling with 2,4-dinitro­
phenylhydrazine; n, after 2il huurs coupling with 2.4-dinitrophenyl­
hydmzine.
FIGUUF.
1
Color III'odliecd vllried unprcdictahly .frull1 tilllC to time.
{cu.
Fur !1nh i hy Ulll
HlI'WThltf~nd(·nt or
S. \..QVE:HNM€ N T
'''HINTING OFFICE:
JhH'ultltlnt!5. U .....;. (iC.l\-·('!'nrn,}rlt;
\~lllshiligtHn ~».
n. C.
U~!jo-tt92943
Prifltin~
fHlicf!.
•
!
i. •
•
.,
I