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CHANGES IN THE APPARENT ASCORBIC ACID OF STFAWBERRIES

DURING FROZEN STORAGE

by

Chuan-huan Wu

AK ABSTRACT

Submitted to the School of Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfullment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Foods and Nutrition

Year 1955

 

Approved ”M £0 WW

Chuan-huan'Wu

CHARGES I? TEE APPARENT ASCORBIC ACID OF STRANBERRIES

DUB HG FROZEJ STORAGE

Changes in the apparent ascorbic acid of strawberries
of the Catskill and Robinson varieties during frozen storage
were studied.

Concentrations of total apparent ascorbic acid, reduced

ascorbic acid, dehydroascorbic acid, 2,3-diketogulonic acid,

reductones, reductic acid, total ascorbic acid, reducing
sugar and total solids were determined in strawberries which
had been frozen for 30 hours and in strawberries during froz-
en storage at -27 to -31 degrees Centigrade. Analyses were
made at monthly intervals for a period of ten months in
strawberries of the Catskill variety and six months in straw-
berries of the Robinson variety. The concentrations of the
various components in the strawberries which had been frozen
for 30 hours were employed as base line to study the changes
of these components during frozen storage.

There was little change in the total apparent ascorbic
acid of the strawberries during the first month of frozen
storage; however, there was a marked decrease in reduced as-
corbic acid and an increase in the concentrations of dehydro-
ascorbic acid and 2,3-diketogulonic acid of the frozen straw-
berries during this period. Fluctuations in concentrations

of the components of the strawberries of both varieties oc-

Chuan-huan Wu 2
Changes in the Apparent Ascorbic Acid of Strawberries

During Frozen Storage
curred throughout the entire period of frozen storage; there
.was a significantly inverse relationship between the concen-
trations of 2,3-diketogulonic acid and total ascorbic acid
of the frozen strawberries.

The influence of the changes of apparent ascorbic acid
in strawberries during frozen storage on the utilization of
ascorbic acid in strawberries was studied with six healthy
young women as subjects. Subjects were given 75 milligrams
crystalline reduced ascorbic acid as a supplement to their
customary diet for seven days before each test period. Test
doses given at four test periods were (a) 200 grams straws
berries of Catskill variety, frozen for 30 hours; (b) an
amount of crystalline reduced ascorbic acid equivalent to
the amount of total apparent ascorbic acid provided in the
test dose of 200 grams of strawberries; (c) an equivalent
amount of dehydroascorbic acid in the form of orange juice
treated with activated charcoal, and (d) 250 grams of straw-
berries, frozen and stored for four months. Blood samples
'were taken before and at hourly intervals after the test
doses for a five-hour period. Urine was collected for a one-
hcur period preceding and for a five-hour period following
each test dose. The blood serum was analyzed for reduced

and total apparent ascorbic acid; urine was analyzed for re-

duced ascorbic acid.

 

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Chuan-huan Wu 3
Changes in the Apparent Ascorbic Acid of Strawberries
uring Frozen Storage

There was not a statistically significant difference
among the maximum concentrations of ascorbic acid in the
blood serum following the test doses, nor in the urinary ex-
cretions of ascorbic acid after the test doses. When the
total apparent ascorbic acid of the strawberries which had
been held in frozen storage was corrected for the amount of
2,3-diketogulonic acid present in the berries, the total
available ascorbic acid appeared to be as well utilized as
that of the strawberries which had been frozen for only 30

hours.

’1

CHAKGES IN Try APPAREHT ASCORBIC ACID or sraiwsaaaias

DURIIG FROZEN STORAGE

BY

Chuan-huan Wu

A TIES IS

Submitted to the School of Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF HILOSOPHY

Department of Foods and Nutrition

1955

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ACKN OW LEDGI~EN TS

The writer wishes to express her grateful appreciation
to Dr. Wilma D. Brewer, who served as both advisor and sub-
ject for this study, for her patient guidance, valuable sug-
gestions, unfailing understanding throughout the investiga-
tion, and for her criticism and advice in the writing of
this thesis, without which this study could not have been
done.

Acknowledgment is made to Mrs. Hazel Amen, Dr. Betty
Hawthorne, Mrs. Deloris Kereluke, Mrs. Mary Mills Neely, and
Mrs. Andrea Wagoner for their participation as cobperative
subjects and for their helpful assistance in the laboratory.

Thanks also are extended to Dr. Pauline C. Paul for her
kindness in supervising the freezing of strawberries.

Grateful acknowledgment is also due to Dr. William D.
Baten of the Hathematicstepartment for his advice in statis-
tical treatment of the data.

The author is greatly indebted to Dr. Eva Hwang for her

friendly help and constant encouragement.

PAGE
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1
REVIEW 0? LITERATURE . . . . . . . . . . . . . . . . . 5
Chemical methods for the determination of ascorbic
acid in foods . . . . . . . . . . . . . . . . . . 5
Changes in the total ascorbic acid content of foods
during storage . . . . . . . . . . . . . . . . . . 15
Possible role of biosynthesis in the ascorbic acid
content of frozen foods . . . . . . . . . . . . . 23
Studies of human utilization of ascorbic acid in
foods . . . . . . . . . . . . . . . . . . . . . . 27
Ascorbic acid in the blood and urine . . . . . . . . 38
EXPJERILEIITAL PEOCEDURB . . . . . . . . . . . . . . . . its
Determination of the apparent ascorbic acid content
of frozen strawberries . . . . . . . . . . . . . . AS
Plan of experiment . . . . . . . . . . . . . . . . MS
Freezing and sampling of strawberries . . . . . . us
Chemical methods . . . . . . . . . . . . . . . . . h?
Reduced ascorbic acid . . . . . . . . . . . . . AB
Total apparent ascorbic acid . . . . . . . . . . M9
Dehydroascorbic acid and 2,3-diketogulonic acid 51
Reductones and reductic acid . . . . . . . . . . S2
Reducing sugar . . . . . . . . . . . . . . . . . 53

Total SOlids . C O O O O O O O O O C O O O O O 0 51+

Studies of the physiological utilization of the

apparent ascorbic acid of frozen strawberries

Plan of experiment . ... . . . . . . . . . .
Subjects . . . . . . . . . . . . . . . . . .
Preparation of test doses . . . . . . . . .
Strawberries . . . . . . . . . . . . . . .
Crystalline ascorbic acid . . . . . . . .
Dehydroascorbic acid . . . . . . . . . . .
Collection and treatment of blood and urine
samples . . . . . . . . . . . . . . . . .
Chemical analysis . . . . . ... . . . . . .
RESULTS AKD DISCUSSION . . . . . . . . . . . . .
Changes of the total apparent ascorbic acid in
frozen strawberries . . . . . . . . . . . .
Reduced ascorbic acid . . . . . . . . . . .
Total apparent ascorbic acid . . . . . . . .
Dehydroascorbic acid . . . . . . . . . . . .
2,3-diketogulonic acid . . . . . . . . . . .
Total ascorbic acid . . . . . . . . . . . .
Reducing sugar . . . . . . . . . . . . . . .

TOtal SOlidS O O O O O O O O O O O O O O I 0

iv

58
6O
61

61
66
70
75a
76
78
79
80

 

 

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Concomitant changes in ascorbic acid and related
compounds in frozen strawberries . . . . . . . . 80
Physiological utilization of the total apparent
ascorbic acid of frozen strawberries . . . . . . . 89
Description of the subjects . . . . . . . . . . . 89
Serum concentration and urinary excretion of
ascorbic acid before test dose . . . . . . . . . 91
Ascorbic acid supplied in test doses . . . . . . . 99

The utilization of ascorbic acid of the various

test doses . . . . . . . . . . . . . . . . . . . 106
Test dose of crystalline reduced ascorbic acid . 112
Test dose of dehydroascorbic acid . . . . . . . 118
Test dose of strawberries, frozen for 30 hours . 120

Test dose of strawberries, frozen and stored for
four months . . . . . . . . . . . . . .p. . . 123
Comparison of the various test doses . . . . . . 126
SUMMARY AND CONCLUSION . . . . . . . . . . . . . . . . lhO

LITERATUFLE CI'PED . O O O O O C O O O 0 O O O C C O O 0 lug

II.

III.

IV.

VI.

LIST or TABLES

The Average Concentration of Ascorbic Acid
and Related Compounds in Strawberries
(Catskill Variety) at Monthly Periods of
Storage . . . . . . . . . . . . . . . . . .
The Average Concentration of Ascorbic Acid
and Related Compounds in Strawberries
(Robinson Variety) at monthly Periods of
Storage . . . . . . . . . . . . . . . . . .
Analysis of Variance of the Concentration of
Reduced Ascorbic Acid in Strawberries
(Catskill Variety) Frozen for 30 Hours,

Four Months and Eight Months . . . . . . . .
Significance of the Difference of the Means of
Reduced Ascorbic Acid in Strawberries -
(Catskill Variety) Frozen for 30 Hours, Four
Months and Right Months . . . . . . . . . .

Analysis of Variance of the Concentration of
Total Apparent Ascorbic Acid in Strawberries
(Catskill Variety) Frozen for 30 Tours, Four
Months and Right Months . . . . . . . . . .

Significance of the Difference of the Means of
Total Apparent Ascorbic Acid in Strawberries

(Catskill Variety) Frozen for 30 Hours, Four

Months and Eight Months . . . . . . . . . . .

PAGE

62

71

71

75

75

TABLE
VII.

VIII.

IX.

XI.

XII.

XIII.

XIV.

Correlation Coefficients of the Various Com-

ponents of Strawberries (Catskill Variety)

During Frozen Storage . . . . . . . . . . . .

Correlation Coefficients of the Various Com-

ponents of Strawberries (Robinson Variety)

During Frozen Storage . . . . . . . . . . . .

Description of Subjects . . . . . . . . . . . .

The Reduced and Total Apparent Ascorbic Acid of

the Blood Serum and the Urinary Excretion of
Ascorbic Acid of the Subjects Preceding the
Administration of the Test Doses for the Suc-

cessive Experimental Periods . . . . . . . .

Analysis of Variance of Concentrations of Serum

Ascorbic Acid Preceding the Administration of

Various Test Doses . . . . . . . . . . . . .

Analysis of Variance of the Concentration of

Reduced Ascorbic Acid in the Urine of Six
Subjects Preceding the Administration of

Various Test Doses . . . . . . . . u . . . .

Ascorbic Acid Content of Individual Test Doses

of 200 Grams of Strawberries, Frozen for 30

Ilours . O O 0 O O O O O O O O O O O O O O O .

Ascorbic Acid Content of Individual Test Doses

of 250 Grams of Strawberries, Frozen for

B1011? l'IOI'lthS o o o o o o o o o o o o o o o o 0

vii

PAGE

8A

92

91+

97

100

102

TABLE PAGE
XV. The Concentration of Ascorbic Acid and Related
Compounds in the Various Test Doses . . . . . 105
XVI. Reduced Ascorbic Acid Concentrations of Blood
Serum of Six Subjects Preceding and at
Periodic Intervals after the Administration
of Various Test Doses . . . . . . . . . . . . 108
XVII. Total Apparent Ascorbic Acid Concentrations of
Blood Serum of Six Subjects Preceding and at
Periodic Intervals after the Administration
of Various Test Dose . . . . . . . . . . . . 110
XVIII. Calculated Increment of Reduced Ascorbic Acid
of Blood Serum of Six Subjects after Admin-
istration of Various Test Doses . . . . . . . 113
XIX. Calculated Increment of Total Apparent Ascorbic
Acid of Blood Serum of Six Subjects after Ad-
ministration of Various Test Doses . . . . . 11h
XX. Average Urinary Excretions of Reduced Ascorbic
Acid before and after Administration of the
Various Test Doses . . . . . . . . . . . . . 115
XXI. Analysis of Variance of the Maximum Concentra-
tions of Ascorbic Acid in the Serum of Six
Subjects after Various Test Doses: F-Values
Adjusted by Analysis of Co-variance to Remove
the Influence of Basal Blood Values on maximum

Concentrations . . . . . . . . . . . . . . . 127

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TABLE PAGE
XXII. Analysis of Variance of Concentration of Re-
duced Ascorbic Acid Excreted per flour after

Different Test Doses . . . . . . . . . . . 133

ix

Graph Showing the Changes in Concentration of

Ascorbic

-"

Acid and

I)
1L3

Solids and Reducin“

let

0
\J

d Compounds, Total

Sugar in Strawberries of

the Catskill Variety during Erozen Storage . . .

Graph Showing the Chanjes in Coacentration of

Ascorbic Acid and Related Compounds, Total

Solids and heducing Sugar in Strawberries of

the Hobi
Concentrat

jects be

‘7

HS on V 8.1“

iety during firozen Storage . . .

ions of Slood Ascorbic Acid of Six Sub-

fore and'after Various Test Doses . . .

Urinary Excretions of Reduced Ascorbic Acid by Six

Subjects Preceding and Eollowing

tion of

Various

Test Doses.

Concentrations of Reduced

Serum of Six Subjects before and

tervals af ter the

Doses .

Ascorbic

Adninistration

the Administra-

‘eriodic In-

of Various Test

Concentrations of Total Apparent Ascorbic Acid of

Blood Serum of Six Subjects before and at

Periodic

Various

Intervals after the

Test Dos

f3
‘1

S

Administration of

63

93

111

- ‘ — 1‘ ' 7.77
v Lu L._.a AV .4

'y.
.—

7. Mean Concentrations or heduced Ascorbic Acid and

Total Apparent Ascorbic Acid of Six Subjects

before and at Periodic Intervals after Various

Test Dose . . . . . . . . . . . . . . . . . . . 129
8. Kean Successive Changes in Concentrations of Re-

duced Ascorbic Acid and Total Apparent Ascorbic

Acid of Blood Serum of Six Subjects after Vari-

Gus friest Doses . O O O O O O O O I C O O O O O O 131

INTRODUCTION

Retention of ascorbic acid in stored or processed foods
is important in providing foods which are of high nutritive
value. Freezing is the most satisfactory method of preser-
vation of ascorbic acid in foods for relatively long periods
of storage, since chemical changes which affect ascorbic
acid take place more slowly in foods held at lower tempera-
tures than at higher temperatures.

It is generally accepted that the concentration of re-
duced ascorbic acid decreases in foods during frozen stor-
age. However, contradictory statements have been found in
the literature relative to the effect of frozen storage on
the total ascorbic acid (reduced ascorbic acid and dehydro-
ascorbic acid) content of foods. Bedford and McGregor ‘
{19u8), Hewston, Fisher, and Orent-Keiles (1951), and Lee
(1951) found that the total ascorbic acid concentrations of
fruits and vegetables were decreased during frozen storage,
although there were fluctuations in values at different time
intervals. On the other hand, Paul, Wiant, and Robertson
(l9h9) reported that increases in the concentration of total
ascorbic acid occurred during frozen storage in a variety of
fruits and vegetables. The highest concentration of total

ascorbic acid was after a three months storage period. An

2
increase in the total ascorbic acid content of strawberries
held in frozen storage for three months as compared with the
total ascorbic acid content of fresh berries also was found
in this laboratory by Einbecker and her co-workers (19h7;
1950).

Apparent differences in the effect of frozen storage on
the total ascorbic acid content of foods as reported from
various laboratories may be due to (a) the possible presence
of substances, related to ascorbic acid, which interfere
with the chemical determination of total ascorbic acid in
certain foods (Wokes, Organ, and Jacoby, 19h3; Snow and
Zilva, l9hh; Purinton, l9h7; and Somers, Kelly, Thacker and
Redder, 1951); (b) the conversion at different rates under
various conditions of the reduced form of the vitamin to its
biologically inactive oxidation product which also is deter-
mined in the analysis of total ascorbic acid (Pijoan and
Gerjovich, 19u6; Hartzler, 19h8); (c) the possible biosyn-
thesis of this vitamin from its precursors during frozen
storage (Isherwood, 1953).

Biologically inactive reductones and reductic acid
Which are derivatives of carbohydrates and which have been
reported to be present in foods (Wokes~lg£.§£-: l9u33 Roe,
Mills, Oestering and Damron, 19MB; and uoldblith and Harris,
19h8), react with 2,Lp-dinitrOphenylhydraZine (Penney and

Zilva, l9h5), which is used as a reagent for the analysis of

3
total ascorbic acid. Changes in the relative concentrations
of these substances during the storage of foods may there-
fore be a possible cause of variations in determined total
ascorbic acid values.

The reduced form of the vitamin reacts rapidly with
various oxygen carriers present in plant tissue to form the
reversible oxidation product, dehydroascorbic acid. Above
pH five dehydroascorbic acid readily undergoes a rearrange-
ment in which the lactone ring is split. The product, 2,3-
diketogulonic acid, is not reducible by sulfhydryl compounds
in cells and is no longer biologically active (Borsook, Dav-
enport, Jeffreys and Warner, 1937; Penney and Zilva, l9h3a).
This substance combines with 2,h-dinitrOphenylhydrazine more
rapidly than does total ascorbic acid to give the character-
istic color of the osazone (Mills, Damron, and Roe, l9h9).
Thus, in a determination of total ascorbic acid content of
foods based upon this color reaction, 2,3-diketogulonic acid
also would be measured if present, and the apparent ascorbic
acid values would exceed the true ascorbic acid concentra-
tion of the material.

Synthesis of l-ascorbic acid from reducing sugars in
plants has been reported by Ray (193A) and Isherwood (1953)
and in animals by Jackal, Mosbach, Burns and King (1950) and
Horowitz, Doerschuk and King (1952). The synthesis of as-

corbic acid in foods from reducing sugar during frozen stor-

h

age has not been demonstrated, but it is possible that bio-
synthesis of the vitamin also may occur under this condition
in fruits which are not blanched before freezing.

This study was planned to investigate the relative con-
centrations of reduced ascorbic acid and total ascorbic acid
of fresh strawberries and of strawberries stored in the
frozen state and further to investigate the various sub-
stances which contribute to the apparent ascorbic acid values
of frozen strawberries. The utilization of the total as-
corbic acid of frozen strawberries by humans also was stud-
ied to investigate whether the changes in apparent ascorbic
acid in strawberries during frozen storage represented true
changes in the vitamin content of the berries.

Strawberries were selected as the food for the investi-
gation of the changes in apparent ascorbic acid during frozen
storage, since strawberries are a relatively rich source of
ascorbic acid and since a marked increase of total ascorbic
acid of strawberries during frozen storage was reported pre-
viously from this laboratory (Paul 33 31., 19h9; Einbecker
£93., 19m; 1950).

REVIEW 0? LITERATURE

Chemical Methods for the Determination of Ascorbic Acid

in Foods

Ascorbic acid values of food are reported usually as
reduced ascorbic acid or as total ascorbic acid. The latter
term has been used to designate both the reduced ascorbic
acid and the oxidized form of ascorbic acid, dehydroascorbic
acid. Both forms of ascorbic acid are biologically avail-
able, although some workers have reported that dehydroascor-
bic acid was not as well utilized as reduced ascorbic acid
(Gould and Schwachman, l9h3; Roe and Baunum, 1936). Early
studies of the vitamin C content of foods by chemical anal-
ysis were based upon oxidation-reduction reactions of re-
duced ascorbic acid with an oxidizing substance as iodine
(Stevens, 1938), methylene blue (Martini, 193A), or 2,6-
dichlorophenoliiniophenol (Bessey and King, 1933). Results
from these analyses correSponded closely with the antiscor-
butic potency of foods as measured by the protective action
of the food against the development of scurvy in guinea pigs
(Penney and Zilva, l9h3a). This relationship existed more
closely, however, with the amount of reduced ascorbic acid
in fresh foods than with the amount of reduced ascorbic acid

in processed foods (Strobecker and Vaubel, 1936; Mack and

6
Tressler, 1937; Hou, 1937). This observation stimulated in-
terest in the development of a method of analysis of ascor—
bic acid in foods which would measure both the reduced as-
corbic acid and the dehydroascorbic acid.

Roe and Keuther (19h2; 19h3) reported a method for the
determination of ascorbic acid. In the method, reduced as-
corbic acid was converted to dehydroascorbic acid by treat-
ment with an activated charcoal. The dehydroascorbic acid
thus formed was coupled with 2,h-dinitrophenylhydrazine to
form an osazone. When this osazone was treated with 85 per-
cent sulfuric acid, a reddish colored product was formed
which absorbed maximally at 500 to 550 millimicrons and 350
to 380 millimicrons.

A year later Roe and 0esterling (l9hh) modified the di-
nitrophenylhydrazine method to differentiate dehydroascorbic
acid from the reduced form of ascorbic acid in plant tissue.
In the modified procedure, tissue extract was treated with
activated charcoal as in the Roe and Keuther method (19h3).
However, when thiourea was added to a second aliquot of tis-
sue extract in an amount to give a one percent solution, the
reduced ascorbic acid was protected by the thiourea in its
reduced state and only the dehydroascorbic acid in the tissue
extract reacted with dinitrophenylhydrazine. By a combina-

tion of the two procedures, then, the authors obtained a

value for the concentration of combined reduced ascorbic
acid and dehydroascorbic acid in the plant extract and a
value also for dehydroascorbic acid alone.

Reductones and reductic acid are related closely in

chemical structure to ascorbic acid.

 

 

 

 

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Reductone Reductic acid Ascorbic acid

The enediol group is common to the three compounds. Carpeni
(1938) has reported that the ultra-violet absorption spectra
of the compounds were similar with respect to three absorp-
tion maxima. Penney and Zilva (19h5) reported that 1-ascor-
bic acid, reductone and reductic acid solutions incubated at
25 degrees Centigrade for fifteen minutes with dinitrophenyl-
hydrazine produced 2.5, A8, and 92 percent respectively of
the color given by ascorbic acid and dinitrophenylhydrazine
after three hours at 37 degrees Centigrade. These authors
suggested that interferences by the reductones and reductic
acid in the determination of ascorbic acid might be con-
trolled by regulating the conditions of incubation. However,
this procedure failed to differentiate the reductones and re-

ductic acid from ascorbic acid when all were present in the

Same extract. Later, Schocken and Roe (1952) devised a

8
method for the determination of reductones and reductic acid
based upon the fact that the reductones and reductic couple
with the 2,h-dinitrophenylhydrazine in 18 normal sulfuric
acid at room temperature, while ascorbic acid, dehydroascor-
bic acid, and diketogulonic acid do not form derivatives
with 2,h-dinitrophenylhydrazine under these conditions.

This procedure made it possible to determine reductones and
reductic acid simultaneously with the determination of as-
corbic acid and to correct ascorbic acid values for inter-
ference by these compounds.

Penney and Zilva (1943) found that 2,3-diketogulonic
acid interfered more markedly than reductones and reductic
acid in the determination of total ascorbic acid with 2,h-
dinitrophenylhydrazine. Two, three-diketogulonic acid is
the irreversible oxidation product of dehydroascorbic acid.
According to Borsook and his co-workers (1937) further oxi-
dation of 2,3-diketogulonic acid yielded oxalic acid and

threonic acid as metabolic products:

 

 

COOH
OC-——- 0 —-—1 COOH
I COOH
H00 0 OC
H O O I oxalic acid
H00 0 O?
l COOH
HC-——- HG———— HTOH l _
I | » HCOH
HOCH HOCH HOCH |
| | | HOCH
CH OH CH OH CH OH |
2 2 2 CHZOH
reduced dehydro- 2,3-diketo-

ascorbic acid ascorbic acid gulonic acid threonic acid

Both Borsook 93 31. (1937) and Penney and Zilva (19h3)
found that 2,3-diketogulonic acid does not possess the bio-
logical activity of ascorbic acid. 'When the 2,3-diketogu-
lonic acid is coupled with the dinitrophenylhydrazine re-
agent, however, the osazone formed cannot be distinguished
from that formed by dehydroascorbic acid. Thus, if diketogu-
.lonic acid is present in food, determination of total ascor-
bic acid by coupling with dinitrophenylhydrazine may give a
higher value for the food than is represented by the two bi-
ologically active forms of the Vitamin. Roe, Hills, Oester-
ling and Damron (l9h8) published details of a procedure
which was developed to correct for the presence of diketogu-
Ionic acid in the tissue extract. In this procedure, tis-
sues were extracted with a mixture of 0.5 percent solution
of stannous chloride in five percent metaphosphoric acid.
Diketogulonic acid was determined by reducing dehydroascor-

bic acid to reduced ascorbic acid with hydrogen sulfide and

10
then only diketogulonic acid was coupled with dinitrophenyl-
hydrazine. ‘When the same procedure was applied to the tis-
sue extract with the omission of the reduction reaction,
values were obtained for both dehydroascorbic acid and diketo-
ggulonic acid. Therefore the difference of the values ob-
tained by the two processes represented the concentration of
dehydroascorbic acid in the tissue extract. The determina-
tion of reduced ascorbic acid was performed on a second ali-
quot of the acid extractant by passing hydrogen sulfide
through the aliquot to remove the stannous ions by the for-
mation of precipitated stannous sulfide. The precipitated
stannous sulfide was separated by filtration and the fil-
trate was treated with bromine to oxidize the reduced ascor-
bic acid before coupling with the 2,u-dinitrophenylhydrazine
reagent. This gave the total concentration of reduced as-
corbic acid, dehydroascorbic acid, and diketogulonic acid.
By subtracting the concentration of dehydroascorbic acid and
diketogulonic acid, the reduced ascorbic acid concentration
of the extract was obtained.

As indicated above, the reduced ascorbic acid fraction
of the total ascorbic acid of foods can be determined by
difference by the method of Roe and Oesterling (l9hh) and
the method of Roe, Hills, Oesterling and Damron (l9h8).
Chemical analyses of reduced ascorbic acid in foods based

upon the oxidation-reduction reaction of reduced ascorbic

11
acid with 2,6-dichlorophenolindophenol have been reported
from many laboratories (Henaker and Guerrant, 1938; Harris
and Olliver, l9h2; Daniel and Hunsell, 1937). This method
was originally developed by Bessey and King (1933) for vis-
ual titration of ascorbic acid of the plant tissue with a
solution of an alkaline salt of 2,6-dichlorophenolindophenol.
In the oxidized state 2,6-dichlorophenolindcphenol is blue
in an alkaline medium and red in an acid medium; in the re-
duced state the 2,6-dichlorophenolindophenol is colorless.
The method of Bessey and King (1933) was developed to apply
these characteristics of the dye, i.e. the change in color
when the solution was changed from an alkaline to an acid
state and the change from a colored to a colorless state
when the dye was reduced.

Since the method was acolorimetric procedure, there was
interference in the determination of ascorbic acid in plant
extracts by water soluble plant pigments. Various adapta-
tions of the method were developed to eliminate the effect
of the plant pigments on the color developed by the reaction
of ascorbic acid and 2,6-dichlorophenolindophenol. These
included the use of water immiscible solvents such as amyl
acetate (Fujita and Numata, 19h1) and xylene (Helson and
Somers, l9h5) to extract the excess 2,6-dichlor0phenolindo-
phenol reagent from the aqueous mixture of 2,6-dichlorophenolr

indophenol and the plant extract. The intensity of the color

12
of the organic solvent then was proportional to the amount
of dye which had not been reduced by the ascorbic acid of
the plant extract.

Since the reaction of reduced ascorbic acid and 2,6-
dichlorophenolindophenol was also an oxidation-reduction re-
action, there was interference with the procedure by various
metal ions and reducing substances present in biological ex-
tractants. The interference of ferrous iron with the deter-
mination of ascorbic acid by the 2,6-dichlorophenolindophe-
nol method has been reported by several workers (Eekelen and
Emmerie, 1936; Basu and Nath, 1938). It was found, however,
that in a sufficiently acid medium small quantities of fer-
rous salts do not interfere with the indophenol titration
(Eekelen and Emmerie, 1936). Ott (1941) reported that fer-
ric salts interfered in the determination of reduced ascor-
bic acid bylinCreasing the rate of oxidation of ascorbic
acid and that metaphOSphoric acid was effective in arresting
the catalytic action of traces of ferric ions. Barron, Guz-
man, and Klemperer (1936) and Dekker and Dickinson (l9h0)
reported that interference might result from the reducing
action of cuprous salts upon the indOphenol dye or through
the catalytic oxidative action of the cupric ion upon ascor-
bic acid. The latter effect was thought to be due to enzyme
systems which contain copper in combination with various

protein groups. Both the reducing property of the cuprous

13
ion and the catalytic effect of cupric ion can be controlled
by compounds such as 8-hydroxyquinoline (Sendroy, 1937),
acetic acid, and metaphosphoric acid (Ott, l9h1).

Reducing substances like hydrogen sulfide, reductones,
reductic acid, glutadfibne, cysteine, which are present in
biological extracts, have been found to reduce the 2,6-di-
chlorophenolindophenol reagent similarly to ascorbic acid.
(King, 1936; Mack and Tressler, 1937). Therefore it was
realized that the presence of these substances in foods
might result in the determination of falsely high values for
reduced ascorbic acid in foods. The effects of these ex-
traneous reducing substances upon the determination of as-
corbic acid were controlled, in part at least, by regulating
the time of the determination, since the rates of reaction
were found to differ for the various reducing substances
with 2,6-dichlorophenolindophenol, and the rate of reaction
of ascorbic acid with 2,6-dichlorophenolindophenol was more
rapid than the reaction rate of many other reducing com-
pounds with the dye. Bessey (1938) described a photocolor-
imetric method for the determination of small quantities of
ascorbic acid and dehydroascorbic acid in turbid and colored
solutions in the presence of other reducing substances. In
principle, this method involved the addition of an excess of
an aqueous 2,6-dichlorophenolindophenol solution to a buf-

fered metaphosphoric acid extract of the unknown and meas-

1h

urements of the percent transmission of light with a photo-
electric colorimeter at 15 and 30 second intervals after the
addition of the dye. Correction for other interfering sub-
stances which reduced the dichlorophenolindophenol at a slow-
er rate than ascorbic acid was made by subtracting the drift
of the galvanometer needle between the 15 and 30 second pe-
riod from the transmission reading obtained after the first
15 seconds. Later, Loeffler and Ponting (19h2) reported
that when plant tissue was macerated in a one percent meta-
phosphoric acid solution, ascorbic acid could be determined
in fruits and vegetables, whether fresh, frozen, or dehy-
drated,by taking the transmission reading on a photoelectric
colorimeter at 15 seconds after the start of the addition of
the dichlorophenolindophenol reagent to the metaphosphoric
acid tissue extract. The authors considered that a single
measurement of percent transmission was adequate since the
amounts of interfering substances in plant tissues were rel-
atively small.

Recognition of the probable interference by various re-
lated substances in the determination of ascorbic acid has
led to the use of the term "apparent" by some investigators
in reports of ascorbic acid values. 'The term "apparent as-
corbic acid" was used originally by Butler and Cushman
(l9hl) to describe ascorbic-acid-like reducing substances in

the white layers of centrifuged blood from leukemic patients

15
and later applied to foods and food products by Wokes, Or-
gan, Duncan and Jacoby (l9h3). These authors referred to a
group of substances which resembled ascorbic acid in chemi-
cal and physical properties but did not possess the anti-
scorbutic properties. Einbecker, Jackson, Paul and Ohlson
(19H7) used the term "apparent total ascorbic acid" for the
amount of ascorbic acid determined by the method of Roe and
Oesterling (l9hu) with 2,h-dinitrophenylhydrazine. Somers,
Kelly, Thacker and Redder (1951) defined "total apparent as-
corbic acid" as a term which referred to the amount of as-
corbic acid equivalent to the total amount of 2,6-dichloro-
phenolindophenol reduced by the buffered aliquot of the tis-
sue extract, whether the dye was reduced by ascorbic acid or
by some other substances. Stewart, Horn and Robson (1952)
referred to the substances in blood which reacted with 2,u-
dinitrophenylhydrazine to form osazones by the method of Roe

and Kuether (l9h3) as "apparent ascorbic acid."

Changes in the Total Ascorbic Acid Content of Foods

During Storage

(The development of methods for the analysis of both de-
hydroascorbic acid and reduced ascorbic acid in foods pro-
vided data from.various laboratories which demonstrated that
a relatively larger amount of ascorbic acid may be present in

the form of dehydroascorbic acid in processed foods than in

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16
fresh foods (Mack and Tressler, 1937; Hou, 1937; Woessner,
Elvehjem, and Schuette, l9u0; Wokes, l9h3). . This ob-
servation stimulated interest in the investigation of the
influence of various factors upon the relative concentra-
tions of reduced ascorbic acid and dehrdroascorbic acid in
foods.

Changes which occurred in the concentrations of reduced
ascorbic acid, dehydroascorbic acid and total ascorbic acid
of cabbage after cutting and shredding were studied by
McMillan and Todhunter (1946). There was a reduction in the
amount of reduced ascorbic acid of the cabbage immediately
after cutting and shredding; however, there was an increase
in the concentration of dehydroascorbic acid of the cabbage
after cutting and shredding. The increased concentration of
dehydroascorbic acid was maintained for 30 minutes, after
which the concentration of dehydroascorbic acid also was re-
duced. At the end of 120 minutes after the cutting and
shredding of the cabbage, values for reduced ascorbic acid,
dehydroascorbic acid and total ascorbic acid were less than
for the fresh cabbage.

Dodds, Price and Moore (19h8) reported studies of the
concentrations of reduced ascorbic acid and dehydroascorbic
acid of sweet potatoes which were cured by storage at a tem-
perature of 80 to 85 degrees Fahrenheit and high humidity

for seven to ten days and then stored at a temperature of 50

to 55 (33???
tion of deh;
after six we
through ut t

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17
to 55 degrees Fahrenheit and low humidity. The concentra-
tion of dehydroascorbic acid of the sweet potatoes increased
after six weeks of storage and then was reduced gradually
throughout the experimental period of 2h weeks. The concen-
tration of reduced ascorbic acid decreased gradually
throughout the entire storage period.

Changes in the concentration of reduced ascorbic acid
and dehydroascorbic acid of vegetables during frozen storage
were reported by Bedford and McGregor (19h8). These authors
found that whereas four to thirteen percent of the ascorbic
acid was present as dehydroascorbic acid in the fresh, green
vegetables, from 22 to 9k percent of the ascorbic acid was
in the form of dehydroascorbic acid in frezen vegetables
which had been held in frozen storage for six months. The
total amount of ascorbic acid of the vegetables was less
after six months of frozen storage than in the fresh vege-
tables.

Wolfe 23 21. (19h9) reported that fresh samples of
muskmelon contained an average of O.h0 milligrams of reduced
ascorbic acid and O.h2 milligrams of total ascorbic acid per
gram; after the muskmelon was frozen and stored, the reten-
tion of ascorbic acid in the frozen melon at successive time
intervals was 36, kl, 39, and 37 percent of the reduced as-
corbic acid and 60, 59, #8 and 5h percent of the total as-

corbic acid at periods of one, three, six and nine months

of :13 froze:
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total assert:

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18
respectively. Thus, although there was a marked reduction
in the concentration of both reduced and total ascorbic acid
after one month of frozen storage, the ascorbic acid content
of the frozen muskmelon remained relatively constant during
the remainder of frozen storage. The average retention of
total ascorbic acid was about 18 percent higher than that of
the reduced ascorbic acid.

Hewston, Fisher, and Orent-Keiles (1951) studied the in-
fluence of time and temperature on changes of both reduced
and total ascorbic acid in several canned fruits, fruit
juices and one vegetable during storage. The temperatures
ranged from u5 to 73 degrees Centigrade and the length of
storage period varied from three to AZ days. Canned grape-
fruit, orange juice, orange-grapefruit juice, tomato juice,
and Spinach, were studied. The total ascorbic acid content
of the grapefruit was reduced approximately 80 percent after
five days of storage at 73 degrees Centigrade. There was a
reduction of 58 percent in the total ascorbic acid of canned
orange juice under the same storage conditions. Considerable
loss of total ascorbic acid also occurred in the canned
orange-grapefruit juice which was stored for seven days at
73 degrees Centigrade. However, there was a loss of only 18
percent of the total ascorbic acid of tomato juice which was
Stored at a temperature of h5 degrees Centigrade for 1h days.

Among the foods stored, only spinach showed an increase in

the concentration of total ascorbic acid during storage. The
canned spinach was stored for h2 days at LS degrees Centi-
grade. At the end of this storage period the total ascorbic
acid concentration had increased from 15.1 milligrams per 100
grams, which was the value before storage, to 20.6 milligrams
per 100 grams.

Paul, Wiant and Robertson (19h9) found that after three
months of frozen storage there was an increase in the total
ascorbic acid concentration of strawberries. The fresh berries
contained h7.5 milligrams of total ascorbic acid per 100 grams;
after three months of frozen storage, the berries contained.83.3
milligrams per 100 grans. The concentration of total ascorbic
acid was lowered to 66.5 milligrams per 100 grams at the endcfi‘
six months of frozen storage and 50.8 milligrams per 100 grams
at the end of nine months of frozen storage. A similar in-
crease in the total ascorbic acid content of red cherries, red
and black raspberries, and rhubarb also was observed after
three months of frozen storage, although the increase was of
less magnitude than that for the frozen strawberries. These
workers did not report separate values of reduced ascorbic
acid and dehydroascorbic acid of the frozen fruits.

Following the work of Paul at El° (l9h9), Einbecker and
co-workers (19h7; 1950) studied the relative concentration of
reduced ascorbic acid and total ascorbic acid of fresh straw-
berries and strawberries which had been frozen and held

in frozen storage over a period ot time. Berries of the

20
Robinson variety were studied. The fresh strawberries con-
tained 65.6 milligrams reduced ascorbic acid and 65.h milli-
grams of total ascorbic acid per 100 grams of strawberries.
After three months of frozen storage, the strawberries con-
tained h5.5 milligrams of reduced ascorbic acid and 78.1 mil-
ligrams of total ascorbic acid per 100 grams. The concen-
tration of reduced ascorbic acid in the frozen strawberries
was no.2 milligrams per 100 grams after six months of stor-
age and 32.3 milligrams per 100 grams after nine months of
storage. Some reduction in the concentration of total as-
corbic acid was found in the frozen strawberries after six
months of storage; the value was 50.7 milligrams per 100
grams. The concentration of total ascorbic acid in the
stored frozen strawberries after nine months of storage was
similar to that after six months of storage.

Since l9h8, when Roe and his co-workers modified the
method for the determination of total ascorbic acid to dif-
ferentiate diketogulonic acid from reduced ascorbic acid and
dehydroascorbic acid, some studies have been reported of
processed foods which have included measurements of diketogu-
lonic acid as well as reduced ascorbic acid and dehydroascor-
bic acid. Mills, Damron and Roe (l9u9) investigated the as-
corbic acid content of 27 vegetables and fruits and reported
that fresh foods as purchased at the market contained some

dehydroascorbic acid and diketogulonic acid but that the

21
amounts were small unless the foods had been allowed to de-
teriorate considerably. Quality of the product also ap-
peared to affect the amount of diketogulonic acid in frozen
foods since frozen strawberries in poor condition contained
no reduced ascorbic acid, 12.7 milligrams dehydroascorbic
acid and 37.5 milligrams of diketogulonic acid per 100 grams,

whereas frozen strawberries of good quality contained 80.0
milligrams of reduced ascorbic acid, 12.0 milligrams of de-
hydroascorbic acid and 3.0 milligrams of diketogulonic acid
per 100 grams.

Fresh plant tissues were reported by Stokstad and Jukes
(l9h9) to contain small amounts of dehydroascorbic acid and
diketogulonic acid. These authors found that frozen’food-
stuffs contained from O to 75 percent of the total ascorbic
acid derivatives in the form of diketogulonic acid.

Changes in the quantitative relationahip of reduced as-
corbic acid, dehydroascorbic acid and diketogulonic acid
during storage were investigated by Mills and her co-workers
(l9u9) in orange juice and in a slurry prepared from pota-
toes. Orange juice and aslurry of white potatoes were
stored in the refrigerator at two degrees Centigrade. Orig-
inally all the ascorbic acid of the orange juice was present
in the reduced form. Analysis on the second day of storage
showed that five percent of the reduced ascorbic acid had

been changed to dehydroascorbic acid. The amount of dehy-

22
droascorbic acid increased gradually throughout the storage
period. On the thirteenth day of the study, diketogulonic
acid was found to be present for the first time. The amount
of diketogulonic acid increased slowly until at the last
analysis on the thirty-eighth day, it comprised the largest
percentage of the substances measured as total ascorbic
acid. The total concentration of the three compounds re-
mained constant for the first 30 days, then decreased slowly
so that by the thirty-eighth day the total ascorbic acid
content was equivalent to approximately 83 percent of the
concentration of ascorbic acid in the fresh orange juice.

A potato slurry was prepared by Mills and her co-
workers by blending potatoes with distilled water (ratio:
one to 50) in the container of an electric blender. The
original slurry contained almost no reduced ascorbic acid,
but about 90 percent of the substances measured as total as-
corbic acid was dehydroascorbic acid and 10 percent was di-
ketogulonic acid. Within six hours, however, diketogulonic
acid comprised over half of the total of the substances
measured as total ascorbic acid. By the nineteenth day, 90
percent of the total amount of the three compounds was in
the form of diketogulonic acid. The values for the total
concentration of dehydroascorbic acid and diketogulonic acid
did not change appreciably during the first week, but later

decreased rapidly until at the last analysis only five per-

23
cent of the concentration of total ascorbic acid of the
freshly prepared slurry was present.

The interference of reductones with the determination
of total ascorbic acid was reported by Hewston gt El' (l9h8)
who found that as much as h5 percent of the apparent ascor-
bic acid in french-fried potatoes was reductones.

The studies which have been reported here indicate that
the oxidation of reduced ascorbic acid to dehydroascorbic
acid and possibly to diketogulonic acid may occur during the
storage of foods. The oxidative process apparently can take
place in foods stored in cold storage, in canned foods, and
in frozen foods. In those studies in which an actual in-
crease in the total ascorbic acid concentration of processed
foods was observed, it would appear that other chemical
changes occurred in the foods during storage, perhaps of an
oxidative nature, which yielded compounds capable of reacting
with the 2,h-dinitrophenylhydrazine. An actual increase in
the total ascorbic acid content of the food would result, it
would seem, only from the possible synthesis of ascorbic

acid during storage.

Possible Role of Biosynthesis in the Ascorbic Acid Content

of Frozen Foods

Storage of foods in the frozen state does not preclude

the possibility of enzymatic reactions occurring in the food.

21;

Inactivation of the enzymes of vegetables is achieved before
freezing by blanching of the vegetables. Many fruits, how-
ever, are frozen in the raw state, without blanching. Sizer
and Josephson (l9h2) reported that the storage of lipase,
trypsin and invertase in aqueous solution with their reSpec-
tive substrates for 27 days at -70 degrees Centigrade did
not affect their activity. Ball and Lineweaver (1938) in
studying the action of enzymes at low temperature concluded
that enzyme activity may continue at low temperatures and
may be an important factor in problems of food preservation.
These studies suggest that certain enzymatic changes may oc-
cur in fruits in the frozen state, and provided a basis for
the hypothesis that biosynthesis of ascorbic acid from its
precursors might occur by enzymatic action in frozen fruit.

The conversion of sugars, i. e., d-glucose, d-fructose,
and d-mannose into l-ascorbic acid, was demonstrated to oc-
cur in cotyledonless pea seedlings by Ray (193A). Ghosh
(19h6) suggested that pyruvic acid is a precursor of ascorbic
acid in the rat, since the incubation of liver tissue with
sodium pyruvate under suitable conditions produced an in-
crease of from 15 to 20 percent ascorbic acid above the
amount originally in the liver tissue. In the presence of
vitamin B1 the increase was more than 30 percent. Horowitz
and his co-workers (1952) have reported that experiments

with Clu-d-glucose indicated that there was a direct conver-

25
sion of glucose to l-ascorbic acid. This observation was
based on the fact that administration of Clu-d-glucose to
rats resulted in the excretion in the urine of labelled
l-ascorbic acid. A year later Horowitz and King (1953) also
found that glucuronic acid might serve as a precursor of as-’
corbic acid in the albino rat. Hath, Belavady, Sahu and
Chitale (1953) found that intramuscular injection of glu-
cose, sodium acetate alone, sodium acetate followed imme-
diately by glucose, or the condensation product of glucose
and ethyl acetoacetate, resulted in a temporary rise in the
plasma concentration of ascorbic acid within an hour follow-
ing the injection. Isherwood (1953) attempted to establish
the nature of the hexose precursor and elucidated the mecha-
nism by which hexose is transformed into l-ascorbic acid by
adding certain compounds postulated as intermediates in the
reaction to the solution in which cress seedlings were grown.
The amount of l-ascorbic acid present in the treated seed-
lings was compared with that in seedlings grown in water.

He found that the r-lactones of d-glucuronic and l-gulonic
acids, when added to cress seedlings, were transformed into
l-ascorbic acid. d-galacturonic acid methyl ester and l-
galactonoar-lactone behaved similarly. Mapson (1953) was
interested to find whether an enzyme extract could be ob-
tained to catalyze the conversion of either l-gulono-r-

lactone or l-galactono-y-lactone to l-ascorbic acid. He

26
found that mitochondria prepared from partially germinated
pea seedlings were able to convert 1-galacton04r-lactone
rapidly to 1-ascorbic acid, but could not effect the corre-
sponding conversion of l-gulono-r-lactone to l-ascorbic acid.
This reaction was catalyzed most efficiently by oxygen; how-
ever, cytochrome oxidase also was involved. The reaction
proceeded smoothly until approximately hO percent of the
lactone had been transformed into l-ascorbic acid and then
ceased, but began again if an additional quantity of the
lactone was added. The disappearance of the lactone, apart
from that portion converted to l-ascorbic acid, was shown to
be due to its conversion into the free galactonic acid, a
reaction which proceeded simultaneously with the enzymically
catalyzed formation of l-ascorbic acid. Since galactonic
acid was not itself converted to l-ascorbic acid, this reac-
tion decreased the yield of l-ascorbic acid obtained from
the‘rblactone. This side reaction indicated that biosyn-
thesis of ascorbic acid from its precursors was not a simple
process.

Thus, Isherwood (1953) and Mapson (1953) have demon-
strated that biosynthesis of ascorbic acid from hexose de-
rivatives may occur in living plant tissues. There is no
evidence at present that similar synthesis of ascorbic acid
from reducing sugars may occur in processed foods, but it is

possible that enzymatic changes occurring in food, even at

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27
the temperature of frozen storage, might result in the syn-

thesis of ascorbic acid from its various precursors.

Studies of Human Utilization of Ascorbic Acid in Foods

Reduced ascorbic acid is a water-soluble vitamin. The
absorption of the vitamin from the intestinal tract has been
found to occur quite rapidly and blood plasma or serum con-
centrations of the vitamin have been found to reflect changes
in the intake of the vitamin in relatively short periods of
time (Todhunter, Robins, and McIntosh, l9h2; Clayton and F01-
som, 19h0; Linkswiler, 195h). Changes in the concentration
of ascorbic acid in the urine also have been found to re-
flect changes in vitamin intake in short periods of time
(Belser, Hauck, and Storvick, 1939; Berryman, French, Harper
and Pollack, 19hh). Therefore changes in the blood and
urine concentrations of ascorbic acid corresponding to
changes in the intake of the vitamin have been used as a
basis for the study of the human utilization of ascorbic
acid in foods.

Early in 1936, Hawley and her co-workers (1936) studied
the urinary excretion of ascorbic acid following the admin-
istration of comparable quantities of the vitamin in orange
juice and in pure crystalline form and found that the vita-

min 0 of orange juice was as well utilized as the pure sub-

stance.

28

Clayton and Folsom (l9h0) compared the blood concentra-
tions and urinary excretions of ascorbic acid of subjects
following the administration of a dietary supplement of po-
‘tatoes which supplied 50 milligrams of ascorbic acid and 25
milligrams of the pure vitamin‘with the blood concentrations
and urinary excretions of the subjects following the admin-
istration of a dietary supplement of 75 milligrams of the
pure vitamin. Each period was continued for four days.
Blood values for two of the four subjects were slightly
higher after the potatoes were given than when the test dose
was crystalline ascorbic acid. Blood values for one subject
were approximately the same for the two experimental periods.
One subject had a slightly lower concentration of vitamin C
in the blood when potato was used as a partial source of the
dietary ascorbic acid than when the ascorbic acid was sup-
plied as the pure vitamin, although the blood concentration
of ascorbic acid was greater than one milligram per 100 mil-
liliters. The authors concluded therefore that the vitamin
from the potato was well utilized. Urinary excretions of
vitamin C also were determined and were found to be compa-
rable in the two test periods. This indicated that there
was good absorption of the ascorbic acid of the potato from
the intestinal tract and that the retentions of the vitamin

were comparable for the two test periods.

29

Todhunter and Fatzer (l9h0) studied the utilization of
ascorbic acid by seven college women. The blood ascorbic
acid values and the urinary excretions of ascorbic acid were
reported to be comparable for each subject after a diet which
contained 20 milligrams of ascorbic acid and a dietary sup-
plement of raspberries containing no milligrams of ascorbic
acid was fed for six days and after the same diet supple-
mented with ho milligrams of crystalline ascorbic acid was
administered for a comparable period of time. The subjects
were saturated with orange juice containing 200 milligrams
of ascorbic acid for three days before each experimental
period. The authors estimated the so-called "utilization inp

dex," Intake - Output
Body weight

 

x/KEE, for each subject; the utiliza-

tion index indicated that the ascorbic acid of the red rasp-
berries was as well utilized as crystalline ascorbic acid.
The ability of the test dose supplement to maintain
tissue saturation and to support plasma ascorbic acid con-
centrations was the criterion which was used by Clayton and
Borden (19h3) in studies of the availability of ascorbic
acid in raw cabbage and canned tomato juice. Four college
students on a basal diet containing ten milligrams ascorbic
acid were given 150 milligrams ascorbic acid daily for eight
days before each experimental period of nine days. During
the experimental periods, subjects were on the basal diet

supplemented with (a) 75 milligrams of ascorbic acid, (b) 25

30
milligrams of ascorbic acid plus an amount of raw cabbage
which provided 50 milligrams of ascorbic acid,and (c) 25
milligrams of ascorbic acid plus an amount of tomato juice
which provided 50 milligrams of ascorbic acid. Ascorbic
acid was determined in a sample of blood taken when the sub-
ject was in a fasting state and on a 2h-hour urine composite.
Both blood and urinary ascorbic acid were determined on the
last day of each experimental period. The concentrations of
ascorbic acid in the blood of the subjects following the ex-
perimental periods when cabbage and tomato juice were given
as dietary sources of the vitamin were comparable to the con-
centration of ascorbic acid in the blood following the period
when the ascorbic acid was supplied in the pure form. The
urinary excretions of ascorbic acid were somewhat lower when
the natural foods were a source of dietary ascorbic acid
than when the dietary supplement was crystalline ascorbic
acid. Therefore, the authors concluded that the ascorbic
acid contained in the raw cabbage and in the canned tomato
juice was as well utilized or even better than ascorbic acid
in the pure form.

Hartzler (l9h5) studied the availability of ascorbic
acid in papayas and in guava juice. On the first day, nine
subjects, four women and five men, were given 300 milligrams
of ascorbic acid. Following this, there was a control pe-

riod of six days in which subjects were given a basal diet

31
plus 75 milligrams of pure ascorbic acid. Each experimental
period was six days in length. The administration of the
massive dose of 300 milligrams of ascorbic acid and the con-
trol period were repeated_preceding each experimental period.
During one experimental period, the subject received 75 mil-
ligrams of crystalline ascorbic acid daily. The test foods
were given as the source of ascorbic acid in the experimen-
tal periods; each test food was given in an amount which
supplied 75 milligrams of ascorbic acid. Urinary excretions
of ascorbic acid were determined daily and blood plasma as-
corbic acid was determined once each week. Application of
analysis of variance to the ccperimental data indicated that
the ascorbic acid of the papayas and of the guava juice was
as well utilized as the pure ascorbic acid.

Elliot and Schuck (19h?) reported that the ascorbic
acid of grapefruit was as well utilized as the crystalline
vitamin. This observation was based upon studies of the
urinary excretion of ascorbic acid by nine subjects during
two three-day test periods and studies of the concentration
of ascorbic acid in whole blood of three subjects at the end
of each test period.

Goldsmith and Ellenger (1939) were among the first to
study the changes of ascorbic acid content in-blood at peri-
odic intervals after the administration of a test dose. The

subjects were 22 persons; three were considered to be normal

32
and nineteen were hospital patients who had no fever, acute
infectious disease or vitamin C deficiency. The blood plas-
ma concentration of reduced ascorbic acid was determined at
intervals of one, three and six hours after the oral admin-
istration of a test dose of 600 milligrams of ascorbic acid.
At the end of one hour, there was a rise in the concentra-
tionscfi'blood plasma ascorbic acid. The highest concentrations
ranged from 1.7 to 2.9 milligrams per 100 milliliters and oc-
curred at the end of three hours after the administration of
the test dose. After six hours had elapsed, there appeared
to be a reduction of the amount of vitamin C in the blood
plasma. The curves of plasma ascorbic acid following the
test dose resembled those for dextrose tolerance tests as
described by Greenberg, Rinehart and Phatak (1936).

In l9h2, Todhunter, Robbins and McIntosh (l9h2) report-
ed a study of the rate of increase of blood plasma ascorbic
acid after ingestion of 50 milligrams of ascorbic acid in
the crystalline form, and after consumption of equivalent
amounts of ascorbic acid in cauliflower, orange sections,
orange juice or strawberries. The ascorbic acid content of
blood plasma began to increase within 30 to 60 minutes after
the test dose was given and returned to the fasting concen-
tration in three or four hours. The maximum increase in
plasma concentration of ascorbic acid was reached within one

and one-half hours after ingestion of 50 milligrams of as-

33

corbic acid either as crystalline ascorbic acid, as orange
juice, or as orange sections. The maximum increase in plas-
ma concentration of ascorbic acid occurred in two hours when
strawberries were the source of ascorbic acid, and in two
and one-half hours when cauliflower was eaten.

Investigation of the rate of increase of plasma reduced
ascorbic acid after ingestion of strawberries and crystalline
ascorbic acid was reported also by Einbecker, Jackson, Paul,
and Ohlson (l9h7). The plasma ascorbic acid concentrations
of five young women were measured in the fasting state and
one-half, one, one and one-half, two and one-half, and three
and one-half hours after an ascorbic acid-free breakfast
which was supplemented with (a) no supplement, (b) 150 grams
unsweetened frozen strawberries, (c) crystalline ascorbic
acid equivalent to the amount of reduced ascorbic acid pro-
vided by the strawberries and (d) crystalline ascorbic acid
equivalent to the amount of apparent total ascorbic acid
provided by the strawberries. The increases in plasma as-
corbic acid obtained after ingestion of the strawberries were
comparable to those obtained when crystalline ascorbic acid
was taken in amounts equivalent to the reduced ascorbic acid
in the strawberries. The maximum concentration occurred one
and one-half hours after ingestion of the berries, while two
and one-half hours passed after the administration of crys-

talline ascorbic acid before the maximum concentration was

3h

reached. Increases in blood ascorbic acid concentration ob-
tained when crystalline ascorbic acid was taken in amounts
equivalent to the apparent total ascorbic acid were greater
than those obtained after the ingestion of strawberries. In
this study, blood plasma ascorbic acid was measured by oxi-
dation of reduced ascorbic acid with 2,6-dichlorophenolindo-
phenol according to the procedure of Farmer and Abt (1936).
Any ascorbic acid which was present in the blood as dehydro-
ascorbic acid would not have been measured by this procedure.
Studies of human utilization of ascorbic acid also have
been concerned with the relative utilization of dehydroascor-
bic acid in comparison with crystalline reduced ascorbic
acid. Earlier reports of studies of the utilization of dew
hyircascorbic acid by guinea pigs indicated that dehydroas-
corbic acid had 25 percent (Roe and Barnum, 1936) and 80
percent (Gould and Schwachman, l9u3) of the potency of re-
duced ascorbic acid. However, Penney and Zilva (l9h3) re-
ported that studies with guinea pigs indicated that dehydro-
ascorbic acid was as well utilized as reduced ascorbic acid.
Borsook, Davenport, Jeffreys, and Warner (1937) studied
the effect with human subjects when orange juice as a source
of reduced ascorbic acid was replaced by charcoal-treated
orange juice as a source of dehydroascorbic acid. These au-
thors found that the concentrations of reduced and total as-

corbic acid in the blood plasma of subjects were comparable

35

after a test dose of fresh orange juice and after a test
dose of orange juice which had been treated with charcoal to
oxidize the ascorbic acid. A similar study was conducted by
Todhunter and her co-workers (1950) on young women. These
authors also used activated charcoal to oxidize the reduced
ascorbic acid in orange juice to dehydroascorbic acid. It
was concluded that when dehydroascorbic acid was fed, the
concentrations of reduced ascorbic acid in blood serum and
in the urine of young women were comparable to the concen-
trations of reduced ascorbic acid in the blood serum and
urine when the subjects received reduced ascorbic acid; this
was considered to be indicative of satisfactory utilization
of the dehydroascorbic acid. Bitter and Cohen (1951) found,
too, that differences between the average urinary excretions
of ascorbic acid after oral doses of reduced ascorbic acid
and after oral doses of dehydroascorbic acid were not sta-
tistically significant.

Linkswiler (195h) also studied the blood ascorbic acid
of subjects following the ingestion of crystalline dehydro-
ascorbic acid (method of preparation not given) and reduced
ascorbic acid. Reduced and total ascorbic acid determinations
were made on the blood serum of five women, 39-h0 years of
age, before and after the oral administration of 50, 150, or
300 milligrams crystalline dehydroascorbic acid or 150 mil-

ligrams reduced ascorbic acid. The concentration of ascor-

36
bic acid in the serum was maximum at a period of one and one-
half hours after the oral dose. The maximum increment in
the reduced ascorbic acid values in blood averaged 0.18,
0.3a, and 0.79 milligrams per 100 milliliters respectively
following the ingestions of 50, 150, and 300 milligrams de-
hydroascorbic acid; the corresponding total ascorbic acid
values in blood averaged 0.21, 0.5h, and 0.91 milligrams per
100 milliliters. The maximum increment following ingestion
of 150 milligrams reduced ascorbic acid was 0.h5 milligrams
of reduced ascorbic acid and 0.53 milligrams of total ascor-
bic acid per 100 milliliters of blood plasma. Thus, al-
though the increment in reduced ascorbic acid in the blood
was higher after the test dose of reduced ascorbic acid than
after the test does of dehydroascorbic acid, the increments
in total ascorbic acid in the blood were comparable after
test doses of reduced ascorbic acid and dehydroascorbic acid.

The availability of ascorbic acid in canteloupe and
grapefruit was studied by Chen and Schuck (1951) by compari-
son of the urinary excretions of dehydroascorbic acid, re-
duced ascorbic acid and diketogulonic acid by five healthy
women on a basal diet supplemented with the test food and on
a basal diet supplemented with crystalline ascorbic acid.
The basal diet supplemented with no milligrams crystalline
ascorbic acid provided 8.h2 milligrams diketogulonic acid,
6.61 milligrams dehydroascorbic acid, and 69.67 milligrams

lull Ia!

37

reduced ascorbic acid per day. The average daily excretions
in urine for the last four days of the six-day experimental
period were h.88 milligrams diketogulonic acid, 6.73 milli-
grams dehydroascorbic acid and 38.91 milligrams reduced as-
corbic acid. When the basal diet was supplemented with can-
taloupe, the daily diet provided 19.78 milligrams diketogu-
lonic acid, 1h.88 dehydroascorbic acid, and 56.2h milligrams
reduced ascorbic acid. The average urinary excretions were
h.72 milligrams diketogulonic acid, 6.00 dehydroascorbic
acid, and h2.21 milligrams reduced ascorbic acid. The basal
diet, supplemented with grapefruit, supplied 9.67 milligrams
diketogulonic acid, 7.15 dehydroascorbic acid, and 73.61 mil-
ligrams reduced ascorbic acid per day. The corresponding
urinary excretions were h.86 milligrams diketogulonic acid,
5.39 milligrams dehydroascorbic acid, and 36.87 milligrams
reduced ascorbic acid. The data indicated that the intake
of the basal diet plus the cantaloupe supplement, which con-
tained higher intakes of dehydroascorbic acid and diketo-
gulonic acid, did not increase the excretion of these two
substances above that observed on the basal diet supplemented
with crystalline ascorbic acid or grapefruit. The authors
postulated that the occurrence of dehydroascorbic acid and
diketogulonic acid in urine might be due in part to oxida-
tion of reduced ascorbic acid during filtration and storage

in the kidneys and bladder.

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38

The studies reported above indicate that, although va-
rious procedures have been used in the study of the utiliza-
tion of ascorbic acid by humans, these procedures have been
based essentially upon the influence of the test food on the
blood and/or urinary concentration of ascorbic acid. The
procedures which have been used may be grouped as follows:

(a) Blood and/or urinary concentrations of ascorbic
acid of subjects maintained on a basal diet, low in ascorbic
acid, and supplemented by pure ascorbic acid or an equivalent
amount of ascorbic acid from the test food.

(b) Blood and/or urinary concentrations of ascorbic acid
of subject at periodic intervals following a test dose of
pure ascorbic acid or an equivalent amount of ascorbic acid
from the test food. This pattern of study has been used both
when subjects were maintained on a diet low in ascorbic acid

and when subjects were on self-selected diets.
Ascorbic Acid in the Blood and Urine

Blood concentrations of ascorbic acid have been used
widely as an index of nutritional status with respect to as-
corbic acid. According to Youmans (19hl), a concentration of
0.7 milligrams of ascorbic acid per 100 milliliters of blood
plasma or serum is indicative of good ascorbic acid nutri-
tion and concentrations of 1.2 milligrams per 100 millili-

ters of serum or above have been observed frequently among

39
well-nourished individuals. Youmans considered that blood
plasma or serum ascorbic acid values within the range of O.h
to 0.7 milligrams of ascorbic acid per 100 milliliters rep-
resented a borderline state of ascorbic acid nutrition, and
that values less than O.h or 0.5 milligrams of ascorbic acid
per 100 milliliters were indicative of a deficiency state
with respect to ascorbic acid. Similar interpretations of
blood ascorbic acid values have been reported by Farmer
(lth), Adamson and co-workers (l9u5), Borsook at 31. (19kb)
and King (1951).

Borsook and his co-workers in 1937 suggested that an
equilibrium.may be maintained between reduced ascorbic acid

and dehydroascorbic acid in the blood:
Ascorbic acid (reduced):Dehydroascorbic acid

and that the glutathione of the cells may be a factor in
maintaining this balance. Penney and Zilva (l9h1), however,
were unable to demonstrate the presence of dehydroascorbic
acid in guinea pig blood after the administration of test
doses, either of 120 milligrams of dehydroascorbic acid or
120 milligrams of 2,3-diketogulonic acid. Todhunter and co-
workers (1950) reported also that human blood did not con-
tain dehydroascorbic acid. In contrast, however, Stewart,
horn and Robson (1952; 1953) reported that the average vita-

min C concentration of fasting plasma of tenlealthy subjects

no
was 0.56 milligrams of reduced ascorbic acid per 100 milli-
liters of plasma as determined by the method of Farmer and
Abt (1936) using 2,o-dichlorophenolindophenol as an oxidiz-
ing agent, and 0.77 milligrams of total ascorbic acid per
100 milliliters of plasma as determined by the method of Roe
and Kuether (l9u3) based on the reaction of oxidized ascor-
bic acid with 2,h-dinitrophenylhydrazine. 'When the plasma
extract was treated with hydrogen sulfide and the ascorbic
acid then determined by titration with 2,6-dichlorophenol-
indophenol, the values were more nearly comparable. The au-
thors interpreted this to indicate the presence of dehydro-
ascorbic acid in the blood plasma in amounts which averaged
about 20 percent of the total ascorbic acid. Davey, Wu, and
Storvick (1952) also found that dehydroascorbic acid was
present in blood.

Relatively little attention has been given to the pos-
sible occurrence of diketogulonic acid in the blood. Penney
and Zilva (19h3) found that the blood plasma of guinea pigs,
after five days on scorbutic diet, contained five milligrams
of diketogulonic acid per 100 milliliters of plasma. Fif-
teen minutes after the oral administration of 120 milligrams
of dehydroascorbic acid to guinea pigs weighing 300-350
grams, the average diketogulonic acid content in the plasma
was five milligrams per 100 milliliters of plasma and re-

mained at this concentration for one hour. When 120 milli-

Ln
grams dehydroascorbic acid were given intramuscularly, the
plasma concentration of diketogulonic acid of the guinea
pigs reached a maximum concentration of 36 milligrams per
100 milliliters within fifteen minutes, then decreased grad-
ually to 17 milligrams per 100 milliliters by one hour after
injection. When 120 milligrams of diketogulonic acid were
administered orally, the plasma diketogulonic acid was from
four to seven milligrams per 100 milliliters. However, when
the same amount of diketogulonic acid was given intramuscu-
larly, the plasma diketogulonic acid increased to 55 milli-
grams per 100 milliliters after 15 minutes and then decreased
gradually to 23 milligrams per 100 milliliters at the end of
one hour. In contrast with the study of Penney and Zilva
(l9h3), Damron, Monier and Roe (1952) did not find diketogu-
lonic acid in the blood offull grown guinea pigs, either on:
a control diet or after five eye on a scorbutic diet. In
human subjects, Chen and Schuck (1951) reported that both
dehydroascorbic acid and diketogulonic acid were found in
fasting blood samples and also in the blood at intervals
after the ingestion of crystalline ascorbic acid. In the
experiment reported by Stewart, horn, and Robson (1953), the
ascorbic acid in the blood plasma was converted to the re-
duced form after treatment with hydrogen sulfide and the
values for total ascorbic acid by the Roe and Kuether (1943)

method and reduced ascorbic acid by the Farmer and Abt

AZ
(1936) method were similar. This indicated that the sub-
stances in the blood which had reacted with the 2,h-dinitro-
phenylhydrazine were reduced ascorbic acid and dehydroascor-
bic acid rather than 2,3-diketogulonic acid, since the 2,3-
diketogulonic acid, if present, would not have been reduced
to ascorbic acid. As reported previously, Chen and Schuck
(1951) found that the average daily urinary concentrations
of diketogulonic acid, dehydroascorbic acid and reduced as-
corbic acid of subjects on a basal diet supplemented with
pure ascorbic acid were u.88, 6.73 and 38.91 milligrams re-
spectively. The concentrations(XTdiketogulonic acid and de-
hydroascorbic acid were relatively constant when canteloupe
was substituted for pure ascorbic acid as the dietary sup-
plement, although the canteloupes supplied approximately 13
milligrams of diketogulonic acid and eight milligrams of de-
hydroascorbic acid. The total ascorbic acid concentration
in urine has been found to be highly correlated with the re-
duced ascorbic acid in the urine (r - 0.9lu, P 5 0.01) (Ber-
ryman, French, Harper, and Pollack, 19AM).

It was recognized early that the amount of vitamin C in
the urine was closely related to the vitamin C intake (Har-
ris and Ray, 19353 Abbasy, 32 31., 1937) and that ascorbic
acid has the characteristics of agrenal threshold substance
(Faulkner and Taylor, 1938; Lewis, at al., l9h3). Thus the

urinary excretion of ascorbic acid is related to the blood

#3

concentration of ascorbic acid,which in turn is dependent
upon the dietary intake of ascorbic acid. Levcowich and
Batchelder (l9h2) reported that ascorbic acid excretions on
a vitamin C free diet ranged from seven to 16 milligrams per
day. According to Haines and co-workers (19h7), the mean
daily urinary excretions of ascorbic acid ranged from 15 to
30 milligrams for subjects on intakes from 33 to 70 milli-
grams of ascorbic acid per day. When test doses of #00 mil-
ligrams of ascorbic acid were administered, the urinary ex-
cretion of ascorbic acid ranged from 15 to 125 milligrams on
the first day and from 152 to 302 milligrams on the third
day that the test dose was given. The authors suggested that
for two subjects who had previously been on a diet supplying
33 milligrams ascorbic acid per day, the tissues were taking
up the available ascorbic acid, so that although the renal
threshold was exceeded for these subjects in the third day
of administration of the 400 milligrams test dose, little
urinary excretion of ascorbic acid occurred. Melnick 33 El.
(l9h5) studied the physiological availability of water sol-
uble vitamins using five male subjects on ordinary adequate
diet. During the experflmental days, subjects were put on a
basal diet which supplied 115 milligrams ascorbic acid for
one day. The average 2h-hour urine excretion was 37 milli-
grams ascorbic acid. After a test dose of 200 milligrams

ascorbic acid was given in addition to the basal diet, the

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urinary excretion of ascorbic acid was 115 milligrams in a
24-hour period.

Todhunter, Robbins, and McIntosh (l9h2) found that as-
corbic acid intakes from 60 to 90 milligrams per day were re-
quired, in addition to a basal diet which supplied 20 milli-
grams of ascorbic acid, to saturate the tissues of three
woman subjects. Tissue saturation was judged on the basis of
urinary excretions of ascorbic acid following test doses of
MOO milligrams ascorbic acid which were administered at the
end of each period of controlled ascorbic acid intake.

Urinary excretion of ascorbic acid following a test
dose has been found to occur in a relatively short period
after the administration of the test dose. The urinary ex-
cretion of ascorbic acid during a four-hour period after the
test dose has been found to give as satisfactory an estimate
of the state of the tissues with respect to ascorbic acid as
in the urinary excretion of ascorbic acid inza 2h-hour peri-
od after the test dose (Helnick, Hochberg and Oser, l9h5;

Engelfried, l9hh; Sigurjonsson, 1951).

EXPERIMENTAL PROCEDURE

Determination of the Apparent Ascorbic Acid Content

of Frozen Strawberries

 

Plan of Experiment

Fresh strawberries of the Catskill and Robinson vari-
eties were sampled at random for the analysis of reduced and
total ascorbic acid. The rest of the berries were packaged
in pint containers, frozen at -27 to -31 degrees Centigrade,
and kept at this temperature for the entire storage period.
It was not possible to analyze all of the components studied
here in the fresh strawberries; therefore analyses of re-
duced ascorbic acid, total apparent ascorbic acid, dehydro-
ascorbic acid, 2,3-diketogulonic acid, reductones and reduc-
tic acid, reducing sugar and total solids were carried out
on strawberries which had been frozen for 30 hours. These
values provided a base line for interpretation of the changes
in these components of the strawberries during frozen stor-
age. The frozen berries were sampled every month and ana-
lyzed for these components. The period of study was six
months for strawberries of the Robinson variety and ten

months for strawberries of the Catskill variety.

a6

Freezing and Sampling of Strawberries

Strawberries of the Catskill and the Robinson varieties
were used in this experiment. They were purchased from the
local market, Lansing, Michigan, in July, 1952. Immediately
after purchase, the strawberries were washed in enamel dish
pans by immersing two quarts at a time in about six quarts
of tap water. The berries were mixed thoroughly by hand and
the water was decanted. This washing was repeated one addi-
tional time. The berries were drained in an enamel colander
for two minutes and spread on towels for removal of any ex-
cess moisture on the surface of the berries. Stems of the
berries were removed by hand and the berries were mixed
again. Samples were taken for the analysis of the reduced
and total ascorbic acid content of fresh strawberries.

Since it has been reported that sugar did not aid in the re-
tention of ascorbic acid (Rabak, 1939), the remainder of the
berries were packed without sugar or sirup in pint carton
boxes.l Two hundred grams of berries were weighed into each
box. The berries were frozen in a home type deep freeze
chest at -27 to -31 degrees Centigrade and stored at this
temperature. Four pints of each variety of the strawberries

were selected at random from the freezer approximately 30

 

1Nestrite carton pint sized box, Lily-Tulip Cup Corp.,
New York, N. Y.

LL?

hours after freezing and the berries were thawed in the car-
tons overnight (12 hours) in a refrigerator. The contents
of the four boxes were mixed thoroughly with a porcelain
spatula in a three-liter beaker. Two 200-gram samples of
the strawberries of the Catskill variety were weighed for
use in the human feeding experiment. The rest of the ber-
ries were used for the chemical analyses of reduced ascorbic
acid, total apparent ascorbic acid, dehydroascorbic acid,
2,3-diketogulonic acid, reductones and reductic acid, reduc-
ing sugar and total solids. Each month following the begin-
ning of the experiment, four pints of each variety of the
berries were thawed and sampled for chemical analyses.
Strawberries of the Catskill variety were stored in the froz-
en state for a total period of ten months. There was not an
adequate supply of strawberries of the Robinson variety on
the local market to permit analyses of the chemical constituy
ents at monthly intervals for ten months; therefore, chemical
analyses of strawberries of the Robinson variety were ter-

minated at the end of a six-month storage period.

Chemical Methods
Chemical analyses were carried out on duplicate samples
of strawberries. The order of procedure for carrying out
the various methods was similar for each series of analyses.
0n the morning after the frozen strawberries had been thawed,

samples were taken for the different procedures and the

 

 

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samples added to the acid extractant or the appropriate dil-
uent, and stored in such a manner that no change in the com-
ponent would be expected to occur which might be attributed
to sampling procedure or delay in handling of sample until
the analyses could be performed.

Reduced ascorbic acid. The determination of reduced

 

ascorbic acid in strawberries was by a modification of the
procedure reported by Loeffler and Ponting (19h2). Fifty
grams of strawberries were added to 100 milliliters of five
percent meteuahosphoric acid in the container of an electric

2 The mixture was blended for two minutes, trans-

blender.
ferred to a 500—milliliter volumetric flask and made to vol-
ume with distilled water. The contents of the flask were
mixed and filtered. Twenty milliliters of the filtrate of
the strawberries of the Robinson variety and fifteen milli-
liters of the filtrate of strawberries of the Catskill vari-
ety were diluted respectively to 100 milliliters with one .
percent metenphosphoric acid. Five milliliters of the di-
luted extract were added from a rapid delivery pipette to
five milliliters of a solution of 2,6-dichlorophenolindophe-

n01 in the cuvette of a spectrophotometer.3 The cuvette was

placed in position and the percent transmission of the solu-

 

2Waring Blender, Central Scientific Co., Chicago, Ill.

3Coleman Spectrophotometer, Model 11, Coleman Electric
Co., Inc., Haywood, Ill.

19
tion was read from the galvanometer exactly fifteen seconds
after the addition of the fruit extract to the dye reagent.
The percent transmission was measured at a wavelength of 5&0
millimicrons. The instrument was standardized at 100 per-
cent transmission against a decolorized solution containing
five milliliters of the dye reagent, five milliliters of the
dilute fruit extract and a few crystals of reduced ascorbic
acid. A series of standard solutions was prepared of vary-
ing concentrations of reduced ascorbic acid in one percent
meta-phosphoric acid. Aliquots of the ascorbic acid solu-
tions were combined with the dye reagent and the percent
transmission determined as described for the fruit extract.
The concentration of the reduced ascorbic acid in the fruit
extract was determined by the application of the Beer-
Lambert law.

Total apparent ascorbic acid. In this study, the au-

 

thor has used the term "apparent ascorbic acid" to represent
the substances in the tissue extract that react similarly to
ascorbic acid in a chemical determination of ascorbic acid
and therefore are analyzed as ascorbic acid. Particularly,
the term "total apparent ascorbic acid" has been used to re-
fer to the substances which form osazones with 2,h-dinitro-
phenylhydrazine by the Roe and Oesterling procedure (19AM).
In comparison, "total ascorbic acid" has been used by the

author with reference to the results of the study to desig-

50
nate the ascorbic acid value of foods obtained by subtrac-
tion of the diketogulonic acid and reductones and reductic
acid from the values obtained by the Roe and Oesterling pro-
cedure (19hh).

The total apparent ascorbic acid content of the straw-
berries was determined by the method of Roe and Oesterling
(l9hh). One hundred and twenty-five grams of a solution of
five percent metaphosphoric acid in ten percent acetic acid
were weighed into 25 grams strawberries in a beaker. This
mixture was blended in electric blender for two minutes.
Four grams of the slurry of the berries of Catskill variety
or five grams of the slurry of the berries of Robinson vari-
ety were made to a volume of 100 milliliters with the meta-
phosphoric-acetic acid solution. One gram of activated
charcoalLL was introduced into this solution and mixed thor-
oughly. After standing for five minutes, the mixture was
centrifuged at 2500 revolutions per minute for 15 minutes.
One milliliter of two percent 2,h-dinitrOphenylhydrazine in
10 normal sulfuric acid was added to four milliliters of the
supernatant liquid in a test tube and the mixture was incu-
bated for six hours at 37 degrees Centigrade. After incuba-
tion, the tube and its contents were cooled in an ice bath

and five milliliters of 65 percent sulfuric acid was added

 

“Merit A, Central Scientific 00., Chicago, Ill.

51
gradually with mixing. The sample then was kept at room
temperature for 30 minutes for maximum color development.
After this, the percent transmission of the solution was de-
termined with a spectrophotometer5 at a wavelength of 5h0
millimicrons.

Dehydroascorbic acid and 2,3-diketogulonic acid. Dehy-
droascorbic acid and 2,3-diketogulonic acid were determined
by the method of Roe, Mills, Oesterling and Damron (l9h8).
The method was modified by the use of twice as much thiourea
as the authors specified and the omission of stannous chlor-
ide. Stannous chloride was omitted to avoid the turbidity
produced by stannous sulfide which is formed when the proce-
dure of Roe at 31. (19MB) is followed. The use of stannous
chloride was suggested by Roe at El' (l9h8) to prevent the
strong oxidant effect of oxy-hemoglobin when present in tis-
sue. Since oxy-hemoglobin was not present in strawberries,
and since thiourea has been reported to be satisfactory for
the protection of reduced ascorbic acid in plant tissue by
Roe and Oesterling (19hh), this modification was adopted.

Twenty-five grams of strawberries were ground in a glass
mortar with 50 milliliters of a solution of five percent
thiourea in five percent metaphosphoric acid. The slurry
was diluted to 250 milliliters with five percent metaphos-

phoric acid and filtered. Twenty-five milliliters of the

 

SColeman Spectrophotometer, Model 11, Coleman Electric
Co., Inc., Maywood, Ill.

52
filtrate were diluted to 200 milliliters with five percent
metaphosphoric acid. Chemical analysis of the diluted acid
extract according to the method for total apparent ascorbic
acid, with the exception of the addition of activated char-
coal, gave the content of dehydroascorbic acid and diketogu-
lonic acid of the strawberries.

One hundred milliliters of the diluted acid extract
were measured into a large test tube; hydrogen sulfide was
passed into the test tube from a Kipp generator for 15 min-
utes and the solution was filtered. Carbon dioxide was
bubbled into an aliquot of MO milliliters of the filtrate in
order to displace the excess hydrogen sulfide. Since dehy-
droascorbic acid had thus been converted to the reduced form
of ascorbic acid, clemical analysis of this solution by the
procedure for total apparent ascorbic acid, again with the
omission of activated charcoal, gave the content of 2,3-di-
ketogulonic acid of the strawberries. The dehydroascorbic
acid content of the strawberries was obtained by subtraction
of the value for 2,3-diketogulonic acid from the value for
2,3-diketogulonic acid and dehydroascorbic acid.

Reductones and reductic acid. Reductones and reductic

 

acid were determined by the method.cfidehocken and Roe (195 ).
The extract which was prepared for the determination of“total
apparent ascorbic acid was used for the determination of re-

ductones and reductic acid. The procedure was identical to

that for the determination of total apparent ascorbic acid

53

with the exception that five milliliters of 65 percent sul-
furic acid were added before the addition of the 2,h-dinitro-
phenylhydrazine solution; this prevented the coupling of the
dehydroascorbic acid and diketogulonic acid with 2,h-dinitro-
phenylhydrazine to form osazones. The method of Schocken and
Roe (1952) was modified to the extent that the incubation pe-
riod for the tissue extract with 2,h-dinitrOphenylhydrazine
was six hours rather than three hours. Preliminary experi-
ments indicated that extension of the incubation period did
not affect the values for reductones and reductic acid, and
the procedures for the determination of total apparent ascorh
bic acid and for reductones and reductic acid were thus car-
ried out simultaneously in the laboratory.

Reducing sugar. Ten grams of strawberries were blended

 

in 100 milliliters of distilled water for two minutes. The
islurry was transferred to a 250 milliliter flask and the con-
tents of the flask were made to a volume of 250 milliliters
with distilled water. The diluted slurry was filtered and
100 milliliters of the filtrate was clarified by the addition
of 10 milliliters of saturated lead acetate. The excess lead
acetate was precipitated by the addition of anhydrous sodium
oxalate and the solution was filtered. Fehling's solution
was added to the filtrate. The cuprous oxide which was
formed was determined gravimetrically. The amount of reducing
sugar equivalent to the cuprous oxide was determined from the
Munson and Walker table for calculating invert sugar from

.weights of cuprous oxide (A. 0. A. 0., 1950).

51;
Total solids. Total solids were determined by the use

6

 

of a semi-automatic moisture tester.

Studies of the Physiological Utilization of the Apparent

 

Ascorbic Acid of Frozen Strawberries

 

Plan of Experiment

This phase of the experiment was planned to study the
utilization of the apparent ascorbic acid of strawberries
frozen for 30 hours and of strawberries frozen and stored
for four months, in comparison with the utilization of crys-
talline ascorbic acid and dehydroascorbic acid. Utilization
of the ascorbic acid was studied by determination of the
blood concentrations of reduced and total ascorbic acid at
periodic intervals after the administration of the test
doses. Reduced ascorbic acid of the urine also was deter-
mined.

There were four tests. Theinterval between the tests
was about one month. The tests were planned as follows:

(1) The determination of blood and urinary ascorbic
acid following the administration of 200 grams of Catskill
variety strawberries which had been frozen for 30 hours.

(2) The determination of blood and urinary ascorbic

acid following the administration of crystalline reduced as-

 

6Brabender Semi-automatic Moisture Tester, Model DF,
Brabender Corporation, Rochelle Park, New Jersey.

SS

corbic acid equivalent to the amount of total apparent as-
corbic acid contained in the test does of strawberries froz-
en for 30 hours.

(3) The determination of blood and urinary ascorbic
acid following administration of orange juice which.had been
treated with activated charcoal to oxidize the ascorbic acid
to dehydroascorbic acid. The amount of orange juice which
was given contained an amount of dehydroascorbic acid equiv-
alent to the amount of total apparent ascorbic acid in the
test dose of strawberries frozen for 30 hours.

(h) The determination of blood and urinary ascorbic
acid following administration of a test dose of strawberries
which’had been frozen for four months and which was estimated
to contain an amount of total ascorbic acid equivalent to
the amount of total apparent ascorbic acid in the test dose
of strawberries frozen for 30 hours.

The time intervals for the collection of blood following
the test doses were one, two, three, four and five hours,
when strawberries or dehydroascorbic acid was the test dose.
Since other workers have indicated that crystalline ascorbic
acid is absorbed quickly into the blood stream (Todhunter
and Fatzer, 19h0), blood samples were taken at one-half,
one, two, three and four hours following the test dose of
reduced ascorbic acid. Urine collections were made during a

four-hour period after the crystalline ascorbic acid test

56
dose and a five-hour period after the other test doses were

given.

Subjects

The subjects were six healthy adult women who were mem-
bers of the research staff or graduate students. The range
of ages was from 2h to 37 years; body weights were from h7.8
to 61.9 kilograms. Seven-day dietary records were collected
from each subject to provide an indication of the usual di-
etary habits and an estimation of the average daily ascorbic
acid intakes of the subjects. The ascorbic acid intakes of
the seven-day dietary records were estimated by calculation
with the use of the food table of Donelson and Leichseuring
(l9u5). Each subject was given 75 milligrams of crystalline
ascorbic acid daily for seven days preceding the administra-
tion of each test dose, in order that a comparable state of
nutrition with respect to ascorbic acid might be attained

for all subjects preceding the various tests.

Preparation of Test Doses

Strawberries. The strawberries wenaof the Catskill va-

 

riety; the berries were purchased on the local market and

washed, drained and packaged as described previously. Only
two subjects were studied at a time; therefore berries used
for the test dose were purchased in three separate lots, so

that all strawberries given the subjects in the initial test

57

period were frozen only 30 hours. Four pints of the straw-
berries of the Catskill variety were selected at random 30
hours after freezing. The berries were thawed in the car-
tons overnight (12 hours) in a refrigerator. The contents
of the four boxes were mixed thoroughly with a porcelain
spatula in a three-liter beaker. Two 200-gram samples of
the strawberries were weighed as test doses. The rest of
the berries were used for the chemical analyses of all the
components reported in this study. The test dose of straw-
berries frozen and stored for four months was 250 grams.

Crystalline ascorbic acid. The test dose of crystal-

 

line ascorbic acid was 169 milligrams. This was the quanti-
ty of total apparent ascorbic acid supplied the subjects in
the test dose of 200 grams of strawberries which had been
frozen for 30 hours. The ascorbic acid was dissolved in 50
milliliters distilled water in a 100 milliliter beaker imme-
diately before it was taken by the subject. The beaker was
rinsed twice with distilled water and the rinsings were

drunk.

Dehydroascorbic acid. At the present time, dehydroas-

 

corbic acid is not commercially available. The dehydroas-
corbic acid used as a test dose was prepared in the labora-

tory. Fresh oranges7 were purchased from the retail market.

 

7Sunkist oranges.

58
The juice was extracted from the orange by a glass reamer,
and filtered with suction through a pad of glass wool in a
Buchner funnel. To every 100 milliliters of the filtered
juice, four grams of activated charcoal was added. This
mixture was shaken for five minutes and then was filtered
under suction with a filter paper8 and a layer of filter
a1d9 in a Bflchner funnel. The filtrate was divided into
portions of 1,000 milliliters and 200 milliliters and frozen
at -30 degrees Centigrade. The sample of 200 milliliters
was analyzed for reduced ascorbic acid and dehydroascorbic
acid on the day preceding the administration of the test
dose. The juice was given in an amount to supply 169 milli-
grams of dehydroascorbic acid. The treated, frozen, and
thawed orange juice thus prepared from three batches con-
tained u0.h to kl.7 milligrams dehydroascorbic acid per 100
milliliters; the reduced ascorbic acid content was found to

be negligible.

Collection and Treatment of Blood and Urine Samples
All the blood samples were taken by finger tip puncture
according to the method of Gybrgy (1950). The blood was cols
lected in 10 x 50 millimetersvials and allowed to stand for
15 minutes in a refrigerator for formation of the blood clot.
8Whatman no. AZ, Central Scientific Co., Chicago, Ill.

9Hyflo super-cel, Johns-Manville, New York, N. Y.

59

The clot was loosened with a fine glass rod and the tube and
its contents were centrifuged. Two-tenths of a milliliter
of the serum was taken for the analysis of reduced ascorbic
acid. Titrations were performed about two hours after depre-
teinization. Ten cubic millimeters of the serum were depre-
teinized, frozen and held at a temperature of -30 degrees
Centigrade for the analysis of total ascorbic acid content.

The subjects were instructed to discard the first void-
ing of urine after rising in the morning of the test day.
The urine, excreted before the test dose was given, was col-
lected in a brown glass, one-liter bottle which contained
twenty milliliters of a solution made up of 100 milliliters
of five normal sulfuric acid and 100 milliliters of a solu-
tion of one percent metaphosphoric acid, containing two_mil-
liliters of 1.5 percent 8-hydroxyquinoline in ethyl alcohol.
The time at which the subject arose in the morning was noted;
the test dose was administered approximately one hour later.
The exact time was recorded and the ascorbic acid content of
the urine of the subject preceding the test dose was calcu-
lated as milligrams per hour.

Urine collections were made for a four-hour period fol-
lowing the test dose of crystalline reduced ascorbic acid and
five-hour periods following the other test doses. The urine

of each subject was composited for the four- or five-hour pe-

c1-

6O
riod. The collection bottles were kept in the refrigerator

throughout the test.

Chemical Analysis

The method of Farmer and Abt (1936) was used to deter-
mine the reduced ascorbic acid of blood serum.

Total apparent ascorbic acid of blood serum was deter-
mined according to the method of Lowry, Lopez, and Bessey
(l9u5). Readings of percent transmission of the solutions
after develOpment of color were made in a SpectrophotometerlO
at a wavelength of ShO millimicrons.

Urinary reduced ascorbic acid was determined by the
method of Evelyn, Malloy,and Rosen (1938). A five-milliliter
aliquot of urine in one percent metaphosphoric acid was
measured into a. cuvette of a spectrophotometer. Five mil-
liliters of a solution of 2,6-dichlorophenolindophenol were
added and readings of the percent transmission of the reac-
tion mixture were taken at five, ten, twenty, and thirty
seconds after the addition of the dye; the transmission
readings were plotted against time on linear graph paper and
extrapolated] to zero time. The zero time reading was used
to determine the ascorbic acid content in urine by the ap-

plication of the Beer-Lambert law.

 

loBeckman Spectrophotometer, model DU, Beckman Instru-
ments, Inc., South Pasadena, Calif.

RESULTS AND DISCUSSION

Changes of the Total Apparent Ascorbic Acid

in Frozen Strawberries

The mean concentrations of reduced ascorbic acid, de-
hydroascorbic acid, 2,3-diketogulonic acid, total apparent
ascorbic acid, total ascorbic acid, reducing sugar and total
solids in fresh frozen strawberries of the Catskill variety
and in strawberries of the same variety during successive
periods of frozen storage are presented in Table I and plot-
ted in Figure 1. Similar data for strawberries of the Bob-
inson variety are given in Table II and Figure 2. Values
are expressed in terms of unit weight of wet matter.

The total apparent ascorbic acid was determined by the
method of Roe and Oesterling (l9uh) and included reduced as-
corbic acid, dehydroascorbic acid, diketogulonic acid, and
any other substances which may have formed osazones with the
2,h-dinitrophenylhydrazine under the conditions of the meth-
od. Values for the 2,3-dikepogulonic acid were subtracted
from the values for total apparent ascorbic acid and the
differences are given under the heading of Total Ascorbic
Acid. Analyses were made for reductdnes and reductic acid
on all samples of the strawberries. However, this group of

compounds was not present in measurable quantities. There-

TABLE I

Th3 AVERAGE COLCEN”RATIOH OF ASCORBIC ACID AID RELATED
COHPOULDS In STRANBJHHIES (CATSLILL VARIETY)
AT mOthLY PERIODS OF STORAGE

 

 

Reduced Total
Storage Total bi Apparent
Period Solids Ascord c Ascorbic
A61 Acid
001. la Col. 2b
% ma./loo gm. mg./100 gm.
Fresh Berries - 81.6 83.0
Frozen, 30 Hrs. 11.55 75.0 80.0
Months
1 10.90 h2.3 86.3
2 11.01 no.0 78.0
3 10.51 filo? 77.5
5 11.21 u2.k 80.0
o 11.ul A9.7 7u.o
7 11.31 h7.2 78.5
8 11.50 50.6 72.5
9 ll.u5 51.u 68.6
10 11.43 51.0 75.0

 

aCol..l-Reduced Ascorbic Acid by Loeffler and Ponting
method (1742).

bCol..2-Total Apparent Ascorbic Acid by Roe and Oester-
ling method (19AM).

0001.5 : Col. 2 - 001. u.
dCOlo-é : (1‘01. 5 - 0010 30

TABLE I (CCnTlNUED)

62

 

!

 

Dehydro- Diiéio- Total Reduced Reducing
Ascorbic gulonic Ascorbic Ascorbic Sugar
Acid ’ Acid Acid Acid
001. 3 001. u 001. 5° C01. 6d
mg./100 gm. mg./100 gm. mg./100 gm. mg./100 gm. %
16.3 12.5 67.5 51.2 8.21;
21.0 29.0 57.3 36.3 7.65
22.5 6.5 71.5 h9.0 6.70
22.0 6.0 71.5 h9.5 6.7h
21.5 20.5 52.5 31.0 6.87
19.5 15.5 éuob 45.0 6.73
10.0 21.5 52.5 h2.5 6.79
16.7 2u°3 Sh-Z 3705 6079
23.5 19.5 53.0 29.5 7.29
12.7 9.5 59-3 ué-é 7-7A
9.h 15.6 59.h 50.0 6.97

 

 

..........

oooooooooo

..........

OOOOOOOOOO

 

 

 

 

Figure 1. Graph showing the changes in concentration of
ascorbic acid and related compounds, total
solids and reducing sugar in strawberries of
the Catskill variety during frozen storage.

CAT5K/LL VARIETY

REDUCED ASCORJIC ACID

I00

     
    
   

0
’ Tor/u. mamas/v 7-

A5 C ORB/C A CID
80

073/ij1».

TOT“ ’9 SC ORB/C
ACID

 

 

 

 

 

 

 

 

 

 

 

 

 

5 A A 1 A l I A__A
O I 2 J 4 r6 7 8 9 IO
P
DEHYDROAJCORa/c 4610 2,3 - DIKETOGU‘O/V/C 4610
5D» 5
, 40- 4
S.
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4 A. I A A A A_J ‘ A A L A A l J 4 L4
"0123431789” Izl436781w

Orange Time ( #10an)

TABLE II

I3 AVERAGE COACENTRATION OE ASCORBIC ACID AND RELATED
COHPOUhDS IN STRAHDERRIES (ROBIASOA VARIdTY)
AT hOthLY PERIODS OE STORAGE

 

 

 

Total
Storage Total Reduced Apparent
Period Solids ASCOPblc Ascorbic
Col. 18 001. 2b
% mg./100 gm. mg./100 gm.
Fresh Berries - h7.6 50.0
Frozen, 30 hrs. 8.25 h6.6 50.0
Honths
l 8.hl 29.7 50.6
2 8.19 2a.? L9.0
3 8.06 23.8 h3.0
A 6.15 20.5 39.0
5 8.35 22.5 h2.0
6 8.30 28.7 39.6

 

 

aCol. l-Reduced Ascorbic Acid by Loeffler and Ponting

method (l9h2). -

ling

b
Col. 2-Total Apparent Ascorbic Acid by Roe and Oester-
method (19%) o

C
001. S : C01. 2 "' 00].. LL.

dCOlo 6 : 001. 5 - C01. 30

6A

TABLE II (COhTInUED)

 

 

 

 

 

. 2 3- I" +- n
Dehydro- Dikéto- 10081 . Ineduced Reducing
ascorbic lulonic Ascorbic Ascorbic Sugar “
Acid Acid acid Acid ‘
001.3 C01. A 001. 5C Col. 69
mg./100 gm. mg./100 gm. mg./100 gm. mg./100 gm. %

6.5 6-3 h3o7 37-2 7.29
18.5 11.0 36.6 18.1 o.09
1807 005 14.805 29.8 5014.6
17.5 5.0 38.0 20.5 5.39
19.8 13.0 26.0 6.2 5.7M
1h.0 12.0 30.0 16.0 5.66

902 901+ 3002 2100 5051

 

 

Figure 2. Graph showing the changes in concentration of

ascorbic acid and related compounds, total
solids and reducing sugar in strawberries of
the Robinson variety during frozen storage.

50.0

 

 

REDUCED

ROB/N5 O/V

£5 C ORB/C ACID

 

 

  

 

 

 

 

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$0 1 . . 1 J _.
O I 2 3 4- 5' 5

66
fore, no values for reductones and reductic acid were in-
cluded in Tables I and II and it was assumed that this group
of compounds did not contribute to the values for total ap-
parent ascorbic acid.

Reduced ascorbic acid. The reduced ascorbic acid con-

 

centrations of the frozen strawberries as determined direct-
ly by reaction with 2,6-dichlorophenolindOphenol and by sub-
traction of the dehydroascorbic acid fraction of the straw-
berries from the total ascorbic acid content both are given
in Tables I and II. There was not good agreerent between
the values for reduced ascorbic acid obtained by the two
methods; nor were the differences consistent between the
values obtained by the two methods. Although the mean dif-
ference in values by the two methods for the strawberries of
the Catskill variety was only 5.h milligrams per 100 grams,
the differences in individual values varied from 1.0 to 23.8
milligrams per 100 grams. 'ne mean difference in average
values for reduced ascorbic acid at monthly time intervals
for strawberries of the Robinson variety was 6.8 milligrams
per 100 grams and the range was from 3.3 to lh.3 milligrams
per 100 grams.

The determination of reduced ascorbic acid in foods by
the two methods used here has been reported by Dodds, Price
and Moore (19h8). These authors also failed to obtain good

agreement between the two methods. However, the differences

,or

67
varied only from 0.9 to 6.3 milligrams ascorbic acid per 100
grams of baked sweet potato and were not as great as the
differences of values shown in Tables I and II. In this
study, most of the values obtained by the 2,6-dichlorophenol-
indophenol procedure exceeded those obtained by the Roe and
Oesterling (lth) procedure. However, the reverse was true
of the results reported by Dodds and co-workers. Analyses
of reduced ascorbic acid of canteloupes reported by Chen,
Elliott and Schuck (l9h8) agreed within 2.0 milligrams per
100 grams for freshly cut canteloupe and for canteloupe
halves which were stored in the refrigerator for twenty four
hours. Analyses of 2,3-diketogulonic acid were not reported
by Dodds and co-workers or by Chen, Elliott and Schuck.

Possible changes in the reduced ascorbic acid concentra-
tions of strawberries during frozen storage have been inter-
preted on the basis of analysis of reduced ascorbic acid by
the 2,6-dichlorophenolindOphenol method of Loeffler and
Ponting (l9h2), since this procedure represents a direct
measurement of the reduced form of ascorbic acid present in
the fruits.

The average reduced ascorbic acid content of the fresh
strawberries was 81.6 milligrams per 100 grams for strawber-
ries of the Catskill variety and h7.6 milligrams per 100
grams for berries of the Robinson variety. The values for

reduced ascorbic acid for both varieties were within the

 

 

 

arll‘lk, laIw 1i£inu¥u11|l

 

68
range of 25 to 1&2 milligrams per 100 grams reported for
strawberries by various investigators (Illyuvieu and Ula-
nova, 1939; Chi and Read, 1935; Burrell and Ebright, 19h0;
Chen and Schuck, 1951). Ezell, Darrow, Wilcox, and Scott
(19h?) found that Catskill berries contained 81.0 milligrams
of reduced ascorbic acid per 100 grams; this was almost

identical with the average values found in this study. These

authors reported that the reduced ascorbic acid in strawber-
ries of the Robinson variety was 71.1 milligrams per 100
grams and Einbecker and her co-workers (19u7; 1950) found
the value to be 65.6 milligrams per 100 grams. Both values
were much higher than those found in this study. Factors
such as degree of ripening (Oliver, 1938) and solar irradia-
tion (McCroy, 1936; Schuphan, 19h23 Hansen and Waldo, 19hh)
havebeen demonstrated to affect the reduced ascorbic acid
content of fruits. Variations of over 100 percent for
samples taken from the same plants on different days have
been reported by Kirk and Tressler (l9h1). The procedure of
handling the berries from the farm to the market also may
affect the ascorbic acid content of the berries. Thus any
of various factors might have been responsible for the rela-
tively low concentration found in this study for the ascor-
bic acid in the strawberries of the Robinson variety.

The reduced ascorbic acid concentration of strawberries

of the Robinson variety, thirty hours after freezing, was

69
similar to the concentration of the fresh strawberries. A
slightly lower value for reduced ascorbic acid was found for
berries of the Catskill variety after 30 hours in the frozen
state than for the fresh berries. The strawberries of the
Catskill variety contained 81.6 milligrams of reduced ascor-
bic acid per 100 grams before freezing and 75.0 milligrams
per 100 grams after freezing. This represented a loss of
about eight percent.

The average concentration of reduced ascorbic acid de-
creased rapidly during the first month of frozen storage for
berries of both varieties. The change in reduced ascorbic
acid was from 75.0 to h2.3 milligrams per 100 grams in ber-
ries of the Catskill variety and h6.6 to 29.? milligrams per
100 grams in berries of the Robinson variety. The concentra-
tion of reduced ascorbic acid in strawberries of both vari-
eties was relatively constant during the second, third and
fourth months of storage. For both varieties an increase in
reduced ascorbic acid concentration was observed after five
months of storage.

Since strawberries of the Catskill variety were used
for the human utilization studies, and since the strawber-
ries were analyzed preliminary to each feeding trial, a
greater number of replications was carried out on strawber-
ries of the Catskill variety which had been frozen for 30

hours and which had been held in frozen storage for four

70
months than at other time intervals of frozen storage or
than on strawberries of the Robinson variety. Therefore, six
additional replications were made after eight months of froz-
en storage of the berries of the Catskill variety, so that
the data for the ascorbic acid concentrations of the berries
frozen 30 hours, four months and eight months could be ana-
lyzed statistically by analysis of variance. The analysis
of variance presented in Table III indicated that the change
in the reduced ascorbic acid of the strawberries during
frozen storage of four and eight months was highly signifi-
cant (p £- 0.01). The significance of the difference of the
means was tested by the t-test (Snecdecor, l9h6). This
analysis is presented in Table II. It is apparent that the
loss of reduced ascorbic acid after four and eight months of
storage was highly significant; there was not a statistical-
ly significant difference between the average reduced ascor-
bic acid content of frozen strawberries stored four months
and that of frozen strawberries stored for eight months.

Total apparent ascorbic acid. The total apparent as-

 

corbic acid contained in the fresh berries of the Robinson
variety was 50.0 milligrams per 100 grams which was compar-
able to that reported by Paul, Wiant and Robertson (l9h9),
who found the average concentration to be h7.5 milligrams
per 100 grams. The average total apparent ascorbic acid

contained in the fresh berries of Catskill variety was 83.0

71
TABLE III
ANALYSIS OF VARIANCE OF THE CONCENTRATION OF REDUCED

ASCORBIC ACID IN STRAWBERRIES (CATSKILL VARIETY)
FROZEN FOR 30 HOURS, FOUR MONTHS AND EIGHT MONTHS

 

 

 

Source of Degree of Sum of Mean F-value
Variation Freedom Squares Squares

Total 23 5033-8

Periods 2 h260.5 2130.3 57.9**
Error 21 713.3 36.8

 

 

*fip é 0.01; highly significant.

TABLE IV

SIGNIFICANCE OF THE DIFFEREN E OR THE MEARS OF REDUCED
ASCORBIC ACID IN STRAWBERRIES (CATSRILL VARIETY)
FROZEN FOR 30 ROURS,FOUR MORTRS ARD EIGHT MONTHS

 

 

 

Comparison Calculated t-value

 

Without storage vs.

h months storage 10.200%*
Without storage vs.

8 months storage 8.367**
A months storage vs.

8 months storage 1.833

 

 

*%p 5 0.1; highly significant.

72
milligrams per 100 grams. No comparison could be made on
the total apparent ascorbic acid content of strawberries of
the Catskill variety with reported values, since no such
values were found in the literature.

The mean concentration of total apparent ascorbic acid
of strawberries of the Robinson variety, 30 hours after
freezing, was similar to the concentration of the fresh ber-
ries. A slightly lower concentration of total apparent as-
corbic acid was found in berries of the Catskill variety
after 30 hours in the frozen state than in the fresh berries.
The value after freezing was foUr percent less than that of
the fresh berries.

There was a slight increase in the concentration of to-
tal apparent ascorbic acid in strawberries of the Catskill
variety during the first month of frozen storage. The values
were 80.0 and 86.3 milligrams per 100 grams for the frozen
strawberries without storage and after one month of storage,
respectively. There was no change in the total apparent as-
corbic acid content of the strawberries of the Robinson va:
riety during the first month of frozen storage (Tables I and
II).

The concentration of total apparent ascorbic acid in
strawberries of both varieties was lowered gradually through
the second, third and fourth months of storage. There was a

decrease of nine percent:h1 the total apparent ascorbic acid

73

content of the berries of the Catskill variety and 22 per-
cent in the total apparent ascorbic acid content of berries
of the Robinson variety after four months of frozen storage.
A slight increase in total apparent ascorbic acid concentra-
tion occurred after the fifth month of storage in comparison
with the average concentration of the berries after four
months frozen storage; this was followed by a decrease after
the sixth month, in berries of both varieties. At the end of
six months of frozen storage there was an average loss of
7.5 percent in total apparent ascorbic acid in strawberries
of the Catskill variety and 21 percent in strawberries of
the Robinson variety in comparison with the values of the
berries frozen for 30 hours. In the Catskill variety, there
was a slight increase in the concentration of total apparent
ascorbic acid from the sixth to the seventh month of storage,
then a reduction was observed after eight and nine months.
The lowest value for total apparent ascorbic acid of the
berries was observed after nine months of frozen storage.
This was 68.8 milligrams per 100 grams of strawberries.
After ten months of storage, the total apparent ascorbic
acid had increased again to a value of 75.0 milligrams per
100 grams of strawberries.

Statistical treatment of the data on the total apparent
ascorbic acid content of strawberries of the Catskill vari-

ety indicated that there was a highly significant change in

71+

total apparent ascorbic acid concentration of the berries
during the storage period (Table V). The 't'-test was ap-
plied to test the significance of the differences of the
means. The results of this test indicated that there was a
highly significant decrease of total apparent ascorbic acid
concentration in strawberries after frozen storage for four
months and eight months, in comparison with frozen strawber-
ries which had been frozen only for 30 hours (Table VI).
There was not a statistically significant difference between
the total apparent ascorbic acid content of strawberries froz-
en and held for four months and that of strawberries frozen
and held for eight months.

The changes in total apparent ascorbic acid content of
strawberries in this study were in accord with the results
of Bedford and McGregor (l9h8), Dodds, Price, and Moore
(l9u8), Hartzler (19h8), Wolfe (19U9), and Mills, Damron and
Roe (19u9). These authors found that the total ascorbic acid
content of foods decreased during storage and that the total
ascorbic acid of the food varied from time to time. The
change in total apparent ascorbic acid of strawberries ob-
served in this study did not follow the pattern of the study
reported by Paul and her co-workers (l9h9), whose data indi-
cated that there was about a 75 percent increase in total
ascorbic acid in strawberries of the Robinson varietytduring

the first three months of frozen storage, in comparison with

75

TABLE v

ANALYSIS OF VARIANCE OF HE CONCENTRATION OF TOTAL APPAREL?r
ASCORBIC ACID IN STRANBERRIES (CATSRILL VARIETY) FROZEN
FOR 30 HOURS, FOUR EONTnS AND EIGHT MONTHS

 

 

 

Source of Degree of Sum of Mean
Variation Freedom Squares Square F-value
Total 23 1,313.u ,
Period 2 830.h h15.2 18.l**
Error 21 N83.0 23.0

 

 

**p g 0.01; highly significant.

TABLE VI

SIGNIFICANCE OF TAE DIFFERENCE OE THE HEARS OF TOTAL
APPARENT ASCORBIC ACID IN STRANBERRIES (CATSKILL
VARIETY) FROZEN FOR 30 HOURS, FOUR HONTHS

AND EIGRT MONTHS

 

\
‘

Comparison Calculated t-value

Without storage vs.

h months storage h.l67**
Without storage vs.

8 months storage S.833**
u months storage vs.

8 months storage 1.667

J
J

**p 5 0.01; highly significant.

 

 

75a

the fresh berries and an increase of no percent after six
months of storage; the total ascorbic acid concentration of
the frozen strawberries was greater than that of the fresh
strawberries after the end of the experimental period of
nine months. The chemical procedure used for the determina-
tion of total apparent ascorbic acid in this study was Roe
and Oesterling (19uu). This was similar to the procedure
used by Hartzler (19h8), Dodds, Price and Moore (19h8),
Mills, Damron and Roe (19h9), and Paul at 31. (19h9) for the
determination of total ascorbic acid of foods during storage.

Dehydroascorbic acid. The dehydroascorbic acid content

 

of the strawberries frozen for thirty hours was 16.3 milli-
grams per 100 grams for the Catskill variety (Table I) and
6.5 milligrams per 100 grams for the Robinson variety (Table
II). A value of 12.0 milligrams of dehydroascorbic acid per
100 grams of strawberries was reported by Mills, Damron and
Roe (l9u9). The variety of the berries studied by these au-
thors was not given.

The concentrations of dehydroascorbic acid in strawber-
ries increased during the first month of storage from 6.5 to
18.5 milligrams per 100 grams in the strawberries of the Rob-
inson variety and from 16.3 to 21.0 milligrams per 100 grams
in strawberries of the Catskill variety. The increase in
concentration of dehydroascorbic acid during the first month

of storage was in reverse of the change which was observed

76
for reduced ascorbic acid. The average concentration of de-
hydroascorbic acid in frozen strawberries of the Robinson
variety remained high from the first to the fourth month of
storage and then was lowered. The average concentration of
dehydroascorbic acid after the fourth month of frozen stor-
age was 19.8 milligrams per 100 grams. At the end of the
sixth month the concentration was lowered to 9.2 milligrams
per 100 grams. A similar pattern was observed for strawber-
ries of the Catskill variety during the first four months of
frozen storage. At the end of four months the dehydroascor-
bic acid concentration was 21.5 milligrams per 100 grams,
which was almost identical with that after the first month.
A decrease of 11.5 milligrams per 100 grams of the dehydro-
ascorbic acid in berries of the Catskill variety was ob-
served from the end of the fourth month of frozen storage to
the end of the sixth month. A subsequent increase in dehy-
droascorbic acid occurred, which reached a peak of 23.5 mil-
ligrams per 100 grams at the end of the eighth month. The
concentration of dehydroascorbic acid was lowered during the
ninth and tenth months; at the end of the tenth month of
frozen storage, there were 9.h milligrams of dehydroascorbic
acid per 100 grams of strawberries of the Catskill variety.

2,3:diketogulonic acid. After 30 hours of frozen stor-

 

age, the average concentration of the 2,3-diketogulonic acid

was 12.5 milligrams per 100 grams for berries of the Catskill

77

variety and 6.3 milligrams per 100 grams for berries of the
Robinson variety. The maximum increase in the concentration
of the diketogulonic acid in the strawberries of the two va-

rieties occurred during tee first month of frozen storage.

This increase was from 12.5 to 29.0 milligrams of diketogulon-

b-

ic acid per 100 grams in bvrries of the Catskill variety and

U

100 trams in berries of the

L

"S

from 6.3 to lh.0 millirrams pe
Robinson variety. A sudden decrease to less than the ori

_,

value was observed in both varieties after the second monthcfi‘
frozen storage. In the Robinson berries, the diketogulonic
acid concentration was relatively low at the end of the third
month, then increased to 13.0 milligrams per 100 grams at the
end of the fourth month. Values at the end of five and six
months of frozen storage were 12.0 and 9.h milligrams of di-
ketogulonic acid per 100 grams of strawberries, respectively.
In strawberries of the Catskill variety, similar changes in
the diketogulonic acid concentration were observed at the

end of three, four and five months. The concentrations were
6.0, 20.5 and 15.5 milligrams of diketogulonic acid per 100
grams of strawberries respectively. There was an increase of
six milligrams of diketogulonic acid per 100 grams of straw-

n

berries during the sixth month of lrozen tora:e and a fur-

(’1

ther increase of three milligrams per 100 grams of berries
during tLe seventh month of frozen storage. A reduction of
diketogulonic acid was observed after the eighth month and

again after the ninth month. of storage. . .A rise in dike-

78
togulonic acid concentration to a value of 15.6 milligrams
per 100 grams of strawberries had occurred by the end of the
tenth month of frozen storage.

The 2,3-diketogulonic acid content of fresh strawberries
was reported by Chen and Schuck (1951) as 1.13 to 5.11 mil-
ligrams per 100 grams. Mills, Damron, and Roe (l9h9)fomxlthat
the diketogulonic acid content in frozen strawberries ranged
from 3.0 to 37.0 milligrams per 100 grams depending upon the
condition of the berries. All values for diketogulonic acid
of the strawberries of this study were within the range re-
ported by Mills and co-workers.

Total ascorbic acid. The total ascorbic acid values

 

which were obtained by subtraction of diketogulonic acid,
reductones and reductic acid from the total apparent ascorbic
acid values also were presented in Table I for berries of the
Catskill variety and in Table II for berries of the Robinson
variety.

The total ascorbic acid concentration of berr'es of the
Catskill variety, frozen for 30 hours, was 67.5 milligrams
per 100 grams. A marked decrease to 57.3 milligrams per 100
grams was observed after the first month of frozen storage.
This value was increased to 71.5 milligrams per 100 grams
after the second month of frozen storage and was maintained
for another month. A decrease in total ascorbic acid con-

tent of berries of the Catskill variety to 52.5 milligrams

79
per 100 grams after four months storage was followed by an
increase to 6h.5 milligrams after five months storage. Dur~
ing the last five months of storage, the total ascorbic acid
values remained relatively constant, that is, from 52.5 to
59.h milligrams per 100 grams of berries.

In berries of the Robinson variety, the total ascorbic
acid content of strawberries frozen for 30 hours was h3.7
milligrams per 100 grams. This value was decreased to 36.0
milligrams per 100 grams after the first month of frozen
storage. An increase to h8.5 milligrams per 100 grams was
observed after the second month of storage; this was followed
by a decrease of 10.5 milligrams per 100 grams after the
third month of storage. By the end of the fourth month of
frozen storage, the total ascorbic acid content of berries
of the Robinson variety was only 26.0 milligrams per 100
grams. Then a slight increase to 30.0 and 30.2 milligrams
per 100 grams after five and six months of storage was found.

Reducing sugar. The concentration of reducing sugar in

 

the berries frozen for 30 hours was 8.2h percent for the
Catskill variety and 7.29 percent for the Robinson variety,
respectively. These values were within the range reported

in the literature for reducing sugar for strawberries (Chat-

H

‘ield and McLaughlin, 1928; Chatfield, 19h0). The concen-
trations of reducing sugar ranged from 6.70 to 8.24 percent

in the berries of the Catskill variety during ten months

80
frozen storage and from 5.39 to 7.29 percent in berries of
the Robinson variety during six months frozen storage. The
greatest decrease in the concentration of reducing sugar in
the berries was observed during the first two months of
frozen storage for both varieties. This decrease was 19
percent in the berries of Catskill variety and 25 percent in
the berries of the Robinson variety.

Total solids. The percentage of total solids was rel-

 

atively constant during the frozen storage period. The av-
erage range was from 10.51 to 11.55 percent in strawberries
of Catskill variety stored for 10 months. The mean percent-
age of total solids ranged from 8.06 to 8.hl percent in Bob-

inson berries for a period of six months frozen storage.

Concomitant Changes in Ascorbic Acid and Related Compounds
in Frozen Strawberries
At the end of the first month of frozen storage, there
was a marked decrease in reduced ascorbic acid concentration
of strawberries of both varieties in comparison with the con-
centration of the berries frozen for thirty hours. A defi-
nite increase in the concentration of dehydroascorbic acid
and 2,3-diketogulonic acid occurred during this period; there
was very little change in the total apparent ascorbic acid
concentration. This indicated that during the first month
of frozen storage some of the reduced ascorbic acid probably

was oxidized to~dehydroascorbic acid and 2,3—diketogulonic

81
acid. The reduction in concentration of reduced ascorbic
acid during the first month of frozen storage for strawber-
ries of the Catskill variety was greater than the correspond-
ing increase in concentration of dehydroascorbic acid and di-
ketogulonic acid. For strawberries of the Robinson variety,
however, the increment in 2,3-diketogulonic acid and dehydro-
ascorbic acid during the first month of frozen storage was
greater than the decrement in reduced ascorbic acid.

During the second month of frozen storage there was fur-
ther reduction of the concentrations of reduced ascorbic
acid in the strawberries, but the concentration of dehydroas-
corbic acid was relatively the same as at the end of the
first month. Considerable reduction in the concentration of
diketogulonic acid, occurred, perhaps due to further oxida-
tion of this acid to oxalic acid and threonic acid.

Although there was a loss of 22.5 milligrams of 2,3-di-
ketogulonic acid per 100 grams of berries of the Catskill va-
riety during the second month of frozen storage, the total
apparent ascorbic acid concentration was reduced only by 7.3
milligrams. Therefore the total ascorbic acid concentration
of the strawberries was higher after the second month than
after the first month of storage. Similarly, there was a
loss of 13.5 milligrams of 2,3-diketogulonic acid per 100
grams of strawberries of the Robinson variety during the

Second month of storage, but a change only of 0.h milligrams

82

of total apparent ascorbic acid per 100 grams of strawber-
ries. Thus, in these berries, too, an increase in total as-
corbic acid was observed at the end of the second month of
frozen storage. The failure of the marked changes in 2,3-
diketogulonic acid during the second month of frozen storage
to be reflected in the values for total apparent ascorbic
acid suggest that other unidentified substances may have
contributed to the determination of total apparent ascorbic
acid. If this were true, then the apparent increase in to-
tal ascorbic acid during the second month of frozen storage
would be an artifact. However, it also is possible that, in
the procedure of Roe and Oesterling (l9hh) for the measure-
ment of the total apparent ascorbic acid, not all of the
2,3-diketogulonic acid of the strawberries formed an osazone
with the 2,h-dinitr0pheny1hydrazine. If this were true,
then the values for total ascorbic acid obtained by differ—
ence of the 2,3-diketogulonic acid and the total apparent
ascorbic acid also would be erroneous. The experimental
pattern of this study does not provide adequate basis for
evaluating either of these hypotheses. However, values for
reduced ascorbic acid and dehydroascorbic acid were rela-
tively constant at the end of the first, second and third
months of frozen storage, and, therefore, there appears to
be support for the second hypothesis, since if other uniden-

tified substances had contributed to the measurement of to-

83
tal apparent ascorbic acid, it would seem that these sub-
stances too might have affected tne determination of dehy-
droascorbic acid.

The data were analyzed statistically in an attempt to
ascertain whether any relationships may have existed among
the substances which were determined in this study. Correla-
tion coefficients were calculated for the various components
and are given in Tables V11 and VIII for strawberries of the
Catskill and Robinson varieties, respectively. There was a
significant relationship between the total apparent ascorbic
acid and total ascorbic acid of the berries of the Robinson
variety (P 5'0.05); however, this relationship was not sig-
nificant for berries of the Catskill variety. A negative
relationship existed between the reduced ascorbic acid and
dehydroascorbic acid of strawberries of the Robinson variety
during the period of frozen storage (P 5'0.05); the relation-
ship between reduced ascorbic acid and dehydroascorbic acid

of strawberries of the Catskill variety was not significant,

although in tLes

(D

berries, too, the values varied inversely,
as indicated by a negative correlation coefficient of 0.h08.
This supports the hypothesis that some of the reduced ascor-
bic acid may have been oxidized to dehydroascorbic acid.

The reduction in dehydroascorbic acid in tne last few months
of storage (Figures 1 and 2) suggest that there was further

oxidation of the dehydroascorbic acid.

84

TABLE VII

. CORRELATION COEFFICIENTS OF THE VARIOUS COMPONENTS OF
STRAWBERRIES (CATSKILL VARIETY) DURING FROZEN STORAGE

 

4 Correlation
Components Coefficient

 

Apparent total ascorbic acid & reduced ascorbic acid -0.00S
H H H H

dehydro " " 0-309
n " " " fl diketogulonic 9 0.31
n H " " " total ascorbic " 0-32
u N " " " reducing sugar 0.133
n N " " " total solids -0-183
Reduced " " " dehydroascorbic acid -O.u08
n " " " diketogulonic " -O.116
" " " " total ascorbic " 0.106
" " " " reducing sugar 0.728*
n " " " total solids 0.598
Dehydroascorbic " " diketogulonic acid -0.0Sl
I " “ total ascorbic." 0.256
H " " reducing sugar -O.105
" " " total solids -O.h68
Diketogulonic " " total ascorbic acid -0.795**
n " " reducing sugar 0.050
n " " total solids 0.129
Total ascorbic " " reducing sugar 0.033
n H " " total solids -0-2u7
Reducing sugar " total solids 0-u70

 

 

*P 6 0.05; significant.

**P 5 0.01; highly significant.

85

TABLE VIII

CORRELATION COEFFICIENTS OF THE VARIOUS COMPONENTS OF
STRAWBERRIES (ROBINSON VARIETY) DURING FROZEN STORAGE

 

}

Correlation
Components Coefficient

 

Apparent total ascorbic acid & reduced ascorbic acid 0.589
N N H H H H

dehydro " -0.050
" " " " " diketogulonic " -0.322
“ " " " " total ascorbic " 0.798*
" " " " " reducing sugar 0.558
" " " " " total solids 0.282
Reduced " " " dehydroascorbic acid -0.763*
" “ " " " diketogulonic " -O.166
" " " " total ascorbic " 0.h65
" " " " reducing sugar 0.920**
" " " " total solids 0.221
Dehydroascorbic " " diketogulonic acid 0.082
' " " total ascorbic " 0.089
" " " reducing sugar -0.600
" " " total solids -0.275
Diketogulonic " " total ascorbic acid -0.822*
" " " reducing sugar -0.96h**
” " " total solids 0.526
Total ascorbic " " reducing sugar 0.302
n u " " total solids -0.161

Reducing sugar ; " total solids 0.200

 

 

 

*P 5 0.05; significant.
**P 5 0.01; highly significant.

 

OOOOOOOOOOOOOOOOOOOOO

 

 

. n n . I II Illlv‘rt‘lllllll
v in II ‘

 

86

There was a significantly inverse relationship between
2,3ediketogulonic acid and total ascorbic acid in strawber-
ries of both varieties. It also is apparent from Figures 1
and 2 that a decrease in total ascorbic acid appeared to oc-
cur simultaneously with an increase in 2,3-diketogulonic acid
in the strawberries. This supports the hypothesis that as-
corbic acid may be oxidized to 2,3-diketogulonic acid during
the storage of frozen fruits. However, as indicated earli-
er, there is not a simple explanation of the observation
that a decrease in total ascorbic acid at certain intervals
of frozen storage paralleled an increase in 2,3-diketogulon-
ic acid concentration, since it has been reported that 2,3-
diketogulonic acid is an irreversible oxidation product of
dehydroascorbic acid.

The inverse relationship between diketogulonic acid and
reducing sugars was highly significant for strawberries of
the Robinson variety (r = -0.96h). However, this inverse
relationship did not exist for strawberries of the Catskill
variety during frozen storage. The high values of 2,3-dike-
togulonic acid during the last three months of frozen stor-
age of the strawberries of the Robinson variety probably ac-
counted for the significantly inverse relationship between
2,3-diketogulonic acid and reducing sugar. Possibly reduc-
ing sugar as well as total ascorbic acid may have been oxi-

dized to 2,3-diketogulonic acid during the last three months

 

 

_ Sum.

I‘ll | .|IIII.|. Il‘ll'lllpi. nllIIl'lll'llll ,ll

 

 

87
of frozen storage of strawberries of the Robinson variety.

Reducing sugar was correlated positively with reduced
ascorbic acid during the period of frozen storage for straw-
berries of both varieties. This does not support the hypoth-
esis that biosynthesis of ascorbic acid may occur from six-
carbon sugar molecules during frozen storage. However, the
increase in reduced ascorbic acid in the frozen strawberries
of the Robinson variety after the fourth month of storage
and in the strawberries of the Catskill variety from the
fourth to the sixth month of storage and again after the
seventh month of storage does indicate that biosynthesis of
ascorbic acid may have occurred during frozen storage or
that dehydroascorbic acid was reduced to the reduced form of
ascorbic acid.

It is of interest that a greater number of relation-
ships existed among the constituents of the frozen strawber-
ries of the Robinson variety than of the strawberries of the
Catskill variety. However, the calculations of the correla-
tion coefficients of the constituents of the strawberries of
the Robinson variety were based only on six months of frozen
storage, whereas the calculations of the correlation coeffi-
cients of the strawberries of the Catskill variety were
based on ten months of frozen storage. Comparison of the
graphs which were presented in Figures 1 and 2 indicates

that the changes in the components of strawberries of the

--

68
Catskill variety paralleled to a considerable extent the
changes in components of the strawberries of the Robinson
variety during the first six months of frozen storage, but
that further changes during the additional four months of
frozen storage were less interdependent among the components
of the strawberries than during the first six months of
storage.

The data presented here support the reports of Bedford
and ncCregor (19h8), Dodds, Price and Moore (19h8), Wolfe,
e__§l. (l9h9) and hills, 3E 51. (19h9) that the concentration
of total ascorbic acid was decreased during frozen storage.
The data are not in accord with the findings of Paul at £1.
(19h9) that an increase in total ascorbic acid concentration
of strawberries occurred after three months of frozen stor-
age, since the chemical method used by Paul 2£.§l° (19h?) was

he Roe and Oesterling (lth) procedure which measured the
ascorbic acid and ascorbic acid-like compounds of strawber-
ries reported in Tables 1 and II of this study as total ap-
parent ascorbic acid. It was found in this study, however,
that the loss of reduced ascorbic acid of the strawberries
during frozen storage was much greater than the loss of to-
tal apparent ascorbic acid or of total ascorbic acid. flore-

over, there did not appear to be a constant relationship be-

tween total ascorbic acid and the combined fractions of re-

89
duced ascorbic acid determined by the 2,6-dichlorophenol-
indOphenol method and dehydroascorbic acid. It would appear
therefore that there is need for further study of the sub-
stances which may interfere with the methods for the deter-
mination of ascorbic acid and for the development of proced-

ures which may eliminate this interference.

Physiological Utilization of the Total Apparent Ascorbic
Acid of Frozen Strawberries

Description of the subjects. A physical description of

 

the six subjects is presented in Table IX. he ages of the
subjects ranfed from 2b to 37 years and the weights ranged
from 47.8 to 61.9 kilograms. The heights of these subjects
varied from 152.5 to 173.5 centimeters.

The average daily intake of ascorbic acid for each sub-
ject on her customary diet was calculated from a seven-day
dietary record kept by the subject between the periods of
administration of the test doses of strawberries frozen 30
hours and strawberries frozen for four months. This repre-
sented the period between July and hovember, 1952. Thelnean
range of ascorbic acid intake by the subjects was from 51 to
118 milligrams per day with an‘average of 96 milligrams, as
shown in Table IX. All of the subjects but one, M. m”kairwan
daily intakes of ascorbic acid which exceeded 70 milligrams
per day, the daily allowance recommended for young women by

the Food and Kutrition Board of the Eational Research Coun-

 

 

 

 

 

 

TABLE IX

 

 

9O

 

Dietary Intake'of

 

Subject Age Weight Height Ascorbic Acid*
yrs. kg. cm. mg??gay mg??§:y

H. A. 26 u7.8 157.5 118 78-1uo
. w. 2h 57.8 159.5 118 90-125
w. B. 37 59.1 165.2 10k 82-112
B. H. 32 61.9 173.5 103 9h-120
w. M. 25 53.u 162.5 51 30-85
D. K. 26 49.0 152.5 80 68-105

 

*Based upon seven-day dietary record.

:-
‘—

91
oil (Shank, 195M). Subject M. M. had an average daily in-
take of 51 milligrams of ascorbic acid with a range of 30 to
85 milligrams per day.

Serum concentration and urinary excretion of ascorbic

 

acid before test dose. Reduced and total apparent ascorbic

 

acid concentrations of the blood serum of six subjects be-
fore the ingestion of each of.the test doses are presented
in Table X and Figure 3. Each value represents the average
of results obtained from tests performed on two consecutive
days. The mean serum reduced ascorbic acid concentrations
before the respective test doses were 1.21 e 0.05, 1.21 t
0.05, 0.89 t 0.03, and 1.00 t 0.08 milligrams per 100 milli-
'liters. The corresponding mean concentrations of total ap-
parent ascorbic acid in the serum were 1.38 1 0.07, 1.51 1
0.06, 1.62 g 0.07, and 1.h2 g 0.06 milligrams per 100 milli-
liters. Statistical treatment of the data by analysis of
variance indicated that there was a significant difference
among the basal ascorbic acid values before the administra-
tion of the different test doses (Table XI). Application of
the Fisher's ‘t'-test indicated that the concentrations of
reduced ascorbic acid in the serum before the test dose of
200 grams strawberries, frozen for 30 hours, and before the
test dose of crystalline ascorbic acid were significantly
higher than before the test doses of charcoal-treated orange

juice and of 250 grams strawberries, frozen for four months.

'0

 

TABLE X

T33 REDUCED AND TOTAL APPAREET ASCORBIC ACID 0? Th3 BLOOD

SERUM AQD THE URINARY EXCRETION OE ASCORBIC ACID 0?

m2?

.LAL-J

SUBJECTS PRECEDIhG Th3 ADhIhISTRATION or T33 TEST_
Dosssfiaoa THE SUCCESSIVE EXPERIEENTAL PERIODS

 

 

 

 

 

 

 

Test I Test II
Serum Urine Serum Urine
Sub- Total Total
ject Reduced apparent Reduced Reduced apparent Reduced
ascorbic ascorbic ascorbic ascorbic ascorbic ascorbic
acid acid acid acid acid acid
mg./1oo m1. mg./hr. mg./100 m1. mg./hr.
H. A.” 1.28 1061 0093 1010 1.33 0097
A. W. 1.38 l.hh 0.h6 1.10 1.50 0.80
w. B. 1.09 1.23 0.80 1.37 1.61 2.22
B. H. 1.29 1.39 1.85 1.28 1.62 7.55
1'1. 1:. 1006 101,4. Ion—O 1012 1033 1.58
D. K. 1.17 1.87 2.9M 1.28 1.67 2.83
I/IEAN 1.211 1.38: 1.14.0: 1.21: 1.51: 2.593
0.05 0.07 0.37 0.05 0.06 1.02

 

*Test Dose: I--200 grams of strawberries frozen for 30

hours.
II--169 milligrams of crystalline reduced
ascorbic acid.

III--l69 milligrams of dehydroascorbic acid
in the form of charcoal-treated orange
juice.

IV--250 grams of strawberries, frozen and
stored for four months.

92

TABLE x (CONT.)

 

 

 

 

 

 

 

Test III Test IV
Serum Urine Serum. Urine
t t
Reduced aggagint Reduced Reduced aggaiint Reduced
ascorbic ascorbic ascorbic ascorbic ascorbic ascorbic
acid acid acid acid acid acid
mg./100 m1. mg./hr. mg./1oo m1. mg./hr.
0.87 1.82 1.50 0.96 1.36 0.68
1.02 1.h5 1.95 1.00 1.81 1.83
0.93 1.83 1.30 0.98 1.60 1.18
0.82 1.77 2.h3 0.95 1.57 0.78
0.83 1.89 , 2.52 1.00 1.19 1.10
0.85 1.76 1.35 1.11 1.h1 0.69
0.89: 1.62; 1.8ht 1.001 1.82: 1.03:
0.03 0.07 0.22 0.08 0.06 0.18

 

 

 

 

IIIIII

......

......

OOOOOO

......

 

 

 

 

 

 

 

 

___..—.-._-.-——'
- __‘______.__ ... “——

-_1_.__.f4fi

Figure 3. Concentrations of blood ascorbic acid of six

subjects before and after various test doses.

Test dose: I--200 grams of strawberries, frozen

for 30 hours.
II--169 milligrams of crystalline re-
duced ascorbic acid.

III-~169 milligrams of dehydroascorbic
acid in the form of charcoal-treatmi
orange juice.

IV--250 grams of strawberries, frozen
and stored for four months.

m REDUCED Ascoealc Ive/0 (IA/CREME/vr)

[:1 REMED acme/d “'10 can“)

WI/I/I/I/A—

 

 

 

m 7074‘. flPPflfiE/V 7' ASCOREIC ACID (YA/CREME” T)

 

 

 

(811551.)

 

- Tor/u. APFWPEIVT weeks/c ACID

 

2.5

( 7w col/'[ul) ppy 31120359 (10499

 

MEAN

fl. W-

.SVJJBT H.R.

911

TABLE x1

ANALYSIS OF VARIANCE OF CONCENTRATIONS OE SERUM
ASCORBIC ACID PRECEDING THE ADMINISTRATION
OF VARIOUS TEST DOSES

 

 

Degree of
Source of Variation Freedom F-value

 

Reduced Ascorbic Acid

 

 

Test Doses 3 21.92**
Subjects 5 2.19
Error 15

Total Apparent Ascorbic Acid "
Test Doses 3 3.53w
Subjects 5 2.53
Error 15

*P 5 0.05; significant
**
P 5 0.01; highly significant

95
The concentration of total apparent ascorbic acid in the
serum was significantly higher before the test dose of char-
coal-treated orange juice than before the test doses of
strawberries. However, all individual serum values for re-
duced and total apparent ascorbic acid preceding the admin-
istration of the different test doses exceeded the value of
0.7 milligrams per 100 milliliters, which has been reported
as a blood concentration that is indicative of a good state
of ascorbic acid nutrition (Moyer, Harrison, Fisher, and
Miller, 1988).

The urinary excretions of ascorbic acid before the dif-
ferent test doses were presented in Table X and have been
plotted in Figure 8. The ranges of ascorbic acid excreted
in the urine before the ingestion of the test doses of
strawberries, frozen for 30 hours, crystalline reduced as-
corbic acid, charcoal-treated orange juice, and strawberries,
frozen for four months, were 0.86 a 2.98, 0.80 a 7.55, 1.30
__ 2.52, and 0.68 1 1.83 milligrams per hour with averages of
1.80 1 0.37, 2.59 t 1.02, 1.88 t 0.22, and 1.03 t 0.18 mil-
ligrams per hour, respectively.’ Statistical treatment of
the data by analysis of variance indicated that there was no
significant difference in the amounts of ascorbic acid which
were excreted in the urine preceding the various test doses

(Table XII).

 

 

— —. ._—.____—-—

__. -.___——-- .«—-
-.__._—.-.———

Figure 8.

Urinary excretions of reduced ascorbic acid by

six subjects preceding and following the admin-
istration of various test doses.

field in (Ir/'00 (”y/bx)

. Assert/c

30'

20'

IO'

 

 

 

 

 

 

 

 

 

 

 

 

 

'2??er z 3 4-
sunscr AM.

 

 

 

 

 

 

 

AM

 

 

 

4'.J1

 

234

Wad.

 

 

 

 

 

 

 

 

- Pfific E DING TEST DOJ’E

1:! FOLLOWING TEST DOSE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I ‘134
MM.

4' 2 3 4-
MEAN

\
e.,:

l
. w
A

ANALYSIS OE VARIANCE OF

ASCORB

IC ACID IN

THE ADMINISTRATION OF

{IT}?
114—.)

TABLE XII

97

THE CONCENTRATION OF REDUCED

URINE OE SIX SUBJECTS PRECEDING

VARIOUS TEST DOSES

 

 

 

Source of Degrees of Sum of Mean fl
Variation Freedom. Squares Square r-value
Total 23 16.27

Test Doses 3 8.13 2.71 1.53
Subjects 5 11.52 2.30 1.30
Error 15 26.62 1.77

 

 

98

The concentration of reduced and total apparent ascor-
bic acid in the serum of the six subjects and the urinary
excretions of reduced ascorbic acid by the subjects indi-
cated that the subjects were in a good state of nutrition
with respect to ascorbic acid preceding the administration
of each of the test doses. It had been anticipated that the
administration of 75 milligrams of crystalline ascorbic acid
to each subject preceding the various tests might establish
a uniform state of ascorbic acid nutrition for the subjects
for all of the tests. Statistical differences in the serum
concentrations of ascorbic acid preceding the different tests
indicated that this was not achieved. Thus it would appear
that the variations in dietary ascorbic acid of the subjects
were great enough to affect the serum concentrations of as-
corbic acid. The differences may have been partly seasonal
in nature, since the mean concentrations of reduced ascorbic
acid in the serum of the subjects preceding the first two
tests were higher than the mean concentrations of reduced
ascorbic acid in the serum of the subjects preceding the
last two tests. The first two tests were conducted in July
and August and the last two tests in September, October and
_Eovember. Seasonal differences, if they existed, probably
reflected the use of a greater amount of fresh fruits and

vegetables in the summer months than in the early fall. Un-

99

fortunately, dietary records were not obtained preceding
each test.

Ascorbic acid supplied in test doses. The total appar-

 

ent ascorbic acid concentration of the test dose of 200 grams
of strawberries, frozen for 30 hours, was the basis for se-
lection of the amount of test doses of crystalline ascorbic
acid, dehydroascorbic acid and strawberries, frozen for four
months. The ascorbic acid content of the individual test
doses of strawberries, frozen for 30 hours, is given in
Table XIII. The total apparent ascorbic acid content of the
test dose of strawberries, frozen for 30 hours, varied from
16u.0 to 176.0 milligrams per subject. The mean concentra-
tion of total apparent ascorbic acid supplied by the straw-
berries, frozen for 30 hours, was 169 milligrams in the test
dose of 200 grams. This represented the total apparent as-
corbic acid of the strawberries, since diketogulonic acid,
reductaxs and reductic acid were not determined separately.
The reduced ascorbic acid of the test dose of strawber-
ries, frozen for 30 hours, ranged from 137.2 to 156.0 milli-
grams for the individual subjects. The mean ascorbic acid
intake supplied by the test dose of strawberries, frozen for
30 hours, was lh8 milligrams. This represented the concen-
tration of reduced ascorbic acid measured by the oxidation-
reduction method of Loeffler and Ponting (l9h2), using 2,6-

dichlorOphenolindophenol as an oxidizing reagent.

kn

100

TABLE XIII

ASCORBIC ACID CONTENT OF INDIVIDUAL TEST DOSES OF ZOO GRAMS
OF STRAWBERRIES, FROZEH FOR 30 HOURS

 

 

Nature of Subject
Ascorbic Mean
Acid H. A. A. W. W. B. B. H. M. M. D. K.

 

Reduced a. a.,
mg. 150.0 150.0 137.2 137.2 156.0 156.0 1&8

Total apparent
a. a., mg. 166.0 166.0 176.0 176.0 16h.0 16u.0 169

 

 

101

Test doses of crystalline reduced ascorbic acid and de-
hydroascorbic acid were given in amounts equivalent to the
average intake of total apparent ascorbic acid of the straw-
berries, frozen for 30 hours. The test dose of crystalline
reduced ascorbic acid and dehydroascorbic acid each supplied
169 milligrams per subject.

The amount of strawberries, frozen for four months,
which was given as a test dose was 250 grams. The increased
amount of strawberries after frozen storage for four months
in comparison with the amount of freshly frozen strawberries
was given to compensate both for the reduction in concentra-
tion of biologically active forms of ascorbic acid and the
increase in d’ketogulonic acid concentration in strawberries,
frozen and stored for four months. It was considered desir-
able to adjust the test dose of strawberries for the increase
in diketogulonic acid concentration, since it has been re-
ported that diketogulonic acid does not possess the biologi-
cal activity of reduced ascorbic acid (Borsook'et a1. 1937).

The total apparent ascorbic acid and the reduced ascorbic

‘l 0

acid of the individual test doses of strawberries after
frozen storage for four months-are given in Table XIV. The
total apparent ascorbic acid of this test dose of strawber-
ries varied from 173.6 to 192.3 milligrams per subject, with

a mean of 183. The reduced ascorbic acid of the test dose

of 250 grams of strawberries, frozen and stored for four

4--

102

TABLE XIV

ASCORBIC ACID COhTENT OE IADIVIDUAL TJST DOSES OF 250 GEARS

OF "TEAWBEHRIES, 330233 FOR FOUR LOETHS

=—

 

_’__ --.- .

 

Nature of
Ascorbic

Subject
Mean

 

Acid H. A. A. w.

154. B. E. II. 1'1. I'i. D. 1:.

 

Reduced a. a.,

Total apparent
a. a., mg. 181.9 181.9

123.8 123.8 97.5 97.5 11h

192.3 192.3 173.6 173.6 183

 

 

103
months, ranged from 97.5 to 123.8 milligrams for the sub-
jects with a mean concentration of 11h milligrams, as deter-
mined by the 2,6-dichlorophenolindophenol method.

, Unfortunately, time did not permit analyses of dehydro-
ascorbic acid, diketogulonic acid and reductones and reduc-
tic acid in the samples of strawberries fed to the subjects.
Therefore these data were obtained from the analyses of the
components of the frozen strawberries of the Catskill vari-
ety which were sampled monthly during storage and reported
in Table I. The total apparent ascorbic acid of the straw-
berries sampled after 30 hours freezing was 80 milligrams
per 100 grams. The average concentration of the strawber-
ries fed in a test dose at this time was 8h.5 milligrams of
total apparent ascorbic acid per 100 grams. The total ap-
parent ascorbic acid of the strawberries sampled after four
months frozen storage was 72.5 milligrams per 100 grams of
strawberries. This agreed closely with the average concen-
tration of the total apparent ascorbic acid of the test
doses of strawberries fed to the subjects, 1. e., 73.2 mil-
ligrams per 100 grams. The similarity of values indicated
that the sampling procedure was satisfactory and the author
was justified in using the data for the various components
reported in Table I for calculation of the concentrations of

these compounds in the test dose of strawberries.

10h

The concentrations of total apparent ascorbic acid,
2,3-diketogulonic acid, reductones and reductic acid, total
ascorbic acid, dehydroascorbic acid, and reduced ascorbic
acid of the test doses of strawberries are given in Table
XV. The total apparent ascorbic acid contained in the test
dose of strawberries, frozen for 30 hours, of crystalline
reduced ascorbic acid, of charcoal-treated orange juice, and
of strawberries, frozen and stored for four months, were 169,
169, 169, and 183 milligrams, respectively. The correspond-
ing concentrations of total ascorbic acid which were computed
by subtraction of the 2,3-diketogulonic acid from the total
apparent ascorbic acid were 1AA, 169, 169, and 132 milligrams,
respectively. The 2,3-diketogulonic acid was not present in
the test dose of crystalline ascorbic acid and dehydroascor-
bic acid, but there were 25 milligrams of 2,3-diketogulonic
acid in the 200 grams of strawberries which had been frozen
for 30 hours and 51 milligrams of 2,3-diketogulonic acid in
the test dose of strawberries which had been frozen and
stored for four months. Reductones and reductic acid were
absent from all of tie test doses. ho dehydroascorbic acid
was present in the test dose of crystalline ascorbic acid,
but there were 169 milligrams of dehydroascorbic acid in the
charcoal-treated orange juice, 33 milligrams in the test
dose of freshly frozen strawberries and 5h milligrams in the

berries which were frozen and stored for four months. The

 

 

TABLE XV
Th3 COLCERTRATIOH OE ASCORBIC ACID AnD RELATED COAPOUHDS
IN THE VARIOUS TflST DOSES

 

 

 

Total
Apparent 2,3-Diket0-
Source of Ascorbic Acid Ascorbic gulonic
Acidl Acid
001. 1 Col. 2
mg. mg.
Strawberries, Frozen 30 Hours 169 25
Crystalline Ascorbic Acid 169
Charcoal-treated Oranfe Juice 169
Strawberries, Frozen Four honths 183 51

 

 

 

.- — —.—___.._

Includes reduced ascorbic acid, dehydroascorbic acid,
diketogulonic acid, and any other substances which may have

formed osazones with 2,h-dinitropheny1hydrazine under the
conditions of the method.

Total apparent ascorbic acid corrected for diketogulon-

ic acid, reductones and reductic acid 001. 1 - (Col. 2 +
Col. 3) .

3Reduced ascorbic acid determined by a modification of
the procedure of Roe and Oesterling (19AM); 001. h - Col. 5 .

LLReduced ascorbic acid determined by 2,6-dichlorophenol-
indOphenol method.

 

105

TABLE xv (cowTIXUED)

’*
L:

Reductones

 

and Total2 Dehydro- Reduced3 Reduced)4
Reductic Ascorbic ascorbic Ascorbic Ascorbic
Acid Acid Acid Acid Acid
Col. 3 Col. u Col. 5 '
mg. mg. mg. mg. mg.
0 11a 33 111 iua
169 169 169
169 169 0 0

o 132 51 78 114

V—fi—

106
reduced ascorbic acid values obtained by the subtraction of
the dehydroascorbic acid and 2,3-diketogulonic acid from the
total apparent ascorbic acid were 111, 169, 0, and 78 milli-
grams in the test dose of strawberries frozen for 30 hours,
crystalline reduced ascorbic acid, charcoal-treated orange
juice, and strawberries, frozen and stored for four months.
The corresponding values for reduced ascorbic acid deter-
mined by the 2,6-dichlorophenolindOphenol method were 1&8,
169, 0, and 11h milligrams, respectively. The reduced as-
corbic acid values of the strawberries as determined by the
2,6-dichlorOphenolindophenol method were 37 and 36 millie
grams higher than the reduced ascorbic acid values of the
two test doses of strawberries as determined by the 2,h-di-
nitrophenylhydrazine method.

The utilization of ascorbic acid of the various test

 

dgggg. The series of tests in which (a) crystalline reduced
ascorbic acid and (b) charcoal-treated orange juice, as a
source of dehydroascorbic acid, were the test doses were in-
cluded in the experiment to provide a basis for the inter-
pretation of the data for blood and urinary ascorbic acid
following the administration of the two test doses of straw-
berries. Therefore the data which were obtained when crys-
talline ascorbic acid and dehydroascorbic acid were the test
doses are presented first, although this was not the order

in which the tests were performed.

107

Table XVI gives the concentrations of reduced ascorbic
acid of the blood serum of the six subjects at periodic in-
tervals after the administration of the various test doses.
The concentrations of reduced ascorbic acid in the blood
serum of the six subjects before the administration of the
test dose, although given previously in Table X, are includ-
ed again in this table so that the increments in serum as-
corbic acid resulting from the test doses can be estimated.
The concentrations of reduced ascorbic acid of the blood
serum of the six subjects preceding and at periodic inter-
vals following the administration of the various test doses
also are shown graphically in Figure 5.

Similar data for the concentration of total apparent
ascorbic acid of the blood serum of the subjects before and
at periodic intervals after the administration of the test
doses are given in Table XVII and shown graphically in Fig-
ure 6. Each series of tests included the measurement of re-
duced and total apparent ascorbic acid in the blood serum
before and after the administration of the test dose. There
was not a statistical difference between the two daily repli-
cations of each test; therefore the blood values for ascor-
bic acid obtained on the two successive days were averaged
for each subject and the values given in Tables XVI and XVII
are the averages of the two successive daily tests for each

subject.

 

 

TABLE XVI

REDUCED ASCORBIC ACID CONCENTRATIONS OF BLOOD SERUM OE SIX
SUBJECTS PRECEDING AhD AT PERIODIC IhTERVALS AFTER
TnE ADHINISTRATION OF VARIOUS TEST DOSES

 

 

Time After

 

Test Dose Ingestion
of Test Dose

 

 

 

 

 

 

1.1. A. A. w.
hrs.
9 1.;8 1.10
" lo I'M-7
159 mg. . . i 1.69 1.62
Reduced Ascorbic ACld 2 1.71 1.5
3 1.60 1.58
h 1.59 1.50
O 0.87 1.02
1 1.12 1.21
Dehydroascorbic Acid 3 1.2M 1.31
h 1.16 1.16
S 1.13 1.11
O 1.28 1.38
l 1.61 1.60
200 grams 2 1.68 1.71
Strawberries, 3 1.69 1.6M
Frozen 30 Hours H 1.66 1.57
5 1.60 1.55
O 0.96 1.00
1 1.2a 1.35
2 O 2 O
250 grams 3 i £2 33: gg
Strawberries, u 1.3u 1.3u
Frozen for Four Honths 5 1.28 1.32

 

wEach value represents the average of two consecutive
days' tests.

108

TABLE XVI (COHTIHUED)

 

 

Reduced Ascorbic Acid*in Blood Serum
mg./100 m1.

 

 

 

 

 

 

Subject
Mean i S E.
W. Bo B. H. 1'1. II. D. K.

1.37 1.28 1.12 1.28 1.21 i 0.05
1-5h 1.59 1.26 l.uh l.u8 t 0.05
1.76 1.79 1.h9 1.61 1.66 t 0.05
1.69 1.65 1.58 1.72 1.67 i 0.02
1.71 1.59 1.66 1.70 1.6A 2 0.02
1.63 1.53 1.62 1.58 1.58 t 0.08
0.93 0.82 0.83 0.85 0.89 t 0.03
1.19 1.0a 0.96 1.11 1.11 2 0.0M
1.3M 1.23 1.16 1.29 1.28 t 0.03
1.30 1.1a 1.05 1.27 1.22 t 0.0M
1.16 1.10 0.99 1.16 1.12 t 0.03
1.16 1.05 0.96 1.16 1.10 2 0.03
1.09 1.29 1.06 1.17 1.21 t 0.05
1.17 1.38 1.36 1.86 1.50 t 0.10
1.h1 1.60 1.39 1.77 1.59 t 0.07
1.60 1.93 1.h1 1.65 1.65 i 0.07
1.u7 1.70 1.51 1.63 1.59 i 0.0M
1.27 1.58 1.h3 1.57 1 50 i 0.05
0.98 0.95 1.00 1.11 1.00 i 0.08
1.27 1.27 1.22 1.u9 1.31 i 0.0M
1.u2 1.59 1.h6 1.75 1.52 t 0.06
1.50 1.38 1.6M 1.60 1.88 I 0.05
1.u0 1.3M 1.53 1.5M 1.u2 I 0.0M
1.23 1.27 1.37 1.32 1.30 t 0.03

 

M...- 1-1

-

 

 

Eigure 5.

Concentrations of reduced ascorbic acid of blood
serum of six subjects before and at periodic in-
tervals after the administration of various test

doses.
Legend:

After test dose of crystalline reduced

ascorbic acid

test does of dehydroascorbic

acid

After test dose of strawberries,
frozen for 30 hours

After test dose of strawberries,
frozen for four months

After

 

wth .3.“ .3xe .9361 S 0835

 

 

 

 

 

 

 

 

 

 

 

 

a. .V m. N a o u. + n N . x 0 hi .V m. N a o
3 1 q 1 1 as 1 1 I d 1 n6 4‘ 1 3 I 3 ”.0
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3 /| |.\ 3: WW
3 re FM.
9
9
a
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a P
g0.” .o.N ¥°.~
b 4. n N 1 o m + m. N a o h w n a . .
. w 4 . . ed . . . - . no .1 . 1 J . . ad
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mp
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a:
0.. a V
a.
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o
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-— .—..—.-—_.-———-.
_.-_._..—._—

TABLE XVII

TOTAL APPAKELT ASCOHSIC ACID COICELTRAHIOXS OF BLOOD SERUJ

03 SIX SUjJECTS

“ETCEDIKG AnD AT PjHIUDIC IATERVALS

I 1’;
M91“: "3:33 A0.-1I1.ISTZ1ATIOA o:- VARIOUS ”.3511 DOSES

Test Dose

Time After

 

 

Ingestion

of Test Dose

 

 

 

 

 

II. A. A. w.
hrs. .

o 1.33 1.50
‘ :12- 1.73 1.611
169 mg. . 1 1.92 1.80
Reduced Ascorbic Acid 2 2.02 1.97
3 1.82 1.78
1 1.711 1.75
O 1.82 l.u5
1 2.05 1.gg
169 mg. 2 2033 1. \
Dehydroascorbic Acid i 2.18 1.85
5 1.95 1.611
0 1.61 1.%u
200 grams % 2:11 1:98
Strawberries, 3 1.82 1.78
Frozen 30 Hours 8 1.86 1.71
5 1.81 1.58
0 1.36 l.%%

1 1.79 l.
w 250 grams 2 2.10 2.12
Strawberries, 7 3 1.74 1.93
Frozen for Four months u 1.62 1.70
5 1.61 1.61

 

“Each value represents
days' tests.

the average of two consecutive

m- ,_ 4.— _=—_- ‘_

110

Mean t S. E.
1.51 t 0.06

K.

3.8 u/O 8 8
3.1251388 7.
O

M. M.

TASLE XVII (CUhTInUED)

/100 m1.

27150140
67022 00

000000
112222

mg.

Subject

1.61
1.76
2.06
1.98
1.95
1.88

‘W. B.

 

 

 

 

 

 

 

Total Apparent Ascorbic Acid* in Blood Serum

7.7600146
000100

c o o o o 0
000000
+_+.¢.-+..T-+-

8101.478
33888 7/0

111111

700 701/0 3
I48 96/06
0 O O C C 0

111111

1.1h
1.89
1.50
1.58
1.78
1.52

91488.
38 0/21

0 O O
1112

1.89
1.92

1.23
1.79
1.83
1.89
1-79
1.62

1.81
1.98
2.08
1.96
1.89
1.75

1.19
1.68
1.82
1.90
1.80
1.78

1.57
2.03
2.28
2.19
2.13
2.05

1.60
1.87
1.96
1.97
1.89
1.73

 

 

 

i -. __.___._--__. 4-
,_ V...—_.——

Figure 6.

Concentrations of total apparent ascorbic acid of
blood serum of six subjects before and at periodh:

intervals after the administration of variouS'Mmt
doses.

Legend:

————— After test dose
bic acid

--------- After test dose
acid

After test dose of strawberries,
frozen for 30 hours

After test dose of strawberries,
frozen for four months

of crystalline ascor-

of dehydroascorbic

 

 

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112
The greatest concentration of redu ed and total appar-
ent ascorbic acid in the blood serum of the subjects after
the administration of the various test doses was used as a

basis for the calculation of the highest increment in blood

serum ascorbic acid for each subject following

_—

the ingestion
of the test doses. The calculated increments of reduced as-
corbic acid of the blood serum of the six subjects after the
administration of the various test doses are given in Table
XVIII and similar data for the total apparent ascorbic acid
of the blood serum of the subjects are given in Table XIX.
The excretions of reduced ascorbic acid by the subjects
before and after the administration of the tee doses are
given in Table XX. Values are expressed as milligrams of
reduced ascorbic acid excreted in the urine per hour. The
values for urinary excretions of ascorbic acid by the sub-

jects also represent averages of two daily replications of

each test.

Test Dose of Crystalline Reduced Ascorbic Acid
The test dose of crystalline reduced ascorbic acid was
169 milligrams. This was equivalent to the amount of total
apparent ascorbic acid in the 200 grams of the strawberries
which were frozen for 30 hours. T18 average concentrations
of reduced ascorbic acid in the blood serum of the subjects

at intervals of one-half, one, two, three and four hours

after the test dose were 1.h8 t 0.05, 1.66 t 0.05, 1.67 t

CALCULATED IhCREHEKT 0?

TABLE XIX

111.}.

TOTAL APPARELT ASCORBIC ACID OF

BLOOD SERUA 0E SIX SUBJECTS AFTJR ADthISTRATION
OF VARIOUS TEST DOSES

 

Increment* of Serum Total Apparent

Ascorbic Acid, mg./100 m1.

 

Test Dose

 

 

 

 

 

Subject
Mean 1 S.E.
HOA. Aow. W.B. BQH. I'TQI‘I. DOK.

169 mg. 2
Reduced 0.69 0.87 0-85 0.73 0.55 0.87 0.56 t 0.05
Ascorbic Acid

169 mg.
Dehydro- 0.51 0.83 0.81 0.35 0.89 0.52 0.85 i 0.03
ascorbic acid

200 grams
Strawberries, , .. . , ,
Frozen for 0.50 0.52 0.66 0.85 0.60 0.50 0.61 t 0.06
30 hours '

250 grams
Strawberries
Frozen for ’0.78 0.71 0.37 0.71 0.71 0.63 0.65 t 0.06

Four Months

 

 

 

*eased on highest concentration

of the subject after the test dose.

of serum ascorbic acid

.__—___..—. .-
, __,..__—._.._—
__.————...

TABLE xx

AVJRAGE URINARY EXCRETIOLS 02 REDUCED ASCORBIC ACID
BEFORE m0 AFTER ADL-iIl-JISTHATIO‘N
ow THE VARIOUS TEST DOSES

Urinary Reduced Ascorbic

 

 

 

 

 

 

 

b-
Test Su
Dose 5. A. A. w. w. B.
Before After Before After Before After
169 mg.
Reduced Ascorbic 0.97 16.73 0.807 80.07 2.22 18.56
Acid
169 mg.
Dehydroascorbic 1.50 18.60 1.95 27.12 1.30 16.95
Acid
200 grams
Strawberries, Frozen 0.93 17.20 0.86 12.80 0.80 22.02
for 30 Hours
250 grams
Strawberries, Frozen 0.68 18.90 1.83 29.01 1.18 18.88
for Four Months

 

 

 

F’””

 

 

TABLE xx (CONT.)

115

 

Acid, mg./hr., Before and After Test Dose

 

 

 

 

 

 

 

ject
. In T'Iean . S. E.
B. 1‘1. Ho M. D. K.
Before After Before After Before After Before After
7.55 18.59 1.58 10.98 2.83 18.13 2'59 1 19°17 1
1.02 8.29
"’ 1 1.8L]. 1. 18071 t
2.83 18.38 2.32 13.00 1.35 18.19 0.22 1.89
1.80 t 16.82 i
, . 1.03 1 16.98 i
0.74 11.78 1.10 13.15 0.69 18.60 0.18 2.60

 

L

 

116

002, 1.6L; 1- 0.02 and 1.58 i 0.08 milligrams per 100 milli-

liters of serum, respectively (Table XVI). The highest

value for the mean concentration of reduced ascorbic acid

in the blood serum occurred two hours after the ingestion of

the test dose. The highest value for serum reduced ascorbic

acid occurred at two hours after the ingestion of the test
dose for three of the subjects, at one hour after the test
dose was given for one subject, and not until three hours

after the ingestion of the test dose for one subject, M. a.
The greatest increase in concentration of reduced ascorbic

acid in the blood serum ranged from 0.39 to 0.61 milligrams

per 100 milliliters for the six subjects; the mean increment

was 0.51 i 0.03 milligrams per 100 milliliters (Table XVIII).
The mean concentrations of total apparent ascorbic acid
in the blood serum of the subjects after the test dose of

crystalline reduced ascorbic acid were 1.70 i 0.07, 1.96 i

0.11, 2.02 i 0.05, 1.9M i: 0.06, and 1.85 i. 0.05 milligrams

per 100 milliliters at time intervals of one-half, one, two,

three and four hours, respectively (Table XVII). The high-
est value for the average concentration of total apparent

ascorbic acid in the blood serum occurred two hours after

the administration of the test dose, as was observed for

seruzn reduced ascorbic acid. The highest values of total

apparent ascorbic acid in the blood serum after the test

doses were given ranged from 1.88 to 2.35 milligrams per 100

117

milliliters for the six subjects. These values corresponded

to increments in serum total apparent ascorbic acid of 0.1.6

to 0.73 milligrams per 100 milliliters (Table XIX).

hm urinary excretions of reduced ascorbic acid of the
six subjects before the ingestion of the test dose of 169

milligrams of pure ascorbic acid ranged from 0.80 to 7.55
milligrams per hour. Two of the subjects had excretions of
less than one milligram of reduced ascorbic acid per hour;

three of the subjects had urinary excretions which ranged

from 1.5 to 2.5 milligrams of reduced ascorbic acid per

hour. Only one subject, B. 11., excreted more than seven

milligrams of reduced ascorbic acid per hour before the ad-

After the test dose of 169

ministration of the test dose.

milligrams of pure ascorbic acid was given, the urinary ex-

cretions of reduced ascorbic acid ranged from 10.911, to h0.07

milligrams per hour; the average urinary excretion was 19.17

:I: 4.29 milligrams of reduced ascorbic acid per hour for the
four-hour period that urinary collections were made. This

represented an average increment of 16.58 milligrams per
hour in the urinary excretion of ascorbic acid. One sub-
ject, A. W., had an unusually high urinary excretion of as-
corbic acid during this period; the value for this subject

was 14.0.07 milligrams per hour. This represented a total

urinary excretion of160 milligrams of reduced ascorbic acid

during the four—hour period, or an amount almost equivalent

118

t0'mmaamount of test dose administered. Since the blood

serum ascorbic acid values of this subject before and after

the test dose of reduced ascorbic acid were comparable to

those of the other subjects, it would seem that the chemical

determination of urinary ascorbic acid may have measured

other reducing substances in the urine of this subject, as

well as reduced ascorbic acid.

Test Dose of Dehydroascorbic Acid
After a test dose of 169 milligrams dehydroascorbic

acid, prepared from fresh orange juice treated with activat-

ed charcoal, was given to the subjects; the average values

in milligrams per 100 milliliters of serum were 1.11 1 0.0h,
1.28 1 0.03, 1.22 1 0.0h, 1.12 1 0.03, and 1.10 i 0.03 for
reduced ascorbic acid and 1.91 i 0.07, 2.03 1 0.07, 1.98 t

0.09, 1.85 i 0.10 and 1.79 1 0.07 for total apparent ascor-

bic acid at time intervals of one, two, three, four and five

hours reapectively.

The maximum concentrations of reduced ascorbic acid in

the blxxxi serum of the six subjects ranged from 1.16 to 1.37

Inilligjuwns per 100 milliliters. Blood serum concentrations

of ITthced ascorbic acid were lower for subject M. M. than

for time other subjects. The highest values of serum reduced

ascorbic acid occurred two hours after the ingestion of the

test dose.

119

Thelfignest values of total apparent ascorbic acid in

'Um blmXiserum of the six subjects ranged from 1.8h to 2,33

millkxems per 100 milliliters after the supplement of 169
ubjects had

11‘
I ree

milligrams of dehydroascorbic acid.
mmnm1values which exceeded two milligrams of total apparent

ascorbic acid per 100 milliliters; the average of the high-

est values of total apparent ascorbic acid in the blood se-

rum of the subjects after the test dose of 169 milligrams of

dehydroascorbic acid was 2.03 i 0.07 milliirams per 100 mil-

hest values for total apparent ascorbic

liliters. The hi3
acid in the serum of the subjects occurred at two hours after
the test dose was given for four of the subjects, at one hour
after the test dose for one subject, 5. 3., and at three

hours after the test dose for one subject, D. I.
The urinary excretion of reduced ascorbic acid by the

[six subjects before the test dose of 169 milligrams of dehy-
chmoascorbic acid ranged from 1.30 to 2.52 milligrams per hour.

rarmxe of urinary excretions of reduced ascorbic acid

TQie

after"mae test dose of 169 milligrams of dehydroascorbic
acid was from 13.00 to 27.12 milligrams per hour; the aver-

agezaexcrwation was 18.71 i 1.89 milligrams of reduced ascor-

bit: acifiljper hour. This represented an average increase of

1f5.87’xni1Lligrans per hour above the basal urinary excretion

of‘:reéhlcexi ascorbic acid, comparable to the average increase
uztinary excretion of reduced ascorbic acid after the

irl tire
The urinary excre-

test dose of crystalline ascorbic acid.

120

tion ofzeduced ascorbic acid by subject A. w. was 27.12
rfllligmmm per hour, less than that after the test dose of

pure mmxnbic acid but greater than the urinary excretions

of the other subjects.

Test Dose of Strawberries, Frozen for 30 Hours

test dose of strawberries which had been frozen for

The
This amount of strawberries

30 hours was two hundred grams.

supplied 169 milligrams of total apparent ascorbic acid, 25
milligrams of 2,3-diketogulonic acid, 1hh milligrams of total
ascorbic acid, 33 milligrams of dehydroascorbic acid, and 1h8

milligrams of reduced ascorbic acid, as determined by the 2,6-
dichlorophenolindophenol method (Table XV). Thus the test

dose of strawberries, frozen for 30 hours, supplied an amount

of total apparent ascorbic acid comparable to that of the

test doses of reduced ascorbic acid and dehydroascorbic acid,

lNIb the amount of total ascorbic acid and of reduced ascor-
bic euxhi present in the test dose of strawberries was less

than tfiuat of the test dose of pure reduCed ascorbic acid or
of"the tuital ascorbic acid equivalent to the dehydroascorbic

zacid 111 the test dose of charcoal-treated orange juice.

TTme average concentrations of reduced ascorbic acid in
the lilocxi serum of the subjects at intervals of one, two,

test dose of strawber-

three, four and five hours after the

ries, frozen for 30 hours, were 1.50 i 0.10, 1.59 i: 0.07,

1.65 :l: 0.07, 1.59 :l 0.01; and 1.50 i 0.05 milligrams per 100

121

nfilltflters, respectively. There was considerable variation

in fixapattern of the response of the subjects after the

frozen for 30 hours, as may be

testdose of strawberries,
The highest value for

seen hithe graphs shown in Figure 5.
serum reduced ascorbic acid after the test dose of strawber-
ries occurred at one hour for subject D. K., and exceeded

the peak concentrations of reduced ascorbic acid in the blood

serum of this subject after the test doses of reduced ascor-
The shape of the graph of

bio and of dehydroascorbic acid.
test

the blood values of reduced ascorbic acid after the

dose of strawberries for subject A. w. corresponded closely

to the shape of the graphs of the blood values of reduced

ascorbic acid after the test doses of reduced ascorbic acid

and dehydroascorbic acid. The highest values of reduced as-

corbic acid in the blood serum of subjects B. n. and'w. B.
did.rnrt occur until the third hour after the test dose of
strawberries was given, and the peak of concentration of re-
dtuxxi ascorbic acid in the blood serum of subject M. M. did
not occur until the fourth hour after the test dose was giv-
en. ffleis indicated that the absorption of the ascorbic acid
01"the sitrawberries did not occur as rapidly for these three
snflejecims as the absorption of the ascorbic acid of the test

doses of reduced ascorbic acid and dehydroascorbic acid.

TEE: infinflease in serum concentration of reduced ascorbic acid

above the basal value after the test dose of strawberries

122

for:nflflect H. A. was comparable to the increase in serum con-
centmnflon of reduced ascorbic acid after the test dose of
gnue ascorbic acid; however, blood values of reduced ascorbic

eudd declined more rapidly for subject n. A. after the test

dose of pure ascorbic acid than after the test dose of straw-

berries.
The average concentrations of total apparent ascorbic

acid in the blood serum at intervals of one,'two,three, four

and five hours after the supplement of frozen strawberries

0.07, 1.89 t 0.08, 1.8a 1 0.10, 1.77 1 0.0u, and

were 1.61 1
The range of the

1.68 1 0.06 milligrams per 100 milliliters.

highest values of total apparent ascorbic acid of the blood

serum of the six subjects was from 1.7h to 2.2h milligrams

‘per 100 milliliters; this range was greater than that observed

after the test doses of reduced ascorbic acid and dehydroas-

corbic acid. The maximum concentration of total apparent as-

corbdx: acid in the blood serum of the subjects occurred at two

.houums after the test dose was given for subjects H.A., A. w.,

and.I).IK.,at three hours after the test dose was given for

and not until four hours after the

subjects B. H. and W. 13.,

testzchose was given for subject M. fl. The graphs which were

gufiesernxxi in Figure 6 indicated that the pattern of response
of'ialocmi values of total apparent ascorbic acid after the
'test;<dosea of strawberries, frozen for 30 hours, was similar

to tfliat.21fter the test dose of pure ascorbic acid for sub-

but differed ooth in the rate of ap-

jects H. A“ mm.A.w.,

123

pearmum of the total apparent ascorbic acid in the blood
semmnand in the correSponding values of serum concentra-
tions of total apparent ascorbic acid for the other subjects.

After the test dose of 200 grams of strawberries, froz-
en for 30 hours, the average urinary excretion of reduced

ascorbic acid was 16.82 i 2.1a milligrams per hour for a

five-hour.period. Two subjects, w. B. and D. K., excreted

quantities greater than 20 milligrams per hour; subject

4. M. excreted only 8.h6 milligrams per hour. The urinary

excretion of subject A. W. was 12.80 milligrams per hour,

considerably less than that after the test doses of reduced

ascorbic acid and of dehydroascorbic acid. The mean incre-

ment in the urinary excretion of ascorbic acid after the

test dose of strawberries was 15.h2 milligrams per hour

above the basal excretion; this was slightly less than the

:mean increment after the test doses of reduced ascorbic acid

and dehydroascorbic acid.

Testilhose of Strawberries Frozen and Stored for Four Months,

The test dose of strawberries, frozen for four months,

was 250 grams. As was shown in Table XV, this test dose

suppdjfixi 183 milligrams of total apparent ascorbic acid, 51
znilligjunns of 2,3-diketogulonic acid, 132 milligrams of to-
tal ascorbic acid, ELL milligrams of dehydroascorbic acid,

armi llh.rnilligrams of reduced ascorbic acid, as measured by

time 2,éwadichlorophenolindophenol method. Thus the total ap-

12h

pammn;ascorbic acid of the test dose of strawberries, froz-

en fln°four;months, exceeded that of the other test doses.

Althmnjlthe amount of total ascorbic acid in the test dose

cfl’strmdmrries, frozen for four months, was 37 milligrams

less than that of the test doses of reduced ascorbic acid

and of dehydroascorbic acid, the difference between the to-
tal ascorbic acid of the test dose of strawberries, frozen

the strawberries, frozen for 30

for four months and that of
hours, was only 12 milligrams. Thus the test dose of straw-
berries, frozen for four months, supplied less total ascor-

bic acid than the other test doses, but a greater quantity
of total apparent ascorbic acid. The amount of reduced as-

corbic acid was less than in the other test doses also, with

the exception of the test dose of dehydroascorbic acid.
The average concentrations of reduced ascorbic acid in

tee blood serum of the subjects after the test dose of

stuanberries, frozen for four months, were 1.31 i 0.0h, 1.5

i O.C%x, 1.h8 1 0.05, l.h2 1 0.0u and 1.30 i 0.03 milligrams
jper ILKDLnilliliters at time intervals of one, two, three,
:four euui five hours, respectively The highest values of

reduced ascorbic acid in the blood serum of the subjects

ingestion of the test dose occurred at two hours

after the
and at three

B. H. and D. K.,

for subjeCtsiL.AU A.W.,
The range of the highest

hours for subjects w. B. and M. n.
'Valnles; of‘ reduced ascorbic acid in the blood serum of the Sub-

jecrts Ives; from l.h2 to 1.75 milligrams per 100 milliliters.

125

{Mm average concentrations of total apparent ascorbic
acid hlthe blood serum of the subjects after the test dose
of smxmberries, frozen for four months, were 1.87 t 0.05,
2.05 i 0.06, 1.95 2|: 0.06, 1.81; i 0.08, and 1.75 i 0.07 mil-
ligramsper 100 milliliters at intervals of one, two, three,

four and five hours after the test dose of strawberries was

Peak concentrations of total apparent ascorbic acid

given.

in the blood serum of the subjects after the test dose of

strawberries occurred at two hours for subjects H. A., A. w.,
and

B. H. and D. K., and at three hours for subjects w. B.
M. M. The highest values of total apparent ascorbic acid in
the blood serum of the subjects after the test dose of

strawberries, frozen for four months, ranged from 1.90 to

2.28 milligrams per 100 milliliters.
The average urinary excretion of reduced ascorbic acid

after time test dose of strawberries, frozen for four months,

runs 16.98 i 2.60 milligrams per hour. This represented an
increnmnit of 15.90 milligrams above the basal urinary excre-

tififlltlf reduced ascorbic acid. Again, the urinary excretion
the av-

of wahiced ascorbic acid was high for subject A. W.;

eragfiaivalue for the five-hour period was about 30 milligrams
per hour, which represented a total excretion of 150 milli-

grwnns cu? reduced ascorbic acid in the five-hour period.

126

Comparison of the Various Test Doses

As indicated earlier, there was a significant differ-

ence in the concentrations of reduced ascorbic acid and of
total apparent ascorbic acid in the blood serum of the six
subjects preceding the administration of the various test

doses. This difference was apparent in the graphs shown in

Figures 5 and 6. There was only one subject, A. w., for

whom the basal values of serum ascorbic acid were closely
comparable preceding the four test doses, and this was only
for the serum concentration of total apparent ascorbic acid.

Therefore, the data for serum concentrations of reduced as-

corbic acid and total apparent ascorbic acid after the vari

ous test doses were analyzed statistically by analysis of

covariance in order that the influence of the basal concen-

tration of ascorbic acid on the maximum concentration of as

The ad

corbic acid in the blood serum might be eliminated.
justed F-values which were obtained after the influence of
the basal concentration of ascorbic acid in the blood serum
on the maximum concentrations of reduced ascorbic acid and
total apparent ascorbic acid had been removed are given in
Table XXI. This analysis indicated that there was not a

si{ufoicant difference in the highest values of either re-

dxuxni ascorbic acid in the blood serum or of total apparent

ascorfidx: acid in the blood serum after the administration of

the various test doses.

127

TABLE XXI

AJALYSLS‘JE VARIANCE 09 THE MAXIMUA CONCENTRATIONS 09
ASCORBIC ACID 1; TEE S3303 0s SIX SUBJECTS spree VARIOUS
EST DOSES: F-VALUES ADJUSTED BY ANALYSIS .0F
CO-VARIANCE TO REMOVE THE INFLUENCE 03 BASAL
BLOOD VALUES 0w MAXIMUM cowcgseaATIowS

 

 

 

 

Degree of Sum of Mean .
Source of Variation Freedom Squares Squares F-value
Reduced Ascorbic Acid:
Test Doses 3 0.08 0.27 1.0h
Subjects 5 0.03 0.006 1.22
Error 1h 0.36 0.26

Total Ascorbic Acid:

Test Doses 3 0.03 0.010 1.11”
Subjects 5 0.1a 0.028 3.11“
Error 1h 0.l2 0.009

 

*P 5 0.05; significant.

128

Interpretation of possible differences in the utiliza-

tioncfiTascorbic acid after various test doses according to

1X6 pattern of this study also is dependent upon considera-
tion of the rate of appearance and disappearance of ascorbic

acid in the blood. The average concentrations of reduced as-
corbic and of total apparent ascorbic acid in the blood se-

veriodic interVals after each of

}

rum of the six subjects at

the test doses have been plotted and are shown in Figure 7.

Since differences among individuals were relatively great,

each point of the graph must be considered to have a high

standard error. There was a more rapid increase in the re-

duced ascorbic acid and total apparent ascorbic acid of the

blood serum after the test dose of reduced ascorbic acid

than after the other test doses. The pattern of response of

total apparent ascorbic acid in the blood serum of the sub-

jects was similar after all of the test doses. There was a

greater ranfe between the mean concentrations of total ap-
;parent ascorbic acid and reduced ascorbic acid in the blood

serunicxf tie subjects after the test dose of dehydroascorbic

acid ttuni after the other test doses. This would indicate

that time dehydroascorbic acid was absorbed and measured in

the blxxxi serum largely as dehydroascorbic acid, although the

increase in concentration of serum reduced ascorbic acid
after'1flme test dose of dehydroascorbic acid appeared to in-

dicmrte thiat some reduction of dehydroascorbic acid to the

 

 

 

.MW
___....- -— fic-’ -

A... - -___————.————-

 

Figure 7. Mean concentrations of reduced ascorbic acid and
total apparent ascorbic acid of six subjects be-

fore and at periodic intervals after various amt
doses.

......... Total apparent ascorbic acid
——————— Reduced ascorbic acid

 

 

 

 

029/100 ml. Jorum

Ascofiblc Aald ,

2.5 I.

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130

reduced form of ascorbic acid occurred within one hour after

the administration of the test dose.

The range between the concentrations of total apparent

ascorbic acid and of reduced ascorbic acid in the blood se-

rum was greater after the test dose of strawberries, frozen

for four months, than after the test dose of strawberries,

frozen 30 hours. This probably reflected the higher concen-

tration of dehydroascorbic acid in the test dose of straw-

berries, frozen for four months,than in the test dose of

strawberries, frozen for 30 hours.
A further attempt was made to compare the blood values
of the subjects at periodic intervals after the administra-
tion of the test dose by calculation of the average changes
in the concentration of reduced ascorbic acid and in the
concentration of total apparent ascorbic acid of the blood
serum at intervals after the administration of the test

doses. The mean increments and/or decrements, based upon

the average fasting blood values of the four tests, have

'been plotted in Figure 8. This graph indicates that the

concentrations of total apparent ascorbic acid and of re-
duced ascorbic acid in the blood serum of the subjects were

greater after the test dose of strawberries, frozen for four

Inoniflns, than after the other test doses. The mean incre-

nunnts 3J1 total apparent ascorbic acid and reduced ascorbic
acid.£xfter the test dose of dehydroascorbic acid were less

than after the other test doses.

 

. A L

———o~o—-4
_,____..

 

 

 

...—....a—. --

4 .J,’_~—-—~A-

Figure 8.

Mean successive changes in concentrations cxf re-
duced ascorbic acid and total apparent ascorbic

acid of blood serum of six subjects after wrarious
test doses.

Legend: _ d
———_.—. After test dose of crystalline J?edu°9
ascorbic acid

---------- After test dose of dehydroascorbic add

After test dose of strawberries ,

frozen for 30 hours
------ After test dose of strawberries ,
frozen for four months

 

 

 

 

Acid In Jerum

raj/loo ml.

Ascorbic

2.4,

2.2.

 

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77099 In ”a or:

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TOT/ll. AWAR EN 7'

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132

The urinary excretions of reduced ascorbic acid after
the four test doses were treated statistically by analysis

of variance. Analysis of covariance was not used to remove

the influence of the basal urinary excretions of reduced as-
corbic acid, since there were not significant differences in

the concentrations of reduced ascorbic acid of the urine of

the subjects preceding; the various test doses. The analysis

of variance of the urinary excretions of reduced ascorbic
acid after the test doses is given in Table XXII. Although

there were significant differences in the urinary excretions
of the six subjects, the analysis indicated that there were
not significant differences in the mean concentrations of

the urinary excretions of reduced ascorbic acid after the

different test doses.
The data which have been presented indicate that the
ascorbic acid of the test doses of strawberries, frozen 30

hours, and strawberries, frozen for four months was as well
utilized as the test doses of crystalline reduced ascorbic

acid and of dehydroascorbic acid. There was some indication

that the test dose of dehydroascorbic acid was not as well
utilized as the ascorbic acid of the other test doses, al-
‘thOtmfln tirere was considerable individual variation in the
utilization of the test dose of dehydroascorbic acid.

The observation that the ascorbic acid of the test dose

of strawberries which had been frozen and stored for four

 

133

TABLE XXII

ANALYSIS OF VARIANCE OE CONCENTRATION OF RJDUCED
ASCORBIC ACID EXCRETED PER HOUR AFTER

DIFFERENT TEST DObno

 

 

 

Source of . Degree of Sum of Mean F 1
Variance Freedom Squares Squares -va ue
Total 23 1,026.39
Test doses 3 25.70 8.5 0.21
Subject 5 537.h0 107.5 3.48*
Error 15 h63.30 30.9

 

 

*P 9 0.05; significant.

13h

monfiuswas as well utilized as the ascorbic acid of the

othertmst doses projects the question of which components

of mr=strawberries which had been frozen and stored for

factors to the concentra—

fourzmxnms were the contributing

tions of reduced ascorbic acid and total apparent ascorbic

acid of the blood serum of the subjects. Although the total

apparent ascorbic acid of the strawberries which had been
frozen and held in storage was greater than that of the other

test doses, this was true only because of the relatively

higher concentration of 2,3-diketogulonic acid in the straw-

berries. Chemical analyses indicated that the reduced as-

corbic acid concentration of the strawberries which had been
held in frozen storage was 33 milligrams less than in the
test dose of the freshly frozen strawberries, as measured by
a.modification of the Roe and Oesterling method (lth) and
jfliznilligrams less as measured by the 2,6-dichloropnenolindo-
The reduced ascorbic acid concentration of the

phenol.
strawberries, which had been held in frozen storage, was 91

znilliguumns less than the test dose of crystalline reduced
ascorbic acid by the method of Roe and Oesterling (19ml) or
55 milligrams less than the test dose of crystalline reduced
ascxxrbix: acid by the method of Loeffler and Ponting (19h2) for

determination of reduced ascorbic acid with 2,6-dichlor0phe-

In Spite of these differences, the blood

nolindophenol.
the subjects after the

values of reduced ascorbic acid of

135

testckme of strawberries which had been held in frozen

stormfiafor four months were comparable to the blood values

aftertmst doses of freshly frozen strawberries or of the

cmwstalline reduced ascorbic acid.
The total ascorbic acid of the strawberries which had

been held in frozen storage for four months also was less

than the total ascorbic acid of the other test doses. There

were 12 milligrams less than in strawberries which were

freshly frozen and 37 milligrams less than in the test doses

of dehydroascorbic acid or of crystalline ascorbic acid. In

spite of these differences, the concentrations of total ap-

parent ascorbic acid of the blood serum of the subjects
after the test dose of the strawberries which had been in
frozen storage were comparable to the concentrations of to-

tal apparent ascorbic acid of the blood serum of the subjects

after the other test doses.
It might be suggested that the measurement of the total

apparent ascorbic acid of the blood serum of the subjects

Inea3tuwxi 2,3-diketogulonic acid in the blood, as well as re-

chiced enscorbic acid and dehydroascorbic acid. In this case,

tine hdidrer concentration of 23-diketogulonic acid in the test

dose of the strawberries, frozen and stored for four months,

theui ir1 the other test doses might be reflected in the meas-

'urwnnerrt cxf the total apparent ascorbic acid of the blood se-

:rur1 oI‘ tkme subjects. however, this would not provide an ex-

136

plmwfidon of the fact that the reduced ascorbic acid concen-
trathnm of the serum of the subjects after the test dose of

strawnnudes which had been in frozen storafe were similar

to firereduced ascorbic acid concentrations of the serum of

the subjects after the other test doses. A more probable

explanation appears to be that the method for study of uti-

lization of ascorbic acid of test doses which was used in
this investigation was not precise enough to demonstrate the

relatively small differences in total ascorbic acid that ex-

isted among the test doses. The variations in patterns of

response to the test doses among individuals would support

this hypothesis.
The failure of the statistical analysis of the data on

the maximum concentrations of ascorbic acid in the blood se-

rum of the individuals after the various test doses to demon-

strate significant differences either in the reduced ascor-

‘bic acid concentration of the blood serum or the concentra-

tion.cfl?'total apparent ascorbic acid of the blood serum

mi{irt'be interpreted to indicate that the reduced ascorbic

acid euui the dehydroascorbic acid of the test doses were re-

versiixuy oxidized and reduced during the process of absorp-

tion and in the blood serum.

flkxihunter, McMillan and Ehmke (1950) reported that

'blcmxi diml not contain dehydroascorbic acid. This is not in

agreement with the results of this study, since values for

137

tmtalznmarent ascorbic acid of the subjects were uniformly

greafin'than values for reduced ascorbic acid in the serum

of mugsubjects. Moreover, the differences between values

of refined ascorbic acid and total apparent ascorbic acid of
tie blood serum were greater after the test doses of dehi-
droascorbic acid and of strawberries, which had been in froz-
en storage for four months and which contained a larger
amount of dehydroascorbic acid than the freshly frozen
strawberries or the test dose of crystalline ascorbic acid.
Higher values for total ascorbic acid than for reduced as-
corbic acid in the blood serum also have been reported by

Borsook and co-workers (1937), Davey and co-workers (1952)

and Linkswiler (l95h). It is unfortunate that time did not

permit the analysis of the dehydroascorbic acid and 2,3-di-
ketogulonic acid of the blood serum of the subjects in this

study. The relative concentrations of reduced ascorbic acid,
dehydroascorbic acid, 2,3-diketogulonic acid of the blood

after'iflme administration of different sources of ascorbic
acicl hwith.different proportions of reduced ascorbic acid

and Ckfliydroascorbic acid in the test doses) is suggested for

further study.

like pattern of this study differed from that reported
by'iiintmecker _t’al. (l9h7; 1950), and direct comparisons of
the experimental findings cannot be made. Einbecker _e_t _a_l_._.

found that the increments in plasma ascorbic acid were com-

138
parable after test doses of crystalline ascorbic acid and
frozen strawberries which supplied equivalent amounts of re-
duced ascorbic acid. In this study, the frozen strawberries
supplied considerably less reduced ascorbic acid than the
test dose of crystalline ascorbic acid, yet the plasma as-
corbic acid curves were similar. It is possible that a
greater amount of the ascorbic acid was in the form of dehy-
droascorbic acid in the strawberries used in this study than
in the strawberries studied by Einbecker and co-workers.

Einbecker gt El‘ (19h7; 1950) found also that the incre-
ment in blood ascorbic acid concentration was less following
a test dose of frozen strawberries than a test dose ofzcrys-
talline ascorbic acid which was equivalent to the apparent
total ascorbic acid of the strawberries. This was not the
case in this study for a test dose of strawberries which had
been frozen for 30 hours.

In this Study, the test dose of strawberries which had
been frozen and stored for four months contained an amount
of total apparent ascorbic acid which was greater than the
test dose of crystalline ascorbic acid. Since the plasma
ascorbic acid concentrations of the subjects after the test
dose of strawberries, frozen for four months, were compar-
able to those after a test dose of a smaller quantity of
crystalline ascorbic acid, the results of the two studies,

in this respect, do not appear to be in conflict.

~

139

The data reported in this study indicate that changes
in the reduced ascorbic acid, dehydroascorbic acid and 2,3-

diketogulonic acid concentrations occurred in strawberries

throughout the period of frozen storage. Information on the

ascorbic acid content of frozen strawberries based on the
analysis of the total ascorbic acid content of the strawber-
ries would appear to be more reliable than information based

on analysis only of the reduced ascorbic acid concentration

of the strawberries. Further study of substances which may

form osazones with 2,h-dinitrophenylhydrazine in the deter-

mination of total ascorbic acid is needed.

SUMMARY AND CONCLUSION

Changes in the apparent ascorbic acid of strawberries

during frozen storage were studied on strawberries of the

Catskill and Robinson varieties. Fresh berries of the Cats-

kill variety contained 83.0 and 81.6 milligrams of total ap-
parent and reduced ascorbic acid, respectively, per 100
grams of strawberries; fresh berries of the Robinson variety
contained 50.0 and h7.6 milligrams of total apparent ascor-
bic acid and reduced ascorbic acid, respectively, per 100
grams of strawberries.

Concentrations of total apparent ascorbic acid, reduced
ascorbic acid, dehydroascorbic acid, 2,3-diketogulonic acid,
reductones and reductic acid, reducing sugar and total sol-
ids were determined in strawberries which had been frozen
for 30 hours and in strawberries during frozen storage at
-27 to +30 degrees Centigrade. Analyses were made at month-
ly intervals for a period of ten months in strawberries of

the Catskill variety and six months in strawberries of the

Robinson variety. The concentrations of the various compo-

rumats in the strawberries which had been frozen for 30 hours
‘were enmoloyed as base line to study the changes of these
conmxnnents during frozen storage. Strawberries of the Cats-

kilJ.‘variety which had been frozen for 30 hours contained

lkl
80.0 milligrams of total apparent ascorbic acid, 75.0 milli-
grams of reduced ascorbic acid, 16.3 milligrams of dehydro-
ascorbic acid, l2.5 milligrams of 2,3-diketogulonic acid,
8.2M grams of reducing sugar, and 11.55 grams of total sol-
ids in 100 grams of berries. Strawberries of the Robinson
variety which had been frozen for 30 hours contained 50.0
milligrams of total apparent ascorbic acid, A6.6 milligrams
of reduced ascorbic acid, 6.5 milligrams of dehydroascorbic
acid, 6.3 milligrams of 2,3-diketogulonic acid, 7.29 grams
of reducing sugar, and 8.25 grams of total solids per 100
grams of strawberries. Reductones and reductic acid were
not present in measurable quantities in the strawberries.
The total ascorbic acid values for the strawberries were ob-
tained by subtracting the concentration of 2,3-diketogulonic
acid from the concentration of total apparent ascorbic acid.
The total ascorbic acid concentration in strawberries which
had been frozen for 30 hours was 67.5 milligrams per 100
grams of strawberries of the Catskill variety and h3.7 mil-
ligrams of strawberries of the Robinson variety.

At the end of the first month of frozen storape, there
was a marked decrease in reduced ascorbic acid concentration
of strawberries of both varieties in comparison with the
concentrations of berries frozen for only 30 hours. An in—
crease in the concentration of dehydroascorbic acid and 2,3-

diketogulonic acid occurred during this period; there was

1&2
very little changefin the total apparent ascorbic acid con-

centrations. The concentration of reducing sugar was de-

creased also in strawberries of both varieties after one

month of frozen storage.

The concentration of 2,3-diketogulonic acid was low

after the second and third month of frozen storage of the

strawberries; however, an increase in the concentration of

2,3-diketoculonic acid occurred during the fourth month of

frozen storage.

There was a loss of total ascorbic acid of the straw-

berries during the first month of frozen storage,but an in-
crease in total ascorbic acid occurred during the second

month.

Fluctuations in concentrations of the components of the

strawberrkmacncurred throughout frozen storage. Statistical

analysis indicated that there was a significantly negative
correlation between the concentrations of reduced ascorbic

acid and dehydroascorbic acid in strawberries of the Robin-

son variety and a significantly inverse relationship between

2,3-diketogulonic acid and total ascorbic acid in strawber-

ries of both varieties; this appeared to indicate that as-

corbic acid was oxidized to 2,3-diketogulonic acid during

the frozen storage. There was a highly significant relay

tflbnship between concentrations of 2,3-diketogulonic acid

mnireducing sugar in strawberries of the Robinson variety

9.

that IMIt in.strawberries of the Catskill variety.

.fiae influence of the ct ges of apparent ascorb

311 strwnfioerries during frozen storage on the utilization of
ascorbic.sufld.in strawberries

was studied with six healt
jmnung'women as subjects. Subjects were given 75 milli

crystalline reduced ascorbic acid as a supplement to their

customary diet for seven days before each test period. ”est
doses given in two successive days at four test

'1'}

eriods were

(a) 200 grams strawberries of Catskill variety, frozen for

30 hours; (b) an amount of crystalline reduced ascorbic acid

equivalent to the amount of total apparent ascorbic acid

contained in the test dose of 200 grams of strawberries

frozen for 30 hours; (c) dehydroascorbic acid in the form of

orange juice treated with activated charcoal, and (d) 250

grams of strawberries, frozen and stored for four months.

The test dose of

N

00 grams strawberries, frozen for 0

hours, contained 169 milligrams of total apparent ascorbic

\JJ

achi,]hfi milligrams of reduced ascorbic acid, 33 milligrams

cfi'dehydroascorbic acid, 25 milligrams of 2,3-diketogulonic

acid,anm.lhh milligrams of total ascorbic acid. The test
dosecflfcrystalline ascorbic acid contained 169 milligrams

dfxeduced ascorbic acid only; the test dose of orange juice
treamxlwith charcoalucontained 169 millijrams of dehydroas-

mnbiczmid only. In the test dose of 250 arams of straw-
J c-

bernkm, frozen for four months, there were 183 milligrams

mu

of total apparent ascorbic acid, 11h milligrams of reduced
ascorbic acid, 5h milligrams of dehydroascorbic acid, 51
milligrams of 2,3-diketopulonic acid, and 132 milligrams of

total ascorbic acid. Blood sanples were taken before and

one-half, one, two, three, and four hours after the adminis-

tration of crystalline ascorbic acid;.and before and one,

two, three, four, and five hours after ingestion of tre
other test doses. A one-hour urine sample was collected

fore each test dose; a composite urine sample was collected

for a four-hour period following the test dose of crystal-

line ascorbic acid and a five-hour period following the

other test doses. The blood serum was analyzed for total

apparent ascorbic acid and reduced ascorbic acid; the urine

was analyzed for reduced ascorbic acid.
There was not a significant difference between the

hirhest values of ascorbic acid in the blood serum of the

¥

subjects after the test doses of frozen strawberries and

after the test doses either of crystalline reduced ascorbic

acid or of ehydroascorbic acid. There were no significant

differences in urinary excretion of ascorbic acid of the

subjects following the various test doses.

The total ascorbic acid of the strawberries, frozen and

stored for four months,appeared to be as well utilized as

‘miat of strawberries frozen for 30 hours only.

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