TEE AVAILABILITY TO THE EAT OP CERTAIN CAROTENES
IN HAW AND COOKED VEGETABLES

3y
Marion A. Wharton

A THESIS
Submitted to the School of Graduate Studies of Michigan State
College of Agriculture and Applied Science in partial
fulfillment of the requirements for the degree
of
DOCTOR OF PHILOSOPHY

Department of Poods and Nutrition
Year

I9 U7

CONTENTS
Page
1

INTRODUCTION
Section 1 - THE ALPHA- AND BETA-CAROTENE CONTENT OE RAW, COOKED
AND FROZEN SPINACH AND CARROTS
Review of Literature

2

Experimental Procedures

4

Results and Discussion

5

Summary and Conclusions

11

Section 2 - THE RELATIVE VALUES OF ALPHA- AND BETA-CAROTENE,
VITAMIN A ALCOHOL, RAW AND COOKED SPINACH, ADD CARROTS FOR
GROWTH OF THE RAT
Review of Literature

12

Experimental Procedures

14-

Re suit 3 and Discussion

16

Summary and Conclusions

20

Section 3 - THE FECAL EXCRETION AID A3SORPTION OF VARIOUS
CAROTENES AND STORAGE AS VITAMIN A IN THE LIVER OF THE EAT
Review of Literature

21

Experimental Procedures

23

Results and Discussion

25

Summary and Conclusions

28

General Summary

29

Tables and Graphs

.31

Acknowledgments

4-5

References

46

THE AVAILABILITY TO THE EAT OF CERTAIN CAROTENES
IN RAW AND COOKED VEGETABLES

The literature presents many discrepancies concerning the utiliza­
tion of carotene as vitamin A.

Guggenheim (l), Frans and Meinke (2) and

Treichler, Kemmerer and Fraps (3) have all reported that the carotene in
different foods is not as well utilized "by the rat for storage of vitamin
A in the liver as is carotene in cottonseed oil.

Many theories have "been

advanced as to the probable causes but the work of Kemmerer and Fraps (H)
has

substantiated that foods which contain similar total amounts but

varying proportions of different carotenes have different biological values.
Human foods have not been studied extensively as to their relative
proportions of the biologically active carotenes and very little has been
done on the effect of cooking on these.

Therefore a study of the effect

of home cooking of spinach and carrots on the concentration of alphar- and
beta-carotene and the biological values of these was undertaken.
This research is reported under the following headings:
1.

The alpha- and beta-carotene content of raw, cooked and frozen stored
spinach and carrots.

2.

The relative values of alpha- and beta-carotene, vitamin A alcohol,
raw and cooked spinach, and carrots for growth of the rat.

3.

The fecal excretion and. absorption of various carotenes and storage
as vitamin A in the liver of the rat.

- 2 -

1.

THE ALPHA- AND BETA-CAROTENE OF RAW, COOKED AMD FROZEN SPINACH AND
CARROTS

Both carrots and spinach or other leafy vegetables may contribute a
large proportion of carotene to the human diet.

This has been especially

true during the war and post war years when the animal sources of vitamin
A have been scarce.

Considerable discrepancy has been reported in the

literature in the carotenoid content of vegetables, as determined by chem­
ical and physical methods, and the actual potencies found by animal feed­
ing experiments.

It appeared possible that, in some cases, the analytical

methods might be unreliable in that non-active carotenoids were determined
and calculated as provitamin A - active pigments.

Kemmerer and co-workers

(l+,5 ) investigated the constituents of the crude carotene of some human
foods.

These investigators, using chromatographic methods, found that jS

per cent of the crude carotene from leafy vegetables was in the form of
beta-carotene, while raw carrots contained 62 per cent of the total caro­
tene in the beta form.

They have shown that the carotene extracts of

plants are complex in nature and may consist of beta-carotene, of impurity
A, of neo-beta-carotenes B and U and alpha-carotenes.

The neo-beta caro­

tenes are stereoisomers of beta-carotene and can be formed from and con­
verted to beta-carotene.
found to range from

The crude carotene content of fresh carrot was
to 1120 p.p.m.

Zscheile and co-workers (6,7) re­

ported that spinach contained 60 micrograms per gram total carotene of
which S7 to 89 per cent was beta-carotene and 11 to 13 per cent neo-betacarotene.

Cooking for ten minutes showed an increase in total carotene

but no change in the percentage beta-carotene.

During frozen storage raw

spinach lost considerable of its total carotene and its percentage of betacarotene waE reduced.

Fraps and Meinke (2) found that spinach contained

- 3 -

6l p.p.m. total carotene of v/hich 52 per cent was "beta-carotene, lU per
cent neo-beta-carotene B, 17 per cent neo-beta-carotene U and 17 per cent
impurity A.
In an investigation of the carotenoid content of l6 varieties and 18
strains of carrots, Harper and Zscheile (8) used both chromatographic and
s'oectrographic means of analysis.

Garden varieties of carrots were found

to average 54 micrograms of carotene per gram of material, of which 1+6
oer cent was alpha-carotene and p4 P er cent was beta-carotene; zetacarotene appeared ra-ther generally and lycopene was present in certain
varieties.

These results indicate a. lower betar*carotene concentration

than is generally assumed.

Kemmerer and Frans (4, 9) have reported values

of 52 to 65 per cent beta-carotene and have accounted for 94 per cent of
the total carotenes as alpha- and beta-carotenes separated chroraatographically.
Harper and Zscheile's findings of 46 per cent alphar-carotene is high­
er than the highest value of 36 oer cent found by Mackinney (10) in a sur­
vey of leaf carotene.

Harper and Zscheile (8) consider that the high per­

centage of alpha-carotene found in carrots is an important factor in the
estimation of provitamin A activity as it could cause errors of 25 P ei*
cent in the biological Tjotency.

However the other carotene constituents

are less than 10 per cent of the total carotenes thus could only influence
vitamin A activity slightly.
The literature contains little on the effect of cooking or frozen
storage on carrots and spinach thus a study of the effect of these on the
total carotene content and the percentage alphas and betar-carotene was
undertaken.

_ u -

EXPERIMENTAL PROCEDURES:

Processing and Sampling:
Approximately 10 pounds of spinach, purchased on the local market in
May and November 19*+5 was washed, midribs removed, cut into small pieces
and mixed.

Lots of ^ 0 0 grams were cooked for 10 minutes in 2.5 times the

\<reight of boiling water.

The drained cooked weights were obtained.

Both

the raw and cooked spinach, in 10 gram lots were packaged and sealed in
.

o

o

cellophane bags and quick frozen at -40 P. and stored at -10 E.

Carrots

purchased on the local market, at the same time, were washed, cut in half
longitudinally, using one-half for the raw sample and the other for the
cooked.

They were then grated crosswise.

The cooking sample was cooked

for nine minutes in 2.5 times its weight of boiling water.

Both the raw

and cooked samples were packaged and stored in the same manner as the
spinach.

This procedure of preparation was used to minimize the sampling

error and to secure uniform sample for animal feeding.
The vegetables were analyzed spectrographically for carotene on the
same day they were processed.

This procedure was repeated at later storage

periods.

Chemical Methods:
Samples of approximately five grams were extracted with absolute ethyl
alcohol in a Waring blendor (a small amount of calcium carbonate was added
to counteract any effects of plant acids which might be liberated).

The

suspension was filtered on a Buechner funnel and the residue washed repeat­
edly with petroleum ether until all pigment was removed.

The filtrate was

transfered to a separatory funnel, adjusted to 85 Per cent alcohol with
water, and the carotene extracted with petroleum ether.

The solution was

washed with 90 per cent methyl alcohol, and the chlorophylls removed "by
shaking for one minute with 20 per cent potassium hydroxide in methanol
(7).

The petroleum ether extract was washed free of alkali, dried over

sodium sulphate, evaporated under reduced pressure and the residue dissolv­
ed in cyclohexane (Eastman's) and made to volume.

Spectrographie analyses

v/ere made on a Beckman quartz photoelectric spectrophotometer.
Absorption values we re determined at wave lengths l+3*+0, ^530. ^700

0

and 1+620 A , which are co-incident points of the curves for alpha- and
beta-carotene in cyclohexane.

Specific absorption coefficients at these

wave lengths for cyclohexane solutions are 1 5 9 , 215 , 175 and- 166 liters
per gram centimeter, respectively.

Wave length 1+530 A° was chosen for

superposition of the characteristic curves of the vegetable extract as
Harper and Zscheile (S) had found this point satisfactory for carrot ex­
tracts.

Absorption at wave length 1+620 A

merization.

Wave lengths 1+620 and 1+900 A

was used as an index of iso­
v/ere used for analyses of ex­

tracts as two-component systems for alpha- and beta-carotene according to
the method of Beadle and Zscheile (7 ).

The ratio of wave lengths given

in Table 2 refer to ratios of relative absorption value at two indicated
wave lengths.
The standard absorption curves and coefficients v/ere determined on
alpha- and beta-carotene (General Biochemicals, Inc.) in cyclohexane
purified for spectrographie determinations (Eastman, Inc.) and shown in
Figure 1 and Table 1.

BESULTS AND DISCUSSION:
Most carotene research has been conducted using hexane as a solvent
but cyclohexane is equally as satisfactory from wave length 3CKX) to 5000 A°
and was more available in 19^5*

Therefore it was chosen in this research

- 6 -

for both the carotene and vitamin A determinations.

Table 1 presents the

specific absorption coefficients used in this study for alphar- and betacarotene at the coincident points and the widely separated points of the
characteristic curves.

The absorption curves are presented in Figure 1

with the curves for raw spinach.
The only references to beta-carotene in cyclohexane are given by
Devine et al (11) and Braude and co-workers (12).
at 4p6o A

with specific absorption coefficients of 24'9 and 24-0 respective.

ly.

They report a maximum

In this study the maximum was at 4530 A

ient of 215.

o

with an absorption coeffic­

An earlier sample of beta-carotene (General Biochemicals, Inc.)

had absorption coefficients which agree better with the previous work with
an absorption value of 240 at the maximum point 4530 A .
Differences in the sample lots purchased were also noted for alphacarotene.

The sample used in this research had a maximum at 4490 A

o

with

an absorption coefficient of 227 1 * Per gm - CE3* while the earlier purchase
had the same maximum point but an absorption coefficient of 357-

Since

there was insufficient of the first lot for repetition of results or the
continuance of the experiment the results from the second lot have been
used as standards throughout this research.
The carotene contents of the raw and cooked spinach and carrots dur­
ing various periods of frozen storage are reported in Table 2.

The effects

of storage on the characteristic curves as compared to the initial product
and to alpha- and beta-carotene are presented for raw and cooked spinach
and ra.w and cooked carrot in Figure 1, 2, 3 and 4 respectively.
Spinach purchased in May contained 4.19 mS- of total carotene per 100 g.,
November spinach 5-9^ mg. and the percentage betar-carotene was 76.4 and 81.6
respectively.

When cooked, May spinach contained 6 .3 3 mg. total carotene

7-

per 100 g., November spinach 6.92 mg., in which the beta-carotene content
was 86.5 and 94.1 per cent respectively.

Carrots purchased in May con­

tained 14.00 rag* of total carotene per 300 g, whereas the November sample
contained 1 0 »53 m g *
spectively.

The percentage beta-carotene was 6 6 . 2 and 76.4 re­

When cooked, the total carotene concentration of the May

sample was 12.79 mg* and the November sample 9*64 with a beta-carotene
concentration of 58.6 and 62.5 per cent respectively.
The total concentrations in Table 2 do not check as determined at
the various coincident points.

This is in agreement with the results of

Harper and Zscheile (8 ) and Kemmerer (12).
calculated at 4340 A
o
4820 A *

o

In every case, the results

are higher than at the wave lengths 4530, 4700 or

This undoubtedly is because the carotene extracts contained not

only alpha- and beta-carotene but also others which Harper and Zscheile
(8 ) identified as zeta-carotene in carrot extracts.

Kemmerer, (13) and

Harper and Zscheile, (8 ) have reported that the results obtained at 4500 A 0
in hexane which compares to 4530 A

o

in cyclohexane are very satisfactory

for total carotenes although the latter workers state that 4782 A° (4820 A°
in cyclohexane) is the most reliable for the content of the combined alphaand beta-carotenes.

This latter point was used by Strain (14) in the anal­

ysis of butterfat carotenoids.

Wave length 4900 A0 , according to Harper

and Zscheile, (8 ) ie satisfactory for estimating alpha- and beta-carotene
contents individually, a.lthough the presence of gamma-csrotene or lycopene
will cause such results to be high in favor of beta-carotene.
Table 2 shows that tho cooking of spinach increases the total carotene
content 45.5 per cent for sample 1 and 23.1 for sample 2.

However, when

these were calcuJ.ated on the total weight of the raw spinach used in cook­
ing and the drained weight of the cooked, then these gains became 1 0 . 1 and

1 . 2 per cent respectively, indicating that these changes were due to the

- S -

inaccuracies involved in obtaining a cornparadive weight of the drained
products.

Cooking of carrots produced losses of 2.1 and 15.1 per cent

respectively hut when calculated on the total amounts cooked then the
losses were 11.0 and ~f.% per cent.
The percentage of heta-carotene (Tahle 2) apoears to he greater in the
cooked than in the raw spinach.

This may he because the absorption at wave

.
o
lengths 1+S20 to 5000 A , Figures 1 and 2, is greater than that of raw
spinach, becoming almost identical with the beta-carotone curve.

Harper

and Zscheile, (£) reported that ratios of absorption values, chosen from
the standard curves of pigments known or thought to he present in the ex­
tract are very helpful as an indication of pigment identity.

Wide depart­

ures of ratios from expected values indicates the presence of additional
pigments.

1+7SO
1+900
In this work, the ratios KggQ and ^ggQ were chosen and these

ratios for pure alpha- and beta-carotene ranged from 1 .0 7 0 to 0.9S9 and
O. 58 I+ to O.Sl+3 respectively.
for the ratio

All the values in Tahle 2 were in this range

except both samoles of cooked spinach which remained

consistently high throughout storage.

This may indicate the presence of

some isomer which has a greater absorption than beta-carotene at wave length

0
1+900 A .

This change in the characteristic absorption curve of cooked spin­

ach may explain the apparent increase in percentage beta-carotene.
Frozen storage of raw spinach caused losses in total carotene content
of b.7 per cent in three months, 1+9.2 in 8 months, 1+3*5 in 10 months and
59.1+ in 15 months.

Zscheile, Beadle and Kraybill (6) reported that raw

spinach (Giant Koble) lost 6 3 .5 P er cent of total carotene in 11 months and
(King of Denmark) 57*1 Per cent in 11 months and 51-7 in 12 months.

These

losses were considerably greater than what was reported for 10 months here'
but in this case the variety was not known and the spinach purchased in

May was probably of a different variety than that "bought in November.
The character of the absorption curve, Figure 1, was markedly changed
after 10 months storage.

In general, the absorption coefficients were

increased above those for raw spinach from *4000 to *+530 A
to *+760 A

0

0

and below it from *4760 to 5000 A .

and from *4560

This indicates isomeriza--

tion and the general shape of the curve resembles that of Zechmeister (15)
for beta-carotene after iodine treatment and that of Zscheile and Porter
(l6) for neo-beta-carctene.
Cooked spinach, on storage, showed an apparent gain in total caro­
tenes but it is questionable whether this is an actual gain or whether it
is due to the change in the character of the absorption curve.

These re­

sults do not compare to the losses of 26. *4 per cent in 11 months reported
by Zscheile and co-workers (6) for blanched spinach (Giant Nobel).

This

may be because cooking for nine minutes in boiling water destroys more en­
zymes than blanching for two minutes.

The changes in the specific absorp­

tion curve due to storage (Figure 2) resemble those of the curve for raw
,

spinach stored 10 months below 4S20 A

0

excent that they are less marked,

and above this wave length it is identical with the freshly cooked product.
Carrots purchased in May contained more total carotene than those
bought in November but the percentage beta-carotene was less.

Both the

total carotene and the percentage beta-carotene are in the range reported
by Earper and Zscheile (3) and Kemmerer and Fraps (3) using a chromato­
graphic method.
The shaTje of the absorption curve of cooked carrot resembles that for

0
raw carrot but its absorption is greater from *4000 to *4*420 A
,
0
from *4770 "to 5000 A .

and less

The percentage beta-carotene is less in the cooked

product than in the raw.

I
- 10 -

Haw carrot during frozen storage showed no changes in total carotene
in three months hut losses of 2o.U per cent in eight months, lU.7 per cent
in 10 months and 4-U. 1 per cent in 15 months.
in the percentage beta-carotene.

There was no marked change

The absorption curve of the stored raw

carrot, (Figure 3) did not show the marked change produced in raw spinach.
o
It was lower than the fresh product from wave length UOOO to ^3*40 A ,
practically identical from UbFQ to I+63 O A°, higher to U77 O A , lower than
, ^
0
o
the fresh to HSoO A and then identical to ^>000 A .
Cooked carrot, during frozen storage, showed no significant change in
total carotene.

The percentage beta-carotene as measured spectrographi-

cally, was increased slightly.

The absorption curve for cooked carrots

stored for 10 months, was similar to that of raw carrots, except slightly
more exaggerated between H65 O and ^77^ A .
When the characteristic curves of the extracts of raw and cooked
spinach and carrots (Figures 1, 2, 3* r-n.d U) , which were not subjected to
,

frozen storage, are superimposed to agree a.t
at U7 OO and ho20 A

for spinach but the raw carrot is slightly higher than

.
0
the cooked at 4S20 A .
and beta-carotene.

0

A , they also agree well

These wave lengths are coincident points for alpha-

When the vegetables v/ere stored 10 months, their coef-

ficients were noticeably increased at 4700 A

0

which indicates the formation

of some of the cis-forms of these pigments but which were not present in
any great concentration in either the raw or the cooked vegetables. This
o
also indicates that Hi 00 A may not be a very accurate point for measuring
total carotene in processed vegetables.

0
At wave lengths below U400 A

the curves for the vegetable extracts

usually are higher than the standard curves of either alphas or beta-caro­
tene especially from wave lengths U 25 O to k-350 A° and tlOOO to bl50 A°.
This is in agreement with the work of Harper and Zscheile (2) on carrots
in which they claim that this increase is due to a carotene, provisionally
named zeta— carotene by Strain and Manning (1 7 ), which has major maxima in

the same position as those of the pigment designated as unnamed carotene 1
"by White, Zscheile, and Brunson (12).

It was prepared from carrots hy

them and also hy Strain (iH) and Strain and Manning (17)Harper and Zscheile (s) reported that the carotenol fraction of most
commercial carrots was a relatively small part of the total carotenoids,
approximately 10 per cent and that cryptoxonthol may form part of this.
The alpba-carotene fraction as determined in the work reported here may
contain some cryptoxenthol.

However, any error in calculation owing to

the analyses of cryptoxenthol as alpha-carotene would he insignificant
from the "biological standpoint because of the similarity in provitamin A
potency and the small quantities involved.

SUMMARY ADD CONCLUSIONS:
Spinach and carrots, purchased on the local market in May and Dovember
were analyzed spectrograohically for total carotenes, alpha- and betacarotene in the raw and cooked states and at various periods after frozen
storage.
Spinach purchased in May contained U. 19 rag. of total carotene per
100 g., November spinach 5*96 mg. and the percentage beta-carotene was 76 .
and SI.6 respectively.

Carrots grown in May contained lU.OO mg. of total

carotene per 100 g. and the November sample 10.53

and the percentage

beta-carotene was 66.2 and ~[G.k respectively.
Frozen storage caused little change in the total carotene content of
either raw spinach or carrots in three months but caused losses up to ap­
proximately 50 per cent by 15 months storage.

There was little change in

the total carotenes of the cooked products.
Absorption curves are presented for alpha- and beta-carotene in cyclohexane and for extracts of the raw and cooked spinach and carrots immediate­
ly after purchase and after 10 months frozen storage.

Cooking produced a

greater change from the raw product in the absorption curve of carrots than

- 12 -

of spinach.

Frozen storage of raw spinach and carrots caused a marked

change in the absorption curves, indicating isomerization.

The changes

in the cooked products were less significant, indicating that this process
protected their carotene content.

2.

THE RELATIVE VALUE OF ALPHA- AND BETA-CAROTENE, VITAMIN A ALCOHOL, RAW
AND COOKED SPINACH AND CARROTS FOR GROWTH OF THE RAT

Considerable attention has been given to the discrepancies between
chemical determinations of carotene and the results of bioassay methods.
Much effort has been expended in trying to discover the cause or causes of
this apparent discrepancy.
Lease and co-workers (19) and Sherman (20, 21) have observed that the
utilization of carotene by the rat in curative growth tests is partially
dependent upon the provitamin carrier.

Smith and Otis, (22) reported that

the quantity of vitamin A in the livers of rats fed carotene, after a de­
pletion period, varied with the source.

Using essentially the same technic,

Guggenheim (l) has demonstrated that the utilization of carotene derived
from various plant materials ranged from 33 to 67 per cent of that noted
when preformed vitamin A was taken.

In the case of lettuce, however,

carotene utilization was found to equal that of the preformed, vitamin.
Kemmerer and Fraps (9, 23 ) explained the variation between the chemi­
cal and biological methods as due to the fact that the total carotenes did
not consist entirely of beta-carotene but usually contained imourity A,
neo-beta-carotenes U and B and sometimes alpha-carotenes, in addition to
the beta-carotene.

Neo-beta-carotene B (9 ) and alpha-carotene (2^) have

50 per cent of the vitamin A potency of beta-carotene; neo-beta-carotene U
25 (25) to 33 per cent (2 6 ) and impurity A (^) no apparent vitamin A

- 13 -

potency.

Using rats as their test animal, Deuel and co-workers (27)

reinvestigated the relative provitamin A potencies of cryptoxanthin and
beta^-carotene and in two independent assays obtained an actual pro-vitamin
ratio of 5*+: 100 and 59 : 100 .
A study of the relative value of carotene in twenty-seven vegetables
and feeds which compared the capacity of each foodstuff to promote growth
in rats was made by Kemmerer and Fraps (25).

At a low level of feeding,

sufficient for moders,te growth, the vitamin A value of the beta-carotene
equivalent in plant materials, except carrots, appeared to be equal to
that of beta-carotene in cottonseed oil.

At high levels of feeding, for

storage of vitamin A in the liver, previous reports have shown that the
beta-carotene in plants is not nearly as well utilized as beta-carotene in
cottonseed oil.

The beta-carotene equivalent was the amount of beta-caro-

tene plus 25 per cent the neo-beta-carotene U plus 50 P er cent the neobeta-carotene B plus 50 P er cent the alpha-carotene.
Oser and Meinick (2S) compared the bioassay data with the colormetric
data on several plant foods, including spinach and carrots.

They conclude

that, on the average 1 microgram of crude carotene (free from xanthophyll
and lycopene) as determined colorimetrically was equal to 1 U.S.P. unit of
vitamin A by bioassay when tocopherols were fed.
Studies during the past several years, Bacharach (29), Davies and
Moore (30) and Harris and co-workers (31) have demonstrated that vitamin
E improves the utilization of carotene by protecting it against oxidation,
primarily in the intestinal tract.

Karrer and Keller (33)

Guggenheim

(l) found better provitamin A utilization in lettuce than other vegetables.
Karrer and Keller (33) showed that lettuce had the greatest amount of
vitamin E of any of the vegetables tested.

Guggenheim (l) demonstrated

-

Ik -

that supplementation of the basal carrot diet with tocopherol doubled the
biological value of the carotene content.

These findings suggest that

this may be one cause of variation in the responses to carotene feedings.
This research reports the relative value of aloha- and beta-carotene,
vitamin A alcohol and total carotenes from raw and cooked spinach and
carrots for a four week growth period of weanling rats.

EXPERIMENTAL PROCEDURES:
Rats, 22 to 23 days of age, from a. colony maintained on a low vitamin
A and vitamin D ration, were housed individually in screen bottomed cages
and fed U.S.P. XII vitamin A free ration ad libitum.

The entire consump­

tion of food for the 23 day experimental period was recorded.

Supplements

of vitamin A or carotene were fed twice weekly in series 1 and four times
weekly in series 2.

Weekly growth records were kept.

sacrificed on the morning of the 2$th day.

The animals were

Gross examinations for abscesses

in the middle ear, base of the tongue, glands of the neck and for abnormal­
ities of the lungs, bladder and kidney were made.
The procedures for uniform sampling, cooking and storing the spinach
and carrots are described in the preceeding section.

The vegetables were

prepared for rat feeding by removing individual frozen packages, weighing
10 grams and grinding in a micro Waring blendor with a heavy hydrolyzed
starch solution.

The resulting heavy suspension was made to volume in

stoppered graduates and fed orally to the rat by measuring the required
dosages in one milliliter hypodermic syringes.
for each day's feedings.

Fresh samples were prepared

The vegetables were fed on the basis of the total

carotene content as determined spectroscopically.
Vitamin A alcohol (Distillation Products, Inc.) was weighed and made
to volume in cyclohexane.

It ivas analyzed spectroscopically for vitamin A

- 15 -

whenever supplements for rat feeding were prepared.

The required volume

of the cyclohexane solution was evaporated under reduced pressure and the
vitamin A dissolved and made to volume in wesson oil which contained the
desired dosage of mixed tocopherols.
Alpha- and hetn-carotenes (General Biochemicals, Inc.) were weighed
and made to volume in wesson oil to which had. been added the required d.osage
of mixed tocopherols.

The concentrated solutions were analyzed regularly

and dilute solutions prepared f o r feeding.
Mixed tocopherols (Vegol, a concentrate in oil obtained from Distill­
ation Products, Inc.) were dissolved in oil and fed in the required concen­
trations to the negative and vegetable groups.
held in refrigerator storage.

All oil supplements were

All the oil supplements were prepared so

that 0.3 ml. of oil was fed orally from a one milliliter hypodermic syringe,
twice weekly to series 1 and four times weekly to series 2.
Series 1 consisted of the following groups (five male and five female
rats in each group); negative control, beta-carotene fed on levels of 0.h5t
0.70, 1-03. I . 3 6 and 2.0 micrograms per rat per day, vitamin A alcohol

O . b S micrograms daily, alpha-carotene 2 .3 6 micrograms per day, raw spinach,
cooked spinach, raw carrot and cooked carrot in amounts supplying 3 »S2
micrograms daily of total carotenes as determined spectrographically.
Series 2 consisted of the following groups of 10 animals each (five males
and five females); negative control, beta-carotene 7 . 0 micrograms daily,
alphar-carotene 2 3 .6 micrograms per day, vitamin A alcohol U.60 micrograms
daily, raw carrot and cooked carrot in amounts supolying 2 0 .0 micrograms
of total carotenes oer day.

A3.1 groups in series 1 received 0 . 5 mg. of

mixed tocopherols and in series 2 , 1 . 0 mg. daily.

- 16 -

The growth data were corrected for the effects of initial weight and
food consumption hy analyses of covariance.

The significance of gains be­

tween groups was determined hy "t" values.

RESULTS AND DISCUSSION:
In this experiment the depletion period was omitted because, in the
preceeding yean, with the U.S.?. vitamin A method over 20 per cent of the
rats died during the experimental period.
maining rats varied greatly.

The growth response of the re­

Weanling rats from a colony maintained on a

low vitamin A ration, which permitted little storage of this vitamin, as
indicated by an average depletion period of 21 to 22 days for males and
22 to 23 days for females, responded more uniformly to vitamin A than did
depleted rats.
Table 3 presents the total carotene content and the percentage alphaand beta-carotene of the various vegetables fed.
from the preceeding section.

These data were taken

Chemical analyses showed that there was no

significant variation during the storage period required for the bioassay.
The relationship of gain in weight to the daily supplements of betaCctroi/Sns is presented in Figure 5 for male and female rats, fed 0.*+5»

I.0 3 , I . 3 6 and 2.00 micrograms per rat per day.

The group receiving 7-0

micrograms per rat per day was not included in the regression data since
the growth response from this greater amount of carotene did not show a
linear relationship to the lower levels fed.

For the regression equation

y ■ a ■}• bx, the regression coefficient b was obtained from the formula

2*xy - (^x) fey)
b -

n_

and a from the formula

a = My - b Mx where y

eTTIanZ2-equals g. increase in weight of rats in four weeks and x equals the daily
dose of beta-carotene in micrograms.

The error of estimate was 2.3^ g.

- 17 -

for the males and 1.^3 g-

females.

In the males, three of the

five groups were within one error of estimate from the regression line;
in the females, four of the five were within one error of estime,te.

These

regression lines were used for interpretating the results of the other sup­
plements in terms of "beta-carotene.
When the relationship of gain in vreight to the daily supplement of
beta.-carotene was calculated hy the method of Coward (3^0 using the log­
arithm of the daily dose practically identical results were obtained.
Tahle H presents the gain in weight, food consumption data, and the
gain in weight adjusted by covariance for initial weight and food intake
for the rats in series 1 and 2.

Figure 6 shows graphically the relation­

ship of the averaged adjusted gains in weight for males and females to the
various supplements fed in series 1 and 2 .
Tahle 5 presents the final covariance tahle of gain in weight, initial
weight and food consumption for the combined groups in series 1 and 2.

It

is noted that both sex and treatment produced highly significant effects
on the growth of the rats.
In series 1 all animals receiving carotene or vitamin A raa.de signifi­
cantly greater gains than the negative animals but there were no signific­
ant differences among groups receiving 0 .^ 5 micrograms, 0 . 7 0 or 1 .0 3 micro­
grams beta-carotene daily.

There were however significant differences in

the gains of rats receiving 0 .^5 . 1 .3 6 an(i 2 .0 0 micrograms of betar-carotene
daily.

These differences became progressively greater with greater dif­

ferences in carotene intake.

The results from these different feedings of

beta-carotene produce the regression growth line (Figure 5)»
When the growth of males and females were averaged together for groups
fed 0 .7 0 micrograms beta-carotene, 2 .3 6 micrograms alpha-carotene and 0 .U6

- IS -

micrograms and vitamin A alcohol, the gain in weight in each case was
98 g. in 28 days.

On the same "basis there was no significant difference

in growth between groups of rats fed raw spinach, cooked spinach or
cooked carrot. Hov/ever, those fed raw spinach gained significantly more
than those on raw carrot.

(P.0 5 )

When beta-carotene was fed at the levels of O . k ’j, 0 . J 0 and 1.03
micrograms per day, there was no significant difference in the gains of
males and females, but at all higher levels of feeding the differences
v/ere significant.

The rate of growth, as shown by the slope of the re­

gression line (Figure 5 ), was greater for males than females and this
difference became significant when the carotene intake was greater than
1.03 micrograms per rat per day.

The literature supplies very little

information regarding the requirement of vitamin A of the female rat
for growth.
When the other supplements are calculated as micrograms of betacarotene on the basis of growth promoting abilities then the ratio of
betar-carotene to vitamin A alcohol, alpha-carotene, the total carotenes
of raw spinach, cooked spinach, raw carrot and cooked carrot for male
rats are 1.00: O.tyj: 2 .0 3 : 2.08: 2 .2 5 : 3-18: 2 .5 6 respectively and for
female rats 1.00: O.7 2 : 5*90: 3*29: 3*6^: 6 .U7 : 3*29 micrograms of betacarotene respectively.
The estimated vitamin equivalent of betar-carotene to vitamin A
alcohol of 1.00: 0.h5 for male rats compares well to that of Harris and
co-workers (31, 32), however he presents no data for female rats.

The

alpha-carotene ratio for male rats of 1 .0 0 : 2.0 3 compares favorably to
data presented by Deuel et al, (2b).

The females however utilized the

alpha-carotene only half as well for growth.

The beta-carotene equiva­

- 19 -

lent of all vegetables was considerably lower than that reported hy Oser
and Melnick (28).

By the chemical method employed the beta-carotene con­

centration of the cooked spinach was considerably greater than in the raw
product.

The bioassay does not substantiate this.

It is possible that

the cooking produced some unidentified isomers which were not measured
by the spectrographie method used.

Raw carrots had a lower beta-carotene

content and their bioassay value was lower than the raw spinach.

Cooked

carrots had a higher biological value but not significantly so, than the
raw although their beta-carotene content was lower.

In this work any

physical effect of the cooking of the vegetables on their digestibility
was removed as all vegetables were ground to a fine suspension in a
Waring blendor.
In series 2 all animals made significantly greater gains than those
fed the lower concentrations of the same supplement in

series

1.

Inthis

series there was no significant difference between the

gains made

carotene, alpha-carotene, raw carrot and cooked carrot

at the

levelsfed.

In the case of vitamin A alcohol, significantly poorer

growth

was

onbeta-

obtain­

ed indicating that at higher levels of intake in series 2 growth was not
stimulated to the same extent as it was when alphar- and beta-carotene
were increased in the same proportions over that fed in series 1 .
The negative group of rats fed 1.0 mg. daily of mixed tocopherols
made significantly greater gains than those fed 0 . 5 micrograms in series

1 , when the gains were corrected for initial weight and food intake.
However, the food intake in series 1 was greater than in series 2, in
fact, so much so that both groups v/ere receiving essentially the same
amount of vitamin E, supplied by the wesson oil (0.0ty$ tocopherols).
Since the ration, itself, contains this nutrient the difference shown by

- 20 -

the adjusted weights are unreliable for gains due to tocopherol intake.
In series 1 the depletion period averaged 21 days for males and 22 for
females, in series 2 , 22 days for males and 23 for females.

SUMMARY Aim CONCLUSIONS:
Weanling rats, from a colony maintained on a. low vitamin A and vita­
min D ration were fed U.S.P. XII vitamin A free ration ad libitum for 28
days.

The first series consisted of negative control, beta-carotene fed

at 0.45, 0.70, 1.03, I .3 6 and 2.00 micrograms per rat per day, vitamin A
alcohol 0.46 micrograms per day, alpha-carotene 2 .3 6 micrograms per day,
raw spinach, cooked spinach, raw carrot and cooked carrot in amounts
supplying 3.82 micrograms daily of total carotenes as determined chemi­
cally.

The second series consisted of the following groups, negative

control, beta-carotene ~J.G micrograms daily, alpha-carotene 2 3 .6 micro­
grams per day, vitamin A alcohol 4.6 micrograms daily, raw carrot and
cooked carrot in amounts supplying 2 0 .0 micrograms of total carotenes per
day.

The growth data were corrected for the effects of initial weight

and food consumption by analyses of covariance.
A regression growth - dosage curve is presented for the lowest five
levels of betar-carotene fed.
When the other sources of vitamin A were calculated to micrograms
of beta-carotene from the growth response the ratio of beta-carotene to
vitamin A alcohol, alphascarotene, the total carotenes of raw spinach,
cooked spinach, raw carrot and cooked carrot for male rats are 1 .0 0 : 0.45:

2 .0 3 : 2.08: 2 .25 : 3-18: 2 .5 6 respectively and for female rats 1 .0 0 : 0 .7 2 :
5 .9 0 : 3 .29 : 3-64: 6 .4 7 : 3 .2 9 micrograms of betar-carotene respectively.
Female rats did not utilize vitamin A alcohol, alphascarotene or the
carotenes from any of the vegetables as well as did the males for growth.

- 21 -

At the lowest levels of feeding there was no significant difference
in the growth of males and females "but at all higher levels there was a
significant difference.
The total carotenes from spinach gave a "better growth response than
those from carrot, however, this difference was not significant.

By

chemical analyses spinach contained more "beta-carotene than carrots.
Cooling of the vegetables produced no significant difference in the bio­
assay.
At the higher levels of feeding, in series 2, there was no signifi­
cant difference between the gains made on

J.O micrograms betar-carotene

per rat per day, 23.6 micrograms alphascarotene or 20.0 micrograms total
carotenes from raw or cooked spinach but vitamin A alcohol fed at 4.60
micrograms per rat per day gave significantly poorer growth although it
had compared favorably when, the supplements were fed at a lower level in
the first series.

3.

THE FECAL EXCRETION AND ABSORPTION OF VARIOUS CAROTENES AND STORAGE
AS VITAMIN A IN THE LIVER OF THE RAT

Researches
the results are

on the absorption of carotenes are limited in number and
conflicting.

and co-workers (37)

Kemmerer and Fraps (35), With (36 ), Bussell

Wilson et al (3^) have reported the presence in

hen and rat feces of yellow pigments other than carotene which may have
interfered with the determination of carotene in feces and explain some
of the variation of results.

Most workers including Wagner (39). and

Morgan and Bentley (Ho) have found that carotenes and vitamin A are ex­
creted in the feces and not in the urine except under abnormal conditions.
Kemmerer and Fraps (35) aacL Treichler et al (3) report that carotene
in oil was more completely utilized by rats than carotene in alfalfa.

- 22 -

However Booher and Csllison (*41) found that the carotenes of vegetables
were utilized "better hy humans than carotene in oil.

The relative digest­

ibility of various carotenes by adult and by weanling rats was studied by
Fraps and Meinke (2, *42).

For adult rats the digestibility of beta-caro-

tene was 57 P er cent, alpha-carotene 6*4, crude carotene of boiled carrot
S, boiled mustard greens 55 s&d. raw carrot *-5 per cent.

The digestibility

of neo-beta-carotene-3 was practically the same as beta-carotene.

The

weanling rs.ts utilized 52 per cent of the beta-carotene and 53 P er cent of
the alpha-carotene when fed 60 micrograms in an oil which contained S3 per
cent beta.-carotene, l6 ner cent aloha-carotene and one per cent impurity A ;
The literature contains many reports on the storage of vitamin A in
the liver when generous or massive dosages of vitamin A or carotene are
fed but there is little information on the liver storagewhen the provita­
min is fed at levels just supporting growth.

Little, ThGmas and Sherman

(*4-3), Caldwell, Mac Leod and Sherman (*4*4), Roher and Sherman (*45) and
Campbell et al (U6 ) reported that ro.ts receiving 3*0

of vitamin A per

gram of dry food or 0.8 I.U. per calorie stored negligible amounts of the
vitamin in the liver.

The vitamin A was measured in these researches by

the Carr-Price or spectrographie method or by bioassay using the single
feeding technic of Sherman and Todhunter.

These workers found that 3*0 I.U.

of vitamin A per gram of food was adequate for normal growth, reproduction
and lactation.

Using the Carr-Price method for determining the vitamin A

in liver, Lewis and co-workers (*47) found no liver storage below 10 I.U,
daily and only slight or doubtful storage at 25 I.U. per day.
Callison and Knowles (*48) renort that rats maintained till 275 or 3&5
days of age stored no measurable amount of vitamin A in the liver when they
received less than 50 to SO I.U. of vitamin A daily per kg. of body weight.
This is about four times the minimum requirement of the rat.

They suggest

- 23 -

that the daily requirement of the rat for vitamin A is really higher than
20 I.U. per kg. of body weight per day and that some considerable need of
the body must be met in an apparently normal animal before liver storage .
occurs.
Praps and Meinke (1+2) studied the relative biological values of the
carotenes of six different foods by determining the amount of the vitamin
stored in the livers of rats over a, fourteen day period.

These authors

found that carotene in vegetables oossessed low biological value while
carotene in butter and beef liver possessed high potency.
In 195]2 Trcichlcr, Kemmerer and Praps (3) found what they termed pseudo
vitamin A in liver.

The vitamin A content of the liver as measured spectro-

graphieally was found to increase significantly during the two weeks follow­
ing weening despite the fact that they were fed a vitamin A free ration.
He found that 60 I.U. daily of vitamin A in cod liver oil was the most ef­
ficient for liver storage, carotene was 55 P er cent as effective and caro­
tene in alfalfa 21 per cent.
The following research reports the absorption by the rat of alpha- and
beta-carotene and carotenes from raw and cooked spinach and carrots at low
and moderate intake.

At the highest levels of intake it was considered

worthwhile to study the vitamin A stores of the liver.

EXPERIMENTAL PROCEDURES:
The digestibility of the various carotenes was studied on groups of
rats fed beta-carotene on levels of 0.1+5, 0.J0, 1.03, 1.3&* 2.0 and J.o
micrograms daily; alpha-carotene 2 .3 6 and 2 3 .6 micrograms; total carotenes
of 3 «S2 micrograms daily from raw and cooked spinach and carrots and 2 0 .0
micrograms from raw and cooked carrot as fed in the previous experiment.
The liver storage of vitamin A was studied on the groups of rats fed betacarotene 7*0 micrograms daily, alpha-carotene 23.6 micrograms, vitamin A

•24-

alcohol 4.6 micrograms and 20.0 micrograms of total carotenes from raw and
cooked carrot.
The feces from each rat were collected daily in+c individuel stoppered
bottles, which contained sufficient ethanol to cover.

The bottle and alcohol

were weighed at the beginning of the experimental period and again after the
last collection of feces thus determining the fecal weight by difference *
The focal specimens were refrigerated during the experimental period and
until analyzed for carotene®
On the 29th day when the animals were sacrificed, the livers were re­
moved, the excess blood absorbed on filter paper, weighed, sealed in cello­
phane begs, labelled, quick frozen and stored until analyzed.

Chemical Methods for Carotene end Yitamin A.
Both the feces and livers were extracted in a faring blendor (micro
sized container) with alcohol, transferred to a low actinic boiling flask,
4 ml3. of 40 per cent potassium hydroxide added, then refluxed for 30 m i n ­
utes (standard taper flasks and condensers were used)®

The solutions were

cooled, filtered, transferred to separatory funnels and the water, alcohol
ratio adjusted.

The solutions were extracted three times with petroleum

ether, washed with 85 per cent ethanol, washed with distilled water until
free of alkali, dried with sodium sulphate-, than evaporated to dryness
under reduced pressure.

The residues

were dissolved in cyclohexane, mads

to volume and read on the Beckman spectrophotometer.

Wave length of 4530 A°

was used for measuring total carotenes and 3260 for vitamin A.
The extract from feces contained a material which absorbed light through

o

the wave lengths 3000 to 5000 A ®
of the feces.

Therefore

This absorption varied with the weight

the E values

values from the feces or the negative

were correctedby subtracting the E
animals on the basis of weight of

-

25 -

feces.

RESULTS AED DISCUSSION:
Figure 7 presents the average absorption spectra of two or more caro­
tene extracts.

The male rat's feces collected during the four week experi­

mental period for the negative group, and the groups fed vitamin A alcohol
4.6 microgrems daily, beta-carotene

"J.O micrograms, alphar-carotene 23.6

micrograms and raw carrot 20.0 micrograms of total carotenes per day were
extracted and diluted to 25 ml.
similar absorption curves.

The corresponding female groups presented

It is noted that the negative and the vitamin

0

,

A alcohol groups both absorbed light throughout the 4000 to 5000 A

area

thus any spectrographic readings for carotene concentration must be cor­
rected for this.

In group 1, the average

value on a 25 ml. extract,

per g. of feces was 0.0131 for male negative control rats and 0.0151 for
females; in group 2, 0.0164 for the male controls and 0.0170 for female
controls.

These values were used to correct all carotene determinations

of feces reported.
This observation is in agreement with the work of Russell et al (37)»
Kemmerer and Fraps (35) a*1** Wilson and co-workers (38).

This absorption

may be due to some material in the ration or some other excretory product,
possibly resulting from the earlier sexual development of the treated rats.
In either case a correction factor based on the weight of the feces would
be more accurate than a total correction ner rat.

The vitamin A alcohol

group presents further evidence for this as the feces extracts absorbed
more light than those from the negative group.

The food consumption of

the former group was greater as was also the growth response and the weight
of the feces.

All groups fed carotene showed absorption maxima character­

istic of carotenoids superimposed upon this broad band.

Therefore, direct

-

26 -

spectrographic readings for total carotenes of feces should present true
values if they axe corrected by a blank prepared from the negative control
group,

Neither of the vitamin A alcohol groups showed any maxima at 3260 A

and in this region all groups including the negative controls absorbed a
very high percentage of the light therefore no quantitative values for
vitamin A excretion could be obtained in this research.
Table 6 presents the excretion and absorption data.

The term absorpt­

ion is used to mean the difference between the amounts of carotene fed and
the amounts excreted.
^
c fV*
S-.oW^
.

~ S*. "h-rr
^

Some of the carotene not excreted may, however, be

r» *vn— rl n +VO.'rs-rv
■ nk
.'** *
.-nMf—V *a*v

+•
V,ViO vi

"«Kwo t y > c

^

mO c + V
. o—r —
!

ora H

o c r j i m-*
i.l— o +•. Gr\
-».•

1

*PV»0
—— —

mechanical effect of feeding vegetables and comparing them to carotene in
oil is practically removed in this research as all vegetables were ground
to a fine pulp in a Waring blendor before oral feeding.
In general there was great variation im the amount of carotene excret­
ed by individual rats fed at the same level and this wa,s more marked at the
low feeding levels than when the animals were receiving adequate amounts.
The per cent carotene absorbed on intakes of O.U5 , O. 7O and 1.03 micrograms
dally, was higher for the female group than for the male but at all higher
feeding levels there was no consistent variation between the utilization
for male and female groups.

On these feeding levels, six female and two

male rats utilized all of the carotene.

In all the groups in series 1 there

were nine female and two male rats which did not excrete carotene.

This may

also indicate that at the lower levels of feeding the females utilized caro­
tene more completely than the males.
There was no marked difference between the percentage of betar-carotene,
alpha-carotene, raw or cooked spinach and carrot absorbed and in all cases
the percentage was better than that reported by Fraps and Meinke (2, 1+2).

The digestibility of the raw vegetables tended to be better than the cooked

and the carotene from spinach was absorbed as well as that from carrot.
Figure S presents the absorption spectra from wave lengths of 3000 to

o
3500 A

of a vitamin A extract of a rat liver from the following groups;

negative, beta-carotene J.00 micrograms per day, alpha -carotene 23.6
micrograms, cooked carrot 20.0 micrograms and vitamin A alcohol 1+.6 micro­
grams per day.

All groups present approximately equal absorption which

decreases as the wave length increases and shows no absorption maximum at

0
32o0 A

thus the presence of vitamin A was questionaole.

This method was

compared to that of Carr-Price using one-half of the extra,ct for each de­
termination on liver from 10 of these animals and no blue color was formed
indicating that no vitamin A was present.
The sensitivity of the absorption method was tested by weighing two
portions of four grams each from a liver and adding vitamin A alcohol to
one before carrying out the analytical procedure described.

The percent­

age recovery ranged from 98 to 107 when 7 to 36 micrograms of vitamin A
alcohol were added.

Therefore this method should be satisfactory when a

suitable blank is used.

0
The absorption, at wave length 3260 A , was in agreement with the work
of Treichler, Kemmerer and Fraps (3) and which they termed pseudo vitamin
A.

The absorption spectra of these livers were studied from 2200 to 5000 A°

i^ave length and the absorption decreased as the wave length increased ex­
cept for an absorption maximum between 2500 to 2600 A°.

With the carotene

groups the extracts were pooled and reduced to a small volume for the read­
ing of the absorption spectra from UOOO to 5000 A° wave length and the ab­
sorption decreased as the wave length increased with no indications of
maxima which might be considered characteristic of carotene.

Therefore no

carotene was stored in the liver when 20.0 micrograms per day was fed for
a four week period.

SUMMARY AND CONCLUSIONS:
The digestibility of carotenes was studied on weanling rats fed betacarotenes at levels of 0.U5, 0 . 70 , 1 .03 , 1 .36 , 2.00 end 7*00 micrograms
per day, alpha-carotene at 2.36 and 23.60 micrograms, raw and cooked
spinach and carrots at 3*82 micrograms of total carotenes and raw and cook­
ed carrots at 20.0 micrograms total carotenes per day.
lected for a 2S day period and analyzed.

The feces were col­

Liver storage of vitamin A was

studied on groups fed the following; beta.-earotene 7*0 micrograms, alph&carotene 23.60 micrograms, vitamin A alcohol U.6 micrograms and raw and
cooked carrot 20.00 micrograms of total carotenes per day.
The absorption spectra of carotene extracts of the feces is presented

0
from wave lengths of 3000 to 5000 A .

Rat feces extracts show a broad con­

tinuous band which decreased in density with increasing wave length.

When

carotenes were fed, there was superimposed upon this broad band absorption
maxima, characteristic of carotenoids.

Total carotene determinations were

o
made by spectrographic readings at b-530 A

a&d corrections made for absorp­

tion not due to carotenes by using extracts from the feces of the negative
controls on the basis of their weight.
There was no marked difference between the percentage of beta-carotene,
alpha-carotene raw or cooked spinach and carrots absorbed.

The range was

from bl to 91 per cent.
At low levels of feeding, there was a great variation in the utiliza­
tion of carotenes within groups.

Female rats absorbed carotenes better

than males at the lowest levels of feeding.
The absorption spectra of extracts of rat livers were studied from

0
wave lengths of 2200 to 5000 A .

The extracts decreased gradually in den­

sity with increasing wave length except for a maximum between 2500 and 2600 A°

- 29 At the levels fed there was no storage in the liver of vitamin A or
carotenes.

GENERAL SUMMARY;
Carotenes were studied under the following headings; 1. the alphaand beta-carotene content of raw, cooked and frozen spinach and carrots,
2. the relative value of alphar- and "beta.-carotene, vitamin A alcohol, raw
and cooked spinach and carrots for growth of the rat,

3- the fecal excre­

tion and absorption of various carotenes and storage as vitamin A in the
liver of the rat.
Spinach and carrots, purchased in the local market were analyzed
spectrographically for total carotenes and alpha- and bet s.-carotene in the
raw and cooked state and at various periods of frozen storage.

Weanling

rats on a vitamin A free ration were fed beta-carotene, alpha-carotene,
vitamin A alcohol and rev; and cooked spinach and carrots at various levels
of intake for a 23 day period.

The growth response, fecal excretion, ab­

sorption and liver storage of vitamin A were studied.
The total carotene content of raw spinach was 4. 19 and 5-96 mg. per
100 g. of vegetable, carrots, 14.00 and 10.53 mg- for vegetables purchased
in May and November respectively.

The percentage beta-carotene was

and 31.6 for raw spinach and 66.2 and
Cooked spinach contained 6.33
12.79

fS.k '

j6 .k for raw carrot respectively.

6.92 mg. total carotene and cooked carrot

9-64 mg. per 100 g. of vegetable with a betar-carotene content of

36.5 and 94.1 for cooked spinach and 58*6 and 62.3 for cooked carrot.
Frozen storage resulted in little change in total carotene content of either
raw spinach or carrots in three months but losses up to 5^ P e^ cent occurred
after 15 months storage.

There was little change due to storage in total

carotenes of the cooked product.

Absorption spectra are presented for alpha-

and beta-carotene in cyclohexane and for extracts of the raw and cooked
spinach and carrots and these products after 10 months frozen storage.

- 30 -

Cooking produced some change in the absorption curves but frozen storage,
of the raw products, for 10 months, caused a marked change in the absorp­
tion curves, indicating isomerization.

The change in the cooked products

were less significant, indicating that cooking stabilized the carotene
content.
In the growth study of weanling rats, when all sources of vitamin A
are calculated to micrograms of beta-carotene from the beta-carotene growth
response curve, the ratio of betar-carotene to vitamin A alcohol, alphacarotene, the total carotenes of raw spinach, cooked spinach, raw carrot
j
tlllU .

1—
i
U U J P . C U

X

j.
vj U

„
l u u j . Si

» . „ 4- A u u b

—
„
C-**A v

i
J- •

n r \ .
^

^

n•

) i k .
"T j •

o

m
c__* w

.
•

o n».
^

i- *

2.56 respectively and for female rats 1.00: 0 t J 2 : ^ . 9 0 :

r>

oc.

7

3-29: 3*6^+:

1

®.■

w

6.U7:

3.29 micrograms of beta-carotene respectively.
The percentage carotene absorbed ranged from 6l to

91 but there was

little difference between betar-carotene, alpha-carotene or the carotenes
of raw or cooked spinach and carrots.

At low feeding levels there was a

great variation in utilization within groups and females in these groups
absorbed carotene better than males.

The absorption spectra of negative

control rat's feces decreased in density with increasing wave length from

0
3000 to 5000 A .

When carotenes were fed, there was superimposed upon

this, absorption maxima, characteristic of carotenoids.

The absorption not

due to carotenoids necessitated the use of a suitable blank obtained, in
this case, from the negative control group.
Eats stored no vitamin A in the liver at any levels fed.

Absorption

0
spectra of extracts of rat livers from wave lengths of 2200 to 5000 A

de­

creased in density with increasing wave length except for a maximum between
2500 and 2600 A .

This absorption would necessitate the use of suitable

blanks for the spectrograohic determination of vitamin A in rat livers.

TABLE 1.

Soecific Absorption Coefficients for Alpha- and Beta-Carotene
in Cyclohexane Solution

Wave length

0

A

Specific Absorption Coefficient
Aloha-car 01 ene

Beta-carotene

1 . per gm. cm.

1 . per gm. cm.

159

\tcr?r\
'-rjj"-'

01 C

159

coinsident points

Oluc
y

c__

0
0

U3 U 0

175

175

coinsident points

1+S20

186

186

coinsident points

i+yso

203

183

far apart points

U900

10s

15S

far apart points

TABLE 2.
Vegetable

Sample
No.

Carotene Content of Raw and Cooked Vegetables Fresh and After Frozen Storage
n otal carotene content

Frozen
Storagei

H530A 0
months
Raw spinach

1

4.39

15

1.89
6 .1 s
5.90
3.77

2

1

10
0

3
Cooked spinach

2

Raw carrot

X

4

Cooked carrot

3

4

g
15
0
3
10
0
8
15
0
3
10
0
8
15
0
3
10

48 20A°

g. fresh weight
mg„ per 100 ;

0
8
0

U700A 0

Carotene
loss

2.30

6.60
6.13
7.58
7.23
6.87
7.92

15.12
10,62
s. 29
11.00
11.Oh
9.70
14.05
13.50

4.19
2.13
1.70
5.96
5.56
3-37
6.33
5.92
7.17

6.92
6.70
7.36

14.00
10.31
7.83
10.53

10.61
8 .9 s

4780A?
4S 20A

6.00
5.51

5.89
5.37

6.71

0.392
1.009
1.021
1.001
1.005

3.60

3.21

43.46

1.015

5.96
5.92
7.36
7.30

5.69
5*83
7.O8

6.48
413.27

6.71
7.72
13.13
10.38
s.04

10.93
10.63

1.001

3.18

7.28
12.86
io.4o
7.82
n.o4

4 6.36

i.oo4

10.63

4 0.76
14.72

9.42

12 .77

12.59
12.76

11.92

11.60

11.52
9.64
10.75

n.39

10.16

9.59
10.84
10 .66

1.020
0.999
0.994
0.997

7.25
6.59

12.79
12.86

12.93

49.16
59-43

11.28
9.37
10.63
9-98

1.023
26.36
44-. 07

Beta.-carotene

0
4820 - 4900A
percent

3.91

2.06
1.60

4900A°
47s20a

percent

3.97
2.15
1.73

8.86

12.77
10.44

4530A 0

Absorption Ratios

1.018

1.026
1.010

1.013
1.023

1.017
4 0.56
9.93

1.018

411.51
4 5.39

1.020
1.022

1.027
1.017

0.812

0.809
0.789
0.827
0.331
0.822
0.814
0.852
0.840
0.843
0.848
0.845
0 .7 S5
0.773
0.782
0.793
0.783
0.796

0.761
0.771
0.772

76.4
79.6
71.7
81,6
38,6
84.8
86.5
97.1

92.2
94.1
95.0
94.2
66.2
65.3

63.0
76.4

69.1
74.4
58.6
64 .5
64.9

0.766

62.3

0.773

64.9
73-5

0.794

All figures represent triplicate analyses.
Samples 1 and 3 were purchased in May and 2 and 4 in November.
All analyses are expressed on the fresh-weight basis.
The total carotene fraction was analyzed soectrographically as a mixture of alpha- and beta^-carotenes.

TABLE 3.

Carotene Content of Vegetables.

Vegetable

Total Carotene
mg. per 100
g. of fresh weight

Betar-Carotene

Alpha-Carotene

percent

percent

Raw spinach

5.96

si. 6

18.1*

Cooked spinach

6.92

9U.1

5.9

10.53

76. k

23.6

9.6**

62.3

37.7

Raw carrot
Cooked carrot

TABLE 1+.

Growth aud food. Intake of Rats Fed Various Carotenes and Vitamin A Alcohol for 28 Day Period.
Females

Males

2
Supplement

Scries 1.

0
betar-carotene
hetai-carotene
beta-carotene
betar-carotene
beta-carotene
Vitamin A
alcohol
alpha- cerotene
Raw spinach
Cooked spinach
Rav7 carrot
Cooked carrot

Daily Initial
Intake Weight

r

Food
Gain in
Intake Weight

Initial
Weight

Food
Gain in
Intake Weight
g.

PV
o»

75.0
90.7

1+2.8
1+8. 1+

21+0

123.S

100.9
100.0
109.1
120.6

1+6.6
1+7.2
1+6 .1+

57.k
89.1+
92 .S
98.1+
10U .0
9 k. 1+

101+.1+

102.8

1.03

129.8

105.3
117.1+
H1+.9

1.16
1.81+
1.70

118.6

106.0

1.20

1+6.8
1+9.8

1.1+9

50.2

g.

g.

0
0 . 1+5
0.70
1.03
1.36
2.00

i+s.6
1+S.1+
1+8.0
51.2
kg .2
1+6.U

25 1+
2o9
288
331
323

59.0

111+.6
121.2

300

0 . 1+6

51.2
1+7.6
50.s
50.2

299
307
3^7
331
321+
331

1+7.8
1+7 .1+

BetaCarotene
equiv.
rj day

g.

g.

3.S2J
3.82.
3.52J
3 » 82

Adjusted
Gain

8

80.8
98.1+

111.0
138.6

126.6

o*

111.1

1+7.0

1+9 .1+
50.1+
1+7.2

289
293
297
310

265
281
302
311
295
298
291+

87.0

Males &
*7 . Females
Adjusted
Adjusted1" Beta-^
Carotene Gains
Gain
equiv.
W d ay
g.
g.
77-7
91.2

76.3
90.9
97.7

92.6

98.2

96.5
96.9
105.1+

103.0
113.1

93.0
101+. 1+
97.0
93.1+

92.2
90.0

0.61+
o.i+o

97.5
97.5

96.9
95.9
91 .s

1.16
1.05

107.2

0.59

98.8

96.6

96.8

1.16

lOl+.O

96.5
117.8

115.2

105.3

Series 2.
0
beta-carotene
alpha- c ar o t ene
Vitamin A
alcohol
Raw carrot
Cooked carrot

0
•7.0

23.6

20.0,
20.0

1+2. S
1+2.8
l+g.o

209
315

1+5.6

29s
296
. 301+

1+1+.1+
1+3.8

329

57.6
138.6
11+7.0

90.5
128. b
132.1+

121.1+

118.8
127.9
122.1

130.0
132.6

1.92

1+1.1+
1+1.6
1+2.2

222
278
253

69.2
113.2
103.6

1+5.0
1+3.1+
1+2.8

273
285
281+

92 .1+
113.0
111.6

93.3
123.5
125.6

118.5
99.8
lll+.o

Each figure represents the average of data on five animals.
2. Gains adjusted by covariance to correct for variation in initial weight and food consumption.
3. Beta-carotene equivalent was determined from the betar-carotene gain - dosage curve, Figure 1.
1+. Total carotene as determined spectrographically.

1.1+9

109 .1+
121.6
120.7

TABLE 5.

Covariance Table of Growth, Initial Weight and Food Int;ike of Bats for a 28 Bay Period.

D.F.

Source

X
s.s

xz
s.s

XY

YZ
S.S

S.S

z
S.S

Y

Error of Estimate
S.S
D.F
M.Sq

179

4,667

3 ,32 b

15,79^

i b s , 31b

119,329

290,873

1

84

1,177

1,313

IS , 387

16 ,4-92

20,502

Treatment

17

i,n4

12

6,675

67,015

63,746

120,167

S x T

17

275

99

zjb

12,088

8,929

20,75s

144

3,13b

2,039

6,932

50,834

30,162

129,446

9,998

161

4,307

2,050

13,608

117,899

93,90s

249,613

32,878

Total
Sex

Within Sex and
Treatment
(error)
Treatment 4
Error

22,380

Treatment
Sex 4 Frror

145

3 ,27S

3,216

8,245

69 ,272

46 ,654

149,945

S x T 4 Error

161

3 ,46g

2,137

7,so6

62,972

39 ,09 0

150,204

*

X - Initial weight in gms.
Y = Gain in weight in gms. in 28 days.
Z = Food intake in gms. in 28 days.
**
The probabilities are 99/X00 that sex and treatment effect growth response.

7 0 .4

17

1,346

19.13 **

1

4,496

63.86 **

17

132

12, 239

2,241

S x T

142

14,494
4,496

Sex

F.

1.87

TABLE 6 .

The Fecal Excretion and Absorption of Carotenes by Eats,,

Carotene Intake

per day
X

beta-carotene
beta-carotene
beta-carotene
beta-carotene
beta-carotene
beta-carotene
aloha-car o tene
aloha-caro tene
raw soinach
cooked spinach
raw carrot
cooked carrot
raw carrot
cooked carrot

Digestibility

Excre tion

Material Fed

0.U5
0.70
1.03
1.36
2.00
7.00
2.36
23.60
2.36
2.36

2.36
2.36
20.00
20.00

To tal in
22 days
Y*

Total in
28 days
Y

Range

12.60
19.60

3 .Us

28.8k

11.18

38.02
56.00
I36 .OO
'66.08

660.20
106.96
106.96
106.96
106.96
560.00
560.00

Females

Males

6.70

5.19
22.12
33.27
S.Ul

Range

Y

V

percent

o-s.50
0-13.18
2.3U-16.15
2 .U3- 9.50
7 .59- 35.59
3U.53-Ul.6U

72. U
65. s
61.2
86.U
60.5
so. 5

1.55

0- 3.31

2. 70

0-3.20

2.55
5.99
17.32

0-10.18
12.09-25.53

3.7S-13.08

37.3
77.0

S7-7
86.2
31.2
8U.3
69.1
7U.2
86.8

127.93

8U.6
77.U
S9.9

16.20
23.60
18 . 23

6O.U7-207.U3
i3.36-2i.89

10.85
18.19

U.26-15.so
l.lU-29.7s
80.U3-1U1.2U
92.77-1U7.76

115.93

Total in
28 days
percent

r

152.0U
16.Us
2k. 17

105.88

Digestibility

Excretion

17.26-33.99

50. SU
8.71

83.0

17.90

81.1
79.3

12U. 73

Each figure represents the average of data from five animals.
Vegetables were fed as total carotenes determined by chemical analyses.

139.75

0-5. Ul

Ul. U2-6U. Ul
0-19.26
S8.59-163.9U
3 .35- 26.08
0-56.35
13.01-21. Ul
6.38-26. Ul
88.73-152.65
107.85-188.38

80.6 J
SU.S

77.9
S3.0
33.3

77.7
75.00

ACKNOWLEDGMENTS

Grateful acknowledgment is made to Dr. M. A. Ohlson, Head of Food
and Nutrition Department, for supervising the research; to Dr. C. A.
Hoppert, Professor of Chemistry, for supplying the rats for the biolo­
gical work; to the Agricultural Chemistry Department for the privilege
of using their Beckman quartz spectrophotometer, to Miss S. L. Bandemer
for instruction in the use of the instrument and to Dr. E. J. Benne for
supplying a micro Waring blendor container for this research; and to
Dr. W. D. Baten, Professor of Statistics for supervising the statis­
tical analyses.

-

46 -

REFERENCES

1.

Guggenheim, K.
The "biological value of carotene from various sources
and the effects of vitamin E on the utilization of carotene and
of vitamin A. Biochem. J. 38, 260-264, 19Mi.

2.

Fraps, G. S. and W. W. Meinke.

Digestibility hy rats of alpha- betar-

and neo-beta-carotene in vegetables.

Arch. Biochem. 6, 323-327,

1945.
3.

Treichler, R . , A. R. Kemmerer and G. S. Fraps.
The utilization of
carotene and vitamin A in the rat.
J. Nutrition, 24, 57- 64, 194-2.

4.

Kemmerer, A. R . ,

carrots.

and G. S. Fraps.

Constituents of crude carotene of

Food Research 10, 4-57-460, 194-5.

5.

Kemmerer, A. R . , G. S. Fraps, and W. W. Meinke.
Constituents of the
crude carotene of certain human foods. Food Research 10, 66-J1,
1943.

6.

Zscheile, F. P., 3. W. Beadle, and H. R. Kraybill.
of fresh and frozen green vegetables.
1943.

Carotene content

Food Research 8, 299-313,

7 . Beadle, B. W. and F. P. Zscheile.

Studies on the carotenoids.
II.
The isomerization of beta-carotene and its relation to carotene
analysis.
J. Biol. Chem. l44, 21-33, 1942.

8.

Harper, R. K. and F. P. Zscheile.
Carotenoid content of carrot varie­
ties and strains.
Food Research 10 , S4-97, 1945*

9.

Kemmerer, A. R . ,
of plants.

and G. S. Fraps.
Constituents of carotene extracts
Ind. Eng. Chem. Anal. Ed. 15, 7l4-7l6, 1943.

10.

Mackinney, G . , Leaf carotenes.

11.

Devine, J. , R. F. Hunter and IT. E. Williams.
The preparation of betacarotene of a high degree of purity. Biochem. J. 39, 5-6, 1945.

12.

J. Biol. Chem. Ill, 75-34, 1935*

Braude, R . , A. S. Foot, K. M. Henry, S. K. Kon, S. Y. Thompson, and
T. E. Mead.
Vitamin A studies with rats and pigs. Biochem. J.

35, 693-707, 1941.
13.

Kemmerer, A. R.

14.

Strain, E. K . , Carotene XI.
Isolation and detection of alpha-carotene
and the carotenes of carrot roots and of butter.
J. Biol. Chem.
127 , 191- 201, 1939.

Report on carotene.

J. of A.O.A.C. 29 , IS-24, 1946.

15. Zechmeister, L.Cis-traas isomerization and stereochemistry of caro­
tenoids and diphenylpolyenes.
Chem. Rev. 34, 267-344, 1944.
1 6.

Zscheile, F. P. and J. W. Porter. Analytical methods for carotenes of
lycopersicon species and strains. Anal. Chem. 19, 47-51, 1947.

-

17.

1+7 -

Strain, H. H . , and W. K. Manning. A unique polyene pigment of the
marine diatom Mavicula Torquatum.
J. Am. Chem. Soc. 65 , 2258,
19^3.

IS.

White, J. W . , F. P. Zscheile, and A. M. Brunson.
The carotenoids of
yellow corn grain.
J. Am. Chem. Soc. 6U, 2603- 2606 , 19*+2.

19 .

Lease, S. J., J. G. Lease, E. Steenbock, and C. A. Bauman.
The bio­
logical value of carotene in various fats. J. Nutrition 17,
91, 1939.

20.

Sherman, ¥. C. Chromatographic identification and biological evaluartion of carotene from mature soybeans. Food Research 5, 13, 19^0

21.

Sherman, V/. C. The effect of certain fats and unsaturated fatty a.cids
upon the utilization of carotene. J. Nutrition 22, 153, 19^1-

22.

Smith, M. C. and L. Otis.
Observations on carotene analyses of vege­
tables and fruits as ». basis for prediction of their vitamin A
value. Food Research 6 , 1U 3-I 5O, 19^1.

23 .

Kemmerer, A. R. and G. S. Fraps.
Nature of carotenes in alfalfa.
Am. Chem. Soc. 66 , 305, 1 9 ^ .

2U.

Deuel, H. J., Jr., 3. Sumner, C. Johnston, A. Polgar, and L. Zechmeister.
Stereochemical configuration and provitamin A activity III. Alltrans-alphascarotene and neo-alpha-carotene U. Arch. Biochem. 6 ,
157-161, 13^5.

J.

25 . Kemmerer, A. R. and G. S. Fraps. Relative value of carotene in veget­
ables for grov/th of the white rat. Arch. Biochem. S, 197-201.
19U5.

26.

Deuel, H. J. , Jr., C. Johnston, E. Sumner, A. Polgar and L. Zechmeister.
Stereochemical configuration and provitamin A activity I. Alltrans-beta- carotene aad neo-betar-csrotene U. Arch. Biochem. 5,
107- 11 U, lghil.

27.

Deuel, E. J. Jr., E. R. Keserve, C. H. Johnston, A. Polgar, and L.
Zechmeister. Re-investigation of the relative provitamin A poten­
cies of cryptoxanthin and betar-carotene. Arch. Biochem. 7, UU 7^50, 19^5.

28.

Oser, B. L. and D. J. Melnick. Physiological availability to the rat
of crude carotene in vegetables.
J. Nutrition 30 , 3S5-3S9, I 9 I+5 .

29 . Bacharach, A. L.

Effect of ingested vitamin 3 (tocopherol) on vitamin
A storage in the liver of the albino rat.
Qjaart. J. Pharm. 13
138-1^9, 191*0 .

30.

Davies, A. W. and T. Moore,
1 U7 , 791t-SOO, 19U 1 .

Inter-action of vitamins A and E.

Nature

-

48

-

31.

Harris, P. L . , M. W. Kaley, and K. C. D. Hickman. Co-vitamin studies
II. The sparing action of natural tocopherol concentrates on
carotene.J. Biol. Chem. 152, 313-320, 1944.

32.

Hickman, K. C.
D . , M. W. Kaley, and P. L. Harris. Co-vitamin Studies I.
The sparing action of natural toconherol concentrates on vitamin A.
J. Biol. Chem. 152 , 303-3H, 194-4. ‘

33.

Karrer, P., and H. Keller.
'“Suantitative Bestimmung der tocopherole in
verschiedenem ansgangsmaterialien, Helv. Chim. Acta 21, II 6I-II 69 ,

193s.

G. A. 33 , 1355-9 , 1539.

34.

Coward, K. E. Biological standardization of the vitamins, p. 22. W.
Wood and Company, Baltimore, 1933.

35.

Kemmerer, A. R. , and G. S. Fraps. Digestibility of carotenes by rats
and chickens.
J. Nutrition l6, 3°9-315, 1333.

7,£

^

V*

AV,

■«->-. + * Qvi

mri £ A h ft! i

v,

r>+q

0

V ^ ^ ‘rrMTliLT' **

C T- T*0 _

tene, with some remarks on the vitamin A requirements. Published
by Einar Munksgaard and Oxford University Press, London, p. 1-269,
19U0 .
37.

Bussell, W. C., M. W. Taylor, H. A. Walker and L. J. Polskin.
The
absorption and retention of carotene and vitamin A by hens on
normal and low fat rations.
J. Hutrition, 24, 199-211, 1942.

3S.

Wilson, B. H . , A. M. Ambrose, P. De Eds, K. J. Hatton and G. P. Bailey.
The content and biological availability of carotene in raw and
dehydrated carrots and other vegetables. Arch. Biochem. 10,
131- 140 , 1946.

39-

Wagner, K. H. The excretion of vitamin A and beta-carotene in the feces
and urine upon the administration of known amounts of vitamin A ^
and beta-carotene.
25. klin. med. 137, 694, 1940.
C. A. 37, 669 ,
19^3.

40.

Morgan, A. F . , and L. S. Bentley.
Storage of vitamin A in the livers
of dogs fed beta-carotene or vitamin A. Fed. Proc. 3 , 1, 1944.

41.

Booher, L. E. , and E. C. Callison. The minimum vitamin A requirement
of normal adults.
II. The utilization of carotene as affected
by certain dietary factors and variation in light exposure.
J.
Kutrit'on IS, 459-471, 1939.

42.

Fraps, G. S. , and W. W. Meinke.
Relative values of carotenes in foods
as measured by storage of vitamin A in livers of rats. Food
Research 10, 187- 196 , 1945.

43.

Little, R. W . , A. W. Thomas- and H. C. Sherman.
Spectrophotometric
studies of the storage of vitamin A in the body.
J. Biol. Chem.
148 , 441-443, 1943.

-

HU.

1+9 -

Caldwell, A. B. , G. MacLeod, and H. C. Sherman. Bodily storage of
vitamin A in relation to diet and age, studied "by means of the
antimony trichloride reaction using a photoelectric colorimeter.
J. Nutrition 30, 3 U9- 353 , 19^+5-

U 5 . Bohrer, A. B. and K. C. Sherman.
The Bodily store of vitamin A as
influenced By age and By food.
J. Nutrition, 25, 605- 609 , 19U3H6.

Campbell, H. L . , M. Udiljalc, H. Yarmolinsky and H. C. Sherman. Bodily
storage of vitamin A in relation to diet and age studied By the
assay method of single feedings.
J. Nutrition 30, 3U3-3US, I9 U5 .

U7.

Lewis, J. M . , 0. Bodansky, F. G. Folk and G. McGuire.
Vitamin A re­
quirement in the rat.
The relation of vitamin A intake to growth
and to concentration of vitamin A in the Blood plasma, liver and
retina.. J. Nutrition 23 , 351 , 19U2.

US.

Callison, E. C. and V. H. Knowles.
Liver reserves of vitamin A and
their relation to the signs of vitamin A deficiency in the albino
rat. Am. J. Physiol. 1U3, UUU-U52, 19U5.

FIGURE 1.

Effect of Froaen Storage on Absorption Spectra of Raw Spinach
( in cyclohexane )

210

-

Specific

absorption

c o e ffic ie n t

190

170

150

130

110

90

70

50

30

— /9 C aro ten e
—oc C a ro te n e
-- Raw spinach
— Raw spinach sto re d 10 mo.
4000

4200

4400
4600
Wavelength A°

4800

5000

230

2 IQ-

absorption

coefficient

190

170

150-

130-

Specific

110-

90-

70/3 C arotene
oc Carotene
Cooked carrot
C ooked c a rro t stored 10 mo.

50-

30

FIGURE 2.

4000

4200

4400
4600
W avelength A

4800

5000

Effect of Frozen Storage on Absorption Spectra of Cooked Spinach
( in cyclohexane )

230i

r\

210

Specific

absorption

coefficient

190-

170-

150

130

10

90

70

—

50

/<? C arotene
OC Carotene
Raw c a rro t
Raw c a rro t stored 10 mo.
\

30

4000
FIGURE 3.

4200

4400
4600
Wavelength A°

4800

5000

Sffect of Frozen Storage on Absorption spectre ox naw uarrot
( in cyclohezane )

230-1

210

-

190-

absorption

coefficient

170-

150-1

130-

Specific

11 0 -

90-

70/<?Garotene
oC C arotene
Cooked spinach
Cooked spinach stored 10 mo.

5030
4000

FIGURE H.

4200

4400
4 o6 0 0
Wavelength A

4800

5000

Effect of Frozen Storage on Absorption Spectra of Cooked Carrot
( in cyclohexane )

Males

/

Females

ymales* 84-25 +18.03 x
0e* 2.34 g
fem ales*86.59 + 8.95 x
*l.43g
20
tO
1.5
x* v*/0 Carotene /day

Relationship of Gain in Weight to Dosage of Beta-carotene for Hale and Female Eats

Gain in weight (g.)
<D

o

~sl

o
_I_

00

o
_I_

CO

oI

o
o

o

ro

o

_l_

OJ

o

Negative
Carotene 0.45 Hday
/Q Carotene 0.70 r/day

LD
CD
CD

cn

/3 Carotene

1.03 r/day

/? Carotene

1.36 r/day

/3 Carotene 2.00 r/day
Vitamin A alcohol 0.46 r/day
O/Garotene 2.36 r/day

Daily supplements

Raw spinach 3.82r/day(Total carotenes)
Cooked spinach 3.82 r/day (Total carotenes)
Raw carrot 3.82r/day (Total carotenes)
Cooked carrot 3.82r/day(1otal carotenes)

Negative

(f)

/? Carotene

7 0 r/day

0 / Carotene

23.6 r/day

CD
CD'

(/)

Vitamin A alcohol 4.6 r/day

ro

Raw carrot

2 0 .0 r/day (Total carotenes)

Cooked carrot

FIGURE 6,

2 0 .0 r/d ay (Total carotenes)

Relationship of Oain in Weight to the Various Supplements Ted to Eaeh Sroup of Rats
( Average of 5 Males and. 5 Eeftisiss )

Negative control
Vitamin A alcohol 4.6 r/day
/? C arotene 7 0 r/d a y
— Raw c a r r o t 2 0 .0 r /d a y
CX. C aro ten e 2 3.6 *•/day
4.00-

Extinction

3 .0 0

2.00 -

0.00

FIGURE 7 .

3000

3400

3800
4200
Wavelength A°

4600

Absorption Spectra of Carotene Extract of Rat Feces

5000

.001

0.80-

Extinction

0.60-

0.40-

— Negative control
— oc Carotene 2 3 .6 r/d ay
— Cooked c a rro t 2 0 .0 r /d a y
Carotene 7 .0 r/d ay
— Vitamin A alcohol 4.6 r/d ay
3000

FIGURE S .

3100

3200
3300
Wavelength A°

3400

3500

Absorption Spectra of Cyclohexane Extract of Rat Livers.