Substances Related to Vitamin A in the Whale Liver Oil
BY
SEIICHI ISHIKAWA,YOSHIMORI OMOTE,HIROSHI OKUDA

The maximum of absorption spectrum of whale liver oil is near 310 mµ,considerably deviating from 328 mµ, that of vitamin A. This point was pursued
to isolate kitol having theabsorption maximum at 290
mµ.1>
Furthermore, substances related to vitamin
A such as anhydro vitam:lnA and subvitamin A have'OOO
been confirmed in ordinary
fish liver oils. Also in thewhale liver oil, these vitamins may ~ expected being
present. It is very interesting that there are some
substances with the struc2000
ture closely related to vitaEvmin A. This paper is concerned with the analyticalmethod of substances related
cto vitamin A, with the-~~thermal change of whale
81000
liver oil, and with theaction of maleic anhydride
upon the whale liver ojJ.

Analysis of Absorption Spectrum of Whale Liver Oil.

250

300350
Wave length,

400

450

m1•

Fig. I. Absorption spectra of vitamin Ai (Ai), vitamin A2 (A2), subvitamin A (S), kitol (K), anhydrovitamin Ai (Ai'), anhydrovitamin A2 (AD and anhydrosubvitamin A (S').

Every substance relatedto vitamin A shows strong
absorption between 250-400mµ. The absorption spectraof hitherto known substances
related to vitamin A aresummarized in Fig. i.1>2>a>

54

SEIICHI ISHIKAWA, YOSHIMORI 0MOTE, HIROSHI OKUDA

An example of absorption spectrum of whale liver oil is shown in curve S inFig. 2. As compared with Fig. I deviation of the maximum of the curve S from328 mµ, the maximum of vitamin A, to shorter wave length was considere::I to bedue to the presence of kitol.
the assumption that additional resultant- ofOnabsorption spectra of vitamin A and kitol would make the curve S, each component
was calculated as shown by the curves A and Kin Fig. 2. A represents the absorption spectrum
20of vitamin Ai, and K shows that of kitol. S' is
the curve obtained by the additional compositionof A and K on the figure. This method istemporarily called AK method.
For the purpose of discussion on reliabilityof this method, using the same liver oil, the250300350

mp.

extinction coefficient of vitamin A at 328 mµcalculated by AK method was compared withresults determined by the following two methods.
1. AKA' method ....... To analyze the original
curve assuming that it is composed of vitamin
Ai, kitol, and anhydro vitamin Ai.
2. Morton-Stubbs's correction 4l.
Fig. 2. AnalySis of the absorption
curve of whale liver oil, (S) sample,(S') resultant, (A) vitamin A, (K)kitol.

Non cotrection

AK-methodMS correction

AKA' method

21.4

17.5

15.9

11.7

Although these corrections are less strict due to calculation on the rough.
assumption, they may present us far nearer value to the true vitamin A than noncorrected extinction coefficient at 328 mµ in the sample, taken for that of vitamin

A.
Thermal Change of Whale Liver Oil.
It has been already known that kitol produces vitamin A by thermal decomposition and that 1 mole of vitamin A is yielded from 1 mole of kitoI. 1Js> Therefore, also in case whale liver oil is heated itself, its containing kitol is expected
to decompose to vitamin A. However, when liver oil is heated open in the air,the decomposition of vitamin A by oxidation makes it impossible to find newl_y
produced vitamin A. So, first of all, whale liver oil was heated in carbon dioxide
at 220°, and co1 liver oil unit was measured with the lapse of time. This
result is shown in Fig. 3.
When heated for 2 minutes, cod liver oil unit of the oil reached about 130 %Substances Related to Vitamin A in the Whale Liver Oil

515of that of original, while after heating for longer time, vitamin A was found todecompose. When heated under high vacuum, its absorption curve was comparedas shown in Fig. 4.

202800300

320340mµ60

4020time (min.)Fig. 3. Change of cod liver oil unit of
the whale liver oil heated in the C02 ·tube.

Fig. 4. Change of the absorption spectrumof whale liver oil heated in vacuum, at220°, for 3 min.; - original sample, ..... .
sample heated.

The extinction coefficients of vitamin A and kitol were calculated with application of AK method, on the basis of which vitamin A produced from I mole ofI&soc0"c..

<c§60-~'"'"

-!

""20

I

0

24Hours.

68Fig. 5. Re3ction of the whale liver oil vitamin Awith maleic anhydride at C•) 50°0, ( x) 25°0.

56

SEIICHI ISHIKAWA, YOSHIMORI 0MOTE, HIROSHI OKUDA

kitol was calculated to be 1.42 mole. Taking the decomposition of vitamin A into
consideration, there is possibility for more increase than the above figure. This

acontradicts with the_ figures previously reported that from 1 mole of kitol 1 mole ofvitamin A was formed. This respect will be persued in future.

Reaction of Whale Liver Oil with Maleic Anhydride.
Baxter and others6l stated a method to analyze vitamin A and neo-vitamin A
by the action of maleic anhydride. The authors observed the reaction of whale
liver oil with maleic anhydride by their method. From this result the relation
between decrease of cod liver oil unit and reaction time was found as shown inFig. 5.
On the assumption th3.t antimony trichloride produced blue color with only
vitamin A and neo-vitamin A, the amount of neo-vitamin A was calculated to be
46.5 % of the total vitamin. This figure is approximately same as th3.t found with common fish liver
20oils.9l
The~·absorption spectrum of whale liver oiliJ

reacted with maleic anhydride was shown in Fig.
6. Absorption of vitamin A nearly disappeared
and rather similar curve to the spectrum of kitol
was found.
Moreover, diene value was measured for thepurpose of the reference to study the reaction of300

250

350

ffii'

Fig. 6. Absorption spectrumof the whale liver oil reactedwith maleic anhydride atroom temperature.

maleic anhydride upon whale liver oil and its
molecular distillates. The results were shown in
Table I, with comparison of cod liver oil unit.

Table I Diene Value3 of Whale Liver Oil and Its
Molecular Distillates
Temperature%I

D~ene value

I

Original sample
Distillate 1.
Distillate 2.
Distillate 3.
Distillate 4.
Distillate 5.
Residue

Cod liveroil unit

1Found[ TheoreticalE(328)

A(328)

I

170°C
200°c
210°C
220°c

2.5I
2.3 II 33.9
6.2 15.1
7.4
3.1
23.5
1.4

1.6
35.6
10.9
5.7

230°C

17.1

1.8

0.9

3.009221.4 II
16.6
2105367 I 360
I631 I 120
1103456358I88191841
109- ! 2.9 I 0II

1.8III

Substances Related to Vitamin A in the Whale Liver Oil

57

Experimental Part.•Experimental 1. Ana/y3is of the Absorption Spectrum of Whale Liver Oil.
(1) AK method ......... The observed curve i3 assumed to consist of absorption
spectrum of pure vitamin A1 and p'.lre kitol only, additionally composed. Letabsorption coefficients of sample at 290 mµ and 330 mµ, being near their absorption
maxima, be represented by E (290) and E (330), and that of pure vitamin A1 at330 mµ in the sample, A (330), and that of pure kitol at 290 mµ, A (290). Thenthe following eqmitions are obtained.
E (330)=A (330)+0.293 K (290)}
E (290)=K (290)+0.390 A (330)'
Factor 0.293 means ratio of pure kitol, K (330)/K (290), and factor 0.390
means ratio of pure vitamin A1, A (290) /A (330), all of which were quoted
from the reference in Fig. 1. The values of A (330) and K (29J) from the above
equations are solved as follows:A (330) = 1.13 E (330)-0.33 E (290) }.
AK equationK (290) = 1.13 E (290)-0.44 E (330)The way of finding A (328) from A (330) is only to multiply it by 1.018. If
AK method is applied to the abs'.>rption spectrum of Antarctic whale liver oil(sample 1), we ca:i get A(330)=17.2, K(290)=13.4 and A(328)=17.5. On thebasis of these figures A and K curves in Fig. 2 were plotted similarly to that ofpure substance.
(2) AKA' method ......... Let absorption coefficients of sample at 290 m,u, 330 mµand 350m,u, be respectively represented by E (290), E (33J) and E (350), that ofvitamin A{ in the sample at 330mµ, A (33J), that of kitol at 290 m,u, K (290),
and that of anhydro vitamin A1 , A' (350), and the following equations can beobtained similarly to (1).
A (330) = 1.452 E (330)-0.383 E (290)-0.595 E (350)K (290) = 1.139 E (290)+0.101 E (350)-0.498 E (330)lAKA' equation

A' (350)=0.112 E (290)+1.304 E (350)-0.728 E (330)The sample 1 was ana!yzed with these equations as follows.
A(330)=15.7, A(328)=15.9, K(290)=14.7, A' (350)=3.2
This fact proves the presence of anhydro vitamin A in the whale liver oil, althoughsmall amount. So, unsaponifiable_matter in this liver oil was treated by chromatography. As expected, anhydro vitamin A fraction could be isolated and confirmed.
(3) MS method ......... This method, which shows how to get corrected E (328) ofvitamin A when absorption spectrum 'of vitamin A is affected by other irrelevant
spectra, is on the basis of the assumption that other spectra can be regarded
linear near the maximum of vitamin A. The wave length corresponding to 6/7

SEII~HI ISHIKAWA, YOSHIMORI 0MOTE, HIROSHI OKUDA

58of the height of the peak in the curve of vitamin Ai in Fig. 1 was found 311 mµand 337 mµ.

Now when the absorption coefficients of the sample at 311 mµ, 328

mµ and 337 mµ. are expressed in E (311), E (328) and E (337), corrected E (328)viz, A (328) is shown in the following equation.
A (328)=7 E (328)-2.42 E (311)-4.58 E (337) ......... MS equation.
A (328) of sample 1 was determined 11.7 in this method.

Experiment 2.
(1)

Thermal Change of Whale Liver Oil.

Whale liver oil (sample 2) was put in the small test tube, the atmospherebeing substituted with carbon dioxide at 10- 2 mm, and sealed in ampoule. 8
ampoules were heated in the oil bath· for l, 2, 4, 8, 16, 32 and 64 minutes andcod liver oil units of. them were determine::! by antimony trichloride method.
resutls are shown in Fig. 3.

The(2) Two ampoules of sample 2 were similarly made, sealed immediately after
degassing at 50°, under the vacuum of 10-4 mm, one of which was heated for
3 minutes at' 220°. The absorption spectra of both ampoules were compared eachother as shwon in Fig. 4.

The amount of vitamin A and kitol in the above twoampoules was calculated by AK method.
I E(330) E(290) A(328) K(2oo)Iil

Before heating

I 53.17After heating

59.08

51.81

43.80

35.15

I f>0.80 I 50.80 I 31.41 /When calculated with molecular weight of vitamin A= 285, E (328) = 1800, 7)
molecular weight of kitol=572, E (290)=7075 ), 1 g. of oil with E=l will containl.94xl0-6 mole of vitamin A at 328mµ and 2.47xl0- 6 mole of kitol at 290mµ.
Then the number of molecules of vitamin A produced from 1 molecule of kitol isshown in the following equation.
.
.
A (328) increased
1.94xl0-6
V1tamm A mole= K (290) decreased X 2.47xl0-6= A (328) increased X

K (290) decreased

0 786
·

The result through application of the above experiment to this equation wasthat 1 mole of kitol produced 1.42 mole of vitamin A.

Experiment 3. Reaction of Maleic Anhydride upon Whale Liver Oil.
(1)

Sample 2 was dissolved in dry benzene to make a solution having the cod

liver oil unit of about 1.0. An aliquot of 5 cc. was added to the same volume ofmaleic anhydride solution (10 g. maleic anhydride per 100 cc. benzene).

After aSubstances Related to Vitamin A in the Whale Liver Oil

59

certain hours' storage in the thermostat at 25° and 50°, an aliquot was thenremoved to determine the cod liver oil unit.
Under the same condition, a comparative experiment was carried on forsample and benzene only instead of maleic anhydride solution.

From both values,

the remaining amount of vitamin A not reacted with maleic anhydride was calculated. ·Assuming that the initial amount of vitamin A was 100, the relation between
the recovery of vitamin A and the reaction time was shown in Fig. 5. Moreover, therecovery after 16 hours' reaction at 25° was 44.5 % . According to Baxter andothers,6l under the same condition, the recovery of vitamin A palmitate was 90%,
and that of neovitamin A palmitate was 5 %. Then the percentage of neovitamin
A in the sample was calculated as follows.

% of neovitamin A= ~.!J~-;(2)5

Xl00=46.5%

0.2 g. of maleic anhydride was let to react with 1 g. of sample 2 in benzene

solution below 50°. When it began to show not blue but reddish purple, in
antimony trichloride reaction, the absorption spectrum of the solution was searched(Fig. 6).
(3) After molecular distillation of sample I, dien<:: values of original sample,
distillates and residual oil were measured. 5 cc. of xylene solution of maleic
anhydride (about 5 %) was added to about 500 mg. of the sample and after sealing under carbon dioxide keeping at 100° for three hours, the residual maleic
anhydride was titrated with alkali in the water solution. · Diene value is shown in
the following equation.
1.269 X normal alkali in cc.
Diene v a l u e = - - - - - - - - - - - sample in g.
As a reference, cod liver oil unit, E (328) and A (328) were also annexed inTable I.
The theoretical diene value in the table was calculated on the basis of. thevalue of A (328), assuming that 2 moles of maleic anhydride react with 1 mole ofvitamin A (viz, theroretical diene value of pure vitamin A is 178).

References~.D. Embree, E.l\L Shanti,

J. Am. Chem. Soc., 65, 910 (1943).
E.}:I. Shantz, Science, 108, 417 (1948).
S.D. Embree, E.:'tf. Shantz, J. Am. Chem. Soc., 65, 9C6 (1943).
R.A. :\Iorton, A.L. Stubbs, Biochem. J., 42, 195 (1948).
F.B. Clough, H.M:. Kascher, C.D. Robeson, J.G. Baxter, Scien~e, 105, 436, (1947).
C.D. Robeson, J.G. Baxter, J. Am. Chem. Soc., 69, 139 (1947).
J.D. Cawley, G.D. Robeson, L. " 7eisler, E.M. Shantz,_ ,N.D. Embree, J.G. Baxter,
Science, 107, 346 (1948).
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