On the simultaneous extraction of vitamin A-D andvitamin B 2 complex from the liver of a fin whale(Nagasu-kujira, Balaenoptera physals L.).

Tadashi Tawara
Since the discovery of vitamin A in 1913, by Osborne and Mender,<1>
studies of vitamin showed extraordinary development. Later, Mellanby,<2>
McCollum,<3> Sherman, Pappenheimerc4> and others distinguished between
vitamins A and D.
Then Kawakami,< 5> Kawaics> and others confirmed that vitamins A andD existed in the liver of fishes widely and in rich concentrations. Material for vitamin A and D is now almost wholly suppliep from the liver ofaquatic animals.
It was also discovered that vitamins A and D existed richly in whaleliver. Whale liver is now one of the most essential resouces of vitamin AD oil, as its volume is very large.
In 1926, vitamin B2 was distinguished from vitamin B, by Goldbergerand others,<7> and so-called " Complexity of Bz" was claimed.
Members of BJ group is now said to contain many factors including
the following: lactoflavin (riboflavin), nicotinic acid, vitamin Bs (adermin),pantothenic acid and other factors. Then, Kuhn, cs> Karrerc9> and others
discovered that liver contained a large amount of vitamin Bi complex.
B2 complex is richly contained in the liver of aquatic animal, especially,in those of tunny (Maguro; Thunnus orientalis), spearfish (Makajiki; Makaira mitsukurii), swordfish (Mekajiki; Xiphias gladius), bonito (Katsuo;Katsuwonus vaganus), fin whale (Nagasu-kujira, Balaenoptera musculus L.),humpback (Zato-kujira ; Megaptera nodosa Bonn), etc.
There are many method of extracting liver oil, principal methods being
as follows:(A) Heating with steam-Liver is heated with steam, and then the oil separated.
(B) Resolving by standing-Liver is left standing, putrefied, resolved, and the oil separated.
(C) Vacum method-

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Tadashi Tawara

Liver is placed in a vacuum kettle, and the oil separatEd by heatingwith low pressure steam or hot water.
(D) Refrigeration method-Liver is pressed while frozen, and the oil separated.
(E) Electrolytic methodLiver cells are destroyed by passing an electric current, and then theoil separated.
(F) Extraction with organic solvents-(i) Dry methodLiver is heated and almost dried. And the oil extracted with organic
solvents such as ether.
(ii) Resolving with caustic soda-Liver is dissolved in caustic soda, and then the oil extracted withorganic solvents, or separated by centrifuge.
At present, the last method is almost always employed because theyield of liver oil is very good, and vitamins A and D are extracted al.mostcompletely without decomposition.
The present method of preparation of liver oil is limited to the obtaining of vitamin A-D oil, and sacrificing of other effective components which
are contained in the liver is unavoidabl.
The author aimed at this point and tried to extract as completely as
possible both the vitamins A-D and B2 from one liver.
Vitamin A is quite stable to alkalis but is sensitive to acids, whilevitamin B2 (lactofiavin) is quite stable in strong mineral acids but is sensitive to alkalis. Vitamin A is sensitive to oxidation, but lactofiavin is quite
stable to agent. On account of these conflicting properties, it is difficult toextract both A-D and B2 completely without decomposition.
Vitamins A and D are fat-soluble vitamins while B2 complex is watersoluble. The author, therefore, tried to extract first, B, complex withwater, and then A-D oil from its residue. Extraction of vitamin B2 complex
on various conditions were tested to observe how the quantity of vitamin
A changed by various conditions, and to discover conditions under which B2complex is extracted completely and decomposition of vitamih A is theleast. Vitamin B:i complex was extracted by various methods and thequantity of lactofiavin in the extract was measured. Its residue was thenresolved with caustic soda, the liver oil extracted with ether, and then theOn the simultaneous extraction of vitamin A·D andvitamin B2 complex from the liver of a fin whale

33

qunntity of vitamin A was measured.
Considerations were given to the effect of air, acids and heat onvitamin A, and to the effect of destruction of cells, and temperature ofextraction on Bitamin H~.
Fin whale liver was used as material.
Experimental
Fin whale liver was. minced in a meat-chopper, mixed homogeneously
and 100 g of it were used for each experiment.
I. Quantitative analysis
A) Quantitative analysis of vitamin A.
Raw liver or the liver from which B2 complex had been extracted wastaken, about 100 cc of water added and warmed in a water-bath which
was later brought to a boil. After the meat had coagulated, solution of
2 g NaOH dissolved in 20 cc of water was added little by little to thecoagulated liver with agitation. In the meantime, about 200 cc of water
was also added to it, little by little.
After the whole quantity of NaOH solution and water were added, theheating was continued until the granules of liver melted completely andturned into a liquid. This liquid was then allowed to cooled, transferred
into a separating funnel, and vitamin A-Doil was extracted five times with
140 cc each of ether. The upper etherial layer was gathered, and washedwith water until the washings did not colour to phenolphthalein. Afterthe etherial solution was .dried with anhydrous Glauber's salt, ether was
distilled off, and vitamin A-D oil was obtained.
The yield of this oil was weighed, and the quantity of vitamin A in
1 g of the oil was measured with spectrophotometer by spectro-graphic
method (solvent, chloroform).
Ei~~ 328 millimicron= 1600c10) was used.
International unit (I. U.) per gram multiplied by the yield of oil gives
the total amount of vitamin A in 100 g of liver.
B) Quantitative analysis of vitamin B2 (lactofiavin)10 g of acid clay (Fuller's earth) was adrled to the liquid by which
vitamin B2 was extracted from 100 g of liver. By shaking this mixture
vigorously, lactoflavin was adsorbed completely, and then filtered. Theadsorbed earth on the filter paper was washed with a small amount ofTadashi Tawara

water,. and dried at 60°C. 50 cc of alcohol (94%) was added to the driedearth, the mixture shaken, filterd and the earth again dried at 60°C.
Lactoflavin was eluted from one-tenth quantity of this adsorbed earth
with 20 cc of .40% alcoholic 0.25 N-NaOH solution. The residual Fuller's
earth was eluted again with 20 cc of 40% alcoholic 0.1 N-NaOH solution,and the two lots of solutions were brought together. This was exactly
neutralized with HCl, concentrated at 40°C under a reduced pressure, andalcohol was then distilated off. This was further concentrcted to about
10 cc, and was made into a 0.5 N-NaOH solution by the addition of 5 NNaOH solution.
This solution was measured by Kuhn's method01 ) 02 ) with PulfrichStufenphotometer (Filter S. 47; E 10n~cc=4.75).
The value thus obtained multiplied by the gives the quantity of lacto:flavin (r) in the extract obtained from 100 g of liver.
II. Extraction of vitamin A-D oil and B2 complex under various conditions.
( 1 ) Quantative analysis of total vitamin A in 100 g of liver.
Total quantity of vitamin A extracted from 100 g of minced liver by
the abovementioned method of resolving with caustic soda, was ·measured.
Vitamin A
720,000 I. U.
( 2) Extraction of vitamin B2 compl.Ex by boiling.
100 g of liver as above was taken. 300 cc of water was added to it,. and the solution was brought to pH 5.0 with the addition of phosphoricacid. This solution was boiled in a water-bath for 2 hours, tilted withfilter-paper while hot. By the above-mentioned method, the quantity ofvitamin A in the total amount of residue, and the quantity of lactofl.avinin the filtrate were measured. In this case, owing to having boiled inacidity, the amount of vitamin A decreased greatly.
Vitamin A
550,000 I. U.
1086 rLactoflavin( 3 ) Extraction by boiling in vacuum.
Since vitamin A is oxidized easily in acidity and on heating, the boiling
was made under a reduced pressure in order to prevent decomposition.
However, the degree of the heating was to such an extent that water inthe water-bath was boiling although, it goes without saying that thetemperature of liver solution did not reach 100°C. Other conditions were

the same as above.

On the simultaneous extraction of vitamin A-D andvitamin B.i complex from the liver of a fin whalelr·.

:35

The quantity of vitamin A in this case, was greater, but, on accountof insufficient heating, the amount of lactoflavin decreased.
690,000 I. U.
Vitamin A
1022 rLactoflavin( 4) Extraction by digesting.
The liver was digested in order to make the the extraction of vitamin
Bi complex better by destruction of liver cells.
100 g of liver as above, was taken, 300 cc of water and 15 cc of 90%
phosphoric acid were added, and then 1 g of saccharated pepsin (J. P.)
added with stirring, and kept at 40°C for 2 hours. The digested solution
thus obtained was filtered, and the same processes as above were carriedout on the filtrate and the residue.
In this case, the extraction of B2 became somewhat better, but owing
to higher acidity the amount of vitamin A obtained was decreased.
576,000 I. u.
Vitamin A
1150 rLactoflavin( 5 ) Extraction by digesting in vacuo.
The digesting process, in this case, was carried out in vacuo. Otherconditions were the same as above.
·vitamin A
690,000 I. u.
863 r
Lactoflavin
The amount of vitamin A became somewhat better, but, on accountof insufficient heating under reduced pressure, the yield of B2 decreasedexceedingly.
( 6) Extraction by digesting and boiling.
In order to extract J.actoflavin more completely, the digested liquid wasboiled for 10 minutes, filtered while hot, and the residue and the filtrate
were treated in the same manner as above. The condition of the digestion
was the same as for (4).
456,000 I. u.
Vitamin A
1788 rLactoflavin( 7 ) Extraction by digesting und boiling in vacuo.
In the above process, vitamin A wa5 oxidized greatly because of boilingthe solution in strong acidity. Hence the same process was carried out
m vacuo.
675,000 I. u.
Vitamin Ailf..

36

Tadashi Tawdra

1563 rLactoflavin( 8 ) Extraction by digesting, neutralizing and boiling.
In the above process satisfactory result could not be obtained on theyields of both A and B2. This must have been due to the fact that vitamin
A was boiled in acid, and also to the reduced pressure.
The liver, accordingly, was digested by the method as in (4), then thesolution exactly neutralized with NaOH solution, boiled for 10 minutes, andfiltered.
645,000 I. u.
Vitamin A
1789 rLactoflavin( 9 ) Extraction by digesting, neutalizing and boiling in vacuo.
The operations as in (8) was carried out in vacuo.
Vitamin A
703,000 I. U.
Lactoflavin1406 rIn all the methods of extractions under various conditions mentioned
above, the vacuum process for the extraction of vitamin A brought a goodresult, but it brought a bad result on B~. The inferior result of the extraction of B2 was due to the failure of the temperature to reach boilingunder reduced pressure.
Hence, it was thought that if the boiling was carried out under oridinarypressure without air, the result would be better. Carbon dioxide gas wasused as an inert gas for this purpose.
(10) Extraction by digesting, neutralizing and boiling in carbon dioxidegas.
C02 gas was used instead of a vacuum, and all the processes of digestion, neutralization and boiling were carried out in C02 gas.
711,000 I. U.
Vitamin A
Lactoflavin2110 r
The loss of vitamin A, as a result, was almost negligible, and theextraction of BJ was better.
(11) Extraction by boiling, digesting, neutralizing and boiling in CO;l gas.
The liver was coagulated by boiling so as to allow pepsin to act easily
on proteins, and cooled to 40°C by adding water, pepsin was then added.
All the process were carried out in C02 gas. Other conditions were theOn the simultaneous extraction of vitamin A-D andvitamin B2 complex from the liver of a fin whale

same as for (10).
Vitamin A
Lactoflavin!17

711,000 I. U.
2849r

SummaryConditions under which vitamin A is decomposed least and vitamin B2
extracted completely from a liver, are as follows:
Minced liver is boiled with water in· C02 gas, cooled to 40°C by adding
water, acidified with phosphoric acid, and the liver substance is digested
with protease such as pepsin at about 40°C. After digestion is completed,the solution is exactly neutralized with NaOH, boiled again and filtered.
All the above process carried out in CO:i gas.
Vitamin B:i is obtained from this filtrate, and vitamin A-Doil from theresidue.
In conclusion, the author expresses his cordial thanks to Dr. T. Maruyama and Mr. T. Nakai for their kind advice and encouragement during
this study.
References( 1)

Osborne T. B. & Mendel L. B.: J. Biol. Chem., 16, 4'...3, (1913).

( 2)

Mellanby: Lancet, 1, 856, (1920).

(3 )McCollum : J. Biol. Chem., 45, 333, (1921).

( 4)

Sherman, pappenheimer: Proc. Exp. Biol. Med. 18, 1S3 (1921).

( 5)

Kawakami Y.: Sci. Pap. I. P. C. R., 11, 968, (1S32).

( 6)

Kawai K.: J. Pharm. Soc. Japan, 708 (1929); 249, 323, 809, &36, 951 (1930).

( 7)

Goldberger etal: U. S. Pub. Health. Rep., 41, 1025 (1926).

( 8)

Kuhn, Wagner-Jauregg: B., 67, 1770 (1S34).

(9)

Karrer Salomon, Schopp : Helv. Chim Act., 17, 419 (1934) ; 13, 1022 (1935).

(10)

Morton R. A. & Heibron I. M. : Bioch. J., 22, 987 (1928).

(11)

Kuhn, Wagner-Jauregg, Kallschmitt: B., 67, 1452 (1934).

(12)

Warburg, Christian: Biochem. Z., 254, 483, (1932); 277, 492 (1933); 2S6, 377 (1933).