Chemistry and Physical Properties of Vitamins B-Factor




CHAPTER VI

chemistry and physical properties of vitamins
B-Factor.

4 Little success has been obtained in the attempts made to isolate the
antiberi-beri substance. Fraser and Stanton by chemical analyses of polished
and unpolished rice hoped to get some clue as to the nature of the substance.
They found that more phosphorus was contained in the whole grain (0-54 per
cent.) than in the polished rice (0-26 per cent.), but the difference was
insufficient to be a sure guide, as it varied in the several samples. They
expressly stated that no significance should be attached to this observation.
Schaumann, however, believed that beri-beri was caused by the absence of some
organic phosphorus compound from the food. Feeding experi­ments with isolated
organic phosphorus compounds have shown that this group of compounds has no
curative action upon beri-beri. Funk cured fowls suffering from polyneuritis
with a preparation which contained no trace of phosphorus, thus proving that
the unknown substance is not a phosphorus compound.

At one time it seemed as if Funk had actually isolated the antiberi-beri
substance; he obtained a small quantity of an active crystalline compound from
rice polishings, yeast, dried milk, ox-brain and lime-juice. The diffi­culties
confronting the chemist are really very great. The substance exists in minute
quantities in food-stuffs, hence very large quantities of material must be
taken at the start. In the absence of knowledge of the chemical properties of
the substance every stage in the process

63



64   VITAMINS 
AND  THE  CHOICE 
OF FOOD

must
be tested upon birds to see if the active substance be still present. In his
first attempt Funk used 54 kilos (= 120 lbs.) of rice polishings, which he
worked up in portions of 1*5 kilos with 4 litres (= 7 pints) of acid alcohol.
The total extract amounted to some 126 litres (= 25 gallons). This
large quantity of liquid had to be carefully evaporated in vacuo; a
residue weighing 347 grams (= 12 oz.) consisting mainly of fat was obtained; it
was treated with water and ether to remove fat and the volume of watery liquid
became 17 litres. From this 17 litres of liquid one dose, corresponding to 20
grms. of the original rice polishings, was required to cure poly-neuritis in
pigeons. Further lengthy and laborious operations gave a solution of which a
dose equivalent to 40 grms. of the original rice polishings was required to
effect a cure. Thus loss occurs at each stage. Eventu­ally from all this bulk
of material Funk obtained a precious 0-5 grm. (= 0-02 oz.) of substance, just
enough to test on birds and analyse. A single dose containing 172 milligrams (=
0-006 oz.) of the substance was sufficient to cure a pigeon with polyneuritis.
The analysis of the substance gave a formula C17H1304N.
Nothing was left after this analysis and curative experiments had been made.
The substance was believed to be an amine, that is a nitrogenous base, and was
called by Funk vitamine.

Funk still persisted in this laborious work, and used even larger
amounts of rice polishings, yeast, milk and other foods, but never got further
than with the first experiment, except that he eventually found his vitamine
was not a pure chemical substance but a mixture of impurities. Only a very
Small quantity of the pure sub­stance can be present in the original
food-stuffs. Workers outside bio-chemical laboratories hardly appreciate the
labour involved in such an operation and the resulting disappointment.

The chemists' difficulties are enhanced by the instability of the
vitamins to heat, chemical reagents, oxidation,



CHEMISTRY AND PHYSICAL PROPERTIES 65

drying,
etc. Of the three known vitamins B-factor is by far the most stable. It is not
destroyed by drying, and dry seeds retain this vitamin for long periods. Heat­ing
to ioo° G. is not harmful, but it is destroyed by heating to 115 to 1200
G. under pressure for several hours.

B-factor is soluble in dilute alcohol, but only slightly soluble in
absolute alcohol, chloroform, ether, benzene and acetone.

It is not easily destroyed by acid, and is extracted from rice
polishings, etc., with acid alcohol. Funk stated that this vitamin withstood
hydrolysis with 20 per cent. sulphuric acid for six to ten hours, but Drummond
found that the antineuritic value of marmite was greatly diminished by heating
for ten hours with 20 per cent. sulphuric acid.

B-factor is more sensitive to the action of alkali, and loss takes place
on working with it in alkaline solutions. The rate of destruction is slow at
room temperature, but heating with 5 per cent, caustic soda for five hours
caused complete destruction. Experiments made by Daniels and McClurg on the
cooking of beans indicate that the addition of soda as commonly used in cooking
does not appreciably diminish the amount of this vitamin.

B-factor is adsorbed and carried down by any pre­cipitate, or by any
finely divided powder, present in a solution; this is a troublesome property
from the chemist's point of view in attempts to isolate the pure substance.
Harden and Zilva showed that the antineuritic factor in yeast extract is
completely adsorbed by a pure prepara­tion of fullers' earth, known as Lloyd's
reagent. This powder can thus be made an active source of B-vitamin ; the
fullers' earth is quite harmless and does not pass through the wall of the gut,
but the B-vitamin is assimilated. Fullers' earth activated by B-factor is a
convenient form in which to administer concentrated doses of B-vitamin. Eddy
has used with good effect an activated preparation of fullers' earth in the
treatment

F



66   VITAMINS AND THE CHOICE OF
FOOD

of
marasmic infants: I grm. of this powder contained the B-factor from 54 grms. of
fresh lamb's pancreas.

On treating a mixture of equal volumes of autolysed yeast and
orange-juice with fullers' earth, the B-factor is removed and the
anti-scorbutic activity of the solution undiminished. By this means it is
therefore possible to separate B-factor from G-factor.

B-factor is destroyed by radium emanations, but is not destroyed by
ultra-violet light.

C-Factor.

The isolation of the antiscorbutic substance presents even greater
difficulties to the chemist than the antiberi-beri substance, as it is more
easily destroyed by oxidation, heating, drying and other processes.

The action of heat upon the antiscorbutic value of i vegetables
and fruit-juices has been carefully studied by Miss Delf at the Lister
Institute :—

Cabbage-leaf heated in water '—

at
6o° C.       for one hour      loses 70 per cent, of its value
at 70-800 C. for             
90       ,,           

at 90° C.       for 20 minutes        
70                       

Cabbage juice heated—

at ioo" C.    
for one hour      loses 80                       ,,

Swede-turnip juice heated—

at ioo° C.    
for one hour      loses 50        ,,              ,,

Orange juice and lemon juice heated—

at 1100 C.                   no
apparent loss.

Hess and other observers have also found that the antiscorbutic value of
orange-juice is not diminished by boiling for short periods. Hess has used
successfully the juice of canned tomatoes in the prevention of infantile
scurvy, so that tomato-juice must also be relatively stable to heat. On the
other hand, the antiscorbutic substance in milk is very easily destroyed by
heating. Like vegetables, milk suffers less loss of this factor on boiling for a few minutes than by gentle heating for
longer periods.
Hess and Fish reported cases of scurvy in New York in infants
following the use of milk pasteurised at 145 ° F.

1 ioo" C. or 2120 Fahr. is
the boiling point of water.



CHEMISTRY  AND  PHYSICAL 
PROPERTIES   67

for
thirty minutes; the infants were cured by substituting unheated milk for the
pasteurised milk. In Berlin,
an outbreak of scurvy was traced to the instalment of a pasteurising plant by
one of the largest dairies; the pasteurisation was discontinued and the number
of cases of scurvy decreased as suddenly as it had increased. The antiscorbutic
value of milk is reduced but not entirely destroyed by pasteurisation; milk
commercially pasteur­ised is usually re-heated in the home and the C-vitamin is
completely destroyed by the second heating.

The sensitiveness of the antiscorbutic substance to heat varies
according to the medium in which it is con­tained. Orange-juice and
tomato-juice are much more acid than cabbage or milk, but the addition of
citric acid to the water in which cabbage is boiled does not lessen the
destruction of this vitamin. Hence acidity is not the protective agent against
destruction; it is possible that the varying sensitiveness to heat is in some
way connected with the amount and nature of the protein in solution.

C-f actor is rapidly destroyed by alkali, such as common washing soda,
sodium bicarbonate or the alkaline citrates. * The destruction is
particularly rapid if heat is applied at the same time; hence the practice of
boiling green vegetables with soda to improve their colour is greatly to be
condemned. The preparation of infants' foods by adding sodium or potassium
carbonate to the milk mixture has proved to be productive of infantile scurvy.
Faber has shown that the antiscorbutic value of milk is seriously reduced by
the addition of 0-25 per cent. of sodium citrate. A case of severe scurvy has
been recorded in a child of ten months fed from birth on raw milk to which
sodium citrate has been added in the pro­portion of one grain to each ounce of
the milk mixture.

C-vitamin disappears during the ageing of food-stuffs. Harden and Zilva
and also Hess have shown that lemon-juice and orange-juice kept in a cold store
for a fortnight diminish in antiscorbutic value.   Milk, especially pasteur-



68   VITAMINS AND THE  CHOICE OF FOOD

ised
milk, deteriorates very rapidly, but the loss of value in sour milk proceeds
more slowly. Although vegetables air-dried in the ordinary way are useless as
antiscorbutics, certain food-stuffs dried with special care retain their
potency, and ageing has less effect upon them in the dry condition. Lemon and
orange juice dried in vacuo by Harden and Robison were found to be still
active after careful storage for two years. Dry and stable forms of
orange-juice should therefore be available in the future in a form suitable for
use on long expeditions.

Orange-juice can be dried by the same commercial processes as are used
for drying milk, but these prepara­tions are not as active as those dried in
vacuo.
A dry preparation of specially neutralised lemon-juice, made by
Harden and Zilva, had a wonderful effect upon a bad case of infantile scurvy in
which the action of ordinary orange-juice was too slow; a quantity equivalent
to the juice of nine lemons was given in one day, and the child then made rapid
improvement.

Desiccation of cabbage in an atmosphere of carbon-dioxide for
thirty-five hours at 650 0. did not prevent the destruction of
C-factor. Cabbage has been suc­cessfully dried by a special process devised by
Hoist and Frohlich; the preparation retained much of its efficacy after storing
for twenty-six months at tropical temperatures in evacuated bottles. They hoped
that cabbage -preserved in this way might be a practicable form of
antiscorbutic for use on Norwegian sailing ships. The investigations of Shorten
and Ray show that mixed vegetables dried in a factory have no antiscorbutic
value, but sun-dried potatoes, tomatoes and cabbage retain some of their
potency. Spinach, turnips, turnip-tops and carrots sun-dried in the same way
lost all their antiscor­butic value. Milk dried by certain commercial processes
retains much of its antiscorbutic value.

The destruction of the C-vitamin during heating, ageing and drying may
be partly due to oxidation. Some of the C-factor in milk is destroyed by
bubbling air



CHEMISTRY  AND  PHYSICAL 
PROPERTIES   
69

through
it, but the destruction is greater if more active oxidising agents, such as
oxygen itself or hydrogen peroxide, are used. Hess has found by experience with
infants that milk loses much of its antiscorbutic value by excessive handling
during transit from the cow to the baby. Many of the processes used in dairies
involve the exposure of large surfaces of milk to air, and frequent passage
from one container to another is practically equivalent to the bubbling of air
through milk.

The antiscorbutic value of foods is not necessarily destroyed by
fermentation. Guinea-pigs are protected from scurvy by the same-sized dose of
fermented lemon-juice as of unfermented juice. Cider, and alcoholic liquors
prepared from freshly germinated grain, if used in large quantities, are
instrumental in curing and pre­venting scurvy. Stefansson, the Arctic explorer,
mentions the cure of scurvy in three days in men who found a dead musk-ox and
ate its very rotten flesh. Eskimos, he reports, often live for months on putrid
meats and fish without getting scurvy. In the old days sauerkraut (fermented
cabbage) had a great reputation as an anti­scorbutic ; in particular, the
relative freedom from scurvy of the Dutch sailors was attributed to the liberal
use of sauerkraut. Possibly it is now prepared by a different process, for
Ellis and his co-workers have recently tested sauerkraut as used in the States
and found it to have no antiscorbutic value. Silage prepared from corn (the
green leaves and stems) also was valueless, but as the silage during the
process of fermentation may rise to a temperature of 300 C. for a
month or more, heat rather than actual fermentation may be the destructive
agent.

The antiscorbutic substance has been shown by Zilva to be unaffected by
exposure to ultra-violet light.

A-Factor.

This vitamin, usually associated with fats, is not a fat. Fats are
saponified (converted into soaps) by treatment with alkali, but A-factor in
butter is not destroyed by



70   VITAMINS AND THE CHOICE  OF FOOD

boiling with 20 per cent, alcoholic
potash for half an hour (Steenbock, Sell and Buell).

The distribution of A-factor and of the yellow pigments in fats often
runs so parallel that it was suggested that the A-factor might be identical
with the yellow pigments called lipochromes in animals and carotinoids in
plants. Both A-factor and yellow pigment in animal fat are derived ultimately
from plant tissues and not synthesised in the animal body. Tomatoes, which are
rich in pigment, were also shown by Osborne and Mendel to be rich in A-factor.
Drummond tried feeding experiments with carotene; neither impure nor pure
crystalline preparations improved the condition of animals suffering from a
deficiency of vitamin. It has also been shown that cod-liver oil, dog fat, the
kidney fat of pigs and other fats are poor in pigment and yet
disproportionately rich in A-vitamin. Miss Stephenson has shown that butter fat
can be decolorised without impairing its vitamin value. Much other evidence has
also been brought forward to disprove the identity of A-factor and carotene;
the substances are not even quantitatively associated in the very plant tissues
in which they are most probably synthesised.

The recorded observations on the effect of heat upon A-factor were at
first very discordant, but now it is gener­ally agreed that the rate of
destruction varies with the exposure to oxidation during the heating process. Hopkins found that
A-factor in butter is resistant to heat alone up to a temperature of about 1200
C, but is rapidly destroyed if air is bubbled through the melted butter during
heating. The substance is therefore decomposed by atmospheric oxygen. The
A-vitamin in milk, green vegetables, grain and carrots, etc., is not destroyed
by heating in an autoclave for three hours at 15 lbs. pressure (about no° C).

A-factor is not easily destroyed by ageing; it suffers little loss if
protected from oxidation. It is not destroyed in the process of making silage
from green fodder.



CHEMISTRY
AND  PHYSICAL  PROPERTIES   
71

The A-vitamin is completely destroyed by the process of hardening oil by
the action of hydrogen, as in the manufacture of margarine from oils.

Zilva has shown that butter is inactivated by exposure in thin layers to
the action of ultra-violet light for five to eight hours, probably by the ozone
produced by the action of the mercury-quartz lamp upon atmospheric oxygen. The
potency of cod-liver oil is destroyed by the action of ozone more rapidly than
by the action of oxygen.

This vitamin is soluble in alcohol, ether or benzene. Osborne and Mendel
made a very active ether extract of dried green leaves; 30 milligrams (o-ooi
oz.) of extract per day supplied sufficient A-vitamin for rats.

Drummond has shown that the amount of A-factor, in seeds is not
increased by germination. A-factor is syn-thesised in the green parts of the
plant, and the white leaves of cabbage contain less A-factor than the green leaves.
Lower plants (marine algae) containing chloro­phyll synthesise this vitamin.
The marine algae are eaten by small marine animals which in turn are eaten by
bigger ones, eventually by codfish. The A-factor in the codfish is chiefly
concentrated in its liver.

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