The Vitamine Manual by Walter H. Eddy
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Walter H. Eddy >> The Vitamine Manual
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_c_. The process of hydrogenation used in hardening fats appears to
completely destroy the vitamine, hence the many lard substitutes now in
use must in general be considered "A" vitamine-free regardless of the
content of "A" in the fats from which they are derived unless they have
been made by blending instead of hydrogenation.
_d_. Acids and alkalies have apparently little effect on this
particular vitamine.
It may be well to state here however that owing to variability in behavior
with variation in conditions it is dangerous to draw too general
conclusions and until a given source has actually been investigated under
specific cooking conditions one should not rely too strongly on analogies
based on comparative experiments. This statement applies to all vitamines
and presents one of the live subjects of investigation for the cooking
schools and the food factories.
_e_. Little has been learned further about the chemistry of this
substance. [Footnote: Since the above was put in type Steenbock has shown
that the A vitamine resists saponification and that by saponifying fats
which contain the A it may be possible to secure a fraction rich in the
vitamine and free of fat.] Butter fat, nitrogen free and phosphorus free
is shown to carry the vitamine and it is therefore assumed that the
vitamine lacks these elements. It has been claimed that it may be removed
from butter fat by prolonged extraction with water but this has not been
confirmed by more recent experimenters. Steenbock was the first to call
attention to the association of the A vitamine with yellow pigment in
plant and animal sources. Butter, egg yolk, carrots, yellow corn contain
it while white corn and white roots are less rich in this vitamine. This
observation suggested the chemical relation between the vitamine and
carotin. It has however been shown by Palmer and others that carotin is
not vitamine A. This association of the pigment with the vitamine is
therefore apparently a coincidence and this clue has failed as yet to
throw light on the chemical nature of vitamine A.
II. THE CHEMICAL PROPERTIES OF VITAMINE "B"
When Funk first studied this substance he conducted all his evaporations
in vacuo from fear that higher temperatures would prove destructive.
Subsequent investigation however has shown that 100 has very little if
any destructive effect if the vitamine is held in acid or neutral
solution. Temperatures between 100 and 120 maintained in an autoclave at
15 pounds above normal pressure do tend to slowly destroy the factor. The
extent of this destruction also varies with the character of the crude
extract. In general, then, there is little fear of injuring this vitamine
in ordinary cooking temperatures if the use of alkali is avoided.
The effect of alkali depends upon the temperature to a very marked degree.
Osborne has recently reinvestigated this matter and finds that in the
presence of a 0.1N solution of alkali at 20 C. there is very little
destruction but that raising the temperature to 90 C. brings about a
marked destruction. Seidell has shown that if the vitamine is absorbed by
Lloyd's reagent and this reagent be then extracted with dilute alkali the
vitamine passes into the alkaline solution. If the latter is neutralized
quickly it is possible to recover most of the vitamine by this method. The
effect of alkali becomes of practical importance to the housewife because
of certain cooking habits. I refer to the well known practice of adding
soda to the water in which vegetables are cooked to soften the vegetable
and accelerate the cooking. Daniels and Loughlin in this country
investigated this matter and came to the conclusion that this procedure
did not produce enough destruction to be dangerous. Later the matter was
studied by Chick and Hume in England and these investigators brought out a
feature that had perhaps been overlooked in the previous work. Their point
was that in ordinary feeding tests the results merely tell whether there
is enough vitamine present to produce normal growth. Hence if the
substance tested has much vitamine, a large part of it might be destroyed
and this fact not appear in the test because enough might still be left to
induce normal growth. By reducing the amount tested so that it was just
adequate for normal growth and then applying the soda-cooking
experimentation they showed that this method of cookery does do serious
harm to the vitamine. From the practical point of view it is of course
sufficient to show that enough is left after a cooking process to suffice
for normal growth when the substance is taken in the portion sizes
ordinarily eaten. The effect of alkali deserves more attention on the part
of cooks and food preparateurs and we need more data concerning the
minimal dose necessary to protect the human animal.
In neutral and acid solution it is perfectly safe to assume little
destruction of this vitamin through heat and it is now common practice to
boil sources with the extracting reagent and to use the steam bath freely
to concentrate and evaporate these extracts. We have recently investigated
the effect upon cabbage of cooking in a pressure cooker at eight pounds
pressure. The cabbage so cooked, when dried and mixed so as to form 10 per
cent of a basal vitamine free diet, yielded all the "B" vitamine necessary
to produce normal growth in rats.
The very name of this vitamine indicates its ready solubility in water. It
is also soluble in 95 per cent alcohol and either of these extractants may
be used to obtain the vitamine. It is not readily soluble in absolute
alcohol and 95 per cent is not as good an extractant as water. Substances
rich in the vitamine apparently yield the latter more readily if they have
first been subjected to autolysis or if the extracting fluid is acidified.
Funk was the first to show that yeast produced a greater yield if it was
allowed to autolyse before extraction with alcohol. However, Osborne and
Wakeman have produced a method of treating fresh yeast by boiling it with
slightly acidified water which seem as efficient as autolysis in the yield
produced.
The various methods of extraction now in vogue have already been discussed
in Chapter II and need not be repeated here. In general it is apparent
that to obtain concentrates of high potency it is permissible to employ
temperatures of 100 C. if we will maintain an acid or neutral reaction but
that alkali should be avoided wherever possible and when its use is
imperative the temperature must be kept below 20 C. or destruction will
result. In applying this rule to cooking operations the results should be
determined by direct tests rather than by assumptions based on these
generalizations. It should also be noted that the alkalinity of a solution
should be determined on the basis of hydrogen ion concentration and not on
amount of alkali added since many substances have a marked buffer
reaction.
The water-soluble "B" is not only soluble in water but can be dissolved in
other reagents. Thus McCollum has shown that while benzene is of little
value as an extractant of this vitamine, if we will first extract the
vitamine with alcohol or water and deposit this on dextrin by evaporation
it is then possible by shaking the activated dextrin with benzene to cause
the vitamine to pass into solution in benzene. Voegtlin and Meyers have
recently shown that it is soluble in olive oil and in oleic acid and their
data suggest a new means of concentrating the substance which may be of
value in tracing its character.
The "B" vitamine is relatively easily absorbed by finely divided
precipitates. We have already referred to the use of fuller's earth for
this purpose by Seidell. This adsorptive power sometimes manifests itself
in the treatment of plant extracts. A watery extract of alfalfa can be
made to throw down its protein complex by diluting it to 40 per cent with
alcohol. Osborne reports however that this process frequently removes the
vitamine also which appears to be thrown down with the precipitated
material. This adsorptive power therefore often appears as a difficulty in
the handling of the substance as well as a means of extraction. We have
used Osborne's method with alfalfa extracts and find the above result is
not by any means invariable, for in some of our extracts we retained the
greater part of the vitamine. Kaolin and ordinary charcoal are not very
good adsorbents but the latter can be activated to serve this purpose.
The elementary nature of the "B" vitamine remains a mystery. Extracts
which contain it show the presence of nitrogen. Funk's earlier researches
on yeast and rice polishings both yielded crystalline complexes which he
analysed. His data on this subject follow:
_A. The yeast complex_
Crystals melting at 233 C. consisting of:
I. A complex melting at 229 C. and forming needles and prisms nearly
insoluble in water and with the apparent formula of C_24H_19O_2N_5.
II. A complex melting at 222 C. and soluble in water. Formula
C_29H_23O_2N_5.
III. Nicotinic acid melting at 235 C. C_6H_5O_2N.
_B. The rice complex_
Crystals melting at 233 C. consisting of:
I. A complex melting at 233 C. and with a formula of C_26H_20O_9N_4.
II. Nicotinic acid melting at 235 C. C_6H_5O_2N.
Funk held at the time that the possible nature of the compound was:
HN
| \
OC C_16H_18O_6
| /
HN
It was this idea that led him to call it an "amine."
We are unable at present to report any nearer approach to the elementary
analysis and all attempts at purification have shown a tendency to make
the active substance either disappear entirely or else distribute itself
over the several fractions instead of concentrating itself in one. Its
basic nature seems to be well established by its behavior with
phosphotungstic acid and its ready adsorption by carbons activated to take
up basic substances.
III. THE CHEMICAL PROPERTIES OF WATER-SOLUBLE "C"
The properties of this newest member of the family are still less defined.
All are agreed that it is much more sensitive to heat and alkali than the
other two. Temperatures above 50 C. are usually destructive though the
time factor is extremely important as well as the reaction. Hess for
example has found that the temperature used to pasteurize milk continued
for some time, is more destructive to the vitamine than boiling water
temperature continued for only a few minutes. The extent to which orange
juice and tomato juice will resist high temperatures indicates the
protective action of acids to be considerable.
Dr. Delf's experiments at the Lister Institute were especially directed to
the behavior of this vitamine in cabbage. She first determined the minimum
close of raw cabbage required to prevent scurvy in guinea pigs and found
that it was less than 1.5 grams and more than 0.5 gram daily. When the
cabbage was heated in water at 60 C. for an hour, symptoms of severe
scurvy were just prevented by 5 grams of the cooked cabbage fed daily. By
heating at 70 , 80 , 90 and 100 for the same length of time the 5 grams
of cooked material could be made non-effective as a preventive. Her
conclusions are that when cabbage is cooked for one hour at temperatures
ranging from 80 to 100 C. the cabbage leaves lose about 90 per cent of
the antiscorbutic power originally held by the raw equivalent. Sixty
minutes at 60 or twenty minutes at 90 to 100 resulted in about 80 per
cent destruction. Dr. Delf calls attention also to the fact that the
effect of the heat is increased to only a slight degree by rise in
temperature. Assuming that the effect of the rise is orderly, a
temperature coefficient of 1.3 is indicated for each rise of 10 C. This
low result suggests to Delf a contradiction to any theory which imputes to
the vitamine enzyme or protein-like qualities and on the other hand
suggests that the substance is much simpler in constitution. Her results
also confirm Hoist and Frohlich as showing its great sensitiveness at
temperatures of 100 and below and obviously have a direct bearing upon
cookery methods.
The substance is soluble in water and passes through a parchment membrane
or a porcelain filter. Unlike the "B" it is apparently not adsorbed by
fine precipitates such as fullers' earth or colloidal iron. Harden and
Zilva showed that when a mixture of equal volumes of autolysed yeast and
orange juice is treated with fuller's earth the "B" is removed and the "C"
left unaltered. Eddy and La Mer have treated orange juice with fullers'
earth and then tested the filtered off juice as cure and preventive of
scurvy in guinea pigs. Their results showed that 6-2/3 cc. of the treated
juice was curative, hence the loss due to adsorption must be less than 60
per cent to 70 per cent. Harden and Zilva were among the first to state
that the vitamine is much more stable in acid than in alkali. They have
shown, that even 1/50 N sodium hydrate at room temperature has a rapidly
destructive effect. On the other hand Delf showed that when 0.5 gm. citric
acid is added to the water in which germinated lentils are boiled, the
loss of the antiscorbutic properties is, if anything, greater than when no
addition of acid is made. She therefore concluded that in cooking
vegetables there should be no addition of either acid or alkali to the
cooking water if one wishes to conserve this vitamine. Sherman, La Mer, and
Campbell have been engaged in experiments bearing on this point throughout
the past two years. Some of their results have recently been published and
their observations are worthy of special attention from their bearing on
the character of reaction of the vitamine in general. They first proceeded
to determine the amount of filtered tomato juice just necessary to produce
scurvy in degrees extending from no protection to complete protection and
they also constructed a basal diet which is apparently optimum in
nutrients and all other factors except the "C" vitamine. They found that
at the natural acidity of tomato juice (pH 4.2) boiling for one hour
destroyed practically 50 per cent of the antiscorbutic power and by
boiling for four hours they destroyed 70 per cent, which indicates that
the curve of the destructive process tends to flatten more than that of a
unimolecular reaction. This result was confirmed by heating experiments
conducted at 60 , 80 and 100 . In all cases the temperature coefficients
are low. (Q_10 equals 1.1-1.3) confirming Delf's results. When the natural
acidity of the juice was first neutralized in whole or in part, the juice
then boiled for an hour and immediately cooled and reacidified, it was
found that at less than half neutralization (pH 5.1-4.9) the destructive
effect of an hour's boiling was increased to 58 per cent. When alkali was
added to an initial pH 11 (about N/40 titratable alkali to
phenolphthalein) which fell to 9 during the hour's boiling the destructive
effect was about 65 per cent. When reacidification was omitted and the
neutralized boiled juice stored in a refrigerator for five days before
using the destruction increased 90 to 95 per cent. These particular
observations seem to confirm the view of Harden and Zilva that the
vitamine is especially sensitive to alkali. Hess has recently reported
that oxygen is destructive to this vitamine.
IV. PHYSIOLOGICAL PROPERTIES OF THE "A" VITAMINE
Most authorities are now agreed that both the "A" and "B" types are
essential to growth. Rohmann still holds out against the vitamine
hypothesis. McCollum has recently pointed out that while rats do not have
scurvy it does not at all follow that the absence of the "C" in their diet
is immaterial, but that the contrary is true. Failure to grow, then, may
manifest itself as a result of the absence of either of the first two
types and possibly is affected by the absence of the "C." We have already
seen how this failure may be utilized to measure the vitamine content of a
source. The absence of the "A" type however may also manifest itself in
another way, viz., by the development of an eye disease which McCollum
first designated as xerophthalmia or dry eye and which the British
authorities prefer to designate as keratomalacia. The failure of this
result to always follow the absence of the "A" type in the diet has led
some to question the specificity of this disease. While the infection of
the eye is due to other agents the sum of the evidence supports McCollum
and points to the absence of "A" as the true predisposing cause of the
disease. Bulley, basing her claims on a study of some 500 rats fed on a
synthetic diet, claims that the eye condition is not primarily due to a
dietary deficiency but to an infection resulting from poor hygienic
conditions. In reply to her contentions Emmett has reviewed his own data
and presents them in the following summation:
_________________________________________________________________________
| | | |
RAT | KIND OF VITAMINE | NUMBER CASES | POSITIVE CASES | PER CENT
GROUPS | ABSENT IN THE RATION | REPORTED | OF XEROPH- | POSITIVE
| | | THALMIA |
_______|______________________|______________|________________|__________
| | | |
A | Fat-soluble "A" | 122 | 120 | 98
B | Water-soluble "B" | 103 | 0 | 0
C | None | 216 | 0 | 0
_______|______________________|______________|________________|__________
In these groups special hygienic measures were taken against infection.
Furthermore repeated attempts were made to transmit the eye disease by
using sterile threads, passing them carefully over the edges of the sore
lids and then carefully inoculating the eyes of other rats. These attempts
resulted negatively in all cases where the inoculated rats had plenty of
the "A" vitamine. Treatment of advanced cases of sore eyes with a
saturated solution of boric acid and also with a silver protein solution
failed to relieve the condition while as little as 2 per cent of an
extract containing the "A" vitamine when added to the ration, speedily
resulted in cure and increase of weight. These results combined with
similar data compiled by Osborne and Mendel seem to refute Bulley's
contentions and to justify our acceptance of xeropthalmia as a specific
vitamine deficiency disease.
_Osborne and Mendel data_
Total No. No. with eye
symptoms
Rats on diets deficient in A vitamine . . . . . . . . 136 69
" on diets " " B " . . . . . . . . 225 0
" on diets otherwise deficient . . . . . . . . . 90 0
" on " experimental but probably adequate . 201 0
" on mixed food . . . . . . . . . . . . . . . . 348 0
____ __
Totals . . . . . . . . . . . . . . . . . . . . . 1000 69
On the other hand all workers know that rats often do develop and grow
well for a considerable period of time on a diet free from the "A" and
without manifesting the eye disease. The British authorities explain this
by assuming that animals have the power to lay down a reserve of this
vitamine on which they can draw in emergency. Sherman and his coworkers
confirm this power to store the vitamine. Others have been led to explain
their results as due to contamination of the basal diet. Daniels and
Loughlin recently maintained that the commercial lard used in basal diets
and assumed to be "A" vitamine-free was supplied with sufficient of the
"A" to produce growth and prevent eye disease. Their views have failed of
confirmation by Osborne and Mendel. It is evident therefore that these
occasional lapses from specific response to absence of the "A" vitamine
need further elucidation. It is equally manifest that in the majority of
cases the absence of the "A" will result in both stunted growth and
xeropthalmia. The appearance of the eye disease may be taken however, as a
sure indication of the absence or deficiency in the "A" vitamine.
V. PHYSIOLOGICAL PROPERTIES OF THE "B" VITAMINE
Beri-beri is a disease that is described clinically as a form of severe
peripheral neuritis and may appear in two well marked forms. In one type
there is great wasting, anesthesia of the skin and finally paralysis of
the limbs. In the other, the most marked symptom is excessive edema which
may affect trunk, limbs and extremities. In severe cases the heart is
usually involved and death may occur suddenly from heart failure.
Most observers assume that the antineuritic vitamine discovered by Funk
and the water-soluble "B" are identical. This view is based on the fact
that when sources which yield the water-soluble "B" in rat feeding are
tested for antineuritic power these sources are apparently parallel in
antineuritic power and growth production. Furthermore rats deprived of the
water-soluble "B" develop polyneuroses identical in symptoms with those
shown by rats and pigeons when the latter are placed on a polished rice
diet. The British Medical Board has compiled the following table to
support this view:
_Table compiled from pages 35 and 86, British Medical Research Committee
Report_
_______________________________________________________________________
| |
| | VALUE AS A SOURCE OF
| VALUE AS A SOURCE OF | THE ANTINEURETIC
| WATER-SOLUBLE "B" | FACTOR OR ANTI-BERI-
FOODSTUFF | (SHOWN BY EXPERI- | BERI FACTOR (SHOWN
| MENTS WITH RATS) | BY EXPERIMENTS
| | WITH BIRDS)
_________________________|______________________|_______________________
| |
Rice germ . . . . . . . | +++ | ++++
Wheat germ . . . . . . . | +++ | +++
Yeast . . . . . . . . . | +++ | +++
Egg yolk . . . . . . . . | ++ | +++
Ox liver . . . . . . . . | ++ | +++
Wheat bran . . . . . . . | + | ++
Meat muscle . . . . . . | + | +
Milk . . . . . . . . . . | +++ | Slight
Potatoes . . . . . . . . | + | +
Meat extract . . . . . . | 0 | 0
White bread or flour . . | 0 | 0
Polished rice . . . . . | 0 | 0
_________________________|______________________|_______________________
_________________________________________________________________________
| |
BEHAVIOR | WATER-SOLUBLE "B" | ANTINEURITIC VITAMINE
______________________|________________________|_________________________
| |
Solubility in water . | Very soluble | Very soluble
Solubility in alcohol,| |
dilute . . . . . . | Very soluble | Very soluble
Solubility in absolute| |
alcohol . . . . . . | Insoluble | Insoluble
Solubility in ether, | |
chloroform and | |
benzene . . . . . . | Insoluble | Unusually insoluble
| | but can be extracted
| | with ether from
| | fatty materials such
| | as egg yolk
Stability to heat . . | Stable at 100 C, | Destroyed very slowly
| destroyed rapidly at | at temperatures below
| 120 (in neutral or | 100 C., more rapid at
| acid solution) | temperatures
| | between 110 and 120 C.
Stability to drying . | Stable | Stable
Stability to acids | |
(hot dilute) . . . | Moderately stable | Stable
Stability to acids | |
(cold dilute) . . . | Stable | Stable
Stability to alkalies | |
(hot dilute) . . . | Rapidly destroyed | ?
Stability to alkalies | |
(cold dilute) . . . | Stable |
In dialysis . . . . . | Passes through | Passes through
| parchment membrane | parchment membrane
In adsorption . . . . | Adsorbed from acid | Adsorbed from neutral
| or neutral solution | solutions by fuller's
| by fuller's earth, | earth, colloidal
| charcoal, etc. | ferric hydroxide,
| | animal charcoal, etc.
______________________|________________________|_________________________
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