The Vitamine Manual Part 5

II. Nicotinic acid melting at 235C. 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 a.n.a.lysis 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 50C. 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 Inst.i.tute 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 60C. 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 100C. the cabbage leaves lose about 90 per cent of the antis...o...b..tic 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. a.s.suming that the effect of the rise is orderly, a temperature coefficient of 1.3 is indicated for each rise of 10C. 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 const.i.tution. 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 pa.s.ses 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 antis...o...b..tic 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 antis...o...b..tic 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 t.i.tratable 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, pa.s.sing 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 a.s.suming 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 a.s.sumed 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 a.s.sume 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 100C, Destroyed very slowly destroyed rapidly at at temperatures below 120 (in neutral or 100C., more rapid at acid solution) temperatures between 110 and 120C.

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 . . . . . Pa.s.ses through Pa.s.ses 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.

______________________ ________________________ _________________________

Emmett has recently opposed this view and suggests that while the antineuritic factor and the growth factor are found in the same sources and have much in common it does not follow that they are identical and that his experiments tend to show that there are marked differences which suggest that the "B" type is not a single ent.i.ty but a group. Mitch.e.l.l has summarized very well the controversial phases of this question with an impartial review of the facts. One of strongest of the opposition arguments lies in the failure of milk to cure beri-beri except when administered in large quant.i.ties. This objection has been partly allayed by data bearing on the relation of the milk content to the food of the cow. Hess, Dutcher, Hart and Steenbock and others have adduced sufficient evidence to show that the vitamine content of the milk of a cow is largely determined by the cow's food and as a consequence the milk may be very poor in vitamine. It is obvious then that the failure of the milk to cure beri-beri in a given case might be due to this cause and not to lack of ident.i.ty of the curative with the growth factor. Osborne and Mendel have also shown that milk in general must not be cla.s.sed among the rich sources of the vitamine, even when the cow's food is rich in vitamine. The princ.i.p.al facts in the controversy have been presented and at present the evidence for regarding the vitamines identical seems to be preponderant.

Recently Auguste Lumiere in Paris has put forth the view that polyneuritis is not merely a vitamine deficiency disease but a nutriment deficiency disease. He reports that he fed birds on a starvation diet, but with plenty of vitamine "B". These birds developed polyneuritis and were cured by adding to the diet plenty of polished rice. The view he wishes us to take is that all factors must be present and that the absence of the nutriment is as important as the absence of the vitamine.

In the field of nutrition the absence of the "B" type is particularly marked by the behavior of the deprived animal. Rats transferred from a vitamine-free diet to one containing the "B" only, make a much more rapid recovery toward normal (even in the absence of the "A") than do animals transferred from the vitamine-free diet to one containing the "A" and not the "B". This initial jump from addition of the "B" will not continue long in the absence of the "A", as a general rule. Hess believes that in some of his infants he was able to show markedly successful growth on the diet deficient in the "A" but rich in the "B". It is not certain however that his diets were sufficiently devoid of the "A" factor to be declared "A"

vitamine-free and we know little of the amount of the "A" necessary to normal infant growth. All results however show that both "A" and "B" are necessary to growth production and though the term growth vitamine was applied to the "A" originally the distinction is one that should be rejected, for both "A" and "B" and possibly "C" are all ent.i.tled to this name.

The manner in which the "B" vitamine acts is still obscure. Voegtlin some time ago tried to demonstrate that it was identical with secretin and stimulated pancreatic flow. Recent work at the Johns Hopkins University by Cowgill and by Aurep and Drummond in England has failed to confirm this.

One of its most marked immediate effects is increase in appet.i.te. Karr in Mendel's laboratory has shown that dogs which refused their basal diet would resume eating it if they were allowed to ingest separately a little dried yeast. Karr studied the metabolism of these dogs as regards nitrogen part.i.tion but the results give little data that is explicatory of the behavior of the vitamine. In 1915 the author was able to bring about marked immediate improvement and the ultimate recovery of a number of infants who were of the marasmic type by merely increasing the "B"

vitamine content of their food. In these cases the vitamine was carried by Lloyd's reagent and administered mixed with cereal, or the crude extract was combined with the milk. The pancreas of the sheep was the source used.

In these cases the growth curve changed abruptly from a decline to a sharp rise and this increase in weight continued and was accompanied by all the other signs of improved nutrition including increase in appet.i.te. The change in the growth curve from decline to rise was accomplished without increasing or changing the basal diet but as the appet.i.te increased the food had naturally to be increased to keep pace. In these cases the effect of the vitamine was to enable the child to utilize its normal food and to increase its appet.i.te for it. This action certainly suggests stimulation of digestive glands. It also showed that even though the diet may contain the vitamine as was the case in the milk fed to these children the addition of the vitamine in concentrated form often gives an upward push that the food mixture fails to accomplish. Daniels and Byfield have recently confirmed the effect of increased "B" in infant growth. Cramer has suggested in a paper published recently in _The American Journal of Physiology_ that the fatty tissue about the suprarenals may be a depository of vitamine and that in the absence of vitamine this tissue loses its supply and that this is the explanation of lessened activity of that gland in certain metabolic disturbances. This idea tends to support the idea that vitamines are gland stimulants or hormones and the word food hormone has been suggested to describe them on that account. A few years ago Calkins and Eddy tried to determine the effect of the vitamine on the single cell by use of the paramecium but the results of the experiments failed to show a vitamine requirement on the part of these animals.

McDougall has recently suggested that the vitamines produce their effect on yeast cells by increasing hydration. Unfortunately nearly all stimuli which produce growth are accompanied by hydration effects and it is difficult to feel that this is a specific vitamine effect although without denying the possibility. Dutcher has tried to show that vitamines have a relation to oxidation effects. He observed that the issues of polyneuritic birds showed a marked reduction in catalase and that this catalase was restorable by curing the birds with vitamine. The main difficulty lies in the conflexity of factors that function between cause and effect.

[Ill.u.s.tration: FIG. 8. THE EFFECT OF VITAMINE B ON A MARASMIC INFANT

_1_. On the twentieth day the patient developed a cough. _2_. On the twenty-first day the cereal was reduced from three times a day to twice a day. The patient cried during the night. _3_. On the twenty- second day the stools showed free starch. _4_. On the twenty-third day an a.n.a.l abscess was opened. The stools continued to show free starch until the twenty-fifth day. _5_. On the twenty-fifth day the stools showed soluble starch but no free starch. _6_. On the twenty-seventh day the appet.i.te was good and there was no starch. _7_. From the twenty-eighth to the forty-third day no starch was observed in the stools.

_8_. On the thirty-first day the patient developed a cough. _9_.

From the forty-ninth day to the time of discharge three tablespoonsful of orange juice were given daily. _10_. On the seventy-third day the patient developed a bronchitis and mustard paste was applied every four hours up to the eighty-fourth day.

_V1_ = From the twenty-first day to the forty-third day the patient received each day 2 grams of Lloyd powder, activated with pancreatic vitamin. The powder was administered by mixing 1 gram. with each cereal feeding. The result was 20 ounces gain in twenty-two days, a normal growth.

_V2_ = After a period of ten days without vitamin, during which the patient settled down to a level growth curve, the treatment described under V1 was resumed. This was continued from the fifty-third to the seventy-sixth day. The result was the resumption of growth but at a slower rate; 8 ounces were gained in twenty-three days. During the latter part of the period the patient developed a bronchitis. At the end of this period the patient was placed on a whole milk formula. From that time to the time of discharge the patient grew normally.--From the _American Journal of Diseases of Children,_ 1917, xiv, 189.]

[Ill.u.s.tration: Effects of Vitamines on Growth FIG. 9]

These views are at best speculations. The literature is singularly lacking in detailed metabolic a.n.a.lyses of excreta of animals during vitamine stimulation and we know nothing of the possibilities of overdosage, for in all the work done it has been generally a.s.sumed that the presence of an amount greater than that necessary to produce normal growth is not material.

The exact manner of the vitamine's action then remains to be determined and it is obvious that this solution will come much more rapidly if we can first identify the substance chemically.

VI. THE PHYSIOLOGICAL PROPERTIES OF THE "C" VITAMINE

The steps that led to the acceptance of scurvy as a vitamine deficiency disease have already been discussed and show how the vitamine acts in such a disease. Practically all the work done with this vitamine to date has been concerned either with dosage or with reaction to heat, drying, etc.

The only paper that we have seen that suggests another function than antis...o...b..tic power for this vitamine is the one by McCollum and Parsons in which they suggest that even in animals where scurvy does not exist, the presence of this factor may be necessary to normal metabolism. The following table gives some of the data compiled by the British workers as to the antis...o...b..tic power of various sources:

_Table compiled from, page 44, British Medical Research Committee Report_

________________________________________________________________________ MINIMUM DAILY FOODSTUFF VALUE AGAINST RATION NECESSARY SCURVY TO PREVENT SCURVY IN GUINEA PIGS _______________________________ _______________ ________________________ _Cereals:_ Whole grains . . . . . . . . 0 Germ . . . . . . . . . . . . 0 Bran . . . . . . . . . . . . 0 Endosperm . . . . . . . . . 0 _Pulses:_ Whole dry . . . . . . . . . 0 Germinated (lentils) . . . . ++ 5.0 grams _Vegetables:_ Cabbage (raw). . . . . . . . ++++ 1.0 gram Cabbage (cooked one-half hour at 100C) . . . . . . ++ 5.0 grams Runner beans (green pods). . +++ 5.0 grams Carrot (juice) . . . . . . . + 20.0 cc.

Beet root (juice). . . . . . + More than 20 cc.

Swede (juice) . . . . . . . +++ 2.5 cc.

Potatoes (cooked one-half hour at 100C . . . . . . + 20.0 grams Onions . . . . . . . . . . . + Desiccated vegetables . . . 0 to + 60.0 grams expressed as equivalent in fresh cabbage _Fruits:_ Lemon juice (fresh) . . . . ++++ 1.5 cc.

Lemon juice (preserved) . . ++ 5.0 cc.

Orange juice (fresh) . . . . ++++ 1.5 cc.

Lime juice (fresh) . . . . . ++ 10.0 cc.

Lime juice (preserved) . . . 0 to + Grapes . . . . . . . . . . . Less than + More than 20.0 grams Apples . . . . . . . . . . . Less than + Apples dried . . . . . . . . Less than + Tamarind dried . . . . . . . Less than + Mango . . . . . . . . . . . Less than + Kok.u.m . . . . . . . . . . . Less than + _Meat:_ Raw, juice . . . . . . . . . Less than + More than 20 cc.

Tinned . . . . . . . . . . . 0 _______________________________ _______________ _______________________

A glance at this table shows the richest sources (see also table on page 59.) To these must be added canned tomato juice which Hess has shown practically equal to orange juice in efficiency and uses with infants in the same quant.i.ty. This discovery is of great value in instances where the cost of orange juice is often prohibitive.