Funk method of yeast test with Eddy and Stevenson modification
1. To a basal diet of 9 cc. of sterile culture medium such as a von Nageli solution [Footnote: von Nageli's solution consists of the following ingredients NH4NO3, 1 gram; Ca3(PO4)2, 0.005 gram; MgSO4, 0.25 gram dextrose 10.0 grams made up to 100 cc. with distilled water. Other culture media may be used and such combinations will be found in any text on yeasts. They all permit a certain amount of growth but all are apparently stimulated by the addition of vitamine extracts.] in a sterile test tube is added 1 cc. of the sterile, neutral, watery extract of the source of the vitamine. A pure culture of Fleischman's yeast (Funk prefers brewer's yeast) is maintained on an agar slant and twenty-four hours before the test is to be made, a transplant is made to a fresh agar slant. One standardized platinum loopful of the twenty-four hour yeast growth is then used to inoculate the contents of the tube, the tube stoppered with cotton and incubated for from twenty-four to seventy-two hours at a temperature of 31C. The seventy-two hour incubation period yields nearly optimum growth for this purpose.
2. At the end of this time the yeasts are killed by plunging the tube in water heated to 80C. and maintained at this temperature for fifteen minutes. The contents of the tubes are then poured into a Hopkins centrifuge tube which has a capillary tip graduated in hundredths of a cubic centimeter. After twenty minutes centrifugating at a speed of about 2400 revolutions per minute the yeasts in the solution have all been packed into the tip and the volume can then be read accurately to thousandths of a cubic centimeter (with the aid of a scale and magnifier). With a control tube containing 9 cc. of the sterile media and 1 cc. of distilled water in place of the 1 cc. of extract a comparison can be obtained which is an accurate measure of the stimulatory effect of the extract. If this stimulus is due purely to vitamine it is obvious that this procedure would enable us to compare extracts of known weights of and arrive at comparisons which would be measures of their vitamine content. In other words the procedure is now in a satisfactory form for testing and its value depends merely upon our ability to show that the stimulus given the yeast is due solely to vitamine "B."
The interest of the vitamine student in this test will be easily understood for it is so simple of manipulation and so rapid in producing results that it is the nearest approach to a chemical test of satisfactory nature yet proposed but unfortunately evidence soon began to accumulate to show that the stimulation produced by extracts of various sources is not a matter of pure vitamine. If we plot a curve of stimulation for various dilutions of a given extract we find that the stimulation is not directly proportional to the concentration of vitamine present but is a composite of several factors. The chart derived from experiments by Eddy and Stevenson shows the general nature of this curve. Other experimenters have reached similar results and some have gone so far as to maintain that the stimulation is not due to vitamine "B" at all. It is therefore evident that until this controversy is settled the yeast test cannot be used for the purpose proposed. Our own experiments at present make us still firm in our belief that one of the factors and perhaps the most important factor in the stimulation effect is the vitamine but until we can devise a basal medium that is comparable to that used in rat feeding experiments, i.e., one that contains all the elements for optimum growth of yeasts except vitamine "B" it will be unsafe to draw conclusions from the test as to vitamine content. It may be possible to so treat our extracts as to eliminate from them all other stimuli except the vitamine or to destroy the vitamine in them and thus permit the comparison of an extract with the vitamine destroyed against one in which it is present and thus arrive at the result desired. At any rate all we can say at present is that the yeast test is unreliable as a measure of vitamine content but that if it can be made quantitative its advantages are so great that it is very much worth while to continue work upon it until it is certain that it cannot be made to produce the desired result.
Another reason for our attention to this test is that if it can be made to show vitamine effect it provides an excellent medium for investigation of vitamine "B" reactions, and a method for studying the effect of the vitamine upon the protoplasm of a single cell.
THE SOURCES OF THE VITAMINE
Having now considered the general principles involved in vitamine testing we may justly ask what information they have yielded us in regard to the distribution of the vitamines in nature. If we must include vitamines in our diets it is important to know how to select foods on this basis, hence a classification of them on the ground of vitamine distribution becomes essential. The newness of the subject and the limited tests that have been made as well as the uncertainty residing in the test results make any classifications presented more or less approximations but we present such attempts as have been made, with the understanding that these tabulations are merely guides and not quantitative measurements in the sense that tables giving calorie values of protein, fat and carbohydrate content are. The following table (1) has been freely copied from a report of the British Medical Research Committee to which acknowledgment is hereby given.
_Pages 50 and 61 of the British Medical Research Committee's report_ _____________ CLASSES OF FOODSTUFFS VITAMINE "A" VITAMINE "B" VITAMINE "C" _______ __ __ __ _Fats and oils:_ Butter . . . . . . . . . . . . + 0 Cream . . . . . . . . . . . . . + 0 Cod-liver oil . . . . . . . . . + 0 Mutton and beef fat or suet . . + Lard . . . . . . . . . . . . . 0 Olive oil . . . . . . . . . . . 0 Cotton seed oil . . . . . . . . 0 Cocoanut oil . . . . . . . . . 0 Cocoa-butter . . . . . . . . . 0 Linseed oil . . . . . . . . . . 0 Fish oil, whale oil, herring oil, etc. . . . . . . . . . . + Hardened fats (hydrogenated) of animal or vegetable origin 0 Margarine from animal fat . . . In propor- tion to animal fat used Margarine from vegetable fat or lard . . . . . . . . . . . 0 Nut butters . . . . . . . . . . + _Meat, fish, etc.:_ Lean meat (beef, mutton, etc.) + + + Liver . . . . . . . . . . . . . + + + Kidneys . . . . . . . . . . . . + + Heart . . . . . . . . . . . . . + + Brain . . . . . . . . . . . . . + + Sweetbreads . . . . . . . . . . + + Fish, white . . . . . . . . . . 0 Very slight if any Fish fat (salmon, herring, etc.) + Very slight if any Fish roe . . . . . . . . . . . + + Tinned meats . . . . . . . . . ? Very slight 0 _Milk, cheese, etc.:_ Milk, cow's whole raw . . . . . + + + Milk, cow's skim . . . . . . . 0 + + Milk, cow's dried whole . . . . Less than + Less than + + Milk, cow's boiled whole . . . ? + Less than + Milk, cow's condensed sweetened + + Cheese, whole milk . . . . . . + Less than + Cheese, skim milk . . . . . . . 0 Eggs, fresh . . . . . . . . . . + + 0? Eggs, dried . . . . . . . . . . + + 0? _Cereals, pulses, etc.:_ Wheat, maize, rice (whole germ) + + 0 Wheat, maize, rice germ . . . . + + 0 Wheat, maize, rice bran . . . . 0 + 0 White wheat flour, pure corn flour, polished rice, etc. . 0 0 0 Custard powders, egg substi- tutes prepared from cereal products . . . . . . . . . . 0 0 0 Linseed, millet . . . . . . . . + + 0 Dried peas, lentils, etc. . . . + Pea-flour, kilned . . . . . . . 0 0 Soy beans, haricot beans . . . + + 0 Germinated pulses or cereals . + + + _Vegetables and fruits:_ Cabbage, fresh, raw . . . . . . + + + Cabbage, fresh, cooked . . . . + + Cabbage, dried . . . . . . . . + + Very slight Cabbage, canned . . . . . . . . Very slight Swedes, raw expressed juice . . + Lettuce . . . . . . . . . . . . + + Spinach, dried . . . . . . . . + + Carrots, fresh, raw . . . . . . + + + Carrots, dried . . . . . . . . Very slight Less than + Beetroot, raw, expressed juice + + Potatoes, raw . . . . . . . . . + Potatoes, cooked . . . . . . . + Beans, fresh scarlet runners raw Lemon juice, fresh . . . . . . + Lemon juice, preserved . . . . Lime juice, fresh . . . . . . . + Lime juice, preserved . . . . . Very slight Orange juice, fresh . . . . . . + Raspberries . . . . . . . . . . + Apples . . . . . . . . . . . . + Bananas . . . . . . . . . . . . + + Very slight Tomatoes, canned . . . . . . . + Nuts . . . . . . . . . . . . . + + _Miscellaneous:_ Yeast dried . . . . . . . . . . ? + Yeast extract and autolysed . . ? + 0 Meat extract . . . . . . . . . 0 0 0 Malt extract . . . . . . . . . + in some specimens Beer . . . . . . . . . . . . . 0 0 Honey . . . . . . . . . . . . . + _______ __ __ __
+ indicates abundant; + relatively large; + present in small amount; 0 absent.
The following table (2) has been compiled from a review of both British and American data and represents a rather more complete classification than the British report. The four plus system has also been used to permit more complete comparisons.
_____________ FOODSTUFF "A" "B" "C" ______ ___ ___ __ _Meats_: Beef heart . . . . . . . . . . . + + ? Brains . . . . . . . . . . . . . + + +? Codfish . . . . . . . . . . . . + + ? Cod testes . . . . . . . . . . . + Fish roe . . . . . . . . . . . . + + ? Herring . . . . . . . . . . . . + + ? Horse meat . . . . . . . . . . . + + Kidney . . . . . . . . . . . . . + + Lean muscle . . . . . . . . . . 0 0 +? Liver . . . . . . . . . . . . . + + +? Pancreas . . . . . . . . . . . . 0 + Pig heart . . . . . . . . . . . + + ? Placenta . . . . . . . . . . . . + Thymus (sweetbreads) . . . . . . 0 0 0 _Vegetables:_ Beet root . . . . . . . . . . . + + + Beet root juice . . . . . . . . ? Little + Cabbage, dried . . . . . . . . . + + + Cabbage, fresh . . . . . . . . . + + + Carrots . . . . . . . . . . . . + + + Cauliflower . . . . . . . . . . + + + Celery . . . . . . . . . . . . . ? + ? Chard . . . . . . . . . . . . . + + ? Dasheens . . . . . . . . . . . . + + ? Lettuce . . . . . . . . . . . . + + + Mangels . . . . . . . . . . . . + + ? Onions . . . . . . . . . . . . . ? + + Parsnips . . . . . . . . . . . . + + Peas (fresh) . . . . . . . . . . + + + Potatoes . . . . . . . . . . . . 0 + + Potatoes (sweet) . . . . . . . . + + ? Rutabaga . . . . . . . . . . . . + Spinach . . . . . . . . . . . . + + + _Cereals:_ Barley . . . . . . . . . . . . . + + ? Bread (white) . . . . . . . . . + +? Bread (whole meal) . . . . . . . + + ? Maize (yellow) . . . . . . . . . + + ? Maize (white) . . . . . . . . . 0 + ? Oats . . . . . . . . . . . . . . + + 0 Rice polished . . . . . . . . . 0 0 0 Rice (whole grain) . . . . . . . + + 0 Rye . . . . . . . . . . . . . . + + 0 Corn embryo . . . . . . . . . . + Corn (kaffir) . . . . . . . . . + Corn (see maize) . . . . . . . . Corn pollen . . . . . . . . . . + Malt extract . . . . . . . . . . 0 0 0 Wheat bran . . . . . . . . . . . 0 + 0 Wheat embryo . . . . . . . . . . + + 0 Wheat endosperm . . . . . . . . 0 0 0 Wheat kernel . . . . . . . . . . + + 0 _Other seeds:_ Beans, kidney . . . . . . . . . + Beans, navy . . . . . . . . . . + 0 Beans, soy . . . . . . . . . . . + + 0 Cotton seed . . . . . . . . . . + + Flaxseed . . . . . . . . . . . . + + Hemp seed . . . . . . . . . . . + + Millet seed . . . . . . . . . . + + Peanuts . . . . . . . . . . . . + + Peas (dry) . . . . . . . . . . . +? + 0 Sun flower seeds . . . . . . . . + _Fruits:_ Apples . . . . . . . . . . . . . + + Bananas . . . . . . . . . . . . ? + + Grapefruit . . . . . . . . . . . + + Grape juice . . . . . . . . . . + + Grapes . . . . . . . . . . . . . 0 + + Lemons . . . . . . . . . . . . . + + Limes . . . . . . . . . . . . . + + Oranges . . . . . . . . . . . . + + Pears . . . . . . . . . . . . . + + Raisins . . . . . . . . . . . . + + Tomatoes . . . . . . . . . . . . + + + _Oils and fats:_ Almond oil . . . . . . . . . . . 0 0 Beef fat . . . . . . . . . . . . + 0 0 Butter . . . . . . . . . . . . . + 0 0 Cocoanut oil . . . . . . . . . . 0 0 0 Cod liver oil . . . . . . . . . + 0 0 Corn oil . . . . . . . . . . . . 0 0 0 Cotton seed oil . . . . . . . . 0? 0 0 Egg yolk fat . . . . . . . . . . + 0 0 Fish oils . . . . . . . . . . . + 0 0 Lard . . . . . . . . . . . . . . 0 0 0 Oleo, animal . . . . . . . . . . + 0 0 Oleo, vegetable. . . . . . . . . 0 0 0 Olive oil . . . . . . . . . . . 0 0 0 Pork fat . . . . . . . . . . . . 0? 0 Tallow . . . . . . . . . . . . . 0 0 0 Vegetable oils . . . . . . . . . 0? 0 0 _Nuts:_ Almonds . . . . . . . . . . . . + + Brazil nut . . . . . . . . . . . + Chestnut . . . . . . . . . . . . + Cocoanut . . . . . . . . . . . . + + English walnuts . . . . . . . . + Filbert . . . . . . . . . . . . + Hickory . . . . . . . . . . . . + + + Pine . . . . . . . . . . . . . . + + + _Dairy products:_ Butter . . . . . . . . . . . . . + 0 0 Cheese . . . . . . . . . . . . . + + ? Condensed milk . . . . . . . . . + + 0 Cream . . . . . . . . . . . . . + + ? Eggs . . . . . . . . . . . . . . + + 0 Milk powder (skim) . . . . . . . + + +? Milk powder (whole) . . . . . . + + +? Milk whole . . . . . . . . . . . + + + Whey . . . . . . . . . . . . . . + + + _Miscellaneous:_ Alfalfa . . . . . . . . . . . . + + ? Blood . . . . . . . . . . . . . Varies with source Clover . . . . . . . . . . . . . + + ? Honey . . . . . . . . . . . . . + 0 Malt extract . . . . . . . . . . 0 0 0 Nectar . . . . . . . . . . . . . 0 0 0 Timothy . . . . . . . . . . . . + + Yeast, brewers . . . . . . . . . 0 + 0 Yeast cakes . . . . . . . . . . 0 + 0 Yeast extract . . . . . . . . . 0 + 0 ______ ___ ___ __
THE CHEMICAL AND PHYSIOLOGICAL PROPERTIES OF THE VITAMINE
While the chemists have not yet been able to isolate and identify the various vitamines they have succeeded in demonstrating many of the properties of these substances and it is the knowledge of these properties that has enabled us to produce concentrates and conduct tests. Another practical consideration involved in this matter of properties lies in the effect of cooking and commercial methods of food preparation, for not only must we learn where the vitamine resides but how to prevent injury or destruction in our utilization of the source.
The properties of the vitamines may therefore be grouped under two heads: first chemical properties and second physiological properties.
I. CHEMICAL PROPERTIES OF VITAMINE "A"
a. This dietary factor's presence in butter fat and egg yolk fat indicates its solubility in the fat and it would naturally follow that the fat solvents would suffice to remove it with the fats when food sources are treated with such a reagent. Experience has shown however that while ether extraction applied to butter or egg yolk removes the vitamine with the fat this process fails when it is applied to vegetable sources such as cotton seed, corn germ, spinach, lettuce, etc. Neither does the cold or hot press method of oil extraction liberate the vitamine with the oil. Recent experiments by Osborne and Mendel, to which we have previously referred, have shown that preliminary treatment of vegetable sources with alcohol seems to loosen the bond between the source and the vitamine and that when this binding is once loosened subsequent ether extraction will take the vitamine out. That the binding is not difficult to break is shown by the fact that when vegetables are eaten as a source of vitamine the body is able to separate the complex. It is further evident that the body does separate this complex and stores it in animal fat from the experiments with cow feeds and feeding. Milk for example is rich or poor in vitamine according to the supply of the latter in the food given to the cow. The only logical conclusion to be drawn from this observation is that the cow does not synthesize this factor but splits it off from the food source and then, since it is fat soluble, is able to mobilize it in the butter fat of the milk or to a more limited extent in the body fat. This observation as to the dependence of milk content upon food has been confirmed in the case of nursing mothers and suggests the need of especial attention to the diet of the mother during the lactating period.
b. It has been generally assumed that the "A" vitamine is comparatively stable to heat. Sherman, MacLeod and Kramer state that "dry heating at a temperature of 100C. with free access of air, only very slowly destroyed fat soluble vitamine." Osborne and Mendel reported that butter fat treated with steam for two hours and a half did not appear to have lost its value as a source of this vitamine. Drummond's earlier work with fish oils and whale oils seemed to confirm this conclusion. Sherman and his co-workers cited above put it this way: "The results thus far obtained emphasize the importance of taking full account of the time as well as the temperature of heating, and of the initial concentration of the vitamine in the food, as well as of the opportunity for previous storage of the vitamine by the test animal." More recent work by Steenbock and his co-workers in America shows that these earlier results are incorrect in the case of butter fat and that twelve hours exposure of butter fat to 100C. may, under certain conditions, destroy the efficiency of that substance as a source of the vitamine. Drummond and other English workers have confirmed Steenbock in later experiments. Their work has shown that the presence or absence of oxygen is a factor, which may determine the extent of destruction of the vitamine. Heat alone is of very limited effect but when sources are heated in the presence of oxygen destruction of the A vitamine may be very rapid. Drummond attributes the absence of the A vitamine in lard to the oxidation that takes place in the commercial rendering of this product. We must conclude therefore that while the vitamine may be destroyed by continuous exposure to a temperature of 100C. the effect is largely determined by the nature of the process and the way the vitamine is held in the source. Cooking of vegetables therefore will not as a rule result in appreciable destruction of this factor.
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 20C. there is very little destruction but that raising the temperature to 90C. 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 100C. 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 20C. 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 233C. consisting of:
I. A complex melting at 229C. and forming needles and prisms nearly insoluble in water and with the apparent formula of C24H19O2N5.
II. A complex melting at 222C. and soluble in water. Formula C29H23O2N5.
III. Nicotinic acid melting at 235C. C_6H_5O_2N.
B. The rice complex
Crystals melting at 233C. consisting of:
I. A complex melting at 233C. and with a formula of C26H20O9N4.
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 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 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 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 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 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 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 constitution. Her results also confirm Hoist and Frhlich 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 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 . . . . . 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.
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 entity but a group. Mitchell 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 quantities. 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 identity of the curative with the growth factor. Osborne and Mendel have also shown that milk in general must not be classed among the rich sources of the vitamine, even when the cow's food is rich in vitamine. The principal 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 entitled 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 appetite. 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 partition 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 appetite. The change in the growth curve from decline to rise was accomplished without increasing or changing the basal diet but as the appetite 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 appetite 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.
[Illustration: 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 anal 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 appetite 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.]
These views are at best speculations. The literature is singularly lacking in detailed metabolic analyses 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 assumed 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 antiscorbutic 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 antiscorbutic 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 + Kokum . . . . . . . . . . . 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 quantity. This discovery is of great value in instances where the cost of orange juice is often prohibitive.
La Mer and Campbell have presented some evidence to show that the antiscorbutic vitamine has a direct effect upon the adrenal glands. In their scurvy cases they find definite evidence of the enlargement or hypertrophy of this organ. Whether it affects other organs or not it remains to be shown.
HOW TO UTILIZE THE VITAMINE IN DIETS
In the preceding chapters it has been the aim to present the findings of the principal workers in the field. In attempting to summarize the work of so widely scattered a group as are now engaged in vitamine research it is impossible to cover completely the many investigations and it is inevitable that some work will have been overlooked, but the foregoing covers at least the principal data on the subject. What is the bearing of all this information on human behavior and what lessons can the layman draw from it that is of direct application to him? Let us first consider this question from the dietary viewpoint.
I. INFANT NUTRITION
The limited character of the infant's diet has made the consideration of vitamine content in his diet much more important than in the case of the adult with the latter's wide variety of choice. It is evident from the previous data that a growing infant must not only be provided with a sufficient supply of calories, nutrients and salts, but must also have a liberal supply of the three vitamines. Milk has in general been classed as adequate in all these features, but the vitamine researches have forced us to reconsider our views in regard to this staple.
The first point to be borne in mind is that the vitamine content of either cow or human milk is dependent primarily upon the food eaten by the producer of the milk. In other words milk is merely a mobilization of the vitamines eaten and if the diet is to yield vitamine-rich milk it must itself be rich in these factors. Many a cow produces milk low in vitamine content and the same is true of nursing mothers. There are many "old wives" prejudices in regard to what food a lactating mother may eat and unfortunately many of these prejudices are extremely injurious and false. One of them is the prejudice against green vegetables. Experience has shown that under ordinary conditions such vegetables are well tolerated by the mother and from their content of vitamine it is evident that they are suppliers of these factors. In the case of the cow the fact that cereals are poor in some of the vitamines and green grasses rich therein, teaches a lesson that bears directly upon winter feeding of cattle if the milk supply is to be used for infants. We need a series of diets and cattle foods for just this purpose of insuring the proper vitamine content in milk. The preceding tables will enable one to develop such diets fairly satisfactorily, but more data is urgently needed.
The second point in regard to milk lies in the effect of pasteurization. This measure is now well nigh universal and in America at least has played a tremendous part in the reduction of infant mortality, especially during the summer months. At present, however, we know that this treatment while removing dangerous germs may also eliminate the antiscorbutic factor. The sensible attitude then is to recognize this fact and if a clean whole milk is not available retain the pasteurization and meet the vitamine deficiency by other agents. Such agents are orange juice and tomato juice and experience has already shown that these juices can be well tolerated by infants much earlier than used to be thought possible.
While the pasteurization does not appreciably affect the content of "A" or "B" vitamines, the variability in content of these vitamines in milk indicates that it may at times be necessary to supplement them in the diet. In this connection it must be borne in mind that cereals vary widely in content and cannot be, as they often are now, considered equivalent in growth stimulation power. This is a subject that needs special attention on the part of vitamine experts and dietitians and finally by the food manufacturers. A good vitamine-rich cereal combination would form an excellent adjuvant to infant dietaries after they reach the age of tolerance to such a diet. But even before that time the expressed juice of various vegetables as well as fruits is found to be well tolerated when mixed with the milk or given separately, and carrot and spinach juice are now being used in this connection with good results. These juices like orange juice contain the B type in abundance and there is no doubt that in their stimulation to the appetite they play an important part in making the desirable daily gain.
Fortunately for the layman he has in the scales a good indicator of the normal progress of his child and so long as growth is normal he can fairly assume that the diet is adequate but if the scales say otherwise it is time for him to seek advice and then he is wise who insures that his medical adviser knows the newer aspects of nutrition. The parent can do this only by proper selection, but with a little knowledge he can soon satisfy himself as to whether his pediatrist is the right sort and it is one of the purposes of this text to bring home to the layman his responsibility in this matter.
There has grown up in this country a great regard for prepared milk substitutes in infant feeding and a wide usage of condensed milks, reinforced milks, diluted milk formulae, etc. All such preparations must be examined anew in the light of the vitamine discoveries and unless the given preparation can show a clean bill of health in vitamine content, it should be either discarded or properly supplemented.
As children grow up, it is fortunate that in their wider choice of dietaries the danger of vitamine deficiency decreases. But even in childhood it is unsafe to rely too much on chance. In this country there are well deserving movements on foot to attract the parents of the community to the necessity of attention to simple standards of growth progress, and clinics for this purpose are appearing in increasing numbers with each year. Such movements are to be most heartily approved. It is also possible in these measures to not only build better children, but to make the children themselves intelligent in their rejection of unsuitable combinations and in that way not only conserve their own health, but provide an educated body of citizens to pass on the knowledge to future generations. In a school in New York City I recently had occasion to discuss the school lunch room and its offerings with the children of the school in the light of vitamine discoveries. The keenness and intelligence shown by the children in the discussion that followed has convinced me that in this matter of vitamines the children themselves can be relied upon to assist materially in the matter of better food combinations and intelligent selection.
Finally it must be noted that one of the most common of infant deficiencies is the failure of the bones to lay down lime. The effect of this failure is commonly described as rickets. The British workers consider that this deficiency is a lack of vitamine "A." Their views have been set forth at greatest length by Mellanby, the principal worker in this subject. While this view is still debatable and in this country it is not yet accepted, one fact has come out in the controversy and that is the remarkable value of cod-liver oil as a preventive of rickets. It may be that the power of the oil is due to its "A" vitamine content in which it is known to be rich, or it may be due to a new vitamine, but the fact that the oil is a preventive in this respect gives the pediatrist another agent to insure normal growth. The various views on the causes of rickets are set forth more in detail in Chapter VIII.
II. ADULT DIETS
A study of the dietary habits of various sections of the United States shows that there is a very general tendency on the part of the majority of the people to confine their foods to a meat, potato, and cereal diet. The use of salads is looked upon by many sections as a foreign affectation and too little attention is paid to the value of eggs, milk and cheese. Enough has been said already to show that these latter articles have much more than an esthetic value and one of the missions of the nutrition expert must be to show the people why dairy products and salads must become features in the every-day meals of the every-day people. And even if the salads are still unappreciated, it is necessary that cooked green vegetables occupy more of a position in the menu than is too often the case.
There has recently appeared a crusade for the eating of yeast cakes. The claim made for their use rests on a perfectly firm basis, they are rich in the "B" vitamine, the proteins of the yeast cake are of good quality and the cake contains no ingredients poisonous to man. Many people are reporting beneficial effects from their use. Is there any lesson to be drawn from this experiment? I feel that the very fact that benefits have resulted from this yeast feeding is excellent evidence of lack of the vitamine in the diets of the people affected and a clear argument that the dietary habits of many people need adjustment to a higher vitamine content. Whether it is necessary to use yeast cakes or any other concentrate of vitamine, depends entirely upon whether the ordinary diet is lacking in these factors and my first advice in the matter would be to make if possible a selection of the vitamine containing foods and see if normal conditions did not result before utilizing foods whose taste is not pleasing or which are taken as medicine. For it is an old experience that medicines will be taken only so long as the patient is sick and perhaps it is just as well so. In other words I believe it is possible with intelligent selection based on such tables as are given in Chapter IV for people to secure from the butcher and the grocer all their requirements of these vitamines as a part of their regular palatable diet. To those who have neglected this selection and find remedy in concentrates, that fact should lead them to reconstruct their diet rather than persist in dependence on the medicine to correct faulty diet. In other words the same arguments apply to the use of medicinal concentrates of vitamines as applies to the use of laxatives. At times these substances are very valuable as cures, but it is better by far to so regulate the dietary habits as to avoid the necessity for their use.
Another phase of this matter that promises to develop in the near future as a result of the vitamine hypothesis is a reform in food manufacture. There has been a strong tendency during the past two decades to "purify" food products. The genesis of this tendency is to be found in a highly laudable ambition to force the manufacturer to eliminate impurities and adulterations and provide clean, wholesome, sanitary food. Unfortunately in attempting to meet this demand on the part of the public, the food manufacturer has sometimes neglected to seek advice from the nutrition expert and the latter has failed to appreciate the need of advice. The net result has been to discover that Nature is often a better chemist than man and has a much better knowledge of what man needs in his diet than the chemist. The chemist employed by the manufacturer has, as a result, gone to such a limit in his development of purification methods as to often eliminate the essential nutrients and the result has been foods that will stand analysis for pure nutrients, but which will not stand Nature's analysis for dietary efficiency. As a secondary result of this tendency we have acquired habits that in many cases must either be broken or must have grafted on to them other habits which shall remedy the defective ones. Take the milling of wheat as an example. Nature put into the wheat grain most of the elements needed by man and in the early days he was content to grind up the whole grain and find it palatable. The craze for purity as expressed by color has gradually replaced this whole meal wheat with a beautiful white product that is largely pure starch with a few of the proteins retained. And the principal protein retained lacks one of the greatest essentials for growth while the vitamines have all been practically eliminated with the grain germ. Intelligence tells us then that if, having formed the habit, we will persist in our appetite for white flour we must see to it that the protein deficiency of the latter and its lack of vitamines is compensated for by supplementing the diet with the food-stuffs in which these are rich. We may in other words retain our bad habits in taste if we will graft on to them the attention to the eliminated factors and their substitution in other form.
In general then, the adult needs to review his feeding habits and analyze them in the light of our new knowledge. For this purpose the tables of Chapter IV supply data useful so far as vitamines are concerned, but it will be perhaps worth while to repeat here some of this data in more generalized form.
a. Sources of the "A" vitamine
Its most abundant sources are milk, butter, egg yolk fat, and the green leaves of plants usually classed as salads. Cabbage, lettuce, spinach and carrots contain this substance in considerable quantity. The germ of cereals is fairly rich in the factor, but the rest of the grain is deficient and white flours are therefore poorer than whole meals in this respect. Cooking temperatures have little effect on this vitamine and hence little attention need be paid to cooking temperatures as far as this vitamine is concerned.
b. Sources of the "B" vitamine
Its principal sources outside of yeast are the seeds of plants and the eggs and milk of animals. Meat contains relatively little of this substance but glandular organs such as the liver and pancreas are fairly rich in it. In the seeds the distribution is general throughout the whole body of the seed in the case of beans, peas, etc., but in the cereal grains it is largely restricted to the embryo portion and hence a high degree of milling tends to reduce the per cent of this factor in any highly milled cereal. White flour and polished rice are notable examples of deficiency of "B" vitamine due to this milling process. Fruits such as oranges, tomatoes, and lemons are good sources and there is a fair amount present in the apples and grapes and other common food fruits. Many vegetables show it in fair abundance, notably potatoes, carrots, and turnips, but the rule is not general for beets are extremely poor in this factor. Nuts are also good sources. Eggs, milk and cheese contain it in fair abundance. Cooking temperatures have little effect on this type if the temperature does not climb above the boiling point and if the cooking water is not "alkaline." In the latter case it becomes necessary to determine the extent of destruction and either eat enough to insure protection, or reform the method of cookery.
c. Sources of the "C" vitamine
Its richest sources are vegetables such as cabbage, swedes, turnips, lettuce and watercress; fruits such as lemons, oranges, raspberries and tomatoes. Certain of the vegetables such as potatoes have a substantial value in this respect, but meat and most prepared milks are low in antiscorbutic values. The susceptibility of this vitamine to drying, heat and alkali, make it necessary to scrutinize your cooking methods very carefully in order not to ruin a good source by a poor preparation of it for the table.
AVITAMINOSES OR THE DISEASES THAT RESULT FROM VITAMINE DEFICIENCIES
A survey of the vitamines would be incomplete without a discussion of the vitamine deficiency diseases in particular, though many of the facts already cited obviously bear on the treatment and prevention of such diseases.
The idea of "avitaminoses" or vitamine deficiency as the cause of a disease of a specific nature was set forth in detail by Funk in his book Die Vitamine. In his discussion of this view he suggests several types that would, he felt, on examination prove to be due to the absence of a vitamine in the diet. Of these predicted types beri-beri was the only one to be established in 1913. Scurvy has now been added to the fold and rickets or rachitis seems well on the way to acceptance though the specific vitamine absent in this case is not yet positively identified. Pellagra still resists the efforts of the vitamine hypothesis to bend it to that theory and its etiology is still obscure.
This disease while specifically confined to the oriental in the mind of the student can be justly considered of much wider distribution for the mild forms of malnutrition associated with a deficiency in the "B" vitamine are less acute manifestations of this disease. The disease is not likely to become marked in well nourished districts in its acute form, but in famine districts its incidence is always possible. It would be more than possible were it not for the fact that famine tends to eliminate the highly milled cereals and throw the people back on to the whole grain, peas and beans, which are rich in the preventive factor. But when for any reason diets become limited extra attention is demanded in regard to their selection and preparation. The main characteristics of this disease have already been fully covered in what precedes and need not be repeated here.
This disease, like beri-beri has already been fully discussed in what precedes. One of the striking discoveries of this subject has been the retreat from favor of the time-honored lime juice which is now found to be much less potent than oranges, lemons, or even canned tomato juice and which on preservation loses practically all its potency. In the modern hospital, cases of scurvy rarely appear outside of occasional infant cases and it might appear that the problem of scurvy prevention is peculiarly that of the sailor, the explorer and the army rationer. Nevertheless an insufficient supply of the "C" vitamine may retard growth and well being in the individual without manifesting itself in its more acute form of scurvy. In a recent review Hess states: "It is hardly an exaggeration to state that in the temperate zones the development or non-development of scurvy depends largely on the potato crop." "This is attributed in part to the fact that the potato is an excellent antiscorbutic, but to a greater extent because it is consumed during the winter in amounts that exceed the combined total of all other vegetables." To the public and to the food purveyor there is a definite problem in how to best supply the preventive and how best to concentrate and preserve the sources of this vitamine without injury to its potency. The following observation is therefore appended as bearing on this point. In the absence of fruits or other high potency sources it is possible to develop this factor in cereal grains by the simple expediency of sprouting. If seeds are soaked in water for twenty-four hours and then kept moist for from one to three days with the free access of air, sprouts will develop whose content of the antiscorbutic vitamine is comparable to that of many fresh vegetables, even though the dry seeds themselves have little of this factor. In other words the germination process is a synthesiser of the vitamine. This observation may be of value where fruits and vegetables are scarce or expensive. On account of cooking effects, it cannot be too often reiterated that raw fruits, vegetables and salads, are of more value than cooked forms of these same sources and that drying processes are extremely destructive where heat enters into the drying process. Vacuum drying seems to be much less destructive and it may be possible to develop the drying of vegetables to a point where retention of this vitamine factor is practical. At present all dried vegetables should be regarded with suspicion as a source of vitamine "C." Expressed juices may often be used where the whole vegetable is scarce or incompatible and this fact is one to be borne in mind by the worker in famine districts.
III. RACHITIS (RICKETS)
This disease is engaging the attention of many workers on both sides of the Atlantic at the present time. In England the principal contributor is Dr. Mellanby, who has accumulated evidence which he believes indicates that the preventive factor is the A vitamine. This view is not yet accepted as conclusive by the American workers. McCollum, Howland, Park, and others at Johns Hopkins University have experimented with various rickets-producing diets and while the principal deficiency in these diets seems to be Ca salts and the A vitamine they do not consider that the disease can as yet be traced to deficiency in any one factor. Hess has called attention to several new features and the significance of some older measures. He has shown on the one hand that cod-liver oil is almost a specific remedy for the disease but that this remedy is not replaceable by other rich sources of the A vitamine. He has also recently shown that hygienic measures may have an influence. Schmorl showed that the disease was seasonal, a high rate maintaining in the winter months and a lower rate in the summer months. Hess has recently reported beneficial results from use of the ultra-violet rays which he uses as a substitute for sunlight. The results seem to confirm Schmorl's view that the sunlight of the summer months is a preventive factor. He has also suggested that the specific effect of the cod-liver oil might be due to a new vitamine, Vitamine D? On the other hand Zilva and Miura in England have recently shown that crude cod-liver oil is something like two hundred and fifty times as rich in vitamine A as butter fat, which tends to support the British view that the A vitamine is the antirachitic factor.
Sherman and Pappenheimer have recently shown that the phosphates exert a marked preventive effect on rickets and suggest that the utilization of the calcium by the individual may be determined in part by this factor.
The views in brief are now in an extremely chaotic state and it is impossible at present to determine whether rickets is a true avitaminose or a consequence of deficiency in a series of factors. It is however certain that the disease in its subacute forms is extremely wide-spread among infants and that its prevention can be most easily secured by the addition of cod-liver oil to the diet. In this procedure warning is necessary that the cod-liver oil be as pure a product of oil as possible, since the market preparations are often almost devoid of the true oil and hence of the curative agent.
This disease has been the subject of exhaustive inquiry and study on this side of the Atlantic and the findings of the various investigating boards have added much to the prevention and cure of the scourge, but have failed as yet to agree on any one etiological factor. The best recent review of the current findings is to be found in an article by Voegtlin published as Reprint 597 of the Public Health Reports of the United States Public Health Service. His conclusions may be quoted in full as representing the latest summary of evidence now extant:
1. The hypothesis that there is a causal relation between pellagra and a restricted vegetable diet has been substantiated by direct proof to this effect and has led to results of considerable practical and scientific value.
2. The metabolism in pellagra shows certain definite changes from the normal, which point to decreased gastric secretion and increased intestinal putrefaction.
3. In the treatment and prevention of pellagra, diet is the essential factor. The disease can be prevented by an appropriate change in diet without changing other sanitary conditions.
4. A diet of the composition used by pellagrins prior to their attack by the disease leads to malnutrition and certain pathological changes in animals, resembling those found in pellagra. A typical pellagrous dermatitis has not been observed in animals. Pellagrous symptoms have been produced in man by the continued consumption of a restricted vegetable diet.
5. The nature of the dietary effect has not been discovered, although certain observations point to a combined deficiency in some of the recognized dietary factors as the cause of the pellagrous syndrome.
In elaborating on conclusion 5 Voegtlin states that:
The conception that pellagra is due to a dietary deficiency is, therefore, not contradicted by the available evidence. This does not imply that the disease is necessarily due to a deficiency of diet in a specific substance such as the hypothetical pellagra vitamine of Funk (1913). It is much more likely that the pellagrous syndrome is caused by a combination of the deficiencies in some of the well recognized food factors.
V. OTHER AVITAMINOSES
The rle of the vitamine in the nutrition and growth of organisms other than the man is becoming a matter of interest in various ways. The construction of culture media for various strains of bacteria and the conditions favorable or unfavorable to their growth, are features of study in which the new hypothesis has demanded attention. It has already been claimed that vitamines are essential to the growth of the meningococcus, the influenza bacillus, the typhoid bacillus, the gonococcus, the pneumococcus Type I, Streptococcus hemolyticus, the diptheria bacillus, the Bacillus pertussis and certain soil organisms. If these views are confirmed it becomes evident that the means for prevention of the development of these forms may lie in the control of the vitamine content of the materials on which these forms thrive and that in the study of these types it may be possible to speed up the incubation of strains and thus hasten diagnostic measures by introducing the necessary vitamines into the culture media. These observations merely suggest the possible widening of the scope of the vitamine study in the service of man and give added reason for our keeping pace with the strides made in this particular field.
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