_________________________________________________________________________| |INGREDIENTS | A ONLY | CONTAINING B ONLY_______________________________|___________|_____________________________| | | | | | || VIII| IX | X | XI | XII | XIII| XIVPurified protein as casein, | | | | | | |lactalbumin, edestin, egg | | | | | | |albumin, etc. . . . . . . | 18.0|18.0 | 18.0| 18.0| | 18.0| 18.0or Meat residue . . . . . | | | | | 19.6| || | | | | | |Carbohydrates in the form of: | | | | | | |Starch . . . . . . . . . . . | 45.0| 45.0| 29.5| 54.0| 52.4| 26.0| 29.0Sucrose . . . . . . . . . . . | | | 15.0| | | || | | | | | |Fat in the form of: | | | | | | |Lard . . . . . . . . . . . | 15.0| 27.0| 30.0| 24.0| 24.0| 28.0| 25.0Butter fat . . . . . . . . . | | | | | | |Egg yolk fat . . . . . . . . | | | | | | |Cod liver oil . . . . . . . . | 18.0| 6.0| | | | || | | | | | |Salts in the form of: | | | | | | |Salt mixture I . . . . . . . | | | 2.5| | | |or Artificial protein-free | | | | | | |milk (Mixt. IV) . . . . . . | 4.0| 4.0| | 4.0| 4.0| |or Protein-free milk . . . | | | | | | 28.0| 28.0| | | | | | |Roughage in the form of: | | | | | | |Agar-agar . . . . . . . . . . | | | 5.0| | | |_______________________________|_____|_____|_____|_____|_____|_____|_____| | || | | Fed Daily| | |_____________________________"B" vitamine in the form of: | | | | | | || | | 0.2 | 0.4 | 0.2 | 0.04|| | | to | gram| to | gram|Dried brewers' yeast | | | 0.6 | | 0.6 | || | | gram| | gram| |_______________________________|_____|_____|_____|_____|_____|_____|_____| | | | | | |Total . . . . . . . . . . . . |100.0|100.0|100.0|100.0|100.0|100.0|100.0_______________________________|_____|_____|_____|_____|_____|_____|_____
[Note. Diets I, III and X have been practically discontinued at the present time. Diets II, V and XI are standard. For data on salt mixtures see Osborne, T. B. and Mendel, J. B. The inorganic elements in nutrition, Jour. Biol. Chem. 1918, xxxiv, 131.]
Salt mixture I (after Rohman)
gramsCa_3(PO_4)_2 . . . . . 10.00K_2HPO_4 . . . . . . . 37.00NaCl . . . . . . . . . 20.00Na citrate . . . . . . 15.00Mg citrate . . . . . . 8.00Ca lactate . . . . . . 8.00Fe citrate . . . . . . 3.00______
Total . . . . . . . . 100.00
Artificial protein-free milk
gramsCaCO_3 . . . . . . . . 134.8MgCO_3 . . . . . . . . 24.2Na_2CO_3 . . . . . . . 34.2K_2CO_3 . . . . . . . . 141.3H_3PO_4 . . . . . . . . 103.2HCl . . . . . . . . . . 53.4H_2SO_4 . . . . . . . . 9.2Citric acid: H_2O . . . 111.1Fe citrate: 1.5H_2O . . 6.34KI . . . . . . . . . . 0.020MnSO_4 . . . . . . . . 0.079NaF . . . . . . . . . . 0.248K_2Al_2(SO_4)_2 . . . . 0.0245
[N.B.—The ingredients of the artificial protein-free milk are mixed as follows: Making proper allowance for the water in the chemicals the acids are first mixed and the carbonates and citrates added. The traces of KI, MnSO_4, NaF, and K_2Al_2(SO_4)_4 are then added as solutions of known concentration. The mixture is then evaporated to dryness in a current of air at 90 to 100° Centigrade and the residue ground to a fine powder.]
e. When brewers' yeast is used as a source of the "B" vitamine it is first dried over night in an oven at 110°C. and then subjected to the same purification process as the casein and the starch to remove all trace of the "A."
The reasons for the special precautions just described have arisen from some recent work of Daniels and Loughlin who claim that commercial lard contains enough "A" vitamine to permit rats to grow, reproduce and rear young. The British authorities explain their results as not due to the presence of the "A" vitamine in the lard but to a reserve store in the bodies of the animals. They hold that animals may thus store the "A" vitamine but that apparently they have no storage powers for the "B" that are comparable to it. Osborne and Mendel repeated the experiments described by Daniels and Loughlin, using the purification methods just described, but failed to obtain similar results with either commercial lard or with the purified fraction. They question the validity of the British explanation but at the same time reiterate their belief that even commercial lard contains no "A" vitamine. Whatever the explanation of this particular phenomenon it is important that the basal diet be of purified materials and the methods just described supply the procedure necessary to attain that end.
Before discussing the application of these diets to vitamine testing, attention is called to other basal diets developed by McCollum. This worker has paid especial attention to the deficiencies of the cereal grains and in particular to their salt deficiencies. In his basal diets, we find, as would be expected, special combinations particularly suited to the detection of vitamines in such cereals. McCollum has also devised a method of extracting substances to obtain their "B" vitamine and of depositing it on dextrin. For that reason he uses dextrin instead of starch for his carbohydrate and when he wishes to introduce the "B" vitamine it can be done by his method without having to recalculate the carbohydrate component. His method consists of first extracting the source with ether and discarding this extract. Pure ether will not remove the "B" vitamine. The residue is then reextracted several times with alcohol and the alcohol extracts combined. If now these alcohol extracts are evaporated down on a weighed quantity of dextrin the activated dextrin can be used not only to supply the carbohydrate of the ration but also to carry the "B" vitamine of a given source that is under investigation. McCollum's basal diets and salt mixtures are tabulated in the following chart:
McCollum's basal diets and salt mixtures
_______________________________________________________________________ | | | INGREDIENTS | VITAMINE FREE |"A" ONLY | "B" ONLY ___________________|___________________|_________|_____________________ | | | | | | Casein . . . . . . |18.0|18.0|18.0|18.0| 18.0 | Same as the vitamine Dextrin . . . . . |57.3|56.3|76.3|78.3| 71.3 | free diet Lactose . . . . . |20.6|20.0| | | | with "B" added Agar . . . . . . . | 2.0| 2.0| 2.0| | 2.0 | as yeasts as Salt mixture 185 . | 2.7| 3.7| 3.7| 3.7| 3.7 | in the Mendel Butter fat . . . . | | | | | 5.0 | diets or as ___________________|____|____|____|____|_________| extracts carried | on the dextrin. | In the latter | case a given | amount of dextrin Lactose was later discarded when it was shown | carries the to be usually contaminated with the "B" vitamine.| extract of a | known weight | of the source of | the "B" _________________________________________________|____________________
Cereal testing combinations______________________________________________________________________| | | | | |Wheat . . . . . . |56.6| | | | 70.0 |Wheat embryo . . . | |13.3| | | |Corn . . . . . . . | | |71.3| | |Oats . . . . . . . | | | |60.0| |Skim milk powder . | | | | | | 6.0Dextrin . . . . . |31.5|76.4|18.0|30.3| 20.0 | 81.0Salt mixture 185 . | | | 3.7| | |Salt mixture 314 . | | 5.3| | | |Salt mixture 318 . | 6.9| | | | 5.0 |Salt mixture 500 . | | | | 4.7| |Salt mixture ? . . | | | | | | 6.0Butter fat . . . . | 5.0| 5.0| 5.0| 5.0| 5.0 | 5.0Agar . . . . . . . | | | 2.0| | | 2.0___________________|____|____|____|____|_________|____________________
Salt mixtures __________________________________________________________________________ | | NUMBER OF MIXTURES |______________________________________________ | | | | | | INGREDIENTS | 185 | 314 | 318 | 500 | 211 | ? ___________________________|_______|_______|_______|_______|_______|______ | | | | | | | grams | grams | grams | grams | grams | grams | | | | | | NaCl . . . . . . . . . . . | 0.173 | 1.067 | 1.400 | 0.5148| 0.520 | 15.00 MgSO_4 anhydrous . . . . . | 0.266 | | | | | 1.90 Na_2HPO_4:H_2O . . . . . . | 0.347 | | | | | K_2HPO_4 . . . . . . . . . | 0.954 | 3.016 | 2.531 | 0.3113| | 34.22 CaH_4(PO_4)_2:H2O . . . . | 0.540 | | | | 0.276 | 0.89 Ca lactate . . . . . . . . | 1.300 | 5.553 | 7.058 | 2.8780| 1.971 | 57.02 Ferrous lactate . . . . . | 0.118 | | | | | K citrate:H_2O . . . . . . | | 0.203 | 0.710 | 0.5562| 0.799 | Na citrate anhydrous . . . | | | | | | 3.70 Ferric citrate . . . . . . | | 0.100 | | | | 2.00 Mg citrate . . . . . . . . | | | | | | 7.00 CaCl_2 . . . . . . . . . . | | 0.386 | | 0.2569| | CaSO_4:2H_2O . . . . . . . | | 0.381 | 0.578 | | | Fe acetate . . . . . . . . | | | | | 0.100 | ___________________________|_______|_______|_______|_______|_______|______
These diets fall as shown, into two classes. The first group correspond to those of Osborne and Mendel and are available for general testing of any unknown. The cereal combinations are so constituted that all deficiencies of salts are covered and the proportions of the cereal are so selected as to provide the right proportions of protein, fat and carbohydrate. By adding enough butter fat to supply the "A" the deficiency in the "B" can be tested and by adjusting the amounts of "B" on the dextrin the cereal deficiency in this vitamine can be obtained. It is obvious that by substituting lard for the butter fat one could use the same mixture properly supplemented with the "B" to determine the "A" deficiencies of the wheat.
The most prominent worker in the field of the "A" vitamine measurement in America is Steenbock. His basal diets are a combination of those already described.
Steenbock's basal dietsper centCasein (washed with water containing acetic acid) . . . . . 18.0Dextrin . . . . . . . . . . . . . . . . . . . . . . . . . . 73.3Ether extracted wheat embryo as source of vitamine "B" . . . 3.0Salt mixture (McCollum, no. 185) . . . . . . . . . . . . . . 3.7Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0
This was his original basal diet but later he modified it by adopting the McCollum method of carrying his "B" vitamine on the dextrin. This was usually the alcohol extract of 20 grams of wheat embryo. In the following diets the presence of this extract is indicated by the letter (x) following the dextrin.
____________________________________________________________________ | | | | | | INGREDIENTS | | | | | | __________________________|______|______|______|______|______|______ | | | | | | Casein . . . . . . . . . | 18.0 | 18.0 | 16.0 | 18.0 | 16.0 | 12.0 Salt 185. . . . . . . . . | 4.0 | 4.0 | | | | Salt 32 . . . . . . . . . | | | 4.0 | 4.0 | 2.0 | 2.0 Salt 35 . . . . . . . . . | | | | | 2.5 | 2.5 Dextrin (x) . . . . . . . | 76.0 | 71.0 | 78.0 | 57.0 | | Butter fat . . . . . . . | | 5.0 | | 5.0 | | Beets . . . . . . . . . . | | | | 15.0 | | Potatoes . . . . . . . . | | | | | 79.5 | Dasheens . . . . . . . . | | | | | | 83.5 Agar . . . . . . . . . . | 2.0 | 2.0 | 2.0 | 1.0 | | __________________________|______|______|______|______|______|______
Steenbock's salt mixtures
McCollum's no. 185; see page 44.No. 32 consisted of:gramsNaCl . . . . . . . . . . . . . . . . . . . . . . . . . 0.202Anhydrous MgSO_4 . . . . . . . . . . . . . . . . . . . 0.311K_2HPO_4 . . . . . . . . . . . . . . . . . . . . . . . 1.115Ca lactate . . . . . . . . . . . . . . . . . . . . . . 0.289Na_2HPO_4:l2H_2O . . . . . . . . . . . . . . . . . . . 0.526Ca_2H_2(PO_4)_2:H_2O . . . . . . . . . . . . . . . . . 1.116Fe citrate . . . . . . . . . . . . . . . . . . . . . . 0.138No. 35 consisted of:NaCl . . . . . . . . . . . . . . . . . . . . . . . . . 1.00CaCO_3 . . . . . . . . . . . . . . . . . . . . . . . . 1.5
The very nature of these basal diets suggests their use. In general however their utilization for testing purposes is based on the following principles: Since the basal diet supplies all the requirements of a food except the vitamine for which one is testing, it is simply necessary to add the unknown substance as a given percent of the diet and observe the results. If the amount added is small it is assumed that its addition will not appreciably effect the optimum concentrations of nutrients, etc., and for such experiments no allowances are made for the constituents in the unknown. For example let us assume that we wish to test the value of a yeast cake as a source of "B" vitamine. We first select a sufficient member of rats of about thirty days age to insure protection from individual variations in the animals. The age given is taken as an age when the rats have been weaned and are capable of development away from the mother and as furnishing the period of most active growth. These rats are now placed on one of the basal diets which in this case supplies all the requirements except the "B" vitamine. In this experiment any of the diets of Osborne and Mendel or of McCollum will do that have been labelled "A"only. After a week or so on this diet they will have cleared the system of the influence of previous diets and their weight curves will be either horizontal or declining. If now we make the diet consist of this basal diet plus say 5 per cent of yeast cake, the weight curve for the next few weeks will show whether that amount supplies enough for normal growth, comparison being made with the normal weight curve for a rat of that age.
In this method it is assumed that the amount of yeast cake added will not derange the proportions of protein fat, etc., in the basal diet enough to affect optimum conditions in these respects. This is a curative type of experiment. If we wish to develop a preventive experiment the yeast cake may be incorporated in the diet from the first and the amount necessary to prevent deviation from the normal curve determined. Both methods are utilized, the one checking the other. If however the amount of the substance necessary to supply the vitamine required for normal development is large such addition would of course disturb the proportions of nutrients in the normal diet and in that case analysis must be made of the substance tested to determine its protein, fat, carbohydrate and salt content and the basal diet corrected from this viewpoint so as to retain the optimum proportions of these factors. McCollum's cereal testing combinations are illustrative of such methods applied to cereals. Still another method is to add a small per cent. of the unknown and then add just enough of the vitamine tested to make sure that normal growth results. Such a method gives the results in terms of a known vitamine carrier. For example, if we add to a basal diet, sufficient in all but the "A" vitamine (Steenbock's mixture for example), a small per cent of a substance whose content in "A" is unknown and note that growth fails to result we can then add butter fat until the amount just produces normal growth. If now we know just what amount of butter fat suffices for this purpose when used alone we can calculate the part of the butter which is replaced by the per cent of unknown used. To put this in terms of figures will perhaps make the idea clearer. Let us assume that 5 per cent of butter fat in a given diet is sufficient to supply the "A" necessary for normal growth. Assume that the addition of 5 grams of the unknown in 100 grams of the butter-free diet fails to produce normal growth but that by adding 2 per cent of butter fat normal growth is reached. It is obvious under these conditions that 5 grams of the unknown is equivalent in "A" vitamine content to 5 minus 2 grams of butter fat, i.e., is equivalent to 3 grams of butter fat or expressed in per cents the substance contains 0.6 or 60 per cent of the "A" found in pure butter fat.
Experience has shown that it is dangerous to draw conclusions from experiments of too short duration or to base them on too few animals. For complete data the experiments should be carried through the complete life cycle of the rat, including the reproductive period. Otherwise it may turn out that the amount in the unknown while apparently sufficient for normal growths is incapable of sustaining the drain made in reproduction. It is this consideration that makes the accumulation of authoritative data on vitamine contents of foodstuffs so slow and tedious and one of the reasons why we lack satisfactory tables in this particular at present. Osborne and Mendel raise another point of methodology and believe that more accurate results will be obtained if the source of the vitamine is fed separately than if mixed with the basal diet. It is easily possible that since one of the effects of lack of vitamine, especially of the "B" type, is poor appetite, the amount necessary to produce normal growth may be smaller than would appear from results obtained by mixing it in the basal diet. When so mixed the animals do not get enough to maintain appetite and really decline because they do not eat enough rather than because the amount of vitamine given is inadequate to growth. Details of this kind are matters however that particularly concern the experimentalist and as our purpose here is to merely describe the methodology we may perhaps turn now to other types of testing. Before doing so it is perhaps unnecessary to suggest that in all experiments it is important that the food intake consumed be measured. Also that in all such experimentation it is necessary to run controls on a complete diet rather than to rely too much on standard figures. For this latter purpose it is merely necessary to add to the basal diets the "A" as butter fat and the "B" as dried yeast or otherwise to make them complete. Various special mixtures have been tested out for this purpose and the data already presented supplies the information necessary to construct such control diets. Professor Sherman has given me the following as a control diet on which he has raised rats at normal growth rate to the fifth generation:
One-third by weight of whole milk powder.Two-thirds by weight of ground whole wheat.Add to the mixture an amount of NaCl equal to 2 per cent of the weightof the wheat.
A control mixture based on Osborne and Mendel's data would have the following components:
Meat residue 19.6 per cent or casein 18 per cent.Starch 52.4 per cent or 49 per cent.Lard 15 per cent or 20 per cent.Artificial protein-free milk 4 per cent.Butter fat 9 per cent.Dried yeast 0.2 to 0.6 gram, daily.
The preceding description has applied especially to testing for the presence of the "A" or the "B" vitamine. When we come to the methods of testing for the "C" type it is necessary to change our animal. Rats do not have scurvy but guinea pigs do. The philosophy of the tests for the antiscorbutic vitamines then will be identical with that of the polyneuritic methods with pigeons, viz., preventive and curative tests with guinea pigs. The "C" vitamine is especially sensitive to heat and this fact enables us to secure a "C" vitamine-free diet. La Mer, Campbell and Sherman describe their methods as follows:
First select guinea pigs of about 300 to 350 grams weight. Test these with the basal diet until you secure pigs that will eat the diet. Those that will not eat it at first are of no use for testing purposes, for a guinea pig will starve to death rather than eat food he doesn't like. Having secured pigs that will eat they should on a suitable basal diet die of acute scurvy in about twenty-eight days. Their basal diet is as follows:
per centSkim milk powder heated for two hours at 110°C. in an air bath to destroy the "C" vitamine that might be present. . 30 Butter fat . . . . . . . . . . . . . . . . . . . . . . . . 10 Ground whole oats . . . . . . . . . . . . . . . . . . . . . 59 NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
They claim that when fruit juice addenda are given in minimal protective doses and calculated to unit weight bases, the results are comparable in precision to those of antitoxin experiments.
Old food should be removed every two days and replaced by new, cups being cleaned at the same time. Since this is a scurvy-producing diet its use is obvious. We can let the pig develop scurvy on it and then test the curative powers of the unknown by adding it to the diet or we can add it to the diet from the first and determine the dose necessary to prevent scurvy; or we can determine its effect in terms of a known antiscorbutic such as orange juice by combining it with measured quantities of the orange juice.
There are other diets that have been given for this purpose, e.g., Holst and Fröhlich induced scurvy by restricting animals to an exclusive diet of cereals (oats or rye or barley or corn). Hess and Unger have used hay, oats and water given ad libitum. All of these and others are subject to criticism on the basis that they are not necessarily adequate in other food factors and may therefore not be fair bases for testing the antiscorbutic powers of the unknown combined with them. Abels has recently shown that scurvy increases susceptibility to infections and believes that the scurvy hemorrhages are brought about by the toxic effects of infection. It is therefore desirable in testing for antiscorbutic power that the basal diet be itself as complete as possible in all factors except the absence of "C."
The study of rickets has already progressed to the stage of calculating rickets-producing diets and the methodology is identical with that for scurvy but this phase of testing still lacks evidence of an antirachitic vitamine and in that uncertainty it is hardly worth while to elaborate these diets here. The British diets are all based on Mellanby's contention that the "A" vitamine is the antirachitic vitamine. This view is not yet accepted by American workers.
In concluding this chapter it is sufficient to state that with our present methodology the accumulation of data for evaluating the vitamine content of various foods is still far from satisfactory and from the chemist's viewpoint the methodology is most unsatisfactory as a means of testing fractional analyses obtained in the search for the nature of the substance, both because of the time consumed in a single test and from the difficulty of using the fractions in feeding experiments when these fractions may themselves be poisonous or otherwise unsuited for mixture in a diet. It is obvious therefore that interest is keen in any possibility of devising a test that will be specific, quick and not require modification of the material tested, because of its unsuitability for feeding. In 1919 Roger J. Williams proposed a method that seemed to offer promise in these respects but which is not yet in the form for quantitative use. It offers promise that entitles it to a special chapter for discussion and the next chapter presents the present status of the so- called yeast test for vitamine "B."
Before turning to this test it is well to call attention here to the importance of the experimental animal. Without the polyneuritic fowls we might never have cured beri-beri, the guinea pig made the solution of the scurvy problem possible and if some way of inducing pellagra in an animal can be devised that scourge may yet be eliminated.
As far back as the days of Pasteur a controversy arose over the power of yeast cells to grow on a synthetic medium composed solely of known constituents. This controversy hinged on a discussion as to whether these media were efficient unless reinforced with something derived from a living organism. In 1901 Wildier in France published an article in which he showed that extracts of organic matter when added to synthetic media had the power to markedly stimulate the growth of yeast organisms. He did not attempt at the time to identify the nature of this stimulatory substance, but since it was derived from living organisms, he called it "Bios." Soon after the discovery of vitamines the bacteriologists began to discover that they or an analogous factor apparently played a part in the growth of certain strains of bacteria, especially the meningococcus. In 1919 Roger Williams working in Chicago University was struck with the bearing of Wildier's work on the vitamine hypothesis and formed the theory that Wildier's "bios" might be the water-soluble vitamine "B." He proceeded to test out this theory and demonstrated that extracts of substances rich in the "B" vitamine had a marked effect on the stimulation of yeast growth. He developed these experiments and devised a method of comparing the growth of yeast cells when stimulated by such extracts. The results were so striking as to appear to justify his view and he then suggested that his method might be used as a test for the measure of "B" vitamine in a given source. William's method consisted essentially in adding the extract of an unknown substance to hanging drops in which were suspended single yeast cells and observing the rate of growth under the microscope. Soon after, Miss Freda Bachman reinvestigated the problem with various types of yeast and found that practically all types of yeast respond to the stimulation of these "bios" extracts. Her method consisted in the use of fermentation tubes and the stimulatory effect was measured by the amount of CO_2 produced in a given time. By this method she confirmed Williams' view that the "bios" of Wildier was apparently identical with vitamine "B" and that most yeasts require this vitamine for their growth. She also suggested that her method might be made the basis of a test for vitamine content. In 1919 Eddy and Stevenson made extended experiments with these two methods in the attempt to improve the technique and make it serve as a quantitative measure. Their experiments served two purposes, first to bring out certain difficulties in the methods of the two authors from the quantitative viewpoint and the development of a technique to correct these difficulties and secondly to add more data bearing on the specificity of the test. Soon after their publication Funk became interested and coming to the same conclusions as to specificity devised a centrifugating method for measuring the yeast growth. Williams also improved his original method and devised a gravimetric method for the same purpose. From the viewpoint of methodology we now have methods which are suitable as quantitive procedures for determining the effect of extracts of unknown substances on yeast growth and hence if the stimulatory substance is vitamine "B," a means of determining within a space of twenty-four hours the approximate content of stimulatory material in a given source. Since the Funk method is the simplest of these and illustrates the principles involved it will suffice to describe that.
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 NH_4NO_3, 1 gram; Ca_3(PO_4)_2, 0.005 gram; MgSO_4, 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 31°C. 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 80°C. 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 thatoneof 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.
[Illustration: FIG. 7. GROWTH RATE OF YEAST UNDER ALFALFA EXTRACTSTIMULATION
This chart shows the effect of varying concentrations of an alfalfa extract on the growth rate of the yeast cell. The rate of growth was determined after the Funk method by centrifuging the cells after seventy- two hours incubation and measuring the volume in cubic centimeters. The shape of the curve shows that this method will not give comparative results unless the extracts tested are dilute enough for the determinations to fall in the steep part of the curve.]
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.
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| 0Milk, 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) | + | + | 0Wheat, maize, rice germ . . . . | ++ | +++ | 0Wheat, maize, rice bran . . . . | 0 | ++ | 0White wheat flour, pure corn | | |flour, polished rice, etc. . | 0 | 0 | 0Custard powders, egg substi- | | |tutes prepared from cereal | | |products . . . . . . . . . . | 0 | 0 | 0Linseed, millet . . . . . . . . | ++ | ++ | 0Dried peas, lentils, etc. . . . | | ++ |Pea-flour, kilned . . . . . . . | | 0 | 0Soy beans, haricot beans . . . | + | ++ | 0Germinated pulses or cereals . | + | ++ | ++Vegetables and fruits:| | |Cabbage, fresh, raw . . . . . . | ++ | + | +++Cabbage, fresh, cooked . . . . | | + | +Cabbage, dried . . . . . . . . | + | + |Very slightCabbage, canned . . . . . . . . | | |Very slightSwedes, 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 slightOrange juice, fresh . . . . . . | | | +++Raspberries . . . . . . . . . . | | | ++Apples . . . . . . . . . . . . | | | +Bananas . . . . . . . . . . . . | + | + |Very slightTomatoes, canned . . . . . . . | | | ++Nuts . . . . . . . . . . . . . | + | ++ |Miscellaneous:| | |Yeast dried . . . . . . . . . . | ? | +++ |Yeast extract and autolysed . . | ? | +++ | 0Meat extract . . . . . . . . . | 0 | 0 | 0Malt extract . . . . . . . . . | | + in some || | specimens |Beer . . . . . . . . . . . . . | | 0 | 0Honey . . . . . . . . . . . . . | | + |___________________________________|____________|____________|____________
+++ 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 | 0Vegetables:| | |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 . . . . . . . . . . . . . . | + | +++ | 0Rice polished . . . . . . . . . | 0 | 0 | 0Rice (whole grain) . . . . . . . | + | +++ | 0Rye . . . . . . . . . . . . . . | + | +++ | 0Corn embryo . . . . . . . . . . | | +++ |Corn (kaffir) . . . . . . . . . | | +++ |Corn (see maize) . . . . . . . . | | |Corn pollen . . . . . . . . . . | | ++ |Malt extract . . . . . . . . . . | 0 | 0 | 0Wheat bran . . . . . . . . . . . | 0 | + | 0Wheat embryo . . . . . . . . . . | ++ | +++ | 0Wheat endosperm . . . . . . . . | 0 | 0 | 0Wheat kernel . . . . . . . . . . | + | +++ | 0Other seeds:| | |Beans, kidney . . . . . . . . . | | +++ |Beans, navy . . . . . . . . . . | | +++ | 0Beans, soy . . . . . . . . . . . | + | +++ | 0Cotton seed . . . . . . . . . . | ++ | +++ |Flaxseed . . . . . . . . . . . . | ++ | +++ |Hemp seed . . . . . . . . . . . | ++ | +++ |Millet seed . . . . . . . . . . | ++ | +++ |Peanuts . . . . . . . . . . . . | + | ++ |Peas (dry) . . . . . . . . . . . | +? | ++ | 0Sun flower seeds . . . . . . . . | + | |Fruits:| | |Apples . . . . . . . . . . . . . | | ++ | ++Bananas . . . . . . . . . . . . | ? | ++ | ++Grapefruit . . . . . . . . . . . | | +++ | +++Grape juice . . . . . . . . . . | | + | +Grapes . . . . . . . . . . . . . | 0 | + | +Lemons . . . . . . . . . . . . . | | +++ | ++++Limes . . . . . . . . . . . . . | | ++ | ++Oranges . . . . . . . . . . . . | | +++ | ++++Pears . . . . . . . . . . . . . | | ++ | ++Raisins . . . . . . . . . . . . | | + | +Tomatoes . . . . . . . . . . . . | ++ | +++ | ++++Oils and fats:| | |Almond oil . . . . . . . . . . . | | 0 | 0Beef fat . . . . . . . . . . . . | + | 0 | 0Butter . . . . . . . . . . . . . | ++++ | 0 | 0Cocoanut oil . . . . . . . . . . | 0 | 0 | 0Cod liver oil . . . . . . . . . | ++++ | 0 | 0Corn oil . . . . . . . . . . . . | 0 | 0 | 0Cotton seed oil . . . . . . . . | 0? | 0 | 0Egg yolk fat . . . . . . . . . . | ++++ | 0 | 0Fish oils . . . . . . . . . . . | ++ | 0 | 0Lard . . . . . . . . . . . . . . | 0 | 0 | 0Oleo, animal . . . . . . . . . . | + | 0 | 0Oleo, vegetable. . . . . . . . . | 0 | 0 | 0Olive oil . . . . . . . . . . . | 0 | 0 | 0Pork fat . . . . . . . . . . . . | 0? | 0 |Tallow . . . . . . . . . . . . . | 0 | 0 | 0Vegetable oils . . . . . . . . . | 0? | 0 | 0Nuts:| | |Almonds . . . . . . . . . . . . | + | +++ |Brazil nut . . . . . . . . . . . | | +++ |Chestnut . . . . . . . . . . . . | | +++ |Cocoanut . . . . . . . . . . . . | ++ | +++ |English walnuts . . . . . . . . | | +++ |Filbert . . . . . . . . . . . . | | +++ |Hickory . . . . . . . . . . . . | + | + | +Pine . . . . . . . . . . . . . . | + | + | +Dairy products:| | |Butter . . . . . . . . . . . . . | ++++ | 0 | 0Cheese . . . . . . . . . . . . . | ++ | + | ?Condensed milk . . . . . . . . . | ++ | + | 0Cream . . . . . . . . . . . . . | +++ | + | ?Eggs . . . . . . . . . . . . . . | ++++ | ++ | 0Milk powder (skim) . . . . . . . | + | +++ | +?Milk powder (whole) . . . . . . | +++ | +++ | +?Milk whole . . . . . . . . . . . | +++ | +++ | ++Whey . . . . . . . . . . . . . . | + | +++ | +Miscellaneous:| | |Alfalfa . . . . . . . . . . . . | +++ | +++ | ?Blood . . . . . . . . . . . . . | Varies with sourceClover . . . . . . . . . . . . . | +++ | ++++ | ?Honey . . . . . . . . . . . . . | | ++ | 0Malt extract . . . . . . . . . . | 0 | 0 | 0Nectar . . . . . . . . . . . . . | 0 | 0 | 0Timothy . . . . . . . . . . . . | ++ | +++ |Yeast, brewers . . . . . . . . . | 0 | ++++ | 0Yeast cakes . . . . . . . . . . | 0 | ++ | 0Yeast extract . . . . . . . . . | 0 | +++ | 0____________________________________|___________|___________|____________
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.
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 100°C. 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 100°C. 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 100°C. 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.
When Funk first studied this substance he conducted all his evaporations in vacuo from fear that higher temperatures would prove destructive. Subsequent investigation however has shown that 100° has very little if any destructive effect if the vitamine is held in acid or neutral solution. Temperatures between 100° and 120° maintained in an autoclave at 15 pounds above normal pressure do tend to slowly destroy the factor. The extent of this destruction also varies with the character of the crude extract. In general, then, there is little fear of injuring this vitamine in ordinary cooking temperatures if the use of alkali is avoided.
The effect of alkali depends upon the temperature to a very marked degree. Osborne has recently reinvestigated this matter and finds that in the presence of a 0.1N solution of alkali at 20°C. there is very little destruction but that raising the temperature to 90°C. brings about a marked destruction. Seidell has shown that if the vitamine is absorbed by Lloyd's reagent and this reagent be then extracted with dilute alkali the vitamine passes into the alkaline solution. If the latter is neutralized quickly it is possible to recover most of the vitamine by this method. The effect of alkali becomes of practical importance to the housewife because of certain cooking habits. I refer to the well known practice of adding soda to the water in which vegetables are cooked to soften the vegetable and accelerate the cooking. Daniels and Loughlin in this country investigated this matter and came to the conclusion that this procedure did not produce enough destruction to be dangerous. Later the matter was studied by Chick and Hume in England and these investigators brought out a feature that had perhaps been overlooked in the previous work. Their point was that in ordinary feeding tests the results merely tell whether there is enough vitamine present to produce normal growth. Hence if the substance tested has much vitamine, a large part of it might be destroyed and this fact not appear in the test because enough might still be left to induce normal growth. By reducing the amount tested so that it was just adequate for normal growth and then applying the soda-cooking experimentation they showed that this method of cookery does do serious harm to the vitamine. From the practical point of view it is of course sufficient to show that enough is left after a cooking process to suffice for normal growth when the substance is taken in the portion sizes ordinarily eaten. The effect of alkali deserves more attention on the part of cooks and food preparateurs and we need more data concerning the minimal dose necessary to protect the human animal.
In neutral and acid solution it is perfectly safe to assume little destruction of this vitamin through heat and it is now common practice to boil sources with the extracting reagent and to use the steam bath freely to concentrate and evaporate these extracts. We have recently investigated the effect upon cabbage of cooking in a pressure cooker at eight pounds pressure. The cabbage so cooked, when dried and mixed so as to form 10 per cent of a basal vitamine free diet, yielded all the "B" vitamine necessary to produce normal growth in rats.
The very name of this vitamine indicates its ready solubility in water. It is also soluble in 95 per cent alcohol and either of these extractants may be used to obtain the vitamine. It is not readily soluble in absolute alcohol and 95 per cent is not as good an extractant as water. Substances rich in the vitamine apparently yield the latter more readily if they have first been subjected to autolysis or if the extracting fluid is acidified. Funk was the first to show that yeast produced a greater yield if it was allowed to autolyse before extraction with alcohol. However, Osborne and Wakeman have produced a method of treating fresh yeast by boiling it with slightly acidified water which seem as efficient as autolysis in the yield produced.
The various methods of extraction now in vogue have already been discussed in Chapter II and need not be repeated here. In general it is apparent that to obtain concentrates of high potency it is permissible to employ temperatures of 100°C. if we will maintain an acid or neutral reaction but that alkali should be avoided wherever possible and when its use is imperative the temperature must be kept below 20°C. or destruction will result. In applying this rule to cooking operations the results should be determined by direct tests rather than by assumptions based on these generalizations. It should also be noted that the alkalinity of a solution should be determined on the basis of hydrogen ion concentration and not on amount of alkali added since many substances have a marked buffer reaction.
The water-soluble "B" is not only soluble in water but can be dissolved in other reagents. Thus McCollum has shown that while benzene is of little value as an extractant of this vitamine, if we will first extract the vitamine with alcohol or water and deposit this on dextrin by evaporation it is then possible by shaking the activated dextrin with benzene to cause the vitamine to pass into solution in benzene. Voegtlin and Meyers have recently shown that it is soluble in olive oil and in oleic acid and their data suggest a new means of concentrating the substance which may be of value in tracing its character.
The "B" vitamine is relatively easily absorbed by finely divided precipitates. We have already referred to the use of fuller's earth for this purpose by Seidell. This adsorptive power sometimes manifests itself in the treatment of plant extracts. A watery extract of alfalfa can be made to throw down its protein complex by diluting it to 40 per cent with alcohol. Osborne reports however that this process frequently removes the vitamine also which appears to be thrown down with the precipitated material. This adsorptive power therefore often appears as a difficulty in the handling of the substance as well as a means of extraction. We have used Osborne's method with alfalfa extracts and find the above result is not by any means invariable, for in some of our extracts we retained the greater part of the vitamine. Kaolin and ordinary charcoal are not very good adsorbents but the latter can be activated to serve this purpose.
The elementary nature of the "B" vitamine remains a mystery. Extracts which contain it show the presence of nitrogen. Funk's earlier researches on yeast and rice polishings both yielded crystalline complexes which he analysed. His data on this subject follow:
A. The yeast complex
Crystals melting at 233°C. consisting of:
I. A complex melting at 229°C. and forming needles and prisms nearly insoluble in water and with the apparent formula of C_24H_19O_2N_5.
II. A complex melting at 222°C. and soluble in water. FormulaC_29H_23O_2N_5.
III. Nicotinic acid melting at 235°C. C_6H_5O_2N.
B. The rice complex
Crystals melting at 233°C. consisting of:
I. A complex melting at 233°C. and with a formula of C_26H_20O_9N_4.
II. Nicotinic acid melting at 235°C. C_6H_5O_2N.
Funk held at the time that the possible nature of the compound was:
HN| \OC C_16H_18O_6| /HN
It was this idea that led him to call it an "amine."
We are unable at present to report any nearer approach to the elementary analysis and all attempts at purification have shown a tendency to make the active substance either disappear entirely or else distribute itself over the several fractions instead of concentrating itself in one. Its basic nature seems to be well established by its behavior with phosphotungstic acid and its ready adsorption by carbons activated to take up basic substances.
The properties of this newest member of the family are still less defined. All are agreed that it is much more sensitive to heat and alkali than the other two. Temperatures above 50°C. are usually destructive though the time factor is extremely important as well as the reaction. Hess for example has found that the temperature used to pasteurize milk continued for some time, is more destructive to the vitamine than boiling water temperature continued for only a few minutes. The extent to which orange juice and tomato juice will resist high temperatures indicates the protective action of acids to be considerable.
Dr. Delf's experiments at the Lister Institute were especially directed to the behavior of this vitamine in cabbage. She first determined the minimum close of raw cabbage required to prevent scurvy in guinea pigs and found that it was less than 1.5 grams and more than 0.5 gram daily. When the cabbage was heated in water at 60°C. for an hour, symptoms of severe scurvy were just prevented by 5 grams of the cooked cabbage fed daily. By heating at 70°, 80°, 90° and 100° for the same length of time the 5 grams of cooked material could be made non-effective as a preventive. Her conclusions are that when cabbage is cooked for one hour at temperatures ranging from 80° to 100°C. the cabbage leaves lose about 90 per cent of the antiscorbutic power originally held by the raw equivalent. Sixty minutes at 60° or twenty minutes at 90° to 100° resulted in about 80 per cent destruction. Dr. Delf calls attention also to the fact that the effect of the heat is increased to only a slight degree by rise in temperature. Assuming that the effect of the rise is orderly, a temperature coefficient of 1.3 is indicated for each rise of 10°C. This low result suggests to Delf a contradiction to any theory which imputes to the vitamine enzyme or protein-like qualities and on the other hand suggests that the substance is much simpler in constitution. Her results also confirm Hoist and Fröhlich 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.
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: