The Vitamine Manual Part 3

Cereal testing combinations ______________________________________________________________________ Wheat . . . . . . 56.6 70.0 Wheat embryo . . . 13.3 Corn . . . . . . . 71.3 Oats . . . . . . . 60.0 Skim milk powder . 6.0 Dextrin . . . . . 31.5 76.4 18.0 30.3 20.0 81.0 Salt mixture 185 . 3.7 Salt mixture 314 . 5.3 Salt mixture 318 . 6.9 5.0 Salt mixture 500 . 4.7 Salt mixture ? . . 6.0 b.u.t.ter fat . . . . 5.0 5.0 5.0 5.0 5.0 5.0 Agar . . . . . . . 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 cla.s.ses. The first group correspond to those of Osborne and Mendel and are available for general testing of any unknown. The cereal combinations are so const.i.tuted 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 b.u.t.ter 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 subst.i.tuting lard for the b.u.t.ter 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 diets_ per cent Casein (washed with water containing acetic acid) . . . . . 18.0 Dextrin . . . . . . . . . . . . . . . . . . . . . . . . . . 73.3 Ether extracted wheat embryo as source of vitamine "B" . . . 3.0 Salt mixture (McCollum, no. 185) . . . . . . . . . . . . . . 3.7 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 b.u.t.ter 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: _grams_ NaCl . . . . . . . . . . . . . . . . . . . . . . . . . 0.202 Anhydrous MgSO_4 . . . . . . . . . . . . . . . . . . . 0.311 K_2HPO_4 . . . . . . . . . . . . . . . . . . . . . . . 1.115 Ca lactate . . . . . . . . . . . . . . . . . . . . . . 0.289 Na_2HPO_4:l2H_2O . . . . . . . . . . . . . . . . . . . 0.526 Ca_2H_2(PO_4)_2:H_2O . . . . . . . . . . . . . . . . . 1.116 Fe citrate . . . . . . . . . . . . . . . . . . . . . . 0.138 No. 35 consisted of: NaCl . . . . . . . . . . . . . . . . . . . . . . . . . 1.00 CaCO_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 a.s.sumed that its addition will not appreciably effect the optimum concentrations of nutrients, etc., and for such experiments no allowances are made for the const.i.tuents in the unknown. For example let us a.s.sume 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 a.s.sumed 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 a.n.a.lysis 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 ill.u.s.trative 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 b.u.t.ter fat until the amount just produces normal growth. If now we know just what amount of b.u.t.ter fat suffices for this purpose when used alone we can calculate the part of the b.u.t.ter 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 a.s.sume that 5 per cent of b.u.t.ter fat in a given diet is sufficient to supply the "A" necessary for normal growth. a.s.sume that the addition of 5 grams of the unknown in 100 grams of the b.u.t.ter-free diet fails to produce normal growth but that by adding 2 per cent of b.u.t.ter 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 b.u.t.ter fat, i.e., is equivalent to 3 grams of b.u.t.ter fat or expressed in per cents the substance contains 0.6 or 60 per cent of the "A" found in pure b.u.t.ter 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 acc.u.mulation 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 appet.i.te, 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 appet.i.te 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 b.u.t.ter 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 weight of 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.

b.u.t.ter 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 antis...o...b..tic 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 cent_ Skim milk powder heated for two hours at 110C. in an air bath to destroy the "C" vitamine that might be present. . 30 b.u.t.ter 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 ant.i.toxin 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 antis...o...b..tic such as orange juice by combining it with measured quant.i.ties of the orange juice.

There are other diets that have been given for this purpose, e.g., Holst and Frohlich 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 antis...o...b..tic 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 antis...o...b..tic 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 acc.u.mulation 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 a.n.a.lyses 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 quant.i.tative use. It offers promise that ent.i.tles 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.

CHAPTER IV

THE YEAST TEST FOR VITAMINE "B"

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 const.i.tuents. 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 a.n.a.logous 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 quant.i.tative measure. Their experiments served two purposes, first to bring out certain difficulties in the methods of the two authors from the quant.i.tative 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 quant.i.tive 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 s.p.a.ce of twenty-four hours the approximate content of stimulatory material in a given source. Since the Funk method is the simplest of these and ill.u.s.trates 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 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 acc.u.mulate 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 quant.i.tative 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.

[Ill.u.s.tration: FIG. 7. GROWTH RATE OF YEAST UNDER ALFALFA EXTRACT STIMULATION

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.

CHAPTER V

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 cla.s.sification 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 cla.s.sifications 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 quant.i.tative 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.

TABLE 1