The Vitamine Manual Part 2

It will be evident that in the absence of exact tests for a substance which is unknown chemically the problem of detecting its presence must be a matter of indirect evidence. When a chemist is presented with a solution and asked to determine the presence or absence of lead in that solution he knows what he is seeking, what its properties are and how to proceed to not only determine its presence but to measure exactly the amount present.

No such possibility is present in a test for vitamines, but this lack of knowledge as to the vitamine structure has not left us helpless. We do know enough of its action to permit us to detect its presence and the technique that has been developed for this purpose is now well standardized and involves no mysteries beyond the comprehension of the layman. In the present chapter is outlined the development of vitamine testing together with a discussion of some of the deficiencies and the problems for the future that these deficiencies suggest.

When Casimir Funk made his original studies of the chemical fractions of an alcohol extract of rice polishings he utilized a discovery of the Dutch chemist Eijkman. We have already referred to this discovery, viz., that by feeding polished rice to fowls or pigeons they could be made to develop a polyneuritis which is identical in symptoms and in response to the curative action of vitamine, to the beri-beri disease. A normal pigeon can be made to eat enough rice normally to develop the disease in about three weeks. The interval can be somewhat shortened by forced feeding. As soon as the symptoms develop the bird is ready to serve as a test for the presence or absence of the antineuritic vitamine. If at this time we have an unknown substance to test it can be administered by pushing down the throat or mixed with the food or an extract can be made and administered intravenously. If the dose is curative, the bird will show the effect by prompt recovery from all the symptoms of the disease in as short a time as six to eight hours. Such a procedure provides a qualitative test which can be made roughly quant.i.tative by varying the dosage until an amount, just necessary to cure the bird in a given time is found and then expressing the vitamine content of the food in terms of this dosage, in such an experiment the value is obviously based on the curative powers of the vitamine source. Another way of applying the test is to determine just how much of the unknown must be added to a diet of polished rice to prevent the onset of polyneuritic symptoms. Such a determination will give the content in terms of preventive dosage. Both methods have been extensively applied and the following tables compiled from the Report of the British Medical Research Committee ill.u.s.trate both the method and some of its results:

_Minimum daily ration that must be added to a diet of polished rice to prevent and to cure polyneuritis in a pigeon of 300 to 400 grams in weight. The weights are given in terms of the natural foodstuff._

____________________________________________________________ AMOUNT NECESSARY FOODSTUFFS AMOUNT NECESSARY FOR DAILY PREVENTION TESTED FOR CURE ______________________ __________________ __________________ _grams_ _grams_ 1.5 Wheat germ (raw) 2.5 2.5 Pressed yeast 3.0-6.0[1]

3.0 Egg yolk 60.0[2]

20.0 Beef muscle 140.0[2]

3.0 Dried lentils 20.0[2]

______________________ __________________ __________________

[Footnote 1: Autolysed.]

[Footnote 2: Alcohol extract.]

These values ill.u.s.trate both the method and its value in comparing sources. Unfortunately experience has shown that polyneuritis is amenable to other curative agents to a greater or less extent and it is difficult to be sure whether the curative or preventive dose represents merely the vitamine content of the unknown or is the sum of all the factors present in the curative or preventive material. In comparing the value of different chemical fractions it probably gives a fair enough basis for evaluating their relative power but it is not entirely satisfactory as a quant.i.tive measure of vitamine content.

In America the comparison of vitamine content has been largely based on feeding experiments with the white rat. No other animal has been so well standardized as this one. Dr. Henry Donaldson of the Wistar Inst.i.tute of Philadelphia has brought together into a book ent.i.tled _The Rat_ the acc.u.mulated record of that Inst.i.tution bearing on this animal. This book provides standards for animal comparisons from every view point; weight relation to age, size and age, weight of organs and age, s.e.x and age and weight, etc. This book together with the experience of many workers as they appear in the literature and especially the observations of Osborne and Mendel have made the rat an extremely reliable animal upon which to base comparative data. The omnivorous appet.i.te of the animal, his ready adjustment to confinement, his relatively short life span, all contribute to his selection for experimental feeding tests. Another important reason for his selection is that being a mammal we may reasonably consider that his reactions to foods will be more typical of the human response than would another type, the bird for example. It is perhaps necessary to sound a warning here, however, and point out the danger of too great faith in this comparability of rat and man or in fact of any animal with man. In the case of the rat he has been found useless for the study of "C"

vitamine for the simple reason that rats do not have scurvy. In general however his food responses to the vitamines, at least of the "A" and "B"

types, have proved, so far as they have been confirmed by infant feeding, to be reasonably comparable.

Provided with the experimental animal the next step was to devise a basal diet which should be complete for growth in every particular except vitamines. Such basal diets have been a process of development. The requirements for such a diet are the following factors:

1. It must be adequate to supply the necessary calories when eaten in amounts normal to the rat's consumption.

2. It must contain the kinds of nutrients that go to make up an adequate diet and in the percents suitable for this purpose.

3. It must contain proteins whose quality is adequate, for growth, i.e., which contain the kinds and amounts of amino acids known to fulfil this function.

4. It must be digestible and palatable.

[Ill.u.s.tration: FIG. 3. TWO TYPES OF EXPERIMENT CAGES DEVISED BY OSBORNE AND MENDEL

These are manufactured by the Herpich Co. of New Haven, Conn.]

5. It must be capable of being supplemented by either or both vitamines in response to the particular test it is devised to meet and when both are present in proper amounts it must produce normal growth and serve as a control.

[Ill.u.s.tration: FIG. 4. A METABOLISM CAGE DEVISED FOR USE IN THE AUTHOR'S LABORATORY

The cages being bottomless are readily cleaned. They are set on circles of wire mesh over galvanized iron funnels permitting urine and feces to pa.s.s through. A second screen over the collecting cup and of fine mesh separates the feces from urine and also collects scattered food.]

In building up such a diet many experiments have been combined and thanks largely to the efforts of Osborne and Mendel and McCollum in this country, we have a thoroughly standardized procedure even extending to types of cages and care best suited to normal growth and development. For clearer appreciation of the nature of these diets and their preparation we have summarized in the following pages the combinations used by the princ.i.p.al contributors to the subject in this country.

[Ill.u.s.tration: FIG. 5. ILl.u.s.tRATING THE USE OF THE CHATILLON SCALE FOR RAPID WEIGHING OF ANIMALS

The dial is so made that it can be set to counterbalance the weight of the cage and the weights read directly. This is also used for weighing food.]

[Ill.u.s.tration: FIG. 6. SAMPLE LABORATORY RECORD]

It is at once obvious from the table that the testing value of these basal diets demands the absence of the two vitamines in the protein, carbohydrates and fat fractions. To make sure of this absence various methods have be devised to attain the maximum purity. The authors recommend the following procedure:

_a_. To purify the casein or other protein used. Boil the protein three successive times (it is a.s.sumed that the original is already as pure as it is possible to obtain it by the usual methods of preparation) for an hour each time, with absolute alcohol, using a reflux condenser to prevent loss of alcohol. Filter off the alcohol each time by suction. This process will take off all the adherent fat and hence all the "A" vitamine that might be present. The casein is then dried and ready for use. In certain experiments the authors use meat residues instead of a single protein.

This they prepare as follows: Fresh lean round of beef is run through a meat chopper and then ground to a paste in a Nixtamal mill, stirred into twice its weight of water and boiled a few minutes. The solid residue is then strained, using cheese cloth, pressed in the hydraulic press and the cake stirred into a large quant.i.ty of boiling water. After repeating this process of washing with hot water the extracted residue is rapidly dried in a current of air at about 60C. This dried residue may then be further purified with the absolute alcohol treatment as described for casein.

_b_. To purify the carbohydrate they treat starch in exactly the same way as the casein.

_c_. To purify the lard. This is melted and poured into absolute alcohol previously heated to 60C., cooled over night and filtered by suction. This process is repeated three times and the resulting solids dried in a ca.s.serole over a steam bath.

_d_. When b.u.t.ter fat is used to provide a source of "A" vitamine it is prepared as follows: b.u.t.ter is melted in a flask on a water bath at 45C. and then centrifugated for an hour at high speed. This results in a separation of the mixture into three layers: (a) Clear fat, containing the "A" vitamine and consisting of 82 to 83 per cent glycerides. This is siphoned off and provides the b.u.t.ter fat named in the diets, (b) An aqueous opalescent layer consisting of water and some of the water-soluble const.i.tuents of the milk. This is rejected. (c) A white solid ma.s.s consisting of cells, bacteria, calcium phosphate and casein particles.

This is also rejected.

_Osborne and Mendel's diet_

(Figures give the per cent of each ingredient in the diet)

_________________________________________________________________________ INGREDIENTS VITAMINE FREE CONTAINING A ONLY _______________________________ _________________ _______________________ I II III IV V VI VII Purified protein as casein, lactalb.u.min, edestin, egg alb.u.min, etc. . . . . . . 18.0 18.0 18.0 18.0 18.0 or Meat residue . . . . . 19.6 19.6 Carbohydrates in the form of: Starch . . . . . . . . . . . 29.5 54.0 52.4 29.5 54.0 54.0 52.4 Sucrose . . . . . . . . . . . 15.0 15.0 Fat in the form of: Lard . . . . . . . . . . . 30.0 24.0 24.0 15.0 15.0 15.0 15.0 b.u.t.ter fat . . . . . . . . . 15.0 9.0 9.0 Egg yolk fat . . . . . . . . 9.0 Cod liver oil . . . . . . . . Salts in the form of: Salt mixture I . . . . . . . 2.5 2.5 or Artificial protein-free milk (Mixt. IV) . . . . . . 4.0 4.0 4.0 4.0 4.0 or Protein-free milk . . . Roughage in the form of: Agar-agar . . . . . . . . . . 5.0 5.0 _______________________________ _____ _____ _____ _____ _____ _____ _____ Total . . . . . . . . . . . . 100.0 100.0 100.0 100.0 100.0 100.0 100.0 _______________________________ _____ _____ _____ _____ _____ _____ _____

_________________________________________________________________________ INGREDIENTS A ONLY CONTAINING B ONLY _______________________________ ___________ _____________________________ VIII IX X XI XII XIII XIV Purified protein as casein, lactalb.u.min, edestin, egg alb.u.min, etc. . . . . . . 18.0 18.0 18.0 18.0 18.0 18.0 or Meat residue . . . . . 19.6 Carbohydrates in the form of: Starch . . . . . . . . . . . 45.0 45.0 29.5 54.0 52.4 26.0 29.0 Sucrose . . . . . . . . . . . 15.0 Fat in the form of: Lard . . . . . . . . . . . 15.0 27.0 30.0 24.0 24.0 28.0 25.0 b.u.t.ter 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, x.x.xiv, 131.]

_Salt mixture I (after Rohman)_

_grams_ Ca_3(PO_4)_2 . . . . . 10.00 K_2HPO_4 . . . . . . . 37.00 NaCl . . . . . . . . . 20.00 Na citrate . . . . . . 15.00 Mg citrate . . . . . . 8.00 Ca lactate . . . . . . 8.00 Fe citrate . . . . . . 3.00 ______

Total . . . . . . . . 100.00

_Artificial protein-free milk_

_grams_ CaCO_3 . . . . . . . . 134.8 MgCO_3 . . . . . . . . 24.2 Na_2CO_3 . . . . . . . 34.2 K_2CO_3 . . . . . . . . 141.3 H_3PO_4 . . . . . . . . 103.2 HCl . . . . . . . . . . 53.4 H_2SO_4 . . . . . . . . 9.2 Citric acid: H_2O . . . 111.1 Fe citrate: 1.5H_2O . . 6.34 KI . . . . . . . . . . 0.020 MnSO_4 . . . . . . . . 0.079 NaF . . . . . . . . . . 0.248 K_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 110C. 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 quant.i.ty 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 b.u.t.ter 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"

_________________________________________________ ____________________