Experimental Biology and Medicine

The term “fat soluble vitamine” or “vitamin A” is here employed to designate the substance or substances occurring in butter fat, egg fat, codliver oil and elsewhere by virtue of which growth is promoted when the diet is otherwise adequate, and the characteristic eye disease, noticed especially in rats by Osborne and Mendel, is prevented and may often be cured. If, as indicated by some recent observations, especially those of Hess, the relations of butter fat and codliver oil to rickets are so different as to suggest that their vitamines are different substances, it becomes conceivable that more than one substance having growth-promoting and “antixerophthalmic” properties may be embraced under the one term “fat-soluble vitamine” or “vitamin A” as now used.

I. Distribution of the Substance or Substances “Vitamin A” between the Fatty and Aqueous Phases in Milk.-Several years ago McCollum stated in a brief note that fat soluble A is about thirty times more soluble in fat than in water, in which case skimmed milk will contain about half as much of this vitamine as whole milk. On the other hand, Mellanby, studying experimental rickets in puppies, and Hess and Unger in their studies of the clinical r81e of the fat soluble vitamine, appear to have assumed that their experimental diets could contain considerable amounts of skimmed milk, either in fluid or solid form, and still be nearly devoid of the fat soluble vitamine. According to our experience, skimmed milk contains a very significant amount of fat soluble vitamine, probably about half as much as whole milk as McCollum's brief statement would imply.

Our experimental evidence of the presence of significant amounts of fat soluble vitamine or “vitamin A” in skimmed milk is two-fold.

From the dietetic standpoint the two main indications for the treatment of gastric ulcer are:

1. To protect the ulcer from mechanical and chemical injury, and

2. To maintain the nutrition of the patient at a level which will favor the healing of the ulcer.

In the author's opinion, none of the diets hitherto employed in the treatment of gastric ulcer fulfills these indications: in none of them do the therapeutic properties of food appear to have received adequate consideration. In general, the frequent administration of small or moderate quantities of milk constitutes the basis of diets for gastric ulcer. Even admitting the economic quality of the secretion called forth by milk, its hourly or two-hourly administration keeps the secretory glands of the stomach in a state of constant activity. Recognizing this some authors advise the neutralization of the gastric contents with magnesia or soda midway between the feedings. Since the majority of the symptoms of gastric ulcer, at least in the earlier stages, and possibly considerable injury, are referrable to hyperacidity, such a diet lacks physiological support. In addition to excessive secretion, the motor activity of the stomach is abnormally increased.

In the diet which I propose the objections referred to have been eliminated. Moreover, I believe that the maximum protection of the ulcer obtainable when food is given by mouth has been achieved. As in some other diets, complete rest is given the stomach for 3 to 5 days through the use of glucose enemata—300 C.C. of a 7 per cent. to 12 per cent. solution by the Murphy-drip method, three or four times a day. The quantity of water is sufficient to prevent thirst. Protein and fat are not added to the enemata because of the doubt of their absorption.

The purpose of this paper is to present a method whereby two neoplasms, that are histologically identical, can be shown, nevertheless, to be different in their physiological reactions. This experiment was begun June, 1920, and is still being continued. Enough data have been accumulated, however, to warrant a preliminary report.

The tumors employed are two adenocarcinomas of the mammary gland, that arose spontaneously in two female mice of a closely inbred strain, the second one arising some three weeks after the first. The mice have been rigidly inbred, brother to sister matings, for about eleven years. One would expect, in that time, that the strain must have become homozygous in all, or nearly all, genetic factors that no doubt underlie morphological and physiological characters. This conclusion is warranted by evidence obtained from implanting bits of the two tumors into mice of this strain. The trochar method of implanting the neoplastic tissue has been employed throughout the experiment. In this preliminary experiment both tumors grew in 100 per cent. of all mice inoculated, irrespective of whether the two tumors were inoculated into the same mouse or into separate individuals. In case the two were inoculated into the same animal, the first (dBrA) was always inoculated into the right axilla, the second (dBrB) into the left axilla. The growth curves for each were charted from weekly observations. There was apparently no effect of one tumor upon the other when growing in the same mouse, either in percentage of indications or rate of growth. They remained entirely distinct. The question naturally arose, “Are the two tumors actually identical?”

The complexity of the genetic factor system that evidently underlies susceptibility to transplantable tumors makes it highly improbable that two identical tumors could arise independently within three weeks of each other, even in the same relatively homozygous strain of mice.

In a paper published by one of us last year we called attention to the fact that it was quite likely that not all the toxic substances produced in cultures by bacteria can be peremptorily classified as either exotoxins or endotoxins. The writers working with a number of different organisms, biologically unrelated, have found poisonous substances of moderate potency developed both on fluid and on solid media which they believe should not be regarded at the present time either as specific or antigenic exotoxins, or as endotoxins. Indeed, it seems quite impossible at the present time to definitely classify these substances, for which reason they are referred to in our laboratory, for the present, as the “X” substances.

It is always dangerous to apply to any species of animal reasoning or theories deduced from experimental observations upon another species. In no phase of immunological work is such deduction more unjustified than in anaphylaxis where we know that the reactions induced in different species by a reinjection of proteins vary from each other in fundamental, physiological mechanism. It is of course unlikely, therefore, that we can justly draw conclusions from monkey experiments to conditions prevailing in human beings. But some of those who have regarded the occurrence of true anaphylaxis in the human being as at least doubtful, have, at the same time, cited the difficulty of producing anaphylactic reactions in monkeys in analogy. The problem is hardly one warranting a great deal of extensive research, but in connection with other work going on in our laboratory, we have found it important to investigate, for ourselves, the true conditions prevailing in the lower monkeys.

The production of antibodies in monkeys has for some time been a matter of controversy. Uhlenhuth 1 injected human serum into Macacus rhesus and found that specific precipitins were formed. Berkeley, 2 in 1913, reinvestigated this question on Macacus rhesus and on a Java monkey, and found that these animals treated with human, horse or dog sera, receiving four injections of 1 to 2 c.c. of these sera, produced neither precipitins nor complement fixing antibodies for the antigens used. He does not believe, therefore, that it would be possible to utilize antisera from lower monkeys for the forensic differentiation of human and monkey sera, as suggested by Uhlenhuth.

There has not been a great deal of systematic work published upon monkey anaphylaxis. Yamanouchi 3 was unable to produce active anaphylaxis in the lower monkeys against horse serum, and found that the serum of the lower monkeys did not sensitize guinea pigs passively.

The species used was Amœba proteus. By means of a micropipette liquids of various kinds were injected and the effect noted.

Oils form spherical droplets which are carried about in the cytoplasmic currents. A large drop is usually expelled. Immediately on being extruded the drop tends to flow over the surface of the Amœba thus partially engulfing it.

Distilled or spring water diffuses through the granular endosarc diluting it. The dilution is followed by a contraction of the endosarc and the massing of a hyaline fluid between the endosarc and the external pellicle of the Amœba. This dilates the area usually termed the ectosarc. The fluid soon accumulates on one side of the Amœba in the form of a blister which is ultimately pinched off.

A number of acid indicators were injected. The color reactions showed that the protoplasm of the Amœba is more acid than its environment. Upon death the colors change to those characteristic of the surrounding medium.

The difference in behavior of living protoplasm to “basic” and to “acid” dyes is striking. The “basic” dyes used were all chlorides of colored basic radicles and the “acid” dyes, potassium or sodium salts of colored acid radicles. In every case the “basic” dyes had a coagulating and the “acid” dyes, a liquefying effect on the protoplasm.

In the case of the “acid” dyes, when the effect is local, the healthy non-colored portion of the endosarc shrinks away from the colored liquefied area. This liquid accumulates under the pellicle in the form of a blister and is ultimately pinched off.

If the “basic” dye be relatively nontoxic its injection results in a coagulated area which is localized as a colored lump of inert material. This lump is carried about in the protoplasmic currents.

The recent World War was instrumental, in connection with the impetus given to the search for antiseptics and parasiticides, in calling attention to the powerful antiseptics and germicidal properties of various essential oils; as for instance so well described by Cavel 1 and Fränkel 2 . This antiseptic action of volatile oils is undoubtedly responsible for the remarkable medicinal virtues of ancient balsams, especially in relation to the treatment and healing of wounds. An ethnological study of the habits and customs of ancient peoples, especially in the Orient, cannot fail to call attention to the extensive employment of incense, perfumes and fumigations among these people. Incense was burned in connection with the religious and sacrificial offerings on the one hand, and for esthetic purposes in private homes on the other. Again, powerfully odoriferous substances are in great vogue in the Orient as perfumes and not only are such drugs applied to clothing but very frequently the orientals fumigate their naked bodies directly with the smoke of aromatic herbs and spices, burned over glowing coals. These circumstances suggested to the authors the possibility that such perfumes and fumes may serve a hygienic as well as esthetic purpose by exerting an inhibitory effect on the growth and spread of microörganisms. Accordingly some experiments were undertaken in order to ascertain the value of such an hypothesis.

The authors subjected a number of gums, spices and other odoriferous substances to destructive dry distillation by heat and studied the effects of the fumes produced thereby on bacterial cultures. The following substances were investigated; gum olibanum, gum glabanum, storax, myrrh, saffron, cinnamon, benzoinum and various samples of domestic and Japanese incense. The various substances were heated in glass test tubes over a flame and the fumes were allowed to come in contact with various microorganisms, care being taken to exclude the germicidal effects of steam.

Dutcher 1 concluded from experiments on pigeons that honey contained a small but negligible amount of antineuritic vitamine. Faber 2 did not find honey to protect against scurvy in guinea pigs.

The present authors carried out feeding experiments on albino rats, to determine whether the growth promoting accessories fat-soluble A and water-soluble B were present in white clover honey or in a mixed strained honey, and whether these honeys protected guinea pigs against scurvy.

Rats fed a diet lacking water-soluble B when compared with rats fed the same diet except that half of the carbohydrate was replaced by an isodynamic equivalent of either of these honeys, showed in five weeks an average gain in weight of only five grams in favor of the honey-fed rats.

Similar experiments on the addition of the strained honey to diets deficient in fat-soluble A showed almost similar failure of growth. The addition of comb honey, however, brought about cessation of decline and distinct gains in weight.

The addition of twenty per cent. of honey to the diets of guinea pigs did not prevent, or appreciably delay, the development of scurvy in these animals.

H. D. Moore 1 has described a race of guinea pigs that are naturally deficient in complement: the deficiency is inherited. A number of these animals were obtained from Dr. F. A. Rich of the Vermont State Agricultural Experiment Station and the sera of four were separately examined as to the presence of the components of complement. The findings were identical in all of the sera.

Both the mid-piece and the end-piece of complement are present. There is lacking only the so-called “third-piece,” which is the thermostable element of complement that is destroyed by cobra-venom and absorbed by yeast cells and bacteria.

By itself, the complement-deficient serum produces no hemolysis when used in a quantity 40 times that of the minimal completely heomlytic quantity of normal serum. When mixed with a small quantity of inactivated normal serum (guinea pig or human) the complement-deficient serum hemolyses in about three times the minimal hemolytic quantity of normal guinea pig's serum.

The third piece of complement is not identical with the lipoid cytozyme (thrombokinase), since the blood of the complementdeficient guinea pigs clots normally. The third piece of complement is not absorbed out of normal serum by six volumes of ether.

Our interest in the potassium content of human blood was aroused some time ago by the observation of Smillie 1 that poisoning may result from the administration of potassium salts to nephritic patients, and later confirmed in experimental uranium nephritis.

Observations on the potassium content of serum and whole blood have been made in fifteen cases, including four normal subjects, several cases of nephritis with marked nitrogen retention and a few miscellaneous cases. The potassium estimation was carried out by the cobaltinitrite method of Drushel essentially as described by Myers 2 in 1909 for spinal fluid.

The results obtained for serum in the four normal cases were somewhat less than 20 mg. per 100 c.c. calculated as K, and for whole blood about 8 to 10 times this amount. In five cases of nephritis with marked nitrogen retention the figures for the serum varied from 10 to 19 mg., in one mild case the potassium was 28 mg. while in a case of double polycystic kidney 35 mg. were found. The potassium content of the whole blood in these cases ranged from 52 to 148 mg. with an average of 100 mg. per 100 c.c. In all of these cases there was an associated secondary anemia and the total solids were diminished. No significant variation was found in a case of pure hypertension or a case of diabetic coma. Figures obtained for whole blood in two cases of pernicious anemia were low owing to the diminution in the red cell content.

In general the potassium content of whole blood tends to vary directly with the red cell content and the percentage of total solids. The few observations reported on cases of nephritis with marked nitrogen retention do not appear to support the suggestion that possibly some of the symptoms of uremia are due to a potassium poisoning as a result of retention of this element.

In two recent publications 1 , 2 on the toxic effect of mercuric chloride in normal and in naturally nephropathic animals, observations have been made concerning the relationship between the development of an acid intoxication and the acute kidney injury. In these animals the poison was given in large doses, 15 mgs. per kilogram, which eliminated the study of the development of the intoxication in the early stages.

The following preliminary note has as its object a study of the early stages of the development of an acid intoxication from mercuric chloride and the relationship of such a disturbance to renal function as is shown by the elimination of phenolsulphonephthalein.

Fourteen normal dogs have been used in the study. The animals were kept in metabolism cages, fed on bread with a small amount of cooked meat and given 500 C.C. of water by stomach tube daily. The animals were catheterized once a day and the urine examined for albumin and glucose. Cen trifugalized samples were examined for casts. The phenolsulphonephthalein test was conducted according to the technique of Rowntree and Geraghty. Blood urea determinations were made by the method of Marshall as modified by Van Slyke and Cullen. The reserve alkali of the blood (R.p.H.) was determined by the method of Marriott. After the commencement of the intoxication the urine was examined for the presence of mercury by the method of Elliott. Following two days of normal observations, the animals were given 4 mgs. per kilogram of mercuric chloride by stomach tube. Prior to administering the mercury the dogs were partially narcotized by a subcutaneous injection of 0.25 C.C. of a 4 per cent. solution of morphine sulphate per kilogram. Such a procedure prevents the vomiting of the mercury.

The early studies of G. Cohn on saligenin and homosaligenin and the recent investigations of D. I. Macht on benzyl alcohol have demonstrated that these substances are mild antiseptics. We have studied the action of saligenin,

and have found that all of these substances are weak antiseptics. One or two per cent. solutions kill the bacillus coli, staphylococcus albus, streptococcus hemolyticus pneumococcus and gonococcus after 60 minutes exposure. Piperonyl alcohol,

whose sodium salt is water soluble. Solutions of this compound possess about the the same antiseptic power as HgCl₂, i.e., I :10,000 solutions kill staphylococcus, bacillus coli and gonococcus in five minutes and streptococcus hemolyticus in ten minutes in bouillon cultures, while in beef serum bouillon I :500 solutions kill in five minutes. Mercury saligenin is not a dye, and it is much less irritant to mucous membranes than is HgC1₂, so that a I :I000 soluion can be held in the urethra for five minutes without burning or subsequent irritation. We are therefore testing it clinically in anterior gonorrheal urethritis with encouraging results.

has also been prepared. Its water soluble sodium salt has the same antiseptic action as that of the mercury saligenin, but no greater. We have also prepared the mercury compound of parahydroxymetanitrophenylcarbinol which yields a monomercury derivative, probably

The sodium salt of this compound has the same antiseptic strength as to the other two mercury compounds.

Attempts at the chemotherapy of trypanosomiasis (brucei) and spirillosis (obermeyeri) in rats by the use of saligenin, piperonyl alcohol and mercury saligenin have thus far yielded only negative results.