Experimental Biology and Medicine

In a paper dealing with the effect of opium alkaloids on the ureter, 1 the author has shown that the opium alkaloids in respect to their action on that organ can be divided sharply into two classes: the pyridin-phenanthrene group, of which morphin is the principal member on the one hand, and the benzyl-isoquinoline group, of which papaverin is the principal representative, on the other. The author showed that morphin and all its derivatives, with the exception of peronin or benzyl-morphin, all stimulate the contractions of the ureteral rings and increase their tonicity, while all the members of the papaverin group, namely, papaverin, narcotin and narcein inhibit the contractions of the ureter and lower its tonus.

In a subsequent paper 2 the author further analyzed the action of the opium alkaloids Qn the ureter and also studied a large number of other alkaloids and chemical compounds allied to them and was led to conclude that, as far as the stimulating action of the morphin group on the ureter is concerned, that stimulating effect is to be ascribed to the pyridin part of the morphin molecule. Furthermore, the author showed that the inhibitory and tonuslowering action of papaverin and its allied alkaloids appeared to be due to the presence of the benzyl grouping in the papaverin molecule. The skeleton structure of the morphin alkaloids and of the papevarin alkaloids is represented by the following structural formulæ:

It was further shown that when a combination of various opium alkaloids is used, the effect of the benzyl-isoquinolin members predominates in most cases. This explains also the anomalous effect of peronin or benzyl-morphin which, unlike all other morphin alkaloids, inhibits the contractions of the isolated ureter. 1

Following the studies of the effect of the opium alkaloids upon the smooth muscle of the ureter, the author investigated the action of the same alkaloids upon other kinds of smooth muscle.

Givens and Mendel 1 have found that “Administration of hydrochloric acid produced no significant effect upon the balance of N, Ca, and Mg in the dog.” Stehle 2 has stated that “the administration of hydrochloric acid by mouth to the dog causes an increased excretion of calcium and magnesium as well as of sodium and potassium.” New experiments have been conducted on two dogs which received a diet poor in lime, consisting of meat, cracker meal, lard, and agar. During two long periods each animal received daily 2 gm. hydrochloric acid. The results with such a diet corroborate our former statement and do not support the conclusion of Stehle.

Similar experiments were undertaken with these dogs on a diet enriched in lime by the addition of dried milk. With one animal a negative calcium balance was appreciably increased. It seems unlikely, however, that this outcome is a neutralization phenomenon. It has repeatedly been shown that when acid is introduced into the organism the increased production of ammonia is sufficient to neutralize it.

When sodium chloride instead of hydrochloric acid was added to the diet rich in lime the urinary calcium output was increased and the calcium balance was favorably affected.

The magnesium balance and the partition of this element between the different paths of excretion was not significantly altered in any of the experiments just described.

The experiments here reported are preliminary observations designed to test the suitability of a certain bacterial characteristic, the curve of viability in water, for use in more extensive studies of salt action planned for the future.

The organisms used were typical colon bacilli of the B. communis (sucrose negative), type, isolated from a polluted stream in the autumn of 1916. These bacteria were grown on standard nutrient agar slants at 37° C. for 16-18 hours. The growth from the surface was washed off in pure water, shaken for 5 minutes to break up clumps and added in I c.c. portions to the bottles of sterilized pure water or salt solution in which the viability was to be tested. These solutions had been warmed to 37° before seeding. Plates were made on agar one minute after seeding and the bottles were then replaced in the 37° incubator and kept there for 24-52 hours, the number of surviving organisms being determined at stated intervals. Counts were made on standard agar after incubation for 24 hours at 37° C.

The “pure water” used was twice redistilled in glass vessels and again redistilled in a block tin condenser. It gave a negative test for ammonia with Nessler's reagent. The sodium chloride was purified by treatment with calcium hydroxide (to remove magnesium), and with sodium carbonate (to remove calcium and barium), and was then recrystallized from pure hydrochloric acid and from pure water. The calcium chloride was twice recrystallized and the crystals dried in a current of air and in the drying oven.

The solutions to which the bacteria were exposed contained respectively, calciuni chloride alone, sodium chloride alone, and a mixture of the two salts in the ratio of five molecules of sodium to one modecule of calcium. The tonicity varied from nothing to a ten times isotonic condition.

It is evident that the calcium salt alone exerts a marked lethal action. Even a 0.1 isotonic solution shows a distinct reduction in bacterial numbers and increasing proportions grow more deadly till a 5.0 times isotonic solution produces sterility in 6 hours. The sodium salt is less harmful. Tonicities of 0.5 and 1.0 show a slight effect, and a tonicity of 5.0 a marked effect, but the latter concentration does not completely sterilize even after 52 hours. A mixture of the two salts in proportion of 5 parts of sodium to one of calcium is much more favorable than either salt alone. Up to and including a tonicity of 5.0 the bacteria actually increase in numbers. Line D of Table III shows that 3.5 per cent. sodium chloride plus 1.3 per cent. calcium chloride had no harmful influence, while in Table II .4 per cent. of sodium chloride alone, and in Table I less than .2 per cent. of calcium chloride alone exerted a demonstrable lethal effect.

The toxic effect of calcium and sodium salts and their antagonistic influence upon each other seem to be much the same among the bacteria as in the higher forms of life; and the viability curve in water would seem well suited for the study of such phenomena.

In diabetes there are abnormalities not only in carbohydrate but also in protein and fat metabolism as well. There is frequently a marked loss of protein in this disease, which may be either direct or the result of a failure on the part of carbohydrate to spare protein. Our experiments deal with one phase of protein metabolism in experimental diabetes, the influence of the removal of the major portion of the pancreas on the characteristic protein sparing action of glucose. For this purpose several female dogs weighing between nine and fifteen kilograms each were selected. Both the fasting nitrogen elimination and the effect of glucose feeding on this excretion were determined. During the fasting period the animals received only 40 c.c. of water per kilo. On the sugar days they received in addition 15 gm. of glucose per kilo. An after-period during which the glucose feeding was withdrawn was then introduced. Dr. Allen, of the Rockefeller Institute, then removed surgically about 90 per cent. of the pancreas. The wounds healed aseptically and the animals were allowed from two to five weeks to recuperate. The procedure consisting of fore-glucose and after-periods just described was repeated. In spite of the large loss of pancreatic tissue the protein-sparing action of glucose was not diminished in any of the animals. It appears that pancreatic tissue is provided far in excess of actual requirements or that this gland is not solely responsible for the protein-sparing action of carbohydrates. Dr. Ringer found this function intact in dogs made completely diabetic by means of phlorizin.

The experiments were made on etherized dogs. The intestines and stomach were dislocated and frequently handled. Two manifestations were studied, the blood pressure and the pain sense. We shall state merely the facts obtained.

About one third of the dogs were given a preliminary injection of morphin. Some of the animals were given ether by cone, others by the method of intratracheal insufflation. These factors had no apparent influence on the results.

There were 42 dogs which received surgical narcosis. In 15 the blood pressure never reached a level below 95 mm. even when the experiment lasted 4 hours or longer, and sensation returned as soon as the animals came out of ether. In 13 dogs the blood pressure reached a level below 70 mm. of mercury within two and a half hours and there was no return of pain sense. In 8 dogs the blood pressure sank to a low level but sensation of pain returned when ether was discontinued. In the remaining 6 dogs sensation was lost while the pressure was still above 95 mm. of mercury.

In 10 other dogs etherization was pushed to the point of intoxication. Interpretation of the results of this series is not simple, but it may be stated that the blood pressure, which fell in some instances to 42 mm. often continued to rise for as much as two hours after stopping the ether, eventually reaching a level above 90 mm. Pain sense sometimes returned after an even longer interval, but eventually recovered more completely than the blood pressure.

A recent communication 1 reported that the proteins of meat (dried skeletal muscle) and of a glandular tissue, liver, are adequate for the needs of nutrition in growth. The muscle contains a comparatively small quantity only of the water-soluble vitamine that is an essential dietary factor, whereas liver tissue, on the other hand, was found to be much richer in this. We have further observed a similar adequacy of the proteins of heart, kidney and brain tissue. The growth of rats to adult size has been secured upon diets in which these tissues, used desiccated, furnished the sole source of protein and water-soluble vitamine. In the cases of the liver, kidney and heart, at least, fat-soluble vitamine also seems to be present. The ether extract of pig's liver—liver oil—has manifested growth-promoting properties comparable to those described for butter fat and cod-liver oil; and it appears to be efficient as a curative agent for the type of eye disease which we have described to occur when animals are kept on a diet deficient in the fat-soluble vitamine. The details will be published in the Journal of Biological Chemistry.

In former investigations 1 we found that different strains of mice differ very much in their tumor rate and tumor age; and that these differences in tumor rate and tumor age were maintained and hereditarily transmitted to the following generations throughout the period of observation, notwithstanding the similarity of external conditions under which the animals were kept.

It was of interest to inquire how continued inbreeding of these various strains would affect the tumor rate and tumor age.

We found that while in the majority of strains the differences in tumor rate and tumor age remained constant in the successive generations, in others certain variations became noticeable as a result of the continued propagation of these strains. In a few cases an increase in the tumor rate occurred in the later generations, but in the large majority of cases in which a change was observed there was a tendency towards a decrease in the tumor rate which in some instances was very marked.

These changes in the tumor rate concomitant with continued inbreeding seem to depend on the following two factors: (I) As a result of long-con tinued in breeding in mice certain characteristics of a strain may change, the strain becoming less prolific and less vigorous. These changes may be accompanied by a lowering in the tumor rate. This was especially evident in the case of the “ No. 8 “ strain which had been inbred through the largest number of generations and through a considerable number of years. (2) In other cases it could be shown that in the course of continued propagation certain families, which were more resistant to certain diseases or which were naturally more prolific or otherwise favored by accidental conditions began numerically to preponderate in later generations. In some cases such families or substrains differed in their tumor rate from the main strain and thus as a result of selection within a strain differences in the tumor rate appeared in the course of continued inbreeding.

Our further investigations confirmed and still further emphasized our previous conclusion that on the whole in the mice with a higher tumor rate the tumors appear at a relatively earlier age than in those strains in which the tumor rate is lower.

It was demonstrated by Baumann and others that the toxic carbocyclic radicals split off from the proteins by the growth of intestinal bacteria are conjugated in the liver with sulphuric acid and thus detoxicated. It is also known that a portion of these aryl compounds are detoxicated by conjugation with glycuronic acid.

It is obvious that in testing for the functional capacity of the liver it is essential not only to test the glycogenic, ureogenic, biligenic etc., functions, but also to examine the detoxicating power of the hepatic tissue, in order to ascertain the complete working power of the gland. This is done in the following manner: The patient receives a dose of castor oil to evacuate his bowels. He is then kept on a known diet for two days, during which time the urine is collected, preserved and analyzed for total sulphur and ethereal sulphates. On the third day the patient receives a capsule containing 0.5 gm. thymol. The urine is collected for the next two days, preserved, and again analyzed for total sulphur and for ethereal sulphates.

If all the thymol were absorbed, and if all the thymol were conjugated with sulphuric acid and none with glycuronic acid, the 0.5 gm. thymol would be excreted as 0.766 gm. of thymol sulphuric acid. This would cause a marked increase in the percentage of ethereal sulphates in the urine. If the detoxicating power of the liver were below par, the thymol would not be conjugated, and the percentage of ethereal sulphates would be only slightly different from what it had been on the first two days-before the thymol administration.

We have found that this detoxicating function of the liver usually runs parallel with the other functional derangements of this organ.

The question recently has been raised as to whether scurvy in guinea pigs and in human beings is due to constipation and to the putrefactive activity of the bacteria in the intestinal tract. In order to determine this point a study of the intestinal flora was made in guinea pigs on a normal diet, on a diet which produced scurvy, and again on a diet which cured this disorder. For this purpose cultures and grain stained films were made from the feces, as well as from the different levels of the small and large intestine immediately after chloroforming the animals. This study on guinea pigs is portrayed in Table I.

It will be seen that proteolytic bacteria other than subtloid types were not found in the intestinal tract; that the bacteria which were cultivated were merely those found in the outer world, for example, on dried foodstuffs. Similar organisms, indeed, were cultivated from the hay and oats which were fed these animals. Attention may be called to the fact that with none of the diets did B. coli develop, and that it was isolated from only one animal. The number of viable bacteria will also be noted as remarkably few, generally less than 1,000 per milligram of material. Among these there were hardly any that were actively proteolytic. Furthermore, a point of prime importance, there was no change in the flora on adding orange juice to the diet, although the scorbutic symptoms disappeared. It is our opinion, therefore, that most of the bacteria entering the intestinal tract are destroyed, and that the pabulum is not such as to encourage the growth of native intestinal bacteria.

A similar study was carried out on the stools of three infants suffering from scurvy.

A study has been made of the factors involved in the development of the red-brown color produced on heating solutions containing dextrose, picric acid and sodium carbonate.

The dextrose concentrations chosen corresponded in range to those found in the blood. Temperature levels above 100° C. were attained by heating in an autoclave, and the required accuracy of temperature control was obtained by means of an electric thermo-regulator.

The amount of color produced did not vary directly with the amount of sugar present except at a certain definite concentration of sodium carbonate. At low alkali concentrations small amounts of sugar gave relatively less color than large amounts, while at high alkaline concentrations, the reverse was found. The optimum alkali concentration varied with temperature changes.

With every increase in temperature from 80 to 130° C. there was an increase in color production, and at all temperatures the more prolonged the heating, the deeper was the color.

All observers are agreed that the catalytic activity of the liver is far in excess of that of muscle, and it is generally assumed, I think, that this is because the liver contains more catalase. Assuming the liver to be five times more active than muscle, a mixture of muscle and liver in the proportion of five to one ought to give the same catalytic activity as double the quantity of muscle alone. This is not the case, as the following experiment shows.

A 50 per cent. solution of commercial hydrogen peroxide, representing 1.5 per cent. H₂O₂, was used. The oxygen was collected over water in the usual way, and the volume reduced to 0° and 760 mm. Hg. The time was 10 minutes.

I gm. rat's muscle gave 50 C.C. oxygen.

I gm. rat's liver gave 244 C.C. oxygen.

0.5 gm. muscle + 0.1 gm. liver gave 250 C.C. oxygen.

To be on the safe side, the assumption has here been made that the liver is five times as active as the muscle, yet the mixture, instead of having the activity of the muscle, has the activity of the liver.

Assuming the liver to be ten times as active as muscle, the following combination was made: 0.5 gm. muscle + 0.1 gm. liver. This combination gave 224 C.C. of oxygen.

Blood clot + muscle gives similar results, and to a less extent, spleen also. The difficulty of getting spleen free from blood complicates the matter.

The above facts appear to be certain. The interpretation is not so clear, but it may be the liver secretes an accelerator of catalase, perhaps in the nature of an internal secretion; and it may be it is this accelerator that causes the catalase activity to vary in the different tissues and organs.