Experimental Biology and Medicine

Diplococcus pneumoniœ, Eberthella typhosa, and Escherichia coli were grown 24 hours at 37.5°C in meat infusion or meat extract culture medium enriched with 1% of peptone, 0.7 to 1.8% of Na₂HPO₄. 12 H₂O, and 1% of glucose. The pneumococcus was grown under anaerobic as well as highly aerobic conditions. The principal products were lactic acid, formic acid, acetic acid, and ethyl alcohol. The last 3 products appeared in the approximate ratio of 2 to 1 to 1. The reactions representing their formation most likely are: Glucose → hexose diphosphate → 2 triosephosphate → glycerophosphate and phosphoglycerate. Glycerophosphate → C₂H₅OH + HCOOH. Phosphoglycerate → phosphopyruvate → CH₃COOH + HCOOH. These are anaerobic mechanisms. Only a trace of CO₂ was obtained from pneumococci.

Rapidly growing bacteria may metabolize sugar almost entirely by anaerobic mechanisms when grown under relatively aerobic conditions. The same intermediary reactions are probably also utilized by microörganisms when growing in tissues in the course of an infection.

Wilson 1 in summarizing the work on bacillemia in human tuberculosis up to 1933, concludes that bacillemia, detectable by present methods, is, except as a transitory phenomenon, rarely present until the disease has assumed an acute phase. Because of this discrepancy between the reported rare occurrence of bacillemia and the common demonstration of hematogenous tuberculous lesions at necropsy, it seems important to determine the frequency and significance of bacillemia as a transitory phenomenon in tuberculosis.

Corper, 2 using dogs as experimental animals, reports that large doses of tubercle bacilli given intravenously produced a rapidly fatal bacillemia without the formation of tubercles. Smaller doses of tubercle bacilli produced no bacillemia and no tuberculous lesions. A search of the literature reveals no report of daily blood-cultures over a period of several months, which would seem to be the most adequate means of determining the frequency of transitory or sporadic bacillemia.

In our experiment, dogs were used because of the relative ease of obtaining an adequate quantity of blood daily for culture, and because the relative resistance of the dog to tuberculosis is more closely parallel to that of the human, than is the low resistance of the rabbit and guinea pig.

Experimental tuberculosis was produced in dogs by the intravenous injection of large doses of a virulent human type of tubercle bacilli (H37) in a suspension of Kaolan, mineral oil, and normal saline. This method was found to produce embolic tubercles limited largely to the lungs and tending to heal or to progress slowly.

Tuberculous abscesses were produced by injection of a similar suspension of tubercle bacilli into the chest wall of dogs. These lesions tended to break down and discharge for a time and then heal.

The toxicity of gonococcus and meningococcus cells is appreciably but temporarily reduced by acetylation with ketene.

The administration of APL to infantile female rats causes masculinization of the clitoris consisting of gross enlargement and the development of an os priapi.

Collip and coworkers have recently reported metabolic stimulation in rabbits, guinea pigs, and rats occurring within a few hours following injection of certain pituitary extracts. 1 , 2 , 3 The substance in the extracts producing this stimulation was called the “specific metabolic principle” of the pituitary. From the similarity in chemistry and occurrence in extracts, as well as from certain differences between these and other known pituitary hormones, it was suggested that the specific metabolic principle and the melanophore hormone might be identical. In view of the previous report of Zondek and Krohn 4 that “Intermedin” did not affect the metabolism of rabbits, it seemed worth while to study the subject further.

Accordingly, the effect on oxygen consumption of various melanophore hormone preparations was studied in rats. A modified Benedict spirometer was used to measure oxygen consumption. The rats were kept at room temperature but the metabolism was measured at 28°C. Comparable results were obtained with normal, thyroidectomized and hypophysectomized rats, and with rats recently fed as well as rats fasted 12-18 hours.

The usual procedure was to measure the basal rate in the morning, inject the extract after 2-3 hours, and measure the oxygen consumption over the following period of 8 hours without removing the rat from the machine. Using this technic, basal oxygen consumption for normal rats averaged 211 l/sq.m./day, for thyroidectomized rats 160, and for hypophysectomized rats 137. Control studies are shown in Fig. 1. On the ordinate is the percentage change in oxygen consumption over the basal value. Each column represents the highest reading obtained after injection in one experiment. Fig. 2 summarizes only the positive findings with various pituitary extracts.

The hypertrophy of the liver, kidneys, heart, and spleen caused by hyperthyroidism occurs in the absence of the pituitary; that of the adrenals does not.

Extensive studies on the sleep of the insane by Ladame 1 and Courbon 2 have attempted to correlate particular types of insanity with various types of sleep habits. Muncie 3 and Richter 4 have also made observations on the sleep characteristics of psychotic patients. Forbes 5 and Page 6 have compared the motility during sleep of psychopathic patients with that of normal individuals. Their findings indicate that in several types of psychopathic patients the motility during sleep is fairly comparable to that of normal individuals. The motility of catatonics in Page's study was found to be quite low and more or less uniform throughout the night, while normal individuals and manic-depressives and postencephalitic parkensonians all moved more during successive thirds of the night.

Using recording devices 7 for measuring the time spent in motility, the amount of motility, and the distribution of the night's movements, supplemented by regular hourly observations recorded on sleep charts by nurses and attendants, we studied a number of psychopathic patients and mentally defective individuals.

Psychopathic patients in the Division of Psychiatry of the University of Chicago Clinics (Billings Hospital) whom we studied were divided into 3 groups: psychoneurotics, schizophrenics, and affective psychotics. Only those subjects whose sleep motility was observed for at least 10 days were used in the analysis of the results. The results of 408 nights' sleep of 24 patients are given in Table I. Thus we see that all of these psychopathic patients showed sleep habits and motility quite similar to that found in normal persons. 8 Just as in the normal individual, the incidence of spontaneous awakening in each class of psychopaths was over twice as great in the second half of the night as during the first half of the night's sleep.

The sensitivity of an animal to insulin is usually judged by the extent and duration of the depression of the blood sugar level after the administration of insulin. The factors which are usually considered to exert an important influence upon the insulin depression curve are: the available carbohydrate stores in the liver and the ease with which these stores may be mobilized. 1 The state of the endocrine glands is important in both these respects, and it is well known that adrenalectomy, 2 hypophysectomy, 3 and thyroidectomy 4 increase the sensitivity to insulin. Another important factor in insulin sensitivity, to which less attention has been paid, is the rate of destruction or inactivation of insulin by the tissues of the body. That this factor is significant is shown by the well-established fact that the same amount of insulin becomes more effective when administered in divided doses or by prolonged constant injection than when given in a single dose. 5

The exact mode of insulin inactivation in the body is unknown, but ever since it was shown that insulin is a protein, the supposition has been that it is destroyed by proteolytic enzyme systems. 6 However, it is also known that the physiological action of insulin depends upon the integrity of its S-S groups, 7 , 8 and that the in vitro reduction of these groups by sulphydryl compounds renders the insulin inert upon subsequent injection. 9-13 That a similar mode of inactivation applies to insulin in the living organism is indicated by the work of Jacobs, who has recently demonstrated that the administration of cysteine decreases the reaction to subsequently injected insulin. 14

A combination of type-specific serum, administered intraperitoneally, and sulfapyridine, administered by stomach tube, was more efficacious in the treatment of experimental lobar pneumonia of rats than either alone, when treatment was begun within 4 hours after infection. The optimal dose of sulfapyridine under the conditions of our experiments was 0.5 to 1 g per kilo of rat per day. After the infection was well established, following intrabronchial insufflation of relatively large doses (0.1 cc of 10⁻⁴ and 10⁻⁵ of 18-hour bouillon cultures) of pneumococci, serum was more efficacious than sulfapyridine in preserving life, but a combination of the 2 did not show a significant reduction in mortality even with the smaller infecting dose.

The work of several investigators who have studied the vitamin B₁ content of evaporated milk has been recently reviewed by Daniels. 1 She reported two trends in results: certain workers had found about 50% destruction of vitamin B₁ when evaporated milk was compared with raw milk, whereas others had observed only 0-20% destruction. Using a modification of the 10-day rat growth technic of Schlutz and Knott, 2 Daniels assayed raw Guernsey milk and commercial evaporated milk. Her results indicated 60% less vitamin B₁ in the evaporated milk than was present in the raw milk. Since certain studies in our laboratory differed from the results presented by Daniels, and because she had used repeated assays on the same animals but had not employed an improvement we had reported for our basic ration, 3 we have thought it advisable to investigate further the effect of the process of evaporation upon vitamin B₁.

Fresh raw milk was immediately iced and delivered by special messenger together with evaporated milk prepared from the same lot. By careful refrigeration the raw milk was kept sweet for the duration of the test. The evaporated milk was stored at room temperature.

Sixty-gram rats were fed the following ration: 15.0% vitamin-free casein, 15.0% dried autoclaved liver,∗ 45.5% sucrose, 17.0% Crisco, 3.0% cod liver oil and 4.5% modified Wesson's salts. After 22-25 days when a rat had been depleted of its vitamin B₁ reserve and was losing weight slowly but steadily, it was given a daily supplement of the material to be assayed. The dose was continued for 10 days, the amount being changed from day to day, if necessary, to maintain a gain of about 2 g per day.

A method is described that permits estimation of erythrocytes from a blood smear. A specific optical setting is used yielding a ratio of the “Dry Chamber Unit” to the hemacytometer unit of 1: 0.72. On the blood smear a usable urea is determined. Its boundaries are described. The midpoint fixes the position of the Optimal Micrometer Square. Within this area the erythrocytes are counted in the same manner as done for the hemacytometer method. The average number of erythrocytes of 5 ocular micrometer squares is used for the estimation of the equivalent RBC number found with the hemacytometer method. A total of 2,000 comparative counts have been made on bloods ranging from 720,000 to 8,750,000 cells per mm³. The Dry Chamber Method yields totals that are comparable with that of the hemacytometer. The quantitative error of the Dry Chamber count is within ±200,000, the standard error allowed for the counting chamber.

The merits of the cat, dog, rat and guinea pig as assay animals for adrenal cortical extracts is discussed. The guinea pig is found to be the most satisfactory animal because (1) it is easy to operate and handle; (2) it has no accessory adrenal cortical tissue, and (3) it requires small amounts of extract to maintain it in an apparently normal state.

We wish to thank Dr. Carlson for his advice during the course of this work.

Opossum pouch young, indifferent as to sex at birth, show stimulated responses of both sex ducts from treatment with ointments or injections of androgens or estrogens; duct inhibitions are not evident. Responses are revealed by day 16. Androgenic stimulation of Müllerian, and estrogenic stimulation of Wolffian, ducts are unexpected; female prostate induction occurs with androgenic treatment and male prostate inhibition with estrogenic treatment. Responses differ from those revealed in the free-martin.

Two explanations have been offered for the cyanosis often noted after sulfanilamide administration. The first is that the drug causes formation of methemoglobin, or (secondarily, because of the presence of sulphur) sulfhemoglobin. 1 , 2 The second is that a black 3 or blue pigment 4 derived from sulfanilamide, occurs in the blood and produces cyanosis. We have confirmed Ottenberg and Fox's observation 4 that colorless sulfanilamide solutions are changed to a bluish purple when exposed to ultraviolet light. The results of the present study reveal, however, that a pigment derived from sulfanilamide is not directly responsible for the cyanosis. We are now convinced that the cyanosis due to sulfanilamide is caused in all instances by methemoglobin (rarely sulfhemoglobin). In order to detect methemoglobin in every case it has been necessary to employ a sufficient concentration of laked blood (1:5, tube thickness 2 cm; Zeiss grating spectrometer). With more dilute solutions we have often failed to detect appreciable concentrations of methemoglobin.

The concentration of methemoglobin in the blood has been measured by one or both of two methods in a series of 26 determinations. The first method was a spectrocolorimetric procedure such as used previously for measuring the concentration of porphyrin. 5 Blood laked with distilled water in varying dilutions of from 1:10 to 1:30 (depending upon the relative concentration of methemoglobin), was compared with a 1:100 dilution of the same blood in distilled water, in which all of the hemoglobin was converted to methemoglobin by addition of K₃Fe(CN)₆. (2-3 mg to 100 cc). The second method was spectrophotometric, such as employed by Heilmeyer; 6 certain modifications will be described in detail elsewhere. The results of the study are shown in Table I.

Lyons 1 has recently reported the results of his studies on antibacterial immunity to the staphylococcus. He states that young infusion-broth cultures of toxigenic and non-toxigenic strains of staphylococci are encapsulated; old infusion-cultures contain mostly non-encapsulated cocci; the capsule is not present in young serum-grown cultures; capsules are no longer demonstrable after agitation on a mechanical shaker, but they withstand heating to 100°C for 5 minutes; young (2-hour) broth cultures when exposed to defibrinated blood are only phagocyted in small numbers in 15 minutes, as compared to overnight cultures which are phagocyted in large numbers. In other words, the young encapsulated cocci are resistant to phagocytosis. Lyons indicates that a marked phagocytosis of a young culture is related to a specific factor present in serum. It should also be pointed out that he has reported type-specific agglutinins present in the serum of rabbits immunized with young cultures, whereas immunization with old cultures resulted in a preponderance of group-agglutinins. No swelling of the capsules (“Quellung”) could be demonstrated in young encapsulated organisms when added to this type-specific serum.

Because little is known concerning antibacterial immunity in patients with staphylococcal sepsis, it appeared highly important to extend the observations of Lyons as to whether young cultures of staphylococci isolated from patients possessed a capsule that could be demonstrated morphologically, and whether these young organisms resisted phagocytosis. In the present study, attempts were made to demonstrate an encapsulated phase of several strains of staphylococci. Quantitative phagocytic studies were also carried out with the use of human leukocytes and young and old cultures of staphylococci. Thirty strains of pathogenic staphylococci were used; 27 of which were isolated from patients, and 3 were supplied to us by Dr. A. T. Henrici of the Department of Bacteriology, University of Minnesota.

The view has been expressed that the thyroid hormone passes through the placenta but since thyroglobulin has a molecular weight of 700,000 and thyroxine is practically insoluble in water, we thought more evidence on this question desirable. We have, therefore, determined thyroid hormone iodine in mother's blood at the time of delivery and in infant's blood taken from the umbilical cord.

Table I indicates that mother's blood may be more variable in thyroid hormone than is new-born infant's blood. We do not intend this table to show the limits of variation but we believe that these values cast doubt upon the assumption that thyroid hormone passes freely through the placenta. If it passes through the placenta we think it passes very slowly and that no equilibrium is established. Perhaps the fetus derives its thyroid hormone from its own thyroid.

Measurement of the area of the frontal silhouette of the human heart as registered on an X-ray plate permits calculation of the total heart volume with an average error of less than ±5% (studies on “fresh” cadavers, Keys and Friedell 1 ). By means of the multiple slit roentgenkymograph (cf. Johnson, 2 Roesler 3 ), it is possible to trace the outlines of the living human heart in both (ventricular) systole and diastole of a single cardiac cycle (Keys and Friedell 4 ). From measurement of these areas the volume of the heart in systole and in diastole can be calculated. The error in volume estimation from the frontal area is mainly a reflection of abnormality in the relation between frontal and anterior-posterior shape of the heart. Since these abnormalities must be reflected both in systole and in diastole, the error in the estimation of the difference of volumes should be relatively small.

The difference between the volume of the heart in systole and in diastole should be directly related to the stroke output if the valves allow no back flow. The precise numerical relation, however, could not be predicted a priori because the auricular and ventricular cardiac cycles are not absolutely simultaneous in phase. In addition, the difference between the areas of the systolic and diastolic kymogram outlines must be underestimated because the kymogram only registers excursions in one of the 2 planes of the surface of projection.

We have developed a procedure in which roentgenkymograms are made immediately before and after acetylene rebreathing experiments. By this means we obtain estimates of diastolic and systolic volumes of the heart from the kymograms and of the cardiac stroke output by the method of Grollman. 5

In the preceding paper it was shown that the stroke output of the heart may be measured accurately by calculation from the diastolic and systolic areas traced on a roentgenkymogram. Actually, this is a measure of the amount of blood expelled from the heart; in the subject with normal valves it equals the net amount of blood circulated by the stroke. In the patient with aortic or mitral regurgitation, however, we should expect that such equality would not hold and the amount of blood circulated would be less than the amount expelled from the heart by the amount of regurgitation.

The net amount of blood circulated may be measured by the acetylene-rebreathing method. We have found it possible to make what are essentially simultaneous roentgenkymograms and acetylene cardiac output determinations. Comparison of the two results with this procedure should give an absolute quantitative measure of the extent of the back leakage (or the efficiency) of the mitral and aortic valves.

The equation we have developed for the calculation of stroke volume gives, by definition, an average efficiency of the valves of 100% when subjects with normal valves are studied in this way with the two procedures used simultaneously. We have applied this procedure to a series of cases of aortic, mitral, and double aortic and mitral regurgitation and invariably have obtained results which differ from the normal subjects in the expected way.

The subjects were selected on the basis of a clearly defined clinical condition and ability to cooperate in the acetylene rebreathing. None of the subjects was decompensated, though several were on the verge of decompensation or had been decompensated at some time. The clinical evaluation of the lesions in the various patients differentiated 3 groups: (1) minimal leaks, (2) moderate leaks, (3) severe leaks.

The authors have previously reported a method of decerebration of the dog by means of cephalic vascular stasis. 1 The emphasis at that time was upon the use of the procedure as a method for preparing decerebrate animals. Since that time our interest has shifted to the evidence that could be gained concerning the resistance of various cells in the brain to temporary cessation of blood flow and the correlation of these changes with changes in the behavior of the animal.

The technic has been modified to the following form: Two days after laminectomy at the second cervical level, the animal is atropinized, and a metal tracheal tube is inserted orally. A blood pressure cuff is wrapped about the neck, and the pressure is raised quickly to 700 mm Hg, at which level it is maintained as long as vascular arrest is desired, artificial respiration being administered through the tracheal tube. The completeness of circulatory arrest in the head is determined by ophthalmoscopic examination of the retinal vessels. With this technic, consciousness is lost in a very few seconds, the corneal reflex disappears in from 20 to 40 seconds, and spontaneous respiration ceases in from 40 to 90 seconds.

In 6 adult animals there was complete cessation of cephalic blood flow for periods of from 2 to 10 minutes. In the dogs subjected to 8 minutes or less of compression the lid wink returned in 5 to 8 minutes; in 2 animals treated for 10 minutes it did not return till later. The first gasp occurred in 1 to 5 minutes and satisfactory spontaneous respiration returned in 2 to 11 minutes, the slowest return being after the longest compression. Intermittent spontaneous, high-pitched vocalization, associated with vigorous running movements of all 4 limbs, with the dog lying on his side, often occurred during the first few hours.

In cats whose spinal cords had been asphyxiated for various periods of time, van Harreveld and Marmont 1 found that after recovery the hind legs could show an exaggerated extensor tone which usually stayed until death (in some experiments 3 weeks in total). Considering that after this time the acute effect of asphyxia will have disappeared, it was concluded that the high extensor tone is caused by a more or less selective damage of an inhibiting system present in the cord which normally keeps the tone in check.

A further study of these phenomena was made. A period of asphyxia for 30 minutes was usually followed within a few hours by the development of high extensor tone without abolishment of the flexor reflex. However, the effect of pinching the foot was not the same in all animals; sometimes it caused a regular flexor reflex with flexion in ankle and knee; in other experiments it caused, after the flexion, an extension of the leg, and in a few cases this stimulus caused instead of flexion an increase of the extensor tone.

When the contractions of the M. tibialis anterior and the triceps group were recorded simultaneously, it was often seen that stimulation of the N. peroneus superficialis caused a contraction in both of these antagonists. In other animals it was observed that the stimulation of the sciatic nerve caused a contraction in the quadriceps muscle of a Sherrington preparation instead of inhibiting the tone in this muscle.

By Sherrington's scheme for reciprocal innervation these observations can be best explained. Since, according to this scheme, the extensor motor neurones are inhibited during the flexor reflex, the simultaneous stimulation of afferent nerve fibers for extensor reflexes (e.g., for the extensor thrust) running in the stimulated branch of the sciatic nerve, normally remains without effect.

Heptyl aldehyde was administered to albino rats by stomach tube, mixing with the diet, in the drinking water and injected directly into tumors. No effect was apparent on a malignant, transplantable spindle cell sarcoma with respect to resistance to growth of the tumor, survival time of the rat and regression or liquefaction of the tumor. The tumors of the rats receiving heptyl aldehyde weighed less at death in spite of the similar survival times.

It has been shown in the case of the post-pubertal albino rat that: 1. The variability of Qo₂, based upon wet weight determinations, is no greater than when based upon determination of dry weight. 2. Organs and tissues differ significantly in respect to mean value of wet/dry weight ratio.

(1) The rate of the incisor teeth eruption in newborn rats is markedly accelerated by thyroxine; time of eruption varying with the dosage used. (2) The use of this observation as a method of standardizing the thyrotropic hormone was unsuccessful.

1. Sera of guinea pigs sensitized with extracts of certain graminae pollens contained specific precipitins only for the antigen used. 2. The antigen structure of graminae pollens appears to be specific. 3. These experiments justify the use of a specific pollen therapy.

Krueger and Mundell 1 recently reported a method for demonstrating intracellular phage precursor. The essential step in the method is the preparation of “activated” suspensions of staphylococci; this is accomplished by growing the organisms in a heavily oxygenated medium. The activated cells are separated from the broth, resuspended in Locke's solution and are then maintained at 5°C for 2 hours before they are used. To demonstrate intracellular precursor 4 ml of activated cell suspension containing 5 × 10⁸ bacteria/ml is added to 1 ml of phage diluted with Locke's solution to contain 1 × 10⁹ activity units/ml. The mixture is kept for 5 minutes at 5°C and is promptly titrated for total phage content. The end titer is 2 × 10⁹ activity units/ml, an increase of 500% in phage concentration.

Krueger and Scribner 2 supplied additional evidence for the existence of the intracellular precursor. They made use of the fact that staphylococci activated in the presence of Mn⁺⁺ have a relatively low lytic threshold and require only small amounts of phage to induce lysis. Starting with a small amount of phage it was possible to transform the phage precursor in successive lots of staphylococci into phage and to obtain the newly formed phage free in solution by lysing the precursor-containing cells. The original phage added at the start of the experiment has been diluted at least 1 to one million without any reduction in plaque count or activity titer.

It seemed likely from data already available that the phage precursor is more thermolabile than the bacterial cell which produces it. In the experiments of Krueger and Fong 3 the relationships shown in Table I between bacterial reproduction and phage formation at various temperatures were observed.

To determine the route by which the virus spreads from the portal of entry to the medulla and cord, previous investigators have examined different regions of the central nervous system at varying intervals of time during the preparalytic period for the presence of virus. The results of these and related studies have established that poliomyelitis virus spreads axonally, 1 probably as an intraneural infection.

The work we wish to report represents an extension of these earlier studies in which the presence or absence of virus was merely determined and the amount present was not actually measured. Our investigations deal with the concentrations which are reached by the virus during the preparalytic period. They were prompted in part by certain observations on the kinetics of bacteriophagy which indicate that the lytic agent is at first produced in the absence of recognizable bacterial lysis and that actual lysis is not initiated until a certain critical concentration of the agent has been reached. 2 The mechanism of this prelytic formation of bacteriophage is not yet known and need not concern us here. In the studies here reported it was our purpose merely to determine the concentration of poliomyelitis virus which is reached in different regions of the central nervous system and to relate this, if possible, to the extent of neuronal damage sustained in the particular regions, the guiding hypothesis being that the production of virus and of nerve cell damage do not necessarily parallel each other and that certain neurons, although supplying a pabulum for the production of virus may, nevertheless, be relatively resistant to its action.

Part of our observations was made on intracranially inoculated animals; part on intranasally inoculated animals.

1. A manometric micro-method for the determination of serum proteins by protein carbon estimation has been outlined which gives consistently reproducible results on as little as 0.1 cc of serum. 2. Certain advantages, both technical and theoretical, in the use of carbon rather than nitrogen as the reference element in serum protein determination is discussed.

A method is presented for the direct determination of the carbohydrate fraction of several classes of foods. The principal features of the method are: Materials very low in carbohydrate are concentrated by air-drying below 60° C until the residue can be pulverized. The soluble sugars are separated from the starch by solution in 60% ethyl alcohol. After inversion of the sucrose the sugars are determined before and after fermentation. The starch is separated from the unavailable carbohydrate residue by pancreatin digestion and further acid hydrolysis of the filtrate to convert the starch into glucose which is determined as such. The residue is then dissolved in 21.4 N sulfuric acid and further hydrolyzed to convert the cellulose into glucose, the hemicellulose into non-fermentable sugars. The lignin which is insoluble in dilute acid is determined gravimetrically.

Data obtained by this method and certain conventional procedures in a composite analysis account for more than 98% of the carbohydrate in a variety of common foods. Our results compare favorably in regard to available carbohydrates with those obtained by reliable direct determinations in other laboratories. We know of no data with which to compare our results on the unavailable carbohydrate fraction. In a previous publication 1 we emphasized the errors of the old crude fiber determinations.

A fractionation of the acid-soluble organic phosphoric esters of the small intestine as the uranium salts has been described. Aminoethyl phosphoric ester and adenylic acid have been isolated in crystalline form, each comprising about 1/4 of the acid-soluble organic P of the rabbit small intestine. The occurrence of the former ester in pig intestine has also been demonstrated.

There is considerable evidence that caffeine affects the blood sugar of animals. In dogs, a rise follows caffeine injection 1 amounting in some cases to 40-60% above normal 2 in both arterial and venous blood; 3 a double dose is necessary to produce hyperglycemia during avitaminosis. 4 During work, 5 the sugar tolerance curve rises more rapidly with caffeine than without and stays up longer.

However, caffeine produces no hyperglycemia in castrated female rabbits, 6 nor does it affect the glycogen contents of the heart or liver in rats and guinea pigs. 7

No report could be found regarding the effect of caffeine on the blood sugar or sugar tolerance curve for man.

Eight sugar tolerance curves were determined on the same subject (M.D.) using the sugar method of Folin and Malmros. 8 The nutritional history was nearly the same in all cases. No food was taken after 5 p. m. the day before, the amount of sleep and activity was similar, and the determinations were made at the same time of day, 10 a. m.—1 p. m. Fingertip blood samples were taken every 10 minutes for the first 1 1/2 hours after ingesting the dextrose. All sugar determinations were made in duplicate.

The data and results from 4 of the experiments are shown in Fig. 1.

These results indicate that large doses of caffeine depress the peak of the human sugar-tolerance curve and slightly delay the return to normal. Caffeine alone does not raise the blood sugar appreciably. The amounts commonly ingested in coffee and tea probably do not significantly affect either the human tolerance curve or the blood sugar levels.

The relative effectiveness of ingested and injected manganese in preventing the development of perosis in chicks fed a high calcium-phosphorus diet was studied. The results obtained showed that excess calcium and phosphorus in the diet greatly reduces the availability of manganese in the intestinal tract.

The recent chrome acid-fast culture produces lesions in the rabbit that grossly and microscopically resemble those seen in human leprosy. Here, the lesion is definitely progressive over a period of 4 or more months. During this period it is noteworthy that the exciting microörganism steadily increases in number. The “foamy” cells are crowded with dense colony-like masses of the specific bacillus. The chromogenic acid-fast bacillus was recovered in culture from the lesions of the animals 4 months following the last injection. The isolation of the bacillus used in the experiment was more difficult from the lesions that were 4 months old than from the earlier lesions. Visible growth occurred only after 4 weeks' incubation. In the control animals no gross or microscopic lesions were noted 4 months after the last inoculation. The cultures inoculated with material from these animals remained sterile after 4 weeks' incubation.

The use of vitamin K to combat the lowered plasma prothrombin level in jaundiced bleeders 1 , 2 has created a demand for a simple clotting test. Quick 3 has suggested the use of whole blood as a test for thromboplastic activity; we propose to reverse the procedure, and use thromboplastin, in large amounts, to test the clotting power of whole blood. We also propose to show that deficiency in prothrombin can be compensated in part by changes in other factors.

The test. In a 3 cc tube is placed 0.1 cc thromboplastin. Blood, freshly drawn, is run into the tube to a 1.0 cc mark, then inverted once, and tilted gently every few seconds, and the clotting time observed. The test is repeated on a normal individual, and the unknown expressed in percentage of the normal.

For example, a patient's blood clotted in 60 seconds, a control in 30. Activity, therefore, was 50% of normal.

To prepare thromboplastin, extract 10 g ground brain or lung (ox or rabbit) 2 hours with 10 cc saline; strain, and preserve in ice box. Variable potency does not affect the clotting time ratio of unknown to control. If normal values exceed 60 seconds, the thromboplastin is rejected; if less than 25 seconds one should dilute the thromboplastin with saline.

Results. Table I shows results on 10 cases selected at random from our series. The prothrombin levels obtained by the 2-stage titration procedure developed in this laboratory 4 serve as a standard. It is seen that the values obtained with the new test usually agree within 15% with the true prothrombin levels, indicating that the new test is dependent in large part upon the prothrombin concentration.

1. The severe nerve degeneration characteristic of growing chicks fed a riboflavin-low ration failed to occur in rats maintained on a similar ration. 2. A ration low in riboflavin for the chick had no adverse effect upon reproduction in the rat. 3. This ration apparently lacks a growth-promoting factor for the rat. The addition of nicotinic acid did not improve the ration.

Scharlach red, dissolved in ether and applied to decapitated shoots of Kalanchoe Daigremontiana, produces crown gall-like overgrowths which are characterized by leafy shoots and roots, and which resemble typical crown galls induced by P. tumefaciens.

Other carcinogenic agents as 1,2,5,6-dibenzanthracene, methyl-cholanthrene, and benzpyrene applied in lanolin cause injuries to the treated stem without inducing overgrowths on the Kalanchoe. Roots are produced by the Kalanchoes below and above the areas treated with 1,2,5,6-dibenzanthracene, methylcholanthrene, and benzpyrene. Indole acetic acid induces an abundance of long, white roots on the Kalanchoe, together wth small intumescences which are viable for only short periods. Roots are also produced on injured Kalanchoes when treated with lanolin alone. Root formation on the Kalanchoe induced by substances other than the heteroauxin studied here, results from injury which stimulates the host cells to produce root-forming substances. These substances appear to be transported to parts of the stem below and above the treated areas.

The vitamin C requirement was determined on 2 male adults who were hospitalized during the study and fed diets containing constant minimal amounts of vitamin C. The 24-hour urinary excretion of vitamin C was determined daily and the plasma concentration 3 times a week. The determination of vitamin C in both plasma and urine was done by the indophenol titration method 1 in the first case and in the second case both by titration and by the photoelectric colorimeter. 2 , 3 The use of the photoelectric colorimeter reduces the error due to the small amount of non-vitamin C reducing substances present in the urine. On the diet fed, titration gave urine figures which were consistently higher by 10 mg per day. No significant differences were found in the plasma values.

The first case, L. R., age 57, height 72.5 in., weight 160 lb., was observed for a total of 110 days. In the first period of 19 days he received 50 mg of ascorbic acid daily; during the second period of 53 days he received 100 mg of ascorbic acid daily; during the third period of 22 days, 200 mg daily and during the fourth period of 16 days 350 mg daily. The ascorbic acid was given in divided doses of 50 mg each at regular intervals throughout the day. In the first period the daily excretion of vitamin C averaged 11 mg, the daily retention averaged 39 mg and the blood plasma concentration varied from 0.76 to 0.97 mg %, the average being 0.85 mg %. In the second period the daily excretion averaged 20 mg, the daily retention 80 mg, and the plasma vitamin C varied from 0.93 mg % to 1.22 mg %, the average being 1.12 mg %.

A clinical and pathological investigation of a spontaneous and highly fatal intoxication in pregnant rabbits indicated that the disorder was of endogenous origin and that it was analogous to toxemia of pregnancy in women. 1 , 2 The occurrence of the disease in typical form in pseudopregnant and in post-partum animals eliminated the possibility of a toxic factor arising from the products of conception. On the other hand, the presence of productive changes in the pituitary in contrast to the degenerative character of lesions in other organs suggested that an abnormal secretory activity might be a primary factor in the genesis of the intoxication. Accordingly, attempts were made to induce the disorder in experimental animals with various pituitary preparations.

Pituitary extracts were prepared by first grinding the anterior lobes of fresh beef glands with sterile sand to form a paste. After the addition of 3 cc of distilled water per gram of anterior lobe tissue, the mixture was agitated for 6 hours in a mechanical shaker. Following centrifugation, the supernatant fluid was withdrawn and used immediately or stored for a brief interval at freezing temperature.

Subcutaneous or intraperitoneal injections of 5 cc of this extract, 500 rat units of Antuitrin S∗ or 1 cc of Antuitrin∗ made on the 27th day of gestation and repeated on the 29th day induced a disorder which resembled the spontaneous disease in many particulars. On the other hand, control inoculations of sterile saline or fresh and autolyzed extracts of beef muscle and liver failed to reproduce any feature of the condition.

The clinical pictures and pathological changes induced by beef pituitary preparations, Antuitrin S and Antuitrin were similar but, in general, a less severe disorder followed the administration of Antuitrin S than occurred after injections of the other materials.

We have found that the rate of elimination of divinyl ether from an anesthetized dog does not follow the formula first developed for acetone by Widmark 1 , 2 C_t = C_o.e^—Vλt/m where C_o is the original concentration of volatile substance in the blood; C_t the concentration after time t; t, the time; V, the alveolar ventilation; λ., the partition coefficient between air and blood; and m, the “reduced volume”. A mathematical treatment has also been applied to the elimination of ethyl ether by Henderson and Haggard. 3 , 4 Widmark's simplified formula is C_t = C_o.e ^—αt where α is the “elimination constant” and which in the logarithmic form becomes log C_t = log C_o -αt. If, therefore, the rate of elimination of a substance which follows this formula is plotted on semi-logarithmic paper with log C_t plotted against t a straight line should be obtained the slope of which is determined by the elimination constant.

Curve I in Fig. 1 is the typical straight line obtained for ethyl ether with dogs based on the data of Haggard. 4 Curve II, also from Haggard's data, shows the effect of administering carbon dioxide on the rate of elimination by increasing the alveolar ventilation. At point (A) on the curve the administration of carbon dioxide was stopped and the subsequent portion is essentially parallel to Curve I.

Curve III shows a result obtained in our laboratory for divinyl ether (the technic of these blood concentration studies will be published elsewhere). This curve does not become a straight line until after 30 minutes and hence we must infer that some new factor is in operation which is not accounted for by the Widmark expression.

Experiments have shown that sulfapyridine has a curative action when administered to mice infected with pneumococci Types I, IV, V, VIA and B, VII, XI, XX, XXII, XXIV, XXVII, and XXIX. The drug has little curative action in infections with Types II, III, and VIII, although it does prolong life.

Sulfanilamide by subcutaneous injection in dose of 0.160 g per kilo apparently prolonged the lives of some mice when given in 2 doses 6 hours apart before intraperitoneal inoculation with B. typhosus in a dose fatal in about 48 hours, followed by subsequent doses at 6-hour intervals. But when the compound in the same dose was given by subcutaneous injection immediately after inoculation followed by subsequent doses at 6-hour intervals there was much less evidence of therapeutic activity. However, sulfanilamide was much more effective when given intraperitoneally in the same dose 2 hours after intraperitoneal inoculation followed by a second dose 4 hours later and subsequent doses at 6-hour intervals. Of the total of 22 treated mice in 3 experiments, 3 survived and the lives of 7 were prolonged, whereas all of 8 untreated controls succumbed in 1 to 2 days after inoculation.

Sulfapyridine was less effective. Of 10 mice given 0.160 g by intraperitoneal injection immediately after inoculation, followed by a second dose 4 hours later and subsequent doses at 6-hour intervals, one survived and the lives of 2 were prolonged about 24 hours beyond the survival of 4 untreated controls which succumbed in 24 to 48 hours after inoculation.

We 1 , 2 have discussed the comparative therapeutic efficiency of sulfapyridine and sulfanilamide in the treatment of experimental hemolytic streptococcal and Type I pneumococcal infections in mice. These reports were based upon results obtained in mice the origins of

which were unknown. Recently, we have used pure bred mice (strain CF1) whose genetic formula is ccaabb, as test animals for the study of the effects of chemotherapeutic agents upon various types of experimental infections. We believe the use of pure bred mice eliminates the factor of variations in host susceptibility.

In this report we will present data concerning the comparative therapeutic effects of sulfapyridine and sulfanilamide upon experimental infections in mice produced by several types of microörganisms. With the exception of the Welch bacillus and meningococcus, the strains of organisms were virulent for mice in dilutions of 10⁸ to 10⁹, and the meningococcus was made highly virulent by suspension in mucin. The technic of producing these infections has already been described by us. 3 , 4 The data presented in each instance represent the average of several experiments.

As will be noted in Table I, the chemotherapeutic effect of sulfa-pyridine in experimental Types I, II, and III pneumococcal infections in mice is superior to that of sulfanilamide. The results obtained by us are distinctly inferior to those reported by Whitby, 5 but this may be explained by the higher mouse virulence of the strains of pneumococci which we employed and the fact that we used slightly smaller, though more frequently repeated, doses of sulfapyridine.

Little difference was noted in the chemotherapeutic effects of the two compounds in the control of experimental meningococcal or Welch bacillary infections in mice.

A flocculative reaction between the toxin of Cl. welchii (Type A) and its antitoxin is reported, which shows close correlation to the mouse-protective values of Perfringens antitoxins. Concentrated toxins are used as antigens.

It is true 1 that crystalline trypsin 2 can clot ordinary citrated plasma and can activate prothrombin without added calcium. As these experimental facts appear to conflict with the statement of Northrop and Kunitz 3 that a trace of ionized calcium is necessary, we have reinvestigated the point minutely, with an enzyme preparation kindly supplied by the Rockefeller workers.

It was found (Table I) that the trypsin is much more active in the presence of added calcium salt and that excess of citrate can inhibit its action. Whereas trypsin alone requires amounts of the order of 1-2 mg to coagulate 1 cc of citrated dog plasma and the clots quickly undergo fibrinolysis, much smaller quantities (0.01-0.02 mg) of enzyme, synergized by calcium, produce better clots which do not lyse in 24-48 hours.

The addition of cephalin to clotting systems containing the proteolytic enzyme results in a minor improvement of the thrombic activity developed. For its full demonstration, this requires the presence of calcium.

A minor loss of activity on boiling the crystalline trypsin solution 4 is restored by added cephalin and calcium. It is known, of course, that cruder enzyme preparations are thermolabile.

Lung extracts evince a thromboplastic action exactly analogous to that of dilute trypsin (plus cephalin). The partial retention of the activity in boiled lung extract has been explained on the basis of its phospholipid content. 5 We have a semi-quantitative confirmation of this in tests made with the isolated (total) lung P-lipids. Pending isolation, may we not regard the major, thermolabile factor in thromboplastic tissue extracts as analogous to a weak proteolytic enzyme?

A cytological study has been made of the hypophyses of 2 dogs and 2 sheep which had received prolonged injections of extracts of sheep-pituitary glands,∗ and of 2 dogs that had been injected with antihormone-serum. The striking physical changes in one of the dogs, namely, an atrophy of the thyroid, adrenals and gonads, a cessation of growth, and the development of obesity, have been reported elsewhere. 1

The 2 dogs which were injected subcutaneously with the sheep-pituitary extract for 4 and 7 months respectively, developed anti-hormones which inactivated not only the injected pituitary extract, but also several of the hormones of their own hypophyses.

The 2 dogs which were injected daily with 10 cc of canine antihormone-serum showed signs of moderate inactivation of the gonads, thyroids and adrenals.

Twin ewes were injected with sheep-pituitary extract† daily for 6 months. Their genitalia and mammary glands continually showed signs of activation, and at autopsy their gonads and uteri were found to be stimulated. No gonadotropic antihormone developed. A half-sister of these ewes served as a control.

The hyophyses of the dogs injected with sheep-pituitary extract. The basophile cells show the most striking abnormalities consisting of: (1) a great increase in the size and number of the basophiles; (2) an excessive clumping and liquefaction of cytoplasmic granules; (3) the formation of many vacuoles of 3 different types: (a) clear round spaces containing a non-stainable substance or one removed by the technic employed; (b) scattered clear pale blue vacuoles which in some of the basophiles coalesced to become extensive vacuolar inclusions; (c) scattered deeply basophilic vacuoles which in some cells displaced the cytoplasm almost entirely.

Ovulation in a monkey was apparently delayed by administration of estrin in the first part of the menstrual cycle. Accompanying changes in sexual responsiveness are described.

It has been possible to grow a number of strains of the Park 8 variety of diphtheria bacillus, as well as one or 2 other unrelated strains, on media of entirely known composition. 1 Growth obtained on such media is extremely luxuriant, yet a number of the factors essential for the growth of organisms such as the staphylococci, the streptococci and the pneumococci are absent. It appeared, therefore, that such a medium might be unusually suitable for diagnostic purposes in preparing media for throat cultures to replace the ordinarily used Loeffler medium. A medium based upon Formula A in the paper referred to above has therefore been prepared, solidified by the addition of 2% agar, and used for the cultivation of organisms obtained by throat swab from a considerable number of individuals, both normal and suffering from clinical diphtheria.

The results in general, although encouraging, have been too unsatisfactory to suggest any immediate practical value for the method. The majority of normal throats yield no growth whatever after 24 hours' incubation. A certain number of cases of diphtheria, or cultures taken from the throats of individuals known to harbor the diphtheria bacilli, have given strongly positive growth of practically a pure culture of diphtheria. On the other hand, there are a certain number of normal throats which contain organisms capable of growth on this medium, a matter naturally to be expected, but there are also a number of cultures known to have been taken from positive throats which yielded no growth.

In following up this matter, it appeared that the difficulty lay in the fact that even with strains which grow well on the simple medium, a light inoculum generally fails to grow.

Studying the percutaneous absorption of androgenic hormone (testosterone) it was noted that the hormone dissolved in alcoholic solutions is more readily absorbed than that in oily solutions. Most instructive is this difference in the comb test, because here the hormone can be applied at the very site of its effectiveness.

Using the baby chick comb test, we found that brushing the combs with alcoholic solutions of testosterone is about 5 times as effective as brushing the combs with oily solutions, according to Fussgaenger's method. The method proved to be much more sensitive than the subcutaneous and intramuscular administration of the hormone since it is possible to detect quantities as small as 10×0.05 gamma = 0.5 gamma of testosterone at least. We are recommending the standardization of small quantities of androgenic hormone with the use of this method.

If the recovery test is used, on castrated rats, the effectiveness of the percutaneous method using alcoholic solutions of the hormone is less marked. As a matter of fact, alcoholic testosterone solutions are more readily absorbed than oily solutions percutaneously; in this method, however, the subcutaneous administration of the hormone in oily solution is the most effective (4 times as sensitive as the alcoholic percutaneous method).

Duncan, Huffman and Robinson 1 reported that calves restricted to whole milk diets alone, or supplemented with iron, copper and manganese, eventually manifest tetany which is indistinguishable from tetany in rats on a magnesium-low diet. 2 The tetany was prevented and serum magnesium was restored to normal by the administration of magnesium carbonate or magnesium oxide. 3 Since the content of magnesium in milk appears to be rather low, in relation to the calcium and phosphorus, it is possible that prolonged restriction to milk diets of young of other species might eventuate in magnesium deficiency, as suggested by Schmidt and Greenberg. 4 According to data cited by Cox and Mueller, 5 milk of cows and humans may contain about 0.013 and 0.005% magnesium respectively. Analyses of rat milk gave 0.031% magnesium. 5

But in several investigations rats have been reared to adulthood on cow milk supplemented with iron and copper and in no instance have symptoms been reported which indicate the occurrence of magnesium deficiency. 6 This suggests that rats are either (a) better able than calves to utilize the magnesium of cow milk or (b) their requirement for the element is less than that of calves. Duncan and associates believed that “…. there is a failure in the magnesium metabolism which prevents the animal (calf) from utilizing the available magnesium.” Whatever might be the explanation of these observations, the findings suggest that direct investigation should be made of the adequacy of cow milk as a source of magnesium for rats.

Young rats weighing 40 to 45 g were used. They were kept in individual cages and fed exclusively commercial grade A pasteurized cow milk ad libitum, supplemented with 0.25 mg thiamin per liter.

1. Reduction of the percentage of oxygen in the superambient air of broth cultures of the Type I pneumococcus greatly reduced or prevented bacteriostasis by sulfanilamide.

2. When the oxygen concentration was reduced to 0.04% an actual stimulus of growth by sulfanilamide was found.

3. When the oxygen was further reduced by the presence of pyrogallate or hydrosulfite the inhibition by sulfanilamide reappeared.

4. Hydrogen peroxide was detected only in cultures in equilibrium with atmospheres containing 10% oxygen or more. These concentrations correspond to those permitting effective bacteriostasis and are comparable to those obtainable in the plasma.

5. The lack of inhibition at intermediate concentrations shows that oxygen plays a vital rôle in the action of sulfanilamide. The failure to form peroxide at these same concentrations is taken as evidence that oxygen exerts its influence through the agency of hydrogen peroxide. The stimulus and recurring inhibition at the lowest values is interpreted as evidence of the formation of a toxic reduction compound, possibly a sulfide. The latter type of inhibibition may play a rôle in any bacteriostatic effect against anaerobes.

Griffith's 1 technic for typing Group A hemolytic streptococcus has been applied in this laboratory to the classification of more than one thousand strains recovered from a variety of infections. It was possible to type 70% of the organisms examined in 1935 and 1936 by the procedure described previously. 2 At that time several difficulties were pointed out: (1) the elimination of anti-C cross-reactions; (2) the granular character of matt organisms; and (3) the failure of certain strains to be agglutinated by any of the 28 available type-specific sera.

As shown previously 2 interfering cross-reactions due to anti-C substance in the sera could be eliminated by absorbing the sera with purified C-substance, but the purification of C-substance according to Heidelberger's method was a matter of considerable difficulty. However, C-substance prepared by another method (Fuller 3 ) has has proven quite satisfactory for this purpose 4 so that the preparation of type-specific sera has been greatly simplified.

The granular nature of the suspensions produced by many matt organisms interfered seriously with the typing of these organisms up to 1937. Subsequently two methods have been found to obviate this difficulty. Kodama 5 has made granular suspensions homogeneous by subjecting them to supersonic vibrations. In collaboration with Dr. L. Emmet Holt, Jr., we have confirmed this observation, but found the method impractical. A simpler alternative consists in subculturing granular organisms in broth containing trypsin. The trypsin (Difco-Bacto Trypsin supplied in solution in 10 cc ampoules) is stored at 5°C until used. One cc of the solution is added to 9 cc of phosphate-buffered broth just prior to seeding. A heavy inoculum of culture (about 0.1 cc) added to the mixture and incubated at 37°C usually gives satisfactory growth in 6 to 18 hours.

The ease with which nutritional deficiency states develop in young infants suggests the advisability of studying their prothrombin level, since it is now known that vitamin K is required for the synthesis of this clotting factor.

Blood was obtained by venipuncture from normal healthy infants varying in age from 3 to 7 days. The prothrombin was quantitatively determined by the senior author's method 1 on both the undiluted plasma and on plasma diluted with an equal volume of saline solution. Typical cases of this series are presented in Table I.

The results plainly show that the prothrombin concentration in the blood of the babies studied was essentially the same as that of normal adult blood. It was found, however, that the prothrombin diminished in infants' blood more rapidly than from that of adults, and curiously in a few bloods, such as Case 3, the decrease of prothrombin was strikingly rapid when the plasma was diluted.

These findings are completely at variance with those reported by Brinkhous, Smith and Warner. 2 In their series the prothrombin concentration of the bloods of the 3 babies who were less than 11 days old were 27, 36, and 44% of normal. Furthermore, even in infants 2 months old, they found that the prothrombin was less than

50% of the adult level. In view of the exceedingly low incidence of hemorrhage in very young infants, such low prothrombin values seem rather surprising. It must be remembered that the methods of both Smith and Quick are empirical and based on certain unproved assumptions. Smith and his coworkers take for granted that high dilution of plasma does not alter the prothrombin, but does inactivate the normal antithrombin.

Against a heavy infecting dose of fixed rabies virus administered intracranially to rats, the oral administration of sulfanilamide or of sodium sulfanilylsulfanilate resulted in a slight prolongation of life but failed to save the life of any animal.

The methyl esters of the fatty acids of corn oil are absorbed at the same rate as unaltered corn oil. Adrenalectomy has no significant effect on the absorption of either of these two fats. The rate of absorption of both methyl esters of corn oil fatty acids, and unaltered corn oil is the same for each hour up to 6 hours after feeding.

Although certain protein-like substances have been found to traverse the placenta, it seems that our experiments demonstrate that the guinea pig placenta is not permeable to the toxic protein ricin.

The daily subcutaneous injection of 10 to 25 mg of testosterone depressed the spermatozoa count in a 67-year-old man. After its withdrawal the number of spermatozoa increased and during a second period of testosterone therapy the number of spermatozoa again decreased. The duration of the 2 periods of treatment, the daily dosage, and the variation in the spermatozoa counts are presented in the graph.

The results seem to indicate that the factors for hereditary hairlessness of the rat reside principally in the skin. The effect of operation on the hairless rat is to stimulate a secondary growth of hair, the amount of growth decreasing with the age of the rat. This growth is not confined to the site of operation but occurs over the whole body.

Although the diastatic activity of the blood was known as long ago as 1846 1 it was not until Wohlgemuth 2 developed his method of quantitative estimation that the subject began to receive the attention of clinicians. Since that time other more accurate methods have been devised for the micro-estimation of diastase in blood and urine. 3

Clinical experience with the method in the diagnosis of pancreatic disease has led to the conclusion that it is one of the most valuable diagnostic procedures available particularly in acute pancreatitis. 4 Unfortunately, the value of the test is limited by the fact that the rise and subsequent fall in the concentration of the diastase after mechanical obstruction of the pancreatic ducts takes place over a comparatively short period of time, usually 2 to 3 days to attain a maximum height and 10 to 14 days to recede to normal.

With this restriction in mind, it is obvious that normal values many times cannot be satisfactorily interpreted. Not only is this frequently true in the case of acute pancreatitis which may be seen by the clinician too late to obtain evidence of abnormal diastatic activity, but it is commonly the case, with such lesions as chronic pancreatitis, cysts and neoplasms where the diastase determinations are often of little or no value whatever.

Accordingly, the following experiments were carried out in an effort to overcome this inherent difficulty of the test. Dogs varying from 10 to 15 kg in weight were anesthetized with sodium-pentabarbital (nembutal) gr 1/2 per kg of body weight intravenously and local infiltration with 1% solution of novacain. A mid-line abdominal incision was made and a rubber balloon was introduced through an incision in the stomach wall and placed into the duodenum just past the pyloric ring.

Intravenous injection of 50 units of Antuitrin Growth into adult healthy dogs induced progressive depression of fasting blood sugar for 2 hours. At 24 hours the level approached that found at 1 hour, and after a lapse of 48 hours the fasting level closely approximated the initial level.

1. A procedure is described for the determination of the free volatile acids of tissues. 2. Normal tissues of the dog, with the exception of brain, contain only traces, from 0 to 0.25 mM or cc N, of free volatile acidity per kg. This constitutes from 1 to 3% of the total volatile acids which can be obtained by hydrolysis with 2 N H₂SO₄. About 10%, from 1.5 to 2.0 mM, of all of the volatile acids of the brain are present either as free acid or in some form which is readily hydrolized. 3. The metabolism of ethyl alcohol in the dog does not result in an increase of the free volatile acids of the blood and tissues.

The production of local protection or “local immunity” to micro-organismal infection was first fully described by Wassermann and Citron 1 and interpreted by them as an “Umstimmung” or “retuning” of the local cells. This phenomenon has subsequently been observed but differently explained, especially by Besredka, 2 Gay, 3 and Opie. 4

Bull and McKee 5 and recently Walsh and Cannon 6 demonstrated in rabbits a definite specific resistance of the upper respiratory tract to pneumococcal infection subsequent to specific local intranasal instillation. These observers point out that although the rabbit host seems protected in general, evidences of mouse-protective substances are not demonstrable. Gay and Rhodes, 7 on the other hand, found that only the derm was resistant to the streptococcus following experimental erysipelas whereas infection was produced by other routes of inoculation. Extensive literature in this field is afforded by Pacheco 8 and Gay. 9

We employed both hairy and hairless white mice, and Type I pneumococcus A5-51R obtained through the courtesy of Dr. Kenneth L. Burdon and grown on blood-agar slants. When a saline suspension of the pneumococcus killed with 0.4% formalin was injected intraäbdominally on each of 5 days during a period of one week, the animals then survived for 5 to 6 days subsequent to an intraäbdominal injection of living pneumococci that was fatal to controls over night. In seeking a useful ratio of protecting factors to lethal dose it was found that .05 cc of saline suspension of a 24-hour growth, containing approximately one-half billion microorganisms per cc, killed mice within 24 to 48 hours when injected intraäbdominally, thus representing one minimal lethal dose. A similar dose injected subcutaneously killed in 36 to 72 hours.

A simple piece of apparatus for an accurate and constant injection of small volumes of fluid into the tissues of man is described. It is especially useful when colored solutions, which preclude the reading of the scale on a syringe, are used.

Rogoff and Stewart observed that pregnancy and pseudopregnancy would greatly extend the survival periods of bilaterally adrenalectomized dogs. 1 , 2 This observation has since been confirmed, 3 and more recently it has been shown that when pseudopregnancy is experimentally induced, it is just as effective in alleviating the symptoms of adrenal insufficiency as spontaneous pseudopregnancy. 4 , 5 Experiments on the adrenalectomized, pseudopregnant ferret indicate that it is possible to obtain survivals which are, on the average, 5 times as long as those of the anestrus or male controls. 6

In the experiments reported here, the right adrenal glands were removed from 10 mature female cats. Nine of these animals were driven into estrus by injections of a menopause urine extract (Gamone∗), while the tenth animal came into spontaneous estrus after the operation. Gamone has been found effective in producing estrus and mating reaction in the dog, 7 but for use in the cat it was necessary to run a series of preliminary tests to determine the proper dosage. The results of these experiments indicated that 20 R.U. per kilo body weight per day would produce cornified vaginal smears and typical mating reactions after an injection period of 4 to 6 days (Table I). During the estrus period the cats were mated with a normal male, and in some cases they were greatly stimulated with the glass rod while taking vaginal smears. Thus every attempt was made to secure luteinization of the estrous ovaries by each animal's own pituitary gland secretion.

Immediately at the end of the mating period, the remaining adrenal gland was removed with as much care as possible in order to minimize the chances of shock affecting the survival time.

Normal, whole, neutralized gastric juice which had been passed through a Berkefeld V filter and administered intravenously to patients with pernicious anemia does not produce a hematologic response probably because there is no extrinsic factor as such in the parenteral tissues to provide a substrate for interaction.

A virus capable of inducing fatal pneumonia in Swiss mice has been isolated from normal mouse lungs, and its immunological characteristics are now being studied in detail.

Twenty-one groups of normal Swiss mice were inoculated intra-nasally under ether anesthesia with 0.05 cc of lung-suspensions from uninoculated mice. Serial mouse-passage was carried on with each group, using 10% to 30% lung-suspensions. Passages were made at an average interval of 7 days; usually 6 mice were used in each group. The mice were obtained from 6 different breeders. Initially, passages were made without regard to the breeder from whom the mice had been obtained. Lately, however, passages have been made in mice from each individual breeder in order to determine the source of the virus. Definite areas of pulmonary consolidation were present in 43% of the groups in the third serial passage, and in 52% of the groups at the sixth passage. Death occurred as early as the fourth passage, and by the sixth passage deaths were recorded in 24% of the groups. Cultures of the mouse-lung suspensions were made routinely and were sterile in a great majority of instances. Rabbits were injected intraäbdominally with virus-containing material from various passages. They were bled before injection and again 8 to 10 days afterwards. Their serum was tested for the presence of antibodies capable of neutralizing the various strains of virus in the manner described by Magill and Francis. 1

Fatal pneumonia was caused by 0.05 cc of a 10⁻³ to 10⁻⁴ dilution of infected mouse lung, and definite pulmonary consolidation was produced by 10⁻⁵ to 10⁻⁶ dilutions. The virus was filtrable through Berkefeld V and N candles, passed through graded collodion membranes 2 with an APD of 300 mμ and was retained by a Seitz filter.

Rabbits produce black-widow antivenin which, taking into account the shorter injection period and the relatively small number of spiders necessary, compares reasonably well with that produced in the sheep. This finding may be of importance in the production of antivenin commercially, where only small amounts of readily available serum are desired.

A simple method is described whereby estrogenic substances can be extracted from human urine for bioassay. This method consists of adding 50 cc concentrated HCl (35-37%) per liter of urine; boiling for one hour and then cooling; vigorously shaking on a mechanical shaker for one hour with an equal volume of ethyl ether (U.S.P.); breaking up emulsion with 30-50 cc of sodium taurocholate (10% sol.) and discarding aqueous fraction; adding 6 cc sesame oil to the etherial extract and then evaporating ether on a hot water bath. This method is comparatively accurate and can be accomplished in about 3 1/2 hours, thus making it an excellent routine method in an endocrine laboratory to expedite clinical and experimental work.

The studies of Gage 1 and Pope 2 have shown the life cycle of Triturus viridescens to consist of 3 phases. (1) an aquatic larval stage of about 3 months' duration; (2) an immature, red-colored land stage, lasting from 2 1/2 to 4 years and, finally, (3) an aquatic adult stage. While there are some variations in this typical life cycle, apparently depending upon the locality, 3 , 4 the newt in the Blue Ridge Mountains of extreme Western North Carolina shows exactly the life cycle described by Gage and Pope, with a possible extension of the second stage into a fifth year.

The life cycle of Triturus is very interesting because of the 2 complete changes in habitat that take place. First, at 3 to 6 months of age, the metamorphosis from the aquatic larval to the terrestrial condition with the accompanying migration to land, and second, in the fourth to fifth year, the migration back to water as the animal approaches or reaches sexual maturity with consequent readaptation to a water habitat.

Metamorphosis among the Amphibia has been shown to be essentially an effect of the endocrine glands, particularly of the thyroid and indirectly the pituitary. 5 In our investigations on Triturus we have been interested, not in particular morphological changes, but in the relationship that might possibly exist between the endocrine glands and the phenomenon of migration from land to water and vice versa. Do the pituitary and thyroid glands, in addition to bringing about certain morphological changes, provide the “drive” which induces the change in habitat following metamorphosis? In seeking the answer to this question we sought first to determine the relation of the pituitary, if any, to the return of the land stage of Triturus to water.

When radioactive phosphorus is fed to normal rats, mice or chickens 1-4 in an ordinary diet, a large percentage of the phosphorus is deposited in the bony skeleton. This more or less selective deposition of phosphorus in bone, suggested to us the possibility of using radioactive phosphorus as a source of therapeutic irradiation in leukemia. In preliminary experiments, mice with lymphatic and myelogenous leukemia were fed radiophosphorus in relatively small amounts and there was no definite influence on the course of the disease. However, at autopsy, some of the tissues were assayed for activity and it was unexpectedly found that in leukemia there is an abnormality in the handling of a single dose of tagged phosphorus. Before pursuing this question further, it seemed to us important to determine first the total phosphorus content of leukemic, lymphomatous and normal tissues as a basis for further study. The findings 5 show that per gram wet weight, lymphomatous tissue or tissues infiltrated with lymphoid cells have no greater total phosphorus content than their normal analogues, i. e., lymph glands and various uninfiltrated viscera. We wish here to report observations on normal and lymphomatous mice which show that a single dose of phosphorus is handled differently in the latter group of animals.

The phosphorus used in these experiments was made radioactive in the cyclotron of Lawrence and Cooksey 6 by bombarding red phosphorus with deuterons having an energy of approximately eight million volts. As stated above, P³², the radioactive isotope, is chemically similar to P³¹, the naturally occurring element. It has a half life of about 14.8 days. Because of the beta ray emission, radio-phosphorus can be detected and quantitated by means of an electroscope or a Geiger counter.

It has been shown 1 that the electrical impedance per unit area of the skin may deviate from normal values not only in thyroid and other endocrine conditions but also in disturbances involving the autonomic and central nervous systems. Gross changes in skin capacity have been reported in certain types of mental disease. 2 It appeared to be of interest, therefore, to study a group of mental patients with a view to determining whether or not changes in the impedance properties of the skin could be found.

Impedance determinations were made by the 3-electrode method at 11,160 c.p.s., using a concentric and a disc electrode applied over the region of the biceps and triceps as described elsewhere. 1 , 3

The patients were not selected in any way and included all of the females (54) in the adult wards at the N. Y. State Psychiatric Institute. This group was composed predominantly of schizophrenics and psychoneurotics. The average age was 29 years.

A histogram of the results obtained is shown in Fig. 1 (dotted lines) and represents the distribution of the mean impedances of two 6 cm 2 skin areas measured on the anterior and posterior sides of the upper right arm. A corresponding histogram of the results obtained on 102 normal females in a previous investigation 1 (solid lines) is given for purposes of comparison.

As will be seen from Fig. 1, there is a considerable shift of the mentally diseased group toward low values, their mean impedance m falling well to the left of the normal mean M. The group of patients having impedances lying entirely outside the normal histogram on the low side are of particular interest since it is in this low impedance region that the values for thyrotoxics fall. 1 None of the patients in this low impedance group showed signs of thyrotoxicosis.

A convenient method for the preparation of follicle-stimulating extracts from sheep pituitary powder by use of trypsin is described briefly. These preparations produced follicles only, when tested on normal female rats for 4.5 days, and were free of lactogenic and thyrotropic activities.

The carbohydrate content of our follicle-stimulating and luteinizing preparations is given in terms of glucose calculated from the reducing action of the hydrolysates. Results from enzymatic inactivation and electrodialysis are given which suggest that the follicle-stimulating activity may be associated with a carbohydrate grouping.

Most of the studies on the protective action of sulfanilamide and sulfapyridine on streptococcic and pneumococcic infections have been made on white mice. In the work here reported rabbits were used as the experimental animals. Intracutaneous inoculations were made in order to permit the observation of differences in the local lesions occurring in the treated and the control groups.

Rabbits weighing approximately 2 kilos were given 0.3 cc of a 1-100 dilution of an 8-hour culture of a Type II pneumococcus. The strain used had been transferred alternately through mice and veal broth for more than a year.

Intraperitoneal injections of 0.2 cc into mice, in dilutions of 1:10,000,000, kills 50% of the animals.

Sulfapyridine, 2-(sulfanilamide)-pyridine, was administered orally by suspending in 10% acacia and permitting the suspension to trickle down the throat from a large syringe. The treated animals were given 0.5 g one hour before inoculation, 0.5 g 3 hours after inoculation, and 0.5 g every 12 hours thereafter for a total of 5 g.

In addition to the difference in mortality, there was observed a marked difference in the lesions produced in the treated and control animals. Rhoads and Goodner 1 have reported oedema and a spread of the cutaneous lesion by gravity in rabbits inoculated endermally with pneumococci. These results were evident in our control group. The treated animals showed little or no oedema, and probably because of this, showed little or no spread by gravity. That an infection was present in the skin was evidenced by the area of inflammation in the skin of treated animals. The above authors have also stated that in occasional animals they find signs of hemorrhage in the skin. In the lesions of our control series there were extensive hemorrhages, caused either by the virulence of our strain or by the age of the culture.

The injection of fine silicious particles directly into the splenic vein in the amounts described will produce a progressive cirrhosis of the liver. Secondary to this a state of splenic vein hypertension has been produced with a concomitant congestive splenomegaly.

The effect of the pexis between kidney and the spleen or omentum upon the blood pressure of dogs rendered hypertensive by Goldblatt's 1 method of renal constriction has been studied in this laboratory since 1936. Sixty-five dogs have been employed in the development of 2 suitable technics for establishing organ pexis to such ischemic kidneys. Seven animals survived the various surgical procedures for producing carotid loop, 2 hypertension 1 and the union of the organs.

Only 2 died as the result of the latter operation. Fifty-six dogs died or were discarded because of hemorrhage from the carotid loop employed for blood pressure determination, anesthetic deaths, uremia from infarction of the kidneys, distemper, and operative infection. Postoperative shock was insignificant following the pexis which was performed one to 3 months after the hypertension was established. Control operations in which the omentum and spleen were manipulated did not result in a significant prolonged fall in blood pressure.

The blood pressure of all of the 7 animals was lowered within 4 days following the union. Three of these, following the pexis of encapsulated renal surface with omentum, had temporary reversal of hypertension lasting 6-38 days. (Table I.) The other 4 animals in which the longitudinal cut surface of the spleen was attached to the renal surface or imbedded (Animals Nos. 95 and 116) in an incision through the long axis of the kidney down to the pelvis, showed a continuous decline in blood pressure toward normal, persisting S weeks to 6 months. (Table I.)

To verify the existence of a collateral circulation, India ink was injected into the splenic artery of an excised preparation consisting of an adherent kidney and spleen.

Criegee, Kraft and Rank 1 showed that glycols and related compounds are split by lead tetraäcetate to yield ketones or aldehydes. Threonine (CH₃CHOHCHNH₂COOH) should, therefore, yield acetaldehyde when oxidized by lead tetraäcetate. Eegriwe 2 described a highly sensitive color reaction for the qualitative estimation of lactic acid based upon the oxidation of the acid with hot concentrated sulfuric acid to acetaldehyde; from the acetaldehyde an intense violet color is produced by condensation with p-hydroxydiphenyl. Miller and Muntz 3 modified Eegriwe's method and they have shown that the color is not given by a number of other acids such as glycolic, oxalic, formic, 2,3-dihydroxybutyric, etc. The apparent specificity of the p-hydroxydiphenyl test for acetaldehyde suggested that it would be of value in the quantitative estimation of threonine.

The following method can be used for the determination of threonine alone or in the presence of other amino-acids: 25 cc of glacial acetic acid containing from 0.5 to 1.5 mg of threonine and 1 g of lead tetraäcetate are kept at 30° for one hour. The acetaldehyde is removed from the reaction mixture during the course of the oxidation by moderately rapid aeration and taken up in 10 cc of concentrated sulfuric acid containing 5 drops of water and 100 mg of p-hydroxydiphenyl in suspension. At the end of the hour, the excess p-hydroxydiphenyl is dissolved by warming to 100°. The solution may be diluted to volume with concentrated H₂SO₄. The amount of color is in direct proportion to the quantity of threonine used. Control tests with alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, norleucine, phenylalanine, proline, serine, tryptophane, tyrosine, valine and mixtures of the same were negative.