Israel S. Kleiner

Chemical Nature of Catalase

The chemical nature of catalase has been studied by a number of authors. The experiments of Zeile 1 and of Stern 2 indicate that the catalase molecule is probably a chromoprotein in which the hemin group is related to that of the natural blood pigments. Recently Agner 3 reported that if horse liver catalase is dialyzed against N/10 or N/100 HCl the enzyme splits into 2 inactive components, one of a low molecular weight which dialyses through the cellophane membrane and which is possibly hemin and another component which remains within the bag and which is a protein. If the 2 neutralized components are mixed, an active preparation is again obtained, according to Agner. Having the intention of making certain studies on the basis of Agners report, we first attempted to repeat his experiment. The technique is simple but we were not able to confirm his results. The only difference between his and our technique is in the species of animal from which the liver was derived. Horse liver, which Agner used, is practically unobtainable in our locality. We have, however, tried beef, rabbit, and rat liver, respectively. We followed the experiments of Agner in every detail, dialyzing the purified catalase against N/10 or N/100 HCl, for 10 to 48 hours. No splitting (inactivation) of the catalase could be obtained. With stronger HCl irreversible inactivation of the catalase took place, due to a marked decrease in pH inside the dialyzing bag.

Isolation of a specific ascorbic acid (vitamin C) Oxidase.

An extract of Hubbard squash oxidizes both the synthetic and the natural ascorbic acid (vitamin C)∗ with great rapidity. This is due to an enzyme having an optimum pH of 5.83 to 5.96. It may be obtained and purified by extracting the squash (edible part) with twice its weight of 30% ethyl alcohol for 10 minutes. The centrifuged and filtered fluid is treated with an equal volume of acetone, which causes a yellow sticky substance to precipitate. This may be washed free of yellow pigment with acetone, dissolved in water and reprecipitated with acetone. A third precipitation yields a preparation, which after drying in vacuo over sulphuric acid has an activity 500 times that of the original extract. This preparation is water soluble and gives slight protein tests. Alcohol and saturated solutions of neutral salts, however, do not precipitate it. It is digested (inactivated) by trypsin. A polysaccharide accompanies the enzyme in the above precipitation. We have found no way of removing it thus far. This enzyme differs in various ways from the “hexoxidase” which v. Szent-Györgyi 1 discovered in cabbage leaves, e. g., the hexoxidase is precipitated by saturated (NH4)2SO4 solution; it oxidizes not more than about 25% of the substrate, whereas the enzyme of the squash oxidizes 100% very rapidly. Moreover, the kinetics of our preparation point to the presence of a single enzyme. Substances thus far tested, such as cysteine, tyrosine, glutathione, and phenols, are not affected. We suggest, therefore, that the enzyme responsible be designated “ascorbic acid oxidase”. It requires the presence of atmospheric oxygen, which plays the role of hydrogen acceptor. The oxidized ascorbic acid may be reduced to its original state by hydrogen sulphide. The enzyme is remarkably stable to dialysis, oxygen and carbon monoxid. Hydrogen sulphide, however, inactivates it. For ascorbic acid estimation, Tillmans and associates 2 2,6-di-chlorobenzenoneindophenol method was employed.

An office procedure for the rapid estimation of pepsin and its clinical significance

1. This method is simple, accurate and rapid. It can easily be used as an office procedure.

Relation of Cholane Nucleus to the Female Bitterling Test for Male Hormone.

After demonstrating that the female bitterling test was not a test for pregnancy, 1 we have stated 2 that the fraction of male urine containing the male hormones is responsible for the ovipositor elongating reaction. A positive reaction was taken as a lengthening of the ovipositor from a quiescent stage to a length equal to that of the anterior edge of the anal fin, i. e., the ovipositor must reach the end of the fin. Crystalline theelin and theelol were also tested and although they did not give positive reactions, they sometimes caused a slight lengthening of the ovipositor. We therefore felt it important to determine whether any other substance containing the cholane nucleus would produce this reaction in greater or lesser degree. We have tested ergosterol† and cholesterol emulsions and solutions of sodium taurocholate according to our method. 2 Small doses of all these compounds produced no reactions. These doses were equivalent to, or several times greater than the doses of male hormone which produced pronounced reactions. Larger amounts of cholesterol, however, produced slight effects while the same amounts of sodium taurocholate were fairly active. For example, while approximately 1.7 mg. of the crude male hormone fraction produced a marked positive reaction when added to 4 liters of water containing 2 bitterlings, 40 mg. of cholesterol caused only slight effects. Doses of 8 mg. of sodium taurocholate caused no reaction, while 25 to 50 mg. gave a moderately strong reaction. Thirty animals were used. It is highly improbable that these weak reactions were due to contaminations of our “C.P.”preparations with male hormone. This could be excluded only by the use of synthetic products. Since bile salts occur in urine in appreciable amounts only in cases of obstructive jaundice, it is evident that ordinarily this factor would not interfere with the test.

Fluctuations of the Concentration of “Blood-sugar” in Vitro.∗

In previous reports we have discussed the behavior of the bloodsugar under various conditions. It was first shown 1 that when blood from diabetic dogs was dialyzed against Ringers solution the sugar dialyzed out at an irregular rate as compared with normal blood containing added glucose. (Hirudin, or novirudin, was used as the anticoagulant in all experiments.) In some instances, and especially when the determinations were made at short intervals, the curves were most irregular, sometimes apparently indicating a formation of sugar under the influence of dialysis. We therefore subjected diabetic blood to short periods of dialysis, after which the blood was transferred to a glass vessel and blood-samples were taken every 15 or 20 minutes. These blood-sugar curves also showed irregularities 2 ; sometimes figures were obtained which exceeded the original values before dialysis. We have since studied this phenomenon further and wish to report the observation that this irregularity following dialysis almost invariably occurs in diabetic blood (dog or human) particularly if the initial sugar value is quite high. With concentrations of 270 mg. per 100 cc. or less, the fluctuations are not as marked, or may even be absent. As a matter of routine we then conducted control experiments in which the blood was not subjected to dialysis or any other procedure, expecting a slow, regular, downward trend, due to glycolysis. We were surprised to find similar fluctuations in both dog and human diabetic blood, again noting that often the original bloodsugar value was exceeded and also that the greatest fluctuations occurred at the higher levels. Even normal blood to which glucose was added yielded similar irregular curves, but if no glucose was added there occurred only the regular glycolysis.† The curves are not uniform in character; the peaks and troughs do not fall at corresponding points.

A method for the rapid determination of urea in minute amounts of blood

This method involves the digestion of urea by urease (Marshall-Van Slyke), the precipitation of the proteins (Folin-Wu), the direct Nesslerization of the filtrate (Myers) and determination of the color in the micro-colorimeter previously described. 1 For this color comparison a wedge, containing 1 per cent. potassium dichromate, mounted on a deep yellow ground-glass plate, is used. The technique is as follows: A 0.2 c.c. pipette is rinsed with 20 per cent. potassium oxalate solution. The residual fluid is blown out well and 0.2 c.c. of blood is drawn up from the pricked finger or ear-lobe and discharged into a small test-tube. The pipette is then rinsed twice with exactly 0.2 c.c. of water and the washings are added to the blood. Three or four milligrams (knifepoint) of powdered urease are now added to the blood and, after shaking, a stopper is inserted and the tube is kept at 50° for 10 minutes or at room temperature for 30 minutes or longer. Then 1.0 c.c. of water is added, followed by 0.2 c.c. of 10 per cent. sodium-tungstate solution and 0.2 c.c. of 2/3 N sulphuric acid. The mixture is immediately shaken and, after the precipitate has darkened, it is filtered into another small test-tube, using a 2.5-3 cm. funnel and small thin filter paper. With a dry 1 C.C. pipette, graduated in 1/100ths, a definite volume of the filtrate is discharged into one of the 5 C.C. graduated test-tubes with which the micro-colorimeter is provided. It is convenient to take 0.5 C.C. but one need not wait for this amount to filter through. Two volumes of water are added and one volume of Nesslers solution (BockBenedict formula diluted I :5). After thorough mixing the tube is placed in the micro-colorimeter, and matched to the standard “nitrogen” wedge, described above.

Morphin hyperglycemia as a test for pancreatic deficiency

We found that the subcutaneous injection of one or two milligrams of morphin sulphate per kilo in dogs whose pancreatic substance had been strongly reduced by coagulation in situ 1 or by partial resection, caused a much greater rise in the blood-sugar level than the same dose in normal controls. The following table gives the results of some of our experiments. It will be seen that the animals in which the pancreatic tissue had been reduced (AK5, 32, 37, and BD3) showed an increase in the blood-sugar three to four times greater than that obtained in the controls after the same dose of morphin. As these animals with deficient pancreatic tissue may legitimately be considered in a prediabetic state, the morphin hyperglycemia observed in them may be of importance clinically in detecting individuals with an impaired carbohydrate metabolism. That this impairment need not be great and yet yield a strong hyperglycemia to a small dose of morphin is indicated by the fact that our dogs whose pancreatic tissue had been largely coagulated nevertheless showed a surprisingly good tolerance for sugar. In six tests where 10 grams of dextrose per kilo were fed, and in two where 4 to 5 grams of dextrose per kilo were injected subcutaneously, the amount excreted was nothing in two tests; less than 0.5 gram per kilo in three; less than one gram per kilo in two tests; and 1.5 grams per kilo in one test. Whether the test will yield the same results in the human subject which we have obtained in dogs, can only be determined by trial, and such a trial we believe fully warranted by our findings. The procedure can easily be carried out with less than one cubic centimeter of blood if the Epstein method, for example, is used for determining the blood-sugar.