ment. The final product had the same appearance as in the first experiment. 0.050 gram of the substance gave 0.01659 gram nitrogen by Kjeldahl method. N.. Calculated for Found: 4-6-dioxy-2-5-diamino-pyrimidine, and divicine on condensation with urea under identical conditions did not form uric acid. Analysis of carbohydrate. 10 grams of vicine were hydrolyzed in the usual way. The divicine sulphate was removed by filtration. The filtrate diluted to 300 cc. and the sulphuric acid removed by means of lead carbonate, and the lead by means of hydrogen sulphide. A part of the solution was used for the preparation of the phenyl osazone. It was prepared in the usual way. For analysis it was recrystallized out of an alcohol-acetone mixture. The melting point = 205°C. 0.1187 gram of the substance gave 0.2635 gram CO2 and 0.0652 gram H2O. 0.200 gram of the substance dissolved in a mixture of 2 cc. pyridine and 3 cc. of alcohol rotated in a 0.5 dm. tube in yellow light, a = 0.49° in twenty-four hours, a = 0.39°. 0.39°. 0.200 gram of glucosazone under the same condition, a = twenty-four hours, a = 0.49°; in The remaining part of the solution was oxidized with nitric acid and from the reaction product the acid potassium salt of saccharic acid obtained. For analysis it was once recrystallized out of water. 0.100 gram of the substance gave 0.0354 gram K2SO4. K. Calculated for Found: 15.74 15.90 THE IODINE CONTENT OF TUBERCULOUS TISSUES. BY PAUL A. LEWIS AND ROBERT B. KRAUSS. (From the Henry Phipps Institute of the University of Pennsylvania.) (Received for publication, June 2, 1914.) During the past two years we have prepared a number of compounds of iodine with the dyes Trypan-red1 and Trypan-blue and have studied them in animal experiments with the idea that the well-known action of these dyes as vital stains might be used to distribute physiologically active iodine to tuberculous tissue. A number of the dye-stuff compounds with iodine are vital stains with much the same factors of distribution as the original dyes. As in the case of the mother substances many of these iodized dyes show considerable tendency to concentrate in the experimental tuberculous tissues of the rabbit.2 In working with such compounds, however, it is somewhat difficult to overcome certain theoretical objections which might be raised; for example, many of these compounds are without obvious physiological action in the tissues to which they are distributed. The amounts in question are so small that tests for the excretion of iodine to determine whether or not the compound was broken down and merely the original dye-stuff deposited in the tissue cannot be satisfactorily made. From the chemical point of view, it is difficult to separate completely the iodine compound from the mother dye. There thus remained the possibility that some of the unchanged original dye might be responsible for the vital stain of the tuberculous tissue. It seemed advisable, therefore, to select a typical example for analysis in which the tuberculous tissue in the treated animal could be studied for its iodine content. We are aware of two previous attempts to study the relation of iodine to tuberculous tissue. The first of these was by Loeb and 1 Krauss: Journ. Amer. Chem. Soc., xxxvi, p. 961, 1914. 2 Lewis: Arch. of Int. Med., x, p. 68, 1912. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XVIII, NO. 2 Michaud. These observers used rabbits. One eye was inoculated with tubercle bacilli and after a suitable interval potassium iodide was administered to the animal. Sometime later the animals were killed and the tuberculous eyes were examined for their iodine content in comparison with the normal eyes. It was found that the tuberculous eyes uniformly showed, an appreciably higher iodine content than the normal. In these experiments there would at first sight seem to be a satisfactory control. In other experiments the lymph glands of tuberculous guinea pigs which had been treated with iodoform and ethyl iodide were used. In these cases the presence of iodine at the time of examination was considered by the authors as sufficient evidence that it was derived from the substances administered. Wells and Hedenburg repeated these experiments on a larger scale. Their methods of analysis were improved The results were essentially the same as far as the examination of the tissues was concerned. Our first experiment gave an unexpected result which called attention to a defect in the previous observations. We found in this experiment (see table, p. 315) that in some instances the tuberculous tissue from untreated animals contained appreciable quantities of iodine. This has led us to make some further examination of tuberculous tissue for its iodine content. Method. The tissues for examination were prepared as follows: Healthy rabbits were chosen and the inoculations were made on one cornea with a virulent culture of tubercle bacilli of bovine type. After an interval of about three weeks, when the lesion was well developed, the animals were killed, the eyes removed, washed free of blood, and the cornea separated from the remainder of the eye. The tuberculous cornea was examined for iodine, the normal cornea being examined as control. As a check on the method of analysis in some instances, all of the remainder of the eyes was analyzed. In the instances where the tuberculous lung tissue was examined in comparison with the normal lung tissue, the animals after a corneal inoculation were allowed to live much longer, until the disease had become generalized. In the early stages of generalization after this method of inoculation, discrete foci of tuberculosis varying from 1 to 5 mm. in diameter are found in the lungs * Loeb and Michaud: Biochem. Zeitschr., iii, p. 307, 1907. 4 Wells and Hedenburg: Journ. of Inf. Diseases, xi, p. 349, 1912. while the intervening tissue is practically normal. The separation of the tuberculous tissue from the normal lung tissue is relatively easy. In some instances the thyroids of the same rabbits were analyzed for iodine by the same method. The rabbits during the period of the experiment, were confined in separate cages and were fed on the regular diet consisting of a mixture of stale bread and oats soaked for some time in water, with the addition of fresh cabbage two or three times a week. The method of analysis for iodine was essentially that of Kendall with certain modifications which were for the most part kindly suggested by Kendall himself. The disintegration of the tissue was carried out in a crucible furnace of our own design which we have recently described in connection with the analysis of our Trypan-red iodine compounds. Our results are contained in the following table. The results are expressed as milligrams of iodine per gram of dry tissue. 5 Kendall: Journ. Amer. Chem. Soc., xxxiv, p. 894, 1912. 6 Krauss: loc. cit. |