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In 1953 Nagata and Yamamoto developed a method for carrying out the metachromatic reaction (the term "metachromatic reaction " will be referred to as "MR " in this paper) on the urine. The results of experiments initiated in this department have already been reported by these investigators respectively. In this country the results of follow-up studies on the "MR " have been recorded. Making a study on a substance in the urine which took on a blue color in the presence of Nile blue, J. Desbords et al. of France in 1952 presumed that the substance responsible for the change of color would be a free fatty acid with more than 16 carbon atoms. Previously Yamamoto et al. undertook follow-up studies thereon according to the methods of Inouye and Noda. The methods were modified by Fujii who made a basic study on the basis of the follow-up studies. Tne author using Fujii's modification of the methods has isolated and identified higher fatty acids from the blood, serum and urine of 34 cases of urosis, particularly nephrotuberculosis and 20 healthy subjects as well as from the urine of 15 cases of non-tuberculous urosis At the same time the "MR " was per formed on the urine samples in an attempt to seek a relationship between higher fatty acids and the "MR ". The fat loading test was carried out on one case of nephrotuberculosis and one healthy subject. Some findings were noted when similar observations were made. It is the purpose of this paper to describe the findings. Methods and Materials. In the early morning 7 cc of blood was collected from the cubital vein of 54 subjects on a fasting stomach respectively, including patients and healthy people. Two cc of the blood was used. Serum was isolated from the remaining blood and 1-cc serum samples were prepared. At the same time as the blood was taken, urine was collected and 100 cc of it was concentrated to 5 cc on a boiling water-bath. The urine samples thus prepared were employed. The original method for carrying out the " MR " was applied to a part of the urine samples and the estimation of the results was macroscopically made. Experiments were carried out only on the urine samples obtained from 15 cases of urosis. The fat loading test was performed on cases of nephrotuberculosis who always presented the positive " MR " and on 2 healthy subjects in whom the "MR " was always negative. Each sample obtained from the subjects was placed in a flask used for hydrolysis and 50 ml of a 6% KOH solution was added. The resulting solution was. heated at 100℃ for about 3 hours for saponification. After cooling 20 ml of 7N H_2SO_4 was added to result in the production of free fatty acids. The sample was transferred to a triturium in which the free fatty acids were extracted twice with 30 ml of petroleum ether. The ether extract was washed twice in water and thrice in ethylenglycolglycelin-water (1:1:1) and the washings were discarded. The ether layer was heated to 60-70℃ to remove ether. The residus was dissolved in 0.2 ml of ether alcohol by heating. In a capillary tube about 0.02 ml of the resultant solution was collected and drops of it were applied on the original point of defatted and dried filter paper No. 50, Toyo Filter Paper Co., Ltd. Paper chromatography was thus, performed accordiding to Fujii's modification of the methods. The suspected fatty acids were identified by comparing the Rf values thus obtained from each sample with the Rf values of high fatty acids standardized by the author. These suspected fatty acids will be referred to as fatty acids in this paper. Results. 1) Group of healthy subjects: Two or three spots representing each fatty acid were demonstrated when paper chromatography was carried out using the blood and serum samples from this group. There were 26-spots representing 2 saturated fatty acids, including petargonic acid; 26 spots representing 3: unsaturated fatty acids, including elaidic acid; and 20 spots which could not be identified. The urine samples from this group did not show any spot representing fatty acids. The "MR" was negative. 2) Cases of non-tuberculous urosis: Twelve cases of non-tuberculous urosis were examined. The blood and serum samples respectively exhibited 1-4 spots representing each fatty acid. There were noticed 13 spots representing 5 saturated fatty acids, including pelargonic acid; 28 spots representing 5 unsaturated fatty acids, including oleic acid ; and 14 spors which could not be identified. The "MR " was positive or doubtful positive in 8 cases in which fatty acids were detected; whereas it was negative in the rest in which fatty acids could not be detected. 3) Cases of nephrotuberculosis: Paper chromatography was performed on the blood and serum samples obtained from 20 cases of nephrotuberculosis and 2-5 spots representing each fatty acid were detected. There were 32 spots representing 9 saturated fatty acids ; 47 spots representing 5 unsaturated fatty acids , and 47 spots which could not be Identified The "MR " was all positive in this group. The urine samples chromatographically revealed 11 spots representing 6 saturated fatty acids, including caproic acid ; 19 spots representing 5 unsaturated fatty acids, including oleic acid ; and 21 spots which could not be identified. 4) Two cases of epididymal tuberculosis: Paper chromatography was carried out using the blood and serum samples. There were perceived 6 spots representing 2 saturated fatty acids, including pelargonic acid, and 3 spots. representing 3 unsaturated fatty acids, including erucic acid. This group presented the doubtful positive " MR ". 5) Cases of non-tuberculous urosis: When the urine samples obtained from the cases were chromatographed, they met with negative results except for one case of urinary bladder cancer whose urine sample showed 2 spots representing 2 unsaturated fatty acids, including elaidic acid. The "MR " was positive only in the cases in whose urine samples fatty acids were detected. the remaining cases presented the negative " MR ". 6) The fat loading test: Before the fat loading test paper chromatography was carried out on the blood samples obtained from healthy subjects and 2 spots representing palmitic acid (saturated fatty acid) were detected. Until 6 hours after the loading there were observed 12 spots representing 4 saturated fatty acids, including caproic acid; 16 spots representing 3 unsaturated fatty acids, including elaidic acid; and 20 spots which could not be identified. The " MR " was negative in the healthy subjects before and after the fat loading. 7) When blood samples obtained from cases of nephrotuberculosis were chromatographed prior to the fat loading, there were detected 4 spots representing 2 saturated fatty acid, including stearic acid and 2 spots representing linoleic acid (unsaturated fatty acid). In contrast to this, there were 11 spots representing 5 saturated fatty acids, including pelargonic acid; 10 spots representing 4 unsaturated fatty acids, including elaidic acid ; and 27 spots which could not be identified. paper chromatography was carried out using the urine samples. Before the loading 2 spots representing 2 unsaturated fatty acids, including linolenic acid, were demonstratable and the "MR " was positive. On the other hand, after the loading there were noted 3 spots representing 2 saturated fatty acids, including pelargonic acid, 8 spots representing 4 unsaturated fatty acids, including elaidic acid, and 11 spots which could not be identified the "MR " was positive in all the cases. 8) The "MR " was positive or doubtful positive in all the subjects whose urine samples showed the presence of higher fatty acids; while it was negative in the remaining subjects in whose urine samples fatty acids could not be detected. 9) The author believes that the substances which render the "MR " positive are probably higher fatty acids. For this reason the author agrees with J. Desbords et al, who have pointed out that higher fatty acids are substances in urine which assumes a blue color in the presence of Nile blue.
