PAPER CHROMATOGRAPHICAL STUDIES ON HIGHER FATTY ACIDS IN THE BLOOD AND URINE CASES OF UROSIS WITH SPECIAL REFERENCE TO THE METACHROMATIC REACTION IN THE URINE

Abstract

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 7cc 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 100cc of it was concentrated to 5cc 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 50ml of a 6% KOH solution was added. The resulting solution was heated at 100°C for about 3 hours for saponification. After cooling 20ml of 7N H₂SO₄ 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 30ml 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°C to remove ether. The residus was dissolved in 0.2ml of ether alcohol by heating. In a capillary tube about 0.02ml 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. u