Toluene, Xylene 吸入時, 尿中に排泄される馬尿酸, メチル馬尿酸の新定量法について : Thinner 中毒 第4報,有機溶剤中毒 第8報

Abstract

For elucidation of the relationship between toluene concentration in the air and amount of hippuric acid (abbreviated as H.A.), and that between xylene consentration in the air and amount of methyl hippuric acid (abbreviated as M.H.A.) excreted in the urine as metabolites following work, in a workshop where toluene and xylene are used, we have succeeded in measuring minute amounts of urinary H.A. and M.H.A. by a new paperchromatographic method, and also by a simple mass screening examination method which we devised. A. Method of procedure. 1) Measurement of H.A. by paperchromatography; For the determination of H.A. in the urine, 0.1 ml of urine to be tested is spotted on the filter paper (40×40 cm) and is developped in the solvent system; n-buthanol-acetic acid-water, 4:1:1. After drying, the color reaction is brought to completion by spraying with 4% solutionof p-dimethylaminobenzaldehyde in acetic anhydride saturated with sodium acetate (abbreviated as D.A.B.), and by heating it in a drying oven for one minute at 135°C. The coloerd portion of the above paper is cut into small pieces and the elution is conducted three times with 3ml. 2ml, and 2ml of ethanol. The optical density of orange color is measured at 460 mμ by spectrophotometer and the concentration of H.A. is calculated with standard curve. 2) Measurement of M.H.A. by paperchromatography: One ml of the sample urine is mixed with HCI (adjusted to pH 2.0) and saturated with 0.3g of NaCl. To this mixture 2ml of ethyl acetate is added. Then 0.1ml of this extract is spotted on a filter paper (40×40 cm), developd in the solvent system, tolueneacetic acid-water (100:50:2.5). After drying, the color reaction is brought to completion by spraying with D.A.B. reagents and by heating. And Rf. values of H.A., m-M.H.A and p-M.H.A. are 0.38, 0.45 and 0.43 respectively. Each spot of the paper is cut into small pieces. Elution and measurement of H.A. and M.H.A. are conducted as described above. 3) Spot method: The method of extraction of H.A. and M.H.A. from urine is the same as that used in paperchromatography of M.H.A. except that 4 ml ethyl ether containing 10% ethanol is used instead of ethyl acatate. Thereafter 0.03 ml of this eluate mixture is spotted on a filter (Toyo Filter paper, No.51) 4 cm apart with micro pipette and dried, Color reaction is the same as described in paperchromatographic measurement of H.A. For the determination of H.A. from colored spots, the intensity of the developed color is compared with standard paper, on which H.A. at known concentrations has been spotted and the color development has been doneexactly with the same manner. 4) Silicagel method; H.A and M.H.A are extracted from urine as descried in the spot method. Then one ml of this extract is poured on the 0.5g of silicagel in a test tube.Next the ethyl ether is evaporated under reduced pressure. Dry silicagel powder containing H.A. or M.H.A. is heated at 135°C for 3 minutes with D.A.B. reagents in an oil bath. Azlactone formed is eluted twice successively with 4 ml and 3 ml of ethanol, and spectrophotometric determination is conducted as described above. B. Application. Spot method is convenient for screening H.A. and M.H.A. Paperchromatographic method developed by buthanol-acetic acid-water, is useful for the determination of H.A. or M.H.A. And paperchromatography using toluene-acetic acid-water as developing solvent is also available for the separate determination of H.A.and M.H.A from urine containing both. C. Stability of H.A.in various couditions for storing. Of H.A. in the urine about 13% is decomposed in the room temperature, about 17% in the cold after 72 hours, and 11% in the freezing temperature after 240 hours, In the case of the urine spotted and dried on the filter paper, H.A. is well kept without decomposition in the room temperature for 360 hours.