STUDIES ON MUSCLE OF AQUATIC ANIMALS-XXXVII:Separation and Determination of Creatine and Creatinine

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In order to determine creatine and creatinine in tissue extractives, ion exchange procedures prior to colorimetry were found to be applicable. The diacetyl-α-naphthol method for the determination of creatine and the alkali-picrate method for creatinine were re-examined. From the results obtained, the following way was found to be recommendable for the estimation of creatine and creatinine: 1. Creatine estimation. (A) After loading the extractives on 0.9×100cm. column of Dowex 50-X8, the column is operated at room temperature with citrate buffer solution (pH 4.25) at flow rate of 6ml. per hour. Each effluent is collected in 1ml. fraction. Creatine is eluted between the 120th and the 138th fraction, and completely separated from other compounds (Fig. 1). (B) 1.5ml. of distilled water, 0.5ml. of 0.05% diacetyl (Fig. 4) and 2ml. of 2.5% α-naphthol in alkaline solution (Fig. 2) are added to each 1ml. fraction of creatine and adequately shaken. After standing for 20-30min. at 17°C. or 15min. at 22°C. in dark (Fig. 5), the mixture is diluted to 10ml. with distilled water and is measured at 535mμ. 2. Creatinine estimation (A) After loading the extractives on 0.9×15cm column of Dowex 50-X8, the column is operated at room temperature and flow rate of 6ml. per hour with 20ml. of citrate buffer (pH 5.0) at first, and followed with phosphate buffer (pH 6.8). Each effluent is collected in 1ml. fraction. Creatinine is eluted between the 34th and the 40th fraction, and is completely separated from other compounds (Fig. 6). (B) 1ml. of 0.5N NaOH (Fig. 7) and 4ml. of 1% picric acid (Table 1) are added to each 1ml. fraction of creatinine and adequately shaken. After standing for 25min. at 18°C. (Fig. 7), the mixture is diluted to 20ml. with distilled water and is measured at 535mμ. 3. Recoveries of creatine and creatinine added to the tissue extractives by the present methods were 99.0-102.4% (Table 2).
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