Precipitation of Apparent Creatinine from Serum Ultrafiltrates

In earlier isolation experiments 1 , 2 on blood creatinine, the use of protein precipitants, of adsorbents, and evaporation in acid solution all made the interpretation of results difficult. In the case of sera showing various degrees of nitrogen retention a simpler path which avoids these difficulties is open. In the following experiments cellophane No. 300 was used as the membrane in an ultra-filter operated by a nitrogen pressure of 400 lb. per square inch. A few drops of toluene were added to the serum and the receiving vessel.

To 10 cc. of ultrafiltrate are added 250 mg. of pure picric acid. This is dissolved by shaking the tube under the hot water tap, and the solution is cooled to about 25° in cold water. One-tenth cc. of 10% potassium chloride solution is added, and the solution is mixed at once. An excess of picric acid may precipitate at this point, but unless the amount of apparent creatinine is very large, one can centrifugalize and decant without any loss of chromogenic substance. A small crop of crystals then forms on the bottom or wall of the tube containing the decanted liquid. These crystals contain the apparent creatinine, but free picric acid and a small amount of potassium picrate are still present. If the concentration of apparent creatinine is very high, crystallization may go to completion in a few hours, but as a rule 24 hours are required, and with amounts below 4 mg. per 100 cc. the time required may be still longer.

The apparent creatinine values in the ultrafiltrate before and after carrying out the above precipitation are shown in Table I. Unless otherwise stated, 24 hours were allowed for precipitation.