The estimation of creatine

Abstract

The [alpha]-naphthol-diacetyl reaction for the estimation of creatine was investigated. The inhibition, which has been noted by other workers to occur when the estimation of the creatine content of trichloracetic acid extracts of liver is attempted, was shown to be due to the presence of sulfhydryl compounds and, by inference, to the presence of glutathione. The inhibition by sulfhydryl compounds depends on the molar ratio of the sulfhydryl compound to creatine, and is therefore most marked in the liver where the creatine content is low and the glutathione content high. The ratio of sulfhydryl compounds to creatine, in several tissues investigated, is sufficiently high to produce very gross errors in the estimation of creatine by this method. Exceptions are found in voluntary and cardiac muscle, brain and testis, but even here the method is subject to error. The presence of disulfide compounds such as GSSG also produces an inhibition due, presumably, to the tendency of the disulfide linkage to split in alkaline soln. The inhibition produced by sulfhydryl compounds can be almost completely removed by the addition of p-chloromercuribenzoic acid; both this compound and that formed by its reaction with the sulfhydryl compounds possess weak inhibitory powers. The creatine content of various tissues, blood and urine was determined both in the presence and in the absence of p-chloromercuri-benzoic acid and an assessment made of the errors introduced by the omission of this compound. The recoveries of creatine added to extracts of various tissues, blood and urine are mostly, in excess of 90%, thus indicating the general applicability of the modified method.