A microprocedure for extracting tissue nucleotides for analysis by high-performance liquid chromatography

A method is described for the preparation of concentrated tissue extracts for nucleotideanalysis by high-performance liquid chromatography (hplc). Ten to one hundred milligrams of tissue was extracted in a combined weighing-homogenizing-centrifuge tube using a trichloracetic acid (TCA)-methanol extractant containing a radioactive internal standard. This extractant eliminated nucleotide interconversion which was found to occur when TCA alone was employed. High ATP/ADP and ATP/AMP ratios were observed and recoveries of greater than 97% were obtained with exogenous radioactive nucleotides. The method has been applied successfully in studies on muscle, heart, liver, kidney, lung, brain, and subcellular fractions.