EFFECTS OF STORAGE AND HOMOGENIZATION METHODS ON THE HEPATIC RECOVERYOF DEXTRANS DETERMINED BY SIZE-EXCLUSION CHROMATOGRAPHY

The effects of storage and homogenization methods on the analytical re covery of dextran macromolecules from rat livers were investigated usi ng a high-performance size-exclusion chromatographic (HPSEC) method. L ivers were collected from rats dosed with fluorescein-labeled dextrans with molecular weights of 150 or 70 kD. Subsequently, the livers were subjected to different methods to study the effects of the following parameters on the hepatic recovery of dextrans: storage method (freezi ng the livers before homogenization or freezing the homogenates); cont ents of the homogenization buffer (addition of 1% Triton X-100), and s ample type (HPSEC analysis of the whole homogenate or the supernatant after centrifugation). It is shown that in the absence of Triton in th e homogenization buffer, the hepatic recovery of dextrans is substanti ally affected by all the factors studied. However, in the presence of 1% Triton in the buffer, the hepatic recoveries were maximal and indep endent of the storage method or sample type. These studies suggest tha t for optimal recovery of dextran macromolecules from the liver, a sam ple preparation method capable of disrupting the subcellular membranes should be used.