Effects of freezing and storage on subcellular fractionation of guinea pig and human brain.

Abstract

Subcellular fractionations were carried out on guinea pig and human brains. Distributions of protein marker enzymes, and galactolipids were examined with guinea pig cerebral cortex that was (Group I) homogenized immediately; (Group II) stored 3 to 5 days at -70 degrees C prior to homogenization; (Group III) stored 3 to 6 months; (Group IV) homogenized after 3 h at R.T. and 16 to 18 h at 4 degrees C and then stored at -70 degrees C for 7 to 9 months. Human frontal lobe obtained at autopsy was fractionated immediately (Group V) or stored at -70 degrees C for 5 to 8 months prior to fractionation (Group VI). Protein recoveries in myelin, microsomal, synaptosomal, and supernatant fractions were decreased in brains that were not frozen for several hours prior to storage (Groups IV-VI). SDH and MAO recoveries in the nuclear and free mitochondrial fractions were increased in these groups. AChE, a membrane marker, was also increased in the free mitochondrial fractions in Groups IV-VI, suggesting increased contamination of mitochondria by synaptosomal membrane fragments. Arylsulfatase, a lysosomal enzyme, was decreased in the free mitochondrial fraction with freezing, but the distributions in tissues not frozen for several hours showed only an increase in the nuclear fraction and a decrease in the microsomal fraction. Freezing brought about an increase in supernatant LDH and a decrease in this enzyme in the free mitochondrial fractions. Total galactolipid contents in synaptosomal and free mitochondrial fractions were increased by freezing and storage. Though some redistribution of enzymes takes place, meaningful subcellular fractions can be obtained after storage of fresh and postmortem brain tissues.