Preparation of subcellular fractions suitable for biochemical analyses from human subcutaneous adipose tissue obtained by needle biopsy: I. Isolation of mitochondria on a microscale

Abstract

1. 1. A technique has been developed for the isolation of subcellular organelles on a microscale which can be used to examine small samples of tissue (10 to 40 mg). Separation is accomplished with a series of Millipore filters of appropriate pore size. 2. 2. Details of the method for the isolation of mitochondria are presented. Evaluation of the nature and purity of the mitochondrial fraction was made by following the localization of a mitochondrial marker enzyme (glutamate dehydrogenase, GLDH) and by examination of the structure of the particles with light and electron microscopy. 3. 3. The method was applied to the study of a mitochondrial enzyme (mitochondrial malate dehydrogenase, M-MDH) in human neonatal adipose tissue in the first week of life. M-MDH activity in healthy fullterm infants increased with age; between 3 and 7 days of age, M-MDH activity in premature infants was decreased in comparison with fullterm infants and directly correlated with gestational age. 4. 4. The same approach can be used to isolate subcellular particles other than mitochondria and for the preparation of several different subcellular fractions from the same tissue sample. The isolated material is in native form (unfixed) and is suitable not only for ultrastructural but also for biochemical investigations, such as the determination of enzyme profiles or of the metabolism of labeled substrates.