Organic Composition of Bone : Localization of Isocitric Dehydrogenase in Femurs

THE original observation of Dickens 1 that calcined tissues contain the majority of the body's citric acid has raised the question of the mechanism by which this occurs. Currently, hypotheses to explain bone citrate levels are based on the findings of Dixon and Perkins 2 , who observed relatively little isocitric dehydrogenase in rabbit femurs. We felt that these values 2 might be minimal, since manganese ions and triphosphopyridine nucleotide were not used as supplements. Furthermore, citric, rather than isocitric, acid was used as the substrate in the assay system. In the present work, tissues were assayed 3 for isocitric dehydrogenase activity using a spectrophotometric method. Extracts of soft tissues (weight × 5 of 0.1 M tris , p H 7.4 ; 15 min. at l,100 g ) from exsanguinated animals were assayed. The femurs were frozen in ‘dry ice’, cleaned of adhering muscle, connective tissue and periosteum, then sawn into five transverse sections. The marrow was removed from the shaft marrow space. Each section was, crushed in ‘dry ice’ to an eight mesh. 1-gm. samples were extracted in the refrigerator with 2.0 ml. of 0.1 M tris , p H 7.4 for 4 hr., then centrifuged. The extracts were assayed and the rate of reaction was determined from the linear portion of the reaction curve using the molecular extinction coefficient 4 for reduced triphosphopyridine nucleotide of 6.22 × 10 6 cm. 2 /mole.