Measurement of ATP synthesis and flocculent matrix densities in mitochondria as a function of 'in vitro' ischemia in the heart and liver of rats.

We intended to determine the levels of adenosine triphosphate (ATP) synthesis at the time when mitochondria ultrastructurally show flocculent densities in the matrix space. For this purpose, mitochondria were isolated from rat heart and rat liver after the tissues have been maintained under controlled ischemic conditions in vitro at 37 degrees C for intervals of 15, 30, 45, 60, 120, 180, and 240 (heart) min. The isolated mitochondria were tested for new ATP synthesis by luciferin/luciferase luminescence in the presence of substrate and adenosine 5'-diphosphate (ADP). The luminescence peaks were standardized and related to an external measure by measuring absorbance of ATP at 259 nm where the extinction coefficient is 15,400. Mitochondrial yield was monitored by measuring succinate dehydrogenase activity in the first homogenate and in the final mitochondrial pellet. Alternatively, cytochrome oxidase activity was used and the protein in the mitochondrial pellet was also determined. We found that the yield of mitochondria was above 53-54% in both liver and heart at 2 h of ischemia. Longer intervals were accompanied by lower yields. The ability to synthesize new ATP declined at different time intervals in ischemia of the heart compared to the liver. After 30 min ischemia, the synthesis in heart mitochondria is 18% of control, while the synthesis of liver mitochondria reaches 16% of control after 45 min of in vitro ischemia. Flocculent densities in heart mitochondria appeared at 45 min ischemia in vitro and in vivo, and at 60 min in liver mitochondria. We conclude that the decline of ATP synthesis is a significant early change in mitochondria and antedates the appearance of flocculent densities.