Measurement of ATP Synthesis and Flocculent Matrix Densities in Mitochondria as a Function of ‘in vitro’ Ischemia in the Heart and Liver of Rats

Abstract

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° 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.

Oxidative stress causes reversible changes in mitochondrial permeability and structure

“…This discrepancy prompted additional investigations to elucidate the basis of the discordance. While we found that it resulted from a permeabilization difference in fixed cells, the changes in mitochondrial structure and function may have relevance in aging or pathological conditions in which ROS production is increased (Costa et al, 1990; Chiba et al, 2005; Oberley et al, 2008). …”

“…The swollen nature of the mitochondria suggests that osmotic effects may be one cause of this morphology. In addition, intramitochondrial amorphous deposits that likely represent matrix precipitates and degenerating cristae, that is, flocculent or “woolly” densities, were common (arrows) (Costa et al, 1990; Chiba et al, 2005; Oberley et al, 2008). The outer and inner mitochondrial membranes appeared intact in hydrogen peroxide-treated cells, with no gross morphological alterations that would suggest easy access of antibodies to matrix antigens.…”

Intracellular ATP Levels Are a Pivotal Determinant of Chemoresistance in Colon Cancer Cells

“…A 0-0.25 nM ATP standard curve was generated using a standard solution of 10 μM ATP. ATP stock concentration was determined at 259 nm using E mM = 15.4 mM −1 cm −1 (14). The value of ATP produced by mitochondria within 1 min was normalized by the protein content of the mitochondria, which is expressed as “nmol ATP/min/mg protein”.…”