Delta 5 desaturase activity in rat kidney microsomes.

Rat kidney microsomal fraction is able to catalyze the enzymatic desaturation of eicosatrienoic acid (20:3n-6) to arachidonic acid (20:4n-6) by the delta 5 desaturase pathway, in the presence of reduced nicotinamide adenine dinucleotide (NADH), adenosinetriphosphate (ATP) and coenzyme A (CoA). The substrate of the reaction [1-14C]eicosa-8,11,14-trienoic acid (20:3n-6), was separated from the product [1-14C]eicosa-5,8,11,14-tetraenoic acid (20:4n-6) by reverse phase high-pressure liquid chromatography (RP-HPLC). These fatty acids were individually collected by monitoring the eluent at 205 nm and their radioactivity was measured by liquid scintillation counting. The delta 5 desaturase activity in kidney microsomes increased linearly with the substrate concentration up to 20 microM. Enzymatic activity was sensitive to pH with the maximum at 7.0 and was proportional with incubation time up to 10 min. The apparent Km and Vmax of delta 5 desaturase were 56 microM and 60 pmoles.min-1.mg-1 microsomal protein, respectively. Neither the cytosolic renal fraction nor the cytosolic liver fraction enhanced the delta 5 desaturase activity. Contrary to a report but in accordance to others, the present results suggest that rat kidneys can synthesize arachidonic acid at least to satisfy partially their needs for eicosanoid production.

Mitochondrial membrane fluidity changes in renal ischemia.

The fluorescence polarization technique with 1,6-Diphenyl-1,3,5-hexatriene as a probe, was used to determine the lipid rotational mobility (LRM) measured by fluorescence anisotropy of isolated whole mitochondria of the rat kidney following normothermic ischemia of 30, 45, 60 and 90 minutes and upon reperfusion for 24 hours. The LRM of mitochondrial membrane lipids of the ischemic kidney decreased steadily with increasing ischemic times (0.1590 vs. 0.1705, 0.01 less than P less than 0.001 at 60 minutes). Following 24 hours reflow, there were no significant differences in the LRM of mitochondria between ischemic and control groups up to 45 minutes of ischemia, (0.1688 vs. 0.1705, 0.5 less than P less than 0.6). However, when kidney was subjected to ischemic periods longer than 60 minutes, the decreased LRM remained fixed even after reperfusion (0.1783 vs. 0.1738, 0.5 less than P less than 0.6). This suggests that 60 minutes of ischemia probably produces irreversible damage to the mitochondrial membrane whereas lesser degrees of ischemic injury is reversible upon reperfusion.

Effect of ischemia and 24 hour reperfusion on ATP synthesis in the rat kidney.

The ability of renal tissue to synthesize ATP was examined in adult Sprague Dawley Rats immediately following normothermic ischemia of 30, 45, 60 and 90 minutes and upon reperfusion for 24 hours. Following ischemia the rate of ATP synthesis decreased progressively. It was 64.5% of the control at 45 minutes and 10.4% after 90 minutes of ischemia. Reperfusion of the ischemic kidneys for 24 hours restored ATP biosynthesis to control, nonischemic levels in kidneys subjected to ischemia up to 45 minutes (101.8 +/- 13.9% vs 64.5 +/- 2.5% p less than 0.02). However, after 60 minutes of ischemia, reperfusion had no effect (59.3 +/- 4.4% vs 51.7 +/- 7.5%) and reperfusion following 90 minutes of ischemia was associated with decrease ATP synthesis (10.4 +/- 2.2% vs 3.3 +/- 0.9% p less than .001). We conclude that mitochondrial function is restored by reperfusion when normothermic ischemic interval is 45 minutes or less. However, ischemic intervals longer than 45 minutes produce non-reversible impairment of ATP synthesis and the marked reduction following 90 minutes of ischemia signifies possible transition to a non-viable state.

Fatty acid metabolism in renal ischemia.

The increase in free fatty acids in the ischemic tissue is a consistent observation and these free fatty acids are considered to play a role in the cellular toxicity. To elucidate the cause of higher levels of free fatty acids in ischemic tissue, we examined the catabolism of fatty acids. The beta-oxidation of lignoceric (24:0), palmitic (16:0) and octanoic (8:0) acids and the peroxidation of fatty acids were measured at different times of renal ischemia in whole kidney homogenate. The enzymatic activities for the oxidation of fatty acids decreased with the increase in ischemia time. However, the lipid peroxide levels increased 2.5-fold of control with ischemic injury. Sixty min of ischemia reduced the rate of oxidation of octanoic, palmitic and lignoceric acids by 57, 59 and 69%, respectively. Almost similar loss of fatty acid oxidation activity was observed in the peroxisomes and mitochondria. These data suggest that loss of mitochondrial and peroxisomal fatty acid beta-oxidation enzyme activities from ischemic injury may be one of the factors responsible for the higher levels of free fatty acids.

Occurrence of a 22∶2 nonmethylene interrupted dienoic fatty acid and its seasonal distribution among lipids and tissues of the fresh water bivalveDiplodon delodontus from an isolated environment.

Realatively high levels of a non-methylene-interrupted dienoic fatty acid were detected in the freshwater molluscDiplodon delodontus. The (7,13) 22∶2 NMID fatty acid was separated from total fatty acids by TLC, and its structure was determined by GLC and reductive ozonolysis. Its seasonal distribution was investigated in different tissues and lipids of the mollusc. High concentrations of this acid were found in polar lipids. The absence of the 22∶2 NMID fatty acid in the lipids of plankton and sediment in the same habitat suggests that it may be biosynthesized by themollusc. Possible synthesis and functions of the NMID fatty acids are discussed.