Some Effects of Methyl Linoleate Hydroperoxide on Oxidative Phosphorylation in Rat Liver Mitochondria. ∗

Abstract

The effects of methyl linoleate hydroperoxide (MLHP), a peroxidation product of methyl linoleate, on the denaturation of the lipoproteins (Nishida and Kummerow(9)) and on certain pathologic disorders in animals simulating vitamin E deficiency is well known. Both the denaturation phenomenon and the pathologic disorders are alleviated by αtocopherol supplementation (Nishida and Kummerow(10)).

Evidence is reported here which indicates that MLHP has an inhibiting effect on the coupling of phosphorylation with oxidation in rat liver mitochondria.

Materials and methods. Mitochondria were prepared from the livers of normal adult Sprague-Dawley male rats on a stock laboratory diet by the Hogeboom-method (Hoge-boom(4)), using 0.25 M sucrose.

The conventional Warburg procedure was used to measure oxygen uptake over a 30-minute period at 30° following a 6-minute preincubation (see footnote to Table I for composition of media).

Incubation time was terminated by addition of 5% trichloroacetic acid (TCA). The inorganic phosphate remaining in each vessel was determined by Lowry's method(6). The P/O ratio was calculated from the values observed.

The protein content of each tissue sample was determined by the use of phenol reagent (Lowry and Lopez(7)).

The extent of peroxidation in mitochondria was determined by the barbituric acid (TBA) reaction method of Corwin(1), with the exception that 0.5 ml of the mitochondrial preparation was used in place of whole liver homogenate.

To prepare MLHP, 50 g of methyl linoleate urea complex (Hormel Institute, Univ. of Minnesota) was extracted with 100 ml of hot 1 N HC1, and the methyl linoleate was washed 3-4 times, each time with 100 ml of water. The pure methyl linoleate was extracted with 30 ml of Skelly Solve F (SSF). After evaporation of the SSF under nitrogen (yield 13 g), 1 g of the oily methyl linoleate residue was incubated overnight at 0°C in 100 ml of 0.2 M phosphate buffer, pH 8, containing 20 mg lipoxidase (10,000 μ/mg, Sigma) with constant stirring.