Inhibition of Substrate Oxidation by Endotoxin in Vitro 1

Bacterial endotoxins are lipopolysaccharides producing profound vascular changes in experimental animals (1). However, a number of studies indicate that endotoxin produces alterations in tissue metabolism independent of alterations in vascular resistance, blood flow, or tissue oxygenation (1-3). The present studies were undertaken to determine whether endotoxin itself might inhibit oxidative metabolism in liver, similar to the effects of staphylococcal alpha-toxin on toad bladder (4) and diphtheria toxin on myocardium (5).

Materials and Methods. E. coli, S. abortus equi, and S. enteriditis endotoxins (Difco Laboratories) were suspended in isotonic saline immediately before use. Radioactive substrates were obtained commercially in 98% or greater purity. All were dissolved in water and diluted with nonradioactive substrate. The dissolution of 1-¹⁴C-palmitic acid was achieved by dropwise addition of NH₄OH to pH 9.0.

Mice weighing 20-25 g (Charles River Breeding Laboratories) were fed Purina Chow ad libitum, fasted 18 hr, and killed by a blow on the head. Livers were removed at once, blotted, weighed, and homogenized in 2.5 vol of oxygenated Krebs-Ringer phosphate buffer (pH 7.4). Aliquots of 0.25 ml of homogenate were added to 25-ml incubation flasks containing radioactive substrate, 2.0 ml oxygenated KRP, and 0.25 ml of either saline (control) or endotoxin in saline (experimental). Substrate concentrations were: 1-¹⁴C-palmitate 100 μM, 1-¹⁴C-hexanoate 100 μM, 2-¹⁴C-pyruvate 4 mM, and 1,5-¹⁴C-citrate 10 mM.

Flasks were sealed with rubber stoppers holding hanging plastic cups and incubated for 1 hr, with shaking, at 37°. At that time 0.25 ml of saline was injected into experimental flasks and 0.25 ml endotoxin (in saline) into control flasks. Hyamine (0.2 ml) was immediately injected into the plastic cups to collect the ¹⁴CO₂, and the medium was acidified by the injection of 0.3 ml of 4 N H₂SO₄.