Mechanism of antagonizing cyanide-induced lethality by alpha-ketoglutaric acid

alpha-Ketoglutaric acid (alpha-KG) has been shown to be an effective antagonist for cyanide-induced lethality. The mechanism of this antagonism is hypothesized to result from alpha-KG binding with cyanide. Several investigative approaches were taken to determine the existence of this binding. First, mixtures of various molar ratios of alpha-KG:cyanide were injected into a high pressure liquid chromatograph. The addition of cyanide reduced the peak area of alpha-KG at a molar ratio of greater than 1:5. Second, blood from naive male ICR mice was spiked with alpha-KG and cyanide. Headspace above these blood samples was injected into a gas chromatograph and analyzed for released hydrogen cyanide. alpha-KG reduced the peak area of hydrogen cyanide released into the headspace at molar ratios of greater than 1:2.5. Third, the effect of cyanide on the ultraviolet spectrum of alpha-KG was determined as an indication of binding. In the presence of cyanide the absorption peak at 316 nm for alpha-KG was eliminated. Inhibition of cytochrome oxidase is an accepted target enzyme for cyanide-induced lethality. Fourth, further evidence of alpha-KG's mechanism was determined by the effect of alpha-KG on brain cytochrome oxidase (BRCYTOX) and its ability to antagonize cyanide-induced inhibition of BRCYTOX. BRCYTOX activity was determined in the presence of alpha-KG and was found to be unaffected between 0.01 and 0.06 M of alpha-KG. Greater concentrations of alpha-KG inhibited BRCYTOX activity. The complete inhibition of BRCYTOX activity by 10(-5) M cyanide was prevented with 0.05 and 0.06 M alpha-KG. Fifth, BRCYTOX activity of animals pretreated with saline and then an LD80 dose (8.5 mg/kg) of cyanide was 80% inhibited, while BRCYTOX activity of animals pretreated with 2 g alpha-KG/kg, i.p., and then an LD80 dose (7.75 mg/kg) of cyanide was not different from control values. Thus, these data suggest that alpha-KG does bind with cyanide, and this binding can account for the antagonism of cyanide-induced lethality.