COMPARATIVE METABOLISM OF LINAMARIN (2β-D- GLUCOPYRANOSYL OXY) -2METHYLPROPANE NITRILE) IN RATS AND MICE

Abstract

Cyanide interferes, acutely, with cellular respiration by inhibiting cytsochrome oxidase activity. Living in the tropics increased vulnerability to cyanide mainly through ingestion of free Hydrocyanic Acid (HCN) derived from cassava roots that contain cyanogenic glucosides, especially linamarin. Though toxicity of free HCN is well established, there is no biochemical evaluation of species variation in the metabolism of linamarin in animals. Therefore, this study was carried out to evaluate the metabolism, disposition and toxicity of linamarin and its cyanide adjuncts in rats and mice. Three groups, each of five adult male albino Wistar rats (average weight:160g) and Swiss mice (average weight: 44g) were given oral doses of 5, 10 and 50 mg/kg of linamarin, respectively. The fourth group (control) received distilled water. Samples of 24-hour urine, faeces, blood and liver tissues were collected. Residual linamarin and its metabolites were determined in urine, faeces and plasma post exposure. Reduced glutathione (GSH) was determined in whole blood and liver homogenate while liver function test was done with plasma. Three similar animal groupings were given the same linamarin dose regimen intravenously and the elimination of the compound was monitored spectrophotometrically in blood samples collected at intervals of one and thirty minutes post exposure. Liver samples from the animals were homogenized in phosphate buffer solution and centrifuged at 105,000g. In vitro degradation of linamarin and the concomitant appearance of cyanide derivatives by the resultant microsomal pellet were determined. Statistical analyses were done using Student's t-test and ANOVA at p≤ 0.05. The degradation of linamarin in vivo was dose-dependent and resulted in significantly higher plasma; total cyanide concentration of 0.052 ± 0.00g/ml in rats than 0.039±0.0g/ml in mice at 50ml dose. It also gave significantly higher mean faecal and urine total cyanide values of 0.046±0.00g/ml and 0.079±0.00g/ml in rats than 0.024±0,00g/ml and 0.016±0.005g/ml in mice respectively. Blood and liver GSH concentrations were lowered in rats but increased in mice relative to control; thus suggesting greater susceptibility of rats to linamarin toxicity. The mean plasma alkaline phosphatase activity decreased significantly by 31.8% and by 22.9% in rats and mice respectively indicating greater susceptibility of rats to cyanide toxicity. Conversely, aspartate aminotransferase activity increased significantly with dose by xii 82.40% and 72.3% in rats and mice while alanine aminotransferase activity increased by 97.4% and 153.5% in rats and mice respectively. The blood biological half-life of linamarin in rats and mice calculated from semi-log plots was 4.0 and 8.0 mins respectively, suggesting a faster metabolism in rats. In vitro analysis of linamarin metabolites gave a transient high level of thiocyanate at 0.4mM linamarin; Vmax (mg CN/mg protein/min.) and Km (mM) values of 66.7x10-6 and 0.1mM for rats and 3.33xl0-5 and 0.2mM for mice respectively, thus, suggesting a higher degradation rate of the substrate in rats and inhibition of enzymatic conversion of CN- to thiocyanate. The rat degrades linamarin faster than the mouse both in vivo and in vitro, as indicated by a higher excretion rate of cyanide, Vmax and toxicity as well as shorter biological half-life of substrate and Km.