Emmanuel N. Maduagwu

Effect of Thiamin Status on the Metabolism of Linamarin in Rats

The effect of graded levels of thiamin on the metabolism of linamarin was investigated in rats. It was observed that on a diet deficient in thiamin, a large amount of linamarin was recovered unchanged in the urine, together with significantly more thiocyanate (SCN-) relative to the control. The least amount of thiocyanate (p less than 0.05 relative to control) was found in animals receiving the highest amount (twice daily requirement) of thiamin in the diet; but the amount of unmetabolized linamarin was similar to the control. It is suggested that thiamin deficiency may be implicated in the aetiology of tropical ataxic neuropathy (TAN) through the thiocyanate overload in people eating large amounts of cassava and cassava derivatives which contain linamarin.

Differential effects on the cyanogenic glycoside content of fermenting cassava root pulp by β-glucosidase and microbial activities

The degradation of cyanogenic glycosides was studied in spontaneously fermenting cassava root pulp and in fresh pulp samples pretreated to prevent either endogenous beta-glycosidase activity, fermentation, or both. The rate of disappearance of the glycosides, as measured by hydrocyanic acid (HCN) production in situ, in membrane-sterilised media or in samples containing 1% sodium iodoacetate, was comparable with the untreated control in which 85% of the substrate was broken down within 72 h. Pretreatment of the fresh pulp with the beta-glucosidase inhibitor 1,5-gluconolactone (1%) markedly reduced the rate of disappearance of the cyanogens while inclusion of glucose in this test medium at the 3% level appeared to induce some hydrolysis. Loss of bound (glycosidic) cyanide in sterilised medium containing the glucosidase inhibitor was negligible. The results suggest that the contribution of the fermentation process in cyanide detoxification of pulped cassava roots is minimal.

Metabolism of linamarin in rats

The metabolism of linamarin [2(beta-D-glucopyranosyloxy)isobutyronitrile] was investigated in male albino Wistar rats and using rat liver microsomal preparations. In the in vitro experiments incubations of varying concentrations of linamarin at pH 6.0-6.5 with liver microsomal preparations resulted in rapid degradation of the substrate without concomitant production of any detectable amount of hydrogen cyanide (HCN) or of thiocyanate, its detoxication derivative. Boiled incubation medium did not degrade linamarin. Mathematical treatment of the degradation data generated theoretical HCN values that were used to construct a Lineweaver-Burke plot, which gave apparent Km and Vmax values of 3.3 mM-linamarin and 0.017 mg HCN/min/mg protein, respectively. In the in vivo experiments excretion of glucosidic cyanide (linamarin) in rat urine was found, within the range of applied oral doses 10-350 mg/kg body weight, to be dose dependent. Urinary excretion of HCN and thiocyanate did not show this correlation. Following administration (iv) of 10, 50 or 100 mg linamarin, elimination of the test substance from rat blood was observed to occur exponentially, and the half-life was estimated at about 90 min for all three dose levels.

Fate of ingested linamarin in malnourished rats

Pure linamarin at a dose level of 30 g per 100 g body weight was administered in food to a group of Wistar rats maintained on vitamin B2-deficient, sufficient and excess diets for 5 weeks and to another group of kwashiorkor rats. Free and total cyanide, intact linamarin and thiocyanate levels were estimated in urine and faeces obtained at 0-, 24-, 48- and 72-h periods and in blood samples obtained in the seventy-second hour after the drug had been administered. There was no detectable cyanide or intact linamarin in the faecal samples. Vitamin B2-sufficient and excess groups of rats excreted higher total and free cyanide than the respective vitamin B2-deficient groups. Most of the linamarin was degraded after the first 24h. The rate of breakdown of the glucoside within the first 24 h was slowest for the zero and half normal vitamin B2 status, respectively, as evidenced by its appearance in large quantities in the urine. The kwashiorkor rats, on the other hand, excreted less thiocyanate than the controls. In addition, their control group excreted most of the thiocyanate (SCN−) in the first 24 h whilst the kwashiorkor rats excreted theirs in the first 48 h. Dietary protein deficiency prolongs the time of metabolism and hence increases the toxicity of cyanogenic glycoside in the body. It is also suggested that excessive exposure of malnourished humans to cyanide could be a contributory factor in the rampant cases of tropical ataxic neuropathy (TAN).

Cyanide content of gari

500 random samples of gari (a cassava by-product) being hawked for sale in Ibadan markets, in Nigeria, were assayed for hydrocyanic acid (HCN) content. Most of the samples contained detectable amounts of HCN, ranging between 0.8 and 38 mg/kg, and only a very small proportion (0.4%) contained no cyanide. The limit of HCN detection was 0.1 ppm. There was no significant difference (P < 0.01) between the mean total cyanide (glycoside-bound plus non-glycosidic) and free cyanide (non-glycosidic) contents, 8.0 ± 6.9 mg HCN/kg and 7.2 ± 5.8 mg HCN/kg, respectively; indicating that an overwhelming proportion of the residual cyanide content of commercial gari could be present in the “free” form. The toxicity implications of free cyanide in cassava diets is briefly discussed.

Preformed volatile nitrosamines in some Nigerian foodstuffs

Some common Nigerian foodstuffs were assessed for their content of preformed volatile nitrosamine by chemiluminescence detection following gas chromatographic separation. Nitrosodimethylamine levels of between 0.4 and 4.6 ppb were detected in 75% of the samples analysed. The highest value was found in Brassica oleraceae, while Vernonia amygdalina contained the lowest detectable level. These data suggest that Nigerians may be exposed to chronic but very low levels of carcinogenic nitrosamines in their foods.

Potential of endogenous formation of volatile nitrosamines from Nigerian vegetables and spices.

Nitrosatability of dried Nigerian vegetables and spices was investigated under simulated gastric conditions. N-Nitroso-dimethylamine (NDMA) was the only volatile nitrosamine found above the preformed level except in ugwu where N-nitroso-piperidine (2.3 ppb) was also detected. The lowest NDMA value of 0.4 ppb was found in bitterleaf while onions had the highest level (14.7 ppb) of nitrosation. The results suggest that, under endogenous conditions, nitrosation of these foodstuffs could be an important contributory factor in cancer aetiology.

Nitrosamine metabolism in kwashiorkor rats

The in vitro metabolism of nitrosodimethylamine (NDMA) was studied in liver tissue obtained from male weanling kwashiorkor wistar rats. The elimination of this compound and that of nitrosomorpholine (NMOR) from the blood, after a single intravenous dose, was also investigated. N-demethylase activity in liver microsomes of the test animals was not significantly different from that of the controls although the activity of this enzyme per gram wet liver tissue was considerably reduced in the model animals. On the other hand, the glutathione (GSH) content in liver cytosol of the kwashiorkor animals was much higher than that of the controls. The elimination of NDMA and NMOR from the blood of the experimental animals over 8 hr following i.v. administration of the carcinogens, showed that the clearance rate of each nitrosamine was significantly lower in the kwashiorkor rats.

Biliary excretion of linamarin in the wistar rat after a single dose

The biliary excretion of linamarin (2[beta-D-glucopyranosyloxy]isobutyronitrile) was studied in male albino Wistar rats injected i.p. with single doses of 300 mg linamarin/kg following cannulation of the bile duct in vivo; 24 hr faeces of uncannulated rats, similarly dosed, was examined for excretory products. Enzymatic and spectrophotometric analyses of the bile exudate showed that glucosidic cyanide (linamarin, and non-glucosidic cyanide were excreted; the elimination of both cyanide forms exhibits biphasic kinetics. Thiocyanate ion was undetectable. T.l.c. of the test bile followed by enzymatic and chemical investigation of the chromatogram confirmed the presence of unchanged linamarin, and four different u.v. fluorescent non-glucosidic cyanide metabolites. Neither linamarin nor cyanide ion was detectable in faeces of the uncannulated rats.

A comparative assessment of toxic effects of dimethylnitrosamine in six different species

The hepatotoxicity which usually accompanies the oral administration of dimethylnitrosamine (DMN) to laboratory animals was studied in male rats, guinea pigs, cats, lizards, ducks, and monkeys. A single dosage of 50 mg/kg, which is necrotizing to rat liver, in most cases produced within 30 hr more acute liver damage in cats than in guinea pigs, rats, or monkeys, in that order of susceptibility, but was not toxic to lizards or ducks. DMN (5 mg/kg), given daily, induced the same degree of liver damage in guinea pigs and rats as did a single dose of the same compound. However, in cats and monkeys the effect of a single dose of 50 mg/kg was less severe than that obtained after the administration of 5 mg/kg over a period of 5–11 days. DMN (1 mg/kg), administered daily over a period of 30 days, was particularly harmful to cats and lizards. Clinicopathologic signs of overt toxicity were also monitored. Our results would support the view that species differences and dosage rates are both critical factors determining the different susceptibilities of various animals to nitrosamine toxicity.