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1644. Biochemical effects of caffeine
674
NATURAL PRODUCTS
active only during June-October and sainfoin to a very limited extent only in May and August. Mouse bioassay confirmed oestrogenic activity in red clover and in one sample only of white clover, but not in any samples of lucerne. This study clearly shows that seasonal variation in oestrogen content and the type of hormone assay used may explain the conflicting results that have been obtained m the past. Evidence is provided by Lightfoot et al. (cited above) that reduced fertility in ewes grazing on clover may be due to impaired sperm penetration into the female genital tract, a condition possibly brought about by the creation of a hostile cervical mucus, although uterine inertia resulting from the effect of oestrogens on the hypothalamo-neurohypophyseal system may be directly involved. Matsui et al. (cited above) screened 122 natural products used in Chinese and Hawaiian medicine for the presence of antifertility agents. Extracts of each product were administered subcutaneously to female mice and the effect on fertility was determined. Reduced fertility (i.e. a decrease in litters of at least 50 %) unaccompanied by general systemic toxicity was recorded for 16 of the products, and the effect appeared to be mediated by interference with tubal transport of the ova or with implantation rather than with ovulation.
1643. Factors affecting caffeine toxicity Peters, J. M. & Boyd, E. M. (1967). The influence of a cachexigenic diet on caffeine toxicity. Toxic. appl. Pharmac. 11, 121. Colton, T., Gosselin, R. E. & Smith, R. P. (1968). The tolerance of coffee drinkers to caffeine. Clin. Pharmac. Ther. 9, 3 I. It is common to find an enhancement in the toxicity of a compound in animals subjected to stress. In the case of caffeine, a 50 % reduction in food intake was necessary to achieve this effect (Cited in F.C.T. 1967, 5, 424). Peters & Boyd (cited above) studied the effect on caffeine toxicity of a biotin-deficient raw egg-white diet that became rancid on prolonged standing. This diet was capable in its own right of producing severe cachexia, with loss in body weight, reduced food intake, diarrhoea and diuresis in rats. Caffeine-treated rats succumbed more easily to the rancid diet than to standard Purina Laboratory Chow diet. This was to be expected, but rather surprisingly, the biotin-deficient diet was more toxic to caffeine-treated rats when presented in a fresh state than when it was rancid. In other words, rancidity appeared to confer a protective effect. The authors conclude that malnutrition does not necessarily accentuate toxicity. Do regular coffee drinkers develop a tolerance towards caffeine ? This question has been investigated by Colton et al. (cited above) in over 130 medical students who were divided into two groups: those who drank more than one cup of coffee or tea or one bottle of cola a day and those who did not. After a 150-mg oral dose of caffeine, the response of these two groups in respect of pulse rate, sleeping pattern, 'morning-after' and other symptoms was determined. The habitual drinker of caffeinated beverages was significantly less sensitive than the occasional drinker to the pulse rate-reducing action and sleep-disturbing effects of caffeine. Nevertheless the tolerance of the habitual drinker towards caffeine was considered to be of a low order.
1644. Biochemical effects of caffeine Mitoma, C., Sorich, T. J., II & Neubauer, Susanna E. (1968). The effect of caffeine on drug metabolism. Life Sci. 7, 145.
NATURALPRODUCTS
675
Bellet, S., Feinberg, L. J., Sandberg, H. & Hirabayashi, M. (1968). The effects of caffeine on free fatty acids and blood coagulation parameters of dogs. J. Pharmac. exp. Ther. 159, 250. The biological effects of caffeine have aroused much attention, not least among tea and coffee drinkers (Cited in F.C.T. 1968, 6, 389; see also previous abstract). In the first of two biochemical studies reported, Mitoma et al. (cited above) have demonstrated the ability of caffeine, as well as tea and coffee, to induce the synthesis of processing enzymes in the rat. The enzymes studied were acetanilide hydroxylase and o-nitroanisole demethylase. Three daily oral doses of 40 mg caffeine/kg increased acetanilide hydroxylase activity by 60 % of the control value and almost twice this dose (75 mg/kg) doubled enzyme activity. The effect on o-nitroanisole was not so marked. Ethionine and puromycin, inhibitors of protein synthesis, suppressed these increases in enzyme activity, indicating that the effect of caffeine on enzyme activity is one of induction and not activation. Pharmacologically-active theophylline and theobromine, also present in the beverages, were less strong enzyme inducers than caffeine. When the rats joined the tea and coffee set, an intake equivalent to about 75 mg caffeine/kg/day for 3 days sufficed to increase enzyme activity. It would appear that a man would have to drink about 35 cups of coffee/day before his microsomal-enzyme activity came under the influence of caffeine. The second study (Bellet et al. cited above) was prompted by the observation that longchain fatty acids modify various tests for blood coagulation and involved the simultaneous investigation of the effects of caffeine on serum free fatty acids and on blood-clotting parameters in the dog. The rise in serum free fatty acids following an intravenous dose of 25 mg caffeine/kg was greatest between 3 and 4 hr after dosing and it was during this period that the prothrombin, heparin-tolerance and recalcification times were significantly shortened. It was considered possible that the elevation of free fatty acids produced by caffeine may be responsible for the changes in the blood-clotting mechanism. 1645. Refsum and ct-oxidation
Stokke, O., Try, K. & Eldjarn, L. (1967). a-Oxidation as an alternative pathway for the degradation of branched-chain fatty acids in man, and its failure in patients with Refsum's disease. Biochim. biophys. Acta 144, 271. Try, K. & Eldjarn, L. (1967). Normalization of the Tricaprin Test for oJ-oxidation in Refsum's disease upon lowering of serum phytanic acid. Scand. J. clin. Lab. Invest. 20, 294. Eldjarn, L. (1968). Hereditary ataxic polyneuritis: Biochemical and dietary studies. Clin. Biochem. 1, 273. We have discussed at length how patients with Refsum's disease accumulate phytanic acid (I) in their tissues due to a defect in the process of oxidative metabolism (Cited in F.C.T. 1967, 5, 95). Although the normal degradation of I in man is not fully understood, a-oxidation, o~-oxidation followed by/3-oxidation, or the isovaleric acid pathway appear to be involved. An alternative pathway has now been suggested by Stokke et al. (cited above), who took advantage of the fact that the compound 3,6-dimethyl[8-14C]octanoic acid retains a methyl group in the/3-position to the carboxyl group even after an initial w-oxidation. Healthy men degraded this compound by an initial a-decarboxylation which facilitated the subsequent attack by/3-oxidation, but patients with Refsum's disease failed to degrade this/3-methyl fatty acid by an a-decarboxylation. Try & Eldjarn (cited above) consider unlikely the possibility that the primary metabolic defect in Refsum's disease lies in the oJ-oxidation mechanism. By limiting the amount of
NATURAL PRODUCTS
active only during June-October and sainfoin to a very limited extent only in May and August. Mouse bioassay confirmed oestrogenic activity in red clover and in one sample only of white clover, but not in any samples of lucerne. This study clearly shows that seasonal variation in oestrogen content and the type of hormone assay used may explain the conflicting results that have been obtained m the past. Evidence is provided by Lightfoot et al. (cited above) that reduced fertility in ewes grazing on clover may be due to impaired sperm penetration into the female genital tract, a condition possibly brought about by the creation of a hostile cervical mucus, although uterine inertia resulting from the effect of oestrogens on the hypothalamo-neurohypophyseal system may be directly involved. Matsui et al. (cited above) screened 122 natural products used in Chinese and Hawaiian medicine for the presence of antifertility agents. Extracts of each product were administered subcutaneously to female mice and the effect on fertility was determined. Reduced fertility (i.e. a decrease in litters of at least 50 %) unaccompanied by general systemic toxicity was recorded for 16 of the products, and the effect appeared to be mediated by interference with tubal transport of the ova or with implantation rather than with ovulation.
1643. Factors affecting caffeine toxicity Peters, J. M. & Boyd, E. M. (1967). The influence of a cachexigenic diet on caffeine toxicity. Toxic. appl. Pharmac. 11, 121. Colton, T., Gosselin, R. E. & Smith, R. P. (1968). The tolerance of coffee drinkers to caffeine. Clin. Pharmac. Ther. 9, 3 I. It is common to find an enhancement in the toxicity of a compound in animals subjected to stress. In the case of caffeine, a 50 % reduction in food intake was necessary to achieve this effect (Cited in F.C.T. 1967, 5, 424). Peters & Boyd (cited above) studied the effect on caffeine toxicity of a biotin-deficient raw egg-white diet that became rancid on prolonged standing. This diet was capable in its own right of producing severe cachexia, with loss in body weight, reduced food intake, diarrhoea and diuresis in rats. Caffeine-treated rats succumbed more easily to the rancid diet than to standard Purina Laboratory Chow diet. This was to be expected, but rather surprisingly, the biotin-deficient diet was more toxic to caffeine-treated rats when presented in a fresh state than when it was rancid. In other words, rancidity appeared to confer a protective effect. The authors conclude that malnutrition does not necessarily accentuate toxicity. Do regular coffee drinkers develop a tolerance towards caffeine ? This question has been investigated by Colton et al. (cited above) in over 130 medical students who were divided into two groups: those who drank more than one cup of coffee or tea or one bottle of cola a day and those who did not. After a 150-mg oral dose of caffeine, the response of these two groups in respect of pulse rate, sleeping pattern, 'morning-after' and other symptoms was determined. The habitual drinker of caffeinated beverages was significantly less sensitive than the occasional drinker to the pulse rate-reducing action and sleep-disturbing effects of caffeine. Nevertheless the tolerance of the habitual drinker towards caffeine was considered to be of a low order.
1644. Biochemical effects of caffeine Mitoma, C., Sorich, T. J., II & Neubauer, Susanna E. (1968). The effect of caffeine on drug metabolism. Life Sci. 7, 145.
NATURALPRODUCTS
675
Bellet, S., Feinberg, L. J., Sandberg, H. & Hirabayashi, M. (1968). The effects of caffeine on free fatty acids and blood coagulation parameters of dogs. J. Pharmac. exp. Ther. 159, 250. The biological effects of caffeine have aroused much attention, not least among tea and coffee drinkers (Cited in F.C.T. 1968, 6, 389; see also previous abstract). In the first of two biochemical studies reported, Mitoma et al. (cited above) have demonstrated the ability of caffeine, as well as tea and coffee, to induce the synthesis of processing enzymes in the rat. The enzymes studied were acetanilide hydroxylase and o-nitroanisole demethylase. Three daily oral doses of 40 mg caffeine/kg increased acetanilide hydroxylase activity by 60 % of the control value and almost twice this dose (75 mg/kg) doubled enzyme activity. The effect on o-nitroanisole was not so marked. Ethionine and puromycin, inhibitors of protein synthesis, suppressed these increases in enzyme activity, indicating that the effect of caffeine on enzyme activity is one of induction and not activation. Pharmacologically-active theophylline and theobromine, also present in the beverages, were less strong enzyme inducers than caffeine. When the rats joined the tea and coffee set, an intake equivalent to about 75 mg caffeine/kg/day for 3 days sufficed to increase enzyme activity. It would appear that a man would have to drink about 35 cups of coffee/day before his microsomal-enzyme activity came under the influence of caffeine. The second study (Bellet et al. cited above) was prompted by the observation that longchain fatty acids modify various tests for blood coagulation and involved the simultaneous investigation of the effects of caffeine on serum free fatty acids and on blood-clotting parameters in the dog. The rise in serum free fatty acids following an intravenous dose of 25 mg caffeine/kg was greatest between 3 and 4 hr after dosing and it was during this period that the prothrombin, heparin-tolerance and recalcification times were significantly shortened. It was considered possible that the elevation of free fatty acids produced by caffeine may be responsible for the changes in the blood-clotting mechanism. 1645. Refsum and ct-oxidation
Stokke, O., Try, K. & Eldjarn, L. (1967). a-Oxidation as an alternative pathway for the degradation of branched-chain fatty acids in man, and its failure in patients with Refsum's disease. Biochim. biophys. Acta 144, 271. Try, K. & Eldjarn, L. (1967). Normalization of the Tricaprin Test for oJ-oxidation in Refsum's disease upon lowering of serum phytanic acid. Scand. J. clin. Lab. Invest. 20, 294. Eldjarn, L. (1968). Hereditary ataxic polyneuritis: Biochemical and dietary studies. Clin. Biochem. 1, 273. We have discussed at length how patients with Refsum's disease accumulate phytanic acid (I) in their tissues due to a defect in the process of oxidative metabolism (Cited in F.C.T. 1967, 5, 95). Although the normal degradation of I in man is not fully understood, a-oxidation, o~-oxidation followed by/3-oxidation, or the isovaleric acid pathway appear to be involved. An alternative pathway has now been suggested by Stokke et al. (cited above), who took advantage of the fact that the compound 3,6-dimethyl[8-14C]octanoic acid retains a methyl group in the/3-position to the carboxyl group even after an initial w-oxidation. Healthy men degraded this compound by an initial a-decarboxylation which facilitated the subsequent attack by/3-oxidation, but patients with Refsum's disease failed to degrade this/3-methyl fatty acid by an a-decarboxylation. Try & Eldjarn (cited above) consider unlikely the possibility that the primary metabolic defect in Refsum's disease lies in the oJ-oxidation mechanism. By limiting the amount of