Mimosine Content Research Articles

Toxicity of Leucaena leucocephala in ruminants: the effect of supplemental thyroxine on goats fed on a sole diet of Leucaena

Daily injections of thyroxine were ineffective in overcoming the toxic effects of feeding Leucaena to goats. The treated goats did not differ from the controls in liveweight change or body condition over the 15 weeks of feeding Leucaena and both groups of animals developed oesophageal lesions. Treated animals maintained normal serum thyroxine (T4) and triiodothyronine (T3) levels and did not exhibit the thyroid hyperplasia of the controls. An increase in the serum T4 and T3 levels of three of the control animals after 5 weeks of feeding was associated with declining mimosine concentration in the feed and the ability of the hyperplastic thyroids to produce sufficient T4. Serum analyses suggested zinc deficiency in the control animals, associated with their low thyroid status. It was concluded that the goitrogenicity of 3-hydroxy-4(1H)-pyridone (DHP) is only partly responsible for the toxicity of Leucaena to ruminants, and that the low feed intakes and low liveweight gains are related to other effects of DHP. The evidence for extra-ruminal metabolism of DHP is discussed.

Comparative investigations of the action of mimosine on nucleic acid synthesis in Ehrlich ascites-tumour cells

Mimosine (leucenol; 3 [ 3 -hydroxy-rl-oxo1 (4H)-pyridinyllalanine } is a non-protein amino acid of plant origin (see Fowden et al., 1967). Several studies .have shown that mimosinecontaining plants and mimosine itself are toxic to animals (Matsumoto et al., 1951; Owen, 1958; Hegarty et al., 1964). The toxicity of mimosine has also been studied in cell culture (Tsai & Ling, 1971; Prabhakaran et al., 1973). Exposure of cells in culture for relatively long periods causes inhibition of DNA synthesis (Tsai & Ling, 1971, 1972; Hegarty et al., 1978). In a previous report we showed that mimosine had an inhibitory effect on RNA synthesis in Ehrlich ascites-tumour cells. The inhibition was concentration-dependent in the range of 0.05-1 mwmimosine. It was found also that the inhibition was much more pronounced when the cells were preincubated with mimosine before their capacity for RNA synthesis was determined (Itzhaki & Abdulla, 1982). The work was extended to study the action of mimosine in a wider range of concentration on the synthesis of both RNA and DNA. Ehrlich ascites-tumour cells were grown in mice (Itzhaki, 1972), harvested, washed with 0.9% NaCl and suspended in the incubation medium. RNA synthesis was measured as the rate of [5-3H]uridine incorporation into RNA of cells incubated at 37OC with the labelled precursor for 20min (Harwood & Itzhaki, 1973). In a similar set of determinations DNA synthesis was measured as the rate of incorporation of Irneth~l-~Hlthymidine into DNA. Comparison was made in all cases between cells which were incubated with mimosine for 1 h before the determination of RNA or DNA synthesis and those where the effect of mimosine was tested directly without a period of preincubation. The results show that for RNA synthesis, mimosine has a biphasic action in the preincubated series of experiments. A gradual increase in inhibition of RNA synthesis is observed when the concentration of mimosine is increased from 0 . 0 2 m ~ to about 0.5 mM, reaching a maximum inhibition of 75%. When the concentration of mimosine is raised from 1 to 6 m ~ , the inhibition of RNA synthesis gradually decreased, such that the rate of RNA synthesis returned to almost 80% of the control value. However, when RNA synthesis was measured directly in a 20min period of incubation without a period of preincubation, a slight inhibitory effect was observed at the higher range of mimosine concentrations. The results also confirm the earlier observations (Itzhaki & Abdulla, 1982) that a preincubation period is required to detect the action of mimosine. The effect of mimosine on DNA synthesis was quite different. Here, no noticeable effect was obtained with 0.02-0.2 m ~ mimosine. As the concentration was raised there was a gradual stimulation of DNA synthesis, with a broad peak around 3-4m~-mimosine, when the rate of DNA synthesis reached a value of 40-50% above that of control. Moreover, the pattern of thymidine incorporation into DNA is similar in both the preincubated and the non-preincubated series. The results indicate clearly that the requirement for a period of contact of the cells with mimosine for the latter to act on RNA synthesis may be related to its metabolism inside the cell during the duration of preincubation. The metabolism of mimosine in animal tissues has been studied, and among the products found to be formed are mimosinic acid and mimosinamine (Tang & Ling, 1977). It is possible that the action of mimosine on RNA synthesis observed here is due to a metabolite, or it may be brought about by a combination of effects of more than one metabolite of mimosine. In contrast. mimosine may itself exert the stimulatory action on DNA synthesis observed here; however, we have no indication as to its mechanism of action.

Effect of mimosine on RNA synthesis in Ehrlich ascites-tumour cells

Mimosine {leucenol; fiIN-(3-hydroxypyrid-4-one)l-a-aminopropionic acid; 3-I3-hydroxy-4-oxo- 1 (4H)-pyridinyl lalanine} is a non-protein amino acid found in the foliage and seeds of plants of the two legume genera Leucaena and Mimosa (see Thompson et al., 1969). Its toxicity has been studied in animals (Owen, 1958; Hylin & Lichton, 1965; Dewreede & Wayman, 1970) and in cells in culture (Tsai & Ling, 1971, 1972; Hegarty et al., 1978; Reisner & Bucholtz, 1979). In particular, mimosine has an inhibitory effect on hair growth (Montagna & Yun, 1963), which may be related to the reported inhibition of DNA synthesis in hair-follicular cells (Ishibashi & Seiji, 1976; Ward & Harris, 1976). It has been suggested that the biological effects of mimosine may be due to its action as a tyrosine antagonist (Crounse et al., 1962; Lin et al., 1964; Prabhakaran et al., 1973), but the exact mechanism of its action has not been elucidated. The purpose of this study is to investigate the action of mimosine on macromolecular synthesis in a single cell type. We here describe the effect of mimosine on RNA synthesis in Ehrlich ascites-tumour cells. These cells were grown in mice (Itzhaki, 1972), harvested, washed with 0.9% NaCl and suspended in phosphate-buffered saline. RNA synthesis was measured as the rate of [5-'H]uridine incorporation into RNA of cells incubated at 37°C with the labelled precursor for predetermined periods (Harwood & Itzhaki, 1973). The results show that in the presence of 1-5 mwmimosine the rate of RNA synthesis is inhibited by about 25% and slows down greatly after 1&15min, reaching almost a plateau after 20-30 min depending on the concentration of mimosine. There is also an indication that longer periods of incabation of the cells with mimosine slow down RNA synthesis to even greater extents. Therefore we investigated the effect of longer periods of contact of cells with mimosine. This was done by preincubating cells with mimosine for 1 h, followed by the addition of [ .'H]uridine and further incubation for 20min. The results from this series of experiments were compared with those where the effect of mimosine was tested directly without a period of preincubation. Investigations in the concentration range of 0.05-1 mwmimosine show that in the preincubation series the inhibition of RNA synthesis by mimosine is concentrationdependent, reaching an inhibition of up to 75%, whereas in the tests where mimosine and [ 'Hluridine were added together maximum inhibition of only 20% was observed. Further experiments showed that even shorter periods of preincubation with mimosine of 30min or less enhanced the inhibitory action of mimosine, but the effect was not as pronounced as in the 1-lfh period of preincubation. The interpretation of these results is that mimosine is converted in the cell into a metabolite which may be the actual inhibitor; therefore a minimum period of exposure of the cells to mimosine is required before the pronounced inhibitory effect of mimosine on RNA synthesis is observed. Indeed, mimosine is known to be metabolized, though its metabolism has been studied mainly in plants; however, in animal tissues as well, several metabolites have been detected (Tang & Ling, 1977). Crounse, R. G., Maxwell, J. D. & Blank, H. (1962) Nature (London)

An automated colorimetric method for mimosine in Leucaena leaves

AbstractSimplified extraction and colorimetric techniques have been developed for the determination of the toxic amino acid mimosine in Leucaena leucocephala. Fresh leaf material is extracted with dilute hydrochloric acid containing activated carbon and the concentration of mimosine in the clarified extract determined with an Auto‐Analyser. Satisfactory recoveries of added mimosine were obtained, and the results were in agreement with a manual method which was based on electrophoretic separation.

Mimosine, administered orally, and two related compounds of chemical defleecing agents for sheep

Oral doses of mimosine (ranging from 400 to 800 mg/kg body weight) were given to 71 sheep, either as two successive daily doses (10 sheep) or as a single dose (61 sheep). The effectiveness of these treatments for defleecing was assessed and in some sheep measurements were made of the concentration of mimosine in plasma after dosing, and of the rate of wool growth before and after dosing. In addition, the effectiveness as defleecing agents of two related compounds (an analogue of mimosine, 'isomimosine', and a metabolite, 3,4-dihydroxypyridine (DHP)) was assessed. Two successive daily doses of mimosine (300 mg/kg/day) allowed all sheep to be defleeced. With sheep consuming a daily ration of 600 g, a single oral dose of mimosine (400 mg/kg) was sufficient to allow defleecing of most sheep; when the daily ration was 1200 g, a dose of 600 mg/kg was required to allow defleecing of most sheep. The effectiveness of oral doses for defleecing could be explained by the absorption of mimosine over a period in excess of 24 hr. Concentrations of mimosine in plasma were usually above 100 µmoles/l 24 hr after an effective dose. Failure to achieve complete cessation of fibre growth occurred occasionally, irrespective of dose level, and two sheep died after dosing. Fasting prior to dosing appeared to obviate the effects of previous high nutrition, but increased the risk of toxic effects. Fasting resulted in exceptionally high concentrations of mimosine in plasma (200–300 µmoles/l) 1 and 2 days after dosing. Both fibre diameter and mass of wool grown were increased in the early regrowth after dosing with mimosine. 'Isomimosine' had a similar potency to mimosine for stopping fibre growth in sheep, whether given as an intravenous infusion or as a single oral dose. DHP, given as an intravenous infusion, was ineffective as a defleecing agent.

Effectiveness of intravenous and abomasal doses of mimosine for defleecing sheep and effects on subsequent wool growth

Four Merino sheep were given intravenous infusions of mimosine for 2 days at a rate of 110–120 mg/kg/day. Wool fibre growth stopped by about 1 day after the start of the infusions, and the sheep were subsequently manually defleeced. It was estimated that, on average, fibre growth stopped for about 12 days. Wool growth rates were above the pre-infusion rates in the early regrowth (3–5 weeks after dosing), and the mean fibre diameter was still about 2 µm above pre-infusion values at 11 weeks after dosing. Groups of Merino sheep were given intravenous infusions of mimosine for 2 days at rates ranging over 40–320 mg/kg/day. The minimal rate of infusion required to produce consistent defleecing was 80 mg/kg/day; infusions at a rate of 60 mg/kg/day were sometimes effective for defleecing, but at 40 mg/kg/day produced no discernible effects on the strength of wool fibres. No adverse effects were observed at a mimosine infusion rate of 80 mg/kg/day, but one out of four sheep died when dosed at a rate of 160 mg/kg/day, and higher rates (240 and 320 mg/kg/day) were lethal. The concentration of mimosine in plasma was related to the rate of infusion of mimosine. Consistent defleecing was associated with a concentration of mimosine in plasma approaching 100 µmoles/l; lethal doses resulted in plasma mimosine concentrations above 300 µmoles/l. Abomasal infusions of mimosine at a rate of 80 mg/kg/day were equivalent to intravenous infusions. Single injections of mimosine into the abomasum did not influence the strength of wool fibres; pIasma concentrations foIlowing injections indicated rapid absorption of mimosine and rapid removal from the body.

The influence of nutrition on the effectiveness of mimosine for defleecing sheep

The effectiveness of mimosine as a chemical defleecing agent was assessed in adult sheep that were subjected to a variety of nutritional treatments prior to administration of mimosine, and in lambs receiving relatively high feed intakes. Mimosine was given as a continuous intravenous infusion over a period of 2 days. Sheep receiving 600 g of a roughage-based diet per day were consistently defleeced following an infusion of mimosine at the rate of 80 mg/kg/day (designated the standard infusion). The prior feeding of a reduced intake of this diet (300 g/day) did not alter the amount of mimosine required to defleece sheep. Likewise, the prior abomasal infusion of an imbalanced mixture of amino acids, which depressed the wool growth rate, did not allow defleecing with a reduced amount of mimosine. A 4-day fast, commenced 3 days before the start of mimosine infusion, approximately halved the amount of mimosine required to defleece sheep. The provision of a high intake of energy, together with large amounts of amino acids available for absorption from the small intestines (supplied by casein), for at least 1 week prior to mimosine infusion, completely prevented defleecing with the standard infusion of mimosine. The effects of these nutritional treatments could be obviated by a 4-day fast as described above. Also, increasing the rate of infusion of mimosine to 120 mg/kg/day largely overcame the effects of previous high nutrition. Relatively greater amounts of mimosine were required to defleece lambs than adults. The concentration of mimosine in plasma was related to the rate of infusion of mimosine. However, fasting and a low dietary intake tended to enhance the concentration of mimosine in plasma for a given rate of infusion. Infusion of mimosine at the standard rate resulted in plasma mimosine concentrations of the order of 100 µmoles/l, but this concentration did not ensure that defleecing would be possible.

Studies on the specificities of the phenylalanyl- and tyrosyl-sRNA synthetases from plants

The phenylalanyl- and tyrosyl-sRNA synthetases of mung bean seed have been partially purified and their ability to utilize a range of amino acid analogues as substrates determined. The substrate specificity of the phenylalanyl-enzyme seems to be less exacting than that of the tyrosyl-enzyme, a finding in agreement with previous observations using animal and microbial enzymes. Mimosine, a toxic amino acid present in Mimosa and Leucaena species, served as a substrate for the phenylalanyl-, but not the tyrosyl-sRNA synthetase from mung bean. However, the transfer of 14C-phenylalanine to tRNA catalysed by its synthetase enzyme was not impaired by the presence of high concentrations of mimosine; Leucaena phenylalanyl-sRNA synthetase also activated mimosine, but it is concluded that mimosine-induced toxicities do not arise primarily by interference with phenylalanine incorporation into protein molecules. 2-Amino-4-methylhex-4-enoic acid, a natural product of Aesculus californica, behaved as a phenylalanine analogue, being activated by the phenylalanyl-sRNA synthetase of mung bean at rates comparable to that determined for phenylalanine itself.

Leucaena Cytogenetics in Relation to the Breeding of Low Mimosine Lines1

Cytological and biochemical studies were conducted in the genus Leucaena in relation to the breeding of low mimosine, high yielding forage types of L. leucocephala (Lam.) de Wit. Meiotic and mitotic chromosomes were studied in the following species; L. leucocephala and L. pulverulenta (Schlecht) Benth. (2n = 56); L. trichodes Benth., L. lanceolata (S. Wats.), L. stenocarpa (Urban), and accessions designated “L. buitenzorg” (2n = 52) and “L. esculenta” (2n = 104).All species of Leucaena hybridized freely, insofar as they were tested. The 80‐chromosome hybrids of L. pulverulenta ✕ L. leucocephala were grown and studied in detail. Seed recovery and yields of these hybrids were high, despite meiotic chromosome pairing of 26 bivalents and 28 univalents. F2 plants ranged widely in chromosome numbers.Assays for the toxic alkaloid, mimosine, were conducted on F1, F2, and backcross progenies from a cross of high ✕ low mimosine strains of L. leucocephala. Despite the apparent polyploidy of the species, wide variations were observed in mimosine contents of the segregating generations, enabling the selection of plants with less than 30% of the normal mimosine values (ca. 4% dry weight) of tropical forage strains. The correlation between mimosine and protein contents for related F2 segregants from high ✕ low mimosine crosses was not significant (r = .60).

The Effect of elevated temperatures on the mimosine content and toxicity of koa haole ( Leucaena glauca)

When koa haole leaves were stored at elevated temperatures the mimosine content was decreased. The effect was most pronounced and rapid when the temperature was over 70 °C. in the presence of moisture. A similar effect occurred in seeds when sufficient moisture was present. The phenomenon did not take place in dry leaves. Steam was effective in extracting mimosine from koa haole leaves. Heated koa haole leaf meal has been demonstrated to be less toxic than unheated koa haole leaf meal when the meals are fed to albino rats as part of their rations. The development of alopecia in rats fed unheated koa haole meal has been described, and the growth curves of rats fed heated and unheated leaf meals and basal rations have been compared. Ferrous sulfate added to rations containing unheated koa haole leaf meal has been shown to be effective in reducing mimosine toxicity.

The Use of Fresh Green Koa Haole Leaves in Chick Rations

KOA HAOLE (Leucaena glauca) is a small tree or woody shrub which grows in abundance in Hawaii. Throughout the year considerable quantities of the leaves are available for animal feeding, but heretofore limited use had been made of this product because of its mimosine content. The purpose of this investigation was to determine whether fresh green koa haole leaves would be injurious when fed to chicks, and, if not, to determine the age at which the optimum amount for growth could be fed to growing chicks.As far as is known, no literature has been published on the value of fresh green koa haole leaves as feed for poultry. However, dehydrated koa haole leaves and koa haole seed meal have been fed to different experimental subjects including chickens with varying results. Bice (1942) reported that laying pullets maintained 50 percent production when 5 percent of their ration was koa haole .