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Indospicine is a hepatotoxic amino acid that accumulates in the meat of horses that consume the legume Indigofera linnaei. A method to determine indospicine concentration in biological samples using an amino acid analyser has been reported, but the analysis time is long and therefore not suited to the analysis of large numbers of samples. A rapid and reliable method was developed for the analysis of indospicine in horsemeat and serum using High Performance Liquid Chromatography. Horsemeat and serum were extracted with either water or 0.01 N hydrochloric acid, respectively, and deproteinized by ultrafiltration. Precolumn derivatization of samples with phenylisothiocyanate was followed by separation of indospicine from other amino acids on a Pico-Tag C 18 column and UV detection at 254 nm. The calibration curves for indospicine in horsemeat extract were linear over the concentration range 0.4 microg ml(-1) to 20 microg ml(-1), while for indospicine in serum, the linear range was from 0.17 microg ml(-1) to 16.67 microg ml(-1). The mean recovery of indospicine in horsemeat extract was 87.2 +/- 6.8% and in serum was 97.3 +/- 9.9%. Analysis time for indospicine in horsemeat samples was 31 min and in serum samples was 36 min.

The ability of the naturally occurring non-protein toxic amino acids indospicine and canavanine to inhibit nitric oxide synthesis was tested in isolated rat aorta and cultured rat peritoneal macrophages. Both compounds inhibited acetylcholine induced relaxation of rat aorta contracted with noradrenaline, a process mediated by nitric oxide generated in vascular endothelium. Nitric oxide is generated in vascular endothelium from arginine by a constitutive nitric oxide synthase. Indospicine and canavanine also increased superoxide mediated reduction of cytochrome c by phorbol myristate acetate stimulated rat peritoneal macrophages. The increase in superoxide under these conditions was due to decreased nitric oxide synthesis. Macrophage synthesis of nitric oxide is mediated by an inducible form of nitric oxide synthase. It is concluded that indospicine and canavanine are inhibitors of constitutive and inducible nitric oxide synthases and it is suggested that the toxicity associated with these compounds could be related to this activity.

Previous studies of saliva from engorged female cattle ticks revealed a component which induced contration of some isolated smooth muscles. Fractionation and further characterisation have shown that this substance is of the "slow-reacting" type, but that it is neither a bradykinin nor slow-reacting substance of anaphylaxis. The substance is deactivated by incubation with 15-hydroxprostaglandin dehydrogenase and its pharmacological properties also support its classification as a prostaglandin. A second pharmacologically-active component has now been found in the saliva but has not yet been characterised.

Chronic active hepatitis was selectively induced in mice by the feeding of a diet containing 3-hydroxy-4-pyrone (0.5% by weight) for periods of 6 weeks and longer. This model should be of particular value in elucidating the pathogenesis of drug-induced forms of chronic active hepatitis. Maltol (3-hydroxy-2-methyl-4-pyrone) did not produce any liver lesion.

Tropolone (TR) and 3-hydroxy-4-pyrone were investigated for antithyroid activity following the finding that the 2-hydroxy-oxo pyridine, 3-hydroxy-4(1H)-pyridone (DHP, I), is goitrogenic. Both compounds inhibited the thyroidal uptake of radioiodine in rats and resembled the thioamide drugs in inhibiting the organic binding of iodine by the thyroid gland rather than the trapping of iodide, but were weaker binding inhibitors than 6-methyl-2-thiouracil (MeTU). Both compounds also inhibited the iodination of bovine serum albumin and thyroglobulin, catalyzed by thyroidperoxidase (TPO), lactoperoxidase (LPO), chloroperoxidase (CPO) and horseradish peroxidase (HPO) in vitro. The inhibitory effect of TR but not that of 3-hydroxy-4-pyrone was antagonized by ferrous ions. When fed to mice at levels of intake expected to produce goitre both compounds were toxic and caused severe liver damage. Thyroid enlargement was not observed in any of these feeiding experiments, but the thyroids of mice fed 0.1% TR showed moderate hyperplasia. It was concluded that both compounds are weakly goitrogenic. Hyperactivity was observed in the mice fed TR which may be associated with inhibition of catechol methyl transferase (COMT).

3,4-Dihydroxypyridine (3,4-DHP), a goitrogenic derivative of the plant amino acid mimosine, has no SH-group, in contrast to conventional antithyroid agents such as methimazole (MMI) and propylthiouracil (PTU). The current in vitro study shows that 3,4-DHP, like MMI and PTU, inhibits iodination of human thyroglobulin and interferes with mitogenic activation of human lymphocytes. This, together with a very low murine bone marrow toxicity, probably related to the absence of an SH-group, makes 3,4-DHP a potential antithyroid drug.

The non-protein amino acid indospicine, which occurs in the free state in high concentration in the tropical pasture legume Indigofera spicata, causes toxic liver lesions in ruminants. Indospicine is a specific antagonist of arginine and an inhibitor of protein synthesis. The liver lesion was studied in rats at four dose levels by feeding diets containing 96, 48, 24 and 15 per cent. of the seed. The too higher levels caused death of most animals in 2-6 wk. Females were more susceptible than males. The rats fed the 24 per cent. seed diet developed a nodular cirrhosis by 6 wk and survived up to 18 wk. The rats fed the 15 per cent. seed diet developed cirrhosis at 16 wk and survived up to 28 wk. Prior to the onset of cirrhosis the liver showed a characteristic lesion consisting of hepatomegaly, periportal fatty change, portal cellularity due to proliferation of ovoid and cuboidal duct cells, gross enlargement of the hepatocyte cytoplasm nuclei and nucleoli in spite of brisk mitotic activity, and focal centrilobular necrosis. The lesion was interpreted as a restricted hepatic response to a growth stimulus, possibly the mobilisation of tissue amino acids. Improvement occurred in the rats fed the two lower dosage levels after nodular cirrhosis developed, producing a new parenchyma. An attempt is made to relate the lesions to the biochemical derangement induced by a specific amino acid antagonist.