Detection of equine atypical myopathy-associated hypoglycin A in plant material: Optimisation and validation of a novel LC-MS based method without derivatisation.

Hypoglycin A (HGA) toxicity, following ingestion of material from certain plants, is linked to an acquired multiple acyl-CoA dehydrogenase deficiency known as atypical myopathy, a commonly fatal form of equine rhabdomyolysis seen worldwide. Whilst some plants are known to contain this toxin, little is known about its function or the mechanisms that lead to varied HGA concentrations between plants. Consequently, reliable tools to detect this amino acid in plant samples are needed. Analytical methods for HGA detection have previously been validated for the food industry, however, these techniques rely on chemical derivatisation to obtain accurate results at low HGA concentrations. In this work, we describe and validate a novel method, without need for chemical derivatisation (accuracy = 84-94%; precision = 3-16%; reproducibility = 3-6%; mean linear range R2 = 0.999). The current limit of quantitation for HGA in plant material was halved (from 1µg/g in previous studies) to 0.5µg/g. The method was tested in Acer pseudoplatanus material and other tree and plant species. We confirm that A. pseudoplatanus is most likely the only source of HGA in trees found within European pastures.

Atypical myopathy in Père David's deer (Elaphurus davidianus) associated with ingestion of hypoglycin A.

From 2004 until 2016, 21 Père David's deer (Elaphurus davidianus) have died for unknown reason at Zoo Duisburg. These deer, also known as milu, have succumbed from a myopathy that occurred seasonally in autumn and in spring. The clinical signs shown by the animals closely resembles those of a disease called equine atypical myopathy (EAM), which is formerly known in horses. The cause for EAM in Europe was found in the ingestion of hypoglycin A, contained in samaras and seedlings of the sycamore maple tree (Acer pseudoplatanus). To test the hypothesis that the mortality of milus was caused by ingestion of hypoglycin A, 79 sera from all zoos and wildlife parks that have kept milus in Germany and Austria, including 19 diseased and 60 healthy animals, were used. Selected biochemical values and additionally hypoglycin A, methylenecyclopropyl acetic acid-carnitine (MCPA-carnitine), and acylcarnitines, which have been found in horses suffering from EAM, were determined. The results showed greater values of serum activities of creatine kinase (P < 0.001) and aspartate aminotransferase (P < 0.001) in diseased milus comparing to healthy ones confirming a myopathy in affected animals. Moreover, hypoglycin A and MCPA-carnitine were found in the blood of Père David's deer and thus, hypoglycin A intoxication was considered to be a potential cause for the myopathies by ingestion of sycamore maple samaras that were present in the enclosure of the affected animals. Hypoglycin A values were greater in diseased animals (P < 0.01) as well as MCPA-carnitine levels (P < 0.05). Additionally, affected milus showed greater C5-OH-carnitine (P < 0.01) and C6-carnitine (P < 0.001) values. Until now hypoglycin A intoxication was only known in the family of Equidae, in humans, and in laboratory rats, and it has not been previously described in other zoological families. Comparing to horses, ruminants do have a different digestive tract and it will need further investigation to find out if several factors are involved to trigger an outbreak in ruminants.

Protection of rats by clofibrate against the hypoglycaemic and toxic effects of hypoglycin and pent-4-enoate. An ultrastructural and biochemical study.

An ultrastructural and biochemical study of the toxic and hypoglycaemic effects of hypoglycin and pent-4-enoate was made on the livers of normal and clofibrate-fed rats. Injection of hypoglycin to rats doubles (from 22% to 44%) the volume fraction of mitochondria and decreases (from 1.05% to 0.26%) the volume fraction of peroxisomes in hepatocytes. The fast-acting toxin pent-4-enoate causes few ultrastructural changes except for the accumulation of lipids. In male adult rats fed with 0.5% clofibrate in their diet for 1-2 months, the volume fraction occupied by peroxisomes and mitochondria in hepatocytes rose to 6.26% and 29.5% respectively. Clofibrate feeding apparently protected the animals against the toxic, hypoglycaemic and hypothermic effects of hypoglycin and of pent-4-enoate, and completely prevented the ultrastructural damage caused by hypoglycin. After hypoglycin administration, hepatic mitochondrial butyryl-CoA dehydrogenase activity was inhibited by more than 90% and, surprisingly, the activity of the peroxisomal enzymes studied was largely preserved. When hypoglycin was given to rats fed on a clofibrate-containing diet, the oxidation of decanoylcarnitine, which was incomplete after hypoglycin treatment alone, remained incomplete with uncoupled mitochondria, but became apparently complete with coupled mitochondria. In the latter condition, there was a slowing of the rate during the last quarter of the pulse of oxygen uptake. Further, butyryl-CoA dehydrogenase activity was much less affected by hypoglycin in clofibrate-fed animals. Pent-4-enoate does not inhibit beta-oxidation in coupled mitochondria from clofibrate-treated rats.

[Reye syndrome--a status review of current concepts of its pathogenesis]. / Reye-Syndrom--Eine Bestandsaufnahme derzeitiger Vorstellungen zur Pathogenese.

In Europe Reye's syndrome is a rather rare but often fatal disease affecting children and teenagers. Whereas an association with an antecedent viral illness has been documented, our knowledge concerning etiology and pathogeneseis remains sparse. A significant association between salicylate-medication during prodromal illness and RS has well been documented without demonstration of a causal relationship. The metabolic disturbance appears to stem from an acute mitochondrial lesion following a so far not fully understood viral, toxic or multimodal attack. Similar mitochondrial lesions are encountered in hypoglycin- or aflatoxin-intoxication. Consequently the intramitochondrial steps of the urea-cycle are blocked leading to its breakdown in the scene of a tremendous nitrogen-load flooding the organism with ammonia. A number of suspected pathogens are being discussed of which ammonia seems most capable of inducing the neurologic symptoms of RS probably in conjunction with elevated free fatty acids.

Ackee poisoning: relationship to vomiting sickness (abstract only)

The toxic effect of Hypoglycin A, a polypeptide isolated from the ackee, on animals is presented. The outstanding features are vomiting, drowsiness,coma and death, accompanied by hypo-glycaemia and low level glycogen. These findings resemble those observed in some cases of so-called "vomiting sickness" of Jamaica. Animal experiments have also shown that rats fed with a low protein high carbohydrate "Jamaican diet" were more sensitive to Hypoglycin A than rats on a normal diet. The significance of these experiments will be discussed. The final proof that "vomiting sickness" could be due to ackee poisoning would be to identify the ackee toxin from the "vomiting sickness" patients by chemical, biochemical and biological methods. (AU)

Teratogenicity of hypoglycin-A: some morphological and biochemical aspects

Hypoglycin-A is a toxic, non-proteinogenic amino acid of considerable biochemical interest. It is obtained fron the fruit of Blighia sapida K., and is the causative factor of the Jamaican "vomiting sickness". The toxicity of hypoglycin-A is attributed to the formation of a metabolic methylenecycclopropaneacetic acid which inhibits the oxidation of long-chain fatty acids. Hypoglycin-A induced in pregnant rats a significantly high incidence of congenital abnormalities and reabsorption. It did not reduce the fertility in mice, malformations were absent and only a small increase in reabsorption sites was observed after the adminstration of large doses. Hypoglycin-A administered to pregnant rabbits resulted in a high incidence of foetal reabsorption and overall stunting. Injected into the yolk sac of 24 and 48 hour chick embryos, hypoglycin-A was not teratogenic. Leucine, administered to pregnant rats simutaneously with hypoglycin-A, afforded no protection against the teratogenic action of hypoglycin-A. Leucine was shown to be highly teratogenic and exaggerated the teratogenicity of riboflavin and hypoglycin-A to pregnant rats the occurrence of congenital abnormalities. Inhibition of long-chain fatty acid oxidation may represent a basic cellular mechanism involved in the teratogenicity of hypoglycin-A, because of its influence on oxidative phosphorylation and the electron transport system. Reversal of the hypoglycin-induced teratogenic effects by riboflavin, suggests that inhibition of the acyl dehydrogenase flavin-dependent-oxidation reaction, occurring during the degradation of fatty acids, is the site of action of hypoglycin-A (AU)

Some biological observations of hypoglycin A in presence of leucine - abstract

The present work was carried out to investigate the effect of leucine on the biological activity of hypoglycin A. Leucine-free hypoglycin A, prepared from the ackee (Blighia sapida) seeds, has been shown to cause inhibition of the growth of broad bean radicles. When supplied simultaneously, leucine had no effect on the degree of inhibition produced by hypoglycin A even when the leucine concentration was four times that of hypoglycin A. Although the acceped structure of hypoglycin A bears a relationship to that of leucine, yet leucine does not antagonise the inhibitory action of hypoglycin A. When injected intraperitoneally into rats, fasted for 48 hours to ensure complete depletion of their liver glycogen, hypoglycin A induced hypoglycemia. This hypoglycemia was potentiated by leucine when administered one hour before hypoglycin A. These findings indicate that the hypooglycemia induced by hypoglycin A is not a result of the exhaustion of the liver glycogen as was suggested by Patrick (1954). They also indicate that the liver is not the principal site of action as thought by Chen et al. (1957).(AU)

The effects of hypoglycin-A on thyroid activity (abstract only)

The effects of hypoglycin-A (L-a-amino-á-methylene cyclopropane propprionic acid) the active principle of the fruit blighia sapida, ("ackee"), on the thyroid function of the rat have been studied. At the end of twenty-four hours hypoglycin-A (5-40 mg/kg. body weight) depressed the level of the protein bound iodine (PBI) in the plasma. With doses of hypoglycin-A in the range of 5-10 mg/kg body weight, there is some increase in the urinary excretion of radioactive iodine (131I), while larger doses (20-40 mg/kg) definitely decreased the urinary excretion of 131I. It is assumed the at hypoglycin-A acts on the thyroid function like sodium salycylate in causing an acute decrease in the level of the PBI, and like thiouracil, by causing a decrease deiodination by the liver and the thyroid gland. In a time course study, the effect of hypoglycin-A, 10 mg/kg on injected 131I, there appear to be two active metabolic components of hypoglycin-A, which cause depression of the uptake 131I by the thyroid, kidney and liver, one having an effect within one hour, the other acting four hours after administration. The effect of hypoglycin-A on thyroid function appears not to be specific, but a part of its general antimetabolic action (AU)

Differential species susceptibility to the teratogenic response of hypoglycin-A (abstract only)

Hypoglycin-A, the active principle of the fruit of Blighia sapida (called "ackee" in Jamaica), produced a high incidence of foetal abnormalities in the rat. Prenatal treatment of pregnant rats with hypoglycin-A (30 mg/kg.) daily during the first five days of gestation resulted in 95 percent of the foetuses being retarded in development, syndactyly in 87 percent and encephalocoele in 89 percentof the foetuses. Foetal resorption occurred in 5 percent of all implantations. In order to extend the experience reported above, the teratogenic effect of hypoglycin-A was further investigated in the mouse embryo. Hypoglycin-A did not reduce fertility in the mice. No gross malformations were observed, and even in doses as high as 120 mg/kg., there was only a small increase in resorption sites. The differences in species susceptibility to teratogenesis may be due to particular metabolic pathways involving the formation of toxic metabolites in some species but not in others(AU)

Foetal abnormalities caused by the active principle of the fruit of Blighia Sapida (Ackee)

The teratogenic effects of hypoglycin-A on the rat embryo was investigated. The high incidence of foetal resorptions and malformations (encephalocele,syndactyly and stunted growth) suggest that hypoglycin-A is a potent teratogen. Histological studies on the foetal organs show signs of developmental arrest. The embryotoxic effects of this substance is probably due to the inhibition of fatty acid oxidation by methylenecyclopropaneacetic acid, a degradation product of hypoglycin-A (AU)

Some disease associated with protein malnutrition, as seen in Jamaica

Three diseases which occur in Jamaica, akee poisoning, veno-occlusive disease, and kwashiorkor are described. It is thought that previous protein malnutrition is necessary for these disease to appear (AU)