Commentary on a review on the mechanism of Ackee-induced vomiting sickness

A recent review article concluded that glutamic acid probable plays a central role in the vomiting and neurological features of ackee poisoning. The present article draws attention to misconceptions in the basis of that hypothesis, and reviews important evidence suppporting a different role (AU)

Effects of ackee fruit extracts on bronchomotor tone in rats

The unripe ackee fruit, when eaten, is known to cause serious clinical manifestations, including vomiting, hypoglycaemia and acidosis. The effects, of various extracts from the arilli of the unripe fruit (including hypoglyin-A) on the lungs from rats were examined in an in vitro preparation. All the extracts were found to induce moderately severe broncho-constriction, indicating a possible contribution of these effects to the observed toxicity of ackee (AU)

The determination of hypoglycin-A in blood and plasma

Hypoglycin-A, the causative agent of vomiting sickness, was added to samples of human blood plasma and whole haemolysed blood and the quantitative recovery of the compound by means of comparison of the amino acid profiles before and after bromination of the samples was investigated. The method was found to be suitable for the estimation of the compound in blood or plasma down to a level of about 1 æmole/100ml. Analyses of blood samples from cases of suspected ackee poisoning, using the method reported, proved negative. The implications of this are discussed (AU)

Jamaican vomiting sickness: biochemical investigation of two cases

We identified methylenecyclopropylacetic acid, a known metabolite of hypoglycin A, in the urine of two patients with Jamaican vomiting sickness. Excretion of unusual dicarboxylic acids such as 2-ethylmalonic, 2-methylsuccinic, glutaric, adipic and dicarboxylic acids with eight and 10 carbon chains were also detected in both patients. The amounts of these dicarboxylic acids were 70 to 1000 times higher than normal. These metabolities have also been identified in urine of hypoglycin-treated rats. This evidence links hypoglycin A to Jamaican vomiting sickness as its causative agent. Urinary excretion of short-chain fatty acids was also increased up to 300 times higher than normal. These results indicate that, despite their clinical and histological similarities, the cause and biochemical mechanisms of Jamaican vomiting sickness differ distinctly from those of Reye's syndrome in which these abnormal urinary metabolities are not appreciably increased.(AU)

Counteraction of glucocorticoid - induced hyperglycaemia by hypoglycin

Hypoglycin, B-(methylenecyclopropyl) alanine), administered to starved rats pretreated with corticosterone, caused within 6 hr. a marked decline in blood sugar levels. The findings are evidence that the hypoglycaemic agent imposed a restriction on the rate of gluconeogenesis, against which the multiple actions of the hormone were effective. This conclusion emphasizes the crucial nature of the inhibition which hypoglycin produces in the metabolic sequence of gluconeogenesis (AU)

Improved method for isolation of hypoglycins A and B from fruit of Blighia sapida

This report describes an isolation procedure for obtaining á-(methylenecyclopropyl) alanine, and its ç-glutamyl peptide (hypoglycins A and B) from ackee fruit or seeds (Blighia sapida) that is less laborious than previous methos (Hassall & Ryle, 1955; Ellington, Hassall & others, 1959; West, 1968) and gives leucine-free hypoglycin. One kg of arilli from immature ackee fruit were blended in batches with a total of 2 litres of 80 percent ethanol. After filtration through muslin and overnight settling the ethanolic extract was dried under vacuum, and the portion soluble in 0.1n HCI was applied to a column (90 x 4.5 cm) of Dowex 50 (x8) resin in the (H+) form. Several column volumes of 0.1n HCI and of water were passed through the column, after which elution with 1.0M pyridine until the ninhydrin test was negative removed neutral amino-acids with hypoglycin A, as well as acidic amino-acids and hypoglycin B. The eluate was dried by evaporation under vacuum. The residue was taken up in 0.5n acetic acid and placed on a column (70 x 41/2 cm) of Dowex 1 (x8) resin in the acitate form. Elution with 0.5N acetic acid rapidly removed a mixture of neutral amino-acids with hypoglycin A as the main component. This was recovered from the eluate after removing excess solvent by evaporation under vacuum. Crystallization, repeated twice from 50 percent ethanol, routinely yeilded 0.8 to 1.2 g of nearly pure product per kg of starting material. Continued elution with 0.5N acetic acid removed a second zone of ninhydrin-positive material, well separated from the neutral amino-acids. This contained mainly hypoglycin B, present in seeds but not in arilli. This, in turn, was well separated from glutamic and aspartic acids, which appeared subsequently. The identity of hypoglycin A was checked by nmr (Millington & Sheppard, 1968), and paper and thin-layer chromatography using a variety of solvent systems. However, for establishing the level of contamination by leucine and isoleucine, which exhibit similar solubility and chromatographic properties to those of hypoglycin A, it was necessary to perform chromatography of the derivatives formed with dimethylaminonaphtalene-5-sulphonly chloride, using either filter paper (Abrahams & Kean, 1969 or polyamide layer (Woods & Wang, 1967). This technique was used to confirm that, by two or three recrystallizations of hypoglycin A derived from ackee arillus, contamination by these amino-acids could reduced to less than 1 percent. In this respect, seeds were a less desirable starting material, in that the crude product contained relatively larger amounts of leucine and other amino-acids. Hypoglycin B was identified from the products of hydrolysis (boiling with 8N acetic acid for 4 h), which were shown to be hypoglyucin A and glutamic acid. Hypoglycin A with no detectable impurity was thus obtained in yields of up to 0.04 percent (based on the fresh weight of seeds used), by passage of the concentrated hydrolysate through a column of Dowex 1 (acetate form) as described above. Identification of relevant compounds was readily achieved by thin-layer chromatography on silica gel (Eastman Chromatogram Sheets). The solvent system propanol-water (7:3) gave the Rf values: hypoglycin A, 0.73, hypoglycin B 0.51, glutamic acid 0.30. The utility of the procedure lies in the selective elution, by dilute pyridine, of the neutral amino-acid and acidic peptide components in the extract, after adsorption on a strongly acidic cation-exchange resin. This fractionation could not be achieved with dilute ammonia. A solution of 1.0M pyridine is only weakly alkaline (pH 8.0) and this, together with the possibility of graded selectivity of the resin for differentiations, including pyridinium, might explain the retention of the basic amino-acids. Additionally, advantage was taken of the convenient fractionation of hypoglycins A (one amino and one carboxyl group free) and B (one amino and two carboxyl groups free), on a strongly basic anion-exchange resin by dilute acetic acid; this was based on the work of Hirs, Moore & Stein (1954) (AU)

The protective effects of feeding on the hepatic ultrastructure of rats treated with hypoglycin

Hypoglycin is a toxic amino acid found in the fruit of the unripe ackee (Blighia sapida), and associated with Jamaican "vomiting sickness". It produces profound hypoglycaemia when administered to starved rats, and this is accompanied by gross mitochondrial swelling in liver cells. This ultrastructural change is reduced or absent in animals that have been allowed to feed ad libitum, provided that normal blood glucose levels are maintained. Mitochondrial swelling is not a feature of acute hypoglycaemia induced by insulin, under the conditions of this experiment.(AU)

Hypoglycin toxicity in rats. II. Modification by riboflavin of mitochondrial changes in liver

Hypoglycin (L-O-amino-á-methylenecyclopropanepropionic acid) is a toxin found in the unripe fruit of the ackee, and associated with Jamaican "vomiting sickness." It produces profound hypoglycemia due to interference with long-chain fatty acid oxidation and gluconeogenesis, and is accompanied by mitochondrial swelling in rat hepatocytes. The hypoglycemic effect may be prevented or reversed by riboflavin. The present study indicates that mitochondrial swelling is reduced in rats given riboflavin prior to hypoglycin, compared with rats given hypoglycin alone. Riboflavin appears less effective if given 10 minutes after hypoglycin.(AU)

Studies on hypoglycin toxicity in rats. I. Changes in hepatic ultrastructure

Hypoglycin is a toxic amino acid found in the unripe ackee fruit. The ackee is a popular item of diet in Jamaica and has been proposed as a cause of the so-called vomiting sickness. Hypoglycin is thought to act by inhibiting the oxidation of long-chain fatty acids, uncoupling oxidative phosphorylation, and interfering with gluconeogenesis. Hypoglycin was given intraperitoneally to rats in a dose of 10 mg/100 g, and samples of liver taken at hourly intervals up to 5 hr were studied with the electron microscope and compared with controls. The major ultrastructural findings in the hypoglycin-treated rats were progressive mitochondrial swelling with loss of granules and pallor of the matrix, followed by incorporation into autophagic vacuoles. These findings correlate well with the reported biochemical mechanisms.(AU)

Hypoglycin A in biological material: its detection and assay

A procedure for detection and measurement of Hypoglycin A in biological material is described, based on reaction of the amino acid with 1-dimethylaminonaphthalene-5-sulphonyl chloride (Dansyl chloride). The desired flourescent derivative can be separated from others by paper chromatography and measured by flourimetry after elution. Less than 0.2 microgram of hypoglycin A is detectable on chromatograms (AU)