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)

Chemical analyses of some Jamaican preparations of cannabis

The major cannabinoids present in Jamaican samples of herbal cannabis and derived resin were determined by gas-liquid chromatography. Their qualitative and quantative profiles were not unusual, but in 3 of the 4 samples, there was an unexpectedly low content of 1 tetrahydrocannabinol, the main psychoactive ingredient (AU)

Fatty acids generate dicarboxylic acids in hypoglycin poisoning - abstract

We investigated the metabolic origin of certain dicarboxylic acids which appears in urine of hypoglycin poisoned subjects (Jamaican vomiting sickness). These include hexanedioic (adipic), octanedioic (suberic) and decanedioic (sebacic) acids which, with unsaturated variants, are also excreted in some rare congenital diseases (glutaric acidaemia Type II, generalized dicarboxlic acidaemias), and in ketosis. Long-chain fatty acids have been established as precursors only in the case of diabetic, ketotic rats. In one report, evidence for this origin was negative for the case of hypoglycin poisoning, but no alternative precursors appear likely and the problem has remained unresolved since 1972. The present work utilized palmitic acid labelled either with Tritium, or with 14c at various atoms of the molecule as a tracer in hypoglycin-treated rats. Suberate and sebacate, isolated from urines by gas liquid chromatography, were found to be radioactively labelled, and hence, significant conversion of fatty acid to dicarboxylic acid was demonstrated. A further conclusion emerged from the relative labelling yielded by [1-14C] - and [16-14C] palmitic acid. After chain shortening by 3 -4 cycles of fatty acid á-oxidation, w-oxidation appears to intervene as a consequence of inhibition of the former process by the hypoglycin metabolite methylenecyclopropylacetyl-CoA. This sequence is in contrast to the ketotic animal, in which initial w-oxidation of fatty acid apparently precedes bilateral á-oxidation. In fasted hypoglycin-poisoned rats, excretion of each of these compounds is not insignificant, being about 7 - 33 mg/24 hr(1 - 4 mg/mg creatinine) (AU)

Biosynthesis of L-á-(methylenecyclopropyl)-alanine (hypoglycin) in Blighia sapida

Threonine and C(1) units from methionine contribute to form an intermediate in the biosynthesis of L-á-(methylenecyclopropyl)-alanine, the toxic amino acid hypoglycin in Blighia sapida. Thereafter, the pathway followed duplicates that for leucine biosynthesis, utilizing identical or analogous enzymes

ç-Glutamyl transpeptidase of the ackee plant

The enzyme ç-glutamyl transpeptidase was purified from seeds of immature ackee fruit (Blighia sapida; Sapindaceae) by salt fractionation and gel filtration on Biogel P-10 and P-200. The procedure, which differs from an an earlier one applied to kidney bean fruit, achieves 9.8 percent yield and 577-fold purification. The enzyme is also present in other parts of the fruit and in leaves. A MW of 12 500 was found by SDS-polyacrylamide gel electrophoresis, a value much lower than that reported for the enzyme from kidney bean fruit. Neutral or amino sugar accounts for 10 percent of the dry weight. In vitro, the enzyme catalysed synthesis of an unusual ç-glutamyl dipeptide which occurs in ackee seeds, using glutathione as glutamyl group donor. The enzyme mechanism was of the double displacement (ping-pong) type. (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)

Identification of ethylmalonic acid in urine of two patients with the vomiting sickness of Jamaica

Large amounts of ethylmalonic acid have been identified in urines from two patients with the vomiting sickness of Jamaica. The amounts were 178 and 882æg per mg creatinine which are 70 and 350 times, respectively, over control values. Other short and medium chain dicarboxylic acids including glutaric and adipic acids and those with eight and ten carbon chain, saturated and cis-unsaturated, were also detected in large quantities as in the case of hypoglycin treated rats' urine. However, the large increase of urinary ethylmalonic acid in these two human cases is in a sharp contrast to the findings in hypoglycin treated rats in which urinary ethylmalonic acid increased only 3 times over control. It appears that ethylmalonic acid is produced in the cases with the vomiting sickness of Jamaica by carboxylation of n-butyryl-CoA which is not oxidised further due to the inhibition by hypoglycin A. In case of hypoglycin-treated rats, n-butyryl-CoA is mainly conjugated with glycine or deacylated to free butyric acid.(Summary)

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)

Rhein, a selective inhibitor of the DPNH-flavin step in mitochondrial electron transport

Previous findings in the literature that rhein inhibits DPNH-linked mitochondrial oxidations by acting in the DPNH dehydrogenase region of the respiratory chain have been confirmed and extended. In the micromolar range rhein inhibits DPNH oxidase and DPNH-ferricyanide activities and the energy-linked reduction of DPNH by succinate in membrane preparations from heart, as wellas the DPNH dehydrogenase and transhydrogenase activities of the soluble, purified enzyme. The inhibition of the activities of the soluble enzyme are purely competitive with respect to substrate. These facts localize the primary inhibition site of rhein between substrate and FMN. In heart ETP a second noncompetitive inhibition is also present but is detectable only at very low (<10æM) rhein concentrations. Rhein also inhibits DPNH dehydrogenase in Candida utilis mitochondria and the purified enzyme from liver. On conversion of the heart enzyme to the low molecule weight DPNH-cytochrome reductase the typical effect of rhein disappears and is replaced by a slight stimulation or inhibition, depending on the electron acceptor used, showing that the substrate binding site is modified in this form of the enzyme. In beef liver mitochondria DPNH oxidation may appear insensitive to rhein, probably because of the strong binding of rhein to other proteins. To a lesser extent unspecific binding of rhein and resultant interference with the inhibition of DPNH dehydrogenase is also shown by BSA and by proteins in heart ETP. Rhein also inhibits transhydrogenations in mitochondria and at higher concentrations lactate and malate dehydrogenases but has no effect on sccinate, alcohol (liver nad yeast), and glucose-6-p dehydrogenases or on Neuospora DPN-ase, glucose-6-phosphatase, and amine oxidase. (SUMMARY)