Organic aciduria in rats made resistant to hypoglycin toxicity by pretreatment with clofibrate.

1. The lethal, hypoglycaemic and hypothermic effects of hypoglycin in fasted rats are prevented if the rats had been fed on a diet containing clofibrate (0.5% w/w). 2. Injection of hypoglycin into fasted rats maintained on a standard diet caused severe prostration, hypothermia and a massive dicarboxylic aciduria [Tanaka (1972) J. Biol. Chem. 247, 7465-7478]. 3. Rats maintained on a diet containing clofibrate appeared normal after injection of hypoglycin, but had a marked dicarboxylic aciduria which was less than that induced in rats on a normal diet. 4. After administration of hypoglycin, butyryl-CoA and decanoyl-CoA, but not palmitoyl-CoA, dehydrogenase activities were strongly inhibited (80-95%) in the livers of animals on a standard diet. 5. Clofibrate feeding decreased the inhibition of these dehydrogenases to about 40-60%. 6. It was concluded that although clofibrate protects against the toxic effects of hypoglycin, some enzyme inhibitions as indicated by dicarboxylic aciduria are only partly prevented.     

Antidyslipidemic and antioxidant activity of an unusual amino acid (2-amino-5-hydroxyhexanoic acid) isolated from the seeds of Crotalaria juncea

In continuation of our drug discovery programme on Indian medicinal plants, we isolated an unusual amino acid, i.e. 2-amino-5-hydroxyhexanoic acid (1) from the seeds of Crotalaria juncea. The 2-amino-5-hydroxyhexanoic acid (1) showed dose dependent lipid lowering activity in the in vivo experiments and also showed good in vitro antioxidant activity. The cyclized compound, 3-amino-6-methyltetrahydro-2H-pyran-2-one (2) showed better lipid lowering and antioxidant profile than the parent compound 1.     

Simultaneous analysis of the di(2-ethylhexyl)phthalate metabolites 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC–MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1000 μg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1±13.5 μg/l for 2-ethylhexanoic acid, 104.8± 80.6 μg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2± 389.5 μg/l for 2-ethyl-3-oxohexanoic acid.     

Hazard Assessment of Ackee Fruit (Blighia sapida)

Ackee toxicity is associated with consumption of the fruit of the tree Blighia sapida. The problem is endemic in Jamaica, and a number of cases have been reported in the U.S. among Jamaican immigrants. Illness is associated with the method of preparation of the fruit and its ripeness. Malnourished individuals and children appear to be the most susceptible. Levels of the toxic compound, hypoglycin, which are found in the arils and seeds of the fruit, significantly decrease in the arils with ripeness (from 1000 ppm to <0.1?ppm). Symptoms of ackee poisoning in humans occur 6 to 48 hours after ingestion and include vomiting, muscular and mental exhaustion, hypoglycemia, coma and death. Intravenous glucose relieves the hypoglycemia. The most likely mechanism of action occurs through the incorporation of hypoglycin into fatty acid metabolic pathways. Hypoglycin or its primary metabolite methylenecyclopropyl-acetyl-CoA inhibits the oxidation of fatty acids and leucine and the activity of acyl-CoA dehydrogenases. The dose required to elicit acute responses is not known with any precision, nor is it possible to eliminate the likelihood of adverse effects with long-term ingestion of the toxin. Ingestion of unripe aril or pod and seeds represents a significant health hazard; this hazard diminishes considerably with the consumption of properly processed or prepared ripe fruit.     

Selective inhibition of acyl-CoA dehydrogenases by a metabolite of hypoglycin.

Extracts of liver mitochondria from donor rats given hypoglycin, the toxic amino acid from the ackee plant (Blighia sapida) showed drastically reduced levels of acyl-CoA dehydrogenase activity with butyryl-CoA as substrate. Activity with octanoyl- and palmitoyl-CoA was unaffected. Evidence that the active agent is methylenecyclopropylacetyl-CoA, a hypoglycin metabolite, was obtained by observing effects of the compound on a partially purified enzyme mixture prepared from rabbit liver. At 13 muM concentration, it strongly inhibited butyryl-CoA dehydrogenase (EC 1.3.99.2) with butyryl-CoA as substrate; it was far less effective with palmitoyl-CoA as substrate for the other similar enzymes present in the preparation. Unlike normal substrates of the acyl-CoA dehydrogenases, the compound itself, and not a reaction product, is inhibitory. The observed effect is consistent with quite general inhibition of fatty acid beta-oxidation by hypoglycin.     

Enzymatic synthesis of L-6-hydroxynorleucine

L-6-Hydroxynorleucine, a key chiral intermediate used for synthesis of a vasopeptidase inhibitor, was prepared in 89% yield and > 99% optical purity by reductive amination of 2-keto-6-hydroxyhexanoic acid using glutamate dehydrogenase from beef liver. In an alternate process, racemic 6-hydroxynorleucine produced by hydrolysis of 5-(4-hydroxybutyl)hydantoin was treated with D-amino acid oxidase to prepare a mixture containing 2-keto-6-hydroxyhexanoic acid and L-6-hydroxynorleucine followed by the reductive amination procedure to convert the mixture entirely to L-6-hydroxynorleucine, with yields of 91 to 97% and optical purities of > 99%.     

Identification of 5-hydroxyhexanoic acid, 4-hydroxyheptanoic acid and 4-hydroxyoctanoic acid as new constituents of bacterial polyhydroxyalkanoic acids

 A recombinant strain of Pseudomonas putida GPp104 (pHP1014::E146), which expressed the polyhydroxyalkanoic acid (PHA) synthase of Thiocapsa pfennigii exhibiting an unusual substrate specificity at a high level was incubated in two-stage batch or fed-batch accumulation experiments with 5-hydroxyhexanoic acid (5HHx) as carbon source in the second cultivation phase, copolyesters of 3-hydroxybutyric acid (3HB) plus 5HHx, or of 3HB, 3-hydroxyhexanoic acid (3HHx) plus 5HHx were accumulated as revealed by gas-chromatographic and ¹³C-NMR spectroscopic analysis. When the recombinant P. putida GPp104 was incubated with 4-hydroxyheptanoic acid (4HHp) as carbon source in the second cultivation phase, a copolyester consisting of 3HB, 3-hydroxyvaleric acid and 3- and 4-hydroxyheptanoic acid accumulated. Providing 4-hydroxyoctanoic acid as carbon source in the second cultivation phase led to the accumulation of a polyester that contained 1–2 mol% 4-hydroxyoctanoic acid besides 3-hydroxyoctanoic acid, 3HHx, 3-hydroxyvaleric acid and 3HB. In addition to PHA containing these new constituents, PHA with 4-hydroxyvaleric acid was accumulated from laevulinic acid. Eleven strains from five genera have been also analysed for their ability to utilize different carbon sources for colony growth, which might serve as potential precursors for the biosynthesis of PHA with unusual constituents. Although most of the carbon sources were utilized by some strains for colony growth, accumulation experiments gave no evidence for the accumulation of new PHA by these wild-type strains. Received: 22 April/Received revision: 23 May 1996/Accepted: 2 June 1996     

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.     

Metabolic origin of urinary 3-hydroxy dicarboxylic acids

3-Hydroxy dicarboxylic acids with chain lengths ranging from 6 to 14 carbons are excreted in human urine. The urinary excretion of these acids is increased in conditions of increased mobilization of fatty acids or inhibited fatty acid oxidation. Similar urinary profiles of 3-hydroxy dicarboxylic acids were also observed in fasting rats. The metabolic genesis of these urinary 3-hydroxy dicarboxylic acids was investigated in vitro with rat liver postmitochondrial and mitochondrial fractions. 3-Hydroxy monocarboxylic acids ranging from 3-hydroxyhexanoic acid to 3-hydroxyhexadecanoic acid were synthesized. In the rat liver postmitochondrial fraction fortified with NADPH, these 3-hydroxy fatty acids with carbon chains equal to or longer than 10 were oxidized to (omega - 1)- and omega-hydroxy metabolites as well as to the corresponding 3-hydroxy dicarboxylic acids. 3-Hydroxyhexanoic (3OHMC6) and 3-hydroxyoctanoic (3OHMC8) acids were not metabolized. Upon the addition of mitochondria together with ATP, CoA, carnitine, and MgCl2, the 3-hydroxy dicarboxylic acids were converted to 3-hydroxyoctanedioic, trans-2-hexenedioic, suberic, and adipic acids. In the urine of children with elevated 3-hydroxy dicarboxylic acid levels, 3OHMC6, 3OHMC8, 3-hydroxydecanoic, 3,10-dihydroxydecanoic, 3,9-dihydroxydecanoic, and 3,11-dihydroxydodecanoic acids were identified. On the basis of these data, we propose that the urinary 3-hydroxy dicarboxylic acids are derived from the omega-oxidation of 3-hydroxy fatty acids and the subsequent beta-oxidation of longer chain 3-hydroxy dicarboxylic acids. These urinary 3-hydroxy dicarboxylic acids are not derived from the beta-oxidation of unsubstituted dicarboxylic acids.     

ESI-MS/MS study of acylcarnitine profiles in urine from patients with organic acidemias and fatty acid oxidation disorders

Acylcarnitines in urine from 45 patients with organic acidemias and fatty acid oxidation disorders were evaluated using ESI-MS/MS. The urinary acylcarnitine profiles in organic acidemias, SCAD deficiency and MCAD deficiency were compatible with blood acylcarnitine profiles, and abnormalities in urinary acylcarnitine profiles in these conditions were enhanced following carnitine loading. Urinary acylcarnitine profiles were not helpful for characterization of long-chain fatty acid disorders, but a combination of urine and blood acylcarnitine analysis was useful for differential diagnosis of carnitine deficit.     

Stereoisomeric flavour compounds XXXV: Chiral aroma compounds of sherry I. Optically pure stereoisomers of solerone and solerole

The enantioseparation of the sherry aroma components 5-oxo-4-hydroxyhexanoic acid γ-lactone (solerone) and 4,5-dihydroxyhexanoic acid γ-lactone (solerole) is achieved, using Chiraspher (Merck) as the chiral HPLC phase and the optical purity ascertained directly by HRGC with heptakis(3-O-acetyl-2,6-di-O-pentyl)-β-cyclodextrin (Lipodex D) as the chiral stationary phase. The absolute configurations of 4,5-dihydroxyhexanoic acid γ-lactones are assigned by ¹H-NMR spectral data of diastereomeric α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) esters, according to Mosher's model. Sensory qualities of the isomers are given.     

Abnormal urinary excretion of unsaturated dicarboxylic acids in patients with medium-chain acyl-CoA dehydrogenase deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently described metabolic disorder of fatty acid oxidation in humans. Acute episodes are usually characterized biochemically by the appearance of nonketotic dicarboxylic aciduria. In addition, other abnormal metabolites, such as suberylglycine, n-hexanoylglycine, 3-phenylpropionylglycine, and octanoylcarnitine, are excreted in the urine. Urinary organic acids were determined using dual capillary column gas-liquid chromatography and gas-liquid chromatography/mass spectrometry. In three cases of MCAD deficiency we observed a disproportionate increase in the excretion of unsaturated dicarboxylic acids compared to either fasting control children with expected ketotic dicarboxylic aciduria or patients with nonketotic dicarboxylic aciduria not associated with MCAD deficiency. The most significant increase was in the urinary excretion of cis-4-decendioic acid. Additionally, the urinary excretions of cis-3-octenedioic and cis-5-decenedioic acids were slightly decreased whereas the excretion of cis-5-dodecenedioic acid was increased. These data are consistent with the notion that as a result of MCAD deficiency the metabolic oxidation of unsaturated fatty acids such as linoleate and oleate is inhibited more than saturated fatty acids.     

Specific determination of threonine in biological samples by gas chromatography with electron capture detection

Threonine was oxidized into acetaldehyde at 0 degrees C for 30 min with periodic acid. The acetaldehyde formed was converted to a hydrazone with 2,4-dinitrophenyhydrazine. The hydrazone was extracted with n-heptane and quantified by gas liquid chromatography with electron capture detection. An internal standard, 2-amino-3-hydroxyhexanoic acid, was used. The calibration curve of threonine was linear up to 200 nmol in 200 microl sample solution and the determination limit of threonine was 1 nmol in 200 microl sample solution. The recoveries were 100.0, 94.0 and 100.0% from homogenates of octopus tentacles and blood plasma and rat livers, respectively. This method was applied to the determination of threonine in tissues of rats given threonine and starved octopuses. This threonine determination method has been used for studies on the metabolism of d-lactate.     

Effect of chenodeoxycholic acid on biliary and urinary bile acids and bile alcohols in cerebrotendinous xanthomatosis; monitoring by high performance liquid chromatography

Biliary and urinary bile alcohol and bile acid composition has been determined by high performance liquid chromatography in patients with cerebrotendinous xanthomatosis before and after treatment with chenodeoxycholic acid. Most of the bile acids and bile alcohols in the bile and urine were separated in less than 30 min using a radial pack C18 muBondapak 5 micron particle size column with a mobile phase of acetonitrile-water-methanol-acetic acid 70:70:20:1 (v/v/v/v) at a flow rate of 2 ml/min, and a refractive index detector. Before treatment, cholic acid (49%) and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol (27%) were the major biliary bile acid and bile alcohol, respectively, but were not detected in the urine of five patients. 5 beta-Cholestane-pentols were, instead, the major urinary bile alcohols with 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23 xi, 25-pentol (56%) predominating. Whereas 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24S,25-pentol was not detected in the bile, it was isolated in the urine of all patients (27%). The only urinary bile acid isolated by high performance liquid chromatography was nor-cholic acid. After 1 month of treatment with chenodeoxycholic acid, 0.75 g/day, chenodeoxycholic acid became the major bile acid in the bile of all patients (71%) along with its metabolite, ursodeoxycholic acid (21%). Cholic acid and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol were drastically reduced and were only 3% each. The excretion of 5 beta-cholestane-pentols in the urine was also drastically reduced from 130 mg/day to 15 mg/day.     

Identification of urinary 3-ethoxy-4-hydroxybenzoic and 3-ethoxy-4-hydroxymandelic acids after dietary intake of ethyl vanillin

It has been discovered recently that several patients undergoing urinary organic acid profiling excrete high concentrations of 3-ethoxy-4-hydroxybenzoic acid and traces of 3-ethoxy-4-hydroxymandelic acid. These are believed to be the consequences of feeding the patients with synthetic diets flavoured with 3-ethoxy-4-hydroxybenzaldehyde, a vanilla-like artificial flavouring added to improve patient acceptance and taste. The syntheses, mass spectra and gas chromatographic behaviour of these and related compounds are presented.     

Quantification of urinary zwitterionic organic acids using weak-anion exchange chromatography with tandem MS detection

A rapid and accurate quantitative method was developed and validated for the analysis of four urinary organic acids with nitrogen containing functional groups, formiminoglutamic acid (FIGLU), pyroglutamic acid (PYRGLU), 5-hydroxyindoleacetic acid (5-HIAA), and 2-methylhippuric acid (2-METHIP) by liquid chromatography tandem mass spectrometry (LC/MS/MS). The chromatography was developed using a weak anion-exchange amino column that provided mixed-mode retention of the analytes. The elution gradient relied on changes in mobile phase pH over a concave gradient, without the use of counter-ions or concentrated salt buffers. A simple sample preparation was used, only requiring the dilution of urine prior to instrumental analysis. The method was validated based on linearity (r2>or=0.995), accuracy (85-115%), precision (C.V.<12%), sample preparation stability (相似文献   

Hypokalemic metabolic alkalosis caused by surreptitious vomiting: report of four cases.

Four women, aged 22 to 40 years, presented with severe hypokalemia and metabolic alkalosis. Three had related neuromuscular symptoms. All four patients denied vomiting or diuretic ingestion, and a diagnosis of Bartter''s syndrome was entertained. A diagnosis of surreptitious vomiting was suspected from the characteristic urine electrolyte pattern: high values for sodium and potassium, and a chloride concentration of less than 5 mmol/l. Three patients excreted sodium and potassium primarily with bicarbonate and had an alkaline urine; the fourth patient excreted these cations primarily with an organic anion and had an acid urine (pH 5.5). Since self-induced vomiting may be a common method of weight reduction in young women, recognition of this characteristic urine electrolyte pattern will assist in the rapid diagnosis of hypokalemia and metabolic alkalosis of obscure cause.     

Metabolism of n-Alkylcyclohexanes with an even number of carbon atoms in the side chain by Micrococcus sp. RCO-4M

The metabolism of n-alkyleyclohexanes with an even number of carbon atoms (6, 8, 10, 12, 14) in the side chain by Micrococcus sp. RCO-4M was investigated. Evidence for the formation of cyclohexanecarboxylic acid (I), cyclohexaneacetic acid (II), 1-cyclohexenecarboxylic acid (III), 6-hydroxyhexanoic acid (IV), adipic acid (V), and trans-4-hydroxycyclohexaneacetic acid (VI) is presented. The presence of products (II), (IV) and (V) especially represent the complete degradation of n-alkylcyclohexanes with an even number of carbon atoms in the side chain by a single organism. The occurrence of a newly-identified product (VI) suggests that a new metabolic pathway for n-alkylcyclohexanes with even-carbon-number side chain operates in this organism.     

Inhibition of gluconeogenesis by hypoglycin in the rat. Evidence for inhibition of glucose-6-phosphatase in vivo.

Treatment of rats with hypoglycaemic doses of hypoglycin has been shown to abolish the relative detritiation of [2-3H,U-14C]glucose [Osmundsen, Billington, Taylor & Sherratt (1978) Biochem. J. 170, 337-342], indicating that both the Cori and the glucose/glucose 6-phosphate cycles were inhibited in vivo. This inhibition was confirmed and, in addition, it was shown that the conversion in vivo of both [14C]lactate and [14C]fructose into glucose was decreased after hypoglycin treatment. These results suggest that hypoglycin poisoning results in the inhibition in vivo of glucose-6-phosphatase activity, which participates in the overall inhibition of gluconeogenesis and hypoglycaemia. Clofibrate feeding apparently protected the rats against the inhibition of the fructose-to-glucose conversion by hypoglycin. However, in isolated hepatocytes prepared from hypoglycin-treated rats, the conversion of [14C]fructose into glucose and the recycling of [2-3H,U-14C]glucose were not different from that in control hepatocytes. This suggests that the inhibition was lost during preparation of the hepatocytes. The direct measurement of glucose-6-phosphatase activity showed that it was inhibited when measured in concentrated, but not dilute, homogenates prepared from hypoglycin-treated rats.     

A method for the quantitative determination of hypoglycin A in the presence of leucine.

A spectrophotometric method for the quantitative determination of the toxic amino acid hypoglycin A in mixtures with leucine is reported. Hypoglycin A is the hypoglycemic principle found in the arilli of the fruit of Blighia sapida (ackee).