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Type 1 vanilloid receptors (VR1) have been identified recently in the brain, in which they serve as yet primarily undetermined purposes. The endocannabinoid anandamide (AEA) and some of its oxidative metabolites are ligands for VR1, and AEA has been shown to afford protection against ouabain-induced in vivo excitotoxicity, in a manner that is only in part dependent on the type 1 cannabinoid (CB1) receptor. In the present study, we assessed whether VR1 is involved in neuroprotection by AEA and by arvanil, a hydrolysis-stable AEA analog that is a ligand for both VR1 and CB1. Furthermore, we assessed the putative involvement of lipoxygenase metabolites of AEA in conveying neuroprotection. Using HPLC and gas chromatography/mass spectroscopy, we demonstrated that rat brain and blood cells converted AEA into 12-hydroxy-N-arachidoylethanolamine (12-HAEA) and 15-hydroxy-N-arachidonoylethanolamine (15-HAEA) and that this conversion was blocked by addition of the lipoxygenase inhibitor nordihydroguaiaretic acid. Using magnetic resonance imaging we show the following: (1) pretreatment with the reduced 12-lipoxygenase metabolite of AEA, 12-HAEA, attenuated cytotoxic edema formation in a CB1 receptor-independent manner in the acute phase after intracranial injection of the Na+/K+-ATPase inhibitor ouabain; (2) the reduced 15-lipoxygenase metabolite, 15-HAEA, enhanced the neuroprotective effect of AEA in the acute phase; (3) modulation of VR1, as tested using arvanil, the VR1 agonist capsaicin, and the antagonist capsazepine, leads to neuroprotective effects in this model, and arvanil is a potent neuroprotectant, acting at both CB1 and VR1; and (4) the in vivo neuroprotective effects of AEA are mediated by CB1 but not by lipoxygenase metabolites or VR1.

A method for the assay of acidic catecholamine metabolites in biological fluids using capillary gas chromatography—electron-capture negative-ion mass spectrometry is described. The method combines acetylation of phenolic hydroxy groups in buffered aqueous solution followed by pentafluorobenzyl ester formation and acetylation of aliphatic hydroxy groups under anhydrous conditions. The resulting per-O-acetyl carboxypentafluorobenzyl esters provided excellent negative-ion mass spectra with intense and diagnostic anions. The sensitivity of the analysis using electron-capture negative-ion mass spectrometry exceeds that using electron-impact mass spectrometry by two to three orders of magnitude. Analysis of acidic catecholamine metabolites in human lumbar cerebrospinal fluid and plasma were performed with good precision (srel

Two patients with a deficiency of 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) lyase have been described? This enzyme catalyzes the final step of leucine degradation and plays a key role in ketone body formation. Clinically the defect is associated with metabolic acidosis and hypoglycemia. The biochemical diagnosis is based on the finding of abnormal organic aciduria with highly increased urinary excretion of 3-hydroxy-3-methylglutaric acid, 3-methylglutaconic acid, 3- methylglutaric acid, and 3-hydroxyisovaleric acid The enzyme can be measured in various tissues, including leukocytes and fibroblasts.

Rapid, sensitive and accurate stable isotope dilution assays were developed for the measurement of orotic acid and uracil in amniotic fluid. The method utilizes [15N2]orotic acid and [15N2]uracil as internal standards, isolation by liquid partition chromatography and quantitation by chemical ionization selected ion monitoring gas chromatography-mass spectrometry. Orotic acid at a concentration of 0.26 ± 0.05 μmol/1 and uracil at a concentration of 0.55 ± 0.13 μmol/l were detectable in normal amniotic fluid. As affected fetuses with argininosuccinate synthetase or ornithine carbamoyl transferase deficiency showed no significant elevation of orotic acid and/or uracil in their surrounding amniotic fluids, this method unfortunately seemed not to be useful for prenatal diagnosis of these inherited disorders. Nevertheless, it provides significant advantage over available methods for the quantitation of orotic acid and uracil in which the analysis of these compounds must be very accurate, highly specific and sensitive (e.g. detection of heterozygosity for omithine carbamoyl transferase deficiency).

Two brothers, aged 7 and 5 years, who excreted large amounts of the leucine metabolites 3-methylglutaconic acid, 3-methylglutaric acid, and 3-hydroxyisovaleric acid, are described. The excretion of these metabolites could be enhanced by increasing the leucine intake. Restriction of the protein intake resulted in a marked reduction of the metabolite excretion. However, the excretion of the ultimate leucine metabolite, 3-hydroxy-3-methylglutaric acid, remained unchanged at a low level. The only clinical abnormality was speech retardation. A (partial) deficiency of 3-methylglutaconyl coenzyme A hydratase is proposed to be the most likely underlying defect.