James A. Yergey

Quinolinic Acid Concentrations in Brain and Cerebrospinal Fluid of Patients with Intractable Complex Partial Seizures

Summary Quinolinic acid (QUIN) is a neurotoxin and convulsant when injected directly into the brains of experimental animals and as such has been implicated in the etiology of human seizure disorders. In the present study, we quantified QUIN in cerebrospinal fluid (CSF) and in spiking (focus) and nonspiking (nonfocus) regions of surgically resected human temporal neocortex. l‐tryptophan (L‐TRP), the putative precursor of QUIN, was also measured in brain, along with CSF concentrations of L‐TRP, 5‐hydroxyindoleacetic acid (5‐HIAA), and homovanillic acid (HVA). In brain tissue, no differences were found in the concentrations of QUIN and L‐TRP between focus and nonfocus regions in 15 pairs of samples. No differences were found in CSF, L‐TRP, 5‐HIAA, or HVA concentrations between 11 neurologically normal controls and 15 interictal (no seizures for >24 h) and 20 postictal (within 50 min of seizure) samples from epileptic patients. However, CSF QUIN concentrations were significantly lower (32%) in the epileptic patients as compared with controls, which may indicate a generalized disturbance in brain QUIN metabolism or perhaps a response to antiepileptic drugs.

Brain Eicosanoid Formation following Acute Penetration Injury as Studied by in vivo Microdialysis

Formation of eicosanoids has been implicated in the pathological changes that follow brain injuries. In the present study, we used a microdialysis probe to both induce acute penetration injury and also sample extracellular fluid concentrations of eicosanoids. Formation of prostaglandin (PG) D2, PGF2a, and thromboxane B2 was highest in the first hour following introduction of the probe into rat striatum. In contrast, the level of PGE2 was highest during the sixth hour of collection, while 6-keto-PGF1a remained stable throughout the sampling period. We conclude that in vivo microdialysis may be useful in the evaluation of the time course of the effects of acute penetration injury of the brain on the local production of eicosanoids.

Determination of eicosanoids, phospholipids and related compounds by thermospray liquid chromatography-mass spectometry

Thermospray mass spectrometry has proven to be a useful technique for analyzing various biological compounds including eicosanoids and phospholipids. Molecular ions as well as fragment ions which reveal useful structural information are produced for underivatized eicosanoids and phospholipids using filament-off or filament-on thermospray mass spectrometry, respectively. In conjunction with on-line chromatographic separation, complex mixtures of biological samples can be rapidly analyzed with great reliability. Data will be presented concerning the analysis of prostaglandins, other eicosanoids and molecular species of phospholipids as well as the application of these methodologies to complex biological samples.

Prostaglandins in cerebrospinal fluid of healthy human volunteers, abstinent alcoholics and rhesus monkeys

A sensitive and selective assay for measuring prostaglandins in cerebrospinal fluid has been developed, based on the selected-ion-monitoring, electron-capture negative ionization GC/MS detection for the MO-PFB-TMS derivatives of prostaglandins E2, E1, F2 alpha, F1 alpha, and 6-keto-F1 alpha. Improvements over previously published assay procedures have been made, and the new assay has been applied to measurement of prostaglandin concentrations in lumbar CSF of healthy human volunteers, abstinent alcoholic patients, in cisternal CSF of Rhesus monkeys, and continuously sampled lumbar CSF of awake Rhesus monkeys. Results indicated that the concentrations of PGE2, PGE1, PGF1 alpha, and 6-keto-PGF1 alpha were below 15 pg/mL CSF in lumbar CSF of healthy humans and abstinent alcoholics, and in cisternal CSF of Rhesus monkeys. In contrast, continuously sampled lumbar CSF of awake Rhesus monkeys contained more than 200 pg/mL of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha, probably present as a result of local production.

Mass Spectrometric Determinations of Tryptophan and its Metabolites

The unique character of a chemical compound is determined by its molecular architecture. That architecture is a sum not only of all of the atoms in the compound and their masses, but of their particular relationship in space, i.e., the molecular bonds, sub-structural components, and their stereochemistry. Consequently, a physical tool which can measure both the summed masses of the elements in a compound and reflect the subtleties of their arrangement is very useful in both quantitative and qualitative investigations in biochemistry.

Stable Isotope-Labeled Tryptophan as a Precursor for Studying the Disposition of Quinolinic Acid in Rabbits

The utility of stable isotope-labeled tryptophan as a precursor for studying the disposition of quinolinic acid was investigated. TRP-D5 at doses of 50, 25 or 10 mg/kg was administered to rabbits. Blood and CSF samples were taken for up to 6 hours. There was no loss of deuterium from the tryptophan and the specifically tri-deuterated quinolinic acid measured in plasma and CSF. CSF levels of QUIN-D3 remained elevated 6 hours following TRP-D5 administration. Further studies of the CNS disposition of quinolinic acid and other metabolites of the kynurenine pathway employing stable isotope-labeled tryptophan as precursor at appropriate doses and with extended sampling are in progress.

Implications of Alcohol‐Induced Alterations in the Prostaglandin Profile of the Vascular System

Epidemiologic studies have suggested that low rates of alcohol consumption may lower the incidence of atherosclerosis-related cardiovascular pathology, whereas high exposure correlates with a high incidence of vasospastic disorders. We have examined the proposition that the prostaglandin (PG) profile in rat aorta, cerebral microvasculature and plasma is modulated by alcohol dose and the duration of exposure and thereby contributes to changes in the cardiovascular state. Isolated rat aorta were either exposed to alcohol in vitro (FIGURE 1) or by an inhalation technique using our automated apparatus for precise control of alcohol vapor and blood alcohol concentrations (BAC) (FIGURE 2).* A wide range of mean BAC (50450 mg%) were obtained based upon multiple blood ethanol determinations for each rat throughout the exposure period. An acute exposure to moderate alcohol levels in vitro decreases the amplitude of contraction to a thromboxane-mimic, whereas higher levels do not effect the response. The observed depression in aortic contractility is associated with a

Smooth Muscle Pharmacology of Hydroxylated Docosanoids

It has been suggested that subjects whose diets are high in fish oils have a low risk of cardiovascular disease. Fish oils are rich in eicosapentaenoic acid, as well as docosahexaenoic acid (DHA). We have investigated the proposition that metabolites of DHA may be biologically active and be one of the active components of fish fat. Metabolism of DHA to hydroxy derivatives has been observed in many mammalian tissues, including basophils, the macrophage, platelets, the retina, and the brain, in vitro. In this work we have biosynthesized and purified various hydroxylated derivatives of DHA and characterized their structures as well as their smooth muscle pharmacology. Platelets are known to produce 12-lipoxygenase products of DHA. Fresh platelets ( lo8 cells/ml x 1 ml from human, monkey, dog, rabbit, guinea pig, or rat) were washed twice and incubated for 20 min at 37 C with 0.25 MCi of 1-IC-DHA and 1-100 pM unlabelled DHA. Samples were extracted twice with dichloromethane, evaporated, and dissolved in methanol for HPLC analysis. A 5-micron Axxichrom ODS column (4.6 mm x 25 cm) was used with a mobile phase of acetonitrile and 0.1 M ammonium acetate, pH 7.0, using both UV and radioactivity detectors. The two major metabolites of DHA formed by platelets were collected by monitoring their UV absorbance at 235 nm, and then they were extracted, evaporated, and dissolved in Krebs Ringer bicarbonate for subsequent determination of their biological activities. Simultaneously, a portion of these collected fractions were subjected to GC/MS analysis after methylation and trirnethylsilylation, and the structures were confirmed to be 11and 14-hydroxydocosahexaenoic acid (HDHE). Both 5,8,11,14-eicosatetraynoic acid

Brain and cerebrospinal fluid quinolinic acid concentrations in patients with intractable complex partial seizures.

Excitatory amino acids have been implicated in development and maintenance of seizures (Anderson et al., 1987; Meldrum 1987). In accordance with this notion are the observations that antagonists of excitatory amino acid receptors, including the NMDA receptor, are potent anticonvulsants (Meldrum et al., 1988). In the widely used model of kindling, electrophysiologic studies have demonstrated an increased sensitivity of NMDA receptors at stages IV and V (Mody et al., 1988). In man, increases in the concentrations of glutamate and aspartate, but not of GABA, have been reported in the focal compared to non-focal regions of cerebral cortex (Nadi et al., 1987; Sherwin et al., 1988). Additional studies have shown an increased number of NMDA receptors in the epileptic focus (Wyler et al., 1987). Studies in experimental animals have shown that local application of quinolinic acid (QUIN) increases neuronal activity (Perkins and Stone, 1983) and may cause seizures (Lapin, 1982). Conceivably, QUIN may therefore contribute to increased neuronal excitability in human seizure disorders. Direct measures of QUIN in human epilepsy have not been reported, although decreases in the degradative enzyme of QUIN, quinolinic acid phosphoribosyltransferase (QPRT), have been published (Feldblum et al., 1988), possibly indicating an impaired ability to catabolize QUIN.