ECNI GC-MS analysis of picolinic and quinolinic acids and their amides in human plasma, CSF, and brain tissue.

To study the complex inter-relationships between inflammatory and apoptotic responses and the kynurenine pathway, we have utilized electron-capture negative ion mass spectrometry to develop trace analyses to concurrently quantify nicotinic acid (NIC), picolinic acid (PIC) and quinolinic acid (QUIN) in biological samples. We have shown that NIC and its amide nicotinamide (NAM) can be separately quantified by analyzing samples pre- and post-acid hydrolysis. We have now examined human plasma, CSF and brain tissue samples for the presence of putative picolinamide (PAM) and quinolinamide (QAM) by comparing PIC and QUIN concentrations pre- and post- gas phase hydrolysis. We report for the first time that, with respect to the free acids, relatively high concentrations of the amides (or, at least, hydrolysable precursors of the acids) are present in plasma and brain with marked relative increases in CSF. In normal control subjects (n=22) pre-hydrolysis plasma levels (+/- sem) of PIC and QUIN were 0.299 +/- 0.034 and 0.47 +/- 0.047 micromol/L respectively. Following hydrolysis the concentrations rose more than 4-fold to 1.33 +/- 0.115 and 2.2 +/- 0.27 micromol/L respectively. In CSF samples from patients with no sign of brain injury or pathology (n=10) pre-hydrolysis concentrations of PIC and QUIN were 0.017 +/- 0.005 and 0.018 +/- 0.006 micromol/L, respectively, which rose to 0.30 +/- 0.06 and 0.06 +/- 0.008 micromol/L respectively, after hydrolysis. In CSF samples from patients with a range of brain oedema or injury (eg subdural haemorrage, motor vehicle accident) (n=6) pre-hydrolysis concentrations of PIC and QUIN were 0.053 +/- 0.03 and 0.29 +/- 0.12 micromol/L, respectively. Following hydrolysis the concentrations were markedly increased to 6.06 +/- 1.5 and 0.94 +/- 0.63 micromol/L, respectively. The present investigation has shown for the first time that PAM and QAM are present endogenously with PAM being relatively higher than QAM, especially in CSF samples from patients with presumed brain inflammation. The site and mechanism of amidation of PIC and QUIN needs investigation.

[The presence of quinolinic acid in the structures of the rat brain]. / Issledovanie nalichiia khinolinovoi kisloty v strukturakh golovnogo mozga.

The brain tissue extracts from chronically alcoholized (15% ethanol intake for more than 18 months) rats were studied by mass spectrometry. The mass spectra for the striatum of control and alcohol-consuming rats were identical, while those for the hippocampus showed a significant difference: a great increase in the intensity of peaks typical of mass spectra for quinolinic acid.

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.

Regional brain and cerebrospinal fluid quinolinic acid concentrations in Huntington's disease.

Many of the characteristics neuroanatomical and neurochemical features of Huntington's disease (HD) are produced in experimental animals by an intrastriatal injection of the endogenous N-methyl-D-aspartate receptor agonist quinolinic acid (QUIN). Conceivably, a chronic over-production of QUIN in brain could be involved in the pathogenesis of HD. To investigate this hypothesis, concentrations of QUIN were measured both in cerebrospinal fluid (CSF) and postmortem tissue from patients with HD and neurologically normal age-matched controls. CSF QUIN concentrations were slightly lower in patients with HD, however the changes were not significant. Mean concentrations of QUIN tended to be lower in HD putamen, dentate nucleus and several cortical regions, although significant reductions were found only in Brodmann areas 17, 20 and 28. The mechanisms responsible for these small reductions in brain QUIN concentrations remain to be determined. These results do not support the hypothesis that a chronic increase of QUIN production is responsible for neurodengeneration in HD.

Quinolinic acid concentrations in brain and cerebrospinal fluid of patients with intractable complex partial seizures.

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 greater than 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.

Quantification of quinolinic acid in rat brain, whole blood, and plasma by gas chromatography and negative chemical ionization mass spectrometry: effects of systemic L-tryptophan administration on brain and blood quinolinic acid concentrations.

A gas chromatography/mass spectrometry assay is described to quantify the endogenous neurotoxin quinolinic acid (QUIN) in brain, whole blood, and plasma. High specificity and high sensitivity were obtained by using negative chemical ionization and accuracy was achieved by using [18O]QUIN as internal standard. Neutralized perchloric acid extracts were washed with chloroform, applied to Dowex 1 x 8 (formate form), and eluted with 6 M formic acid. After lyophilization, QUIN and [18O]QUIN were esterified with hexafluoroisopropanol (to mass 467 and 471, respectively) using trifluoroacetylimidazole as catalyst. The esters were extracted into heptane and injected onto a gas chromatograph, DB-5 capillary column. QUIN and [18O]QUIN were quantified by selected ion monitoring of QUIN-specific anion currents from the molecular anions (m/z 467 and 471, respectively) and a specific anion fragment (m/z 316 from QUIN and m/z 320 from [18O]QUIN). Minimum sensitivity was 3 fmol, intraassay variability was 3.2%, and interassay variability was 8.1% QUIN concentrations in frontal cortex from over 200 rats ranged from 20 to 180 fmol/mg wet wt. Two hours after systemic L-tryptophan (L-Trp; 0.370 mmol/kg) administration, QUIN increased in whole blood 134.8-fold and in plasma, 74.3-fold. In frontal cortex, increases in QUIN (22.6-fold, corrected for QUIN in blood) exceeded increases in cortical L-Trp (2.54-fold), 5-HT (1.35-fold), and 5-HIAA (1.74-fold). These studies demonstrate that QUIN is present in brain and is sensitive to the availability of systemic L-Trp.

(18O)quinolinic acid: its esterification without back exchange for use as internal standard in the quantification of brain and CSF quinolinic acid.

In the process of developing a high-sensitivity negative chemical ionization gas chromatographic/mass spectrometric assay for brain and cerebrospinal fluid (CSF) levels of quinolinic acid (QUIN, 2,3-pyridine dicarboxylic acid), (18O4)QUIN was prepared. Its properties as an internal standard were compared with those of the structural isomer 2,4-pyridine decarboxylic acid (2,4-PDC) previously used by others. All oxygen atoms in QUIN were labeled by heating in 3 N HCl/(18O)water for 48 h at 80 degrees C. Back-exchange of (18O4)QUIN was prevented during derivatization to an electron-capturing dihexafluoropropanol ester by using trifluoroacetylimidazole as catalyst instead of perfluroacyl anhydrides. When mixtures of QUIN and (18O4)QUIN and/or 2,4-PDC were followed through a procedure to isolate and quantify brain and CSF QUIN, the variability in the ratio of QUIN:2,4-PDC was greater than for QUIN: (18O)QUIN. We conclude that (18O)QUIN is the preferred internal standard in gas chromatographic/mass spectrometric quantification of brain and CSF QUIN, and that (18O)-labeled carboxylic acids may be esterified effectively without back-exchange using acylimidazole reagents.

Increase in the content of quinolinic acid in cerebrospinal fluid and frontal cortex of patients with hepatic failure.

Quinolinic acid (QUIN), an excitotoxic tryptophan metabolite, has been identified and measured in human cerebrospinal fluid (CSF) using a mass-fragmentographic method. Furthermore, its content has been evaluated in frontal cortex obtained at autopsy from the cadavers of patients who died after hepatic coma. During the coma, the concentration of QUIN in the CSF was 152 +/- 38 pmol ml-1. In contrast, the concentration in control patients affected by different pathologies was 22 +/- 7 pmol ml-1. In the frontal cortex of patients who died after episodes of hepatic encephalopathy, the content of QUIN was three times higher than in controls (2.6 +/- 0.6 versus 0.80 +/- 0.08 nmol/g wet weight). As a result of these investigations we are now able to extend our previous observations on the increase of QUIN in the brains of rats used as experimental models of hepatic encephalopathy to man. QUIN should therefore be added to the list of compounds possibly involved in the pathogenesis and symptomatology of brain disorders associated with liver failure.

A radioenzymatic assay for quinolinic acid.

A new and rapid method for the determination of the excitotoxic tryptophan metabolite quinolinic acid is based on its enzymatic conversion to nicotinic acid mononucleotide and, in a second step utilizing [3H]ATP, further to [3H] deamido-NAD. Specificity of the assay is assured by using a highly purified preparation of the specific quinolinic acid-catabolizing enzyme, quinolinic acid phosphoribosyltransferase, in the initial step. The limit of sensitivity was found to be 2.5 pmol of quinolinic acid, sufficient to conveniently determine quinolinic acid levels in small volumes of human urine and blood plasma.

Senile dementia and Alzheimer's disease: lack of changes of the cortical content of quinolinic acid.

The content of Quinolinic Acid (QUIN) was fragmentographically measured in the frontal, parietal and temporal cortex obtained at autopsy from patients affected by Alzheimer's disease-senile dementia Alzheimer type (AD/SDAT) or matched controls. The density of large cholinergic neurons in the nucleus basalis magnocellularis and the density of plaques in the hippocampal formation, parietal and frontal cortex of these patients was also evaluated in order to obtain a quantitative estimation of the Alzheimer type changes. In the three cortical areas studied, the content of QUIN was similar in AD/SDAT patients and age matched controls. The AD/SDAT patients had an important reduction of the number of large cholinergic neurons in the nucleus basalis magnocellularis and a much higher density of plaques in cortex and in hippocampus than age matched controls. The data reported here do not support the possibility than an accumulation of QUIN plays a role in the neuronal degeneration occurring in the cortex of patients affected by AD/SDAT.

The excitotoxin quinolinic acid is present and unevenly distributed in the rat brain.

The presence of quinolinic acid (2,3-pyridinedicarboxylic acid, QA) in the rat brain has been demonstrated using a mass-spectrometric method. Distribution studies indicate that this molecule is more concentrated in the cortex (2.1 nmol/g wet weight) than in other brain areas. Tryptophan, a possible QA precursor, administered in large doses, increases the cortical content of QA. The contrary occurs when rats are pretreated with p-chlorophenylalanine, a drug capable of decreasing brain tryptophan concentration. The neurotoxin 5,7-dihydroxytryptamine is inactive. Our findings support the idea that QA merits special attention as a potential transmitter and as an endogenous excitotoxin in brain.

Identification of quinolinic acid in rat and human brain tissue.

An analytical technique for the determination of the excitotoxic compound quinolinic acid (2,3-pyridine dicarboxylic acid) in brain tissue has been developed. Following sample prepurification by ion exchange and high pressure liquid chromatography, quinolinic acid is converted to the dihexafluoroisopropyl ester and the derivative is analyzed by mass fragmentography. Using the present technique quinolinic acid has been identified in both rat and human brain tissue.

A new fluorometric assay method for quinolinic acid.

A new fluorometric assay method for quinolinic acid is introduced in this study. Quinolinic acid-hydrazine complex, a stable fluorescent compound, is formed after heating quinolinic acid with hydrazine at 215-220 degrees C for 2 min. Fluorescence excitation and emission maxima of the complex are at 285 and 380 nm, respectively. This assay method is rapid and rather sensitive. It takes about 30 min to ascertain the amount of quinolinic acid as low as 50 ng. Specificity of this method is high among biological compounds. An ultrasensitive assay method for quinolinic acid (as low as 20 pg) with diphenylhydrazine instead of hydrazine is also found. After separating the quinolinic acid-diphenylhydrazine complex from residual diphenylhydrazine, this ultrasensitive assay method may be practically applicable.