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Title: Radioenzymatic assay for quinolinic acid

Abstract

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 (/sup 3/H)ATP, further to (/sup 3/H) 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.

Authors:
; ; ;
Publication Date:
Research Org.:
Maryland Psychiatric Research Center, Baltimore
OSTI Identifier:
6846673
Resource Type:
Journal Article
Resource Relation:
Journal Name: Anal. Biochem.; (United States); Journal Volume: 1
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; METABOLITES; RADIOENZYMATIC ASSAY; PHOSPHOTRANSFERASES; ENZYME ACTIVITY; ATP; LABELLED COMPOUNDS; NAD; PYRIDINES; TRACER TECHNIQUES; TRITIUM COMPOUNDS; TRYPTOPHAN; AMINO ACIDS; AROMATICS; AZAARENES; AZINES; AZOLES; CARBOXYLIC ACIDS; COENZYMES; ENZYMES; HETEROCYCLIC ACIDS; HETEROCYCLIC COMPOUNDS; INDOLES; ISOTOPE APPLICATIONS; NUCLEOTIDES; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PHOSPHORUS-GROUP TRANSFERASES; PYRROLES; TRANSFERASES 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Foster, A.C., Okuno, E., Brougher, D.S., and Schwarcz, R. Radioenzymatic assay for quinolinic acid. United States: N. p., 1986. Web. doi:10.1016/0003-2697(86)90595-6.
Foster, A.C., Okuno, E., Brougher, D.S., & Schwarcz, R. Radioenzymatic assay for quinolinic acid. United States. doi:10.1016/0003-2697(86)90595-6.
Foster, A.C., Okuno, E., Brougher, D.S., and Schwarcz, R. 1986. "Radioenzymatic assay for quinolinic acid". United States. doi:10.1016/0003-2697(86)90595-6.
@article{osti_6846673,
title = {Radioenzymatic assay for quinolinic acid},
author = {Foster, A.C. and Okuno, E. and Brougher, D.S. and Schwarcz, R.},
abstractNote = {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 (/sup 3/H)ATP, further to (/sup 3/H) 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.},
doi = {10.1016/0003-2697(86)90595-6},
journal = {Anal. Biochem.; (United States)},
number = ,
volume = 1,
place = {United States},
year = 1986,
month =
}
  • A radioenzymatic assay for the activity of acetohydroxy acid synthase on one of its substrates, ..cap alpha..-ketobutyrate, was developed. Advantage was taken of the fact that acidification of the product of the enzymatic reaction releases /sup 14/CO/sub 2/, which is derived from the carboxyl group of ..cap alpha..-ketobutyrate when ..cap alpha..-(1-/sup 14/C)ketobutyrate is used as the substrate. The /sup 14/CO/sub 2/ evolved is collected and measured in a simple apparatus which is described in the text.
  • The relative brain tissue concentrations of dopamine (DA) and its deaminated metabolite, dihydroxyphenylacetic acid (DOPAC), appears to be a reliable index of the functional activity of dopaminergic neurons. In order to apply this approach to the assessment of dopaminergic neuronal activity in small regions of brain, we have developed a sensitive radioenzymatic assay for simultaneous measurement of DA and DOPAC. The sensitivity of the assay for DA is approximately 10 pg and for DOPAC 100 pg. In addition, the assay is highly specific, simple, and relatively inexpensive. The concurrent estimation of tissue DA and DOPAC concentrations seems to be amore » reliable means of evaluating the rate of DA turnover or release in behavioral, electrical stimulation, and certain drug paradigms. However, the release or turnover of DA as induced by D-amphetamine (and perhaps other indirectly-acting dopaminemimetic drugs) cannot be meaningfully assessed by measurement of DA and DOPAC alone.« less
  • Intravenous injection of 450 mg/kg quinolinic acid (Quin), an endogenous kynurenine metabolite with excitotoxic properties, induced only minor electroencephalographic (EEG) modifications and no neurotoxicity in rats with a mature blood-brain barrier (BBB). BBB permeability was altered in rats by focal unilateral irradiation of the cortex (7 mm in diameter and 5 mm in depth) with protons (60 Gy, 9 Gy/min). Three days after irradiation, Evans blue dye staining showed BBB breakdown in the dorsal hippocampus of the irradiated hemisphere. No neurotoxic or convulsant effects were observed as a consequence of the radiation itself. When BBB-lesioned rats were challenged with 225more » mg/kg Quin iv, epileptiform activity was observed on EEG analysis. Tonic-clonic seizures were induced by 225-450 mg/kg Quin. Light microscopic analysis showed a dose-related excitotoxic type of lesion restricted to the hippocampus ipsilateral to the irradiated side. Neuro-degeneration was prevented by local injection of 120 nmol D(-)2-amino-7-phosphonoheptanoic acid, a selective N-methyl-D-aspartate receptor antagonist. No lesions or EEG or behavioral modifications occurred after 450 mg/kg nicotinic acid, an inactive analog of Quin. The potential neurotoxic and convulsant effects of increased blood levels of Quin under conditions of altered BBB permeability are discussed.« less