Determination of Leucine- and Methionine-Enkephalins in Rat Brains by High Performance Liquid Chromatography with Precolumn Fluorescence Derivatization

Abstract

Fluorometrically reactive peptides of leucine-and methionine-enkephalins in rat brain tissues such as cortex, striatum and hypothalamus were simultaneously assayed by reversed-phase high performance liquid chromatography with fluorometric detection, based on precolumn derivatization specific for N-terminal tyrosine-containing peptides. The enkephalin peptides extracted from the tissues were converted into fluorescent derivatives by reaction with hydroxylamine, cobalt(II) ion and borate in a weakly alkaline aqueous solution (pH 8.5). The fluorescent derivatives of the peptides were separated on a reversed-phase column (TSKgel ODS-120T) by gradient elution of acetonitrile in the mobile phase containing borate buffer (pH 8.5) and tetrabutylammonium chloride, and then determined by fluorometry. The peaks of both the enkephalins in the tissue sample were not observed in the chromatogram after the enzymatic degradation with carboxypeptidase A. The determined concentrations of the leucine-and methionine-enkepalins in the tissues were 20-238 pmol and 80-75 pmol per g of the tissue, respectively. The method was sensitive enough to determine the endogenous enkephalins at concentrations as low as Ca. 6 pmol per g of the brain tissues. KeywordsEnkephalin, rat brain, fluorometry, high performance liquid chromatography, precolumn derivatizationEnkephalins, leucine-enkephalin (LE; Tyr-Gly-GlyPhe-Leu) and methionine-enkephalin (ME; Tyr-GlyGly-Phe-Met), have opiate-like activity in nervous systems.' Recently, opioid peptides have been found to act in vivo as neurotransmitters via interaction with opiate receptors.2Most of the opioid peptides involving LE and ME have a tyrosyl residue at the Nterminus in their molecules.During the studies on the physiological mechanism of the opioid peptides, many methods have been developed for the quantitative determination of the opioid peptides in biological samples. These methods include bioassay3, radioimmunoassay (RIA)4'5, enzyme-immunoassay6, radioreceptorassay' and mass spectrometry8, either alone or in combination with high performance liquid chromatography (HPLC).HPLC can simultaneously separate many synthetic and biological peptides9"0, and has the advantages of superior reproducibility, practicability and speed in the analysis. However, the quantification of the endogenous opioid peptides in brain tissues by the current HPLC methods with spectrophotometric'° and electrochemical" detections is hampered by problems of insufficient sensitivity and selectivity for the peptides.To resolve the problems in the HPLC, we previously developed a unique chemical reaction'2 for N-terminal Tyr-containing peptides, by which the peptides yields intense fluorescences under mild conditions in the presence of hydroxylamine, cobalt(II) ion and borate. This reaction provided a single fluorescent product for each of the synthetic peptides and did not permit the production of fluorescent derivatives for peptides having no N-terminal tyrosyl residue (e.g. Gly-Tyr, Phe-Arg-Gly and Arg-Val-Tyr-Ile-His-Pro-Phe...

High Performance Liquid Chromatography of Bioactive Substances Using Bifunctional Fluorogenic Reagents for Derivatization A Review

“…Methionine-enkephalin and leucine-enkephalin in rat brain tissues such as cortex, striatum and hypothalamus have been simultaneously measured by the precolumn method using [D-A1a2'3]-methionine-enkephalin as an internal standard (detection limits, 6 pmol/ g tissue). 91 The clean-up of the sample homogenate is attained by deproteinization with perchloric acid followed by a solid-phase extraction with an ODS cartridge.…”

HPLC/MS/MS for quantification of two types of neurotransmitters in rat brain and application: Myocardial ischemia and protection of Sheng-Mai-San

“…Currently many bioanalytical techniques have been developed for the analysis of monoaminergic neurotransmitters and neuropeptides. GC [8,9], CE [10,22], LC-UV [11,12] and LCelectrochemical [13,14] fluorescent detection [14][15][16][17] have been reported to determine monoaminergic neurotransmitter and neuropeptide levels. However, most of these methods require derivatization of the analytes, the use of ion-pairing reagents, and large quantities of samples.…”

Determination of Seven Opioid Peptides in Rat Brain by High Performance Liquid Chromatography with On-Line Postcolumn Fluorescence Derivatization

“…16 This reaction produced no fluorescent derivative from peptides without having an N-terminal tyrosyl residue or from many other biological substances such as amino acids other than Tyr, sugars, steroids, purine and pyrimidine bases.17,18 Though this reaction was utilized for a precolumn fluorescence derivatization method of HPLC of the opioid peptides in rat brain tissue, the method only allowed assays of LE and ME. 18 In the present study the fluorescence reaction was applied to a postcolumn fluorescence derivatization method of HPLC in the hope that many endogenous opioid peptides in rat brain could be quantified. In this work the optimum HPLC separation and reaction conditions were studied.…”