ethyl-lysine and Alcoholism

N(ε) -ethyllysine (NEL) is a major stable adduct formed by the reaction of acetaldehyde (AA) with lysine residues in proteins. However, its occurrence and levels in biological specimens and its relationship with AA/alcohol exposure-associated disorders have not been fully elucidated. In this study, we have developed a sensitive and specific method to quantitate NEL levels in human plasma proteins.. The method consists of (1) purification of the protein fraction of interest by Sephadex G-15 to remove low molecular substances, (2) hydrolysis of proteins with Pronase E in the presence of stable isotope-labeled internal standards, (3) derivatization of amino acids with pentafluorobenzyl (PFB) bromide, and (4) quantification of the PFB derivatives of NEL and l-lysine using gas chromatography-negative ion chemical ionization/mass spectrometry in a selected ion monitoring mode.. Using the above method, the NEL levels in human plasma proteins obtained from 10 each of control subjects and alcoholic patients were measured. NEL was detected in all samples analyzed, the average level of NEL in the plasma proteins of alcoholic patients (1.17 ± 0.36 NEL/1,000 l-lysine) being significantly higher than that of control subjects (0.26 ± 0.07 NEL/1,000 l-lysine).. The method could be applied to molecular epidemiological studies to investigate possible associations between the NEL levels in human tissue proteins and human diseases associated with exposure to AA and alcohol.

Topics: Acetaldehyde; Adult; Aged; Alcoholism; Biomarkers; Carbon Isotopes; Case-Control Studies; Central Nervous System Depressants; Ethanol; Gas Chromatography-Mass Spectrometry; Humans; Isotope Labeling; Lysine; Male; Middle Aged; Nitrogen Isotopes

The Maillard reaction that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients, in aging and in neurodegenerative processes. We hypothesize that acetaldehyde (AA), one of the main metabolites of alcohol, may be involved in alcohol-induced neurotoxicity in vivo by formation of AA-derived AGEs (AA-AGE) with brain proteins.. AA-AGE-bovine serum albumin (BSA) and AA-AGE-rabbit serum albumin (RSA) were prepared as described previously. Antibody specific for AA-AGE was isolated from rabbit antiserum by affinity chromatography. Primary cortical neuronal cell cultures were prepared as described previously.. Incubation of cortical neurons with AA-AGE produced a dose-dependent increase in neuronal cell-death, and the neurotoxicity of AA-AGE was neutralized by the addition of an anti-AA-AGE specific antibody, but not by anti-N-ethyllysine (NEL) antibody. The AA-AGE epitope was detected in human brain of alcoholism.. We propose that the structural epitope AA-AGE is an important toxic moiety for neuronal cells in alcoholism.

Topics: Acetaldehyde; Alcoholism; Animals; Antibodies; Cells, Cultured; Central Nervous System Depressants; Cerebral Cortex; Chromatography, Affinity; Epitopes; Ethanol; Glycation End Products, Advanced; Humans; Immunoblotting; Lysine; Nerve Degeneration; Neurons; Rats