Analysis of <scp><i>N</i></scp>^ε‐Ethyllysine in Human Plasma Proteins by Gas Chromatography–Negative Ion Chemical Ionization/Mass Spectrometry as a Biomarker for Exposure to Acetaldehyde and Alcohol

<jats:sec><jats:title>Background</jats:title><jats:p><jats:styled-content style="fixed-case"><jats:italic>N</jats:italic></jats:styled-content><jats:sup>ε</jats:sup>‐ethyllysine (<jats:styled-content style="fixed-case">NEL</jats:styled-content>) 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 <jats:styled-content style="fixed-case">NEL</jats:styled-content> levels in human plasma proteins.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>The method consists of (1) purification of the protein fraction of interest by <jats:styled-content style="fixed-case">S</jats:styled-content>ephadex <jats:styled-content style="fixed-case">G</jats:styled-content>‐15 to remove low molecular substances, (2) hydrolysis of proteins with <jats:styled-content style="fixed-case">P</jats:styled-content>ronase <jats:styled-content style="fixed-case">E</jats:styled-content> in the presence of stable isotope–labeled internal standards, (3) derivatization of amino acids with pentafluorobenzyl (<jats:styled-content style="fixed-case">PFB</jats:styled-content>) bromide, and (4) quantification of the <jats:styled-content style="fixed-case">PFB</jats:styled-content> derivatives of <jats:styled-content style="fixed-case">NEL</jats:styled-content> and <jats:sc>l</jats:sc>‐lysine using gas chromatography–negative ion chemical ionization/mass spectrometry in a selected ion monitoring mode.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Using the above method, the <jats:styled-content style="fixed-case">NEL</jats:styled-content> levels in human plasma proteins obtained from 10 each of control subjects and alcoholic patients were measured. <jats:styled-content style="fixed-case">NEL</jats:styled-content> was detected in all samples analyzed, the average level of <jats:styled-content style="fixed-case">NEL</jats:styled-content> in the plasma proteins of alcoholic patients (1.17 ± 0.36 <jats:styled-content style="fixed-case">NEL</jats:styled-content>/1,000 <jats:sc>l</jats:sc>‐lysine) being significantly higher than that of control subjects (0.26 ± 0.07 <jats:styled-content style="fixed-case">NEL</jats:styled-content>/1,000 <jats:sc>l</jats:sc>‐lysine).</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>The method could be applied to molecular epidemiological studies to investigate possible associations between the <jats:styled-content style="fixed-case">NEL</jats:styled-content> levels in human tissue proteins and human diseases associated with exposure to AA and alcohol.</jats:p></jats:sec>