A new CE‐ESI‐MS method for the detection of stable hemoglobin acetaldehyde adducts, potential biomarkers of alcohol abuse

Abstract

A new CE-ESI-MS method was developed to provide a simple way to study changes to hemoglobin (HbA) induced by acetaldehyde (Ach) in vitro. Instrumental parameters were univariately optimized in order to maximize the sensitivity of the CE-ESI-MS method. The electrophoretic separations were carried out in poly-E323-coated capillaries using 60 mM formic acid raised to pH 3.0 with ammonia and containing 5% 2-propanol while the sheath liquid, 2-propanol/water (30:70) with 0.1% formic acid, was delivered at 1.0 microL/min through a coaxial sheath flow electrospray interface. The HbA was incubated with Ach for intervals up to 24 h at concentration varying in the window 0.2-20 mM. Four stable Ach-hemoglobin adducts in the hemoglobin tryptic digest were observed at the submillimolar Ach concentration and characterized by MS/MS experiments: although the alpha and beta N-amino terminal modifications were expected, the two internal ones arising, respectively, from the condensation of Ach molecules on the histidine residue in position 4 in alpha4 (i.e. the fourth peptide after tryptic digestion of alpha chain starting from amino terminal) and on the asparagine residue in position 2 in beta3, were identified for the first time. During the in vitro experiments higher concentrations of Ach were also used; however, it was not possible to identify any other stable modification of hemoglobin. Interestingly, those stable modifications are the only ones in vivo identified in the hemoglobin of moderate alcohol drinkers.

Determination of maternal-fetal biomarkers of prenatal exposure to ethanol: A review

“…Unfortunately, these assays have poor specificity, because of the difficulties in the selection of specific APAs for the development of antibodies. Recently, a new capillary electrophoresis method coupled to electrospray ionization mass spectrometry (CE-ESI-MS) has been developed for the detection of hemoglobin-associated acetaldehyde (HAA) [49].…”

CE-MS in Forensic Sciences with Focus on Forensic Toxicology

“…De Benedetto et al focused their attention on the development of an efficient CZE-ESI-ion trap MS method in positive ion mode for the detection of Ach adducts of hemoglobin [117]. The method employed in-house coated silica capillaries to hinder protein adsorption and a coaxial sheath liquid interface.…”

Protein biomarkers of alcohol abuse

“…Its concentration after alcohol intake is highly variable with a life-time of approximately 3 h [63]. Circulating acetaldehyde has long been known to react with various proteins, resulting in aldehyde-protein adduct formation [64].…”

“…Some of these adducts have been detected up to 3 weeks after alcohol consumption. MS-based approaches aimed at detecting these for their use as biological markers of alcohol abuse have recently been developed [63]. Alternatively, circulating antibodies against acetaldehyde adducts have been directly measured as biomarkers of alcohol intake.…”