A new CE-ESI-MS method for the detection of stable hemoglobin acetaldehyde adducts, potential biomarkers of alcohol abuse.
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.
Laboratorio di Analisi Chimiche per l'Ambiente e i Beni Culturali, Dipartimento dei Beni delle Arti e della Storia, Università del Salento, Lecce, Italy. giuseppe.debenedetto@unile.it
Giuseppe E
GE
De Benedetto
Matteo
M
Fanigliulo
D000079:Acetaldehyde / Q000032:analysis* / Q000378:metabolism; D000437:Alcoholism / Q000378:metabolism*; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D006454:Hemoglobins / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D015415:Biomarkers; D006454:Hemoglobins; D000079:Acetaldehyde