title: A quantitative method for the analysis of glycated and glutathionylated hemoglobin by matrix-assisted laser desorption ionization-time of flight mass spectrometry.
abstract: The quantization of glycated isoforms of hemoglobin has been increasingly used in clinical practice in recent years. Glycated hemoglobin is currently considered the most important measurement for long-term control of the glycemic state and it has become a reference tool for the management of diabetes. Glutathionylated hemoglobin is an increasingly clinically relevant covalent adduct of glutathione with beta chain of the globin and its concentration has been correlated with oxidative stress. We have developed an innovative technique based on linear mode matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for quantitative analysis of hemoglobin species. This method was applied to the quantification of glycated and glutathionylated hemoglobin. A rigorous comparison was pursued to evaluate the analytical performances in quantifying glycated hemoglobin in comparison to an established high-performance liquid chromatography method. Our results indicated a complete equivalence between the two methods. The same analysis enabled the quantitative determination of the glutathionylated hemoglobin fraction. This isoform was investigated in an adult Italian population (184 individuals, 101 males and 83 females), indicating a bimodal distribution of this species. In fact 65.22% of screened individuals had glutathionylated hemoglobin levels lower than 0.50% while 34.78% had glutathionylated hemoglobin levels higher than 0.50%. A semiautomatic robotic procedure was developed for fast analysis of a large number of samples. This is the first report of a quantitative application of linear MALDI-TOF mass spectrometry for the determination of glutathionylated hemoglobin in blood samples. This method allows fast screening of this hemoglobin isoform, therefore opening the route to explore its specificity and sensitivity as a molecular biomarker.
authors.affiliation: Clinical Biochemistry Laboratory, Children's Hospital Bambino Gesù-IRCCS, Rome (Vatican State), Piazza Sant'Onofrio 4, Rome 00165, Italy.
authors.forename: Antonino
authors.initials: A
authors.lastname: Biroccio
authors.forename: Andrea
authors.initials: A
authors.lastname: Urbani
authors.forename: Renato
authors.initials: R
authors.lastname: Massoud
authors.forename: Carmine
authors.initials: C
authors.lastname: di Ilio
authors.forename: Paolo
authors.initials: P
authors.lastname: Sacchetta
authors.forename: Sergio
authors.initials: S
authors.lastname: Bernardini
authors.forename: Claudio
authors.initials: C
authors.lastname: Cortese
authors.forename: Giorgio
authors.initials: G
authors.lastname: Federici
mesh_terms: D002851:Chromatography, High Pressure Liquid; D005260:Female; D005978:Glutathione / Q000097:blood* / Q000737:chemistry; D006442:Glycated Hemoglobin / Q000032:analysis* / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D008297:Male; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D005978:Glutathione; D006442:Glycated Hemoglobin; D006454:Hemoglobins; C404464:glutathionyl hemoglobin