title: Evidence by chromatography and mass spectrometry that inorganic nitrite induces S-glutathionylation of hemoglobin in human red blood cells.
abstract: Previously we found by HPLC with fluorescence detection that inorganic nitrite induces oxidation of glutathione (GSH) to its disulfide (GSSG) in intact and more abundantly in lyzed red blood cells (RBCs) from healthy humans. In the present work, we performed MS-based protein analysis and observed that nitrite (range, 0-20mM) induces formation of S-glutathionyl hemoglobin (HbSSG) at cysteine (Cys) β93 and β112 of oxyhemoglobin (HbO2) in lyzed human RBCs (range, 6-8mM HbO2). Hemoglobin species were isolated from incubation mixtures of nitrite in lyzed RBCs by ultrafiltration or affinity chromatography and analyzed by HPLC and LC-MS/MS. The mechanism likely involves inhibition of catalase activity by nitrite (IC50, 9 μM), which allows H2O2 to accumulate and oxidize Cys moieties of oxyhemoglobin and erythrocytic GSH to form HbSSG in addition to GSSG. In freshly prepared hemolysate samples, nitrite induced release of superoxide and molecular oxygen. In the presence of paracetamol and nitrite in hemolysate samples, 3-nitro-paracetamol was detected. Nitrite also induced S-nitroso hemoglobin (HbSNO) formation in low yield (i.e., 0.1%). Synthetic cysteine (Cys), glutathione (GSH), N-acetylcysteine (NAC) and N-acetylcysteine ethyl ester (NACET) inhibited nitrite-induced modifications of oxyhemoglobin including methemoglobin, HbSSG (CysSH >> NACET >> GSH ≈ NAC; thiol concentration, 50 μM) and HbSNO. Nitrite-induced oxidative modifications may alter physiological hemoglobin functions and may require alternative treatments for conditions associated with oxidized hemoglobin like in nitrite-induced methemoglobinemia. Accumulation of soluble Cys in RBCs via oral administration of NACET could be a new promising strategy to prevent nitrite-induced methemoglobinemia by nitrite and other oxidants.
authors.affiliation: Centre of Pharmacology and Toxicology, Hannover Medical School, 30623 Hannover, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Böhmer
authors.affiliation: Institute of Toxicology and Core Unit Proteomics, Hannover Medical School, 30623 Hannover, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Pich
authors.affiliation: Centre of Pharmacology and Toxicology, Hannover Medical School, 30623 Hannover, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Schmidt
authors.affiliation: Department of Cardiology and Angiology, Hannover Medical School, 30623 Hannover, Germany.
authors.forename: Arash
authors.initials: A
authors.lastname: Haghikia
authors.affiliation: Centre of Pharmacology and Toxicology, Hannover Medical School, 30623 Hannover, Germany. Electronic address: tsikas.dimitros@mh-hannover.de.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Tsikas
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D005260:Female; D005978:Glutathione / Q000097:blood / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008297:Male; D009573:Nitrites / Q000097:blood / Q000737:chemistry*; D010084:Oxidation-Reduction; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006454:Hemoglobins; D009573:Nitrites; D005978:Glutathione
keywords: Catalase; Erythrocytes; Hemoglobin; Mass spectrometry; Methemoglobin; Nitrite; S-Glutathionylation; Thiols