3-Nitrotyrosine in Sera of Patients with Myelodysplastic Syndromes, a Preliminary Study
Authors: Novak Jaromir , Suttnar Jiri , Chrastinova Leona , Stikarova Jana , Hlavackova Alzbeta , Pastva Ondrej , Kotlin Roman , Cermak Jaroslav , Dyr Jan E 
Published: October 2019

Abstract
Introduction:
Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoesis, associated with cytopenias and high risk of leukemic transformations with common morbidity. MDS are hematological malignancies of unclear etiology where oxidative/nitrative stress may contribute to the pathogenesis1. The posttranslational oxidative modifications of proteins and low molecular weight compounds are induced, revealing dysbalance of redox systems in vivo. Nitration of tyrosine either in free form or bound in proteins is important marker of nitric oxide synthase (NOS) activity shift in the presence of oxidative stress in favour of superoxide formation. The aim of this work was to assess whether 3-nitrotyrosine (3-NT) serum concentrations are enhanced also in MDS patients.
Methods:
Serum samples were obtained using blood of either MDS patients or healthy donors. All tested individuals agreed to the study at the time of blood collection. We proposed HPLC-MS/MS method to estimate 3-NT concentration in serum samples using QTRAP 4000 mass spectrometer (ABSciex, Prague, Czech Republic). Serum proteins were precipitated using ethanol, supernatants were evaporated, reconstituted in 0.1% HCOOH/2% methanol and injected onto HALO C18 microcolumn 100x0.5 mm (ABSciex, Prague, Czech Republic). Oxidative stress in MDS patients and controls was assessed by serum malondialdehyde concentrations measured by HPLC of 2-thiobarbituric acid MDA derivative using UV detection.
Results:
The sensitivity of method proposed for analysis of 3-NT in sera was sufficient for estimation of differences of 3-NT in patients and control samples. We have found enhanced concentrations of both MDA and 3-nitrotyrosine in serum of MDS patients as compared with healthy donors.
Discussion:
Enhanced MDA concentrations in MDS patients confirmed the presence of oxidative stress in MDS patients. The reactive oxygen species may oxidize tetrahydrobiopterin, important cofactor of NOS, resulting into nitric oxide synthase uncoupling with enhanced superoxide and consequently peroxynitrite production2. It is known that methylarginines, naturally occurring inhibitors of NOS, can profoundly increase superoxide generation from uncoupled NOS. Recently, we have found significantly enhanced concentration of asymmetric dimethylarginine in a serum of middle age patients with myelodysplastic syndrome3. The observed increased concentrations of 3-NT in MDS patients correspond with assumed enhanced peroxynitrite formation as compared with controls. 3-nitrotyrosine concentrations thus could serve as a new criterion of NOS changed activity in MDS patients.
Literature:
1. Farquhar MJ, Bowen DT. Oxidative stress and the myelodysplastic syndromes. Int J Hematol. 2003;77:342-350.
2. Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev. 2007;87:315-424.
3. Štikarová J, Suttnar J, Pimková K, Chrastinová-Mášová L, Čermák J, Dyr JE. Enhanced levels of asymmetric dimethylarginine in a serum of middle age patients with myelodysplastic syndrome. Journal of Hematology & Oncology. 2013;6:58.

Disclosures
No relevant conflicts of interest to declare.