Abstract
Introduction
Refractory anemia with ring sideroblasts (RARS), as a form of acquired primary sideroblastic anemia, represents about 11% of myelodysplastic syndromes (MDS) and is classified as a low risk MDS. It´s defined by the WHO as a pure dyserythropoietic disorder with presence of >15% ringed sideroblasts in the bone marrow. Abnormal expression of several genes of heme synthesis in this MDS subgroup and excessive accumulation of iron especially in mitochondria, the main place of reactive oxygen species (ROS) formation, contributes to the elevated oxidative stress. Oxidative stress is also known as one of the factors involved in the pathogenesis of MDS. High iron concentrations catalyse a Fenton reaction, where a hydroxyl radical is produced from hydrogen peroxide and causes an increase in ROS which may lead to the oxidation of DNA, lipids, and proteins, thereby causing cell damage.
The aim of this study was to find a useful method for detection and identification of oxidatively modified proteins in plasma unique for RARS patients.
Methods
Carbonylated protein levels were determined spectrophotometrically using dinitrophenylhydrazine (DNPH) derivatization. Oxidatively modified proteins of plasma samples were derivatized with biotin hydrazide. The dialyzed biotin hydrazide labeled samples and negative controls were mixed with monomeric avidin resin. Captured carbonylated proteins were digested by trypsin and then identified by MS/MS mass spectrometry coupled to a nano-LC system. Mascot (Matrix Science, London, UK) was used for database searching (Swiss-Prot). Two unique peptides (with a higher Mascot score than the minimum for identification, P<0.05) were necessary to successfully identify a protein.
Serum iron (Fe), serum ferritin, transferrin, total iron binding capacity (TIBC), and iron saturation were estimated in MDS patients in the Central National Biochemical Laboratory at the Institute of Hematology and Blood Transfusion.
Results
We have compared plasma of RARS patients with healthy controls or with RCMD patients. We have found significant differences in the measured carbonyl levels between all three groups (***P=0.00036). Furthermore, carbonylated protein levels were significantly elevated in RARS patients (n=10; 2.63±0.58 nmol/mg protein) compared to healthy donors (n=20; 1.80±0.42 nmol/mg protein) (***P<0.001) and to RCMD patients (n=10; 1.83±0.58 nmol/mg protein) (**P=0.00298). We have identified a total number of 27 carbonylated proteins unique for RARS patients which were generated by the effect of ROS. Serotransferrin was found as one of the oxidatively modified proteins. We have also found a significant decrease in TIBC in RARS patients compared to RCMD patients (*P=0.03078). TIBC moderately negatively correlated with carbonyl levels (r=-0.56, P=0.04864) in two investigated subgroups of MDS.
Conclusions
We have shown that there is a clear difference in the effect of oxidative stress between RARS, RCMD patients and healthy controls. Moreover, RARS patients, as a low risk MDS, are characterized by significantly higher protein carbonyl levels in comparison to healthy controls and to RCMD patients. The various sensibility of proteins to oxidation (carbonylation) depends both on their plasma concentration and on their susceptibility to oxidative stress as metal-binding sites or structural characteristics of the proteins. Modification in molecular structure of transferrin could be associated with decreased TIBC. This is in agreement with our data of oxidative modification of serotransferrin in RARS patients and could explain the decreased TIBC levels. Our results suggest that measurement of plasma carbonyl levels and the isolation and identification of carbonylated plasma proteins could serve as a potential diagnostic and prognostic tool in MDS.
Acknowledgment
This work was supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.16/3.1.00/28007.
Disclosures
No relevant conflicts of interest to declare.
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