Heightened Sulfur Amino Acid Oxidation in Plasma and Erythrocytes in β-Thalassemia Major.
Authors: Agrawal Anurag K. , Suh Jung H. , Ames Bruce N. , Vichinsky Elliott P. , Lal Ashutosh 
Published: October 2019

Abstract
Abstract 4065
Poster Board III-1000

Background
Oxidative stress in β-thalassemia major is a well documented problem thought in part to be due to transfusional iron overload. Sulfur amino acid (SAA)-derived metabolites, chiefly glutathione (GSH), are critical components of cellular antioxidant defense. Inability to adequately regulate endogenous antioxidant status may lead to further exacerbation of oxidative stress. To characterize whether alteration in SAA metabolism contributes to heightened oxidative stress in thalassemia, we utilized a novel liquid chromatography linked electrospray positive tandem mass spectrometric (LC-MS/MS) technique to simultaneously quantify the redox states of cysteine (Cys) and GSH as well as the concentrations of major amino acid-derived metabolites in plasma and erythrocytes.

Methods
After institutional review board approval, adult and pediatric subjects with β-thalassemia major were recruited for fasting blood samples drawn immediately prior to the next scheduled blood transfusion. Plasma and red blood cell samples were analyzed using the LC-MS/MS technique (J Chromatogr B 2009;in press) and compared with healthy controls.

Results
Twelve subjects with β-thalassemia major were recruited. The median age of the group was 26.5 years (range 11-41). The median liver iron concentration (LIC) determined by ferritometer was 11.9 mg/g dry-wt (range 1.0-34.8). The median ferritin level for the group was 1980 ng/mL (379-4730). Evidence of myocardial iron overload (T2* <20 msec) was present in 5 of 11 subjects. Plasma SAA redox analysis in these subjects showed significant oxidation of Cys and GSH. Total Cys redox status (Cysteine/2*Cystine ratios) decreased from 0.1 ± 0.01 in healthy controls to 0.08 ± 0.02 in thalassemia (p=0.02). Similarly, the plasma GSH redox state (GSH/2*GSSG ratio) decreased from 38.9 ± 13.7 in healthy controls to 6.7 ± 5.3 in thalassemia subjects (p=0.005). The total GSH and total Cys pools in the plasma did not differ between the two groups. Mirroring the patterns observed in the plasma compartment, the proportion of erythrocyte GSSG was also significantly elevated in thalassemia (0.65% vs. 0.04%, p=0.009), without a concomitant decline in total erythrocyte GSH pool. A significant 3-fold elevation in plasma cystathionine and S-adenosylmethionine levels suggests that key SAA metabolic pathways to augment synthesis of GSH may be up-regulated in thalassemia. In addition to alterations in SAA metabolite profiles, the thalassemia group was >3-fold deficient in plasma valine, spermine and citrulline compared with controls. We found that within this small group, the level of iron overload measured by serum ferritin, liver iron concentration and cardiac MRI T2* showed no correlation with the amount of oxidative stress measured by glutathione redox.

Discussion
Assessment of SAA redox states and metabolic pathways has the potential to be a novel important marker in β-thalassemia major. The alterations in both Cys and GSH redox states without concomitant decline in their total concentration suggests either a significant increased rate of SAA oxidation and/or a decline in the capacity for NADPH-dependent reduction of oxidized disulfides. The latter may be supported by the evidence of profound hypocitrullinemia in thalassemia, which has been noted in subjects with inherited mitochondrial disorders (Proc Nat Acad Sci U S A 2009;106:3941-5). Finally, we show that the oxidative environment in the plasma affects the transfused erythrocytes which acquire characteristics of abnormal thiol metabolites observed in the plasma compartment. The demonstration of significant oxidative stress even in subjects with low body iron burden suggests that currently acceptable LIC levels may not be optimal, or that other contributors to the pro-oxidant state should be sought in thalassemia.

Disclosures:
No relevant conflicts of interest to declare.