S-Glutathionyl hemoglobin is a proposed marker of oxidative stress in patients with diabetes, renal failure, and other diseases. Sickle cell disease (SCD) is a condition of oxidative stress, but S-glutathionyl hemoglobin levels have not been studied in SCD patients. The purpose of these experiments was to determine if S-glutathionyl hemoglobin S (HbS-SG) levels are elevated in patients with SCD. Blood was collected from patients attending pediatric sickle cell clinic for routine care. Hemolysates were prepared by adding 5 μL of erythrocytes to 100 μL of hemolyzing reagent. A standard containing HbS-SG was prepared by incubating hemolysate from a SCD patient with glutathione disulfide (10 mmol/L) for 24 hours at 37°C. Hemoglobin variants, including HbS-SG, were then identified and quantified by capillary isoelectric focusing. Analysis of the standard showed that HbS-SG has an isoelectric point similar to that of fetal hemoglobin (HbF) and could not be differentiated from HbF. S-Glutathionyl hemoglobin can be converted to the unmodified form by incubating with reducing agent. Hemolysates from SCD patients were analyzed before and after incubation with dithiothreitol (DTT), and the level of HbS-SG was calculated as the quantitative difference in the amount of HbF in untreated and DTT-treated hemolysates. The results showed that HbF levels in hemolysates from SCD patients were significantly lower after treatment with DTT. HbF levels in blood from infants with normal hemoglobin phenotype were not decreased following treatment with DTT. Hemolysates stored at −20°C, but not −70°C, contained progressively higher levels of HbS-SG over time. We conclude that HbS-SG was present at low levels in patients with SCD. Since HbS-SG and HbF have similar charge properties, HbF levels may be overestimated in samples containing HbS-SG when measured using conventional charge-based separation methods such as cellulose acetate electrophoresis, especially when samples are stored under suboptimal conditions. The possibility that HbS-SG could interfere with measurement of other hemoglobin variants by other separation methods is also under investigation.
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