Toxic Unbound Iron and Membrane Injury in b-Thalassemia and Sickle Cell Disease: Elevated Non-Transferrin Bound Iron (NTBI) and Malondialdehyde (MDA).
Authors: Walter Patrick B. , Fung Ellen , Killilea David W. , Jiang Qing , Ames Bruce N. , Madden Jacqueline , Butensky Ellen , Porter John , Evans Patricia , Harmatz Paul , Vichinsky Elliott P. 
Published: October 2019

Objective: Chronic red blood cell transfusion therapy is life-saving for patients with β-thalassemia (THL) and sickle cell disease (SCD), but often results in severe iron overload. Clinical observations suggest that organ dysfunction (heart, liver, endocrine dysfunction, bone disease) resulting from iron overload is seen more often in patients with THL than SCD. This study examines the possible correlation between increased organ injury in these patients and oxidative stress, iron metabolism, inflammation and plasma vitamin E.

Methods: Markers of oxidant stress were compared in 18 subjects with THL (7M, 24 ± 9 yrs) and 11 with SCD (7M, 13 ± 4 yrs) with 10 disease-free controls (5M, 27 ± 12 yrs). All THL and SCD patients admitted to the study were healthy and had not had a recent medical event including vascular crises for SCD within the last 4 months. Blood was drawn from fasted subjects, prior to blood transfusion, and plasma, serum and cells were separated by centrifugation. Plasma levels of malondialdehyde (MDA), a marker of lipid peroxidation, were determined by GC-MS. Non-transferrin bound iron (NTBI), protein carbonyls and tocopherol content were measured by HPLC. C Reactive Protein (CRP) was determined by ELISA. Liver iron content was analyzed by ICP-mass spectrometry from disease patients.

Results: Table 1

Values are means ± SD (subject n). Within a row, underlined values are significantly different from control and starred values are significantly different between THL and SCD (P<0.05).

Parameter Control THL SCD Values are means ± SD (subject n). With* in a row, daggered † values are significantly different from control and starred * values are significantly different between THL and SCD (P<0.05) Ferritin, ng/ml 65.0 ± 70.9 (10) 1915.0 ± 1030.6 (18)† 2514.7 ± 1152.6 (11)† NTBI, μM −1.0 ± 0.4 (9) 4.0 ± 1.6 (16) *† 1.9 ± 2.1 (11) (c)† MDA, pmol/ml 20.3 ± 14.2 (9) 36.4 ± 20.5 (18)† 27.0 ± 11.4 (11) ALT, U/l 34.9 ± 7.7 (9) 59.2 ± 31.1 (18) *† 36.1 ± 11.0 (11)† α-tocopherol, μM 22.7 ± 3.4 (10) 14.9 ± 4.6 (17)† 15.3 ± 4.0 (11)† γ-tocopherol, μM 1.8 ± 0.7 (10) 3.5 ± 2.2 (17)† 5.5 ± 1.4 (11) *† CRP, mg/L 0.8 ± 0.7 (11) 1.1 ± 1.2 (18) 2.7 ± 3.2 (10)† carbonyls, nmol/mg 0.6 ± 0.1 (10) 0.7 ± 0.3 (17) 0.6 ± 0.1 (11)

MDA, ALT, and NTBI, were higher in THL despite evidence for lower body iron burden in THL relative to SCD (liver iron: 9 ± 7.1 vs. 15 ± 5.6 mg/g dry weight). In contrast, evidence for inflammation such as ferritin and CRP were significantly higher in SCD. g-tocopherol was significantly higher in SCD relative to THL.

Conclusions: These preliminary findings suggest that THL patients have higher levels of toxic free iron and more evidence for tissue injury than SCD. SCD patients may be protected, in part, by greater inflammation and unique antioxidant reserves. The prospective Multicenter Study of Iron Overload in SCD and THL will determine whether these biomarkers are predictors of differences in end organ failure in these two diseases.