title: Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices.
abstract: Cell-free heme (CFH) and hemoglobin (Hb) have emerged as distinct mediators of acute injury characterized by inflammation and microcirculatory dysfunction in hemolytic conditions and critical illness. Several reports have shown changes in Hb and CFH in specific pathophysiological settings. Using PBS, plasma from patients with sickle cell disease, acute respiratory distress syndrome (ARDS) patients and supernatants from red cells units, we found that commonly used assays and commercially available kits do not distinguish between CFH and Hb. Furthermore, they suffer from a variety of false-positive interferences and limitations (including from bilirubin) that lead to either over- or underestimation of CFH and/or Hb. Moreover, commonly used protocols to separate CFH and Hb based on molecular weight (MWt) are inefficient due to CFH hydrophobicity. In this study, we developed and validated a new approach based on absorbance spectrum deconvolution with least square fitting analyses that overcomes these limitations and simultaneously measures CFH and Hb in simple aqueous buffers, plasma or when associated with red cell derived microvesicles. We show how incorporating other plasma factors that absorb light over the visible wavelength range (specifically bilirubin), coupled with truncating the wavelength range analyzed, or addition of mild detergent significantly improves fits allowing measurement of oxyHb, CFH and metHb with >90% accuracy. When this approach was applied to samples from SCD patients, we observed that CFH levels are higher than previously reported and of similar magnitude to Hb.
authors: Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors: Joo-Yeun
authors: JY
authors: Oh
authors: Departments of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors: Jennifer
authors: J
authors: Hamm
authors: Departments of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Program in Protease and Matrix Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors: Xin
authors: X
authors: Xu
authors: Departments of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Program in Protease and Matrix Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors: Kristopher
authors: K
authors: Genschmer
authors: Departments of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Department of Cardiology, Qili Hospital of Shandong University, China.
authors: Ming
authors: M
authors: Zhong
authors: Departments of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors: Jeffrey
authors: J
authors: Lebensburger
authors: Departments of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors: Marisa B
authors: MB
authors: Marques
authors: Departments of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Birmingham VA Medical Center, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors: Jeffrey D
authors: JD
authors: Kerby
authors: Departments of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors: Jean-Francois
authors: JF
authors: Pittet
authors: Departments of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Program in Protease and Matrix Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Birmingham VA Medical Center, University of Alabama at Birmingham, Birmingham, AL 35294, United States.
authors: Amit
authors: A
authors: Gaggar
authors: Departments of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States. Electronic address: rakeshpatel@uabmc.edu.
authors: Rakesh P
authors: RP
authors: Patel
mesh_terms: D000755:Anemia, Sickle Cell / Q000097:blood / Q000378:metabolism; D015415:Biomarkers; D055252:Cell-Derived Microparticles / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D055354:Exosomes / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010108:Oxyhemoglobins / Q000378:metabolism; D014947:Wounds and Injuries / Q000097:blood / Q000378:metabolism
chemical_list: D015415:Biomarkers; D006454:Hemoglobins; D010108:Oxyhemoglobins; D006418:Heme
keywords: Exosomes; Hemin; Microparticles