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.
journal_name
Redox Bioljournal_title
Redox biologyauthors
Oh JY,Hamm J,Xu X,Genschmer K,Zhong M,Lebensburger J,Marques MB,Kerby JD,Pittet JF,Gaggar A,Patel RPdoi
10.1016/j.redox.2016.08.003subject
Has Abstractpub_date
2016-10-01 00:00:00pages
167-177issn
2213-2317pii
S2213-2317(16)30080-5journal_volume
9pub_type
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