Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices
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...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2016-10-01
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| Series: | Redox Biology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2213231716300805 |
| _version_ | 1828161299232587776 |
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| author | Joo-Yeun Oh Jennifer Hamm Xin Xu Kristopher Genschmer Ming Zhong Jeffrey Lebensburger Marisa B. Marques Jeffrey D. Kerby Jean-Francois Pittet Amit Gaggar Rakesh P. Patel |
| author_facet | Joo-Yeun Oh Jennifer Hamm Xin Xu Kristopher Genschmer Ming Zhong Jeffrey Lebensburger Marisa B. Marques Jeffrey D. Kerby Jean-Francois Pittet Amit Gaggar Rakesh P. Patel |
| author_sort | Joo-Yeun Oh |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T00:36:25Z |
| format | Article |
| id | doaj.art-89cd8963160d4e71ba8c2e87f47c8c89 |
| institution | Directory Open Access Journal |
| issn | 2213-2317 |
| language | English |
| last_indexed | 2024-04-12T00:36:25Z |
| publishDate | 2016-10-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Redox Biology |
| spelling | doaj.art-89cd8963160d4e71ba8c2e87f47c8c892022-12-22T03:55:09ZengElsevierRedox Biology2213-23172016-10-019C16717710.1016/j.redox.2016.08.003Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matricesJoo-Yeun Oh0Jennifer Hamm1Xin Xu2Kristopher Genschmer3Ming Zhong4Jeffrey Lebensburger5Marisa B. Marques6Jeffrey D. Kerby7Jean-Francois Pittet8Amit Gaggar9Rakesh P. Patel10Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United StatesDepartments of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35294, United StatesDepartments of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United StatesDepartments of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United StatesDepartments of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United StatesDepartments of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35294, United StatesDepartments of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, United StatesDepartments of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, United StatesDepartments of Anesthesiology and Perioperative Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United StatesDepartments of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United StatesDepartments of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, United StatesCell-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.http://www.sciencedirect.com/science/article/pii/S2213231716300805ExosomesMicroparticlesHemin |
| spellingShingle | Joo-Yeun Oh Jennifer Hamm Xin Xu Kristopher Genschmer Ming Zhong Jeffrey Lebensburger Marisa B. Marques Jeffrey D. Kerby Jean-Francois Pittet Amit Gaggar Rakesh P. Patel Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices Redox Biology Exosomes Microparticles Hemin |
| title | Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices |
| title_full | Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices |
| title_fullStr | Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices |
| title_full_unstemmed | Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices |
| title_short | Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices |
| title_sort | absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices |
| topic | Exosomes Microparticles Hemin |
| url | http://www.sciencedirect.com/science/article/pii/S2213231716300805 |
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