Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices.
Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices.
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DOI：
10.1016/j.redox.2016.08.003
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发表时间：
2016-10
期刊：
影响因子：
11.4
通讯作者：
Patel, Rakesh P.
中科院分区：
文献类型：
--
作者：
Oh, Joo-Yeun;Hamm, Jennifer;Xu, Xin;Genschmer, Kristopher;Zhong, Ming;Lebensburger, Jeffrey;Marques, Marisa B.;Kerby, Jeffrey D.;Pittet, Jean-Francois;Gaggar, Amit;Patel, Rakesh P.
无细胞血红素(CFH)和血红蛋白(Hb)已经成为急性损伤的不同介质，其特征是溶血性条件下的炎症和微循环功能障碍。一些报告显示，在特定的病理生理环境中，Hb和CFH发生了变化。使用PBS、镰状细胞病患者的血浆、急性呼吸窘迫综合征(ARDS)患者的血浆和红细胞单位的培养上清，我们发现常用的检测方法和商用试剂盒不能区分CFH和HB。此外，他们还受到各种假阳性干扰和限制(包括胆红素)，导致对CFH和/或Hb的高估或低估。此外，常用的根据分子量(MWt)分离CFH和Hb的方法由于CFH的疏水性而效率低下。在这项研究中，我们开发并验证了一种基于吸收光谱去卷积和最小二乘拟合分析的新方法，该方法克服了这些限制，并同时测量了简单的含水缓冲液、血浆或与红细胞来源的微囊相关的CFH和Hb。我们展示了如何结合吸收可见光波长范围内的光的其他等离子体因素(特别是胆红素)，再加上截断所分析的波长范围，或者添加温和的洗涤剂来显著改善匹配，从而允许以90%的准确度测量氧合Hb、CFH和metHb。当这种方法应用于SCD患者的样本时，我们观察到CFH水平高于先前报道的水平，与Hb的水平相似。在多种疾病中，血红素和血红蛋白是氧化损伤的氧化还原活性介质。强调了当前方法对游离血红蛋白和血红素测量的局限性。提出了一种基于光谱去卷积同时测定游离血红蛋白和血红素的新方法。提高了对储存的红细胞中的血红素测量的准确性，并显示了比报道的镰刀细胞病更高的水平。
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. Heme and hemoglobin are redox active mediators of oxidative damage in multiple diseases. Limitations with current methods for free hemoglobin and heme measures are highlighted. New protocols for measuring free hemoglobin and heme simultaneously based on spectral deconvolution are provided. Improved accuracy for heme measures in stored red cells and levels higher than reported in Sickle cell disease is shown.
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影响因子：
6.6
作者：
Cooper, Chris E.;Schaer, Dominik J.;Alayash, Abdu I.
通讯作者：
Alayash, Abdu I.
DOI：
10.1186/cc11425
发表时间：
2012-07-16
期刊：
Critical care (London, England)
影响因子：
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作者：
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DOI：
10.4049/jimmunol.1103623
发表时间：
2012-08-15
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
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作者：
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影响因子：
8.8
作者：
Janz DR;Bastarache JA;Peterson JF;Sills G;Wickersham N;May AK;Roberts LJ 2nd;Ware LB
通讯作者：
Ware LB
影响因子：
5.6
作者：
Chiabrando D;Vinchi F;Fiorito V;Mercurio S;Tolosano E
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Tolosano E