Chakkumpulakkal Puthan Veettil Thulya, Alves Diana, Vongsvivut Jitraporn, Sparrow Rosemary L, Wood Bayden R, Garnier Gil
Centre for Biospectroscopy, Monash University, Clayton, Victoria 3800, Australia.
Centre for Sustainable and Circular Technologies (CSCT), University of Bath, BA2 7AY, UK.
Analyst. 2023 Mar 27;148(7):1595-1602. doi: 10.1039/d2an02001g.
Oxidative treatment of human red blood cells (RBCs) prior to freeze-drying appears to stabilize the RBCs to withstand dried storage at room temperature. To better understand the effects of oxidation and freeze-drying/rehydration on RBC lipids and proteins, single-cell measurements were performed by synchrotron-based Fourier transform infrared (FTIR) microspectroscopy 'live-cell' (unfixed) analysis. Lipid and protein spectral data of -butyl hydroperoxide (TBHP)-oxidized RBCs (oxRBCs), FDoxRBCs and control (untreated) RBCs were compared using principal component analysis (PCA) and band integration ratios. The oxRBCs and FDoxRBCs samples had similar spectral profiles that were clearly different to control RBCs. Spectral changes in the CH stretching region of oxRBCs and FDoxRBCs indicated the presence of increased saturated and shorter-chain lipids, consistent with lipid peroxidation and stiffening of the RBC membrane compared to control RBCs. The PCA loadings plot for the fingerprint region of control RBCs corresponding to the α-helical structure of hemoglobin, shows that oxRBCs and FDoxRBCs have conformational changes in the protein secondary structure to β-pleated sheets and β-turns. Finally, the freeze-drying process did not appear to compound or induce additional changes. In this context, FDoxRBCs could become a stable source of reagent RBCs for pre-transfusion blood serology testing. The synchrotron FTIR microspectroscopic live-cell protocol provides a powerful analytical tool to characterize and contrast the effects of different treatments on RBC chemical composition at the single cell level.
在冻干之前对人红细胞（RBC）进行氧化处理似乎能使红细胞稳定，以承受室温下的干燥储存。为了更好地理解氧化以及冻干/复水对红细胞脂质和蛋白质的影响，通过基于同步加速器的傅里叶变换红外（FTIR）显微光谱“活细胞”（未固定）分析进行了单细胞测量。使用主成分分析（PCA）和谱带积分比，比较了叔丁基过氧化氢（TBHP）氧化的红细胞（oxRBC）、冻干氧化红细胞（FDoxRBC）和对照（未处理）红细胞的脂质和蛋白质光谱数据。oxRBC和FDoxRBC样品具有相似的光谱特征，明显不同于对照红细胞。oxRBC和FDoxRBC的CH伸缩振动区域的光谱变化表明，饱和脂质和短链脂质增加，这与脂质过氧化以及与对照红细胞相比红细胞膜变硬一致。对照红细胞指纹区域对应血红蛋白α螺旋结构的PCA载荷图显示，oxRBC和FDoxRBC在蛋白质二级结构上发生了构象变化，转变为β折叠片层和β转角。最后，冻干过程似乎不会加剧或引发其他变化。在这种情况下，FDoxRBC可能成为输血前血液血清学检测中试剂红细胞的稳定来源。同步加速器FTIR显微光谱活细胞实验方案提供了一个强大的分析工具，用于在单细胞水平上表征和对比不同处理对红细胞化学成分的影响。
