Effect of hydroxyurea (HU) on gelatinization mechanism of type I collagen suspensions

ABSTRACT Mutant hemoglobins thermodynamic phase transitions (HbS, HbC, etc.) have been the causes of serious hemoglobinopathies that have been affecting mankind since the dawn of human race. HbS polymerization is the best known of these transitions and the original cause of sickle cell anemia. Hydroxyurea (HU, NH2CONHOH) is widely used as the main drug administered for the treatment of Sickle Cell Anemia (SCA). Several hypotheses have been raised on the effects of HU on SCA. The most widespread and accepted one points to the production of fetal hemoglobin by the bone marrow, hindering the polymerization of deoxygenated HbS. Moreover, recent studies have reported that the short term in vivo effects of the HU have not been elucidated. By ordinary techniques of Materials Science (scanning electron microscopy, viscoelasticity, viscosity measurements, DSC and weight loss) we assessed the anti-nucleating, depolymerizing, and antigelling direct effects of HU on suspensions and type I bovine collagen gels. Scanning electron microscopy (SEM) revealed that the addition of HU results in the disappearance of the percolating three-dimensional network typical of collagen gels. Vane Rheometry tests pointed to a reduction in the yield stress (σ0) by adding HU into collagen gels with a concentration of 2.2 % w/v. In the liquid phase (sol), suspensions increased viscosity over time but decreased with the addition of HU, highlighting the depolymerizing effect of this substance. The weight loss of the sols is also affect by the addition of HU probably related to the lowering of the interactions between collagen/water as HU is added. The DSC essays indicated changes in the Cp curves as HU is added to the gel, decreasing the structural relaxations commonly observed in those gels. As a result, HU showed an effect on the phase transition of the studied gel, strengthening the hypothesis of anti-nucleating and anti-gelling action thereof on physical, reversible gels, and possibly on physical, reversible gels of mutant hemoglobin (HbS).

摘要 突变血红蛋白的热力学相变（HbS、HbC等）是引发严重血红蛋白病的元凶，自人类诞生以来便长期困扰着全人类。其中HbS聚合是研究最为深入的一类相变，也是镰状细胞贫血（Sickle Cell Anemia, SCA）的根本致病原因。羟基脲（Hydroxyurea, HU, NH2CONHOH）目前被广泛用作镰状细胞贫血的主流治疗药物。学界已针对羟基脲治疗镰状细胞贫血的作用机制提出多种假说，其中最广为接受的观点认为，羟基脲可通过骨髓诱导胎儿血红蛋白生成，从而抑制脱氧HbS的聚合过程。此外，近期研究表明，羟基脲在体内的短期作用机制仍未被完全阐明。本研究借助材料科学常用技术（扫描电子显微镜、粘弹性测试、粘度测定、差示扫描量热法（Differential Scanning Calorimetry, DSC）以及重量损失分析），评估了羟基脲对悬浮液与I型牛源胶原蛋白凝胶的直接抗成核、解聚及抗凝胶化作用。扫描电子显微镜（Scanning Electron Microscopy, SEM）观测结果显示，添加羟基脲后，胶原蛋白凝胶典型的连通性三维网络结构消失。叶片流变测试结果表明，在浓度为2.2% w/v的胶原蛋白凝胶中添加羟基脲，可使其屈服应力（σ0）降低。在液相（溶胶，sol）中，悬浮液的粘度随时间推移逐渐升高，但添加羟基脲后粘度下降，直接印证了该物质的解聚作用。溶胶的重量损失同样受羟基脲添加的影响，这可能与羟基脲加入后胶原蛋白与水分子之间的相互作用减弱有关。差示扫描量热实验结果显示，向凝胶中添加羟基脲会改变其热容（Cp）曲线，降低这类凝胶中常见的结构弛豫现象。综上，羟基脲对本次研究的凝胶的相变过程产生了显著影响，进一步支持了其对物理可逆凝胶具有抗成核与抗凝胶化作用的假说，同时也为其对突变血红蛋白（HbS）物理可逆凝胶的类似作用提供了理论支撑。