Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City 14080, Mexico.
Emergency Department, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City 14080, Mexico.
Int J Mol Sci. 2021 Jul 22;22(15):7819. doi: 10.3390/ijms22157819.
High-density lipoproteins' (HDL) stability is a determinant of their residence times in plasma and consequently an important parameter that influences the beneficial properties of these lipoproteins. Since there are no accessible procedures for this purpose, here, we describe the methodological conditions to assess the stability of the HDL based on the redshift of the fluorescence spectrum of tryptophans contained in the structure of HDL-apolipoproteins during incubation with urea 8M. Along the HDL denaturation kinetics, the main variations of fluorescence were observed at the wavelengths of 330, 344, and 365 nm at room temperature. Therefore, HDL denaturation was estimated using the tryptophan (Trp)-ratio of fluorescence intensity (rfi) at such wavelengths. By setting 100% of the measurable denaturation at 26 h, HDL reached 50% after 8 h of incubation with urea. Then, for further analyses we determined the percentage of HDL denaturation at 8 h as an estimation of the stability of these lipoproteins. To explore the potential usefulness of this test, we analyzed the stability of HDL isolated from the plasma of 24 patients diagnosed with acute coronary syndrome (ACS). These HDL presented significantly higher percentages of denaturation (64.9% (58.7-78.4)) than HDLs of healthy individuals (23.3% (20.3-27.0)). These results indicate that HDL in ACS are less stable than in control subjects. Moreover, the percentage of denaturation of HDL correlated with body mass index and aspartate transaminase plasma activity. Furthermore, apo-I, HDL-cholesterol, HDL-triglycerides, and apo A-I-to-triglycerides ratio correlated with the percentage of HDL denaturation, suggesting that the lipoprotein composition is a main determinant of HDL stability. Finally, the percentage of HDL denaturation is the parameter that predicted the presence of ACS as determined by a machine learning procedure and logistic regression analysis. In conclusion, we established the methodological conditions to assess the stability of HDL by a fluorescence-based method that merits exploration in prospective studies for evaluating the coronary artery disease risk.
高密度脂蛋白（HDL）的稳定性是其在血浆中停留时间的决定因素，也是影响这些脂蛋白有益特性的一个重要参数。由于目前尚无可行的方法来评估其稳定性，在这里，我们描述了一种基于 HDL 载脂蛋白结构中色氨酸荧光光谱红移来评估 HDL 稳定性的方法学条件，该方法学条件是在孵育尿素 8M 的过程中获得的。在 HDL 变性动力学过程中，在室温下，在 330nm、344nm 和 365nm 处观察到荧光的主要变化。因此，通过在这些波长处使用色氨酸（Trp）荧光强度比（rfi）来估计 HDL 变性。将 26 小时时可测量的变性设定为 100%，则 HDL 在孵育尿素 8 小时后达到 50%。然后，为了进一步分析，我们将 8 小时时 HDL 的变性百分比作为这些脂蛋白稳定性的估计值。为了探索该测试的潜在用途，我们分析了从 24 名急性冠状动脉综合征（ACS）患者血浆中分离的 HDL 的稳定性。这些 HDL 的变性百分比明显高于健康个体（23.3%（20.3-27.0））（64.9%（58.7-78.4））。这些结果表明，ACS 患者的 HDL 不如对照个体稳定。此外，HDL 的变性百分比与体重指数和天冬氨酸转氨酶血浆活性相关。此外，apo-I、HDL-胆固醇、HDL-甘油三酯和 apo A-I 与甘油三酯的比值与 HDL 变性的百分比相关，表明脂蛋白组成是 HDL 稳定性的主要决定因素。最后，HDL 变性的百分比是通过机器学习程序和逻辑回归分析确定的 ACS 存在的预测参数。总之，我们通过荧光法建立了评估 HDL 稳定性的方法学条件，值得在前瞻性研究中进一步探索，以评估患冠状动脉疾病的风险。
