Miller Children's Hospital, Long Beach Memorial-University of California Irvine, Long Beach, CA, United States.
Department of Pediatrics, University of California, Irvine, CA, United States.
Nitric Oxide. 2018 Jan 30;72:41-45. doi: 10.1016/j.niox.2017.11.002. Epub 2017 Nov 9.
Assessment of nitric oxide (NO) dynamics in immune cells, commonly measured using NO surrogates such as inducible nitric oxide synthase (iNOS) rather than NO itself, has been effective in understanding pathophysiology across a wide range of diseases. Although the intracellular measurement of NO is now feasible, many technical issues remain unresolved. The principle aim of our study was to determine the effect of storage time of whole blood on nitric oxide (NO) level expression in leukocytes. This is important because immune cells remain chemically dynamic even after they are removed from the circulation, and the impact of storage time must be known to optimally quantify the effect of a disease or condition on NO dynamics in circulating leukocytes. We measured NO levels using the fluorescent probe, diaminofluorescein (DAF-2DA), and flow cytometry in monocytes, neutrophils, and natural killer cells from healthy subjects immediately after blood draw (Time 0) and 30, 60, and 120 min following the blood draw. There was no significant difference among the 4 study time points in NO (DAF-2) levels, though there was wide intra-subject variability at all time points. Using LPS stimulation, we compared iNOS (the more traditional surrogate marker of NO dynamics) with NO (by DAF-2) in natural killer cells and monocytes and, we found no difference in the response patterns. In summary, we did find that within a 2-hour interval from blood draw to sample processing, there was a remarkably wide intra-subject variability in expression of intracellular NO (DAF-2) in leukocytes of healthy individuals at baseline and over time. The mechanism(s) for these differences are not known but could clearly confound efforts to detect changes in NO metabolism in white blood cells. We speculate that rapid pulsatility of NO could explain the wide variability seen.
评估免疫细胞中的一氧化氮（NO）动态，通常使用诱导型一氧化氮合酶（iNOS）等 NO 替代物而不是 NO 本身来进行，这对于理解广泛疾病的病理生理学非常有效。尽管现在可以进行细胞内 NO 的测量，但仍有许多技术问题尚未解决。我们研究的主要目的是确定全血储存时间对白细胞中一氧化氮（NO）水平表达的影响。这一点很重要，因为即使免疫细胞从循环中被移除，它们仍然保持化学动态，并且必须了解储存时间的影响，以最佳量化疾病或状况对循环白细胞中 NO 动态的影响。我们使用荧光探针二氨基荧光素（DAF-2DA）和流式细胞术，在采血后立即（时间 0）以及采血后 30、60 和 120 分钟，测量来自健康受试者的单核细胞、中性粒细胞和自然杀伤细胞中的 NO 水平。尽管在所有时间点都存在广泛的个体内变异性，但在 4 个研究时间点之间，NO（DAF-2）水平没有显著差异。使用 LPS 刺激，我们比较了自然杀伤细胞和单核细胞中 iNOS（更传统的 NO 动态替代标志物）与 NO（通过 DAF-2），发现反应模式没有差异。总之，我们确实发现，在从采血到样本处理的 2 小时时间间隔内，健康个体白细胞内细胞内 NO（DAF-2）的表达存在显著的个体内变异性，且随时间变化。这些差异的机制尚不清楚，但可能会严重干扰对白细胞中 NO 代谢变化的检测。我们推测，NO 的快速脉动性可以解释所观察到的广泛变异性。
