烟酰胺腺嘌呤二核苷酸(NAD+),500多种酶的辅酶,在能源生产中起着核心作用,新陈代谢,细胞信号,DNA修复直到最近,NAD+主要被认为是细胞内分子(iNAD+),然而,其细胞外物种(eNAD+)最近已被发现,并且此后已与多种病理状况相关联。因此,准确定量血浆等体液中的eNAD+对于回答重要的研究问题至关重要。为了创造一种有临床意义和可靠的定量方法,我们分析了细胞裂解的关系,常规临床实验室参数,采血技术,和具有测量的血浆eNAD+浓度的预分析处理步骤。最初,在细胞内和细胞外评估NAD+水平。有趣的是,发现血浆中eNAD+的浓度比外周血单核细胞中的iNAD+低约500倍(0.253±0.02μMvs.131.8±27.4μM,分别为p=0.007)。这种鲜明的对比表明细胞损伤或细胞裂解可能潜在地影响血浆中的eNAD+水平。然而,患者血浆中的全身性乳酸脱氢酶,细胞损伤的标志,与eNAD+无显著相关性(n=33;r=-0.397;p=0.102)。此外,eNAD+与C反应蛋白升高呈负相关(CRP,n=33;r=-0.451;p=0.020),而eNAD+与血红蛋白升高呈正相关(n=33;r=0.482;p=0.005)。接下来,抽血的变化,检查了样品处理和分析前过程。样品在4°C(0-120分钟)下的存储时间,温度(0°C至25°C),用于采血的套管大小和止血带时间(0-120s)对eNAD+无统计学意义(p>0.05).另一方面,长时间离心(>5分钟)和离心转子的更快制动模式(<4分钟)导致eNAD水平显着降低(p<0.05)。一起来看,CRP和血红蛋白似乎与eNAD+水平轻度相关,而细胞损伤与eNAD+水平没有显著相关。抽血试验未显示对eNAD+有任何影响,相比之下,分析前的步骤需要标准化以进行准确的eNAD+测量。这项工作为健壮的eNAD+测量铺平了道路,用于未来的临床和转化研究,并为血浆中可靠的eNAD+定量提供优化的实践方案。
Nicotinamide adenine dinucleotide (NAD+), a coenzyme for more than 500 enzymes, plays a central role in energy production, metabolism, cellular signaling, and DNA repair. Until recently, NAD+ was primarily considered to be an intracellular molecule (iNAD+), however, its
extracellular species (eNAD+) has recently been discovered and has since been associated with a multitude of pathological conditions. Therefore, accurate quantification of eNAD+ in bodily fluids such as plasma is paramount to answer important research questions. In order to create a clinically meaningful and reliable quantitation method, we analyzed the relationship of cell lysis, routine clinical laboratory parameters, blood collection techniques, and pre-analytical processing steps with measured plasma eNAD+ concentrations. Initially, NAD+ levels were assessed both intracellularly and extracellularly. Intriguingly, the concentration of eNAD+ in plasma was found to be approximately 500 times lower than iNAD+ in peripheral blood mononuclear cells (0.253 ± 0.02 μM vs. 131.8 ± 27.4 μM, p = 0.007, respectively). This stark contrast suggests that cellular damage or cell lysis could potentially affect the levels of eNAD+ in plasma. However, systemic lactate dehydrogenase in patient plasma, a marker of cell damage, did not significantly correlate with eNAD+ (n = 33; r = -0.397; p = 0.102). Furthermore, eNAD+ was negatively correlated with increasing c-reactive protein (CRP, n = 33; r = -0.451; p = 0.020), while eNAD+ was positively correlated with increasing hemoglobin (n = 33; r = 0.482; p = 0.005). Next, variations in blood drawing, sample handling and pre-analytical processes were examined. Sample storage durations at 4°C (0-120 min), temperature (0° to 25°C), cannula sizes for blood collection and tourniquet times (0 - 120 s) had no statistically significant effect on eNAD+ (p > 0.05). On the other hand, prolonged centrifugation (> 5 min) and a faster braking mode of the centrifuge rotor (< 4 min) resulted in a significant decrease in eNAD+ levels (p < 0.05). Taken together, CRP and hemoglobin appeared to be mildly correlated with eNAD+ levels whereas cell damage was not correlated significantly to eNAD+ levels. The blood drawing trial did not show any influence on eNAD+, in contrast, the preanalytical steps need to be standardized for accurate eNAD+ measurement. This work paves the way towards robust eNAD+ measurements, for use in future clinical and translational research, and provides an optimized hands-on protocol for reliable eNAD+ quantification in plasma.