Ishima Tamaki, Kimura Natsuka, Kobayashi Mizuki, Watanabe Chika, Jimbo Eriko F, Kobayashi Ryosuke, Horii Takuro, Hatada Izuho, Murayama Kei, Ohtake Akira, Nagai Ryozo, Osaka Hitoshi, Aizawa Kenichi
Division of Clinical Pharmacology, Department of Pharmacology, Jichi Medical University, Shimotsuke 329-0498, Japan.
Department of Pediatrics, Jichi Medical University, Shimotsuke 329-0498, Japan.
Biomolecules. 2024 Dec 31;15(1):38. doi: 10.3390/biom15010038.
Nicotinamide adenine dinucleotide (NAD) is a critical cofactor in mitochondrial energy production. The NADH/NAD ratio, reflecting the balance between NADH (reduced) and NADoxidized, is a key marker for the severity of mitochondrial diseases. We recently developed a streamlined LC-MS/MS method for the precise measurement of NADH and NAD. Utilizing this technique, we quantified NADH and NAD levels in fibroblasts derived from pediatric patients and in a Leigh syndrome mouse model in which mitochondrial respiratory chain complex I subunit is knocked out (KO). In patient-derived fibroblasts, NAD levels did not differ significantly from those of healthy controls ( = 0.79); however, NADH levels were significantly elevated ( = 0.04), indicating increased NADH reductive stress. This increase, observed despite comparable total NAD(H) levels between the groups, was attributed to elevated NADH levels. Similarly, in the mouse model, NADH levels were significantly increased in the KO group ( = 0.002), further suggesting that NADH elevation drives reductive stress. This precise method for NADH measurement is expected to outperform conventional assays, such as those for lactate, providing a simpler and more reliable means of assessing disease progression.
烟酰胺腺嘌呤二核苷酸（NAD）是线粒体能量产生中的关键辅助因子。NADH/NAD比率反映了NADH（还原型）和NAD（氧化型）之间的平衡，是线粒体疾病严重程度的关键标志物。我们最近开发了一种简化的液相色谱-串联质谱法（LC-MS/MS），用于精确测量NADH和NAD。利用这项技术，我们对来自儿科患者的成纤维细胞以及线粒体呼吸链复合体I亚基敲除（KO）的Leigh综合征小鼠模型中的NADH和NAD水平进行了定量。在患者来源的成纤维细胞中，NAD水平与健康对照组相比无显著差异（P = 0.79）；然而，NADH水平显著升高（P = 0.04），表明NADH还原应激增加。尽管两组之间总的NAD（H）水平相当，但观察到的这种增加归因于NADH水平升高。同样，在小鼠模型中，KO组的NADH水平显著增加（P = 0.002），进一步表明NADH升高驱动还原应激。这种精确测量NADH的方法有望优于传统检测方法，如乳酸检测方法，为评估疾病进展提供一种更简单、更可靠的手段。
