Short Audrey H, Al Aayedi Nazar, Gaire Madhu, Kreider Max, Wong Chong Kai, Urayama Paul
Department of Physics, Miami University Oxford OH 45056 USA
RSC Adv. 2021 May 24;11(31):18757-18767. doi: 10.1039/d1ra02648h.
NADPH and NADH are well known for their role in antioxidant defense and energy metabolism, respectively, however distinguishing their cellular autofluorescence signals is a challenge due to their nearly identical optical properties. Recent studies applying spectral phasor analysis to autofluorescence emission during chemically induced metabolic responses showed that two-component spectral behavior, , spectral change acting as a superposition of two spectra, depended on whether one or multiple metabolic pathways were affected. Here, we use this property of spectral behavior to show that metabolic responses primarily involving NADPH or NADH can be distinguished. We start by observing that the cyanide-induced response at micro- and millimolar concentrations does not follow mutual two-component spectral behavior, suggesting their response mechanisms differ. While respiratory inhibition at millimolar cyanide concentration is well known and associated with the NADH pool, we find the autofluorescence response at micromolar cyanide concentration exhibits two-component spectral behavior with NADPH-linked EGCG- and peroxide-induced responses, suggesting an association with the NADPH pool. What emerges is a spectral phasor map useful for distinguishing cellular autofluorescence responses related to oxidative stress cellular respiration.
NADPH和NADH分别因其在抗氧化防御和能量代谢中的作用而广为人知，然而，由于它们几乎相同的光学特性，区分它们的细胞自发荧光信号是一项挑战。最近的研究将光谱相量分析应用于化学诱导的代谢反应过程中的自发荧光发射，结果表明，双组分光谱行为，即光谱变化表现为两个光谱的叠加，取决于一条还是多条代谢途径受到影响。在这里，我们利用这种光谱行为特性来表明，主要涉及NADPH或NADH的代谢反应是可以区分的。我们首先观察到，微摩尔和毫摩尔浓度的氰化物诱导的反应并不遵循相互的双组分光谱行为，这表明它们的反应机制不同。虽然毫摩尔浓度的氰化物会引起呼吸抑制，这是众所周知的，且与NADH池有关，但我们发现微摩尔浓度的氰化物诱导的自发荧光反应与NADPH相关的表没食子儿茶素没食子酸酯（EGCG）和过氧化物诱导的反应表现出双组分光谱行为，这表明与NADPH池有关。由此产生了一个光谱相量图，可用于区分与氧化应激和细胞呼吸相关的细胞自发荧光反应。
