Trammell Samuel Aj, Brenner Charles
Department of Biochemistry ; Interdisciplinary Graduate Program in Genetics Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
Comput Struct Biotechnol J. 2013 May 27;4:e201301012. doi: 10.5936/csbj.201301012. eCollection 2013.
Nicotinamide adenine dinucleotide (NAD(+)) is a coenzyme for hydride transfer reactions and a substrate for sirtuins and other NAD(+)-consuming enzymes. The abundance of NAD (+), NAD(+) biosynthetic intermediates, and related nucleotides reflects the metabolic state of cells and tissues. High performance liquid chromatography (HPLC) followed by ultraviolet-visible (UV-Vis) spectroscopic analysis of NAD(+) metabolites does not offer the specificity and sensitivity necessary for robust quantification of complex samples. Thus, we developed a targeted, quantitative assay of the NAD(+) metabolome with the use of HPLC coupled to mass spectrometry. Here we discuss NAD(+) metabolism as well as the technical challenges required for reliable quantification of the NAD(+) metabolites. The new method incorporates new separations and improves upon a previously published method that suffered from the problem of ionization suppression for particular compounds.
烟酰胺腺嘌呤二核苷酸（NAD(+)）是氢化物转移反应的辅酶，也是去乙酰化酶和其他消耗NAD(+)的酶的底物。NAD(+)、NAD(+)生物合成中间体及相关核苷酸的丰度反映了细胞和组织的代谢状态。对NAD(+)代谢物进行高效液相色谱（HPLC）分析，随后进行紫外可见（UV-Vis）光谱分析，无法为复杂样品的可靠定量提供所需的特异性和灵敏度。因此，我们开发了一种使用HPLC与质谱联用的靶向定量分析NAD(+)代谢组的方法。在此，我们讨论NAD(+)代谢以及可靠定量NAD(+)代谢物所需的技术挑战。新方法采用了新的分离技术，并改进了先前发表的方法，该方法存在特定化合物电离抑制的问题。
