Simultaneous measurement of NAD metabolome in aged mice tissue using liquid chromatography tandem‐mass spectrometry

Abstract

Nicotinamide adenine dinucleotide (NAD) is a major co-factor that mediates multiple biological processes including redox reaction and gene expression. Recently, NAD metabolism has received considerable attention because administration of NAD precursors exhibited beneficial effects against aging-related metabolic disorders in animals. Although numerous studies have reported that NAD levels decline with aging in multiple animal tissues, the pathway and kinetics of NAD metabolism in aged organs are not completely understood. To determine the NAD metabolism upon aging, we developed targeted metabolomics based on an LC/MS/MS system. Our method is simple and applicable to crude biological samples, including culture cells and animal tissues. Unlike a conventional enzymatic cycling assay, our approach can determine NAD and NADH (reduced form of NAD) by performing a single sample preparation. Further, we validated our method using biological samples and investigated the alteration of the NAD metabolome during aging. Consistent with previous reports, the NAD levels in the liver and skeletal muscle decreased with aging. Further, we detected a significant increase in nicotinamide mononucleotide and nicotinamide riboside in the kidney upon aging. The LC/MS/MS-based NAD metabolomics that we have developed is extensively applicable to biomedical studies, and the results will present innovative ideas for the aging studies, especially for that of NAD metabolism.

NAD+ homeostasis in renal health and disease

“…Although little is known about the relationship between declining NAD + levels and CKD, it is plausible that augmentation of NAD + may also have a therapeutic role in slowing CKD progression. More work is needed to develop methods that can interrogate NAD + , NADH, NADP + , NADPH, their respective precursors and their intermediates in kidney tissue to understand how NAD + metabolism is impacted by ageing, both in experimental organisms and in humans 99 .…”

Implications of altered NAD metabolism in metabolic disorders

“…1 months old)Enzymatic[22]↓Male C57BL/6 N mice (24 months old v.s. 3 months old)LC/MS[19]Adipose tissue↓C57BL/6 mice (25–31 months old v.s. 3–6 months old)HPLC[8]↓Male C57BL/6 mice (32 months old v.s.…”

Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age‐associated insulin resistance

“…We also determined levels of the NAD precursor NAM in these mice and observed no changes due to HFD feeding in WT and Nmnat3 Tg mice (Supporting information Figure S1A). Because many studies have indicated that skeletal muscle NAD levels decline with aging (Frederick et al., 2016; Yaku, Okabe, & Nakagawa, 2018; Zhang et al., 2016), we determined NAD levels in WT and Nmnat3 Tg mice at the ages of 4, 18, and 24 months. Nmnat3 Tg mice had twofold higher NAD levels than WT mice at 3 months of age.…”

“…MS settings and chromatographic conditions were used as described previously (Yaku et al., 2018). TCA cycle intermediates were analyzed by selected ion monitoring (SIM) using Agilent 5977 MSD Single Quad mass spectrometer coupled to Agilent 7890 Gas Chromatography.…”