Nitrosamine impurities have provoked numerous global medicine recalls due to their possible presence during drug manufacturing or storage. Regarding formulation of nitrosamine impurities, a key risk involves reactions between nitrosating agents (nitrite) in excipients and vulnerable amines as impurities or degradants. Rapid detection across various sample types is essential to support pharmaceutical manufacturing. In this study, two methods were developed to detect nitrite in excipients and crucial secondary amines in active ingredient metformin hydrochloride at trace levels, respectively. The former method was developed based on the reaction of nitrite ions with 2,3-diaminonaphthalene to form 1-[H]-naphthotriazole (NAT), whereas the latter was based on amine tosylation. Mass spectrometric conditions were optimized using electrospray ionization in the positive mode. Multiple reaction monitoring transitions were determined at m/z 170 -> 115 for NAT, and m/z 200.1 -> 91 for dimethylamine (DMA) and 228.1 -> 91 for diethylamine (DEA). These methods were validated using selected eight excipients or metformin hydrochloride in terms of specificity, linearity, accuracy, precision, robustness, limit of quantification (LOQ), and limit of determination according to the ICH guidelines. The results of the validation were within the acceptable criteria. Applicability of the methods was evaluated using 170 pharmaceutical samples donated by industries. The nitrite content in the excipients ranged from

Sphingosine 1-phosphate (S1P) is a signaling lipid molecule involved in various cellular processes. It is important to develop a quantitative method for S1P to determine endogenous levels and to investigate its functions. As S1P is a tiny lipid component of most biological samples, highly sensitive analysis by LC-MS/MS is required. The main challenge in S1P analysis by chromatography is peak-broadening due to the presence of a polar phosphate and the fact that S1P is indeed a zwitterion itself. In this study, we used hydrogen fluoride (HF) to efficiently remove a phosphate and then analyzed the surrogate, sphingosine, as a sharp peak by LC-ESI-MS/MS. We optimized the dephosphorylation reaction in terms of temperature and reaction time. Multiple reaction monitoring (MRM) for a dephosphorylated form of S1P and C17-S1P as an internal standard at m/z transition 300.4 > 282.4 (quantification ion), 300.4 > 262.4 (qualification ion), 286.3 > 268.2 (internal standard) was conducted. This method was validated by essential parameters such as specificity, linearity, range, LOQ, LOD, accuracy, precision, and repeatability. To confirm this new method, we quantified S1P levels in various serum products (100.0~284.4 nM). In the sample pretreatment conditions for extracting S1P, the concern about potential sphingosine contamination in serum was negligible. The dephosphorylation efficiency by this method was about two-fold higher than that of alkaline phosphatase (APase). To apply the method in vivo, we analyzed S1P in plasma and kidney tissues obtained from a chronic kidney disease (CKD) mouse model. S1P levels were increased only in CKD kidney tissue but not in plasma. In conclusion, by applying the dephosphorylation step with HF, we established a new, sensitive LC-MS/MS quantitative method for S1P that can be applied to biological samples.

Levocetirizine is one of the widely used antihistamines and has the potential to form N-nitrosopiperazine (NPZ) during drug synthesis, manufacturing, or storage.

Breath volatile organic compound (VOC) analysis is a non-invasive tool for assessing health status; the compositional profile of these compounds in the breath of patients with chronic kidney disease is believed to change with decreasing renal function. We aimed to identify breath VOCs for recognizing patients with chronic kidney disease. Using thermal desorption–gas chromatography/mass spectrometry, untargeted analysis of breath markers was performed using breath samples of healthy controls (n = 18) versus non-dialysis (n = 21) and hemodialysis (n = 12) patients with chronic kidney disease in this cross-sectional study. A total of 303 VOCs alongside 12 clinical variables were used to determine the breath VOC profile. Metabolomic analysis revealed that age, systolic blood pressure, and fifty-eight breath VOCs differed significantly between the chronic kidney disease group (non-dialysis + hemodialysis) and healthy controls. Thirty-six VOCs and two clinical variables that showed significant associations with chronic kidney disease in the univariate analysis were further analyzed. Different spectra of breath volatile organic compounds between the control and chronic kidney disease groups were obtained. A multivariate model incorporating age, 2-methyl-pentane, and cyclohexanone showed high performance (accuracy, 86%) in identifying patients with chronic kidney disease with odds ratios of 0.18 (95% CI, 0.07–2.49, p = 0.013); 2.10 (0.94–2.24, p = 0.025); and 2.31 (0.88–2.64, p = 0.008), respectively. Hence, this study showed that renal dysfunction induces a characteristic profile of breath VOCs that can be used as non-invasive potential biomarkers in screening tests for CKD.

Supplementary Figure S1 from Activation of p38 Mitogen-Activated Protein Kinase Is Required for Death Receptor–Independent Caspase-8 Activation and Cell Death in Response to Sphingosine

Zidovudine is an antiretroviral agent prescribed for the prevention and treatment of human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS). It is typically recommended to be used in combination with other antiretroviral drugs. Zidovudine has the potential to generate Nnitrosodimethylamine (NDMA) in the presence of dimethylamine and nitrite salt under acidic reaction conditions during the drug manufacturing process. NDMA is a potent human carcinogen that may be detected in drug substances or drug products. An analytical method was developed to determine NDMA in pharmaceuticals including zidovudine using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS). The analysis involved reversed-phase chromatography on a Kinetex F5 column with a mobile phase comprising water-acetonitrile mixtures. The detection of positively charged ions was conducted using atmospheric pressure chemical ionization (APCI). The calibration curve demonstrated excellent linearity (r = 0.9997) across the range of 1-50 ng/mL with a highly sensitive limit of detection (LOD) at 0.3 ng/mL. The developed method underwent thorough validation for specificity, linearity, accuracy, precision, robustness, and system suitability. This sensitive and specific analytical method was applied for detecting NDMA in zidovudine drug substance and its formulation currently available in the market, indicating its suitability for drug quality management purposes.

This study presents a solvent-free headspace gas chromatography–mass spectrometry (SF-HS-GC/MS) method for robustly screening benzyl chloride, a mutagenic carcinogen, impurities in active pharmaceutical ingredients (APIs) and drug products. The SF-HS-GC/MS method simplifies analysis by eliminating solvent use, reducing matrix interference. Optimized headspace parameters include incubation temperature, time, and sample amount. Validation, aligned with Q2(R1) ICH guidelines and ICH M7 recommendations, covers selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, system suitability, and robustness. Employing a DB-5MS column (30 m × 0.25 mm, 0.25 µm) with solvent-free split injection, the method’s calibration curve (0.05–5 μg/g) exhibits a strong correlation (>0.9998). The LOQ was 0.1 μg/g, with precision (%CV) consistently <5% and accuracy within 95–105%. Furthermore, an investigation confirmed the absence of artefactual benzyl chloride formation in drug products under headspace conditions. The developed SF-HS-GC/MS method successfully screened benzyl chloride in cinnarizine drug substances and products.

This study aimed to evaluate skin health’s functional improvement, such as wrinkles, elasticity, moisture, and whitening, and safety following the consumption of “edible bird’s nest extract” for 12 weeks by women. This single-center, double-blinded, parallel-group, placebo-controlled study included women aged 40–60 years. Our primary purpose was to assess improvement in skin wrinkles, elasticity, and moisture after 12 weeks using an SV700, cutometer, and corneometer, respectively, compared to baseline measurements. Our secondary purpose was to evaluate skin wrinkle, elasticity, and moisture changes at 4 and 8 weeks from baseline using the aforementioned equipment, and measure transdermal water loss and melanin and erythema indexes using a tewameter and mexameter, respectively. Experts performed the visual evaluation of skin wrinkles at 4, 8, and 12 weeks from baseline. The participants were randomly allocated in a 1:1 ratio into the edible bird’s nest extract or the placebo group with 43 participants each, where they consumed 100 mg of the extract or placebo, respectively, daily for 12 weeks. The outcomes were measured at every visit. In this study, upon comparing changes in the skin elasticity value between the two intake groups at 12 weeks of ingestion, skin elasticity in the edible bird’s nest extract group decreased significantly compared with that in the placebo group. Adverse reactions were absent in both groups. In the case of laboratory test results, changes before and after the ingestion of the extract were within the normal range, thus indicating no clinically significant difference. The edible bird’s nest extract was effective in improving skin wrinkles. Moreover, it is beneficial for skin health and can be used as a skin nutritional supplement. Compared with the placebo, the edible bird’s nest extract was identified as safe. Clinical Trial Registration: https://cris.nih.go.kr/cris/search/detailSearch.do?search_lang=E&search_page=M&pageSize=10&page=undefined&seq=21007&status=5&seq_group=20330, identifier KCT0006558.

BACKGROUND AND OBJECTIVE:Colorectal cancer (CRC) is the fourth leading cause of cancer- related death globally, with a high incidence rate in economically fast-growing countries. Sphingosine- 1-phosphate (S1P) is a bioactive lipid mediator that plays critical roles in cancer cell proliferation, migration, and angiogenesis converted by the isoforms of sphingosine kinase (SK1 and SK2). SK1 is highly expressed in colorectal cancer; its inhibitors suppress the formation of S1P and increase ceramide levels having a pro-apoptotic function. RB005 is a selective SK1 inhibitor and a structural analog of PP2A activator FTY720. The purpose of this study is to test whether RB005, an SK1 inhibitor, can be used as an anticancer agent by inhibiting the growth of colon cancer cells.METHODS:We performed MTT and colony-forming assay using colon cancer cell lines HT29 and HCT116 cells to examine the cell toxicity effect of RB005. To determine whether apoptosis of RB005 in colon cancer cell line is due to SK1 inhibition or other mechanisms due to its structural similarity with FTY720, we conducted LC/MS, siRNA knockdown, and PP2A activity experiments.RESULTS:RB005 notably inhibited CRC cell growth and proliferation compared to PF543 and ABC294640 by inducing the mitochondria-mediated intrinsic apoptotic pathway. Apoptotic cell death is caused by increased mitochondrial permeability Initiated by the activation of pro-apoptotic protein BAX, increased ceramides, and activation of PP2A. Also, RB005 treatment in HT29 cells did not change the expression level of SK1, but strikingly decreased SK1 activity and S1P levels. All these events of cell death and apoptosis were less effective when SK1 was knocked down by siRNA.CONCLUSION:This result indicates that RB005 shows the in-vitro anti-CRC effect by inhibiting SK1 activity and PP2A activation, increasing proapoptotic ceramide levels following the activation of the intrinsic apoptotic pathway.

This study intends to develop an essential simultaneous analysis method for the stability study of the combination drugs hydrochlorothiazide, (S)-amlodipine besylate, and olmesartan medoxomil and identify degradation products. A stress test of the combination drug was performed under thermal/humidity conditions to identify decomposition products expected to be generated under long-term storage conditions. Six unidentified degradation products were generated from (S)-amlodipine besylate and olmesartan medoxomil, except for those identified. Unspecified degradation products were identified by degradation prediction software with LC-MS/MS, and genotoxicity was predicted by toxicity prediction software. In silico toxicity, six degradation products were expected to be nonmutagenic. This study describes the development of an analytical method to predict degradation products and determine whether the degradation products are genotoxic. This process will he helpful in further forced degradation studies to predict the toxicity of potential impurities.

Sphingosine-1-phosphate (S1P) is a unique lipid ligand binding to S1P receptors to transduce various cell survival or proliferation signals via small G proteins. S1P lyase (S1PL) is the specific enzyme that degrades S1P to phosphoethanolamine and (2E)-hexadecenal and therefore regulates S1P levels. S1PL also degrades dihydrosphingosine-1-phosphate (Sa1P), with a higher affinity to produce hexadecanal. Here, we developed a newly designed assay using a C17-Sa1P substrate that degrades into pentadecanal and phosphoethanolamine. For higher sensitivity in pentadecanal analysis, we developed a quantitative protocol as well as a 5,5-dimethyl cyclohexanedione (5,5-dimethyl CHD) derivatization method. The derivatization conditions were optimized for the reaction time, temperature, and concentrations of the 5,5-dimethyl CHD reagent, acetic acid, and ammonium acetate. The S1PL reaction in the cell lysate after spiking 20 µM of C17-Sa1P for 20 min was linear to the total protein concentrations of 50 µg. The S1PL levels (4 pmol/mg/min) were readily detected in this HPLC with fluorescence detection (λex = 366 nm, λem = 455 nm). The S1PL-catalyzed reaction was linear over 30 min and yielded a Km value of 2.68 μM for C17-Sa1P. This new method was validated to measure the S1PL activity of mouse embryonal carcinoma cell lines of the standard cell (F9-0), S1PL knockdown cells (F9-2), and S1PL-overexpressed cells (F9-4). Furthermore, we treated F9-4 cells with different S1PL inhibitors such as FTY720, 4-deoxypyridoxine (DOP), and the deletion of pyridoxal-5-phosphate (P5P), an essential cofactor for S1PL activity, and observed a significant decrease in pentadecanal relative to the untreated cells. In conclusion, we developed a highly sensitive S1PL assay using a C17-Sa1P substrate for pentadecanal quantification for application in the characterization of S1PL activity in vitro.

Hair loss by excessive stress from work and lifestyle changes has become a growing concern, particularly among young individuals. However, most drugs for alopecia impose a plethora of side effects. We have found the powerful impact of Malva verticillata seed extracts on alleviating hair loss. This study further isolated effective chemicals in M. verticillata seed extracts by liquid silica gel column chromatography. Under the screening for the growth rate (%) of human follicles dermal papilla cells (HFDPCs), we identified linoleic acid (LA) and oleic acid in n-hexane of M. verticillate (MH)2 fraction. LA treatment activated Wnt/β-catenin signaling and induced HFDPCs growth by increasing the expression of cell cycle proteins such as cyclin D1 and cyclin-dependent kinase 2. LA treatment also increased several growth factors, such as vascular endothelial growth factor, insulin-like growth factor-1, hepatocyte growth factor, and keratinocyte growth factor, in a dose-dependent manner. Besides, LA significantly inhibited Dickkopf-related protein expression (DKK-1), a primary alopecia signaling by dihydrotestosterone. Our findings suggest that LA treatment may alleviate a testosterone-induced signaling molecule and induces HFDPCs growth by activating Wnt/β-catenin signaling.

Four known germacranolide sesquiterpene lactones, 2α,5-epoxy-5,10-dihydroxy-6α-angeloy-loxy-9β-isobutyloxy-germacran-8α,12-olide (1), 2α,5-epoxy-5,10-dihydroxy-6α, 9β-diangeloy-loxy-germacran-8α,12-olide (2, divaricin B), 2α,5-epoxy-5,10-dihydroxy-6α-angeloyloxy-9β-(2-methylbutyloxy)-germacran-8α,12-olide (3) and 2α,5-epoxy-5,10-dihydroxy-6α-angeloyloxy-9β-(3-methylbutyloxy)-germacran-8α,12-olide (4), were isolated from the chloroform-soluble fraction of the whole plants ofCarpesium triste var.manshuricum. Their chemical structures were determined using spectroscopic methods, including 2D-NMR. All the isolates showed significant cytotoxicities (ED50 value: 4.3–16.8 μM) against five human tumor cell lines; A549, SK-OV-3, SK-MEL-2, XF498 and HCT15.

A solvent-free headspace gas chromatography-mass spectrometry (SF-HS-GC/MS) method was developed and validated for screening N-nitrosodimethylamine (NDMA) in various active pharmaceutical ingredients (APIs) and drug products. Experimental parameters such as incubation temperature, incubation time, and sample volume in solvent-free headspace conditions were optimized. The developed SF-HS-GC/MS method was validated in terms of linearity, limit of quantification (LOQ), precision, and accuracy. The results indicated excellent linearity from 5 to 500 ng g(-1) with correlation coefficients higher than 0.9999. The LOQ of this method was 5 ng g(-1) and matrix effects ranged from 0.97 to 1.11. The accuracy ranged from 92.77 to 106.54% and the precision RSDs were below 5.94%. No significant matrix effect was observed for any of the drug products. Also, artefactual NDMA formation in ranitidine, nizatidine, and metformin was investigated under HS conditions. Adjusted (mild) SF-HS conditions were suggested for precise quantification of NDMA in positive drug products by GC/MS. The present SF-HS-GC/MS method is a promising tool for the screening and determination of toxic NDMA in APIs and drug products.

Sphingosine-1-phosphate (S1P) synthesized by sphingosine kinase (SPHK) is a signaling molecule, involved in cell proliferation, growth, differentiation, and survival. Indeed, a sharp increase of S1P is linked to a pathological outcome with inflammation, cancer metastasis, or angiogenesis, etc. In this regard, SPHK/S1P axis regulation has been a specific issue in the anticancer strategy to turn accumulated sphingosine (SPN) into cytotoxic ceramides (Cers). For these purposes, there have been numerous chemicals synthesized for SPHK inhibition. In this study, we investigated the comparative efficiency of dansylated PF-543 (DPF-543) on the Cers synthesis along with PF-543. DPF-543 deserved attention in strong cytotoxicity, due to the cytotoxic Cers accumulation by ceramide synthase (CerSs). DPF-543 exhibited dual actions on Cers synthesis by enhancing serine palmitoyltransferase (SPT) activity, and by inhibiting SPHKs, which eventually induced an unusual environment with a high amount of 3-ketosphinganine and sphinganine (SPA). SPA in turn was consumed to synthesize Cers via de novo pathway. Interestingly, PF-543 increased only the SPN level, but not for SPA. In addition, DPF-543 mildly activates acid sphingomyelinase (aSMase), which contributes a partial increase in Cers. Collectively, a dansyl-modified DPF-543 relatively enhanced Cers accumulation via de novo pathway which was not observed in PF-543. Our results demonstrated that the structural modification on SPHK inhibitors is still an attractive anticancer strategy by regulating sphingolipid metabolism.

Chronic kidney disease (CKD) is a global health problem. Our study employed Raman spectroscopy for the first time to analyze potential biomarker such as tryptophan (Trp) on kidney failure and show the difference between normal and CKD groups. Adenine model of CKD characterized most of the physiological changes observed in human CKD. For that, 6-week-old Wistar male rats were assigned to the two groups: the control group, 0.5% carboxymethyl cellulose (CMC) was administered; the CKD group, adenine was administered to rats at a dose 600 mg/kg for 10 days. The Raman spectra were obtained in the spectral range of 100-2500 cm(-1). The excitation source was 532 nm, the laser power was similar to 40 mW, and the analyzed tissue area was 10.02 x 82.12 mu m(2). The results showed a significant reduction in the case of Raman peak intensities at 748, 1359, 1554, and 1636 cm(-1), which can be assigned as Trp. Moreover, it was shown that tyrosine at 1169 cm(-1) and phenylalanine at 1001 cm(-1) decreased in the CKD group. Trp and kynurenine (Kyn) levels in kidney tissue and plasma were analyzed by using the high-performance liquid chromatography (HPLC). The Trp concentrations in plasma and kidney tissue significantly decreased in CKD 7.4 mu g/ml and 0.1 mu g/mg, respectively. However, Kyn level was not a statistically significant change in kidney tissue. In this work, we showed that Raman spectroscopy usage in CKD model and Raman spectroscopy can be considered a promising technique for semi-quantitative analysis of potential biomarkers such as phenylalanine, tyrosine, and Trp of renal function because of the little or no need for sample preparation, and it is easily observing several biomarkers at the same time. The utilization of multivariate analysis method, such as partial least square analysis can be considered as a promising tool for the discrimination of control and CKD groups.

Chemosensitization of cancer cells with small molecules may improve the therapeutic index of antitumoral agents by making tumor cells sensitive to the drug regimen and thus overcome the treatment resistance and side effects of single therapy. Cell membrane lipid rafts are known to transduce various signaling events in cell proliferation. Sensitizing cancer cells may cause modulation of membrane lipid rafts which may potentially be used in improving anticancer drug response. Cedrol, a natural sesquiterpene alcohol, was used to treat human leukemia K562 and colon cancer HT-29 cell lines, and effects were observed. Cedrol decreased the cell viability by inducing apoptosis in both cell lines by activation of pro-apoptosis protein BID and inhibition of anti-apoptosis proteins Bcl-XL, Bcl-2, and XIAP. Cedrol activated the caspase-9-dependent mitochondrial intrinsic pathway of apoptosis. Furthermore, cedrol inhibited the levels of pAKT, pERK, and pmTOR proteins as well as nuclear and cytoplasmic levels of the p65 subunit of NF-κB. Cedrol caused redistribution of cholesterol and sphingomyelin contents from membrane lipid raft, which was confirmed by a combined additive effect with methyl-β-cyclodextrin (lipid raft-disrupting agent). Lipid raft destabilization by cedrol led to the increased production of ceramides and inhibition of membrane-bound NADPH oxidase 2 enzyme activity. Cholesterol/sphingomyelin-redistributing abilities of cedrol appear as a novel mechanism of growth inhibition of cancer cells. Cedrol can be classified as a natural lipid raft-disrupting agent with possibilities to be used in general studies involving membrane lipid raft modifications.

Illegal dietary supplements adulterated with phosphodiesterase type 5 inhibitors (PDE-5i) are increasingly widely distributed through internet markets and underground routes. For this reason, it demands development of reliable screening methods to determine a wide range of PDE-5i drugs in various types of dietary supplements. Herein, we developed a screening method using gas chromatography-mass spectrometry (GC-MS) for simultaneous detection of 53 PDE-5i drugs in supplements. Common formulations (such as capsule, powder, pill, and tablet) of supplements with complicated matrices were treated by simple liquid-liquid extraction and trimethylsilyl (TMS) derivatization. With the aid of TMS derivatization, 53 PDE-5i drugs could be successfully separated and detected within 15 min, using a short microbore GC column (15 m). Moreover, owing to enhanced detection sensitivity and selectivity of PDE-5i TMS derivatives, 0.5 mg of sample was sufficient to screen and confirm targeted PDE-5i drugs. In this study, specific common ions according to structural characteristics of PDE-5i drugs were found under the electron ionization (EI) of their TMS derivatives. These specific common fragments could reflect the common pharmacophores for 4 classes of PDE-5i drugs (sildenafil, other sildenafil, vardenafil, and tadalafil analogues). Based on characteristic EI fragment ions, extracted common ion chromatograms (ECICs) and discriminant analysis (DA) were effectively used for reliable screening and classification of various types of PDE-5i drugs. Specific ECICs and DA using characteristic EI fragments here will aid in identification of newly emerging PDE-5i counterfeits in supplements. This study will be helpful to supervise illegal adulteration of PDE-5i drugs in dietary supplements to protect public health and consumer safety.

An in vitro cell transformation assay (CTA) is useful for the detection of non-genotoxic carcinogens (NGTXCs); however, it does not provide information on their modes of action. In this study, to pursue a mechanism-based approach in the risk assessment of NGTXCs, we aimed to develop an integrated strategy comprising an in vitro Bhas 42 CTA and global DNA methylation analysis. For this purpose, 10 NGTXCs, which were also predicted to be negative through Derek/Sarah structure–activity relationship analysis, were first tested for transforming activity in Bhas 42 cells. Methylation profiles using reduced representation bisulfite sequencing were generated for seven NGTXCs that were positive in CTAs. In general, the differentially methylated regions (DMRs) within promoter regions showed slightly more bias toward hypermethylation than the DMRs across the whole genome. We also identified 13 genes associated with overlapping DMRs within the promoter regions in four NGTXCs, of which seven were hypermethylated and six were hypomethylated. Using ingenuity pathway analysis, the genes with DMRs at the CpG sites were found to be enriched in cancer-related categories, including “cell-to-cell signaling and interaction” as well as “cell death and survival”. Moreover, the networks related to “cell death and survival”, which were considered to be associated with carcinogenesis, were identified in six NGTXCs. These results suggest that epigenetic changes supporting cell transformation processes occur during non-genotoxic carcinogenesis. Taken together, our combined system can become an attractive component for an integrated approach for the testing and assessment of NGTXCs.

Declining tissue nicotinamide adenine dinucleotide (NAD) levels are linked to ageing and its associated diseases. However, the mechanism for this decline is unclear. Here, we show that pro-inflammatory M1-like macrophages, but not naive or M2 macrophages, accumulate in metabolic tissues, including visceral white adipose tissue and liver, during ageing and acute responses to inflammation. These M1-like macrophages express high levels of the NAD-consuming enzyme CD38 and have enhanced CD38-dependent NADase activity, thereby reducing tissue NAD levels. We also find that senescent cells progressively accumulate in visceral white adipose tissue and liver during ageing and that inflammatory cytokines secreted by senescent cells (the senescence-associated secretory phenotype, SASP) induce macrophages to proliferate and express CD38. These results uncover a new causal link among resident tissue macrophages, cellular senescence and tissue NAD decline during ageing and offer novel therapeutic opportunities to maintain NAD levels during ageing.