Spatiotemporal pharmacometabolomics based on ambient mass spectrometry imaging to evaluate the metabolism and hepatotoxicity of amiodarone in HepG2 spheroids

Three-dimensional(3D)cell spheroid models combined with mass spectrometry imaging(MSI)enables innovative investigation of in vivo-like biological processes under different physiological and pathological conditions.Herein,airflow-assisted desorption electrospray ionization-MSI(AFADESI-MSI)was coupled with 3D HepG2 spheroids to assess the metabolism and hepatotoxicity of amiodarone(AMI).High-coverage imaging of>1100 endogenous metabolites in hepatocyte spheroids was achieved using AFADESI-MSI.Following AMI treatment at different times,15 metabolites of AMI involved in Ndesethylation,hydroxylation,deiodination,and desaturation metabolic reactions were identified,and according to their spatiotemporal dynamics features,the metabolic pathways of AMI were proposed.Subsequently,the temporal and spatial changes in metabolic disturbance within spheroids caused by drug exposure were obtained via metabolomic analysis.The main dysregulated metabolic pathways included arachidonic acid and glycerophospholipid metabolism,providing considerable evidence for the mechanism of AMI hepatotoxicity.In addition,a biomarker group of eight fatty acids was selected that provided improved indication of cell viability and could characterize the hepatotoxicity of AMI.The combination of AFADESI-MSI and HepG2 spheroids can simultaneously obtain spatiotemporal information for drugs,drug metabolites,and endogenous metabolites after AMI treatment,providing an effective tool for in vitro drug hepatotoxicity evaluation.

Integrated mass spectrometry imaging reveals spatial-metabolic alteration in diabetic cardiomyopathy and the intervention effects of ferulic acid

Diabetic cardiomyopathy(DCM)is a metabolic disease and a leading cause of heart failure among people with diabetes.Mass spectrometry imaging(MSI)is a versatile technique capable of combining the molecular specificity of mass spectrometry(MS)with the spatial information of imaging.In this study,we used MSI to visualize metabolites in the rat heart with high spatial resolution and sensitivity.We optimized the air flow-assisted desorption electrospray ionization(AFADESI)-MSI platform to detect a wide range of metabolites,and then used matrix-assisted laser desorption ionization(MALDI)-MSI for increasing metabolic coverage and improving localization resolution.AFADESI-MSI detected 214 and 149 metabolites in positive and negative analyses of rat heart sections,respectively,while MALDI-MSI detected 61 metabolites in negative analysis.Our study revealed the heterogenous metabolic profile of the heart in a DCM model,with over 105 region-specific changes in the levels of a wide range of metabolite classes,including carbohydrates,amino acids,nucleotides,and their derivatives,fatty acids,glycerol phospholipids,carnitines,and metal ions.The repeated oral administration of ferulic acid during 20 weeks significantly improved most of the metabolic disorders in the DCM model.Our findings provide novel insights into the molecular mechanisms underlying DCM and the potential of ferulic acid as a therapeutic agent for treating this condition.

On-tissue chemical derivatization enables spatiotemporal heterogeneity visualization of oxylipins in esophageal cancer xenograft via ambient mass spectrometry imaging

On-tissue chemical derivatization(OTCD)effectively enhances ionization efficiency of low abundant and poorly ionized functional molecules to improve detection sensitivity and coverage of mass spectrometry imaging(MSI).Combination OTCD and MSI provides a novel strategy for visualizing previously undisclosed metabolic heterogeneity in tumor.Herein,we present a method to visualize heterogeneous metabolism of oxylipins within tumor by coupling OTCD with airflow-assisted desorption electrospray ionization(AFADESI)-MSI.Taking Girard’s P as a derivatization reagent,easily ionized hydrazide and quaternary amine groups were introduced into the structure of carbonyl metabolites via condensation reaction.Oxylipins,including 127 fatty aldehydes(FALs)and 71 oxo fatty acids(FAs),were detected and imaged in esophageal cancer xenograft with AFADESI-MSI after OTCD.Then t-distributed stochastic neighbor embedding and random forest were exploited to precisely locate the distribution of oxylipins in heterogeneous tumor tissue.With this method,we surprisingly found almost all FALs and oxo FAs significantly accumulated in the core region of tumor,and exhibited a gradual increase trend in tumor over time.These results reveal spatiotemporal heterogeneity of oxylipins in tumor progression,highlighting the value of OTCD combined with MSI to gain deeper insights into understanding tumor metabolism.

4-Nitrocatechol as a novel matrix for low-molecular-weight compounds in situ detection and imaging in biological tissues by MALDI-MSI

Low-molecular-weight(LMW)compounds are ubiquitous in living organisms and play essential roles in biological processes.The direct analysis of LMW compounds in biological tissues by matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI)could provide a more comprehensive understanding of their essential functions.Here,we evaluated 4-nitrocatechol(4-NC)as a novel positive-ion matrix for enhancing in situ detection and imaging of LMW compounds from the rat liver,brain,and germinating Chinese-yew seed by MALDI-MS.Our results showed that the 4-NC possessed remarkable features,including strong ultraviolet absorption,uniform matrix crystal,excellent chemical stability,and fewer matrix-related background peaks.The use of 4-NC led to the successful detection of 232,218,and193 LMW compounds from the three abovementioned tissue sections,respectively.Also,the use of 4-NC improved the imaging quality of LMW compounds in tissue sections through MALDI-MSI and has the potential as a matrix for MALDI tissue imaging of LMW compounds.

Spatial-resolved metabolomics reveals tissue-specific metabolic reprogramming in diabetic nephropathy by using mass spectrometry imaging

Detailed knowledge on tissue-specific metabolic reprogramming in diabetic nephropathy(DN)is vital for more accurate understanding the molecular pathological signature and developing novel therapeutic strategies.In the present study,a spatial-resolved metabolomics approach based on air flowassisted desorption electrospray ionization(AFADESI)and matrix-assisted laser desorption ionization(MALDI)integrated mass spectrometry imaging(MSI)was proposed to investigate tissue-specific metabolic alterations in the kidneys of high-fat diet-fed and streptozotocin(STZ)-treated DN rats and the therapeutic effect of astragalosideⅣ,a potential anti-diabetic drug,against DN.As a result,a wide range of functional metabolites including sugars,amino acids,nucleotides and their derivatives,fatty acids,phospholipids,sphingolipids,glycerides,carnitine and its derivatives,vitamins,peptides,and metal ions associated with DN were identified and their unique distribution patterns in the rat kidney were visualized with high chemical specificity and high spatial resolution.These region-specific metabolic disturbances were ameliorated by repeated oral administration of astragaloside Ⅳ(100 mg/kg)for 12 weeks.This study provided more comprehensive and detailed information about the tissue-specific metabolic reprogramming and molecular pathological signature in the kidney of diabetic rats.These findings highlighted the promising potential of AFADESI and MALDI integrated MSI based metabolomics approach for application in metabolic kidney diseases.

Spatiotemporally resolved metabolomics and isotope tracing reveal CNS drug targets

Deconvolution of potential drug targets of the central nervous system(CNS)is particularly challenging because of the complicated structure and function of the brain.Here,a spatiotemporally resolved metabolomics and isotope tracing strategy was proposed and demonstrated to be powerful for deconvoluting and localizing potential targets of CNS drugs by using ambient mass spectrometry imaging.This strategy can map various substances including exogenous drugs,isotopically labeled metabolites,and various types of endogenous metabolites in the brain tissue sections to illustrate their microregional distribution pattern in the brain and locate drug action-related metabolic nodes and pathways.The strategy revealed that the sedative-hypnotic drug candidate YZG-331 was prominently distributed in the pineal gland and entered the thalamus and hypothalamus in relatively small amounts,and can increase glutamate decarboxylase activity to elevateγ-aminobutyric acid(GABA)levels in the hypothalamus,agonize organic cation transporter 3 to release extracellular histamine into peripheral circulation.These findings emphasize the promising capability of spatiotemporally resolved metabolomics and isotope tracing to help elucidate the multiple targets and the mechanisms of action of CNS drugs.

Comprehensive chemical profiling and quantitative analysis of ethnic Yi medicine Miao-Fu-Zhi-Tong granules using UHPLC-MS/MS

Developing analytical methods for the chemical components of natural medicines remains a challenge due to its diversity and complexity.Miao-Fu-Zhi-Tong(MFZT)granules,an ethnic Yi herbal prescription,comprises 10 herbs and has been clinically applied for gouty arthritis(GA)therapy.Herein,a series of chemical profiling strategies including in-house library matching,molecular networking and MS/MS fragmentation behavior validation based on ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)were developed for qualitative analysis of MFZT granules.A total of 207 compounds were identified or characterized in which several rare guanidines were discovered and profiled into alkyl substituted or cyclic subtypes.Moreover,network pharmacology analysis indicated that MFZT’s anti-gout mechanism was mostly associated with the nuclear factor kappa-B(NF-κB)signaling,nucleotide oligomerization domain(NOD)-like signaling and rheumatoid arthritis pathways,along with the synergistic effect of 84 potential active compounds.In addition,a quantitative analytical method was developed to simultaneously determine the 29 potential effective components.Among them,berberine,pellodendrine,3-feruloylquinic acid,neoastilbin,isoacteoside and chlorogenic acid derivatives at higher concentrations were considered as the chemical markers for quality control.These findings provide a holistic chemical basis for MFZT granules and will support the development of effective analytical methods for the herbal formulas of natural medicines.