LC-MS/MS assay for N1-methylnicotinamide in humans, an endogenous probe for renal transporters.

BACKGROUND: N1-methylnicotinamide (1-NMN) has been proposed as a potential clinical biomarker to assess drug-drug interactions involving organic cation transporters (OCT2) and multidrug and toxin extrusion protein transporters. RESULTS: A hydrophilic interaction liquid chromatography-MS/MS assay, to quantify 1-NMN, in human plasma and urine is reported. MATERIALS & METHODS: A hydrophilic interaction chromatography (HILIC)-tandem mass spectrometry (MS/MS) assay to quantify 1-NMN in human plasma and urine is reported. The basal 1-NMN levels in plasma and urine were 4-120 and 2000-15,000 ng/ml, respectively. CONCLUSION: 1-NMN plasma AUCs increased two- to fourfold versus placebo following the administration of a clinical candidate that in vitro experiments indicated was an OCT2 inhibitor. The described hydrophilic interaction liquid chromatography-MS/MS assay can be used to assess a clinical compound candidate for the inhibition of OCT2 and multidrug and toxin extrusion protein transporter in first-in-human studies.

Imaging mass spectrometry of frontal white matter lipid changes in human alcoholics.

BACKGROUND: Chronic alcohol use disorders (AUD) are associated with white matter (WM) degeneration with altered myelin integrity. Matrix assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) enables high throughput analysis of myelin lipid biochemical histopathology to help characterize disease mechanisms. PURPOSE: This study utilized MALDI-IMS to investigate frontal lobe WM myelin lipid abnormalities in AUD. METHODS: Standardized cores of formalin-fixed WM from Brodmann Area 4 (BA4) and BA8/9 of 20 postmortem AUD and 19 control adult human brains were embedded in carboxymethyl-cellulose, cryo-sectioned (8 µm), thaw-mounted onto indium tin oxide (ITO) -coated glass slides, and sublimed with 2,5-dihydroxybenzxoic acid (DHB) matrix. Lipids were imaged by MALDI-time of flight in the negative ionization mode. Data were visualized with FlexImaging software v4.0 and analyzed with ClinProTools v3.0. RESULTS: Principal component analysis (PCA) and data bar plots of MALDI-IMS data differentiated AUD from control WM. The dominant effect of AUD was to broadly reduce expression of sphingolipids (sulfatides and ceramides) and phospholipids. Data bar plots demonstrated overall similar responses to AUD in BA4 and BA8/9. However, differential regional effects of AUD on WM lipid profiles were manifested by non-overlapping expression or discordant responses to AUD for a subset of lipid ions. CONCLUSIONS: Human AUD is associated with substantial inhibition of frontal lobe WM lipid expression with regional variability in these effects. MALDI-IMS can be used to characterize the nature of AUD-associated lipid biochemical abnormalities for correlation with lifetime exposures and WM degeneration, altered gene expression, and responses to abstinence or treatment.

Ethanol-Induced White Matter Atrophy Is Associated with Impaired Expression of Aspartyl-Asparaginyl-ß-Hydroxylase (ASPH) and Notch Signaling in an Experimental Rat Model.

Alcohol-induced white matter (WM) degeneration is linked to cognitive-motor deficits and impairs insulin/insulin-like growth factor (IGF) and Notch networks regulating oligodendrocyte function. Ethanol downregulates Aspartyl-Asparaginyl-ß-Hydroxylase (ASPH) which drives Notch. These experiments determined if alcohol-related WM degeneration was linked to inhibition of ASPH and Notch. Adult Long Evans rats were fed for 3, 6 or 8 weeks with liquid diets containing 26% ethanol (caloric) and in the last two weeks prior to each endpoint they were binged with 2 g/kg ethanol, 3×/week. Controls were studied in parallel. Histological sections of the frontal lobe and cerebellar vermis were used for image analysis. Frontal WM proteins were used for Western blotting and duplex ELISAs. The ethanol exposures caused progressive reductions in frontal and cerebellar WM. Ethanol-mediated frontal WM atrophy was associated with reduced expression of ASPH, Jagged 1, HES-1, and HIF-1α. These findings link ethanol-induced WM atrophy to inhibition of ASPH expression and signaling through Notch networks, including HIF-1α.

Altered temporal lobe white matter lipid ion profiles in an experimental model of sporadic Alzheimer's disease.

BACKGROUND: White matter is an early and important yet under-evaluated target of Alzheimer's disease (AD). Metabolic impairments due to insulin and insulin-like growth factor resistance contribute to white matter degeneration because corresponding signal transduction pathways maintain oligodendrocyte function and survival. METHODS: This study utilized a model of sporadic AD in which adult Long Evans rats administered intracerebral streptozotocin (i.c. STZ) developed AD-type neurodegeneration. Temporal lobe white matter lipid ion profiles were characterized by matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). RESULTS: Although the lipid ion species expressed in the i.c. STZ and control groups were virtually identical, i.c. STZ mainly altered the abundances of various lipid ions. Correspondingly, the i.c. STZ group was distinguished from control by principal component analysis and data bar plots. i.c. STZ mainly reduced expression of lipid ions with low m/z's (less than 810) as well as the upper range m/z lipids (m/z 964-986), and increased expression of lipid ions with m/z's between 888 and 937. Phospholipids were mainly included among the clusters inhibited by i.c. STZ, while both sulfatides and phospholipids were increased by i.c. STZ. However, Chi-Square analysis demonstrated significant i.c. STZ-induced trend reductions in phospholipids and increases in sulfatides (P<0.00001). CONCLUSIONS: The i.c. STZ model of sporadic AD is associated with broad and sustained abnormalities in temporal lobe white matter lipids. The findings suggest that the i.c. STZ model could be used for pre-clinical studies to assess therapeutic measures for their ability to restore white matter integrity in AD.

Cigarette Smoke-Induced Alterations in Frontal White Matter Lipid Profiles Demonstrated by MALDI-Imaging Mass Spectrometry: Relevance to Alzheimer's Disease.

BACKGROUND: Meta-analysis has shown that smokers have significantly increased risks for Alzheimer's disease (AD), and neuroimaging studies showed that smoking alters white matter (WM) structural integrity. OBJECTIVE: Herein, we characterize the effects of cigarette smoke (CS) exposures and withdrawal on WM myelin lipid composition using matrix assisted laser desorption and ionization-imaging mass spectrometry (MALDI-IMS). METHODS: Young adult male A/J mice were exposed to air (8 weeks; A8), CS (4 or 8 weeks; CS4, CS8), or CS8 followed by 2 weeks recovery (CS8 + R). Frontal lobe WM was examined for indices of lipid and protein oxidation and lipid profile alterations by MALDI-IMS. Lipid ions were identified by MS/MS with the LIPID MAPS prediction tools database. Inter-group comparisons were made using principal component analysis and R-generated heatmap. RESULTS: CS increased lipid and protein adducts such that higher levels were present in CS8 compared with CS4 samples. CS8 + R reversed CS8 effects and normalized the levels of oxidative stress. MALDI-IMS demonstrated striking CS-associated alterations in WM lipid profiles characterized by either reductions or increases in phospholipids (phosphatidylinositol, phosphatidylserine, phosphatidylcholine, or phosphatidylethanolamine) and sphingolipids (sulfatides), and partial reversal of CS's inhibitory effects with recovery. The heatmap hierarchical dendrogram and PCA distinguished CS exposure, duration, and withdrawal effects on WM lipid profiles. CONCLUSION: CS-mediated WM degeneration is associated with lipid peroxidation, protein oxidative injury, and alterations in myelin lipid composition, including shifts in phospholipids and sphingolipids needed for membrane integrity, plasticity, and intracellular signaling. Future goals are to delineate WM abnormalities in AD using MALDI-IMS, and couple the findings with MRI-mass spectroscopy to improve in vivo diagnostics and early detection of brain biochemical responses to treatment.

Comparative Analysis of Lipid Extracts and Imaging Mass Spectrometry for Evaluating Cerebral White Matter Biochemical Pathology in an Experimental Second-Hand Cigarette Smoke Exposure Model.

BACKGROUND: White matter injury and degeneration are common features of developmental and aging-associated diseases, yet their pathobiological bases are poorly understood. However, recent advances in Matrix-Assisted Laser Desorption Ionization (MALDI) instruments and chemistry have provided critical tools for myelin-lipid analytical research. DESIGN: This study characterizes Cigarette Smoke (CS) exposure effects on frontal lobe lipid ion profiles in adult male A/J mice that had been exposed to air for 8 weeks (A8), CS for 4 (CS4) or 8 weeks (CS8), or CS8 followed by 2 weeks recovery (CS8+R). MALDI data acquired by analysis of lipid extracts plated onto a ground steel target (high through-put) were compared with Imaging Mass Spectrometry (IMS). RESULTS: MALDI-time-of-flight (TOF) detected 120 lipid ions with m/z's of 600 to 1300 (phospholipids and sulfatides) in samples plated onto the steel target or analyzed by IMS, but just 25 ions (18%) were detected by both methods. IMS more effectively detected ions in the highest m/z range, whereas the extracts had abundant middle-range m/z ions. The experimental groups were better discriminated by PCA and R-generated heat map hierarchical clustering of IMS data than lipid extract data. On the other hand, both methods clearly delineated the CS4, CS8 and CS8+R experimental groups from control. CONCLUSIONS: MALDI analysis of brain lipid extracts plated onto a ground steel target for high through-put studies, or imaged directly in tissue can be used to assess biochemical pathology of white matter neurodegeneration and responses to treatment.