Alzheimer’s Disease (AD) currently affects more than 5 million Americans, with numbers expected to grow dramatically as the population ages. The pathophysiological changes in AD patients begin decades before the onset of dementia, highlighting the urgent need for the development of early diagnostic methods. Compelling data demonstrate that increased levels of amyloid-beta compromise multiple cellular pathways; thus, the investigation of changes in various cellular networks is essential to advance our understanding of early disease mechanisms and to identify novel therapeutic targets. We applied a liquid chromatography/mass spectrometry-based non-targeted metabolomics approach to determine global metabolic changes in plasma and cerebrospinal fluid (CSF) from the same individuals with different AD severity. Metabolic profiling detected a total of significantly altered 342 plasma and 351 CSF metabolites, of which 22% were identified. Based on the changes of >150 metabolites, we found 23 altered canonical pathways in plasma and 20 in CSF in mild cognitive impairment (MCI) vs. cognitively normal (CN) individuals with a false discovery rate <0.05. The number of affected pathways increased with disease severity in both fluids. Lysine metabolism in plasma and the Krebs cycle in CSF were significantly affected in MCI vs. CN. Cholesterol and sphingolipids transport was altered in both CSF and plasma of AD vs. CN. Other 30 canonical pathways significantly disturbed in MCI and AD patients included energy metabolism, Krebs cycle, mitochondrial function, neurotransmitter and amino acid metabolism, and lipid biosynthesis. Pathways in plasma that discriminated between all groups included polyamine, lysine, tryptophan metabolism, and aminoacyl-tRNA biosynthesis; and in CSF involved cortisone and prostaglandin 2 biosynthesis and metabolism. Our data suggest metabolomics could advance our understanding of the early disease mechanisms shared in progression from CN to MCI and to AD.

rates of FFA release under basal ( 5 ) and postprandial ( 6 ) conditions, are characteristic features of obesity. Plasma FFA concentrations are an imperfect indicator of FFA kinetics, as evidenced by the greater lipolysis rates in women than men at comparable FFA concentrations ( 7 ) and the divergence of FFA concentrations and fl ux during exercise ( 8, 9 ). Robust isotope dilution techniques have been developed to measure FFA kinetics, including radiotracer methods using high-performance liquid chromatography (HPLC) ( 10 ), gas chromatography-mass spectrometry (GC/MS) ( 11 ), and gas chromatography-combustionisotope ratio mass spectrometry (GC/C/IRMS) ( 12 ).Each of these methods requires an isolation and derivatization step followed by a relatively time-consuming chromatography separation, potentially limiting sample throughput. To improve effi ciency, we developed a new method for simultaneous measurement of concentration and stable isotopic enrichment of plasma FFA. Our method uses HPLC electrospray ionization (ESI) quadrupole mass spectrometry in the selected ion monitoring (SIM) mode. The method is simple and reliable for monitoring changes in plasma FFA concentration and enrichment. By using HPLC for the separation, we can avoid the derivatization step, allowing more rapid sample processing. In addition, by employing a tandem HPLC injection system, we are able to obtain sample data every 5 min. The results compare favorably with the GC/C/IRMS ( 12 ) approach for measuring palmitate fl ux using ultra-low tracer infusion rates. MATERIALS AND METHODS SuppliesPalmitic acid, sodium palmitate, sodium heptadecanoate, essentially fatty acid free albumin, potassium phosphate, potassium biphosphate, heptane, 2.0 M (trimethylsilyl)diazomethane solution, sulfuric acid, and ammonium acetate were purchased form Sigma-Aldrich Chemicals (St. Louis, MO). [ 13 C 16 ]palmitic acid was supplied by Cambridge Isotope Laboratories (Andover, MA). HPLC-grade acetonitrile, isooctane, isopropanol, methanol, and water were obtained from Burdick and Jackson Chemicals Abstract Measurements of plasma free fatty acids (FFA) concentration and isotopic enrichment are commonly used to evaluate FFA metabolism. Until now, gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/ IRMS) was the best method to measure isotopic enrichment in the methyl derivatives of 13 C-labeled fatty acids. Although IRMS is excellent for analyzing enrichment, it requires time-consuming derivatization steps and is not optimal for measuring FFA concentrations. We developed a new, rapid, and reliable method for simultaneous quantifi cation of 13 C-labeled fatty acids in plasma using high-performance liquid chromatography-mass spectrometry (HPLC/MS). This method involves a very quick Dole extraction procedure and direct injection of the samples on the HPLC system. After chromatographic separation, the samples are directed to the mass spectrometer for electrospray ionization (ESI) and analysis in the negative mode using single ion monitoring. By employi...

RATIONALE Sphingolipids are important components of cell membranes that serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. De novo ceramide biosynthesis depends on fatty acid availability, but whether muscle uses circulating free fatty acids or pre-existing intracellular stores is unknown. Our goal was to develop a method to detect the incorporation of intravenously infused [U-13C]palmitate into intramyocellular ceramides. METHODS We used a liquid chromatography/tandem mass spectrometry (LC/MS/MS) to measure the concentrations of different sphingolipids species and 13C isotopic enrichment of 16:0-ceramide. Chromatographic separation was performed using ultra performance liquid chromatography. The analysis was performed on triple quadrupole mass spectrometer using positive ion electrospray ionization source with selected reaction monitoring (SRM). RESULTS The sphingolipids ions, except enriched ceramide, were monitored as [M+2+H]+. The [13C16]16:0-ceramide was monitored as [M+16+H]+. By monitoring 2 different transitions of the [13C16]16:0-Ceramide (554/536 and 554/264) we could indirectly measure enrichment of the palmitate that is not a part of the sphingoid base. Concentration and enrichment could be measured using 20 mg of muscle obtained from volunteers receiving a low dose [U-13C]palmitate infusion. CONCLUSIONS LC/MS/MS can be used to detect the incorporation of plasma palmitate into muscle ceramides in humans, in vivo.

Insulin regulates many cellular processes, but the full impact of insulin deficiency on cellular functions remains to be defined. Applying a mass spectrometry–based nontargeted metabolomics approach, we report here alterations of 330 plasma metabolites representing 33 metabolic pathways during an 8-h insulin deprivation in type 1 diabetic individuals. These pathways included those known to be affected by insulin such as glucose, amino acid and lipid metabolism, Krebs cycle, and immune responses and those hitherto unknown to be altered including prostaglandin, arachidonic acid, leukotrienes, neurotransmitters, nucleotides, and anti-inflammatory responses. A significant concordance of metabolome and skeletal muscle transcriptome–based pathways supports an assumption that plasma metabolites are chemical fingerprints of cellular events. Although insulin treatment normalized plasma glucose and many other metabolites, there were 71 metabolites and 24 pathways that differed between nondiabetes and insulin-treated type 1 diabetes. Confirmation of many known pathways altered by insulin using a single blood test offers confidence in the current approach. Future research needs to be focused on newly discovered pathways affected by insulin deficiency and systemic insulin treatment to determine whether they contribute to the high morbidity and mortality in T1D despite insulin treatment.

Comprehensive metabolomic profiling identified extensive metabolomic abnormalities in T1D with poor glycemic control. Chronic good glycemic control failed to normalize many of these perturbations, suggesting a potential role for these persistent abnormalities in many complications in T1D.