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Metabolic Assessment Core

Concept

We hypothesize that genetic and environmental changes that increase life expectancy will manifest in young adults as altered concentrations of certain small metabolites; and that, of such predictors of longevity, those that generalize across phyla are likely to relate also to human health.

The Metabolic Assessment Core ("the MAC") assists Project investigators and other scientists to define pathways and mechanisms that regulate aging rate and lifelong metabolic output in several species. It seeks to establish a “metabolic profile of longevity” that may be applied across taxa. This profile will comprise select assays, results of which in young adults are good predictors of future longevity. The MAC develops rational sample-preparation and archiving protocols compatible with such assays.

Methods

The MAC accepts from Project laboratories various cryospecimens: various mouse organs; whole Drosophila flies and pellets of Caenorhabditis elegans nematodes and Saccharomyces yeast. Compared to nucleic acids and proteins, concentrations of endogenous small molecules can change rapidly in response to uncontrolled variables; we strongly encourage Project scientists to be mindful of this during growth and treatment of these organisms and during harvests of specimens, and to annotate deviations from uniform procedure, such annotations to be included in the database. Our objectives demand that multiple assays be performed on a specimen; since it is not feasible to conduct many assays in parallel, our first task upon receipt of specimens is to render them homogeneous so random samples can be removed, however, using methods that preserve specimens from precipitous metabolic and degradative change. The Core is equipped to do this by computer-controlled lyophilization, preceded or followed by cryo-pulverization in a mortar, but recent evidence suggests that freeze drying does not halt degradation, e.g., of DNA, unless the specimen has been or subsequently is de-lipidized. The Core is equipped for and experienced with lipid extraction by the Folch procedure, but we have not yet coupled this with freeze-drying; presently, specimens needed for multiple assays are pulverized while frozen, in a mortar on dry ice. Powders are cryo-stored in gas-tight vials under pure nitrogen gas. Specimens intended for single procedures are either pulverized in this fashion, blade-homogenized as suspensions in chloroform or homogenized in cold aqueous suspensions using sample-tube pestles or a bead beater. Internal quantification standards are added early in homogenization.

Equipment

Analytical instruments at the MAC include an HP 5890 Series IIplus gas chromatograph (GC) with a Diplomat-interfaced [Horizon Instrument Group] AOC20i/s autoinjector [Shimadzu]. GC eluents enter the positive-ion electron-impact interface of an HP5792 quadrupole mass spectrometer (MS).

For liquid chromatography (LC), the MAC has an API3000 triple-quadrupole MS [Applied Biosystems/MDS-Sciex]. Its TurboIon electrospray ionization source can be infused by syringe pump (Harvard), but more typically receives the unsplit flow from a VP Series 10 LC system [Shimadzu] comprising a controller, four ultra-low-pulsation ADVP pumps and a chilled autosampler modified for low carryover and low flow. The facility also has two HP1050 HPLC systems with variable-wavelength and photodiode-array UV detectors [Agilent]. One is equipped for chilled sampling and is used with a CHI812B bipotentiostat nanoammeter [CH Instruments] for both DC and pulsed electrochemical detection and cyclic voltammetry. For respirometry, the MAC has an Oxygraph 2K oxygen meter [Oroboros] to measure mitochondrial and suspension-culture dissolved oxygen consumption and a TR-1 Respirometer [Sable Systems] to measure both oxygen consumption and carbon dioxide production by Drosophila fruit flies, Caenorabditis nematodes and mice.

For specimen and sample handling and processing, the laboratory is equipped with an Advantage EL freeze dryer [VirTis], a Model 1025 anaerobic system [Forma] plumbed with high purity nitrogen and containing an MX5 analytical microbalance [Mettler], a Rotavapor rotary evaporator [Buchi], a nitrogen gas blow-down manifold [Organomation], a positive-pressure solid-phase extraction manifold [Pierce], a multi-tube pulsatile vortexer [LabTech] and a Reacti-Manifold [Pierce] for oxygen- and moisture-sensitive derivatization reactions and evaporations.

Software

Software for GC-MS Metabolomics

Assays Currently Running

Thiol Panel (GSH, GSSG, cysteine, cystine, cysteine-glycine, homocysteine and homocystine)

Energy Panel (ATP, ADP, AMP, NADP/H, NAD/H)

Lipid Peroxidation Panel (F2-8-isoprostanes, six distinct HETEs and two HODEs)

Metabolomics assays (broad-capture metabolite scans) GC-MS mass chromatographic features have been shown by permutation-based significance analysis (SAM) to correlate with strain life expectancy in Caenorhabditis elegans and with coat color class in Project 4 mouse liver. LC-LC-MS metabolomics is under development.

Team

John J. Thaden, Ph.D., Core Director

Lulu Xu, Ph.D., Chemist

William Starrett, M.T., Research Technologist

Last updated: Monday 17 July 2006 06:00.00 (CDT)