General Methods
Nucleotides: AMP, ADP, ATP, ZMP, and cAMP
An HPLC method was developed for the separation and quantitation of AMP, ADP, ATP and ZMP in tissue samples. Separation is achieved within 30 minutes on a LC18-T chromatography column. Detection is carried out using a Waters 490 UV detector. cAMP is measured in a similar manner. These methods are unique and sensitive. Assays are carried out sporadically as the need arises.
Amino Acids and Gluconeogenic Assessment
The Water's Acutag method is used to measure amino acid profiles (20+ amino acid concentrations) and select amino acid profiles (branch chain or gluconeogenic amino acids). SA of leucine and alanine are determined using cation exchange columns. Specific activity of hepatic intermediates, including UDPG, UDPGal, UDPG-N-acetylgal UDPG-N-acetylglu and PEP (phosphoenolpyruvate) are likewise determined. The separation of glucose production into its glycogenolytic and gluconeogenic components in vivo is difficult. The latter technique allows a sensitive estimate of the two rates to be made following the analysis of small amounts of liver tissue taken during or at the end of an experiment by biopsy. While the number of samples assayed per year is small (~200) the technical effort required is significant. These assays are run sporadically in response to user need.
Catecholamines
Plasma norepinephrine and epinephrine are routinely measured by HPLC via electrochemical detection. Plasma is absorbed onto alumina at a ph of 8.6, eluted with dilute perchloric acid, and auto-injected onto a c18 reversed-phase column. An internal standard (dehydroxylbenzylamine/DHBA) is included with each extraction to monitor recovery and aid in quantitation. Results are quantitated through a chromatography data station. This service is widely used and provides a precise and sensitive estimate of catecholamine levels in biologic tissues and fluids.
Insulin, Glucagon, Leptin, Growth Hormone, C-peptide, Cortisol, IGF-1, Pancreatic Polypeptide, etc.
Hormones are assayed via RIA using a double antibody procedure. Radioimmunoassay allows for the detection of minute concentrations of biological (hormones) or pharmacological substances in blood or other fluid samples using antigen/antibody reactions. The method provides a very sensitive, inexpensive method of measurement. Final analysis is accomplished by quantifying the bound radioactive counts with a Packard Gamma counter connected to a computerized data reduction station. These assays are high volume and their major benefit is in accuracy and cost savings
| Human/Canine | Mouse |
|---|---|
| Basic Cytokines | Basic Cytokines |
| IL-1Β, IL-6, IL-8, TNFα | IL-1Β, IL-6, IL-8, TNFα |
| Endocrine Panel | Endocrine Panel |
| INS, LEP, GGN, Cpep, Active GLP-1 | INS, LEP, GGN, Cpep, Active GLP-1 |
| Cardiovascular Panels | Cardiovascular Panels |
| MPO, PAI-1, E-selectin, ICAM, VCAM | MMP-9, PAI-1, E-selectin, ICAM, VCAM |
| IL-1Β, IL-6, IL-8, TNFα, MCP-1, VEGF | |
| Human Adipokine | Mouse Adipokine |
| Adiponectin/Resisten, PAI-1 | IL-6, MCP-1, PAI-1, INS, LEP, Resistin |
| Adiponectin single-plex | |
| Other assays available, contact the core | |
Luminex Applications: cytokines (Il-6, TNF Alpha, MCP-1); adipokines (adiponectin and resistin); cardiac markers
These are multi-plexed assays using x-map technology via the Luminex100 system. The reactants (antibodies, oligonucleotides, substrates, etc.) in these assays are attached to the surfaces of tiny, fluorescent, microspheres. Each set of microspheres carry a unique biological reagent distinguishable by internal dye ratios. Identification of an analyte is based upon specific fluorescent emission spectra of the bead associated with the analyte. Two laser beams with high speed digital signal processors and computer algorithms distinguish which assay is being carried on each microsphere while quantifying the reaction based on fluorescent reporter signals. This instrumentation allows for the analysis of multiple analytes from a single 25µl aliquot of sample. The sensitivity for luminex detection however is, however, less than conventional RIA. The core personnel perform adiponectin and resistin assays using this technology. In addition, the Core provides access to the instrumentation for analysis of cytokines and other inflammatory markers. When such is the case the investigator is responsible for the purchase of the reagents, and all aspects of the analysis. The core provides training for use of the equipment and advice regarding quality control and data reduction. This is a new service that became available following the purchase of the necessary equipment. This technology allows us to measure adiponectin and resistin and to help investigators assay other molecules of interest when reliable RIAs are not available or sample volume is limited.
Lipids/Lipoproteins
A wide range of analytical measurements important for studies related to lipid metabolism are offered. These include total plasma levels of non-esterified fatty acids, triglycerides, HDL cholesterol, cholesterol, LDL cholesterol. In addition fatty acids can be separated into different lipids classes. Likewise tissue triglycerides, cholesterol and phospholipid analysis can be carried out. This is also a new service and we expect its use to grow significantly in the next few years.
Isoprostanes
A highly sensitive and specific method for the analysis of isoprostanes has been developed. The methodology is based primarily upon capillary column gas chromatography (GC)/negative ion chemical ionization (NICI)mass spectrometry (MS) or liquid chromatography (LC)/MS using stable isotope dilution techniques employing deuterated or [18O]-labeled internal standards. The assays offered are parent F2- 2-isoprostanes and the major urinary isoprostane metabolite (2,3-dinor-5,6,-dihydro-15-F2t-IsoP,F2-isoprostane-M).
