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Authors: Mari Golub
Group: Mouse Metabolic Phenotyping Centers
Summary: The radial arm water maze (RAWM) measures spatial learning by combining both a water maze and radial arm maze (RAM). This test measures both working and reference memory through a reward system, in this case escape from water. This method is advantageous to other RAM which use food as a reward in that it may be utilized with mice on specialized diets. Reference: Behavioral consequences of ovarian atrophy and estrogen replacement in the APPsswe mouse. Golub et al. Neurobiol Aging (2008) 29(10): 1512-1523.

Proper citation: Mari Golub 2019. UC Davis - Radial Arm Water Maze. protocols.io https://dx.doi.org/10.17504/protocols.io.yv2fw8e Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Aspartate transaminase (AST) also known as aspartate aminotransferase or (sGOT) is a metabolic enzyme expressed primarily in the liver. Elevation of AST levels is an indication of liver damage and has been associated with liver injury. AST levels are monitored routinely in patients with liver diseases. AST is also a very useful tool for preclinical investigation of experimental drug formulations and AST levels are commonly used to monitor and attenuate the hepatotoxic effects of experimental drugs in rodents.

Proper citation: Peter Havel 2019. UC Davis - Aspartate Aminotransferase. protocols.io https://dx.doi.org/10.17504/protocols.io.ybvfsn6 Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Triglyceride levels may be measured in serum, plasma, and tissue samples. Serum and tissue triglyceride levels are affected by alterations in systemic lipid metabolism, lipid digestion/absorption, and lipid clearance. Serum and tissue triglyceride levels are altered in obesity, insulin resistance, type 2 diabetes, alcoholic steatohepatitis, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis.

Proper citation: Jason Kim 2019. U Mass - Triglyceride. protocols.io https://dx.doi.org/10.17504/protocols.io.x5pfq5n Copy   

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Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of HDL cholesterol in blood, serum, and plasma. HDL Cholesterol is determined in a two-step procedure. First chylomicrons, VLDL, and LDL are selectively reacted with cholesterol esterase and eliminated from the reaction. In the second step, the remaining HDL-cholesterol is assayed as described for total cholesterol.

Proper citation: John Stack, Gary Cline 2019. Yale - HDL Cholesterol. protocols.io https://dx.doi.org/10.17504/protocols.io.y3ufynw Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer.

Proper citation: Jason Kim 2019. U Mass - Amylase. protocols.io https://dx.doi.org/10.17504/protocols.io.xq4fmyw Copy   

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Authors: Jeff Hodgin, Jharna Saha
Group: Mouse Metabolic Phenotyping Centers
Summary: This protocol is used to stain and count podocyte nuclei in mouse glomeruli for diabetic nephropathy study.

Proper citation: Jeff Hodgin, Jharna Saha 2019. U Michigan - Podocyte Counting And Density Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.565g9g6 Copy   

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Authors: Trina Knotts
Group: Mouse Metabolic Phenotyping Centers, Microbiome Centers
Summary: The work of Gordon and colleagues (i.e., Nature. 2006 Dec 21;444(7122):1027-31) has shown that obesity can result in marked shifts in the gut microbiome in mice and other models including humans. While the role of the microbiome remains to be fully elucidated, the gut microbiota can no longer be ignored as a potentially important factor when assessing metabolic phenotype. This service involves 16S gene variable region (V3-V4 using primer pair 515F-806R) pair end sequencing (2x300bp) by Illumina (20,000 avg seq read depth) of feces, cecal, or other GI contents. Sequences can be processed through a bioinformatics pipeline (Qiime) to taxonomically classify them and to assess alpha and beta diversity of the community. In addition, Principal Components Analysis (PCA) or partial least squares- discriminant analysis (PLS-DA) can leverage variances in the relative microbial abundances to better understand how specific microbes contribute to separation by group. Correlational analyses can identify which variables of host metadata associate with specific microbes. The Core’s gut microbiome assay will employ this approach to uncover unique microbiota fingerprints in test mice. One caveat is that with fecal samples, patterns are only a surrogate for actual gut microbiota patterns, and may not exactly reflect the intestinal populations.

Proper citation: Trina Knotts 2019. UC Davis - Gut Microbiome Analysis (Illumina-20K reads, 2X300bp). protocols.io https://dx.doi.org/10.17504/protocols.io.ynpfvdn Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Triglycerides are enzymatically hydrolyzed by lipase to free fatty acids and glycerol. The glycerol is phosphorylated by adenosine triphosphate (ATP) with glycerol kinase (GK) to produce glycerol-3- phosphate and adenosine diphosphate. Glycerol-3-phosphate is oxidized by dihydroxyacetone phosphate (DAP) by glycerolphosphate oxidase producing hydrogen peroxide (H2O2). In a Trinder5 type color reaction catalyzed by peroxidase, the H2O2 reacts with 4-aminoantipyrine (4-AAP) and 3,5-dichloro-2- hydroxybenzene sulfonate (DHBS) to produce a red colored dye. The absorbance of this dye is proportional to the concentration of triglycerides present in the sample.

Proper citation: Peter Havel 2019. UC Davis - Triglyceride Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yw3fxgn Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum uric acid levels are affected by alterations in systemic protein and nitrogen metabolism. Serum uric acid levels are altered in kidney failure and renal complications of diabetes.

Proper citation: Jason Kim 2019. U Mass - Uric Acid. protocols.io https://dx.doi.org/10.17504/protocols.io.x5sfq6e Copy   

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Authors: Lin Zhong, Jeffrey Rottman, Chee Lim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:Hypertension in man can be modeled in the mouse by introducing a constriction in the major artery (aorta) thereby obstructing outflow and increasing the afterload to the heart. This protocol describes the procedure for aortic banding aka transverse aortic constriction (TAC).

Proper citation: Lin Zhong, Jeffrey Rottman, Chee Lim 2019. Vandy - Transverse Aortic Constriction. protocols.io https://dx.doi.org/10.17504/protocols.io.6xdhfi6 Copy   

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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum alanine transferase levels reflect liver function.

Proper citation: Jason Kim 2019. U Mass - Alanine Transferase. protocols.io https://dx.doi.org/10.17504/protocols.io.xpmfmk6 Copy   

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Authors: Jennifer Rutkowsky
Group: Mouse Metabolic Phenotyping Centers
Summary: BODIPY® 493/503 can be used as a stain for neutral lipids and as a tracer for oil and other nonpolar lipids. It has uses for identifying and quantifying lipid droplets in various cell and tissue type. Abnormal lipid droplet accumulation can be associated with various metabolic disorders such as Diabetes, cardiovascular disease and cognitive impairment.

Proper citation: Jennifer Rutkowsky 2019. UC Davis - Immunohistochemistry BODIPY. protocols.io https://dx.doi.org/10.17504/protocols.io.56rg9d6 Copy   

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Authors: Stephen M. Griffey
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Gross necropsy with documentation of changes present. Collection and histologic processing (paraffin blocks/H&E slides) and interpretation of kidneys, liver, spleen, pancreas, heart, lungs, esophagus, trachea, thymus, mesenteric lymph nodes, GI Tract, cerebrum, cerebellum, urinary bladder, and reproductive tract and additional tissues with gross changes. This includes photodocumentation of significant findings. This assay can be customized to include other target organs based on findings from other MMPC assays. This procedure is to be followed by trained Comparative Pathology Laboratory (CPL) staff responsible for performing necrospy diagnostics procedures at CPL.

Proper citation: Stephen M. Griffey 2019. UC Davis - Mouse Gross Necropsy with Histology. protocols.io https://dx.doi.org/10.17504/protocols.io.yusfwwe Copy   

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Authors: Bingle Bracy
Group: Mouse Metabolic Phenotyping Centers
Summary: Study of the unstressed mouse requires prior implantation of catheters in the carotid artery and jugular vein. The arterial catheter is used to sample blood and the venous catheter is used as an infusion port. The surgical procedure is performed under sterile conditions in accordance with AAALAC standards.

Proper citation: Bingle Bracy 2019. Vandy – Chronic Catheterization of carotid artery and jugular vein. protocols.io https://dx.doi.org/10.17504/protocols.io.zddf226 Copy   

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Authors: Eva Feldman
Group: Mouse Metabolic Phenotyping Centers
Summary: This protocol measures and quantifies the pain in rodent hind paw. These measurements can be used to evaluate the pathology present in the nervous system.

Proper citation: Eva Feldman 2019. U Michigan - Hind Paw Withdrawal for Rodents. protocols.io https://dx.doi.org/10.17504/protocols.io.x67frhn Copy   

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Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of NEFA in blood, serum, and plasma. NEFA; Free fatty acids are measured in a multistep reaction to form an colored adduct of 3-methyl-Nethyl-N-(b-hydroxy-ethyl)-analine and 4-aminoantipyridine monitored at 560 nm.

Proper citation: John Stack, Gary Cline 2019. Yale - Non-Esterified Fatty Acids. protocols.io https://dx.doi.org/10.17504/protocols.io.y38fyrw Copy   

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Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers
Summary: Analysis of the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) using gas chromatography mass spectrometry (GC-MS) is an analytical approach to identifying unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labelled molecules (eg. 2H- or 13C-labelled) and the naturally occurring most abundant molecules. The use of metabolic tracers (eg. 2H- or 13C-labelled) can offer additional information that can lead to the understanding of interrelationships between the pools of intermediary metabolism, such as with the CAC, and specific metabolic pathways. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions. The following SOP presents a relatively simple method that is sensitive for simultaneously measuring concentrations and heavy labelled molecules of CAC intermediates (relative and absolute) and other related intermediates of energy metabolism using GC-MS technology. Note: the following extraction procedure can also be used parallel analysis for acyl-CoA’s and lipids (for details on isolating and mass spectrometry assay procedures see, refs 4-6).References: 1. Yang, L., Kasumov, T., Kombu, R. S., Zhu, S. H., Cendrowski, A. V., David, F., Anderson, V. E., Kelleher, J. K., and Brunengraber, H. Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle: II. Heterogeneity of metabolite labeling pattern. J. Biol. Chem. (2008) 283, 21988-21996 2. Yang, L., Kombu, R. S., Kasumov, T., Zhu, S. H., Cendrowski, A. V., David, F., Anderson, V. E., Kelleher, J. K., and Brunengraber, H. Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. I. Interrelation between gluconeogenesis and cataplerosis; formation of methoxamates from aminooxyacetate and ketoacids. J. Biol. Chem. (2008) 283, 21978-21987 3. Kombu RS, Brunengraber H, and Puchowicz MA. Analysis of the Citric Acid Cycle Intermediates using Gas Chromatography-Mass Spectrometry In: Methods Molec Biol, “Metabolic Profiling: Methods and Protocols” (ed. Metz,TO), Humana Press, (2011) 708:14757 4. Zhang Y, Zhang S, Marin-Valencia I, Puchowicz MA. Decreased Carbon Shunting From Glucose Towards Oxidative Metabolism In Diet-Induced Ketotic Rat Brain. J Neurochem. (2015) 132(3): 301-12 5. Harris SR, Zhang GF, Sadhukhan S, Wang H, Shi C, Puchowicz MA, Anderson VE, Salomon RG, Tochtrop GP, Brunengraber H. Metabolomics and Mass Isotopomer Analysis as a Strategy for Pathway Discovery: Pyrrolyl and Cyclopentenyl Derivatives of the Pro-Drug of Abuse, Levulinate. Chem Res Toxicol. (2012) 26(2): 213-20 6. Zhang, G. F., Kombu, R. S., Kasumov, T., Han, Y., Sadhukhan, S., Zhang, J., Sayre, L. M., Ray, D., Gibson, K. M., Anderson, V. A., Tochtrop, G. P., and Brunengraber, H. Catabolism of 4-hydroxyacids and 4-hydroxynonenal via 4-hydroxy-4-phosphoacyl-CoAs. J. Biol. Chem. (2009) 284, 33521-33534

Proper citation: Henri Brunengraber 2019. Case - Citric Acid Cycle and Related Intermediates. protocols.io https://dx.doi.org/10.17504/protocols.io.ydbfs2n Copy   

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Authors: Trina Knotts
Group: Mouse Metabolic Phenotyping Centers
Summary: Plasma samples will be assayed for lipopolysaccharide binding protein (LBP) as surrogate for bacterial LPS/measure of endotoxemia via ELISA.

Proper citation: Trina Knotts 2019. UC Davis - Lipoprotein Binding Protein (LBP)-Endotoxemia Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.yrhfv36 Copy   

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Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of inorganic phosphorus in blood, serum, and plasma. Phosphorus (Inorganic) is measured as the complex with molybdate and monitored at 340nm.

Proper citation: John Stack, Gary Cline 2019. Yale - Blood and Urine Inorganic Phosphorous. protocols.io https://dx.doi.org/10.17504/protocols.io.y2gfybw Copy   

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Authors: Gary Cline, John Stack
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to determine the concentration of β-Hydroxybutyrate in blood, serum, and plasma. β-Hydroxybutyrate is measured by the oxidation to acetoacetate, followed by reduction of an indicator dye (monitored at 505 nm) by NADH.

Proper citation: Gary Cline, John Stack 2019. Yale - Beta hydroxybutyrate (Cobas). protocols.io https://dx.doi.org/10.17504/protocols.io.y2bfyan Copy   

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