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Hart, Anna

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LOUIS  University of Alabama in Huntsville

Metabolomic Analysis of Urine From a Rat Model Relevant to Diabetes Using NMRAnna Hart, James Wolfsberger, Dr. Bernhard VoglerDepartment of Chemistry Overview ResultsConclusionsAcknowledgementsThe metabolism is very complex with a numerous amount ofpathways. In those pathways there are hundreds of metabolites withmany present in urine which may be detected using NMRspectroscopy. Diabetes is an autoimmune metabolic disorder whichcauses certain metabolites to often be seen in higher concentrations,which are denoted as taller peaks on nuclear magnetic resonance(NMR) spectroscopy. Identifying these metabolites and theirconcentrations may give a better understanding of Diabetes and theearly biomarkers of the disease.Figure 2: Spike study to evaluate the change in glucose shift relative to the concentration of urine. Shown is the anomeric proton of a-D-glucose.References1. Mordes, John P et al. LEW.1WR1 Rats Develop Autoimmune Diabetes Spontaneously and in Response to Environmental Perturbation, Diabetes; New York Vol. 54, Iss. 9,  (Sep 2005): 2727-33. 2. Human Metabolome Database [Online], http://www.hmdb.ca/, Accessed August 2019.I, Anna Hart, would like to thank God, the UAH Office of the Provost, the UAH Office of the Vice President for Research and Economic Development, theAlabama Space Grant Consortium, Dave Cook, Dr. Love-Rutledge, Sumedha Bobba, and all those who helped with animal care, sample collection,preparation, and analysis.Research and Creative Experience for Undergraduates Program (RCEU)Summer 2019Methods• Urine was obtained from LEW.1WR1 and Wistar Furth Rats.LEW.1WR1s are susceptible to Type 1 Diabetes (T1D). Urinewas collected after 4 and 8 hour fasts in both dark and lightcycles. It was normalized using the fluorescence and lyophilizedand stored at -20°C.• For urine only sample prep, it was reconstituted with 160 mL of100 mM pH=7 H2O K-phosphate buffer and mixed with 40 mL ofDSS and placed in 3 mm NMR tubes.• For spike study sample prep, urine was reconstituted with 600 mLof 100 mM, pH=7, H2O K-phosphate buffer and mixed with 150mL of DSS and placed in 5 mm NMR tubes. After baseline run,the urine was spiked with 50 mL of desired metabolite solutionsuch as glucose.• Samples were ran on NMR using the NOESY experiment.Analysis and quantification were done using MestReNovasoftware (version 14.01).• The spike studies performed helped determine the peak positionof some key metabolites such as glucose and 3-hyroxybutyricacid, this knowledge assisted with quantification.• Some key metabolites including glucose, 3-hyroxybutyric acid,and li-acetoacetate all showed relatively high concentrations indiabetic rat urine when compared to non-diabetic, this may be anearly indicator of T1D.Lew.1WR1 Rat in Metabolic Cage for Urine CollectionWistar Furth Rat in Metabolic Cage for Urine CollectionFigure 1: Diabetic LEW.1WR1 rat in comparison to a set of 27 non diabetic rats consisting of both LEW.1WR1 and SsHNsd.CH31CH3 CH22OOHOHCH31OOHOOH1OHHHHHOHOHOHOH3-hydroxybutyric acid"3-HBA"acetoacidic acid"AcAc"a-glucose"A-Glu"

Metabolomic Analysis of Urine From a Rat Model Relevant to Diabetes Using NMR

https://louis.uah.edu/cgi/viewcontent.cgi?article=1119&context=rceu-hcr

Metabolomic Analysis of Urine From a Rat Model Relevant to Diabetes Using NMR

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LOUIS University of Alabama in Huntsville