Medicine (St Vincent's) - Theses

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Subject: diabetes × Author: Garlick, John ×

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    Mass spectrometry based examination of ADMA/DDAH modification in diabetes and cardiac disease
    Garlick, John ( 2016)
    Nitric Oxide (NO) is an important mediator in the prevention of monocyte recruitment and endothelial dysfunction, which drive the development of atherosclerosis. Asymmetric Dimethylarginine (ADMA) but not its isomer Symmetric Dimethylarignine (SDMA) has been identified as a circulating inhibitor of Nitric Oxide Synthase (NOS), with submiromolar increases of ADMA previously been demonstrated associated with an increase in cardiovascular disease risk. Currently ADMA has been measured with low reproducibility in most analytical techniques other than the gold standard HPLC-MS/MS. This work shows the separation that can be achieved with the emerging technique of Ion Mobility Spectrometry coupled with traditional mass spectrometry. Also using a more traditional HPLC based separation we were able to develop a mass spectrometry based measurement of metabolites ADMA, SDMA, Arginine, Ornithine and Citrulline critical in the NOS/ADMA pathway and the Urea cycle. This assay was used to measure changes across cardiac surgery within the circulation as well as generation from the myocardium. We demonstrated our technique had high reproducibility (>7% CV for ADMA) and showed that ADMA but not SDMA significantly decreases after cardiac surgery. This was the first such study to perform measurements of ADMA and SDMA alongside Urea cycle metabolites and with clinical data also collected will provide the foundation to further work. Our assay was also used in a cell culture model whereby DDAH the enzyme responsible for the majority (80%) of ADMA metabolism has previously been shown to have increased expression and activity with high glucose treatment. Our results demonstrate that ADMA is highly regulated compared to SDMA and the change with high glucose treatment does not correspond to a decrease in intracellular ADMA concentration or from the treatment media. Also tested was the efficacy of the proton pump inhibitor Omeprazole which has been recently demonstrated to inhibit DDAH in endothelial cells. This work demonstrated that Omeprazole does not inhibit DDAH within our HepG2 cell culture model in low or high glucose concentrations.