Analysis of nitric oxide in biological fluids and muscle growthChem 4101Andrew XayamongkhonDate: 12/9/11http://www.furiouslyfit.com/wp-content/uploads/2010/04/No-Xplode.jpg New hit in body building because the manufactures claim that supplemental nitric oxide will deliver more amino acids and nutrients to working muscles Increase muscle mass, muscle repair rate and vascularity Misleading Most weightlifting supplements are not FDA approved The product may not contain labeled ingredients and may be harmful to consumers Cost of nitric oxide-based bodybuilding supplements is generally higher compared to other alternatives such as whey-isolate proteinProblem/ImportanceBackground N.O. has a very short half life in vivo (half-life<5 sec)1because N.O. readily oxidizes with biological fluids to form nitrite and nitrate Need method to detect: NO (MW: 30.01 g mol−1)NO−1(MW: 46.00 g mol−1)NO−1(MW: 62.01 g mol−1) Can use nitrite and nitrate as markers for N.O. production2NitriteNitrate3Hypothesis The consumption of N.O. products does not increase the level of circulating N.O. in the human body to induce muscle growth. To investigate this, the proper analytical method can be used to determine the amount of N.O. in biological fluids.Methods to determine NO in biological fluidsMethodAdvantagesDisadvantagesNitrate reductase/Griessreaction ($185.00, Cayman Chemical Company, Ann Arbor Michigan)• Low cost• Easy to use• Azo (N=N) compound is absorbed at 540-550 nm• Griess reaction reacts with free biogenic amines - Produce false positive results• Limited in detecting N.O. metabolic pathwaysGC-MS• High sensitivity• High resolution• High sample throughput•Wide applicability• Extensive derivatization of N.O. from blood to increase volatility• Thermal degradation once inside the GCMethodAdvantagesDisadvantagesAnion exchange HPLC-fluorescence detector(Spherecone™ column with P-based eluent)• High sensitivity• High reproducibility• High sample throughput• LOD30.1 uM/L• No degration of N.O.• Needs ultrafiltration to reduce protein and salt contaminants• Column optimizationMicrochip capillary electrophoresis –linear photodiode array detector (UV-VIS)• Cheap to manufacture• Requires little sample• High sample throughput• LOD2.5 µM and 1.4 µM (Nitrite and Nitrate)• S/N = 3• Routine analysis• MCE is a relatively new field and the surface chemistry of microchip devices need more researchMethods to determine NO in biological fluids How to make microchips: Microchips are based on microfrabicationtechniques. One can use inexpensive soda lime glass to high quality quartz (Shimadzu). Glass is most often used due to its good optical properties One can also used polymer fabricated microchipso Low manufacturing cost Use photolitography or micromolding to form channels in the chipMethod of choice: Microchip capillary electrophoresis–linear photodiode array (Reference 6) Microchip layout: Most common layout is using a cross-type layout with four reservoirs. This can be made with commercially available computer-aided design tools Sample injection: Electrokinetic with 0.00, 0.40, 0.20 and 0.20 kV to reservoirs 1-4 respectively Separation phase: 0.15, 0.15, 0.00 and 1.80 kV to reservoirs 1-4 respectively The sample migrates from 1 to 2, with the analytes diffusing at the intersection towards 4 due to the buffer composition. Separation is based on electroosmotic flowReference (2)Reference (2) Buffer composition: To decrease Cl−ions in human serum analysis, an artificial human based serum can be made with standards and be used as a running buffer at pH 7.4 (Milli-Q Gradient A10 (Millipore, MA, USA)) Detector: MCE apparatus (MCE-2010, Shimadzu)at 214 nm (Shimadzu SPD-M10AVP PhotdiodeArray Detector, $999.99) Since UV does not rely on chemical reaction, N.O. does not need to be pre-treated Linearly positioned photodiode array detector to optimize separation channel length To increase the resolution, the artificial buffer can be operated without an electroosmotic flow modifier This forces sample ions to migrate againstthe EOF, lengthening the separationchannel to obtain complete peaksReference (2)Reference (2)Sample preparation Deprotonate pooled human serum with sequential centrifugal ultrafiltration at 200g (Biomax-100K, Biomax-30K, and Biomax-5K filtration units (Millipore, MA, USA) in this order) Both the human serum sample and standard sample (nitrate and nitrite) would be diluted 10-folds with distilled water Dilute to 10-folds to increase: Stacking effects The difference of the electric field strength between the sample and the running buffer This increases resolution and peak area Reported data: The artificial serum and running buffer can be prepared with standards Fumaric acid would be added as an internal standardConcentration of artificial serum (running buffer)1Sum of the peak area110%.4850%.2590%.25Expected data analysis and figures of meritReference (2)Reference (2) Use standard samples to construct calibration curves for nitrate and nitrite and calculate concentration based on area Reported1calibration curves (R):Correlation (R) from standard curvesNitriteNitratePeak height0.99580.9948Peak area0.9909 0.9941Conclusion Microchip capillary electrophoresis (MCE) can separate and identify nitric oxide metabolites in biological fluids within 6.5 seconds MCE can be used on a day-to-day basis with a standard deviation of less than 10%1 MCE-linear photodiode array was chosen because the detector has a high scanning rate, which works well with the high sample throughput It is affordable and small sample is required for analysis On site analysis can be performed To improve this method: Needs high sensitivity (factor > 50) due to the blood matrix Increase sample injection volume by active control of voltage Investigate into new chip designs and materials Type-T (has increased sensitivity but small sample injection volume If there is a significant increase in N.O. in vivo compared to basal levels Study the metabolic pathways of N.O. and determine whether or not it correlates with muscle growth Develop better and cheaper bodybuilding supplementsReferences1. Allen, Jason, and J. D. DAllen. "Nitrite, NO and hypoxic vasodilation." British Journal of Pharmacology 158.7 (2009):1653. 2. J, Bloomer,
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