Analysis of nitric oxide in biological fluids and muscle growth

1. Analysis of nitric oxide in biological fluids and muscle growth http://www.furiouslyfit.com/wp-content/uploads/2010/04/No-Xplode.jpg Chem 4101 Andrew Xayamongkhon Date: 12/9/11

2. Problem/Importance • 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 protein

3. Background • N.O. has a very short half life in vivo (half-life<5 sec)1 because 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. production Nitrite 2 3 Nitrate Hypothesis • 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.

4. Methods to determine NO in biological fluids

5. Methods to determine NO in biological fluids

6. Method of choice: Microchip capillary electrophoresis–linear photodiode array • How to make microchips: • Microchips are based on microfrabication techniques. 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 microchips • Low manufacturing cost • Use photolitography or micromolding to form channels in the chip (Reference 6)

7. 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 flow Reference (2) Reference (2)

8. 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 Photdiode Array 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 against the EOF, lengthening the separation channel to obtain complete peaks Reference (2) Reference (2)

9. 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 standard

10. Expected data analysis and figures of merit • Use standard samples to construct calibration curves for nitrate and nitrite and calculate concentration based on area • Reported1 calibration curves (R): Reference (2) Reference (2)

11. Conclusion • 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 supplements

12. References • Allen, Jason, and J. D. DAllen. "Nitrite, NO and hypoxic vasodilation." British Journal of Pharmacology 158.7 (2009):1653. • J, Bloomer, and Bloomer Richard J. "Acute effect of nitric oxide supplement on blood nitrate/nitrite and hemodynamic variables in resistance trained men." Journal of Strength and Conditioning Research 24.10 (2010):2587. • Bharadwaj, Santiago, Mohammadi. “Design and optimization of chip capillary electrophoresis.” Anal. Chem. 2002, 71, 2729-2744. • Miyado, Takashi, TMIYADO, and Miyado. "High-throughput nitric oxide assay in biological fluids using microchip capillary electrophoresis." Journal of chromatography 1109.2 (2006):174. • Sharma, Arun et al. "Determination of nitric oxide metabolites, nitrate and nitrite, in Anopheles culicifacies mosquito midgut and haemolymph by anion exchange high-performance liquid chromatography: plausible mechanism of refractoriness." Malaria Journal 7 (2008) : 71. • Tsikas, Dimitrios, and DTsikas. "Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids." Free radical research 39.8 (2005):797.