Andrew-Xayamongkhon-Analytical Problem- Nitric oxide and muscle growth
Performance enhancing supplement is a multibillion dollar industry and is endorsed by bodybuilders from all around the world. Surprisingly, the majority of these sport supplements, which are being sold at your local nutritional stores, are not regulated by the FDA. The topic of interest is the nitric oxide (NO) compound in sport supplements. NO is relatively new in bodybuilding supplementation and not much is known about its correlation with muscle growth. Brands such as N.O.-Xplode 2.0TM, SuperPump250 TM, NO Beta TM, Epozine-02 NT TM, etc..., have all claimed an increase in muscle pump or muscle density from the use of NO. The synthesis of NO is simple and natural in the human body. NO can be obtained from L-arginine, which one could obtain from eating chicken. The amount of L-arginine consumed by humans per day is less than 1g. More research is needed to determine the conversion factor from L-arginine to NO. Surprisingly, the best selling NO product does not list the amount of L-arginine, but it does contain approximately 19.6g of NO. In the body, NO is synthesized by the nitric oxide synthase, a complex enzyme, which reacts with oxygen, L-arginine, and NADPH to produce NO, citrulline, and NADP+. NO in vivo has a very short half life and undergoes reactions with other biological molecules in the blood fluid to create, for example, NO3- and NO2-. Therefore, the nitrate/nitrate ratio would give the best indication of NO production.
During exercise, the blood vessels dilate naturally due to the usage of ATP and the production of metabolic byproduct. Also, an in increase blood vessel diameter also allows an increase in blood flow and oxygen to the needed muscles. The maximum dilation of the blood vessel in a normal human needs further research. One question to ask is if the blood vessel is expanded to the max during a regular workout, would excess NO be needed? Because of the principle of increase blood and oxygen flow during vasodilation, nutritional supplement manufactures claim that the added NO in powders would add to the effect of vasodilation and increase muscle density. The role of NO itself to increase muscle density is not known, however, NO is known to work with other molecules that are also provided with the product such as the essential amino acids, microfractions, alpha-lactalbumin, beta-lactoglobulin, glutamine, glutamine precursors, etc, etc, etc... (whatever they want to print on the label), to increase muscle density. One molecule that is proven to increase power and muscle mass in proper resistance training is whey isolate protein. Whey isolate protein is a more purified form of whey protein which is also costly. A brand, for example, that has no NO but has whey isolate protein is the Gold Standard Whey TM. It is proven that NO is a vasodilator, play an important in neurotransmission, and is a precursor to photochemical smog (2), but the claim that NO itself has a correlation with increased muscle density is just a hype and is used to increase the market price of the product.
The central hypothesis in this study is that NO does not promote muscle growth. There are two major parts to testing this hypothesis. The first part is analyzing the amount of NO in blood. This could be done by taking the plasma nitrate/nitrite and performing nitrate reductase to convert it into nitrite. In the nitrite form, Griess reaction could be applied to turn it into a deep purple azo color where the nitrite composition could be determined with UV-Vis absorption spectrometry at about 540-550nm. The chemical compound responsible for the color is the azo structure. The solvents that could be used include Milli-Q water or the provided Assay Buffer from the colorimetric assay kit (item No. 780001). The analytes of interest is the plasma nitrate/nitrate and the matrix includes all of the other components found in blood including the unwanted byproducts in the Griess Reaction (3).
Similar Analytical Problem
The analytical problems similar to mine are of Matthew Marah and Andrew Szeliga because our analytical problems deal with a blood matrix. Andrew Szeliga's topic is about perfluoroocatanoic acid levels in human blood. His hypothesis is that people who have close exposure to sources of PFOA dumping will exhibit elevated levels of PFOA in their bloodstream. The matrix would be the blood and the relevance of the study is of health concerns. Matthew Marah's topic is about Cadmium levels in people. His central hypothesis is that people who live close to plants (ie. Industrial plants) will have higher levels of Cd in their blood. The matrix would also be the blood and the relevance of this study is also of health concerns. These are similar to my analytical problem because the matrix is the blood. However, it is also different because our analyte is different. The absorption ranges for each analyte are different. Moreover, pure Cd doesn't seem to have a UV-Vis range. If an oxidized form of Cd, for example, gives the best indication of Cd in the blood, it would be very similar to my testing procedure because I would be using nitrate/nitrite as an indicator for NO due to NO's biological reactions in vivo.
(1) Allen, Jason, and J D DAllen. "Nitrite, NO and hypoxic vasodilation." British Journal of Pharmacology 158.7 (2009):1653.
(2) Nyberg, Michael, and MNyberg. "Interstitial and Plasma Adenosine Stimulate Nitric Oxide and Prostacyclin Formation in Human Skeletal Muscle." Hypertension 56.6 (2010):1102.
(3) 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.
Chemical stricture and standards
The three chemical reactions below represent the production of the analyte of interest:
NO + O_2^- →ONO_2^- + H_^+ →NO_3^- + H_^+
2NO + O_2^ →N_2 O_4 + H2O →NO_2^- + NO_3^-
NO + NO_2^ →N_2 O_3 + H2O →〖2NO〗_2^-
Because NO is highly reactive with other biological fluids in vivo, the sum of NO_2^- and NO_3^- concentrations would be the best representation of NO production. In this example, the possible analytes will all be converted to nitrite via nitrate reductase which will then be reacted with Griess reagent to produce the desired azo product. The chemical structure shown below illustrates the product after nitrate reductase, which is a representation of the analyte of interest before the Griess reagent reaction. Otherwise, the analytes of interest are simply NO, NO_2^- and NO_3^-.
At a cost of $185.00, Cayman Chemical Company, located in Ann Arbor Michigan, can provide a Nitrate/Nitrite Colorimetric Assay Kit (item No. 780001) that contains standards, 1 vial per standard, for both nitrate (item No. 780014) and nitrite (item No. 780016) for the construction of calibration curves. The catalog No. is 760871.
"Nitrate/Nitrite Colorimetric Assay Kit." Cayman Chemical. 30 March 2011. Web. 23 October 2011.
Mass spec of NO
1. From class lectures and scholarly journals, the type of chromatography that is best suited for my analytical problem is a high performance anion exchange chromatography. One could use a GC-MS-validated anion-paring HPLC with ultraviolet absorbance detection at 205 nm, however, this method is not suitable to measure nitrate in human plasma. One could use HPIC do determine nitric oxide in human plasma however large amounts of Cl ions would result in poor peak resolution. HPLC, CE or other MS-based techniques are also suitable methods for the analysis of nitrite and nitrate in human biological fluids, however, high performance anion exchange chromatography would be most suitable.
2. High performance anion exchange chromatography (HPAE) would be my first choice because this technique is rapid, very sensitive, and has an accurate method with a LOD for nitrate of 0.1 microM/L. It doesn't require much precolumn derivatization and that nitric oxide, nitrate, and nitrite can be measured directly. It was also found to be suited for analyzing and separating nitric oxide and nitric oxide derivatives in human biological fluids.
3. A commercial column that is available is the Sphereclone column provided by Phenomenex. The column that would be used would be a 250 x 4.6 mm, Sphereclone column with a 5 micro SAX stationary phase with an anion exchanger (SEM based silica). The stationary phase would be comprised of SAX and has a fully porous silica as a solid support with a pore size of 80 angstrom. The composition of the mobile phase that would be used is 5mM k^-2HPO^-4 and 25MM KH^-2PO^-4, with a pH of 3.0. The mobile phase would be pumped at 1.5 ml/min and the effluent would be monitored at 214nm. The injection volume would be at 20 microL and the column could be kept at 35 degrees C and the samples could be kept at 4 degrees C. The part number is 00G-4149-E0.
4. The composition of the mobile phase that would be used is 5mM k^-2HPO^-4 and 25MM KH^-2PO^-4, with a pH of 3.0. The mobile phase would be pumped at 1.5 ml/min.
5. The recommended detector would be the UV-VIS detector SPD 10 AV. This detector would be practical because it has a high visible sensitivity, high scanning speed, and has low noise. The cell volume for this detector is 12 microL and at 210nm, one would have to use the deuterium lamp (190 - 370nm).
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.
Phenomenex. 2010. 10 November 2011.
Specification Sheet SPD-20A/SPD-20AV. 2006. 10 November 2011.
Capillary electrophoresis techniques
1. Out of CZE, MEKC, cIEF, or CGE, CZE would be most suitable to separate my analytes from other matrix components. MEKC would not be applicable because we are not interested in separating uncharged species despite that MEKC also separates charged species. Nitric oxide is not an amphiprotic specie, therefore cIEF would not be useful. CGE is useful for larger, macromolecules and would not be suitable because NO is a small molecule. However, from lecture 30, microchip-CE would be the most adequate technique for separating NO from biological matrices.
2. Microchip-CE would be my first choice because only a small sample is required, it has a high sample throughput, and has a high sensitivity with a UV-Vis detector. My second choice would be CZE because it is good for small anionic separation, has high resolution, it is cost efficient, and has a high sample throughput.
3. For the MCE, an artificial serum (Na2HPO4, NaHCO3, KCl, NaCl, sodium lactate, urea, glucose, sodium sulfate) would be prepared to a pH of 7.4 and the running buffer (same composition) would also be prepared to a pH of 7.4 with 0.1M HCL. The voltage that would be used would be 0.15kV because this was found to be the optimal voltage from Miyado's study (1). Because the separation channel in the microchip that would be used is short (Type-U, 35mm x 12mm with 50um wide x 20um deep), one can operate without an EOF modifier. This would allow the sample ions to flow against the EOF, lengthening the separation channel. A slightly higher injection volume would then be required to fix low sensitivity and resolution.
For CZE, buffer would include arginine, borate and TTAOH with tetradecytrimethyl-ammonium hydroxide modifying solution. Alkaline species for buffer solutions would be used because it would induce the formation of charged coupled binary layers with the inner surface of the fused silica capillary tube. The pH would be adjusted to 9.5 via 1M NaOH and the running buffer would be operated at -20kV. In both the MCE and CZE, a sample injector with pressure would be used as a sample loading method.
4. I would be using a UV-Vis detector for both methods. For the MCE, I would be using a UV-Vis detector at 214nm with a linear photodiode array detector because nitric oxide fluoresce well. For CZE, I would use a conductivity detector to detect UV because it is 10-folds better in LOD compared to UV-light-based detection. However, in both methods, the only difficulty is that it is not easy to analyze anions in biological fluids.
(1) Miyado, Takashi, TMIYADO, andMiyado. "High-throughput nitric oxide assay in biological fluids using microchip capillary electrophoresis." Journal of chromatography 1109.2 (2006):174.
(2) Y, B o u d k o, andBoudko Dmitri Y. "High-resolution capillary electrophoresis of nitrite and nitrate in biological samples." Methods in molecular biology 279(2004):9.
1. Yes nitric oxide is electroactive
2. With electrochemistry, selective electrodes could be used to identify nitric oxide. Materials to construct the working electrode could include platinum, carbon fiber, glassy carbon, or gold. Akin to other cases, there would always be interfering species and in this case, for example, interference from gaseous oxygen would be a concern if nitric oxide is being measured by means of electroreduction. To identify nitric oxide, interfering species could be reduced by the type of sensor, applied potential, characteristics of the permselective membrane and the biological location. From (1), a bare Pt microelectrode modified with Ni tetrasulfonated phthalocyanine could be used in situ, however, this would involve the electrode to be within the human for in situ analysis.
3. The size of the electrode and the distance at which it is measuring the source of nitric oxide affects NO quantification in biological systems. From the study of (1), it was found that larger working electrodes have larger NO sensitivity and that the NO concentration would decay rapidly as the distance increased from the NO source. Because my analytes are in blood, a larger electrode would be used for bulk NO measurement.
(1) Privett, Benjamin, and B J JPrivett. "Electrochemical nitric oxide sensors for physiological measurements." Chemical Society reviews 39.6 (2010):1925.