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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. .

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Mass spec of NO
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Chromatographic techniques
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.

Analytical electrochemistry

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.


Blog 13? -1 pt.

Blog 9. Answers are correct. However, it is unclear how you may analyze NO by ion exchange chromatography. This analyte is not ionic (-0.1 pt).

Blog 10. NO cannot be directly directed using a fluorescence detector. Some information is missing. (-0.25 pt).

Blog 11. The answer for the detector does not seem right. It seems that you are mixing UV absorption and fluorescence detection. Also, see comment for Blog 10. Based on the buffer composition, you seem to be using a surfactant as an additive. This would imply that you are doing MEKC, unless the surfactant concentration is below the CMC. (-.025 pt).

Blog 10

1. Before the introduction of CE, my preferred technique would have been using HPLC-anion exchange.

2. Allison Blonski’s presentation technique to her analytical problem is very similar to mine. Allison’s analytical problem investigates the taurine levels in energy drinks. Her presented chromatographic technique was using a reversed phase chromatography with a fluorometric detector. For my chromatographic choice, I would be using a HPLC anion exchange technique with a fluorometric detector.

You've done a lot of research on the topic, but I am unclear on your procedure. Nitric oxide will be a gas and will not likely be retained in your sample prep. Nitrate and nitrite will not likely be analyzed by reverse phase HPLC, but by ion exchange chromatography. How soluble are these anions in acetonitrile? (-0.5 pt).

In this answer you were not expected to post instrumentation conditions that you propose to use.

If nitric oxide, nitrate, and nitrite in biological fluids were to be analyzed by high-performance ion chromatography:

1. 50-microL aliqout of the plasma sample would be pipetted into a 300-microL glass tube (Sci-Vi, Chromacol, Welwyn, UK).
2. 50-microL of acetonitrile would then be added, capped and centrifuged at 2000g for 2 mins.
3. To be used in the reversed phase, 50-microLs of the sample would then be pipetted into 1.5-mL polypropylene tubes (Sarstedt, Leicester, UK), mixed with 400-microL of acetonitrile, capped, and then centrifuged at 10,000g for 1 min.
4. The supernatant would then be decanted into a 1.5-mL tube, washed with 400-microL of acetonitrile, and the combined extracts would then be dried under nitrogen and reconstituted in 200-microL of water.
5. Ultrafiltration would then be used. [microcentrifuge tube filters, cellulose triacetate, 20,000 molecular mass cut-off (Whatman, Maidstone, UK), Centricon-10 10,000 cut-off (Amicon, Stonehouse, UK)]. The samples would then be washed with Milli-Q water and dried.
6. The sample would then be centrifuged at 9500g for 5 mins (Whatman and Alltech) and at 5000g for 30 mins (Amicon).
7. The sample would then be transfered to an autosampler vial for injection
8. The system used for this analysis would be the Millennium chromatograph and data system. It is equipped with 616 pumps and has a WISP autosampler. The column is an IonPac AS9-SC ion-exchange column, 250 mm x 4 mm (Dionex). The eluent would be 5mM K^-2HPO^-4, 25 mM KH^-2PO^-4 with a flow rate of 1.5mL/min. The detection would be by absorbance at 214 nm via 486 detector. The water that would be used would be freshly drawn from a Milli-Q system.

Everett, Steven, and S A AEverett. "Nitric oxide in biological fluids: analysis of nitrite and nitrate by high-performance ion chromatography." Journal of chromatography 706.1-2 (1995):437.

Blogs 6 and 7. Good answers. For clarity you may write chemical reactions in word or powerpoint and then save as images.

Atomic spectrometry can be applied to my analytes, however, because the analytes are in a blood serum matrix that contain other biological fluids, it would be easier to quantitatively analyze the nitrite and nitrate with either colorimetric and uv-spectrophotometric methods or gas chromatography-mass spectrometry. The ionization sources and mass analyzers that could be used include ESI-Q, ESI-linear trap, or ESI-LTQ. The ionization source and mass analyzer I would use would be ESI-LTQ because the LTQ would work well with GC-MS. HPLC would work well with GC-MS too.

Molecular weights of analytes and mass spectrum:
Nitric Oxide = 30.0061 g*mol^-1
(I cannot find the instrumental conditions under which this spectrum was taken)
The mass spectrum of NO is linked under “Mass spec of NO”
Nitrate = 62.0049 g*mol^-1
Nitrite = 46.0055 g*mol^-1

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.

Spectral Database for Organic Compounds SDBS. 2010. 27 October 2011 .

Answers to both Blog 4 and 5 are ok. In Blog 4, where is equation 9? In Blog 5, NO does not fluoresce, it is the reaction product that fluoresces

The maximum excitation and emission of nitric oxide is 495 and 515nm respectively with the reagents 4-amino-5-methylamino-2’7-difluorescein (DAF-FM) or 4-amino-5-methylamino-2’7-difluorescein diacetate (DAF-FM diacetate). DAF-FM can be detected by any instruments that can detect fluorscein, for example, flow cytometers, microscopes, fluorescent microplate readers, and fluorometers. Using DAF-FM as a reagent can be beneficial because the spectra of the NO adduct would not be effected at a pH above 5.5 and that DAF-FM is also highly sensitive

“Nitric Oxide Indicators: DAF-FM and DAF-FM Diacetate.” Molecular Probes. 16 May 2001. Web. 17 October 2011. .

Hypothesis: Nitric oxide does not promote muscle growth
Studies: (A) Test a certain powder (the same supplement that our participations would use) to confirm that the amount of nitric oxide in the 1 serving size is what they claim. (B) Obtain a large sample size of men and women from around the country (U.S.) of different backgrounds who has at least 3 years of resistance training and plan on to continue resistance training in the near future. The individuals need to be healthy and have not have any cardiovascular diseases. (C) Before and after each workout, a blood pressure reading would be administered and venous blood sample would be drawn from each individual to measure pre and post-NO levels. (D) Each individual would follow the resistance training plan (slightly modified for the women but the same workout routine for each gender). (E) The participants must agree not to take additional supplements as it would interfere with the research. (They must also not take supplements 1 month prior to the experiment). (F) The supplement with NO would be distributed in a double-blinded distribution with a placebo containing no NO (We will try to find a way so that we can match all of the other ingredients that are included with the brand that has the NO). (G) Before and after the workout program, all individuals would be subjected to a BMI test for pre and post-muscle density recording. (H) We would compare muscle density and NO levels between the pre and post resistance training program for both the placebo and the supplement containing NO. (I) The program will last for 6 months with compensation.
Alternative study: (J) If the results are statically significant in that NO does promote muscle growth, then we need to investigate the biological process of NO in the body.

Analyte levels in the matrix.
In the study of the Estimation of Nitric Oxide Concentration in Blood for Different Rates of Generation by Liu, Yan, Baskerville, and Zweier, they found that the membrane permeability, based on equation 9, equals 4.5 cm/s. This indicated that NO generation must be greater than ~80 uM/s because it needs to reach a steady-state NO concentration of 10nM within the human body. However, the NO generation rate is too high within the human body but it is too low to result in signaling for vasodilation. It was also found that the NO responsible for vasodilation was produced from nitrite reduction in the presence of deoxyhemoglobin (in vitro). Moreover, NO was not the primary product from this reduction and other compounds are also involved in signaling for vasodilation. The reported rate of NO generation from this reaction of 200 um nitrite with 1 mM deoxy-redbloodcells Hb was less than 50 pM/s. This rate is even smaller than reported before. The study suggested that NO is not the primary agent for signaling vasodilation. They believe that derivatives of NO, such as N2O3, that are generated from the red blood cells are involved in nitrite-induced vasodilation.

Liu Xiaoping; Yan Qingtao; Baskerville Kim L. “Estimation of nitric oxide concentration in blood for different rates of generation - Evidence that intravascular nitric oxide levels are too low to exert physiological effects.” Journal of Biological Chemistry 282.12 (2007): 8831-8836

see instructions for BLOG 3. You need to modify your entry. If you do not know how to do it, consult with Chad. Once you have modified your entry your grade will be released. Please notify Chad when this is done. Thanks. Edgar
Regarding your answers....

- (a) Good choice of problem. Consider other problems that focus on analytes in the gas phase (e.g. CO).
You are not answering parts (b) and (c). (-0.5 pt).

An analytical problem similar to mine would be of Osman Jamshed. Both of our analytes/matrix is found in the blood. I am not sure if our analytical solutions would be exactly the same because Osman's analytical problem involves prions, which are misfolded proteins, and mine is just detecting nitrate/nitrite. However, the matrix for both of our analytical problem is the same.

My problem detecting prions by analytical methods problem is similar to your problem as we have the same matrix, blood and there are interactions between the nitro group and my analyte. The compound we are detecting have nitrogen in them too which makes them fall in almost the same range in UV-vis (540-600 nm.)

Both of our problems examine dietary supplements and possible negative outcomes of added substances. Additionally, both of our problems examine whether active ingredients in such products have their intended effect.

Some clarification is needed. Is NO released from the compounds during physical activity and then metabolized to nitrate/nitrites?

The info about the colorimetric kit may be relevant for answers to the second Blog Post.