Analysis of nitric oxide in biological fluids and muscle growth

Chem 4101Andrew XayamongkhonDate: 12/9/11

http://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 musclesIncrease muscle mass, muscle repair rate and

vascularityMisleading

Most weightlifting supplements are not FDA approvedThe 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

Problem/Importance

BackgroundN.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 nitrateNeed 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

2

N

ONitric oxideNitrite

N

O O

N

O O

N

O

OO

NO

O

O

N

O

O O

Nitrate

3

HypothesisThe 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 fluids

Method Advantages Disadvantages

Nitrate reductase/Griess reaction

($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 pathways

GC-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 GC

Method Advantages Disadvantages

Anion exchange HPLC-fluorescence detector

(Spherecone™ column with P-based eluent)

• High sensitivity• High reproducibility• High sample throughput• LOD3 0.1 uM/L• No degration of N.O.

• Needs ultrafiltration to reduce protein and salt contaminants• Column optimization

Microchip 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 research

Methods to determine NO in biological fluids

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 microchipso Low manufacturing cost

Use photolitography or micromolding to form channels in the chip

Method 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 respectivelyThe 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)

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-

treatedLinearly 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 peaks

Reference (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 standard

Concentration of artificial

serum (running buffer)1

Sum of the

peak area1

10% .48

50% .25

90% .25

Expected data analysis and figures of merit

Reference (2)Reference (2)

Use standard samples to construct calibration curves for nitrate and nitrite and calculate concentration based on area

Reported1 calibration curves (R):

Correlation (R) from standard curves

Nitrite Nitrate

Peak height 0.9958 0.9948

Peak area 0.9909 0.9941

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 matrixIncrease sample injection volume by active control of voltage

Investigate into new chip designs and materialsType-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

References1. Allen, Jason, and J. D. DAllen. "Nitrite, NO and hypoxic vasodilation."

British Journal of Pharmacology 158.7 (2009):1653.

2. 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.

3. Bharadwaj, Santiago, Mohammadi. “Design and optimization of chip capillary electrophoresis.” Anal. Chem. 2002, 71, 2729-2744.

4. Miyado, Takashi, TMIYADO, and Miyado. "High-throughput nitric oxide assay in biological fluids using microchip capillary electrophoresis." Journal of chromatography 1109.2 (2006):174.

5. 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.

6. 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.