This work was supported by intramural NIH/NIDDK grant ZIA DK 0251041-14 to Alan N Schechter, MD.

Animal protocol was approved by NIDDK Animal Care and Use Committee (ASP K049-MMB-20). Animals were handled and treated according the current Guide for the Care and Use of Laboratory Animals freely available on AAALAC website.
1. Rat skeletal muscle collection
<ol>
	<li>While a rat is under deep anesthesia (5% isoflurane, confirmed by absent reaction to tail/leg pinch), start perfusion with saline containing heparin by placing a 19 G needle into an apex of the left ventricle and making a nick on the right atrium. Allow saline with heparin to perfuse through the internal organs until at least 1.5 liter/kg have flowed through the tissue. At this point, animals are dead from exsanguination and carcasses are ready to be processed for sample collection. 
	NOTE: Achieving good perfusion is critical, especially for accurate measurements of nitrite, because nitrite is oxidized into nitrate by any remaining hemoglobin.</li>
	<li>Identify target muscle tissues and excise them from the hind legs22 using clean surgical instruments. Remove as much fat and connective tissues from the muscle tissues as possible.</li>
	<li>Place the desired amount of muscle into a microcentrifuge tube and then place on dry ice. Store the tubes filled with tissue in the -80 &#176;C freezer. 
	&#8203;NOTE: In case of human biopsy samples, thoroughly blot them immediately upon collection with clean gauze to remove excess blood.</li>
</ol>
2. Preparation for homogenization
<ol>
	<li>Preparation of nitrite-preserving solution (stop solution)
	<ol>
		<li>Prepare a clear yellow solution containing 890 mM potassium ferricyanide (K3Fe(CN)6) and 118 mM NEM (N-ethylmaleimide) in distilled water, ensuring no crystals. Add non-ionic surfactant (detergent) in a 1:9 ratio (v/v, Table of Materials), and mix gently to avoid foaming.</li>
		<li>Dilute the stop solution in a 1:9 ratio with distilled water. Place the diluted stop solution (1:5 ratio of muscle tissue to diluted stop solution) in the homogenization tube. 
		NOTE: Twenty milligrams of tissue will require 100 &#956;L of stop solution, and 200 mg of tissue will require 1000 &#956;L of stop solution in the tube. Presence of detergent in added solution is critical for disruption of cell membranes. Any non-ionic detergent can be used, but care must be taken to verify that it does not interfere with chemiluminescence method.</li>
	</ol>
	</li>
	<li>Tissue preparation
	<ol>
		<li>Take the tissue out of the -80 &#176;C freezer and slowly thaw in ice. Remove the remaining fat and connective tissue from the skeletal muscle. Cut off pieces of skeletal muscle and blot on gauze to dry.</li>
		<li>Weigh out the amount of tissue (20, 50, and 200 mg). Place the pre-weighed skeletal muscle in the stop-solution in the homogenization tubes or place the pre-weighed tissue in a clean microcentrifuge tube for later use.</li>
	</ol>
	</li>
</ol>
3. Homogenization
<ol>
	<li>Rotary homogenizer (Figure 1)
	<ol>
		<li>Place the M-type tube containing the pre-weighed skeletal muscle and pre-measured stop solution into the machine. Homogenize each sample twice (setting on the most destructive homogenization cycle) and place the tube on ice immediately after each homogenization for 5 min to cool down.</li>
		<li>Centrifuge briefly at 2,000 &#215; g and 4 &#176;C for 5 min. Place the full tube back on ice and add the appropriate volume of methanol (&#8805; 99.9 %, 10x of tissue weight). Vortex thoroughly for 15 s. 
		&#8203;NOTE: For 20 mg of tissue, use 200 &#956;L of methanol. Methanol is used to precipitate proteins from tissue homogenate and does not interfere with chemiluminescence method. If other protein precipitation method is used, test its compatibility with chemiluminescence.</li>
		<li>Homogenize once more and incubate on ice for 30 min. Centrifuge the samples for 35 min at 4 &#176;C and 3,500 &#215; g. Aspirate the supernatant, and measure nitrite/nitrate levels, or store at -80 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Bead homogenizer (Figure 2)
	<ol>
		<li>Place the skeletal muscle tissue in a bead-containing tube (1:5 ratio for tissue:diluted stop solution) and homogenize twice for 45 s at the highest speedavailable on the instrument used. Place the tube on ice immediately after each homogenization for 5 min to cool down.</li>
		<li>Briefly centrifuge using a small desktop centrifuge (2,000 x g) for 5 sec. Place the tube back on ice and add an appropriate volume of methanol (purity &#8805; 99.9 %, 10x of tissue weight). Vortex thoroughly for 15 s. 
		NOTE 1: For 20 mg of tissue, use 200 &#956;L of methanol. 
		NOTE 2: Methanol is used to precipitate proteins from tissue homogenate and does not interfere with chemiluminescence method. If other protein precipitation method is used, test its compatibility with chemiluminescence.</li>
		<li>Homogenize once more for 45 s at the highest speed available on the instrument used. Incubate on ice for 30 min. Centrifuge at 17,000 x g, 4 &#176;C, 30 min. Aspirate the supernatant, and measure nitrite/nitrate levels, or store at -80 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Pulverizer (Figure 3)
	<ol>
		<li>Prepare tubes containing diluted stop solution (5x of tissue weight) and weigh them. Record the weight (tube + stop solution).</li>
		<li>Place the liquid nitrogen pulverizer tool on dry ice and wait for approximately 30 min for it to reach the desired temperature.</li>
		<li>Using tweezers chilled in liquid nitrogen, transfer one sample (tissue weight measured already) to the pulverizer. Add a small spoonful of liquid nitrogen to ensure the tissue is at liquid nitrogen temperature.</li>
		<li>After 95% of the liquid nitrogen has vaporized, place the crushing tool on top of the tissue, and press firmly. You should feel the sample crush. Using the mallet, strike the crushing tool 3-5x.</li>
		<li>Check the sample for any remaining chunks using a spoon cooled in liquid nitrogen. After cooling in liquid nitrogen, use a piece of tissue paper to wipe away any frozen water before touching the tissue. If a chunk is present, move it into the center, and then strike 5-6 more times with the mallet.</li>
		<li>When the whole sample has been pulverized, use the liquid nitrogen-cooled spoon to directly transfer the crushed tissue into the pre-weighed tube containing the diluted stop solution (step 3.3.1). Be sure to perform this step quickly as when the crushed skeletal muscle heats up, it gets sticky and difficult to transfer.</li>
		<li>Vortex for 15 s. Check that no tissue is stuck at the top of the tube by opening the tube. If there is, try to dislodge it and then vortex again.</li>
		<li>Centrifuge the sample for 2-3 s using a small desktop centrifuge. Weigh the tube again. Calculate the tissue weight by deducting the original tube weight (step 3.3.1) from this new weight. Place the tube on ice. 
		NOTE: The exact weights will help determine the exact tissue weight and how much tissue was lost during pulverization.</li>
		<li>Once all samples have been processed up to step 3.3.8, add an appropriate volume of methanol (&#8805; 99.9 %, 10x of tissue weight). Vortex thoroughly for 15 s, and incubate on ice for 30 min. 
		NOTE: For 20 mg of tissue, use 200 &#956;L of methanol. Methanol is used to precipitate proteins from tissue homogenate and does not interfere with chemiluminescence method. If other protein precipitation method is used, test its compatibility with chemiluminescence.</li>
		<li>Centrifuge at 17,000 &#215; g, 4 &#176;C, 30 min. Aspirate the supernatant and measure nitrite/nitrate levels, or store at -80 &#176;C for later use.</li>
	</ol>
	</li>
</ol>
4. Nitrite/nitrate measurement with nitric oxide analyzer (NOA)
<ol>
	<li>Prepare all samples by either of three different homogenization methods described above and inject them into an NOA for nitrate and nitrite measurement. 
	NOTE: Detailed protocols for NOA use were published previously19.</li>
</ol>

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biological+lifetime+of+nitric+oxide:+implications+for+the+perivascular+dynamics+of+NO+and+O2.">The biological lifetime of nitric oxide: implications for the perivascular dynamics of NO and O2.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (1), 355-360 (2001).</li><li>Lundberg, J. O., Weitzberg, E., Gladwin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nitrate-nitrite-nitric+oxide+pathway+in+physiology+and+therapeutics.">The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics.</a> Nature Reviews Drug Discovery. 7 (2), 156-167 (2008).</li><li>Govoni, M., Jansson, E. A., Weitzberg, E., Lundberg, J. 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M., Alving, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intragastric+nitric+oxide+production+in+humans:+measurements+in+expelled+air.">Intragastric nitric oxide production in humans: measurements in expelled air.</a> Gut. 35 (11), 1543-1546 (1994).</li><li>Larsen, F. J., Ekblom, B., Sahlin, K., Lundberg, J. O., Weitzberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+dietary+nitrate+on+blood+pressure+in+healthy+volunteers.">Effects of dietary nitrate on blood pressure in healthy volunteers.</a> New England Journal of Medicine. 355 (26), 2792-2793 (2006).</li><li>Kapil, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inorganic+nitrate+supplementation+lowers+blood+pressure+in+humans:+role+for+nitrite-derived+NO.">Inorganic nitrate supplementation lowers blood pressure in humans: role for nitrite-derived NO.</a> Hypertension. 56 (2), 274-281 (2010).</li><li>Jones, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+nitrate+supplementation+and+exercise+performance.">Dietary nitrate supplementation and exercise performance.</a> Sports Medicine. 44, 35-45 (2014).</li><li>Piknova, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+as+an+endogenous+nitrate+reservoir.">Skeletal muscle as an endogenous nitrate reservoir.</a> Nitric Oxide. 47, 10-16 (2015).</li><li>Wylie, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skeletal+muscle+nitrate+store:+influence+of+dietary+nitrate+supplementation+and+exercise.">Human skeletal muscle nitrate store: influence of dietary nitrate supplementation and exercise.</a> Journal of Physiology. 597 (23), 5565-5576 (2019).</li><li>Piknova, B., Schechter, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+nitrite+in+blood+samples+using+the+ferricyanide-based+hemoglobin+oxidation+assay.">Measurement of nitrite in blood samples using the ferricyanide-based hemoglobin oxidation assay.</a> Methods in Molecular Biology. 704, 39-56 (2011).</li><li>Piknova, B., Park, J. W., Cassel, K. S., Gilliard, C. N., Schechter, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Nitrite+and+Nitrate,+Metabolites+in+the+Nitric+Oxide+Pathway,+in+Biological+Materials+using+the+Chemiluminescence+Method.">Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method.</a> Journal of Visualized Experiments. (118), e54879 (2016).</li><li>Nyakayiru, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+nitrate+ingestion+increases+skeletal+muscle+nitrate+content+in+humans.">Sodium nitrate ingestion increases skeletal muscle nitrate content in humans.</a> Journal of Applied Physiology. 123 (3), 637-644 (2017).</li><li>Troutman, A. D., Gallardo, E. J., Brown, M. B., Coggan, A. 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The authors declare they have no conflicts of interest. Alan N. Schechter is listed as a co-inventor on several patents issued to the National Institutes of Health for the use of nitrite salts for the treatment of cardiovascular diseases. He receives royalties based on NIH licensing of these patents for clinical development but no other compensation. These arrangements do not affect his adherence to JoVE journal policies.

To monitor changes in the NO metabolites, nitrate and nitrite, as a function of physiological interventions, it is imperative to measure the levels of these ions in the different organs that are critical in their metabolism. As hemoglobin in blood will react with NO and its metabolites, it is also important to remove blood quickly from tissue samples as much as possible. Thus animals were perfused with saline before collecting skeletal muscle tissues (gluteus, TA, EDL, gastrocnemius muscle), and connective tissue and fat around the target muscle were immediately removed. For the homogenization solution, a &#39;stop solution&#39; was specially formulated with ferricyanide, NEM, and non-ionic surfactant to preserve nitrite in various tissues19. The presence of non-ionic detergent is critical for full disruption of cell membranes. In the case of the two homogenization machines used in this study, the rotary homogenizer and bead homogenizer, the volume of stop solution required for homogenization was 5x the tissue weight so that the whole tissue was fully submerged in the solution and processed homogeneously.
The homogenization process was performed three times in total (twice before adding methanol and one more time after adding methanol to precipitate proteins), each time using a programmed setting determined by the instrument manufacturers. It is important to place samples on ice immediately after each homogenization to prevent sample deterioration by the mild heating, which may be generated during homogenization. For the pulverization method, tissue samples were initially crushed into powder while keeping them frozen using liquid nitrogen, then samples were mixed with stop solution (5x tissue weight). In all three methods, methanol (10x tissue weight) was added to the samples, and the mixture was incubated on ice for 30 min to precipitate proteins and fully extract nitrate and nitrite from the tissues. The presence of proteins in sample can cause excessive foaming of the reaction solution in the chamber when using chemiluminescence, so it is necessary to precipitate proteins from the samples. Any precipitation agent can be used, but it is necessary to confirm that such agents will not interfere either with the chemiluminescence method or cause any changes in nitrite/nitrate concentrations by reacting with these ions.
Nitrate levels in all gluteus samples prepared by the three methods were very similar (39.6-45.2 nmol/g, Figure 4A), suggesting these three homogenization methods are equally applicable to preparing muscle samples for nitrate analysis. Although nitrite levels were slightly higher in samples prepared by pulverization compared to the two other methods (Figure 4B), all measured values were in a limited range (0.64-0.99 nmol/g). The effect of tissue sample size on nitrate and nitrite values in skeletal muscle samples was examined because researchers often deal with very small samples, particularly from small animals or human muscle biopsies. Neither nitrate nor nitrite levels were significantly different in the range from 20 to 200 mg of skeletal muscle tissue in these experiments (Figure 5).
However, it is noteworthy that, although not statistically significant, nitrate levels tend to be slightly lower when measured in larger samples (Figure 5). We have noticed this phenomenon over the course of our published and unpublished studies, but we do not understand the reasons for it. Therefore, it is probably important to consider the sample weights and ideally keep them consistent throughout a study when preparing homogenates from multiple tissue samples. Another consideration when working with smaller samples (20 mg) is that the final total volume of processed sample is only ~300 &#181;L, which significantly reduces the possibility to measure the same sample several times. However, especially for human studies, 20 mg is the usual acceptable size for muscle biopsies.
To examine whether different muscles in rat legs would have different nitrate/nitrite contents, the concentrations of those ions were compared in four different leg muscles (Figure 6). Both nitrate and nitrite levels were highest in the gluteus muscle compared to three other muscle tissues, although the differences did not reach statistical significance in this study, suggesting that nitrate metabolism in various muscle types may be governed differently. This phenomenon is being investigated in more detail currently.
Other research groups have also reported skeletal muscle nitrate concentrations, revealing significant variability in those values. The Coggan group showed that nitrate levels in Sprague Dawley rat soleus muscle vary from 62 to 124 nmol/g depending on muscle preparation methods21. The same group reported nitrate values in SD rat vastus lateralis (approximately 60 nmol/g) and soleus (approximately 215 nmol/g) in another study23. Ohtake et al. measured nitrate concentrations in mouse gastrocnemius muscle of &#62;300 nmol/g; however, precise methods for muscle homogenate preparation were not described24. The Verdijk group reported nitrate contents in human muscle biopsies (vastus lateralis) ranging from 54 to 80 nmol/g depending on participant age20; in a similar study, Wylie et al. report 226 nmol/g for the same muscle group17. These results suggest that the concentrations of nitrate/nitrite in skeletal muscle samples reflect many factors-physiological (e.g., exercise status), environmental (e.g., diet), and technical (assay details)-all of which presumably contribute to measurement variability.
We determined the coefficient of variability (CV) for all three methods presented here - see attached supplemental file. Even if CV are far from ideal, and vary among the methods used, our CV values are comparable with those published by Troutman et al. 21 using similar methods with different treatment of samples. Unfortunately, there is still no clear explanation why such high variability persists; this paper is the only published detailed protocol we know of for processing muscle samples for nitrate and nitrite determination.
We also measured linearity and degree of nitrate recovery from nitrate-spiked muscle samples (see attached supplemental file). When we added three different concentrations of nitrate into gluteus samples for homogenization, we obtained a good linear response in increase of nitrate concentrations with ~80% recovery of added nitrate for both bead and rotary homogenizers. These results demonstrate that both homogenization methods can be used for determination of nitrate and nitrite levels in biological samples with good confidence. The loss of 20% of added nitrate is likely happening during the deproteinization and centrifugation of muscle tissue homogenate when some ions might co-precipitate with charged proteins or other cell parts.
In general, we hope that our proposed method of muscle sample preparation for measurements of nitrate and nitrite will prove useful not only into the exercise research field, but also into clinical studies. There are neuromuscular and/or metabolic disorders affecting large populations that might relate to nitric oxide cycle malfunctioning and could benefit from nitrate supply. However, one has first to establish if NO deficiency, in fact, exists and if it can be corrected by dietary intervention. We hope that the method described here will enable the fate of nitrate and other metabolites of the NO cycle to be monitored in skeletal muscle and other organs
We are aware that, at this point of its development, methods of skeletal muscle preparation for nitrate and nitrite measurements using chemiluminescence (the most quantitative of all determinations of the nitrogen oxides themselves) still has unknown variables, some of which are discussed above. However, even with its limitations, the described methods allow reasonably accurate comparison of nitrate/nitrite levels of different organs, including different skeletal muscles, and should allow formulation of hypotheses that can be then tested with reasonable accuracy.

Nitric oxide (NO), a small gaseous signaling molecule, plays a critical role in physiological and pathophysiological processes1. NO can be produced from L-arginine by endogenous enzymes of the nitric oxide synthase (NOS) family before undergoing rapid oxidation to nitrate (NO3-) and, possibly, nitrite (NO2-) in blood and tissues2,3. Recently, these anions have been shown to be reduced back to NO in mammalian systems4. Nitrate is converted to nitrite, mainly by commensal bacterial nitrate reductases in the oral cavity acting on ions secreted by the salivary glands and directly ingested 5, and to some extent, by mammalian enzymes such as xanthine oxidoreductase6,7. Nitrite can be further reduced to NO by several mechanisms including deoxyhemoglobin8, deoxymyoglobin9, molybdenum-containing enzymes10, and non-enzymatic reduction in the presence of protons11,12.
This nitrate-nitrite-NO pathway is enhanced under hypoxic conditions wherein NOS activity is diminished because NOS requires oxygen for NO generation4. Many recent studies have reported beneficial effects of dietary nitrate on blood pressure regulation and exercise performance, suggesting that nitrate reduction pathways contribute to the augmentation of NO signaling13,14,15. Previous studies have shown that some skeletal muscles are likely the major nitrate storage places in the body16. Compared to blood or other internal organs such as liver, skeletal muscle (gluteus maximus) contains significantly higher levels of nitrate and has a substantial mass in the mammalian body. Treadmill exercise was shown to enhance nitrate reduction to nitrite and to NO in gluteus in a rat model7. These results imply that some skeletal muscles could be important sources for NO through nitrate reduction pathways in physiological situations. More recent studies suggest that these findings, including changes in muscle nitrate levels during exercise, also occurs in humans17.
Two of the current authors had previously established a method to measure nitrate and nitrite levels in blood and other liquid samples18. However, when the levels of these anions in tissue homogenates were initially analyzed, detailed protocols were not available. To understand the nitrate-nitrite-NO dynamics in several different organs, our goal was to develop an accurate and efficient method to measure nitrate and nitrite levels in mammalian tissues including skeletal muscle. In earlier studies, rodent tissues were used to develop reliable homogenization processes and then analyze nitrate and nitrite contents in those homogenates7,16,19. The usage of this homogenization method was extended to human skeletal muscle biopsy samples, whereby the values were confirmed, and importantly, the values observed for muscle compared to blood/plasma were in similar ranges and ratios to those observed in rodents17. In recent years, other groups also started measuring nitrate and nitrite levels in skeletal muscle homogenates, reporting comparable values to the ones reported by our group20,21.
The aim of this protocol paper is to describe in detail the preparation of skeletal muscle homogenates using three different homogenization methods for subsequent measurement of nitrate and nitrite levels. Additionally, the effects of tissue weight used for homogenization on values of nitrate and nitrite in skeletal muscle samples were examined. We believe that these methods can be easily applied to other types of mammalian tissues. Recently, especially in the field of exercise physiology, attention had been paid to the possible differences in nitrate/nitrite/NO physiology according to muscle groups. We also report the amounts of nitrate and nitrite in four different rodent muscles and find a nonuniform distribution of both ions among these different muscles; an observation which requires further study.

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			<td>gentleMACS dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td></td>
		</tr>
		<tr>
			<td>gentle MACS M tube</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-236</td>
			<td>Length: 87 mm; Diameter: 30 mm</td>
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			<td>Heparin Sodium</td>
			<td>Hospira</td>
			<td>NDC-0409-7620-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>NDC-10019-360-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>646377</td>
			<td></td>
		</tr>
		<tr>
			<td>Minilys bead homogenizer</td>
			<td>Bertin Instruments</td>
			<td>P000673-MLYS0-A</td>
			<td></td>
		</tr>
		<tr>
			<td>NEM; N-ethylmaleimide</td>
			<td>Sigma</td>
			<td>4260</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric Oxide analyzer</td>
			<td>GE</td>
			<td>Sievers NOA 280i</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40; 4-Nonylphenylpolyethylene glycol</td>
			<td>Sigma</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferricyanide; K3Fe(CN)6</td>
			<td>Sigma</td>
			<td>702587</td>
			<td></td>
		</tr>
		<tr>
			<td>Precellys lysing kit</td>
			<td>Bertin Instruments</td>
			<td>P000911-LYSK0-A</td>
			<td>contains 2 mL tubes with 2.8 mm ceramic (zirconium oxide) beads for homogenization</td>
		</tr>
		<tr>
			<td>Pulverizer kit</td>
			<td>Cellcrusher</td>
			<td>Cellcrusher kit</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of rat skeletal muscle homogenates for nitrate and nitrite measurements

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

To obtain representative results, skeletal muscle tissues from 8 Wistar rats (males and females, weight 250 &#177; 50 g) were used. Rat skeletal muscle homogenates (50 mg of gluteus maximus muscle for each method) were prepared by three different homogenization tools (rotary homogenizer, bead homogenizer, and pulverizer). The nitrate and nitrite contents of these homogenates were then determined using a nitric oxide analyzer (NOA) (Figure 4). Nitrate levels (Figure 4A) in those three homogenate samples were very similar to each other, ranging from 39.6 to 45.2 nmol/g.
Interestingly, nitrite levels (Figure 4B) in a homogenate sample prepared by a pulverizer showed the highest value (0.99 &#177; 0.15 nmol/g), and this was statistically different from the two other sample values. To test whether the size of tissues would affect the homogenization efficiency and the resulting nitrate and nitrite values, three different skeletal muscle tissue sizes (gluteus muscle tissue of 20, 50, and 200 mg) were used for homogenization by a bead homogenizer (Figure 5). Neither nitrate (Figure 5A) nor nitrite (Figure 5B) levels were significantly different between muscle sample sizes. However, slightly lower nitrate concentrations were observed when sample size was increased, in multiple experiments for reasons that are unclear.
Next, nitrate and nitrite levels in different kinds of rodent leg skeletal muscle tissues were measured (Figure 6). In addition to the gluteus maximus muscle, tibialis anterior (TA), extensor digitorum longus (EDL), and gastrocnemius muscles (50 mg each) were isolated from rat legs and homogenized using a bead homogenizer. Surprisingly, the nitrate levels of gluteus muscle (40.4 nmol/g) were approximately two-fold higher than those of the other three muscle tissues although in this particular experiment these differences did not reach statistical significance (Figure 6A). The nitrite concentration in the gluteus muscle was also higher than that of the other three muscle tissues and was significantly higher than that in the gastrocnemius sample (Figure 6B).
We determined the coefficient of variation for all three methods used and results are in attached table. For comparison, the table also shows coefficients of variation determined by Troutman et al21.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62427/62427fig01.jpg" /> 
Figure 1: Rotary homogenizer. (A) Homogenizer; (B) tube containing tissue and stop solution. Skeletal muscle tissue samples are placed in a tube with diluted stop solution and homogenized three times. Samples should be immediately placed on ice after each homogenization step. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62427/62427fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62427/62427fig02.jpg" /> 
Figure 2: Bead homogenizer. (A) Homogenizer; (B) beads containing tube. Skeletal muscle tissue samples are placed in a bead-containing tube (5 ceramic beads) with diluted stop solution and homogenized three times in total (45 s at highest speed for each time). Samples should be immediately placed on ice after each homogenization step. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62427/62427fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62427/62427fig03.jpg" /> 
Figure 3: Pulverizer. (A) pulverizer parts (B) pulverizer body - mortar and pestle assembled. (C) detail of mortar and pestle assembly. Pulverizer parts are cooled on dry ice for 30 minutes. Liquid nitrogen is put into the mortar and then pre-weighed skeletal muscle tissue is added. After 90 percent of the liquid nitrogen is gone, the top portion - the pestle - is inserted into the basin. The pestle is pounded with a mallet 5-6 times until sample is powdery. The powdered tissue is then transferred to a tube with stop solution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62427/62427fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62427/62427fig04.jpg" /> 
Figure 4: (A) Nitrate and (B) nitrite contents in gluteus muscle homogenates prepared by three different homogenization methods. Fifty milligrams of gluteus muscle were homogenized by three different homogenization methods. Methanol (500 &#956;L) was added to the homogenates to precipitate proteins. A standard chemiluminescence method with vanadium chloride or tri-iodide solution was used to measure nitrate and nitrite levels, respectively, in the clear supernatant after centrifugation; n=7 (nitrate); n=8 (nitrite); data are presented as averages &#177; SD, * p &#60; 0.05 using one-way ANOVA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62427/62427fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62427/62427fig05.jpg" /> 
Figure 5: (A) Nitrate and (B) nitrite contents in different sample sizes of gluteus muscle. Three different sizes of gluteus muscle homogenates were prepared by using a bead homogenizer. A standard chemiluminescence method with vanadium chloride or tri-iodide solution was used to measure nitrate and nitrite levels, respectively, in the clear supernatant after centrifugation; n=7 (nitrate); n=8 (nitrite); data are presented as averages &#177; SD, <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62427/62427fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62427/62427fig06.jpg" /> 
Figure 6: (A) Nitrate and (B) nitrite contents in four different muscles. Each muscle (50 mg) was homogenized by using a bead homogenizer, and a standard chemiluminescence method with vanadium chloride or tri-iodide solution was used to measure nitrate and nitrite levels, respectively; n=4-7 (nitrate); n=3-8 (nitrite); data are presented as averages &#177; SD, * p &#60; 0.05 using one-way ANOVA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62427/62427fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62427/JoVE_Tables.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements at JoVE.com

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...

preparation-rat-skeletal-muscle-homogenates-for-nitrate-nitrite

Nanyang Technological University

Research

JoVE Journal

Biology

2.2K Views.  National Institutes of Health. We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization. 

Video: Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.