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>

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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.

<table><tbody><tr><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></tr><tr><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. Skeletspieren dienen als een belangrijk reservoir van nitraat dat, na de omzetting in nitriet, dient als een bron van NO tijdens het sporten. Hier presenteren we methodologie om deze ionen in spieren en andere weefsels te meten. Bereid om te beginnen de nitraatconserverende of stopoplossing door 890 millimolair kaliumfercyanide en 118 millimolair n-methylmaleimide in gedestilleerd water te combineren, ervoor te zorgen dat er geen kristallen in oplossing achterblijven, voeg vervolgens een niet...

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

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

Biochemistry

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

Medicine

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

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 ...

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 ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

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