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 nic...

<ol>
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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. 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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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</s...

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 ...

Skeletal muscle serves as a major reservoir of nitrate which, after its conversion to nitrite, serves as a source of NO during exercise. Here we present methodology to measure these ions in muscle and other tissues. To begin, prepare the nitrate preserving or stop solution by combining 890 millimolar potassium ferricyanide and 118 millimolar n-methylmaleimide in distilled water, ensuring that no crystals remain in solution, then add a non-ionic surfactant in a 1-to-9 ratio and mix gently to avoid foaming.Dilute the stop solution in a 1-to-9 ratio with distilled water and add the diluted stop solution to the homogenization tube. Next, thaw the previous isolated and frozen rat muscle tissue on ice. Once the tissue has thawed, remove the remaining fat and connective tissue from the skeletal muscle.Cut off pieces of the skeletal muscle and blot them on a gauze to dry. Weigh out the appropriate amount of skeletal muscle tissue, then place the pre-weight tissue in the homogenization tubes containing the stop solution. For homogenization in a rotary homogenizer, place the M-type tube containing the pre-weighed skeletal muscle and pre-measured stop solution into the machine.Homogenize each sample twice, and after each homogenization, place the tube on ice immediately for 5 minutes to cool down. After homogenization, centrifuge the tube briefly. Place the tube back on ice and add the appropriate volume of methanol before vortexing For 15 seconds.Homogenize the sample again and incubate it on ice for 30 minutes, then centrifuge the sample, aspirate the supernatant, and proceed to measuring the nitrite and nitrate levels. For bead homogenization, place the skeletal muscle tissue in a bead-containing tube and homogenize twice for 45 seconds at the highest speed available on the instrument. After each homogenization, immediately place the tube on ice for 5 minutes to cool down, then centrifuge the tube, place the tube back on ice, and add the appropriate volume of methanol before vortexing For 15 seconds.Homogenize the sample once again for 45 seconds then incubate it on ice for 30 minutes. Centrifuge the sample, aspirate the supernatant, and proceed to measuring the nitrite and nitrate levels. For pulverizer-based homogenization, prepare tubes containing the diluted stop solution, then weigh and record the weight of the tubes.After cooling the pulverizer tool on dry ice for 30 minutes, using tweezers chilled in liquid nitrogen, transfer the pre-weighed tissue sample to the pulverizer, then add a small amount of liquid nitrogen to ensure the tissue is at liquid nitrogen temperature. After 95%of the liquid nitrogen has vaporized, place the crushing tool on top of the tissue and press firmly to feel the sample crush, then using a mallet, strike the crushing tool 3 to 5 times. When the whole sample has been pulverized, using a liquid nitrogen-cooled spoon and tweezers, directly transfer the crushed tissue into the pre-weighed tube containing the diluted stop solution.Next, vortex the tube for 15 seconds, then open the tube to check for any tissue stuck in the lid. If there is, try to dislodge it, then vortex again. Briefly centrifuge the sample for 2 to 3 seconds.Weigh the tube again and calculate the tissue weight by deducting the original tube weight from this new weight. Place the tube on ice. Add an appropriate volume of methanol to the tube and vortex the tube thoroughly for 15 seconds before incubating it on ice for 30 minutes, then centrifuge the sample, aspirate the supernatant, and proceed to measuring the nitrite and nitrate levels.Nitrate levels and rat skeletal muscle homogenates prepared using a rotary homogenizer, bead homogenizer, and pulverizer were similar. Interestingly, for nitrite levels, the homogenate sample prepared by a pulverizer showed the highest value, which was statistically different from the other two sample values. A comparison of nitrite and nitrate levels in gluteus homogenates prepared from 20, 50, and 200 milligrams of tissue using a bead homogenizer showed that neither the nitrate nor nitrite levels were significantly different between the muscle sample sizes.Comparing nitrate levels in different rodent leg skeletal muscle tissues revealed that the gluteus muscle had approximately twofold higher nitrate levels than the other three muscle tissues. However, these differences did not reach statistical significance. Similar to the nitrate levels, the nitrate concentration in the gluteus muscle was also higher than that in the other three muscle tissues and was significantly higher than that in the gastrocnemius sample.Our method is relatively simple and well-suitable to small samples while preserving nitrate and nitrite during the sample preparation.

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2.1K visualizações.  National Institutes of Health. O músculo esquelético serve como um importante reservatório de nitrato que, após sua conversão em nitrito, serve como fonte de NO durante o exercício. Aqui apresentamos metodologia para medir esses íons no músculo e outros tecidos. Para começar, prepare a solução de preservação ou parada de nitrato combinando 890 milimolares de ferricianeto de potássio e 118 milimolares de n-metilmaleimida em água destilada, garantindo que nenhum cristal permaneça em solução, em seguida, adi...