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Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Published: July 29th, 2021



1Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 2Sport and Health Sciences, College of Life and Environmental Sciences, St Luke’s Campus, University of Exeter
* These authors contributed equally

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 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.

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 ac....

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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

  1. 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.......

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To obtain representative results, skeletal muscle tissues from 8 Wistar rats (males and females, weight 250 ± 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

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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.......

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


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

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