Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Ji Won Park; Samantha M. Thomas; Lee J. Wylie; Andrew M. Jones; Anni Vanhatalo; Alan N. Schechter; Barbora Piknova

doi:10.3791/62427

Herstellung von Ratten-Skelettmuskelhomogenaten für Nitrat- und Nitritmessungen

JoVE Journal
Biology

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Biology

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Herstellung von Ratten-Skelettmuskelhomogenaten für Nitrat- und Nitritmessungen

Full Text

2,825 Views

07:19 min

July 29, 2021

DOI: 10.3791/62427-v

Ji Won Park*¹, Samantha M. Thomas*¹, Lee J. Wylie², Andrew M. Jones², Anni Vanhatalo², Alan N. Schechter¹, Barbora Piknova¹

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

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study investigates the measurement of nitrate and nitrite levels in rat skeletal muscle tissue, which serve as sources of nitric oxide during exercise. Three homogenization methods are compared to evaluate their effectiveness in preserving these ions and the impact of different tissue weights on the results.

Key Study Components

Research Area

Muscle biology
Metabolic pathways
Analytical chemistry

Background

Nitrate and nitrite are crucial for nitric oxide production.
Skeletal muscle is a significant reservoir for these ions.
Understanding their levels can enhance knowledge of muscle physiology and exercise performance.

Methods Used

Homogenization techniques: rotary homogenizer, bead homogenizer, and pulverizer.
Rat skeletal muscle as the biological system.
Measurement of nitrite and nitrate levels using spectrophotometric methods.

Main Results

Homogenization methods yielded similar nitrate levels.
Pulverizer method produced significantly higher nitrite levels.
No significant difference was found in ion levels across various tissue weights.

Conclusions

The methodologies developed are effective for small samples.
Findings contribute to understanding the role of nitrates and nitrites in muscle function.

Frequently Asked Questions

What is the significance of measuring nitrate and nitrite levels in muscle?

These compounds are key for nitric oxide production, which plays a vital role in muscle physiology and exercise performance.

What methods were compared in this study?

Three methods were compared: rotary homogenizer, bead homogenizer, and pulverizer.

Did tissue sample size affect the results?

No significant differences in nitrate or nitrite levels were found between different sample sizes.

Which homogenization method proved to be the best for nitrite measurement?

The pulverizer method showed the highest nitrite levels compared to the other methods.

What implications do these findings have for future research?

The results can improve experimental designs focused on muscle metabolism and tissue analysis in exercise physiology.

Are the methods applicable to other tissues?

Yes, the protocols can be adapted for measuring nitrate and nitrite in other types of tissues.

Wir präsentieren Protokolle für drei verschiedene Methoden zur Homogenisierung von vier verschiedenen Muskelgruppen von Ratten-Skelettmuskelgewebe, um die Nitrat- und Nitritwerte zu messen und zu vergleichen. Darüber hinaus vergleichen wir verschiedene Probengewichte, um zu untersuchen, ob die Gewebeprobengröße die Ergebnisse der Homogenisierung beeinflusst.

Die Skelettmuskulatur dient als Hauptreservoir für Nitrat, das nach seiner Umwandlung in Nitrit als NO-Quelle während des Trainings dient. Hier stellen wir die Methodik vor, um diese Ionen in Muskel- und anderen Geweben zu messen. Um zu beginnen, bereiten Sie die nitratkonservierende oder stoppende Lösung vor, indem Sie 890 Millimolar Kaliumferricyanid und 118 Millimolar n-Methylmaleimid in destilliertem Wasser kombinieren, um sicherzustellen, dass keine Kristalle in Lösung verbleiben, dann fügen Sie ein nichtionisches Tensid in einem Verhältnis von 1 zu 9 hinzu und mischen Sie vorsichtig, um Schaumbildung zu vermeiden.

Die Stopplösung wird im Verhältnis 1 zu 9 mit destilliertem Wasser verdünnt und die verdünnte Stopplösung in das Homogenisierungsröhrchen gegeben. Als nächstes tauen Sie das zuvor isolierte und gefrorene Rattenmuskelgewebe auf Eis auf. Sobald das Gewebe aufgetaut ist, entfernen Sie das restliche Fett und Bindegewebe aus der Skelettmuskulatur.

View the full transcript and gain access to thousands of scientific videos