Measurement Of Nitrate And Nitrite In Biopsy-Sized Muscle Samples

Studies of rats have indicated that skeletal muscle plays a central role in whole-body nitrate (NO3-)/nitrite (NO3-)/nitric oxide (NO) metabolism. Extending these results to humans, however, is challenging due to the small size of needle biopsy samples. We therefore developed a method to precisely and accurately quantify NO3- and NO2- in biopsy-sized muscle samples. NO3- and NO2- were extracted from rat soleus samples using methanol combined with mechanical homogenization + ultrasound, bead beating, pulverization at liquid N-2 temperature or pulverization + 0.5% Triton X-100. After centrifugation to remove proteins, NO3- and NO2- were measured using HPLC. Mechanical homogenization + ultrasound resulted in the lowest NO3- content (62 +/- 20 pmol/mg), with high variability [coefficient of variation (CV) >50%] across samples from the same muscle. The NO2-/NO3- ratio (0.019 +/- 0.006) was also elevated, suggestive of NO3- reduction during tissue processing. Bead beating or pulverization yielded lower NO2- and slightly higher NO3- levels, but reproducibility was still poor. Pulverization + 0.5% Triton X-100 provided the highest NO3- content (124 +/- 12 pmol/mg) and lowest NO2-/NO3- ratio (0.008 +/- 0.001), with the least variability between duplicate samples (CV similar to 15%). These values are consistent with literature data from larger rat muscle samples analyzed using chemiluminescence. Samples were stable for at least 5 wk at similar to 80 degrees C, provided residual xanthine oxidoreductase activity was blocked using 0.1 mmol/l oxypurinol. We have developed a method capable of measuring NO3- and NO2- in <1 mg of muscle. This method should prove highly useful in investigating the role of skeletal muscle in NO3-/NO2-/NO metabolism in human health and disease. NEW & NOTEWORTHY Measurement of nitrate and especially nitrite in small, i.e., biopsy-sized, muscle samples is analytically challenging. We have developed a precise, accurate, and convenient method for doing so using an affordable commercial HPLC system.