Measurement of nitrate and nitrite in biopsy-sized muscle samples using HPLC

Abstract

Studies of rats have indicated that skeletal muscle plays a central role in whole-body nitrate (NO3)/nitrite (NO2)/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 N2 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 ~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 80°C, provided residual xanthine oxi-doreductase 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.