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 ( NO−3 )/nitrite ( NO−2 )/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 NO−3 and NO−2 in biopsy-sized muscle samples. NO−3 and NO−2 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, NO−3 and NO−2 were measured using HPLC. Mechanical homogenization + ultrasound resulted in the lowest NO−3 content (62 ± 20 pmol/mg), with high variability [coefficient of variation (CV) >50%] across samples from the same muscle. The NO−2 / NO−3 ratio (0.019 ± 0.006) was also elevated, suggestive of NO−3 reduction during tissue processing. Bead beating or pulverization yielded lower NO−2 and slightly higher NO−3 levels, but reproducibility was still poor. Pulverization + 0.5% Triton X-100 provided the highest NO−3 content (124 ± 12 pmol/mg) and lowest NO−2 / NO−3 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 oxidoreductase activity was blocked using 0.1 mmol/l oxypurinol. We have developed a method capable of measuring NO−3 and NO−2 in <1 mg of muscle. This method should prove highly useful in investigating the role of skeletal muscle in NO−3 / NO−2 /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.