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

Studies of rats have indicated that skeletal muscle plays a central role in whole-body nitrate ( $$ )/nitrite ( $$ )/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 $$ and $$ in biopsy-sized muscle samples. $$ and $$ 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, $$ and $$ were measured using HPLC. Mechanical homogenization + ultrasound resulted in the lowest $$ content (62 ± 20 pmol/mg), with high variability [coefficient of variation (CV) >50%] across samples from the same muscle. The $$ / $$ ratio (0.019 ± 0.006) was also elevated, suggestive of $$ reduction during tissue processing. Bead beating or pulverization yielded lower $$ and slightly higher $$ levels, but reproducibility was still poor. Pulverization + 0.5% Triton X-100 provided the highest $$ content (124 ± 12 pmol/mg) and lowest $$ / $$ ratio (0.008 ± 0.001), with the least variability between duplicate samples (CV ~15%). These values are consistent with literature data from larger rat muscle samples analyzed using chemiluminescence. Samples were stable for at least 5 wk at -80°C, provided residual xanthine oxidoreductase activity was blocked using 0.1 mmol/l oxypurinol. We have developed a method capable of measuring $$ and $$ in <1 mg of muscle. This method should prove highly useful in investigating the role of skeletal muscle in $$ / $$ /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.