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 (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{plus minus}20 pmol/mg), with high variability (CV >50%) across samples from the same muscle. The NO2 - :NO3 - ratio (0.019{plus minus}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{plus minus}12 pmol/mg) and lowest NO2 - :NO3 - ratio (0.008{plus minus}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{degree sign} 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.