Measurement Of Nitrate And Nitrite In Biopsy-Sized Muscle Samples

Abstract

Studies of rats have demonstrated that skeletal muscle plays a central role in whole-body nitrate (NO3)/nitrite (NO2-)/nitric oxide (NO) metabolism. The small size of human muscle biopsy samples, however, presents an analytical challenge in this context. Indeed, a recent study by Nyakayiru et al. (J Appl Physiol 2017; 123:637-644) reported that NO - was below the limit of detection (LOD) using the “gold standard” chemiluminescent method even when assaying 40 mg of tissue. PURPOSE: To develop a method to precisely and accurately quantify the NO3- and NO2- content of biopsy-sized muscle samples. METHODS: NO3- and NO2- were extracted from rat soleus muscle samples using methanol combined with mechanical homogenization + ultrasound, bead beating, pulverization, or pulverization + 0.5% Triton X100. After centrifugation to remove precipitated proteins, NO3- and NO2- were measured using a dedicated high performance liquid chromatography analyzer with a LOD of <0.5 pmol. RESULTS: Mechanical homogenization + ultrasound resulted in the lowest NO3- content (62±16 pmol/mg), with high variability (CV >50%) across samples from the same muscle. NO2- content (1.00±0.18 pmol/mg) was also elevated, suggestive of NO3- reduction during tissue processing. Bead beating or pulverization yielded higher NO3- and lower NO2- levels, but reproducibility was still poor. Pulverization + 0.5% Triton X100 provided the highest NO3- (97±15 pmol/mg) and lowest NO2- (0.59±0.16 pmol/mg) contents, with the least variability (CV ~15%). These values are consistent with literature data from larger rat muscle samples analyzed using the chemiluminescent approach. CONCLUSIONS: We have developed a method capable of measuring NO3- and NO2- in muscle samples as small as 10 mg. The theoretical limit is even lower, i.e., 1 mg for both NO3- and NO2-and 5 ng for NO3- alone. This method should prove highly useful in investigating the role of skeletal muscle NO3-/NO2-/NO metabolism in both healthy and diseased subject populations, in response to exercise and dietary interventions, etc.