Reliability of biochemical assays of nitric oxide biomarkers in human skeletal muscle tissue samples

Accurate measurements of nitrate and nitrite concentrations in human skeletal muscle are fundamental to further our understanding of their role in nitric oxide (NO) homeostasis. We investigated the effects of saline wash and immediate processing with a potassium ferricyanide-containing nitrite-preserving stop solution (to remove the effects of haemoglobin from blood surrounding the tissue) on human skeletal muscle nitrate and nitrite concentrations. Skeletal muscle tissue was collected from 24 healthy adults and processed with and without saline wash, and with and without stop solution. Tissue samples (n = 17) washed in saline had significantly lower nitrate concentration (p < 0.01) and lower inter-sample variability (45 ± 23 nmol/g) compared to tissue samples not washed in saline (104 ± 52 nmol/g), with no correlation and high bias (-59 ± 49 nmol/g) between the two methods. Nitrite concentration was not significantly different between tissue samples washed in saline (1.94 ± 0.90 nmol/g) and samples not washed in saline (1.55 ± 0.86 nmol/g), with no correlation and minimal bias (0.39 ± 0.85 nmol/g) between the two methods. Addition of nitrite-preserving stop solution did not result in significant differences in concentrations of nitrate or nitrite. In summary, although our study is limited by the lack of Hb and Mb measurements, saline washing of biopsied tissue samples improved the reliability of nitrate and nitrite concentrations measured in human skeletal muscle.