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. 2018 Nov 1;125(5):1475-1481.
doi: 10.1152/japplphysiol.00625.2018. Epub 2018 Aug 16.

Measurement of Nitrate and Nitrite in Biopsy-Sized Muscle Samples Using HPLC

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Free PMC article

Measurement of Nitrate and Nitrite in Biopsy-Sized Muscle Samples Using HPLC

Ashley D Troutman et al. J Appl Physiol (1985). .
Free PMC article

Abstract

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 ± 20 pmol/mg), with high variability [coefficient of variation (CV) >50%] across samples from the same muscle. The NO2- / NO3- ratio (0.019 ± 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 ± 12 pmol/mg) and lowest NO2- / NO3- 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 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. 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.

Keywords: high performance liquid chromatography; human muscle; nitrate; nitric oxide; nitrite.

Figures

Fig. 1.
Fig. 1.
Representative nitrite (NO2) and nitrate (NO3) standard curves.
Fig. 2.
Fig. 2.
Nitrate (NO3) content (top) and nitrite (NO2)/NO3 ratio (bottom) of rat soleus muscles processed by mechanical homogenization plus ultrasound (n = 4), pulverization at liquid N2 temperature (n = 6), bead homogenization (n = 6), or pulverization plus 0.5% Triton X-100 (n = 14). Data are means ± SE. Significance of differences determined by one-way ANOVA. Homog, homogenization; Pulv, pulverization; US, ultrasound.
Fig. 3.
Fig. 3.
Effect of addition of 0.1 mmol/l oxypurinol to the methanol plus 0.5% Triton X-100 extraction medium on the nitrite (NO2)/nitrate (NO3) ratio of samples stored at −80°C for up to 5 wk. *P < 0.01 versus day 0 value for same samples. Data are means ± SE for n = 6 samples without (w/o) oxypurinol and n = 8 for samples with (w/) oxypurinol. Significance of differences determined by two-way (i.e., treatment × time) ANOVA, with time as a repeated measure.
Fig. 4.
Fig. 4.
Sample HPLC chromatogram showing nitrite (NO2) (inset) and nitrate (NO3) peaks from 6.75 mg of rat soleus muscle processed by pulverization at liquid N2 temperature followed by extraction with 50 µl of methanol containing 0.5% Triton X-100 and 0.1 mmol/l oxypurinol. Ten-microliter injection. Measured NO2 content = 0.482 pmol/mg; NO3 content = 111.1 pmol/mg.

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