Measurement of nitrate and nitrite in biopsy-sized muscle samples using HPLC.
Measurement of nitrate and nitrite in biopsy-sized muscle samples using HPLC.
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使用 HPLC 测量活检大小的肌肉样品中的硝酸盐和亚硝酸盐。
DOI：
10.1152/japplphysiol.00625.2018
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Coggan,AndrewR
中科院分区：
文献类型：
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作者：
Troutman,AshleyD;Gallardo,EdgarJ;Brown,MaryBeth;Coggan,AndrewR
对大鼠的研究表明，骨骼肌在全身硝酸盐 ()/亚硝酸盐 ()/一氧化氮 (NO) 代谢中发挥着核心作用。然而，由于针吸活检样本尺寸较小，将这些结果扩展到人类具有挑战性。因此，我们开发了一种方法来精确定量活检大小的肌肉样本，并使用甲醇结合机械匀浆 + 超声波、珠磨、液氮温度粉碎或粉碎 + 0.5% Triton X-100 从大鼠比目鱼肌样本中提取。离心去除蛋白质后，用HPLC进行测定。机械均质+超声产生最低含量（62±20 pmol/mg），来自同一肌肉的样品具有高变异性[变异系数（CV）> 50%]。 / 比率 (0.019±0.006) 也升高，表明在组织处理过程中减少。珠磨或粉碎产生较低和稍高的水平，但再现性仍然很差。粉碎 + 0.5% Triton X-100 提供最高含量 (124±12 pmol/mg) 和最低比率 (0.008±0.001)，重复样品之间的变异性最小 (CV ~15%)。这些值与使用化学发光分析的较大大鼠肌肉样本的文献数据一致。如果使用 0.1 mmol/l 奥嘌呤醇阻断残余黄嘌呤氧化还原酶活性，则样品在 -80°C 下可稳定至少 5 周。我们开发了一种能够测量 <1 毫克肌肉的方法。这种方法在研究骨骼肌在人类健康和疾病中一氧化氮代谢中的作用方面应该非常有用。新的和值得注意的小样本（即活检大小的肌肉样本）中硝酸盐（尤其是亚硝酸盐）的测量在分析上具有挑战性。我们开发了一种精确、准确且方便的方法，使用经济实惠的商业 HPLC 系统来实现这一目的。
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 quantifyandin biopsy-sized muscle samples.andwere extracted from rat soleus samples using methanol combined with mechanical homogenization + ultrasound, bead beating, pulverization at liquid N2temperature or pulverization + 0.5% Triton X-100. After centrifugation to remove proteins,andwere measured using HPLC. Mechanical homogenization + ultrasound resulted in the lowestcontent (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 ofreduction during tissue processing. Bead beating or pulverization yielded lowerand slightly higherlevels, but reproducibility was still poor. Pulverization + 0.5% Triton X-100 provided the highestcontent (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 measuringandin <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 & NOTEWORTHYMeasurement 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.