College of Pharmacy, Wonkwang University, Iksan, Republic of Korea.
School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea.
J Pharm Biomed Anal. 2019 Feb 20;165:101-111. doi: 10.1016/j.jpba.2018.11.051. Epub 2018 Nov 24.
Although major adverse effects associated with nonsteroidal anti-inflammatory drugs (NSAIDs) are gastric injury, assessment of NSAIDs-induced gastrointestinal adverse effects is mostly dependent on endoscopy due to the lack of plasma biomarkers. Several amino acids associated with collagenase activity and gastric mucosal mass have been suggested as plasma biomarker candidates for gastric injury. Therefore, this study aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the plasma biomarker candidates, i.e., acetylcarnitine, proline, hydroxyproline, citrulline, and arginine and evaluate their potential as a biomarker for NSAIDs-induced gastric injury. The method utilized simple protein precipitation with methanol and D-citrulline as an internal standard (IS). The assay resulted in the lower limit of quantification (LLOQ) of 0.1 μg/mL for acetylcarnitine and 1 μg/mL for proline, hydroxyproline, citrulline, and arginine in the surrogate blank plasma. The intra- and inter-day accuracy ranged 82.5-111.2% for acetylcarnitine, 95.4-103.3% for proline, 98.9-106.4% for hydroxyproline, 99.5-103.5% for citrulline, and 87.4-105.3% for arginine. The precision was within 6.17%, 3.63%, 6.20%, 6.31%, and 6.17% for acetylcarnitine, proline, hydroxyproline, citrulline, and arginine, respectively. The developed assay was successfully applied to monitor the changes of the plasma levels of the five amino acids in rats and Beagle dogs following repeated oral administrations of aceclofenac. In rats, plasma concentrations of proline, hydroxyproline, and citrulline were significantly reduced after 4 days of aceclofenac administration compared to the control group. In dogs, plasma concentrations of proline and citrulline were significantly decreased after 7 days of aceclofenac administration compared to those obtained after the first aceclofenac administration. These data indicate that plasma levels of proline, hydroxyproline, and citrulline may be used as quantitative biomarkers of NSAIDs-induced gastric damage. The present assay could also be utilized to monitor the changes of these amino acids as potential indicators for various physiological and pathophysiological conditions.
虽然与非甾体抗炎药 (NSAIDs) 相关的主要不良反应是胃损伤，但由于缺乏血浆生物标志物，对 NSAIDs 诱导的胃肠道不良反应的评估主要依赖于内窥镜检查。一些与胶原酶活性和胃黏膜质量相关的氨基酸已被提议作为胃损伤的血浆生物标志物候选物。因此，本研究旨在开发一种用于血浆生物标志物候选物，即乙酰肉碱、脯氨酸、羟脯氨酸、瓜氨酸和精氨酸的液相色谱-串联质谱 (LC-MS/MS) 方法，并评估它们作为 NSAIDs 诱导的胃损伤生物标志物的潜力。该方法利用甲醇简单的蛋白沉淀法和 D-瓜氨酸作为内标 (IS)。该方法在替代空白血浆中的乙酰肉碱和脯氨酸、羟脯氨酸、瓜氨酸和精氨酸的定量下限 (LLOQ) 分别为 0.1μg/mL 和 1μg/mL。乙酰肉碱的日内和日间准确度范围为 82.5-111.2%，脯氨酸为 95.4-103.3%，羟脯氨酸为 98.9-106.4%，瓜氨酸为 99.5-103.5%，精氨酸为 87.4-105.3%。精密度分别为乙酰肉碱、脯氨酸、羟脯氨酸、瓜氨酸和精氨酸的 6.17%、3.63%、6.20%、6.31%和 6.17%。该方法已成功应用于监测重复口服给药后大鼠和比格犬五种氨基酸的血浆水平变化。在大鼠中，与对照组相比，连续 4 天给予醋氯芬酸后，脯氨酸、羟脯氨酸和瓜氨酸的血浆浓度显著降低。在犬中，与首次给予醋氯芬酸后相比，连续 7 天给予醋氯芬酸后，脯氨酸和瓜氨酸的血浆浓度显著降低。这些数据表明，脯氨酸、羟脯氨酸和瓜氨酸的血浆水平可能可作为 NSAIDs 诱导的胃损伤的定量生物标志物。本研究建立的方法还可以用于监测这些氨基酸的变化，作为各种生理和病理生理条件的潜在指标。
