TY - JOUR
T1 - Hydroxylation of salicylate by the in vitro diaphragm
T2 - Evidence for hydroxyl radical production during fatigue
AU - Diaz, P. T.
AU - She, Z. W.
AU - Davis, W. B.
AU - Clanton, T. L.
PY - 1993
Y1 - 1993
N2 - There is increasing evidence that oxygen-derived free radicals produced during strenuous work by the diaphragm may contribute to diaphragm fatigue and/or injury. However, the precise identity of these oxygen radicals remains unknown, inasmuch as oxygen free radicals are extremely short lived and their detection in biologic systems is quite difficult. There is recent evidence that the salicylate-trapping method may be a useful means of monitoring tissue production of hydroxyl radical (·OH). This method is predicated on the fact that salicylate's phenolic ring can be attacked by ·OH at the 3 or 5 position to yield 2,3- or 2,5-dihydroxybenzoic acid (DHB). These metabolites are stable and can be identified by high-performance liquid chromatography (HPLC) coupled with electrochemical or ultraviolet detection. To test the hypothesis that hydroxylated salicylates are produced during diaphragm fatigue, we exposed in vitro rat diaphragm strips to a physiological saline solution containing 2.0 mM sodium salicylate for ~15 min. The solution was then removed, and the strips were fatigued (20 Hz, 200- ms train duration, 1 train/s) via phrenic nerve stimulation for 30 s-10 min. The diaphragm strips were subsequently homogenized, and the homogenate was analyzed by HPLC coupled with ultraviolet detection. Levels of 2,3-DHB were significantly higher in fatigued than in control nonfatigued strips. There was also a significant correlation between the amount of 2,3-DHB in the fatigued muscle and the accumulated tension-time product developed during fatigue, 2,5-DHB was not consistently identified in control or experimental strips. We conclude that hydroxylation of salicylates occurs during low- frequency fatigue in the in vitro rat diaphragm; this provides strong evidence for ·OH production in this setting. The analysis of hydroxylated salicylates by HPLC has potential utility in delineating the role of oxidant stress in the generation of diaphragm fatigue and/or injury.
AB - There is increasing evidence that oxygen-derived free radicals produced during strenuous work by the diaphragm may contribute to diaphragm fatigue and/or injury. However, the precise identity of these oxygen radicals remains unknown, inasmuch as oxygen free radicals are extremely short lived and their detection in biologic systems is quite difficult. There is recent evidence that the salicylate-trapping method may be a useful means of monitoring tissue production of hydroxyl radical (·OH). This method is predicated on the fact that salicylate's phenolic ring can be attacked by ·OH at the 3 or 5 position to yield 2,3- or 2,5-dihydroxybenzoic acid (DHB). These metabolites are stable and can be identified by high-performance liquid chromatography (HPLC) coupled with electrochemical or ultraviolet detection. To test the hypothesis that hydroxylated salicylates are produced during diaphragm fatigue, we exposed in vitro rat diaphragm strips to a physiological saline solution containing 2.0 mM sodium salicylate for ~15 min. The solution was then removed, and the strips were fatigued (20 Hz, 200- ms train duration, 1 train/s) via phrenic nerve stimulation for 30 s-10 min. The diaphragm strips were subsequently homogenized, and the homogenate was analyzed by HPLC coupled with ultraviolet detection. Levels of 2,3-DHB were significantly higher in fatigued than in control nonfatigued strips. There was also a significant correlation between the amount of 2,3-DHB in the fatigued muscle and the accumulated tension-time product developed during fatigue, 2,5-DHB was not consistently identified in control or experimental strips. We conclude that hydroxylation of salicylates occurs during low- frequency fatigue in the in vitro rat diaphragm; this provides strong evidence for ·OH production in this setting. The analysis of hydroxylated salicylates by HPLC has potential utility in delineating the role of oxidant stress in the generation of diaphragm fatigue and/or injury.
KW - free radicals
KW - high- performance liquid chromatography
KW - hydroxybenzoic acids
KW - rats
KW - respiratory muscles
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U2 - 10.1152/jappl.1993.75.2.540
DO - 10.1152/jappl.1993.75.2.540
M3 - Article
C2 - 8226451
AN - SCOPUS:0027257542
SN - 8750-7587
VL - 75
SP - 540
EP - 545
JO - Journal of Applied Physiology
JF - Journal of Applied Physiology
IS - 2
ER -