Direct detection and quantification of singlet oxygen during
ischemia and reperfusion in rat hearts.
Zhai, Xiaolin, and Muhammad Ashraf.
Department of Pathology and Laboratory Medicine, University of
Cincinnati Medical Center, Cincinnati, Ohio 45267
APStracts 2:0154H, 1995.
Direct detection and quantification of singlet oxygen (1O2) are
important in understanding its role in the pathogenesis of
postischemic reperfusion injury. To detect 1O2 in the postischemic
reperfused hearts, 5, 8-endoperoxide, which was produced by the
oxidation of [beta]-carotene by 1O2 and used as a marker for 1O2
generation, was quantified using high performance liquid
chromatography (HPLC). After initial perfusion with 25 [mu]M [beta]
-carotene, isolated rat hearts were subjected to various periods of
ischemia for 5, 10, 20, 30, and 60 minutes followed by 10 minute
reperfusion with buffer containing [beta]-carotene. The coronary
effluent was collected, extracted with ether, and injected into the
HPLC unit. Perfusion with buffer containing 25 [mu]M [beta]-carotene
did not affect cardiac function and enzyme release. 5, 8-Endoperoxide
was undetectable in the coronary effluent before ischemia. The
production of 5, 8-endoperoxide was maximum within 120 seconds of
reperfusion and decreased with further reperfusion. The production of
1O2 was progressively increased, as the ischemic time was prolonged.
Maximal accumulated amount of 1O2 during 10 minute reperfusion was
observed in hearts subjected to 60 minute ischemia (36.2 +/- 1.7
nmol/10 min/g). Meanwhile, there was a good correlation between the
amount of 1O2 production and cardiac function after 10 minute
reperfusion at various ischemic time intervals. Treatment with 25 mM
histidine, a 1O2 scavenger, significantly decreased 5, 8-endoperoxide
from 7.02 +/- 0.47 to 0.98 +/- 0.11 nmol/min/g (p < 0.01) and
improved cardiac function in the group with 60 minute ischemia. This
study demonstrates that (1) the present method is useful and reliable
for the measurement of 1O2 in the heart; (2) 1O2 production during
reperfusion is dependent upon the duration of initial ischemia; and
(3) 1O2 is one of the major factors in the postischemic reperfusion
injury.
Received 5 October 1994; accepted in final form 29 March 1995.
APS Manuscript Number H894-4.
Article publication pending Am. J. Physiol. (Heart Circ. Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 25 April 1995.