Pivotal role of glutathione depletion in plasma-induced endothelial oxidative stress during sepsis.
OBJECTIVE
Plasma from septic shock patients can induce production of reactive oxygen species (ROS) by human umbilical vein endothelial cells (HUVEC) in vitro. How endothelial cells defend themselves against ROS under increased oxidative stress has not yet been examined. This study investigates the antioxidant defenses of HUVEC exposed to plasma obtained from either septic shock patients or healthy volunteers.


METHODS
Prospective, observational study.


METHODS
Medical intensive care unit in a university hospital.


METHODS
Twenty-five patients with septic shock and 10 healthy volunteers.


METHODS
Blood samples were collected within the first 24 hrs of septic shock. In vitro HUVEC production of ROS was studied by spectrofluorimetry using 2',7'-dichlorodihydrofluorescein diacetate fluorescent dye. Reactive nitrogen species were also assessed. Intracellular reduced glutathione (GSH) levels were measured using monochlorobimane fluorescent dye. Activity of catalase and superoxide dismutase in HUVEC were also measured. Cell death was assessed using YOPRO fluorescent dye and the MTT assay.


RESULTS
On admission, the septic shock population's mean age was 55 yrs old, the mean Sequential Organ Failure Assessment score was 12, mean simplified acute physiology score was 50, and intensive care unit mortality rate was 45%. Evaluation of HUVEC antioxidant defenses showed a significantly decreased GSH level, increased catalase activity, and unchanged superoxide dismutase activity. ROS levels and cell death were significantly reduced when cells were pretreated with N-acetylcysteine or GSH, but no changes in reactive nitrogen species were observed.


CONCLUSIONS
This study demonstrates that plasma-induced ROS production by HUVEC is associated with an intracellular decrease in reduced GSH. Both ROS levels and cell death decreased when N-acetylcysteine or GSH were added before exposing the cells to plasma. These data suggest a pivotal role of alterations in GSH in damage caused by sepsis-generated ROS in endothelial cell.
Huet
Olivier
O
Département d'anesthésie réanimation, Hôpital Bicêtre, Assistance Publique des Hopitaux de Paris, France.
Cherreau
Christaine
C
Nicco
Carole
C
Dupic
Laurent
L
Conti
Marc
M
Borderie
Didier
D
Pene
Frédéric
F
Vicaut
Eric
E
Benhamou
Dan
D
Mira
Jean-Paul
JP
Duranteau
Jacques
J
Batteux
Frédéric
F
D018806:APACHE; D000111:Acetylcysteine / Q000494:pharmacology; D000328:Adult; D000368:Aged; D016923:Cell Death / Q000187:drug effects*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D016166:Free Radical Scavengers / Q000494:pharmacology; D005978:Glutathione / Q000172:deficiency* / Q000502:physiology*; D006801:Humans; D007362:Intensive Care Units; D008297:Male; D008875:Middle Aged; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012772:Shock, Septic / Q000097:blood* / Q000378:metabolism / Q000401:mortality
D016166:Free Radical Scavengers; D017382:Reactive Oxygen Species; D005978:Glutathione; D000111:Acetylcysteine