Transcript Glutathione peroxidase & subarachnoid hemorrhage

Glutathione peroxidase & subarachnoid hemorrhage:
implications for the role of oxidative stress in cerebral vasospasm
Gail Pyne-Geithman, D.Phil.1,3,4, Danielle Caudell, BA3, Porus Prakash3, Joseph Clark, PhD1,3, Lori Shutter, MD1,2,3,4,5
University of Cincinnati (UC) Neuroscience Institute1: Division of Neurocritical Care2; Department of Neurology3, Department of Neurosurgery4, UC College of Medicine; Mayfield Clinic5, Cincinnati, OH
Introduction
It has long been suspected that reactive oxygen species
(ROS) play a major role in the etiology of CV after SAH.
We hypothesize that glutathione peroxidase (GSH-Px1)
activity is altered between CSF from SAH patients with
CV (CSFV), compared with those without CV (CSFC), and
that the difference in total antioxidant capacity of CSFV
and CSFC will be largely due to GSH-Px1. There are
many ways in which oxidative stress is implicated in
these disease processes (Fig. 1).
Figure 2. Panel A shows
a representative (N=5)
western blot of GSH-Px1
protein in human CSF,
along with a pure protein
control. Panel B is a
densitometric semiquantitation of these
bands , errors are
standard deviation. There
was no difference
between vasospastic and
non-vasospastic SAH
CSF with respect to
protein content when
probed specifically for
human GSH-PX1.
Methods
• CSF was obtained with appropriate local IRB approval.
• GSH-Px1 protein levels were determined using standard
SDS-PAGE and western blotting techniques, as
published by us and many others1.
• GSH-Px1 Activity levels were measured using a
commercially available kit (Zeptometrix Corp., Franklin,
MA).
• Oxygen Radical Absorbance capacity (ORAC) was
measured using a commercially available kit (Cayman
Chem. Co., Ann Arbor, MI)
There was a significant y higher ORAC in the vasospastic
CSF, which may indicate either a greater response in
vasospastic patients, or that a higher ORAC baseline
exists in patients likely to develop CV after SAH. This
observation warrants further investigation.
We observed that a significant portion of the increased
ORAC is due to increased activity, but not increased
amount of protein, of glutathione peroxidase, possible
implication selenium moieties.
Previous data from our laboratory has shown significantly
increased lipid peroxidation in the CSF milieu in
vasospastic patients ‘ CSF2,3. Thus, despite increased
ORAC and GSH-Px1 activity, there is still elevated
oxidative damage associated with CV after SAH. Whether
this elevation occurs before or after the onset of CV after
SAH is as yet unclear.
Conclusions
Figure 3. Panel A
shows the increased
ORAC of the
vasospastic CSF
compared to that from
SAH patients without
vasospasm. Panel B
shows that GSH-Px1
activity is significantly
higher in CSFV than
CSFC.
N=5 for each group, and
errors are standard
deviations. * indicates
significant difference
from control by ANOVA
(p<0.05).
Figure 1. A concept map illustrating the multiple steps in the disease
process of cerebral vasospasm after subarachnoid hemorrhage in
which oxidative stress plays a known role.
Discussion
Results
• Glutathione peroxidase activity, but not
content, was significantly higher, associated
with CV after SAH.
• Total oxygen radical capacity was also higher
associated with CV after SAH, and yet elevated
lipid peroxidation is not prevented in these
patients’ CSF.
References
1. Pyne-Geithman, G. J., S. G. Nair, D. N. Caudell, J. F. Clark. PKC and Rho in
vascular smooth muscle: Activation by BOXes and SAH CSF. Front. Biosci.
2008 13:1526-1534 Review
2. Clark, J. F., M. Loftspring, W. L. Wurster, G. J. Pyne-Geithman Chemical and
biochemical oxidations in spinal fluid after subarachnoid hemorrhage. Front.
Biosci. 2008 13:1806-1812 Review
3. Pyne-Geithman, G. J., C. J. Morgan, K. R. Wagner, E. M. Dulaney, J. A.
Carrozzella, D. S. Kanter, M. Zuccarello, J. F. Clark. Bilirubin production and
oxidation in CSF of patients with cerebral vasospasm after subarachnoid
hemorrhage. J. Cereb. Bl. Fl. Metab. 2005 25: 1070-1077