Concentration changes of malondialdehyde across the cerebral vascular bed and shedding of L-selectin during carotid endarterectomy.
ABSTRACT Oxidative stress has been postulated to account for delayed neuronal death due to ischemia/reperfusion. We investigated cerebral formation of malondialdehyde as an index of lipid peroxidation in relation to different sources of reactive oxygen species in patients undergoing carotid endarterectomy.
In 25 patients undergoing carotid endarterectomy, jugular venous-arterial concentration differences of brain metabolites, malondialdehyde, plasma total antioxidant status, and soluble P-selectin and L-selectin were measured. A carotid artery shunt (n=5) was placed only after complete loss of somatosensory evoked potentials, indicating a focal cerebral blood flow <15 mL/min per 100 g.
As an indication of cerebral lipid peroxidation, jugular venous-arterial malondialdehyde concentration differences were significantly enhanced before reperfusion, and an additional rise was observed 15 minutes after reperfusion. Plasma total antioxidant status significantly decreased during carotid artery occlusion only in patients with carotid artery shunt. This decrease was matched by cerebral formation of adenosine, hypoxanthine, and nitrite/nitrate. While jugular venous-arterial concentration differences of soluble P-selectin showed changes similar to those of malondialdehyde, the concentration difference for soluble L-selectin was enhanced exclusively at 15 minutes after reperfusion.
Short-term incomplete cerebral ischemia/reperfusion significantly enhanced cerebral lipid peroxidation, as indicated by malondialdehyde formation. The generation of reactive oxygen species by xanthine oxidase or nitric oxide metabolism might be involved in the induction of lipid peroxidation. The additional rise in cerebral release of malondialdehyde was found to coincide with a significant activation of polymorphonuclear leukocytes across the cerebral circulation.
- SourceAvailable from: Stefan Hofer[show abstract] [hide abstract]
ABSTRACT: Lipid peroxidation processes (LPO) are evident in many organ failures. Due to their toxic properties, they are causative for cellular dysfunction at the site of their origin and far beyond. This study was conducted to investigate differences in LPO pattern of patients with established acute respiratory distress syndrome (ARDS) and patients with end-stage liver failure undergoing liver transplantation (LTX) as two mayor prototypes of organ failure. In this prospective, nonrandomized, controlled trial, we examined LPO by measuring malondialdehyde (MDA), and the volatile aldehydes hexanal and propanal as LPO-markers. Eighteen patients with ARDS, 16 subjects undergoing liver transplantation due to liver failure, and 8 healthy controls were included to the study. ARDS patients showed significantly higher levels in MDA concentrations than LTX and controls, respectively. However, MDA levels of patients with end-stage liver failure were equal to those of controls. Blood concentrations of hexanal and propanal, specific by-products of lipid peroxidation, were elevated in both patient groups, but significantly higher only in LTX. Unexpectedly, hexanal and propanal concentrations were significantly higher in LTX than in ARDS patients. In both patient groups, MDA showed no differences between arterial and mixed venous blood, whereas volatile aldehydes were higher in arterial than in mixed venous compartment. Both ARDS and LTX-patients showed significant evidence of enhanced LPO. However, proportions of MDA and volatile aldehydes differed substantially between the groups. Thus, for the interpretation of LPO markers, disease-specific factors have to be taken into account. Distinctions might be attributable to differences in the effected lipid components or variations in metabolism.Journal of Surgical Research 06/2011; 168(2):243-52. · 2.02 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Routine shunting to minimize ischemia during carotid endarterectomy (CEA) is controversial. The aim of this study was to stratify the ischemic parameters associated with CEA and evaluate the effect of routine shunting in attempting to mitigate those ischemia. Data from 248 CEAs with routine shunting were retrospectively evaluated. Our assessment included somatosensory evoked potential (SSEP) amplitude reduction more than 50 % and longer than 5 min (SSEP<50%, >5 min), new postoperative diffusion-weighted imaging lesions (new DWI lesions), and severe stenosis as indicated by reduced ipsilateral middle cerebral artery (MCA) signal on preoperative magnetic resonance angiography (MRA asymmetry), as surrogates of hypoperfusion, microembli, and hemodynamic impairment, respectively. SSEP<50%, >5 min occurred in 15 % of CEAs during cross-clamping, and shunting reversed the SSEP changes. New DWI lesions were observed in 4.1 %. Pre-clamping the common and external carotid artery during dissection (pre-clamp method) decreased the rate of new DWI lesions compared to without pre-clamping (3.5 % vs. 7.5 %, P = 0.22). Occlusion time was significantly longer in the pre-clamp method than without pre-clamping (P < 0.0001). However, the incidence of SSEP<50%, >5 min was not increased with the pre-clamp method (p = 1.0) when using information regarding SSEP and collaterals to modify the speed of shunt manipulation. MRA asymmetry was identified in 39 CEAs (15.8 %) with correction of asymmetry postoperatively. MRA asymmetry correlated with symptomatic hyperperfusion (P = 0.0034). Only three CEAs had symptomatic hyperperfusion (1.2 %) with minimal symptoms. Ten CEAs sustained transient ischemia, symptomatic hyperperfusion, or 30-day-stroke (composite postoperative ischemic symptoms). Logistic regression analysis confirmed that SSEP<50%, >5 min (p = 0.009), new DWI lesions (p = 0.004) and MRA asymmetry (p = 0.042) were independent predictors of composite postoperative ischemic symptoms. SSEP<50%, >5 min, new DWI lesions, and MRA asymmetry were able to stratify the ischemic impacts in CEA. Meticulous routine shunting could mitigate those appropriately.Acta Neurochirurgica 08/2013; · 1.55 Impact Factor
Martin and Hubert J. Bardenheuer
Markus A. Weigand, Andreas Laipple, Konstanze Plaschke, Hans-Henning Eckstein, Eike
Shedding of L-Selectin During Carotid Endarterectomy
Concentration Changes of Malondialdehyde Across the Cerebral Vascular Bed and
Print ISSN: 0039-2499. Online ISSN: 1524-4628
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Concentration Changes of Malondialdehyde Across the
Cerebral Vascular Bed and Shedding of L-Selectin During
Markus A. Weigand, MD; Andreas Laipple; Konstanze Plaschke, PhD; Hans-Henning Eckstein, MD;
Eike Martin, MD, FANZCA; Hubert J. Bardenheuer, MD
Background and Purpose—Oxidative stress has been postulated to account for delayed neuronal death due to
ischemia/reperfusion. We investigated cerebral formation of malondialdehyde as an index of lipid peroxidation in
relation to different sources of reactive oxygen species in patients undergoing carotid endarterectomy.
Methods—In 25 patients undergoing carotid endarterectomy, jugular venous–arterial concentration differences of brain
metabolites, malondialdehyde, plasma total antioxidant status, and soluble P-selectin and L-selectin were measured. A
carotid artery shunt (n?5) was placed only after complete loss of somatosensory evoked potentials, indicating a focal
cerebral blood flow ?15 mL/min per 100 g.
Results—As an indication of cerebral lipid peroxidation, jugular venous–arterial malondialdehyde concentration differ-
ences were significantly enhanced before reperfusion, and an additional rise was observed 15 minutes after reperfusion.
Plasma total antioxidant status significantly decreased during carotid artery occlusion only in patients with carotid artery
shunt. This decrease was matched by cerebral formation of adenosine, hypoxanthine, and nitrite/nitrate. While jugular
venous–arterial concentration differences of soluble P-selectin showed changes similar to those of malondialdehyde, the
concentration difference for soluble L-selectin was enhanced exclusively at 15 minutes after reperfusion.
Conclusions—Short-term incomplete cerebral ischemia/reperfusion significantly enhanced cerebral lipid peroxidation, as
indicated by malondialdehyde formation. The generation of reactive oxygen species by xanthine oxidase or nitric oxide
metabolism might be involved in the induction of lipid peroxidation. The additional rise in cerebral release of
malondialdehyde was found to coincide with a significant activation of polymorphonuclear leukocytes across the
cerebral circulation. (Stroke. 1999;30:306-311.)
Key Words: adenosine?adhesion molecules?carotid endarterectomy?lipid peroxidation
?nitric oxide?oxygen radicals
tomatic and asymptomatic carotid stenoses ?70%.1–3On
average, however, 2% to 6% of all patients undergoing
carotid endarterectomy sustain a stroke in the perioperative
period.4Intraoperative embolism and hypoperfusion are pos-
sible causes of a perioperative neurological deficit due to
clamping of the carotid artery.
Two major hypotheses have been developed to account for
the phenomenon of ischemia/reperfusion–induced neuronal
death. The neurotransmitter hypothesis is related to the role of
excitotoxic amino acids and is preferentially aimed at events
during the acute period of ischemia. The free radical hypoth-
esis is directed at events during reperfusion.5The generation
of reactive oxygen species (ROS) initiates a vicious cascade
of tissue injury. In particular, ROS lead to peroxidation of
phospholipids with consecutive alteration of membrane struc-
arotid endarterectomy has been proven to reduce the
incidence of ipsilateral strokes in patients with symp-
ture. These events provide a conceptual basis to explain
delayed neuronal death after periods of ischemia/reperfu-
sion.6In animal studies it has been shown that endothelial
adhesion of polymorphonuclear leukocytes (PMN), which
generate ROS and reactive nitrogen species, significantly
contributes to the pathogenesis of reperfusion injury after
This clinical study investigates the interrelation between
cerebral energy metabolism, nitric oxide (NO) metabolism,
cellular activation, and cerebral lipid peroxidation as indi-
cated by the formation of malondialdehyde (MDA) in pa-
tients undergoing carotid endarterectomy.
Focal cerebral ischemia was induced by acute vascular
occlusion of the common carotid artery, and the extent of
ischemia was verified by monitoring of somatosensory
evoked potentials (SSEP). During carotid surgery a vascular
shunt was placed only under conditions when total loss of
Received October 12, 1998; final revision received November 13, 1998; accepted November 13, 1998.
From the Departments of Anesthesiology and Vascular Surgery (H-H.E.), University of Heidelberg, Heidelberg, Germany.
Correspondence to Hubert J. Bardenheuer, MD, Department of Anesthesiology, University of Heidelberg, Im Neuenheimer Feld 110, D-69120
Heidelberg, Germany. E-mail Hubert_Bardenheuer@ukl.uni-heidelberg.de
© 1999 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
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SSEP amplitude occurred, indicating a regional cerebral
blood flow ?15 mL/min per 100 g. Carotid endarterectomy is
a relevant clinical model to study focal cerebral ischemia/
reperfusion injury in patients.
Subjects and Methods
After institutional approval, informed consent was obtained from 25
patients (mean age, 65?2 years; age range, 40 to 82 years)
undergoing elective carotid endarterectomy. The group included 6
women and 19 men. In all but 5 patients without prior symptoms, the
indication for carotid endarterectomy was symptomatic carotid artery
stenosis ?70%. Thirteen patients took aspirin as antiplatelet medi-
cation, whereas none of the patients took antioxidants. After pre-
medication with midazolam (3.75 to 7.5 mg), anesthesia was induced
with 1 to 3 mg midazolam, 2 to 5 ?g ? kg?1fentanyl, 0.15 to 0.3 mg ?
kg?1etomidate, and 0.5 mg ? kg?1atracurium. After intubation,
anesthesia was maintained with nitrous oxide in oxygen
(N2O:O2?50:50) and 0.2% to 0.6% isoflurane. Atracurium and
fentanyl were administered intraoperatively as necessary. All pa-
tients were mechanically ventilated to maintain normocapnia with
PaCO2of 38 to 41 mm Hg. In each patient, ECG, end-tidal capnom-
etry, and arterial blood pressure changes were continuously recorded.
A thorough neurological examination was performed immediately
after the patient awakened, 1 hour later, and then daily until the
patients were discharged.
Intraoperatively, SSEP were continuously recorded after contralat-
eral median nerve stimulation (Nicolet Spirit) to detect critical
regional hypoperfusion due to carotid cross-clamping. Importantly, a
shunt was placed only after complete loss of the N20/P25 SSEP
amplitude. According to this criterion, intraoperative shunting of the
carotid artery was performed in 5 of 25 patients (shunt group [n?5]
versus no-shunt group [n?20], respectively). In addition, a catheter
was placed intraoperatively into the ipsilateral jugular bulb by the
surgeon to obtain jugular venous blood samples. The correct catheter
position in the jugular bulb was verified by intraoperative angiogra-
phy. Heparin (5000 U) was given intravenously to all patients before
carotid cross-clamping, and hydroxyethyl starch (500 mL) was
regularly infused. Arterial and jugular venous blood samples were
collected regularly before carotid cross-clamping, 10 minutes after
carotid artery occlusion, before reperfusion, and 15 minutes after
reperfusion, respectively. In patients with shunt, however, the end of
shunt placement (6?1 minutes) was taken as the start of the
Soluble P-selectin (sP-selectin) and soluble L-selectin (sL-
selectin) were measured by enzyme-linked immunosorbent assays
(Bender MedSystems). Plasma nitrite/nitrate was assayed by the
Griess reaction9with a commercially available kit (Boehringer
Mannheim). Plasma total antioxidant status was determined spectro-
photometrically (Randox). We measured MDA by high-performance
liquid chromatography (HPLC) using a slight modification of the
method of Lepage et al.10First 250 ?L of distilled water and 10 ?L
of 0.5% butylated hydroxytoluene were added to 250 ?L plasma in
a glass tube. This was followed by the addition of 200 ?L of 0.66N
H2SO4and 150 ?L of 0.3 mol/L Na2WO4. Thereafter, the mixture
was centrifuged at 1000g for 10 minutes. Next 500 ?L of the
supernatant was mixed with 167 ?L of 50 mmol/L thiobarbituric
acid solution. The mixture was then heated at 100°C for 60 minutes.
Twenty microliters of this reaction solution was then used for HPLC
analysis with a Hypersil ODS C-18 column with 5-?m particle size.
The mobile phase consisted of methanol and water in a gradient
mode. After an initial period of 2 minutes with water alone, the
methanol/water gradient was changed from 0% to 50% over a
2-minute period with a hold at that mixture for 6.5 minutes. Finally,
the gradient was reversed to 100% water within 5 minutes. After 11.5
minutes of reequilibration at that level, the next sample was injected.
The flow rate was 0.45 mL/min, and the column eluate was detected
by UV spectrophotometry (Merck) at 532 nm. Purine compounds
were determined as previously described.11In brief, blood samples
(1 mL) were collected in precooled dipyridamole solution (1 mL,
5?10?5mol/L) to prevent nucleoside uptake by red blood cells.
After immediate centrifugation at 4°C, plasma supernatant (1 mL)
was deproteinated with perchloric acid (70%, 0.1 mL). After neu-
tralization (KH2PO4) and centrifugation, nucleosides were deter-
mined by HPLC. Samples (0.1 mL) were automatically injected onto
a C-18 column (Nova-Pak C18, 3.9?150 mm, Waters). The linear
gradient started with 100% KH2PO4(0.001 mol/L, pH 4.0) and
increased to 60% of 60/40 methanol/water (vol/vol) in 15 minutes,
the flow rate being 1.0 mL/min. This was followed by a reversal of
the gradient to initial conditions over the next 3 minutes. Absorbance
of the column eluate was simultaneously monitored at 254 nm for
adenosine and hypoxanthine, respectively, and at 293 nm for uric
acid with photodiode array detection (Waters). Purine compounds
were quantified with a computer-assisted program (Millenium,
Results are expressed as mean?SEM. Differences within or between
the patient groups were examined by ANOVA followed by Scheffe ´
multiple comparisons. Statistical significance is at the P?0.05 level.
In the present study a total loss of SSEP amplitude occurred
in 5 patients at 6?1 minutes after carotid artery occlusion.
Therefore, intraoperative shunt placement was performed in 5
of 25 patients (shunt group [n?5] versus no-shunt group
[n?20], respectively). As can be seen in Table 1, no signif-
icant differences between the groups were obtained in hemo-
dynamic parameters, PaO2, PaCO2, or hemoglobin concentra-
tions throughout the study period. In patients in the shunt
group, SSEP amplitude remained depressed at 15 minutes
after reperfusion despite reperfusion (Table 2). One patient in
the shunt group suffered postoperatively from a transient
TABLE 1.Parameters of Hemodynamic Data and Blood Gas Analysis
No-Shunt Group (n?20)Shunt Group (n?5)
Time of Occlusion, min
MAP, mm Hg
Heart rate, bpm
PaO2, mm Hg
PaCO2, mm Hg
Arterial hemoglobin, mg/dL
Values are mean?SEM. C1indicates before carotid cross-clamping; C2, 15 minutes after reperfusion; and MAP, mean arterial pressure. There were
no significant differences between groups.
Weigand et al February 1999
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neurological deficit. It is important to note that the mean
occlusion time in the no-shunt group was 30?2 minutes,
which was significantly longer than that in the shunt group
(6?1 minutes). In patients in the shunt group, shunt opening
was taken as the start of reperfusion.
Under baseline conditions, there was no significant differ-
ence in jugular venous–arterial lactate concentration differ-
ence (?LAC) between the shunt group and the no-shunt
group (Table 2). In patients with inadequate collateral blood
flow (shunt group), however, ?LAC was significantly in-
creased during carotid artery occlusion and remained elevated
until 15 minutes after reperfusion.
Before carotid artery occlusion, no significant difference in
jugular venous–arterial adenosine difference (?ADO) was
obtained between the groups. Major concentration changes of
adenosine across the cerebral vascular bed were observed
during the clamping period, when ?ADO exhibited a peak
value of 181?37 nmol/L at 6 minutes after carotid cross-
clamping. In contrast to lactate, ?ADO returned to control
levels within 15 minutes after reperfusion. In general, similar
results were also obtained in the case of hypoxanthine and
nitrite/nitrate in the shunt group.
Under baseline conditions, the jugular venous–arterial
difference in plasma total antioxidant status (?TAS) was
higher in patients with inadequate collateral blood flow
(shunt group). While ?TAS remained nearly unchanged in
the no-shunt group, carotid artery clamping induced a signif-
icant decrease in ?TAS in the shunt group.
In patients with adequate collateral blood flow (no-shunt
group), jugular venous–arterial MDA concentration differ-
ences (?MDA) remained almost stable throughout the study
period (Figure). In patients with inadequate collateral blood
flow (shunt group), ?MDA and jugular venous–arterial
concentration difference in sP-selectin (?sP-selectin) were
significantly different from those in patients with adequate
collateral blood flow under control conditions. Furthermore,
in the shunt group significant changes in ?MDA also oc-
curred after cross-clamping of the carotid artery. ?MDA
increased from baseline values (34?26 nmol/L) to 130?49
nmol/L at the end of the occlusion period (6?1 minutes). At
15 minutes of reperfusion, there was an additional rise of
?MDA to 291.0?70.9 nmol/L (P?0.05).
Both ?sP-selectin and jugular venous–arterial concentra-
tion difference in Sl-selectin (?sL-selectin) exhibited only
minor changes throughout the study period in patients with
sufficient collateral perfusion (no-shunt group). Despite a
significantly shorter period of vessel occlusion in the shunt
group, ?sP-selectin was enhanced in parallel with the
changes in MDA during carotid occlusion and reperfusion,
respectively. In contrast, ?sL-selectin exhibited significant
changes only at the end of the study period (15 minutes after
In patients undergoing carotid endarterectomy, the jugular
venous–arterial MDA concentration differences (?MDA)
were significantly higher in patients in whom a total loss of
SSEP amplitude occurred (shunt group). In contrast to pa-
tients with adequate collateral blood flow (no-shunt group),
increased ?MDA was obtained during an occlusion period as
short as 6?1 minutes and exhibited a 6-fold increase at 15
minutes after start of reperfusion.
ROS such as superoxide anions (O2
(H2O2), and the extremely toxic hydroxyl radical (?OH) are
difficult to detect in patients because of their short half-life.
Therefore, byproducts of lipid peroxidation or depletion of
endogenous antioxidants have often been used as indirect
markers for free radical generation.12–14MDA is a 3-carbon
compound, which reflects both auto-oxidation and oxygen
radical–mediated peroxidation of polyunsaturated fatty acids,
in particular, arachidonic acid.14,15However, release of MDA
is not specific for lipid peroxidation, because other sources of
MDA formation have been described. In certain tissues,
MDA can also be formed by nonenzymatic or enzymatic
processes, for example, by human platelet synthetase.15–17
Nevertheless, the significant increase in ?MDA in the shunt
group together with the decrease in ?TAS across the cerebral
circulation is a strong indication that lipid peroxidation takes
place in the cerebral vascular bed even after short periods of
incomplete cerebral ischemia. This is even more evident
•?), hydrogen peroxides
Changes of Jugular Venous–Arterial Concentrations of Lactate, Nucleosides, Nitrite/Nitrate Ratio, and Plasma Total
No-Shunt Group (n?20) Shunt Group (n?5)
Time of Occlusion, min
Values are mean?SEM. C1indicates before carotid cross-clamping; C2, 15 minutes after reperfusion; and ?HYPO, jugular venous–arterial concentration difference
*P?0.05 vs C1; †P?0.05, shunt vs no-shunt group.
Leukocytes and Cerebral Lipid Peroxidation
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because the obtained changes in ?TAS are temporally related
to the occurrence of cerebral ischemia.14,18,19
Clinically, the occurrence of short-term incomplete cere-
bral ischemia in the shunt group was verified by total loss of
SSEP amplitude after carotid cross-clamping. The electro-
physiological changes indicate a local cerebral blood flow
?15 mL/min per 100 g and are associated with a significant
impairment of cellular ion homeostasis.20,21In addition, the
significant increases in ?LAC and ?ADO are further evi-
dence that some degree of cerebral ischemia is present in
patients with a shunt during carotid artery occlusion. In
particular, adenosine has been characterized as a sensitive
indicator of disturbances in tissue oxygenation in several
organs, including the brain.22,23The data indicate that meta-
bolic parameters are altered in close parallelism with the
impairment of cerebral function (SSEP) when inadequate
collateral blood flow is present in patients undergoing carotid
endarterectomy. In patients with shunt, the shunt was placed
to restore cerebral perfusion and to avoid neuronal death due
to ischemia. Since shunt placement was completed at 6?1
minutes after carotid cross-clamping, it is of particular inter-
est that the changes in cerebral lipid peroxidation were also
induced after a relatively short period of focal cerebral
ischemia followed by reperfusion.
Enhanced generation of ROS in the postischemic reperfu-
sion period induces oxidative damage of proteins and lipids24
and impairs mitochondrial function.25In animal experiments
with 2 hours of middle cerebral artery occlusion, both
mitochondrial function and the bioenergetic cellular state
were shown to only partially recover in the first hour after
reperfusion and to deteriorate again within 2 to 4 hours after
reperfusion. Folbergrova ´ et al6have demonstrated that ROS
Changes of jugular venous–arterial concentrations of various parameters in patients with adequate collateral blood flow ([? shunt]) and
patients with inadequate collateral blood flow ([? shunt]). C1indicates before carotid cross-clamping; C2, 15 minutes after reperfusion.
Values are mean?SEM. *P?0.05 vs C1; #P?0.05 [? shunt] vs [? shunt].
Weigand et alFebruary 1999
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are causally involved in the impairment of cellular energy
metabolism because the spin-trapping agent N-tert-butyl-?-
phenylnitrone (PBN) improved mitochondrial function and
reduced infarct volume. The changes in lactate give addi-
tional indirect evidence that impaired cellular energy metab-
olism occurred under conditions of short-term carotid occlu-
sion. In contrast to adenosine, ?LAC remained enhanced in
patients with shunt at 15 minutes after reperfusion. This
ongoing lactate production by the brain could be due to
ROS-dependent postischemic inhibition of the pyruvate de-
hydrogenase complex, which reflects impaired mitochondrial
function.26This hypothesis is further supported by clinical
data demonstrating that the electrophysiological function of
these patients was still depressed at 15 minutes after reper-
fusion, as indicated by changes in SSEP.
Few data have been presented concerning oxidant produc-
tion during cerebral ischemia and reperfusion in patients.
While Soong et al19found an increase in jugular venous
MDA only 60 seconds after carotid clamp removal, Bacon et
al12observed a decrease in the antioxidant capacity across the
cerebral circulation after declamping of the external and
internal carotid arteries. In contrast to our study, focal
cerebral ischemia was documented in neither of the men-
tioned investigations, because a shunt was either generally
inserted in all patients19or none of the subjects required a
In the present study the origin of MDA formation in the
shunt group is difficult to determine. For instance, brain
tissue itself is at particular risk of being injured by oxidant-
mediated triggers because tissue contains large iron stores
and high levels of polyunsaturated lipids but exhibits only
poor antioxidant defenses. In addition, the cerebral vascular
endothelium can also be one source for the rise in MDA.
Interestingly, ?MDA in the shunt group was already elevated
at the end of the ischemic period. This finding demonstrates
that molecular events leading to oxygen radical production
not only occur during reperfusion but also during short-term
and incomplete tissue ischemia.
Biochemically, a potential source of ROS formation is
purine catabolism. During ischemia, accumulation of adeno-
sine and its metabolite hypoxanthine (see Table 2) takes
place. While in the normoxic brain hypoxanthine is metabo-
lized by xanthine dehydrogenase to xanthine and ultimately
to uric acid, the enzyme xanthine dehydrogenase is converted
to xanthine oxidase during ischemia. In contrast to xanthine
dehydrogenase, xanthine oxidase instead uses molecular ox-
ygen of the nucleotide radical of NAD?as its electron
acceptor, thereby catalyzing the formation of O2
reperfusion.5,27Xia and Zweier28have demonstrated that the
free radical formation via xanthine oxidase is substrate
driven. Because adenosine and hypoxanthine accumulate
significantly in patients with shunt before reperfusion, the
substrate-dependent conversion of hypoxanthine/xanthine to
uric acid by xanthine oxidase seems to be an important source
for the initial burst of free radical generation. Interestingly,
this is coincident with a simultaneous decrease in plasma total
Another important source of ROS is the metabolism of NO.
NO reacts with superoxide to yield the peroxynitrite anion
(ONOO?), which decomposes to ?OH.29Furthermore, the
peroxynitrite anion itself is also a highly reactive oxidizing
agent that can cause tissue damage.8Experimental studies
have demonstrated that NO mediates glutamate neurotoxicity
in primary cortical cultures30and that inhibition of NO
generation can reduce infarct volume induced by transient
occlusion of the middle cerebral artery.31
In this clinical study the ratio of nitrite/nitrate was taken as
an indirect marker of NO production.9Interestingly, in this
clinical study the ratio of nitrite/nitrate was actually increased
in the shunt group. Moreover, these results were well
matched with the decrease in plasma total antioxidant status
before reperfusion. Therefore, the observed changes can be
taken as indirect evidence that NO metabolism might con-
tribute to ROS generation in patients undergoing carotid
del Zoppo et al32suggested a pivotal role for PMN in
cerebral ischemia. This hypothesis is supported by the finding
that antibodies to PMN or adhesion molecules ameliorate
infarct volume after transient ischemia in animals.33,34In
addition, Okada et al35have shown in baboon experiments
that P-selectins can be detected on the cerebral endothelium
in the early phase of reperfusion after cerebral artery occlu-
sion. Until now, however, no data have been available
concerning the kinetics of adhesion molecule expression
during short-term ischemia/reperfusion in patients. In this
study we measured ?sP-selectin and ?sL-selectin to charac-
terize the changes in cerebral expression and shedding of both
Similar to the changes in ?MDA, we found a significant
increase in ?sP-selectin in the shunt group before reperfu-
sion, indicating enhanced expression and shedding of
P-selectin. In contrast, at this point ?sL-selectin was nearly
unchanged. P-selectin expression, which is observed within
minutes after endothelial activation, increases the number of
PMN rolling along the endothelium.36Rolling brings PMN
into close proximity to chemoattractants such as platelet-
activating factor, which is also expressed on endothelial cells
in response to ROS.37As a result, strong attachment of PMN
to the endothelium occurs. The marked elevation of ?sL-
selectin in the shunt group at 15 minutes after reperfusion
provides indirect evidence that activation of PMN is likely to
take place within the cerebral vascular bed.
In conclusion, we demonstrate that lipid peroxidation can
occur during short-term and incomplete cerebral ischemia/
reperfusion in patients undergoing carotid endarterectomy.
Although the quantitative role of each compartment cannot be
determined as yet, the data demonstrate that the ATP-
degradation pathway, NO metabolism, as well as cellular
factors such as PMN are likely to contribute to the production
of ROS under conditions of cerebral ischemia/reperfusion.
This study was supported by an institutional research fund of the
Department of Anesthesiology, University of Heidelberg, Heidel-
berg, Germany. The authors thank Dr H. Bauer for statistical
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Weigand et alFebruary 1999
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