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(Stroke. 2001;32:898.)
© 2001 American Heart Association, Inc.
Original Contributions |
Plasma Vitamin C Levels Are Decreased and Correlated With Brain Damage in Patients With Intracranial Hemorrhage or Head Trauma
From the Institute of Gerontology and Geriatrics, Perugia University Hospital, Perugia, Italy (M.C.P., P.M.), and Linus Pauling Institute, Oregon State University, Corvallis, Ore (B.F.).
Correspondence to Maria Cristina Polidori, MD, Institute of Physiological Chemistry I, Heinrich-Heine University, D-40225 Düsseldorf, Germany. E-mail polidori{at}uni-duesseldorf.de
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Background and Purpose—Free radical hyperproduction may play an important role in brain hemorrhage and ischemia/reperfusion injury. The aims of this study were to assess whether antioxidant depletion occurs after intracranial hemorrhage (ICH) and head trauma (HT) and to evaluate the relation between the diameter of the brain lesion, the degree of the neurological impairment, and any observed antioxidant changes.
Methods—We measured
plasma levels of vitamin C (ascorbic acid, AA), uric acid (UA), vitamin
E (
-tocopherol), and ubiquinol-10 in 13 patients with
ICH and 15 patients with HT on the day of the brain injury and
subsequently every other day up to 1 week. Patients were compared with
40 healthy control subjects.
Results—ICH and HT
patients had significantly lower plasma levels of AA compared with
healthy subjects, in contrast to plasma levels of UA,
-tocopherol, and ubiquinol-10. AA levels were
significantly inversely correlated with the severity of the
neurological impairment as assessed by the Glasgow Coma Scale and the
National Institutes of Health Stroke Scale. AA levels were also
significantly inversely correlated with the major diameter of the
lesion. In addition, mean plasma AA levels were lower in jugular
compared with peripheral blood samples obtained from 5
patients.
Conclusions—These findings suggest that a condition of oxidative stress occurs in patients with head trauma and hemorrhagic stroke of recent onset. The consequences of early vitamin C depletion on brain injury as well as the effects of vitamin C supplementation in ICH and HT patients remain to be addressed in further studies.
Key Words: antioxidants • brain hemorrhage • head trauma • oxidative stress • vitamin C
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Increased production of free radicals and reactive oxygen species (ROS) leading to oxidative stress1 appears to play an important role in the pathogenesis of ischemic,2 3 hemorrhagic,4 5 and traumatic brain injury.6 7 Studies in animal models of focal cerebral ischemia and reperfusion have demonstrated the presence of increased levels of ROS.8 9 10 11 ROS also probably contribute to brain injury in head trauma (HT) and intracranial hemorrhage (ICH) in humans, because hemorrhage is associated with the release of hemoglobin-bound heme iron, which can participate in free radical reactions to produce ROS.4 5 12
One possible consequence of excess ROS formation is lipid peroxidation. The brain appears to be particularly vulnerable to oxidative lipid damage because of its high content of polyunsaturated fatty acids.13 Lipid peroxidation may alter the fluidity and permeability of neuronal membranes and thus, cellular functioning, or damage membrane-bound receptors and enzymes.13 In brain hemorrhage, the presence of "free" iron14 may favor the conversion of lipid hydroperoxides to lipid alkoxyl radicals, which can "reinitiate" lipid peroxidation and hence further expand the radical chain reaction.13 In addition, tissue lactic acidosis can dramatically enhance ROS formation and lipid peroxidation in brain tissue, which in turn can increase the dissociation of catalytic iron from proteins.15
Because ROS are short-lived and usually present at low
concentrations, they are difficult to measure in biological
samples.16 However, there
are indirect indexes that can be used to examine sequelae of ROS
production, such as oxidatively modified
macromolecules17 and changes
in the concentration of endogenous
antioxidants,18 19
such as vitamin C (ascorbic acid, AA), uric acid (UA), vitamin E
(-tocopherol), and ubiquinol-10. Vitamin C appears to be
particularly important in limiting oxidative lipid damage in biological
systems.19 Numerous studies
have demonstrated that under many different types of oxidizing
conditions, AA forms the first line of antioxidant defense and
effectively protects the lipids in plasma and lipoproteins against
detectable peroxidative
damage,19 even in the
presence of free, redox-active
iron.18
Therefore, the aims of this study were to (1) determine whether there is evidence of plasma antioxidant depletion in patients with ICH or HT; (2) examine the time course of any observed changes in antioxidant levels; and (3) correlate those changes with the clinical severity of the disease and/or the extent of the brain injury.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Thirteen patients with ICH (6 women and 7 men, 62.4±6.1 years of age), 15 patients with HT (1 woman and 14 men, 44.6±6.9 years of age), and 40 healthy, nonsmoking, normolipidemic subjects, including 20 "young control subjects" (<45 years of age; 10 women and 10 men, 30.7±7.1 years of age) and 20 "adult control subjects" (>45 years of age; 10 women and 10 men, 58.3±5.8 years of age), were studied. Patients were consecutively recruited from the Neurological/Neurosurgical Intensive Care Unit of the Massachusetts General Hospital after approval was obtained from the ethics committee of the hospital and informed consent was obtained from patients or their relatives. In all patients, clinical examination was performed every day, and a CT scan was performed. The National Institutes of Health (NIH) Stroke Scale and Glasgow Coma Scale were assessed on admission and daily until discharge.
Patients with HT and ICH were divided into subgroups
according to the neuroradiological size (CT scan) of the
hemorrhage or contusion. The CT scan used for this purpose was
performed between the second and the fourth days after the
hemorrhage in all patients (mean time, 2.6 days). Groups A, B,
and C, respectively, consisted of patients with small, medium, or
massive hemorrhage or contusion with major diameter 
2 cm,
between 2 and 4 cm, or >4 cm.
Figure 1
shows an example of measurement of the major
diameter of an ICH.
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In 4 HT patients and 1 ICH patient, simultaneous blood samples were obtained from central (jugular) and peripheral lines.
All patients were enrolled in the study within 24 hours from the onset of the injury. A 10-mL tube of heparinized blood was obtained on admission and every other day up to 1 week. Blood was immediately centrifuged, and plasma was separated and stored frozen at -80°C until analysis, which was performed within 1 week. Blood from healthy control subjects was obtained in a single setting at 8 AM after an overnight fast. In all brain-injured patients, blood pressure, white blood cell count, temperature, inflated oxygen flow (FIO2) (if intubated), medications before and during the hospitalization, nutritional status, smoking habit, and alcohol abuse were assessed. Subjects with multiple and/or major organ failure, other neurological or psychiatric disorders, or taking antioxidant vitamin supplements were excluded from the study. In addition, we excluded patients with multiple brain hemorrhages and other traumas.
The determination of plasma AA and UA levels was performed by high-performance liquid chromatography (HPLC) with electrochemical detection.19 A 100-µL aliquot of plasma was extracted with an equal volume of 5% metaphosphoric acid containing 1 mmol/L of the metal chelator diethylenetriaminepentaacetic acid, then vortexed and centrifuged. Twenty microliters of the supernatant was mixed with 6 µL of 2.58 mol/L potassium phosphate (pH 9.8) and 74 µL of the mobile phase (40 mmol/L sodium acetate, 0.54 mmol/L Na2 EDTA, 1.5 mmol/L dodecyl triethylammonium phosphate, 7.5% methanol, pH 4.75). AA was detected at an applied potential of +0.6 V.19
Ubiquinol-10 was quantified by HPLC with chemiluminescence
detection.20 A 250-µL
aliquot of plasma was extracted with 1 mL of ice-cold methanol and 5 mL
of ice-cold hexane. The sample was vortexed and centrifuged.
The hexane extract was dried under N2,
resuspended in 450 µL of methanol/butanol (50:50), and
analyzed with reversed-phase HPLC with chemiluminescence
detection.20 The eluate was
mixed with a reaction solution containing microperoxidase and
isoluminol, and chemiluminescence produced by ubiquinol was measured at
0.01 mA. UV absorbance at 
=210 nm was monitored in series for
quantification of
-tocopherol.20
All values are presented as mean±SD. Plasma
concentrations of AA, UA, -tocopherol, and ubiquinol-10
were compared between the groups of patients by 1-way ANOVA for the
values on day 1 and by 2-way ANOVA for the values over time.
Correlations of AA, UA, -tocopherol, and ubiquinol-10
plasma levels with the NIH Stroke Scale, Glasgow Coma Scale, or the
diameter of the lesions were examined by linear regression or by
Spearman’s correlation as appropriate. Significance was accepted if
the null hypothesis was rejected at the level of
P<0.05.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Eleven ICH patients (84%) and 1 HT patient (6%) were hypertensive, and 2 ICH patients (15%) had diabetes. None of the subjects studied had history of alcohol abuse. Nine HT patients (60%) and 6 ICH patients (46%) were intubated, with a mean FIO2 of 45.5±3.3% and 49.3±2.5%, respectively. The mean white blood cell count in the patients with ICH and HT was 10 220±550 and 11 320±465/mL, respectively. There were no differences of body temperature between patients, and none showed evidence of viral or bacterial infection during the time of the study. No clinical evidence of muscle and tissue injury was observed, and LDH serum levels were in range in all patients. Four ICH patients died, 1 on day 3 and the other 3 on day 7. None of the HT patients died.
All brain-injured patients had significantly
(P<0.002) lower plasma AA
levels on day 1 compared with healthy control subjects
(Table 1). Plasma concentrations of UA,
-tocopherol, and ubiquinol-10 were not different in
patients compared with control subjects
(Table 1). In addition, plasma antioxidant levels did not
significantly change over time in ICH and HT patients
(Figure 2).
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Patients of group A (small hemorrhage or contusion)
had higher mean plasma levels of AA, UA, and -tocopherol
than patients of group B (medium hemorrhage or contusion), who
had higher levels than patients of group C (massive hemorrhage
or large contusion)
(Table 2). However, only AA levels between groups A and C
were significantly different
(P<0.05).
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Plasma AA levels in all patients but not any of the other
antioxidants were significantly inversely correlated with the major
diameter of the hemorrhage or contusion (
=-0.47,
P=0.002, and =-0.54,
P=0.002, respectively)
(Figure 3). Similarly, only plasma AA levels were
significantly negatively correlated with the NIH Stroke Scale
(r=-0.12,
P<0.03) and significantly
positively correlated with the Glasgow Coma Scale
(r=+0.21,
P<0.02). No significant
correlations between antioxidant levels and smoking habit,
hypertension, diabetes, caloric intake,
FIO2,
serum cholesterol levels (total, HDL, and LDL), or white
blood cell count were observed, both in ICH and HT
patients.
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In the 5 patients from whom both peripheral and
central blood was obtained, central (jugular) plasma exhibited lower AA
concentrations than peripheral plasma on 3 of the 4 days
examined, although none of the differences were significant
(Table 3). Two-way ANOVA for values over time also showed no
significant differences.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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This study provides evidence for oxidative stress-induced antioxidant depletion in human ICH and HT, specifically AA depletion. Importantly, the decreased plasma levels of AA were significantly inversely correlated with the major diameter of the brain lesion and with indexes of injury-related clinical impairment, suggesting the presence of oxidative stress in brain trauma and hemorrhage.
We believe that the loss of vitamin C may be due to the brain injury for a number of reasons. First of all, vitamin C depletion was independent from the presence of smoking habit, hypertension, diabetes, and from dietary or FIO2 intake. Second, plasma vitamin C levels were not correlated with serum cholesterol levels, body temperature, or white blood cell count both in ICH and HT patients. These findings, along with the observation of lower AA concentrations in the jugular blood plasma as compared with the peripheral blood plasma of brain-injured patients, do not support the idea of a systemic cause alone of vitamin C depletion.
The present data are consistent with
observations in experimental animals of antioxidant depletion in brain
injury. For example, decreased brain concentrations of AA were observed
in animal models of focal cerebral
ischemia,21 22
and tissue levels of AA and -tocopherol were decreased
after spinal cord impact trauma in several
species.23 In rats,
transient cerebral ischemia and reperfusion caused ROS
production associated with consumption of ubiquinol-9 and
ubiquinol-10.24 Similarly,
decreases in tissue levels of ubiquinol-9 and ubiquinol-10, AA, and
-tocopherol were observed after spinal cord impact
trauma in rats.25 Studies of
oral supplementation with antioxidants in animal models showed that
treatment with vitamin E and selenium before traumatic brain injury
significantly protected the nervous tissue from progressive declines in
white matter blood
flow.26 27
Treatment with AA before the trauma also significantly delayed
posttraumatic spinal cord
hypoperfusion.26 Finally,
several studies found that a low vitamin C status in humans is
associated with increased mortality rates from
stroke,28 29 and
plasma AA concentrations are decreased in stroke and critically ill
patients.30 31 32 33
These data on antioxidant depletion suggest that there is significant
oxidative stress associated with brain injury. In agreement with this
notion, it has been reported that ischemia/reperfusion injury,
global ischemia, and head trauma in experimental animals are
associated with oxidative damage to proteins and
lipids.15 24 34
ROS released during reperfusion of ischemic brain
tissue may also contribute to cerebral edema, vascular wall injury, and
hemorrhage.15
Hemorrhage is a frequent complication of reperfusion in
ischemic brain tissue, and hemorrhagic transformation of an
ischemic cerebral area can occur spontaneously.
Hemorrhage is associated with the release of heme iron normally
bound to hemoglobin, and as a result, Fenton-type reactions may ensue
with production of hydroxyl radicals and consequent initiation
of lipid peroxidation. The time course and intensity of brain hydroxyl
radical generation, as measured by the salicylate-trapping method, were
studied in animal models of moderate or severe head injury. A dramatic
increase in the indexes of hydroxyl radical formation was observed
immediately after head trauma, and this increase was prevented by the
administration of tirilazad
mesylate.10 35 In
addition, a significant increase in the levels of
prostaglandin F2,
thromboxane A2, and
leukotriene C4 was observed after
head trauma.10
Methylprednisolone, which can inhibit lipid
peroxidation,36 37
has shown beneficial therapeutic effects in experimental
models36 37 and
patients38 with acute spinal
cord injury. Interestingly, iron-induced and trauma-induced injuries to
neural tissue are similar in
nature,6 supporting a role of
ROS produced during the conversion of arachidonate to
prostaglandins in trauma-associated microvascular
damage.6 15
Some limitations to this study should be acknowledged. Although this appears to be the first study regarding plasma antioxidant longitudinal changes after brain hemorrhage, antioxidant concentrations before the occurrence of the disease were unknown. This information would facilitate a clearer interpretation of data. Furthermore, the exact relation between plasma and cerebral tissue antioxidant levels in brain hemorrhage is still to be addressed. Finally, the achievement of a conclusion regarding the role of vitamin C loss may be reached through the analysis, in a larger sample of patients, of the relations existing between plasma vitamin C levels and (1) the enlargement of the hemorrhage over time, (2) the functional outcome after 1 week, and (3) the loss of cerebral tissue after 3 months.
In summary, the present study shows that plasma AA levels are significantly lower in brain-injured patients compared with healthy control subjects and are inversely correlated with the major diameter of the brain lesion. The consequences of early vitamin C depletion on brain injury, as well as the effects of vitamin C supplementation in ICH and HT patients, remain to be addressed in further studies.
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Acknowledgments |
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Dr Polidori is an EU Marie-Curie Fellow for the program "Quality of Life and Management of Living Resources" project entitled "Nutritional Health-Sustaining Factors and Determinants of Healthy Aging: Oxidative Stress-Related Biomarkers of Successful Aging and Age-Related Diseases." The authors wish to thank M.F. Beal, MD, W.J. Koroshetz, MD, E.A. Chiocca, MD, PhD, and G. Rordorf, MD, for help with patient recruitment and screening and G. Nelles, MD, for critically revising the manuscript.
Received August 28, 2000; revision received November 6, 2000; accepted November 15, 2000.
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