Article

Assessment of oxidative stress after out-of-hospital cardiac arrest

a b s t r a c t

Introduction: Pathophysiology of cardiac arrest corresponds to a whole body ischemia-reperfusion. This phenom- enon is usually associated with an oxidative stress in varioUS settings, but few data are available on cardiac arrest in human. The aim of the present study was to evaluate different oxidative stress markers in out-of-hospital car- diac arrest (OHCA) patients treated with therapeutic hypothermia.

Materials and methods: We conducted a prospective study assessing oxidative stress markers (thiobarbitUric acid reactive species, carbonyls, thiols, glutathione, and glutathione peroxidase) in OHCA patients treated with ther- apeutic hypothermia. Measurements were performed during the 4 days after admission and compared between good and poor outcome patients according to Cerebral Performance Category.

Results: Thirty-four patients were included, 10 good and 24 poor outcomes at 6 months. Thiobarbituric acid reac- tive species were higher in the poor outcome group on admission and when therapeutic hypothermia was reached. The other markers were not different between groups. No markers seemed modified by the use of ther- apeutic hypothermia in each group.

Conclusions: After OHCA, good outcome patients exhibit lower oxidative stress markers than poor outcome pa- tients. Thiobarbituric acid reactive species appears to be an early prognostic parameter. Oxidative stress markers seem not mitigated by therapeutic hypothermia.

(C) 2016

Introduction

Out-of-hospital cardiac arrest (OHCA) is a major health problem with a poor prognosis [1,2]. After resuscitation, unfavorable outcome re- sults mainly from the complications of postreperfusion syndrome [3]. refractory shock state and multiorgan failure are responsible of early fa- talities. Lately, most of patients die of irreversible brain damage. Patho- physiology of resuscitated cardiac arrest represents a whole body ischemia/reperfusion. This phenomenon leads to an oxidative stress and a generalized nonspecific inflammation [4]. Oxidative stress results from an imbalance between Reactive oxygen species production and antioxidant defenses levels. A massive generation of ROS occurs in the minutes after reperfusion, but few clinical studies confirmed these laboratory data [5]. Actually, ROS are very unstable with a short half- life, and their measurement techniques are cumbersome. Most of the time, oxidative stress markers are measured as a substitute of ROS

* Corresponding author at: Reanimation Polyvalente et Surveillance Continue, Hopital Pasteur, CHU de Nice, 30, voie Romaine, 06006 Nice Cedex, France. Tel.: +33 4 92 03 33 00.

E-mail address: [email protected] (J.-C. Orban).

because they are more stable. They reflect the interaction and the subse- quent injuries of ROS with different cell components. Thiobarbituric acid reactive species (TBARS) and Malondialdehyde are specific of lipid peroxidation, and carbonyls reflect protein oxidation. On the other hand, reductions in antioxidant levels be it enzymatic (superoxide dismutase, catalase, glutathione peroxidase) or nonenzymatic (gluta- thione, thiols, vitamins…) are most frequently measured as indicators of oxidative stress. Thus, glutathione, one of the main antioxidants, is decreased in multiorgan failure patients [6]. The antioxidant enzyme glutathione peroxidase shows an increased activity when facing an ox- idative stress situation. Several laboratory data begin to unravel the as- sociation between cardiac arrest and oxidative stress. For example, plasma lipid peroxidation and DNA damage marker are correlated to myocardial dysfunction in a model of cardiac arrest [7]. In another ani- mal model, the level of oxidative stress due to the percentage of oxygen during reperfusion is associated to outcome [8]. Unfortunately, clinical data exploring the relations between oxidative stress and cardiac arrest are scarce. Endothelial cells incubated with plasma from cardiac arrest patients exhibit the features of oxidative stress with an increased ROS generation and a diminution of antioxidant defenses [9]. In another study, thioredoxin, a protein induced by oxidative stress and inflamma- tion, was higher in nonsurvivor than in survivor after cardiac arrest [10].

http://dx.doi.org/10.1016/j.ajem.2016.05.054

0735-6757/(C) 2016

Fig. 1. Study protocol.

Therapeutic hypothermia has been used decades ago in the manage- ment of cardiac arrest patients, but its utilization waned due to compli- cations [11]. Recent articles brought back this therapy under the light. To date, it is the only intervention improving mortality and neurologic outcome [12,13]. Several hypotheses were suggested to explain these effects: mitigation of the postreperfusion oxidative stress and inflam- mation, a decreased cerebral oxygen consumption, and reduction of excitotoxicity. Laboratory data support some of these assumptions, showing a decrease in oxidative stress correlated to therapeutic hypo- thermia [14,15]. However, few clinical data are available. Recently, it has been shown that temperature changes during therapeutic hypo- thermia and rewarming could affect the course of proinflammatory and anti-inflammatory molecules [16].

Because laboratory and clinical data suggest a temperature mitiga-

tion of oxidative stress, we investigated the changes in oxidative stress markers during therapeutic hypothermia after OHCA and their associa- tion with prognosis.

Materials and methods

Patient selection

After approval from the local ethics committee (Comite de Protec- tion des Personnes Sud Mediterrranee V, study number 06.008), we in- cluded prospectively comatose patients resuscitated from an OHCA treated with therapeutic hypothermia. Written informed consent was obtained from the next of kin before enrollment. As therapeutic hypo- thermia is a standard of care, we considered unethical the comparison to a normothermic group. All patients aged 18 to 80 years admitted for OHCA from cardiac or respiratory causes were eligible. Those with resuscitation longer than 60 minutes, fraction of inspired oxygen higher than 60% 1 hour after admission, refractory shock, and moribund pa- tients were excluded.

Patients’ management

Once the prehospital team obtained return of spontaneous circula- tion, coronary angiography and percutaneous coronary intervention were performed if needed. Then patients were admitted to the intensive care unit of our tertiary care university hospital. According to ILCOR rec- ommendations, patients were cooled to 34?C [17]. To reach this goal, ex- ternal methods were used consisting of ice packs placed on main vascular accesses and torso and fans. Therapeutic hypothermia was maintained during 24 hours, followed by passive rewarming to normo- thermia (37?C). Temperature was continuously monitored by a Foley

Table

Demographic data of the population

Study population

Good outcome (CPC 1 and 2)

Poor outcome (CPC 3-5)

Age (y)

58 (22-77)

60 (46-71)

57 (22-77)

No flow time (min)

10 (5-15)

13 (8-15)

10 (5-15)

Low flow time (min)

15 (10-30)

10 (5-30)

16 (10-30)

catheter with a temperature sensor (Level 1; Smith Medical ASD, Rockland, MA). Patients were given an association of midazolam and fentanyl during hypothermia and paralyzed using continuous infusion of cisatracurium. All patients were intubated and mechanically ventilat- ed aiming at a PaO2 between 75 and 100 mm Hg and PaCO2 between 35 and 45 mm Hg. A central venous catheter was inserted in the subclavian or jugular vein, and an Arterial line was inserted in the radial or femoral artery for monitoring of blood pressure and sampling of arterial blood. Mean arterial blood pressure was maintained greater than 80 mm Hg, and diuresis was aimed greater than 0.5 mL/kg per hour. Patients were given fluid infusion or dobutamine or norepinephrine to reach this goal, according to hemodynamic monitoring data. Hemoglobin con- centration was kept greater than 10 g/dL. Glucose was maintained be- tween 0.8 and 1.2 g/dL by continuous Insulin infusion according to our local protocol.

Study protocol

Assessment of oxidative stress was evaluated by different parame- ters: markers of lipid peroxidation (TBARS) and protein oxidation (carbonyls) and antioxidant defenses (glutathione, thiol radicals, gluta- thione peroxidase activity) (Fig. 1). These measurements were per- formed on arterial blood at different time points: on admission (T0), at 34?C (T1), after 12 hours at 34?C (T2), after 24 hours at 34?C (T3), after rewarming to 37?C (T4), and on day 4 (T5).

Arterial blood samples were collected in lithium heparin vacutainers as an anticoagulant. Four hundred microliters of whole blood was col- lected in 3.6 mL of metaphosphoric acid for the determination of total glutathione. After centrifugation (4000g, 10 minutes, 4?C), total gluta- thione was determined enzymatically in the acidic protein-free super- natant. The rest of whole blood was centrifuged to separate the plasma for thiols, TBARS, glutathione peroxidase, and carbonyls mea- surements. Plasma was collected in Eppendorff sterile tubes and stored at -80?C until assayed. Lipid peroxidation intermediates were mea- sured by the plasma thiobarbituric acid reactive substances. Thiobarbi- turic acid reactive species are products of the oxidative degradation of polyunsaturated fatty acids, in particular MDA. We used the modified method of Ohkawa et al [18], based on the reaction of aldehyde func- tions of MDA released by acid hydrolysis at 95?C with thiobarbituric acid forming a pink-colored complex quantified by fluorimetry. Plasma carbonyl assay is based on the reaction of carbonyl groups in protein with 2,4-dinitrophenylhydrazine to form 2,4-dinitrophenylhydrazone, which was estimated spectrophotometrically at 380 nm after trichloro- Acetic acid precipitation of proteins. Glutathione peroxidase activity was determined by the modified method of Gunzler using tert-butyl hydro- peroxide as substrate [19]. The measurement of plasma thiol groups was performed using Ellman’s reagent and determined spectrophoto- metrically at 412 nm [20]. All reagents were purchased from Sigma (St Louis, MO).

Collected variables included the patient’s demographic and prehospital data on admission (“no flow” and “low flow” durations, drugs infused, defibrillation). Hemodynamic parameters, temperature, and SpO2 were measured continuously. Outcome was assessed using the Cerebral Performance Category scale (CPC) ranging from 1 to 5 at 6 months. Cerebral Performance Category 1 and 2 were considered fa- vorable outcome, and CPC 3 to 5 were considered unfavorable.

Fig. 2. Thiobarbituric acid reactive species at the different times of the study in good (light gray bars) and poor (dark gray bars) outcome groups. $Significant differences between good and poor outcome groups (Pb .05). #Significant decreases between T0 and T2 (Pb .001) and between T0 and T4 in the poor outcome patients (Pb .001). Normal values are 2.13 to 2.86 umol/L.

Statistical analysis

Statistical analysis was performed using XLSTAT version 2013.2.01 (Addinsoft, New York, NY). Data are presented as median with inter- quartile range. Changes over time were analyzed with repeated- measures test and post hoc analysis by Dunn test. Comparisons between groups according to outcome were made using Mann-Whitney U test. Pb .05 was considered statistically significant.

This study is registered in Clinical Trials NCT00888966.

Results

Patient characteristics

Thirty-four patients were enrolled from February 2006 to May 2010. Median age was 58 years, extremes [22-27], 21 were men and 13 women. First recorded rhythm was ventricular fibrillation in 18 pa- tients, asystole in 15, and pulseless activity in 1 case. Six months after

cardiac arrest, outcome was considered good in 10 patients (CPC 1 and 2) and poor in 24 patients (CPC 3-5). Demographic data were not different between groups (Table).

Oxidative stress markers

Thiobarbituric acid reactive species were significantly higher in the poor outcome group patients compared to the good outcome patients on admission (P= .02), at the beginning (P= .03), and at the end of therapeutic hypothermia (P= .05) (Fig. 2). Their values did not change over time in the good outcome group, whereas differences were found in the poor outcome group (P= .003). Paired comparisons showed sta- tistical differences between T0 and T2 (Pb .001) and between T0 and T4 (Pb .001). Carbonyls values were similar according to outcome and did not change during the study in both groups (Fig. 3). Glutathione levels were not different over time in the good outcome group (Fig. 4). In the unfavorable outcome patients, glutathione levels decreased signifi- cantly between T2 and T5 (P= .003). Glutathione peroxidase activity

Fig. 3. Carbonyls at the different times of the study in good (light gray bars) and poor (dark gray bars) outcome groups. No significant differences between groups and between times of the study. Normal values are 0.35 to 0.45 umol/g of protein.

Fig. 4. Glutathione at the different times of the study in good (light gray bars) and poor (dark gray bars) outcome groups. $Significant differences between good and poor outcome groups (Pb .05). #Significant decrease between T2 and T5 in the poor outcome patients (Pb .01). Normal values are 749 to 1228 umol/L.

was not different between good and poor outcome patients and did not change over time in both groups (Fig. 5). Thiol levels decreased signifi- cantly in both groups especially from the return to normothermia, and paired comparisons showed numerous differences (Fig. 6).

Discussion

The main findings of our study are that (1) the oxidative stress markers show different time course after OHCA; (2) therapeutic hypothermia does not shift oxidative stress markers; (3) TBARS, a lipid peroxidation marker, seems associated to the outcome of patients after OHCA.

The oxidative stress markers used in our study exhibited different time course. This result was already shown in different settings such as acute respiratory distress syndrome or brain injuries [21,22]. Clearly, this set of markers introduces different pieces of information. On one hand, markers of the effects of ROS on proteins (carbonyls) and lipids (TBARS) indicate few evidence of oxidative stress. However, on the other hand, the decrease of antioxidant defenses shows indirect signs of oxidative stress. Glutathione is a tripeptide containing a cysteine group that is considered the main intracellular antioxidant. In our

study, its concentration decreases over time showing indirectly the oc- currence of an oxidative stress. Similar finding was reported in severe intensive care unit patients [6]. Interestingly, it is possible to replenish glutathione stock by giving the antioxidant N-acetylcysteine [23]. This strategy could open a new way in Post-cardiac arrest syndrome treat- ment. Thiols represent an antioxidant family of molecules containing a sulfhydryl group scavenging ROS. Similarly to glutathione, thiol diminu- tion is associated to oxidative stress and inflammation [24]. The differ- ent changes could be confusing, but oxidative stress cannot be considered as a unique entity showing the same changes for different patients and diseases. Taken together, these results suggest that OHCA patients present evidence of a significant oxidative stress. This finding is in line with previous studies using other methods or marker [9,10]. Cells incubated with plasma from cardiac arrest patients exhibited a higher mortality compared to control and an increased production of ROS [9]. In another work, Mongardon et al [10] showed that thioredoxin, a protein associated to oxidative stress, was elevated after cardiac arrest.

Therapeutic hypothermia is now recommended in the postresuscitation care of OHCA patients [25]. Two prospective studies demonstrated an improvement in neurologic outcome of comatose patients after OHCA

Fig. 5. Glutathione peroxidase activity at the different times of the study in good (light gray bars) and poor (dark gray bars) outcome groups. No significant differences between groups and between times of the study. Normal values are 299 to 450 U/L.

Fig. 6. Thiols at the different times of the study in good (light gray bars) and poor (dark gray bars) outcome groups. $Significant differences between good and poor outcome groups (Pb

.05). #Significant decreases between T1 and T5 and between T2 and T5 in the good outcome patients (Pb .01). ?Significant decreases between T0 and T4, between T0 and T5, and between T1 and T5 in the poor outcome patients (Pb .01). Normal values are 5.8 to 7.7 umol/g of protein.

[12,13]. Subsequent retrospective studies confirmed these results [26]. However, few data explain the mechanisms underlying the benefits of therapeutic hypothermia. The commonly admitted hypotheses suggest different mechanisms such as a decrease of cerebral metabolic rate of oxygen, a reduction of excitotoxicity, a mitigation of postresuscitation inflammation, and oxidative stress. Only animal data on global brain is- chemia/reperfusion models support the latter hypothesis. Even Short duration of therapeutic hypothermia compared to normothermia dur- ing reperfusion mitigates brain lipid peroxidation production [14]. Du- ration of hypothermia similar to clinical practice has the same effects on cerebral MDA levels [15]. This decrease in brain oxidative stress could be mediated by a preservation of mitochondrial functions after cardiac arrest [27,28]. Because of ethical reasons, it was impossible to compare oxidative stress markers between therapeutic hypothermia and normothermia after reperfusion. Another way to assess the influ- ence of hypothermia is to evaluate the changes in oxidative stress markers according to temperature. These parameters were measured until 96 hours after resuscitation and showed no clear signal of any in- fluence of temperature. The only changes affected TBARS in the poor outcome patients with a decrease after 12 hours of therapeutic hypo- thermia but no change after return to normothermia.

Oxidative stress and inflammation are linked as ROS induce proin- flammatory cytokine expression. Contrary to our results on oxidative stress markers, Bisschops et al [16] demonstrated a shift in the inflam- matory balance induced by therapeutic hypothermia. It is important to note that this change affected only several markers. Nonetheless, this difference could be explained by several factors. First, the target temper- ature during therapeutic hypothermia was different between the 2 studies. Recent guidelines recommend to induce therapeutic hypother- mia between 32?C and 34?C, but experimental and clinical data seem to show different effects according to the hypothermia level [29]. Second, it is possible that inflammatory markers exhibit a different kinetic com- pared to oxidative stress markers.

Neurologic prognostication is a crucial issue in the management of an OHCA. The American Academy of Neurology published a decision al- gorithm to establish a neurologic prognosis [30]. However, in the mean- time, the use of therapeutic hypothermia improved outcome and modified the prognostic value of different parameters. Recommenda- tions from the European Resuscitation Council state that the evidence is limited and prognostication should not be based on a single

parameter [25]. To date, there is no answer to the significant demand of reliable prognosis markers. Our study suggests that TBARS, a lipid peroxidation marker, is higher in poor outcome patients at different times. This result is comparable to previous findings reporting an asso- ciation of oxidative stress markers to prognosis in different settings [31]. Recently, it has been demonstrated that thioredoxin, a protein induced by inflammation and oxidative stress, was associated to outcome after OHCA treated by therapeutic hypothermia [10]. Thiobarbituric acid re- active species, the same oxidative stress marker used in our study, is correlated to the Severity of diseases in multiorgan failure or acute respiratory distress syndrome [31].

Several limitations of our study should be acknowledged. The first limit is the sample size of the population. It was difficult to perform such a complete panel of oxidative stress markers at different times. This is an exploratory work, and further studies are needed to confirm our results. The second limit of the study is the site of oxidative stress as- sessment. We measured the different markers in the plasma reflecting the insults of the whole body. However, the brain lesions are the most important after OHCA as two-thirds of fatalities result from brain death or care withdrawal due to irreversible encephalopathy [32]. Ani- mal data demonstrated the protective effects of hypothermia on brain oxidative stress [15]. However, in human, the correlation between plas- matic levels of oxidative stress markers and the extent of brain injuries is still unknown.

Conclusions

After OHCA, poor outcome patients exhibit higher oxidative stress markers than good outcome patients. In this setting, TBARS seems to be an early prognostic factor. Therapeutic hypothermia does not miti- gate oxidative stress.

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