ARTICLE IN PRESS doi:10.1510/icvts.2006.128561
Interactive CardioVascular and Thoracic Surgery 5 (2006) 387–391 www.icvts.org
Institutional report - Cardiopulmonary bypass
Markers of inflammation and oxidative stress in patients undergoing CABG with CPB with and without ventilation of the lungs: a pilot study Ivo Debliera,噛, Anna M. Sadowskab,*,噛, Annelies Janssensb, Inez Rodrigusa, Wilfried A. De Backera a Department of Cardiac Surgery, University Hospital Antwerp, Antwerp, Belgium Department of Pulmonary Medicine, Universiteitsplein 1, University of Antwerp, Belgium
b
Received 10 January 2006; received in revised form 6 April 2006; accepted 10 April 2006
Abstract Cardiopulmonary bypass triggers systemic inflammation and systemic oxidative stress. Recent reports suggest that continuous ventilation during cardiopulmonary bypass (CPB) can affect the outcome of patients after cardiac surgery. We investigated the influence of lung ventilation on inflammatory and oxidative stress markers during coronary artery bypass graft (CABG) with CPB in 13 patients with (Group 2) or without (Group 1) ventilation of the lungs with small tidal volume (4 mlykg). IL-10 and elastase in blood were elevated in both groups with a peak at the end of CPB (P-0.05) and returned to the baseline at 24 h after surgery. A significant increase in Trolox Equivalent Antioxidant Capacity (TEAC) was observed in both groups (P-0.05). Glutathione peroxidase (GPx) was significantly elevated 24 h after surgery only in Group 1 (P-0.05). There was a significant decrease in alpha-tocopherol 24 h after surgery in both groups (P-0.05). The inflammatory response observed during CPB is not directly influenced by continuous ventilation of the lungs with small tidal volumes. The modulation of antioxidant defense systems by ventilation needs further investigation. 䊚 2006 Published by European Association for Cardio-Thoracic Surgery. All rights reserved. Keywords: CABG; CPB; Inflammation; Oxidative stress; Ventilation
1. Introduction Although open cardiac coronary artery by-pass grafting (CABG) has become a routine procedure worldwide, patient morbidity and mortality due to the adverse post-operative complications are still high. The inflammatory response and systemic oxidative stress are reported to be directly caused by the procedure w1x. The mechanisms explaining these observations may be related to the several events occurring during cardiopulmonary bypass (CPB) which are material dependent (exposure of blood to non-physiologic surfaces) or material independent (surgical trauma, ischemia-reperfusion, and changes in the body temperature). Moreover, CPB influences the structure of the bronchoalveolar tree by inducing atelectasis w2x, which prolonged may facilitate the proinflammatory cytokine production by macrophages w3x. These cytokines, by acting as chemoattractants for neutrophils, may further enhance the inflammatory response. One of the most damaging consequences of the inflammatory cell activation is the formation of reactive oxygen species (ROS) by myocardial cells, neutrophils or endothelial xanthine oxidase which, in turn, may modulate the activity of the redox-sensitive transcription factor such 噛Shared first co-authorship. *Corresponding author. Tel.: q32 38202589; fax: q32 38202574. E-mail address:
[email protected] (A.M. Sadowska).
as NF-kB and AP-1 leading to pro-inflammatory cytokines up-regulation. Some of the harmful events caused by CPB are inherent to the procedure and cannot be avoided. The dangerous consequences of the partial collapse of the lungs, on the other hand, could be minimized by keeping the lungs occupied during the procedure in order to prevent their collapse and avoid the subsequent re-ventilation. Nevertheless, the reports on that matter are equivocal. The aim of this study is to focus on ventilation with small tidal volumes and evaluate its influence on oxidative stress and inflammation in patients undergoing CABG with CPB. Therefore, we looked at markers of inflammation (TNF-a, IL-6, IL-10, elastase, defensins) and oxidative stress (GPx, SOD, TEAC, tocopherol, retinol) in two groups of patients undergoing CABG with CPB without (Group 1) or with (Group 2) mechanical ventilation. 2. Materials and methods 2.1. Study population Thirteen patients (1 female, 12 males) undergoing elective CABG were included in the study and randomized into two groups. Exclusion criteria were emergency surgery, chronic obstructive pulmonary disease, smoking, steroid use. Patients characteristic and perioperative data are
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Table 1 Patient characteristis and perioperative data
Number of patients MaleyFemale BMI (kgym2)b Age (years)a Body surface area (m2)b Recent myocardial infarction LV ejection fraction 0.5 0.5 ) = )0.3 -0.3 New York Heart Association I II III IV Operative time (min) Intubation time (h) Catecholamine support (number of patients) Longest clamp time (min) Cross-clamp time (min) Pump time (min) Reperfusion time (min) Hospitalization (days) O2 demand (ly24 h)
Group 1
Group 2
6 6y0 26.6"2.0 58 (52–76) 2.1"0.1 0
7 6y1 26.9"3.3 66 (60–72) 1.95"0.1 1
5 1 0
6 1 0
0 3 3 0 207"23 9.8"2.2 2
0 3 4 0 203"33 8.2"2.0 4
11"2 31"12 118"16 53.5"11.5 8.2"1.0 4.2"1.6
9"1 25"14 106"22.0 54.2"18.3 8.3"1.9 5.7"4.3
a Data are presented as median (min-max) or b as mean"S.D. Group 1 – without ventilation, Group 2 – with ventilation.
shown in Table 1. The study was in accordance to the principles outlined in the Declaration of Helsinki. Patients signed the informed consent and the Ethical Committee of the University Hospital of Antwerp approved the study protocol. 2.2. CABG without ventilation (Group 1) Administration of pre-medication and aesthesia with endotracheal intubation and transfusion were uniform in all cases. All patients received fentanyl (50 mg), dehydrobenzperidol (2.5 mg), glycopyrrolate (0.2 mg) as premedication. Anesthesia maintenance was achieved with remifentanyl and sevoflurane. No steroids and no anti-fibrinolytic agents have been administered. An Optima Membrane Oxygenator was used. The pump-oxygenator was primed with 1000 ml Voluven䊛 (Fresenius Kabi, Bad Homburg, Germany) and 500 ml Plasma-Lyte A (Baxter Healthcare Corp, Deerfield, IL). If necessary, plasma substitute Gelofusion was administered. Cardiopulmonary bypass was established with a moderate hemodilution and moderate systemic hypothermia (28 8C). Surgery was performed using the intermittent cross-clamp technique under the protection of Lidoflazine (Janssens Pharmaceutica, Belgium). Fifteen minutes after decannulation, heparin was neutralized with protamine chloride (1:1 ratio; Roche, Belgium). 2.3. CABG with ventilation (Group 2) The same procedure as described above with the ventilation of the lungs with small tidal volume of 4 mlykg, fraction of inspired oxygen (FiO2 ) 40%, with zero-end expiratory pressure was performed in Group 2.
2.3.1. Blood samples Blood samples were collected into sterile lithium-heparin and SST (Serum Separator Tubes) with clotting activator (Vacutainer, Becton Dickinson). Samples were collected: 10 min after induction of anaesthesia (pre), 10 min after start CPB (109 CPB), at the end of CPB (end CPB), 10 min after protamine administration (protamine), 4 h (4 h post) and 24 h (24 h post) after surgery. Bronchoalveolar lavage (BAL) was performed twice (109 after induction of anesthesia (BAL 1) and 2 h after surgery (BAL 2). Samples were processed within 10 min after sampling. All obtained data were corrected for hemodilution. Trolox equivalent antioxidant capacity (TEAC) was measured in plasma according to the method of Rice-Evans and Miller. Intra- and inter-assay coefficient of variation (CV) was 5 and 14%. Glutathione peroxidase (GPx) in full blood was measured with a Ransel Glutathione Peroxidase kit (Randox Laboratories Ltd). Intra- and inter-assay CV was 2 and 6%. Alpha-tocoferol and retinol in serum were measured by High Performance Liquid Chromatography (Dionex, HPLC with a 100% methanol mobile phase) with detection at 292 and 325 nm, respectively. Intra- and inter-assay CV was 5 and 13%. IL-6, IL-10, TNF-a were measured with ELISA (Biosource International, Belgium). Neutrophil defensins in BALF were measured with ELISA (Hycult Biotechnology, Uden, The Netherlands). Neutrophil elastase (NE) in BALF and plasma was measured spectrophotometrically using the synthetic substrate methoxysuccinyl-ala-ala-pro-val-paranitroanilide (MeOSAAPVpNa) (Sigma Chemicals). Aliquots of 50 ml of standard or sample were added to wells of a microtiter plate (Life Technologies Ltd, Paisley, UK) followed by MeOSAAPVpNa solution. The reaction was continued for 20 h at 37 8C. The absorbance was read at 405 nm. 2.3.2. Statistical analysis Statistical package GraphPad Prism 4.00 for Windows (GraphPad Software, San Diego, CA, USA) was used. Data are shown as mean"S.E.M. Repeated measures ANOVA with Dunnett’s multiply comparison test was used to assess within group differences throughout the procedure. Between-groups differences were assessed using Student " t-test. Differences were considered significant at a P -0.05. 3. Results 3.1. Inflammatory response TNF-a in blood was significantly elevated after the surgery (4 h post) in both groups with no difference between groups (Fig. 1). IL-10 in blood was significantly elevated in both groups during the surgery with a peak at the end of CPB (end CPB) versus pre-surgery sample (pre) (Fig. 2). Elastase was also increased throughout the surgery in both groups with a peak at the end of CPB (end CPB) versus pre-surgery (pre) and returned to the baseline 24 h after surgery in both groups (Fig. 3).
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Fig. 1. Time-course of changes in TNF-a in blood during CABG *P-0.05 when compared to pre-operation sample.
Fig. 4. Time-course of changes in retinol in blood during CABG *P-0.05 when compared to pre-operation sample.
Fig. 2. Time-course of changes in IL-10 in blood during CABG *P-0.05 when compared to pre-operation sample.
Fig. 5. Time-course of changes in tocopherol in blood during CABG *P-0.05 when compared to pre-operation sample.
Fig. 3. Time-course of changes in elastase in blood during CABG *P-0.05, **P-0.01 when compared to pre-operation sample.
Elastase, IL-6 and neutrophil defensins (HNP) in BALF tended to increase in both groups 2 h after surgery. This increase, however, did not reach significance level. There was no difference observed between groups (data not shown). IL-10 in BALF remained unchanged in both groups (data not shown). 3.2. Oxidative stress Retinol remained unchanged in Group 2 whereas there was significant decrease in Group 1 (Fig. 4). Tocopherol
Fig. 6. Time-course of changes in TEAC in plasma during CABG *P-0.05 when compared to pre-operation sample.
decreased significantly in both groups 24 h after surgery (Fig. 5). Trolox equivalent antioxidant capacity (TEAC) was increased in both groups throughout the surgery with a peak 4 h after surgery and it remained elevated 24 h after surgery in Group 1 (Fig. 6). Glutathione peroxidase did not change throughout the procedure, there was however, an increase 24 h after surgery in Group 1 (24 h post) versus pre-surgery (pre). No difference was observed in Group 2 (Fig. 7).
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Fig. 7. Time-course of changes in GPx in blood during CABG *P-0.05 when compared to pre-operation sample.
4. Discussion The systemic increase in the oxidative stress and inflammation during CPB has been described before w4x. Moreover, in our previous study, we described the increase in the antioxidant enzymes and the decrease in the retinol and tocopherol during CPB without ventilation w5x. Nevertheless, the influence of the mechanical ventilation on the innate antioxidant defense and systemic and local inflammation has not been fully evaluated yet. 4.1. Inflammation We observed an increase in several inflammatory cytokines, neutrophil elastase and neutrophil defensins in plasma and in BALF in both studied groups, indicating that the inflammatory process during CPB is both local and systemic. The increase, however, was not simultaneous. The plasma level of elastase and IL-10 peaked at the end of CPB while TNF-a was the highest 4 h after the surgery. The mechanism of this delayed TNF-a-up-regulation is not very well described but it is plausible that IL-10 antagonizing effect is involved. A sharp increase in the plasma concentration of IL-10 was observed during some inflammatory states including septic shock w6x. During CPB, IL-10 increased simultaneously with neutrophil elastase, possibly counteracting the early TNF-a increase. This observation is in agreement with the study of Matata et al., who observed an increase in TNF-a not ealier than 24 h after the CPB initiation w7x and Fromes et al., who reported an increase in TNF-a after weaning off the CPB w8x. The release of elastase, on the other hand, may be induced by the interaction of chemotactic agents with plasma membrane receptors that leads to actin polymerization and to cytoskeletal re-organization w9x (both calcium dependent processes) which might result in the degranulation and the subsequent release of elastase w10x. 4.2. Effect of ventilation on inflammatory response It has been described that different ventilation methods may lead to the different outcomes. Namely, it has been reported that in contrast to the conventional mechanical ventilation, the protective ventilation with tidal volumes and high positive end-expiratory pressure (PEEP) induced
less cytokine release and caused less pulmonary injury in patients with acute lung injury w11x. On the other hand, high ventilatory volume (40 mlykg) and zero end-expiratory pressure (ZEEP) induced much stronger inflammatory response than moderate (15 mlykg) and tidal volumes (7 mlykg) with either PEEP or ZEEP. The moderate volumes significantly increased TNF-a, IL-1b in BALF in comparison with the tidal volumes, but the increase was less pronounced than the case of high volumes w12x. On the other hand, Koner et al. showed no difference between protective ventilation technique (6 mlykg and PEEP) and the conventional ventilation (10 mlykg and ZEEP or PEEP) in regard to the inflammatory response w13x. In our study, ventilation of the lungs did not inhibit or delay the inflammatory response as the same pattern was observed in both the groups. It may be postulated that the collapsed lungs may not be the main source of the inflammatory response when compared to the potent reaction caused by CPB. Therefore, their ventilation might not be sufficient to counterbalance or diminish the extensive inflammation caused by extracorporeal circulation. 4.3. Effect of ventilation on the antioxidant defenses In our previous study, we observed an increase in the enzymatic antioxidants parallel with the decrease in the antioxidants such as tocopherol and retinol w5x. In that study, however, patients were pretreated with steroids which may have up-regulated the antioxidant enzymes as it was already described before w14x. In the current study, patients did not receive any steroids and consequently, we did not observe the increase in GPx during the procedure except for the non-ventilated group, 24 h after the surgery. TEAC, on the other hand, increased in both the groups during surgery, but it remained higher in the non-ventilated group only. Because the main component of the TEAC value is uric acid, which has been described to increase during CPB w15x, this observation could explain the rise in TEAC level in the beginning of CPB. The up-regulation of GPx, on the other hand, in the non-ventilated group 24 h after the surgery may be caused by the increased status of the oxidative stress caused by re-ventilation of the collapsed lungs and hence subsequent up-regulation of GPx in this group. In contrast, antioxidants which act as free radical scavengers were consumed during oxidative processes. We observed a decrease in tocopherol in both groups supporting our previous results w5x. The significant decrease in retinol after 24 h surgery was observed in the non-ventilated group only. Moreover, both retinol and tocopherol concentrations were slightly higher in the ventilated group throughout the procedure. This difference, however, did not reach a significant level. It seems plausible, that keeping the lungs occupied during the procedure may prevent them from the injurious re-ventilation and hence prevent, at least to some extent, the antioxidant depletion. In summary, this preliminary study, performed in a small number of selected patients, showed that there was a strong inflammatory response during CABG with CPB. This response was both local and systemic and was not influenced by ventilation of the lungs with a small tidal volume. The oxidative stress status, on the other hand, may be
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