Interact CardioVasc Thorac Surg 2009;8:12-16. doi:10.1510/icvts.2008.183285
© 2009 European Association of Cardio-Thoracic Surgery
Work in progress report - Cardiac general |
Elevated resting and agonist-induced whole blood chemiluminescence in patients with active infective endocarditis
Stanislaw Ostrowskia,
Marek Kasielskib,
Jacek Kordiakc and
Dariusz Nowakd,*
a Departament of Cardiosurgery, Medical University of Lodz, Poland
b Bases of Clinical Medicine Teaching Center, Medical University of Lodz, Kopcinskiego St. 20, 90-153 Lodz, Poland
c Department of Chest Surgery, Oncologic and General Surgery, University Hospital No. 2, Medical University of Lodz, Poland
d Department of Clinical Physiology, Medical University of Lodz, Mazowiecka St. 6/8, 92-215 Lodz, Poland
Received 12 May 2008;
received in revised form 9 September 2008;
accepted 16 September 2008
 This study was supported by Medical University of Lodz Institutional Grants No. 502-11-206, No. 502-11-586, and No. 503-0079-1.
*Corresponding author. Tel.: +48-42-6782661; fax: +48-43-6782661.
E-mail address: dnowak{at}zdn.am.lodz.pl (D. Nowak).
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Abstract
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Infective endocarditis (IE) and surgical procedures related to cardiac surgery are accompanied by inflammatory responses that may alter production of oxidants by phagocytes. This study evaluates luminol enhanced whole blood chemiluminescence (LBCL) as a measure of oxidative production by circulating phagocytes in 26 IE patients in comparison to 27 matched patients with acquired valvular heart disease and 25 healthy controls. Blood was collected the day before and 3, 7, 12 and 21 days after valve replacement surgery for LBCL measurement; resting (rCL) and agonist (fMLP)-stimulated total light emission (tCL). Preoperative rCL and tCL with values observed after 3, 7, 14, and 21 days from surgery were higher (P<0.01) in patients with IE than in healthy controls. Median preoperative rCL, and tCL was about 2.5-times higher (P<0.01) in IE group than in patients with valvular heart disease (4.3 vs. 1.7 U/104 phagocytes and 2473 vs. 782 Uxs/104 phagocytes). Three days after valve replacement, LBCL rose three times (P<0.01) in both operated groups. With patient recovery, LBCL decreased and no differences were noted between groups. Patients with IE had elevated LBCL reflecting increased oxidants release from circulating phagocytes that may predispose to the development of oxidative stress.
Key Words: Infective endocarditis; Whole blood chemiluminescence; Valvular heart disease; Valve replacement; Oxidative stress; Circulating phagocytes
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1. Introduction
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Polymorphonuclear leukocytes (PMNs) and monocytes are the main phagocytic and reactive oxygen species (ROS) producing cells in the blood. A multitude of pro-inflammatory cytokines and toxins from invading microbial pathogens may activate these cells thus leading to enhanced ROS release. Exposition of circulating phagocytes in combination with these factors can occur in the course of infective endocarditis (IE), accompanied by a systemic inflammatory response with elevated plasma levels of IL-6 and IL-1beta [1]. These cytokines can activate blood phagocytes with a subsequent rise in ROS production [2]. Due to the presence of microorganism in vegetations as well as the blood stream, phagocytes can be directly exposed to a variety of bacteria-derived products including formylated chemotactic peptides, resulting in cell activation. On the other hand, bacteria can interfere with phagocytes, inhibiting their respiratory burst [3]. Also elevated C-reactive protein in patients with IE [1] was reported to suppress PMNs respiratory burst in vitro [4]. Therefore, it is difficult to predict if IE would be accompanied by enhanced or suppressed ROS release from circulating phagocytes. Meager while at the same time conflicting data concerning the ROS production by blood phagocytes in the course of IE have been published so far [5–7]. Presterl and coworkers [5] did not find any alterations in ROS production by PMNs obtained from patients with streptococcal endocarditis. Contrary to these results, the decreased ability of PMNs to kill Staphylococcus aureus was described in patients with IE [6]. In another study, patients with IE had enhanced ROS production as evaluated by a single measurement of PMNs chemiluminescence [7]. The situation could be more complicated with patients needing cardiac valve replacement surgery with cardiopulmonary bypass since extracorporeal blood circulation and oxygenation are able to enhance ROS release from PMNs [8]. Therefore, we decided to monitor ROS production by circulating phagocytes using a luminol enhanced whole blood chemiluminescence (LBCL) technique [9, 10] in patients with IE before and after cardiac valve replacement surgery in comparison to patients with valvular heart disease (VHD) and healthy subjects.
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2. Material and methods
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2.1. Study population
The study involved 26 patients with active IE and 27 patients with acquired VHD without any signs of infection that underwent surgical treatment (valve replacement) due to cardiac failure (Tables 1 and 2) and 25 healthy controls (age 58±7, median 56, range 26 years, 6 men). The clinical diagnosis of IE was established according to the modified Duke criteria [11]. The inclusion criteria for cardiac valve surgery patients were: presence of IE or VHD of rheumatic etiology, age between 40 and 80 years and a written informed consent.
The exclusion criteria included the presence of any other infectious or inflammatory processes that may affect leukocyte functions, any history of autoimmune-disorders (apart from past rheumatic fever), immunological defects, diabetes, pregnancy, alcohol and illicit drug abuse, chronic renal and liver insufficiency, usage of any food supplements with antioxidant activities within two weeks preceding the study. Healthy control subjects were required to meet all inclusion and exclusion criteria with the exception of any cardiac pathologies.
2.2. Preoperative patient management
Antibiotics that were shown to be effective through blood culture tests were used for 3–4 weeks prior to surgery in patients with IE. When blood cultures were negative, vancomycin alone or in combination with gentamycin was used. Patients with VHD did not receive any antibiotics before surgery. Both patient groups received diuretics, digitalis, and beta-blockers if required. No patient received systemic steroids and non-steroidal anti-inflammatory drugs within three weeks preceding the heart operation.
2.3. Anesthesia and cardiac surgery
Patients with IE and VHD were premedicated with morphine, diazepam and atropine. Anesthesia was induced and maintained with fentanyl, midazolam, pancuronium, and sevoflurane. All patients underwent surgical valve replacement with mechanical valve prostheses using standard cardiopulmonary bypass (CPB) with crystalloid cardioplegia. After sternotomy, patients received 2x106 IU of aprotinine and 400 IU/kg of heparin i.v. before initiating CPB. On CPB a Jostra Quadrox oxygenator and continuous flow was used. The prime consisted of 1000 ml of normal electrolyte solution, 500 ml Voluven® and 250 ml 20% mannitol with the addition 100 IU/kg heparin.
2.4. Postoperative patient management
Treatment with antibiotics was continued for an additional 3–4 weeks in patients with IE. Patients with VHD received third generation cephalosporin for two weeks. All operated patients received low molecular weight heparin until a therapeutic INR was achieved with an oral vitamin K antagonist. Tramadol was used to relieve postoperative pain. Fourteen patients (6 with IE) were cured with methylprednisolone due to postpericardiotomy syndrome for no longer than one week.
2.5. Study design
Venous blood was collected (at 08:30, 5 ml, EDTA-K3 vacuette-tubes) the day before and 3, 7, 12 and 21 days after surgery for blood cell count and LBCL measurement in the patients with IE and VHD. LBCL measurement procedure began no later than 30 min following blood collection. Additional blood specimens were taken the day before surgery for blood chemistry and blood culture (2x30 ml). The Medical University of Lodz Ethics Committee approved the study protocol and all participants provided a written, informed consent.
2.6. Luminol enhanced whole blood chemiluminescence assay
LBCL as a measure of resting and n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated circulating phagocytes activity to produce ROS was determined according to Kukovetz et al. [9] with some modifications [10]. Three µl of blood sample was added to 947 µl of mixture luminol solution [10] which was pre-warmed in darkness up to 37 °C for 60 min. The samples were placed in a 1251 luminometer (Bio-Orbit, Turku, Finland) and incubated for 30 min at 37 °C. Subsequently, the resting chemiluminescence (rCL) was recorded for 1 min and then a 50-µl fMLP solution was added automatically (final concentration of 0.02 mM), furthermore the light emission measurement was continued for an additional 7 min. All individual results were obtained as a mean of four measurements. The following LBCL parameters were assessed: rCL – the average resting chemiluminescence prior to fMLP addition and total light emission (tCL) – the area under the chemiluminescence intensity curve after fMLP addition until its return to baseline. rCL was expressed in arbitrary units (aU) per 104 phagocytes (PMNs and monocytes) present in the assayed sample, while tCL in aUxs/104 phagocytes [10].
2.7. Statistical analysis
All data were expressed as a mean±S.D. and median with range. The differences between groups were assessed using an analysis of variance for independent variables. Repeated measures ANOVA was applied for dependent variables (before vs. after surgery). For data not normally distributed, the Kruskal–Wallis test and Friedman test were used. Pertaining to significance, appropriate post-hoc tests were implicated. An additional comparison between two selected parameters was conducted using the Wilcoxon test (dependent variables) and the Mann–Whitney U-test (independent variables). The Student t-test for dependent variables was used in comparison of two normally distributed parameters. Correlations between LBCL and selected continuous variables were determined using Pearson's r. A P<0.05 was considered significant.
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3. Results
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3.1. LBCL in patients with VHD vs. healthy controls
Tables 3 and 4 show the time course of LBCL parameters in patients with VHD. Three days after cardiac surgery LBCL (rCL) reached the highest intensity (P<0.01) and then gradually decreased to levels not differing from those observed the day prior to surgery. The preoperative rCL and tCL as well as those values observed after three and seven days after surgery were higher than in healthy controls (P<0.01). After 12 and 21 days postoperatively, no differences were noted.
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Table 3 Resting luminol enhanced whole blood chemiluminescence (rCL) in patients with active infective endocarditis and acquired valvular heart disease before and after cardiac valve replacement surgery
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Table 4 Total light emission (tCL) after whole blood stimulation with fMLP in patients with active infective endocarditis and acquired valvular heart disease before and after cardiac valve replacement surgery
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3.2. LBCL in patients with IE vs. healthy controls
Patients with IE presented with elevated LBCL. Both, rCL and tCL measured at all five time points (before and after surgery) were higher than that in healthy controls (P<0.01). Cardiac surgery raised LBCL intensity in IE patients (Tables 3 and 4). Median rCL and tCL three days postoperatively were 3-times higher than preoperative values. However, along with patient recovery, the LBCL decrease noted in addition to rCl and tCL measured at the last time-point were lower than appropriate preoperative records (P<0.01).
3.3. LBCL in patients with IE vs. patients with VHD
Median preoperative rCL and tCL was about 2.5-times higher (P<0.01) in IE group than in patients with VHD (Tables 3 and 4). This difference was even higher (about 3-times) three days following surgery. In the further post-operative course (after 7, 12 and 21 days), no differences in all parameters reflecting LBCL intensity were noted.
3.4. Correlations of LBCL with selected preoperative and operative data
There were negative correlations between ESR and rCL (r=–0.45, P=0.03), and ESR and tCL (r=–0.47, P=0.03) in IE group the day before surgery. CPB time positively correlated (P=0.05) with rCL (r=0.40) and tCL (r=0.39) in IE patients three days following surgery. No associations of ESR and CPB time with any LBCL parameters were found in the operated group of patients with VHD. No other parameters (Tables 1 and 2) correlated significantly with LBCL in both groups.
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4. Discussion
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This is the first study (to the best of our knowledge) analyzing the time course of resting and fMLP-induced LBCL just before and up to 21 days after cardiac surgery in patients with active IE. Patients with IE had a higher resting and fMLP-induced LBCL than those with VHD and healthy subjects. LBCL gradually decreased along with the patients' recovery, at 21 days after surgery, it was lower than the pre-operative value; however, it was still higher than in healthy controls. This indicates that circulating phagocytes-mainly PMNs are activated and produce increased amounts of ROS in IE patients. Since median preoperative rCL in IE patients was 2.5- and 5.4-times higher than corresponding values in patients with VHD and healthy controls, respectively, it may suggest that ROS overproduction in the course of IE may have clinical significance and induce oxidative stress. Although scanty data concerning oxidative stress in IE patients have been published thus far [12], our results suggest that activated blood phagocytes may contribute to these processes while adjuvant medication with antioxidants act in possibly yielding a beneficial effect.
Previous studies on the functions of PMNs and monocytes in IE patients showed a rather decreased ability in these cells to produce ROS [5, 6] that normalized after patient recovery. This, a stark conflict with our results, demonstrating an increase ROS production via blood phagocytes, a product of perhaps the following differences in the study protocol: (A) to avoid any bias related to changes of cell activity due to isolation procedure [13] we applied the LBCL technique for the evaluation of ROS production by blood phagocytes; and (B) the first (preoperative) LBCL measurement was completed after 3–4 weeks of antibiotic therapy, possibly decreasing the intensity of the systemic inflammatory response and thus preventing the suppression of circulatory phagocytic activity.
Alternatively, the negative correlation between preoperative LBCL and ESR seems to be, to some extent, in accordance with these reports. IE patients with the highest ESR values may represent those with the most severe systemic inflammatory response; therefore, their circulating PMNs and monocytes are partially exhausted and produce less ROS (although more than cells from control groups) under resting conditions and after stimulation with fMLP.
Both, IE and VHD patients had highly elevated LBCL three days after cardiac surgery. This may be attributed to the surgical procedure since extracorporeal blood oxygenation and ischemia and reperfusion related to clamping and declamping of the aorta activates PMNs [14]. In patients undergoing open abdominal aneurysm repair with aortic cross-clamping, the blood levels of neutrophil elastase reflecting PMNs activation were elevated until the 6th day postoperatively [15]. A significant positive correlation between a three-day postoperative LBCL and CPB time in IE patients is in accordance with these results.
It seems that patients with IE could be candidates for preventive treatment with antioxidants and anti-inflammatory drugs [14] to attenuate systemic oxidative stress and its augmentation due to CPB. However, further double-blinded clinical trials evaluating effect of such a treatment on ROS release from circulating phagocytes and plasma levels of oxidants-induced secondary products in association with clinical status and recovery of IE patients are required.
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Abstract
1. Introduction
