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Ann Thorac Surg 2004;78:2139-2145
© 2004 The Society of Thoracic Surgeons
a Cardiac Surgery
b , Department of Cardiac Anaesthesia, The General Hospital, Southampton
c Faculty of Biomedical Sciences, University of Portsmouth, Portsmouth, United Kingdom
Accepted for publication May 17, 2004.
* Address reprint requests to Dr Alexiou, Department of Cardiac Surgery, Glenfield General Hospital, Groby Rd, Leicester LE3 9QP, UK (E-mail: alexiou486{at}aol.com).
Presented at the Fortieth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 26–28, 2004.
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Abstract |
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METHODS: One hundred and ten patients with normal respiratory function, undergoing first time coronary artery bypass grafting, were randomized to two groups. Fifty-five patients had an arterial line leukocyte-depleting filter and 55 controls had a standard arterial line filter. Nitric oxide was sampled through an endotracheal Teflon tube after median sternotomy, but before cardiopulmonary bypass and 30 minutes after cardiopulmonary bypass, using a real time chemiluminescence analyzer, during the phase of the alveolar plateau.
RESULTS: There were no significant differences in the precardiopulmonary bypass values of exhaled nitric oxide between the control (2.92 ± 1.51 ppb/s) and the leukodepletion group (3.11 ± 1.53 ppb/s) (p = 0.4). After cardiopulmonary bypass, the rate of alveolar production of exhaled nitric oxide increased in both groups, being, however, significantly higher in the control group (4.68 ± 1.89 vs 3.72 ± 1.33 ppb/s) (p = 0.02).
CONCLUSIONS: Continuous arterial line leukocyte-depletion significantly reduces the rate of alveolar production of exhaled nitric oxide after cardiopulmonary bypass. Changes in the rate of alveolar production of exhaled nitric oxide may be used as a marker of pulmonary inflammation in coronary artery surgery.
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Introduction |
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Pharmacologic agents and surface modifications of the CPB circuit used to address this problem have often been successful in reducing the indices of inflammation but provided inconsistent clinical results. Also, some interventions, such as steroid treatment, cause side effects that can be detrimental for the patient [1–3].
The introduction of arterial line leukocyte depleting filters in cardiac surgery was based on the knowledge of the central role of leukocytes in the end organ injury that occurs during CPB. Because this approach involves no pharmacologic substances, and the bone marrow produces large numbers of leukocytes that replace those removed by the filter during CPB, leukocyte depletion appears to carry no risks for the postoperative patient. Similar to other antiinflammatory strategies, though, use of leukocyte depleting filters had a variable impact on the organ function of patients undergoing cardiac surgery [4].
Nitric oxide (NO), previously known as endothelium derived relaxant factor, is a gas commonly found in atmospheric pollutions. In the human body it is generated from the L-arginine by the enzyme nitric oxide synthase (NOS). There exist three isoforms of NOS; the endothelial (eNOS), the neuronal (nNOS), and the inducible (iNOS), the production of which can be induced in several cell types upon their exposure to the proinflammatory cytokines and endotoxin [5]. Nitric oxide is a highly reactive free radical, able to diffuse easily across the cell walls. Once released it enters the erythrocytes, binds to hemoglobin, and becomes inactive. Nitric oxide has a half-life of only 1 to 30 seconds, and its effects cease rapidly unless more NO is produced. The physiologic effects of NO include vasodilatation, bronchodilatation, and mediation of several stages of the inflammatory reactions [5].
In the lungs, NO is produced by the bronchial epithelium, the vascular endothelium, the alveolar and lung interstitial macrophages, and bacteria within the bronchial tree [6]. Because NO diffuses rapidly through cell membranes it is thought that all pulmonary sources contribute to the amount of NO that is exhaled in the air, although there is controversy as to which of the two main sources, epithelial or endothelial, predominates [7]. It is also known that the upper airways, particularly the nose, contribute the greatest proportion to the NO exhaled in air [8] and that men produce 50% more exhaled NO than women [9].
Nitric oxide can be detected in the exhaled air of animals and humans with a chemiluminescence analyzer at concentrations as low as 2 ppb [9, 10]. Exhaled NO has been shown to increase in patients with inflammatory diseases of the respiratory tract, such as asthma and bronchiectasis [11], secondary to upregulation of iNOS. Recording the changes in the rate of production of exhaled NO in asthmatic patients over time has allowed the monitoring of the disease progress and the effectiveness of treatment with steroids. Exhaled NO has been shown to increase during periods of exacerbation of asthma and decrease upon its remission [12].
The CPB is associated with cytokinemia, endotoxinemia, and pulmonary inflammation, all of which enhance iNOS. The experience gained with the patients having chronic inflammatory lung disorders encouraged researchers to investigate the kinetics of exhaled NO in relation to CPB, with a view to establishing whether exhaled NO could be used as a marker of pulmonary inflammation, and as a monitoring tool of the success of antiinflammatory interventions in cardiac surgery. These investigations, involving small numbers of patients, produced opposing results [13–22]. The aim of this study was to examine the effect of arterial line leukocyte depletion on the rate of alveolar production of eNOS in a relatively large number of patients undergoing surgery for coronary revascularization.
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Material and Methods |
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One hundred and ten patients were divided in two equal groups using a computer generated randomization system. In the first group (n = 55) a leukocyte depleting LG6 filter (Pall, Portsmouth, UK) was attached onto the arterial line of the CPB circuit. In the control group (n = 55), a conventional nonleukocyte depleting filter (D754, Sorin Biomedica, Mirandola, Italy) was used. The management of anesthesia and CPB, and the conduct of the operations were otherwise identical in both groups.
Management of Anesthesia and Conduct of the Operations
The patients were premedicated 1.5 hours before the operation with 10 mg of morphine and 2 mg of lorazepam. Anesthesia was induced with midazolam, fentanyl, and pancuronium, and maintained using intermittent positive pressure ventilation with oxygen-enriched air and isofluorane. During CPB a propofol infusion was used to maintain anesthesia.
The CPB circuit consisted of a Dideco D703 (Compactflo, Mirandola, Italy) microporous hollow fiber membrane oxygenator (containing a heat exchanger) with integral cardiotomy reservoir (Sorin Biomedica). The circuit was primed with 2L lactated Ringer solution that contained 5,000 U of heparin. Before the establishment of CPB, 3 mg/kg body weight of heparin were administered and supplemented as required to maintain an activated clotting time of 480 seconds. Continuous nonpulsatile blood-flow was delivered to the patient using a multiflow roller pump (Stockert SIII, Munich, Germany) at an indexed flow rate of 2.4 · Lm2 · min. During CPB, all patients were systemically cooled to a nasopharyngeal temperature of 33°C.
After aortic clamping electromechanical diastolic arrest was induced with the delivery of cold (4°C) blood cardioplegic solution. One liter was administered initially through the aortic root, followed by 300 mLs boluses every 30 minutes of aortic clamping or earlier if cardiac electrical activity was seen.
Distal anastomoses were completed during a single period of aortic clamping. Proximal anastomoses were performed on a beating heart using an aortic partial occluding clamp. Cardiopulmonary bypass was terminated after the patient was rewarmed, having a nasopharyngeal temperature of 37°C.
Measurement of Exhaled Nitric Oxide
Exhaled NO was measured using a real-time chemiluminesence analyzer (Logan Research, Northampton, UK). This was calibrated in accordance with the manufacturers’ instruction using 100 ppb NO in nitrogen before studying each patient. The exhaled gas samples were drawn at a sampling rate of 250 mL/min, through a Teflon sampling tube sited 1 cm from the tip of the endotracheal tube. Teflon tubing material was used because it does not react with the NO in the exhaled air, thereby ensuring reliable determination of the NO concentration.
All patients required intermittent positive pressure ventilation during surgery before and after CPB. This enabled a sustained exhalation sample to be taken by stopping the ventilator and allowing a prolonged exhalation to occur. As all patients had been given the muscle relaxant pancuronium during the induction of anesthesia, they were unable to make respiratory effort, which might have contaminated the samples.
Three measurements of exhaled NO were taken at two-minute intervals approximately 15 minutes before CPB, and another set of three measurements was taken 30 minutes after the termination of CPB. The increase in NO concentration per second during a sustained exhalation was calculated using the following formula:
NO = [(CF–CS)/tF-S], where 
NO = change in NO (ppb/s), CF = final concentration of NO (ppb), CS = start concentration of NO (ppb), and tF-S = sampling time duration(s).
An average value for the three measurements of production of exhaled NO (ppb/s) was calculated for each patient both before CPB (baseline value) and after CPB. Using this data, the change from the baseline value of exhaled NO production (ppb/s) after CPB was readily determined for the two study groups.
Statistical Analysis
Continuous variables were expressed as mean values ± the standard deviation and the proportions as percentages. The differences between the groups for the categorical variables were compared using 
2 or Fisher’s exact test as appropriate. Continuous variables in the two groups with normally distributed data were compared with an unpaired Student t test. If the data were skewed a nonparametric test (Kruskal-Wallis) was used. A p value of less than or equal to 0.05 was considered statistically significant. Statistical analysis was performed on Excel 2000 (Microsoft Corp, Redmond, WA) and SPSS (SPSS, version 11.5, Chicago, IL) software.
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Results |
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Comment |
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Of the enzymes responsible for the synthesis of NO, the eNOS and the nNOS are constitutive, whereas generation of the iNOS takes place in several cell types that are exposed to cytokines and endotoxin [5]. The application of endotoxin to pulmonary endothelial cells has been shown to increase the production of tumor necrosis factor-alpha (TNF-
) and interleukin-1 (IL-1), and stimulate the production of NO through an autocrine pathway [23]. Rats treated with intravenous endotoxin exhibited a significantly higher increase in serum TNF- and interstitial, air space and total lung injury, which was accompanied by an increase in the amount of exhaled NO compared to untreated rats [24]. On the basis of these findings it has been suggested that exhaled NO could be used as an early marker of lung inflammation in the clinical setting [24].
The rate of production of exhaled NO was seen to increase consistently in chronic inflammatory lung conditions such us asthma, the mechanism responsible for this increase being an up-regulation of iNOS in the bronchial epithelium upon its exposure to TNF- and interleukins [5, 11, 12]. Moreover, administration of steroids during the periods of exacerbation of symptoms led to symptomatic improvement and a decline in the amount of exhaled NO [11, 12]. The rate of NO exhaled in air has, thus, became a reliable marker of pulmonary inflammation and a monitoring tool of the success of treatment in these patients [11, 12].
Working on a rat model of acute lung transplant rejection some researchers found elevated levels of exhaled NO in fulminant acute rejection, which were reduced after partial immunosuppressive therapy, indicating that exhaled NO could serve as a noninvasive measure of acute lung transplant rejection [25]. In stable lung transplant recipients, exhaled NO levels were not elevated but were seen to positively correlate with the neutrophil counts in the airways and the expression of the iNOS in the bronchial epithelium [26]. Lung transplant recipients with bronchiolitis obliterans exhibited higher levels of exhaled NO that correlated positively with the degree of airway neutrophilia and epithelial iNOS [27]. Other investigators have reported increased levels of exhaled NO in lung transplant recipients having conditions associated with the presence of airway inflammation within the graft such as lymphocytic bronchiolitis, early obliterative bronchiolitis, and infection [28]. Taken together, these studies clearly indicate that measurement of exhaled NO may be an early marker of pulmonary allograft dysfunction [25–28].
Cardiac surgery provokes cytokinemia, leukocyte activation, pulmonary inflammation, and various degrees of lung injury [1–3, 29, 30]. The problem is usually subclinical with most patients recovering uneventfully, but in less than 2% of cases lung injury is severe enough to cause the adult respiratory distress syndrome (ARDS), which carries a mortality of 50% [31]. To address this issue several antiinflammatory interventions, including leukocyte depletion, have been used with variable clinical success [1–4, 32, 33]. The impact of these interventions on the pulmonary function has been usually monitored using such endpoints as respiratory index and other surrogate indices of lung injury and clinical outcome [29, 30, 32, 33]. The availability of modern chemiluminescence analyzers made it possible to examine the kinetics of exhaled NO in relation to CPB and to explore whether exhaled NO could be used as a noninvasive marker of pulmonary inflammation and as a monitoring tool of the success of antiinflammatory interventions.
In this study, the rate of exhaled NO increased significantly 30 minutes after CPB. This increase was observed both in the control group and the patients who were operated with arterial line leukocyte depletion. Compared to controls, though, the leukodepleted group had a significantly reduced rate of production of exhaled NO after CPB. The higher production of exhaled NO after CPB is consistent with the inflammatory events that unravel in the lung parenchyma during CPB and is likely to reflect an up-regulation of the iNOS. The attenuation of this increase in the leukodepleted group indicates a protective effect of leukocyte depleting filter on the lung tissues mediated, perhaps, by the removal of activated leukocytes from the circulation [22]. Nevertheless, the increase in the rate of exhaled NO after CPB in the leukodepleted group suggests that although leukodepletion may reduce the pulmonary inflammation it does not eliminate it. In a more limited study, our group has previously found a significant increase of exhaled NO after CABG in the control group and only a marginal increase in the leukodepleted group [22]. The differing findings of the present study in this regard, may, at least partly, be explained by the bigger sample size available for analysis.
A previous prospective randomized study involving patients undergoing CABG has produced convincing evidence linking the systemic inflammation with the levels of exhaled NO released during CPB [13]. The study recorded a concurrent significant rise in the serum levels of IL-6 and TNF- and the levels of exhaled nitric oxide 5, 20, 35, and 50 minutes after initiation of CPB, all of which were significantly reduced following the administration of methylprednisolone [13]. In a further study, the same group of workers reported that an increase in the amount of exhaled NO during CPB was not observed among patients who were smoking up to six months preoperatively [21].
The findings of the present study are in keeping with those of the previous report [13]. It should be emphasized, however, that the serum concentration of proinflammatory cytokines (eg, IL-6 and TNF-) that affect the expression of iNOS and the production of exhaled NO were not measured and that this is an important limiting factor. Thus, although this study firmly suggests the presence of a link between the systemic or pulmonary inflammation and the levels of exhaled NO during CPB, it does not prove it.
On the other hand, there have been clinical studies reporting no increase [14] or a decrease [15, 18, 19] in the amount of exhaled NO after cardiac surgery. It should be noted that, in these studies, included were relatively small numbers of patients having differing pathological conditions, and that exhaled NO was sampled and analyzed at variable perioperative time points with different methodology. Therefore, a valid comparison of the reported results (these are discussed below) would be extremely difficult [17].
One study involving 11 patients having elective CABG reported no changes in the postCPB levels of exhaled NO compared to preCPB values [14]. A nonsignificant trend towards a reduction of exhaled NO from the time of anesthesia to postCPB period was recorded in 7 patients having CABG surgery [15]. Intravenous administration of nitroglycerin in 12 patients also having CABG operations, produced a dose dependent increase in the amount of exhaled NO before CPB, which was, however, significantly reduced 1 and 3 hours after CPB [16]. Other workers measured exhaled NO levels in 16 patients undergoing a wide spectrum of adult cardiac procedures and found that these levels rose during CPB, returned to preCPB levels 1 hour after CPB, and decreased 6 hours postCPB [18]. Exhaled NO was seen to decrease after surgical closure of atrial septal defects (ASD) in children and was seen to increase after ASD closure with transcatheter devices [19]. In contrast, reduced levels of exhaled NO after transcatheter device closure of ASD were described by other authors [20].
Apart from the pulmonary blood flow [20, 34], measurement of exhaled NO produced in the lungs is mainly affected by the NO produced in the nasal airways, the anesthetic gasses, the flow rate and tidal volume of the ventilator, the timing of the sampling within the respiratory cycle, and the presence of atelectasis precluding the participation of certain parts of the lungs in the process of expiration [17]. It is, thus, likely that one or more of these limiting factors might have affected the values of exhaled NO measured before, during, or after the operations in previous studies.
The technique described in the present report would appear to address the above limitations. Disconnecting the patient from the ventilator for a short period allows a sustained exhalation to be taken and eliminates the effect of the anesthetic gasses, the flow rate, and the tidal volume. The pulmonary NO is not mixed with the NO produced by the nasal airways as it is sampled through a Teflon tube positioned at the end of the trachea. Atelectasis is less likely to affect the recorded NO values because the patient is being ventilated with positive pressure up until the ventilator is disconnected, ensuring complete expansion of the lungs at the time of sampling. Moreover, determining the exhaled NO levels only at an early postCPB stage, allows the identification and quantification of the pulmonary inflammation elicited during the operation. The previous comments are relevant for the patients having normal preoperative respiratory function but may not apply for those suffering from pathological processes severely affecting the epithelial or endothelial cellular components of the lungs (own observations, unpublished data).
Other investigators have previously illustrated the difficulties that may be encountered in the interpretation of exhaled NO values in certain clinical conditions. They compared the levels of exhaled NO measured in ventilated ARDS patients and patients ventilated before having CABG operations [35]. It was revealed that the ARDS patients produced significantly lower amounts of exhaled NO compared to the patients awaiting CABG operations. These results were unexpected, given the extreme inflammatory status of the lung in ARDS and the association of inflammation with iNOS. Altered diffusing capacity of NO (DLNO) has been proposed, among others, as a possible explanation for the reduced exhaled NO in this group [36]. Perialveolar hemorrhage is common in ARDS and it has been suggested that NO-hemoglobin binding could increase the DLNO, which would have contributed to the low levels of exhaled NO seen in the ARDS patients [35]. It is, therefore, evident that in patients with severely damaged lungs, exhaled NO values might not reflect the degree of ongoing pulmonary inflammation.
In conclusion, this study shows that the rate of production of exhaled NO increases after CPB and that this increase is attenuated by continuous arterial line leukodepletion. It also suggests that the rate of production of exhaled NO may be a useful marker of pulmonary inflammation in low risk patients undergoing CABG surgery and having normal preoperative lung function.
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Discussion |
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DR ALEXIOU: Yes, we looked at it, but there were no significant differences in the bypass time from patient to patient. The mean number of distal anastomoses performed in each group was approximately three. As a result, comparing the control versus leukodepletion, there was no difference.
DR CONTE: But is there a linear relationship between the length of time on bypass versus production? Is it linear or does it level off at a certain point?
DR ALEXIOU: No, we did not identify such a relationship.
DR JOHN W. HAMMON (Winston-Salem, NC): Thank you very much for alerting us to this very innovative way of monitoring our patients, and I congratulate you for carefully studying this group. I have two questions. The first is, one of the big knocks against leukodepletion filters is that they clog up at that particular site. Did you have to interrupt cardiopulmonary bypass at any time because of the filter being clogged, or, number two, did you verify that the leukodepletion filter was working; did you have some measure that it was trapping leucocytes?
DR ALEXIOU: Regarding the first question, we did not have a case of a filter being blocked, and I am not aware of such a case, not only for the duration of this study, but for the time these filters have been used in our institution. As for the second question, white cell counts were not measured in this study. There are two reasons for this. First, we have previously performed a more limited study and found that the white cell count during bypass was lower with leukocyte depleting filters. But what was more interesting was that there was a significant difference in the activated white cell counts in favor of the leukocyte depletion group. So, what is really happening is that although the total white cell count does not change much with leukocyte depletion, there is increasing evidence, and we were able to see this in previous studies, that the activated leukocyte count changes. So, the leukodepleting filters may preferentially remove activated leukocytes reducing the activated leukocyte counts. This might explain the beneficial effects reported following the use of this antiinflammatory strategy.
DR TIMOTHY HALL (Morgantown, WV): My congratulations to the authors, they have created a nice study. To place this work in perspective, we introduced this technique at Hopkins almost twenty years ago for control of lung reperfusion damage. At that time, we demonstrated limiting lung injury with very low leukocyte counts. A problem that still exists today, that is particularly pertinent to clinical studies, is the lack of identification of the highest threshold for circulating white cells before losing efficacy. I have two questions: first did you perform a clinical correlation with your end points to determine clinical efficacy. Second, can you extrapolate a target for circulating white cell counts that would be clinically effective?
DR ALEXIOU: As I just said we have not counted white cells in this study and I think my answer to the previous discussant should cover, partly, your question too. The leukocyte-depleting filters have been inconsistent in reducing the white cell counts. This is recognized and has to be accepted. However, these filters have demonstrated ability to remove activated leukocytes. So, although you may find that the total white cell counts remain unchanged, the activated leukocyte counts do get reduced. In this regard, we performed several studies in our institution, and consistently found significantly reduced activated leukocyte counts following leukocyte depletion.
DR PAUL KURLANSKY (Miami Beach, FL): In your initial slides regarding hypotheses, you showed that in asthma, exhaled nitric oxide production was due to induction of INOS. INOS induction requires DNA activation, RNA synthesis, protein synthesis, et cetera. That clearly is not going on during cardiopulmonary bypass sufficient to demonstrate the results. So that I am wondering if you had any sort of functional hypothesis as to what it is that leads to the increased production of exhaled nitric oxide during cardiopulmonary bypass and how the white cell depletion is ameliorating that process?
DR ALEXIOU: Thank you for this very interesting question. There is good evidence to suggest that the increase in the amount of exhaled nitric oxide in inflammatory lung conditions is brought about by the upregulation of the inducible nitric oxide synthase. Studies have shown that in asthmatic patients during the period of exacerbation, and in lung transplant recipients who have lymphocytic bronchiolitis or infection or, indeed, grade 1 bronchiolitis obliterans syndrome, the inducible nitric oxide synthase is upregulated. In addition, in transplanted patients, the upregulation of inducible nitric oxide synthase in lung biopsy specimens was shown to correlate positively with the numbers of leukocytes in bronchoalveolar lavage samples and the levels of exhaled nitric oxide. There seems to be, therefore, a clear connection between leukocytes, inducible nitric oxide synthase, and production of exhaled nitric oxide. It is, thus, conceivable that leukocyte depletion may provide some benefits by reducing the activated leukocyte counts in the lung parenchyma. This is the hypothesis. Also, a study conducted here in the United States, showed that exhaled nitric oxide increases during bypass, and that this increase is attenuated with the administration of intravenous steroids.
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Acknowledgments |
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This article has been cited by other articles:
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  O. Warren, C. Alexiou, R. Massey, D. Leff, S. Purkayastha, J. Kinross, A. Darzi, and T. Athanasiou The effects of various leukocyte filtration strategies in cardiac surgery Eur. J. Cardiothorac. Surg., April 1, 2007; 31(4): 665 - 676. [Abstract] [Full Text] [PDF]
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 R. Zegdi and J.-N. Fabiani Exhaled Nitric Oxide After Cardiopulmonary Bypass Ann. Thorac. Surg., November 1, 2005; 80(5): 1977 - 1977. [Full Text] [PDF]
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C. Alexiou, A. T.M. Tang, D. C. Smith, S. V. Sheppard, M. P. Haw, and R. Gibbs Reply Ann. Thorac. Surg., November 1, 2005; 80(5): 1977 - 1978. [Full Text] [PDF]
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= control; 
= leukodepletion.)