Preservation of Endothelium Nitric Oxide ... - Wiley Online Library

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*Ettore Lanzarone, †Fabrizio Gelmini, ‡Maddalena Tessari, ‡Tiziano Menon,. §Hisanori Suzuki, †Marina Carini, *Maria Laura Costantino, *Roberto Fumero,.
Artificial Organs 33(11):926–934, Wiley Periodicals, Inc. © 2009, Copyright the Authors Journal compilation © 2009, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Preservation of Endothelium Nitric Oxide Release by Pulsatile Flow Cardiopulmonary Bypass When Compared With Continuous Flow *Ettore Lanzarone, †Fabrizio Gelmini, ‡Maddalena Tessari, ‡Tiziano Menon, §Hisanori Suzuki, †Marina Carini, *Maria Laura Costantino, *Roberto Fumero, ‡Giovanni Battista Luciani, and ‡Giuseppe Faggian *Laboratory of Mechanical Biological Structures, Department of Structural Engineering, Politecnico di Milano; †Department of Pharmaceutical Sciences “Pietro Pratesi,” Università degli Studi di Milano, Milan; ‡Division of Cardiac Surgery, University Hospital of Verona; and §Department of Morphologic and Biomedical Sciences, Università degli Studi di Verona, Verona, Italy

Abstract: The aim of this work is to analyze endothelium nitric oxide (NO) release in patients undergoing continuous or pulsatile flow cardiopulmonary bypass (CPB). Nine patients operated under continuous flow CPB, and nine patients on pulsatile flow CPB were enrolled. Plasma samples were withdrawn for the chemiluminescence detection of nitrite and nitrate. Moreover the cellular component was withdrawn for the detection of nitric oxide synthase (NOS) activity in the erythrocytes, and an estimation of systemic inflammatory response was carried out. Significant reduction in the intraoperative concentration with respect to the preoperative was observed only under continuous flow CPB for both nitrite and NOx (nitrite + nitrate) concentration (P = 0.010 and P = 0.016, respectively). Signifi-

cant difference in intraoperative nitrite concentration was also observed between the groups (P = 0.012). Finally, erythrocytes showed a certain endothelial NOS activity, which did not differ between the groups, and no differences in the inflammatory response were pointed out. The significant reduction of NO2- concentration under continuous perfusion revealed the strong connection among perfusion modality, endothelial NO release, and plasmatic nitrite concentration. The similar erythrocyte eNOS activity between the groups revealed that the differences in blood NO metabolites are mainly ascribable to the endothelium release. Key Words: Vascular dilation—Nitric oxide release—Cardiopulmonary bypass—Continuous flow— Pulsatile flow.

A pulsatile flow during cardiac surgery, when a cardioplegic cardiac arrest is required, seems to be beneficial in terms of better district perfusion with respect to the continuous perfusion (1–3). Nevertheless the majority of the adopted cardiopulmonary bypass (CPB) circuits include roller or centrifugal pumps, supplying continuous flow. Under continuous perfusion, nonpulsatile shear stress generated on the

vessel walls results in a lower endothelium nitric oxide (NO) release from vascular endothelium (4,5). As the main effect of NO endothelial release is vasodilation, this reduced NO release seems to be the cause of vasoconstriction and poor organ perfusion during CPB. In the literature, a large number of studies dealt with both ex vivo or in vivo analysis of the endothelial NO release (6–10). In the majority, different perfusion modes are given to the same subject following one another, but this change in perfusion modality affects other mechanisms involved in NO release (11–13), and consequently alters NO release measurement. Moreover, only few studies concern the analysis of human patients undergoing cardiac surgery (14–16). Therefore, the aim of this study is analyzing endothelial NO release in patients undergoing cardiac

doi:10.1111/j.1525-1594.2009.00888.x Received May 2009; revised June 2009. Address correspondence and reprint requests to Dr. Ettore Lanzarone, P.za Leonardo da Vinci 32, 20133 Milan, Italy, E-mail: [email protected] Presented in part at the 5th International Conference on Pediatric Mechanical Circulatory Support Systems & Pediatric Cardiopulmonary Perfusion held May 27–30, 2009 in Dallas, TX, USA.

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NITRIC OXIDE PRESERVATION UNDER PULSATILE FLOW CPB surgery under continuous or pulsatile perfusion. Although pulsatile devices approved for clinical CPB do not accurately reproduce the physiological waveform provided by the left ventricle, the literature stresses the importance of reproducing the left ventricle waveform in the pulsatile group to point out the possible beneficial effects of pulsatile perfusion (1,2). We consequently decided to analyze pulsatile perfusion in the presence of both a physiological flow waveform or the CPB circuit. In our parallel study we analyzed the different endothelial NO release between patients undergoing continuous flow CPB or beating-heart surgery, observing a NO release reduction only in the first group (unpublished results). Nevertheless, this pulsatile group differs from the continuous one not only in terms of perfusion modality, but also for the presence of the CPB circuit (present only in the continuous group). The circuit itself induces systemic inflammation and complement activation (11–13); this could interact with the endothelial regulation and influence the obtained results about the dependence of NO release influence on perfusion modality. Therefore, it is also important to compare NO release under continuous or pulsatile perfusion in the presence of the CPB circuit in both the groups, even if the flow waveform supplied by the CPB devices approved for clinical use differs from the physiological. The aim of this work is thus to compare endothelial NO release in patients undergoing continuous or pulsatile flow CPB. Endothelial NO release can be quantified based on blood NO concentration. Nevertheless, a direct measurement of blood NO concentration is very difficult under basal conditions (17), due to its ultra-short half-life (18) and radical character. Nitrosylhemoglobin is a bioactive storage form of NO in circulation. However, we found that the endogenous production of nitrosylhemoglobin in human blood is not detectable by currently available instrumentation, and it is negligible with respect to the other NO metabolite concentrations (unpublished results). Therefore, the nitrosylhemoglobin concentration analysis was excluded in this work. The majority of released NO is rapidly oxidized to nitrite NO2-, and then to nitrate NO3-. Thus, endogenous NO formation may be measured via the determination of its oxidative products, as in the majority of literature works (17). The most widespread method for the NO2-analysis is the Griess colorimetric assay (14); nevertheless it has poor sensitivity, it suffers from interferences by endogenous amines, and the acidic conditions favor the formation of nitrosothiols from nitrite and reduced thiols (17).The most simple, rapid,

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and sensitive method for determining nitrite and nitrate in plasma is based on reductive chemiluminescence, whose limit of detection is approximately 100 fM. Therefore, the chemiluminescence assay was chosen for detecting NO2- and NOx (NO2- + NO3-) concentrations, to assure sensitivity. Moreover, the enzymatic NO synthase (NOS) activity in the erythrocytes was analyzed. Erythrocytes have been demonstrated to express an active and functional endothelial-type NOS (eNOS), to metabolize, reversibly bind, transport, and release NO within the cardiovascular system (19). Consequently the analysis of NOS activity in erythrocytes is crucial to determine the influence of erythrocyte release on measured plasmatic NO2- and NOx concentrations. Finally, a preliminary evaluation of the systemic inflammatory response, based on clinical parameters (11), and an analysis of plasmatic NO2- and NOx concentrations deriving from both this work and the parallel study (unpublished results) were carried out. MATERIALS AND METHODS Patient enrollment Eighteen patients were enrolled at the Division of Cardiac Surgery, University Hospital of Verona (Verona, Italy) after approval by the institutional review board and informed consent: 1 Continuous group: nine patients subject to continuous flow CPB for coronary artery bypass. 2 Pulsatile group: nine patients subject to pulsatile flow CPB for coronary artery bypass. Inclusion and exclusion criteria were defined to render the population uniform, and to avoid considerable alterations of the flow waveform supplied by the left ventricle. The age of patients ranged between 49 and 78 years, and patients with alcohol and drug dependence were excluded. Patients suffering from copathologies besides the one related to the analyzed surgery, such as diabetes or vasculopathies, were excluded. The absence of previous open-heart operations was a prerequisite. The ejection fraction was requested to be at least 50%, to exclude patients with compromised cardiac output (especially alterations of flow waveform from the physiological). Moreover patients in which adverse events occurred during surgery (as elevated mean flow variations during CPB or vasodilatative drug administration) were excluded. The homogeneity of the two groups was verified. Age, sex, body surface area (20), and surgery duration (from anesthesia induction to protamine infuArtif Organs, Vol. 33, No. 11, 2009

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E. LANZARONE ET AL. TABLE 1. Homogeneity check between continuous and pulsatile group

Age (years) Sex (Male/Female) BSA (m2) (20) Surgery duration (min) Supplied to nominal flow ratio (adim) Mean pressure during surgery (mm Hg)

Continuous group

Pulsatile group

P value

64.2 ⫾ 9.2 8/1 1.90 ⫾ 0.19 179 ⫾ 40 0.96 ⫾ 0.03 68.8 ⫾ 7.4

66.8 ⫾ 8.7 5/4 1.75 ⫾ 0.13 160 ⫾ 45 0.92 ⫾ 0.06 59.4 ⫾ 6.5

0.577 — 0.104 0.392 0.085 0.016

Averaged values of each population (mean value ⫾ standard deviation) and P values of the performed t-tests are reported. For each patient, supplied to nominal flow ratio was obtained as the ratio between the cardiopulmonary bypass (CPB) supplied flow (quite constant during CPB period) and the nominal patient flow (equal to BSA ¥ 2.4). The mean pressure was computed as the average of mean pressures at sample withdrawing. BSA, body surface area.

sion) were averaged within each group and compared performing unpaired t-tests (Table 1). No significant difference was pointed out for all the variables. Moreover, the homogeneity of flow levels during surgery was verified. Under the same flow level, the mean pressure during surgery was significantly lower under pulsatile flow CPB (P = 0.016). This pointed out the vessel dilatory effect of pulsatile perfusion; therefore, aiming at homogeneous flow levels between the groups, it resulted into nonhomogeneous pressure levels.

CPB circuits Commercial CPB circuits equipped with roller pump S3 (Stöckert, Munich, Germany) and 0.5 in. internal diameter silicon tubing were adopted in both the groups. The noncontinuous flow of the pulsatile group was obtained by adopting the S3 Pulsatile Flow Control (Stöckert) connected to the pump. This controller allows us to regulate the pulsatility of flow by imposing three parameters: 1 simulated frequency (frequency of the simulated heart cycle); 2 pulse duration (percentage of systole during the simulated heart cycle); 3 basal flow (percentage of mean flow supplied during the diastole). In the admissible range of the machine, parameter values were set to adapt the supplied flow waveform to the physiological one (21); particularly the basal flow was minimized in a physiological range of the other parameters, to get near a null flow diastole. Simulated frequency was set at 70 bpm, pulse duration at 35%, and basal flow at 30%. Moreover, before using the pulsatile flow CPB circuit, in vitro tests Artif Organs, Vol. 33, No. 11, 2009

were provided to verify the supplied flow in terms of mean value and waveform. Blood sample acquisition Venous blood samples were withdrawn at defined times. A first basal sample was taken the day before surgery from a peripheral venous access, and a second one from the central venous access just after the anesthesia induction. Starting from the heparin infusion, samples were withdrawn upstream of the CPB reservoir each half an hour, until the protamine infusion. The last three samples were withdrawn from the central venous access just after, 1 h, and 2 h after the protamine infusion, respectively. For each patient, the overall number of samples varied from 7 to 10, depending on the different surgery duration. Each sample (8 mL of venous blood) was withdrawn with a disposable syringe and immediately transferred in a prerefrigerated duran glass centrifuge tube (Colaver srl, Vimodrone, Milan, Italy). The tube was centrifuged at 6000 rpm and 4°C for 5 min, to separate the cellular component from plasma. Then they were separately withdrawn with a Gilson pipette and inserted in cryogenic vials; six vials containing at least 400 mL of plasma and two vials containing at least 400 mL of cellular component were obtained for each acquired blood sample. All the vials were stored in liquid nitrogen. Material compatibility with NO was tested to verify the absence of absorption/desorption or radical-aging phenomena; all the utilized components were disposable and prewashed with Milli-Q water (water purification systems, Millipore SAS, Molsheim, France). Plasma samples were used for the chemiluminescence detection of NO2- and NOx, whereas the cellular component was used for the evaluation of NOS activity in erythrocytes.

NITRIC OXIDE PRESERVATION UNDER PULSATILE FLOW CPB NO2- and NOx concentration The chemiluminescence system consisted of a helium carrier gas (set at 23–24 instrumental au), and an external purge vessel with condensers and a NO analyzer (CLD88 exhalizer) (18). Sodium nitrite, sodium nitrate, sodium hydroxide pellets, acetic acid, potassium iodide, and vials in boron silicate glass were purchased from Sigma-Aldrich srl (Milan, Italy). Test tubes in boron-silicate glass with teflon cap, to prevent nitrite oxidation phenomenon, were purchased from Colaver srl (Vimodrone). Helium (5.5) and NO (9.4 ppm) were provided by Sapio Industry srl (Monza, Italy). NO2- and NOx detection were performed with a high sensibility chemiluminometer CLD88 Exhalyzer (ECO MEDICS AG, Dürnten, Switzerland). Data integration was computed with PowerChrom software (AD Instruments, Bella Vista, New South Wales,Australia). All the samples were analyzed three times in three different days, and each concentration was expressed as the mean value and the standard deviation. Erythrocyte NOS activity Endothelial NOS activity was estimated by measuring the conversion of L-[3H]-arginine to L[3H]-citrulline, according to the literature (22). Erythrocytes were separated from the cellular component via differential centrifugation, lysed with a buffer (containing 50 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid [HEPES] pH 7.4, 1 mM dithiothreitol, 1 mM ethylenediaminetetraacetic acid [EDTA], 10 mg/mL leupeptin, 10 mg/mL soybean trypsin inhibitor, 10 mg/mL antipain, and 1 mM phenylmethylsulfonyl fluoride) and then centrifuged (at 10 000 rpm and 4°C for 30 min). The pellet was washed twice with HEPES buffer, solubilized with 20 mM 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate and submitted to three cycles of freezing and thawing. The supernatant of the solubilized pellet, obtained after centrifugation at 10 000 rpm and 4°C for 30 min, was purified by affinity chromatography on 2′-5′-ADPagarose using 50 mM HEPES, containing 10 mM NADPH as elution buffer. Aliquots of the purified fractions were added to a reaction mixture of a final volume of 100 mL containing 50 mM HEPES, pH 7.4, 20 nM [3H]-arginine, 1 mM arginine, 1 mM EDTA, 1.2 mM CaCl2, 1 mg/mL calmodulin, 10 mM flavin adenine dinucleotide, 0.1 mM (6R)-5,6,7,8tetrahydro-1-biopterin, and 1 mM dithiothreitol. The reactions were stopped by adding 0.4 mL (1:1) of slurry of Dowex AG 50 W X8 (Na+ form) (Bio-Rad, Hercules, CA, USA) in 50 mM HEPES, pH 5.5. After 15 min of shaking, radioactivity in the superna-

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tant was measured. Specific enzyme activity was expressed as pmol of citrulline formed in 1 min by 1 mg of protein. Protein concentration in the samples was determined according to Bradford method (23). Data analysis A first analysis of temporal patterns of concentration was carried out. Then the concentrations were averaged in order to obtain three values, relative to the different patient phases: 1 PRE: mean of basal sample and the sample taken after anaesthesia induction; 2 INTRA: mean of two last samples intra surgery, excluding the one after protamine infusion; 3 POST: mean of the samples taken 1 and 2 h after the protamine infusion. The mean value of PRE phase was assumed as the reference value (100%) for each patient, and the averages of INTRA and POST phases were expressed as a percentage of the respective reference value. Statistical analyses were separately performed for each type of percent concentration. Comparisons between pulsatile and continuous flow were performed with the unpaired t-test and comparison between two phases within the same group with the paired t-test. Differences were considered to be significant when the P value was lower than 0.05. Tests were computed using Minitab 15 (Minitab, Inc., State College, PA, USA). Systemic inflammatory response A set of clinical parameters was chosen to estimate the inflammatory response in each patient, according to Luciani et al. (11), where a comparison between classical continuous flow CPB and continuous flow CPB associated to modified ultrafiltration was carried out. Duration of aorta cross-clamping, CPB, intensive care intubation, total intensive care duration, hemoglobin levels across the recovery, bleeding after surgery, diuresis during and after surgery, hemoderivates, and physiological saline solution infusion were taken into account. Values were averaged all over each group, and unpaired t-tests were performed for each parameter. Data analysis from both this work and the parallel study Plasmatic NO2- and NOx concentrations from both this work and our parallel study (unpublished results) were considered together. Therefore four different Artif Organs, Vol. 33, No. 11, 2009

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populations were analyzed (pulsatile flow CPB, beating-heart surgery, continuous flow CPB of this work, and of the parallel study). Statistical analyses were separately performed for NO2- and NOx. The homogeneity of the two continuous populations was firstly investigated by means of two t-tests between the INTRA and between the POST values. Therefore, all the populations were compared with analysis of variance (ANOVA; Tukey method for pairwise comparisons) among the INTRA values and among the POST values. In this analysis the two continuous populations were put together, as the homogeneity was assessed. Numerical results were computed using Minitab 15 (Minitab, Inc.).

RESULTS NO2- and NOx concentration The two populations were homogeneous in terms of basal value (Table 2A,B), as unpaired t-tests did not reveal any significant difference (P = 0.669 for NO2- and P = 0.395 for NOx). Moreover, basal values were in accordance with the literature, particularly with our parallel study (unpublished results); only a small number of patients showed high basal values with respect to the others (probably connected with the cardiac disease itself). Nevertheless, these cases were present in both the groups and did not affect the homogeneity. Temporal patterns showed a reduction during surgery only under continuous flow CPB, with a decreasing trend among the samples. In the pulsatile group the reduction was quite absent or at least lower.A slower recovery was also observed after continuous flow CPB. Sample values were averaged as described in the section Data Analysis (Table 2A,B). Standard deviations were computed including both the contributions intra- and inter-samples; the intra-sample values were always lower than 10%. Lower percent NO2- and NOx concentrations were observed in the continuous group with respect to the pulsatile one, especially concerning NO2(Fig. 1). A significant difference between the INTRA values was observed concerning NO2- concentration (P = 0.012), whereas no significant difference was pointed out for NOx concentration (P = 0.107). A significant reduction in the INTRA value with respect to the PRE was also observed only under continuous flow CPB, for both NO2- and NOx concentration (P = 0.010 and P = 0.016, respectively) (Table 3A). Artif Organs, Vol. 33, No. 11, 2009

Erythrocyte NOS activity The analysis of NOS activity by enzyme assay revealed a certain eNOS activity of erythrocyte in NO production and release. The two populations were homogeneous in terms of basal values (Table 2C). No significant differences were pointed out in the comparison between continuous and pulsatile flow CPB. A general increasing of erythrocyte eNOS activity INTRA surgery and a recovery of the basal value POST surgery were observed. Two patients of the continuous group presented a higher INTRA surgery increase with respect to the others (Fig. 1); a certain significance could be found excluding them, even if a high variability of data is present. Compared to the significant difference of NO2-, the similar NOS activity suggested that the observed differences in plasmatic NO metabolites do not depend on erythrocyte production. Consequently they are mainly ascribable to the endothelium release, connected with the vessel dilatory activity. Systemic inflammatory response The analysis of the acquired clinical parameters revealed no differences between the groups. Nevertheless, a limited population number was considered, as we associated the estimation of the systemic inflammatory response to quantitative analyses of blood samples. On the contrary, the analysis of Luciani et al. (11) dealt with a much larger population than in our study. Therefore, this absence of significant difference could be connected to an inadequate number of the studied population. Data analysis from both this work and the parallel study The comparison among the four populations revealed a larger plasmatic NO2- and NOx reduction in the continuous groups, especially for NO2concentration. Statistical analysis between the continuous populations revealed no significant differences for both NO2- and NOx concentrations. Particularly, the P values concerning the INTRA (P = 0.074 for NO2- and P = 0.249 for NOx) and the POST value comparisons (P = 0.830 for NO2- and P = 0.852 for NOx) revealed a similar reduction. Therefore these two groups were put together, and the ANOVA tests were performed considering three populations (continuous flow CPB, pulsatile flow CPB, beating-heart surgery). ANOVA pairwise comparisons revealed significant differences in the INTRA NO2- concentration between the continuous flow CPB and each one of the pulsatile groups, whereas the pulsatile flow CPB

100.00 ⫾ 0.00

61.32 ⫾ 30.59

Mean

PRE (%) 100.00 ⫾ 43.50 100.00 ⫾ 56.26 100.00 ⫾ 23.81 100.00 ⫾ 31.88 100.00 ⫾ 23.66 100.00 ⫾ 50.00 100.00 ⫾ 57.14 100.00 ⫾ 77.78 100.00 ⫾ 0.22 100.00 ⫾ 0.00

PRE (pmol of citrulline/mg/min)

122.65 ⫾ 53.35 33.15 ⫾ 18.65 10.50 ⫾ 2.50 66.35 ⫾ 21.15 47.03 ⫾ 11.13 44.00 ⫾ 22.00 12.60 ⫾ 7.20 45.00 ⫾ 35.00 4.45 ⫾ 0.01

42.86 ⫾ 36.18

1 2 3 4 5 6 7 8 9

Mean

149.61 ⫾ 138.11

35.06 ⫾ 0.00 426.47 ⫾ 145.48 323.81 ⫾ 74.08 89.07 ⫾ 17.48 57.81 ⫾ 13.75 121.59 ⫾ 69.32 54.37 ⫾ 5.95 53.33 ⫾ 8.89 184.94 ⫾ 88.31

INTRA (%)

122.30 ⫾ 16.63

109.66 ⫾ 92.54 255.35 ⫾ 97.59 209.52 ⫾ 28.57 88.70 ⫾ 6.56 96.23 ⫾ 16.48 51.14 ⫾ 1.14 81.75 ⫾ 53.97 83.33 ⫾ 10.00 125.06 ⫾ 44.61

POST (%)

82.37 ⫾ 20.45

92.67 ⫾ 5.72 87.56 ⫾ 3.85 40.17 ⫾ 3.88 83.65 ⫾ 4.63 84.27 ⫾ 4.17 93.60 ⫾ 9.83 57.74 ⫾ 5.87 98.31 ⫾ 10.75 103.37 ⫾ 17.84

POST (%)

95.96 ⫾ 7.46 89.61 ⫾ 4.30 125.03 ⫾ 8.18 93.79 ⫾ 8.72 87.30 ⫾ 9.33 100.61 ⫾ 6.14 78.08 ⫾ 10.35 76.17 ⫾ 3.24 84.88 ⫾ 23.31 92.38 ⫾ 14.60

POST (%)

35.65 ⫾ 48.24

159.22 ⫾ 0.22 13.00 ⫾ 7.00 45.55 ⫾ 3.75 12.64 ⫾ 4.34 22.80 ⫾ 1.10 11.55 ⫾ 7.25 37.70 ⫾ 0.90 14.65 ⫾ 6.65 3.78 ⫾ 1.79

PRE (pmol of citrulline/mg/min)

74.33 ⫾ 32.41

7.89 ⫾ 0.72 68.37 ⫾ 0.78 82.06 ⫾ 4.05 49.29 ⫾ 3.53 115.25 ⫾ 23.68 83.94 ⫾ 11.59 69.42 ⫾ 5.90 80.48 ⫾ 16.96 112.28 ⫾ 31.73

PRE (mM)

0.26 ⫾ 0.02 4.38 ⫾ 0.61 2.10 ⫾ 0.08 1.62 ⫾ 0.11 6.30 ⫾ 1.31 3.58 ⫾ 0.22 0.21 ⫾ 0.03 0.71 ⫾ 0.04 4.60 ⫾ 0.25 2.64 ⫾ 2.17

PRE (mM)

Values of each patient and the mean values of all the population are reported (mean value ⫾ standard deviation). CPB, cardiopulmonary bypass; eNOS, endothelial nitric oxide synthase.

Patient

78.27 ⫾ 21.56

90.17 ⫾ 7.11 87.80 ⫾ 24.98 47.92 ⫾ 3.62 70.17 ⫾ 4.72 79.91 ⫾ 7.91 112.39 ⫾ 15.50 59.35 ⫾ 4.03 56.30 ⫾ 3.82 100.42 ⫾ 6.88

INTRA (%)

eNOS activity continuous flow CPB

100.00 ⫾ 6.36 100.00 ⫾ 9.97 100.00 ⫾ 20.91 100.00 ⫾ 3.94 100.00 ⫾ 17.54 100.00 ⫾ 7.33 100.00 ⫾ 27.51 100.00 ⫾ 7.39 100.00 ⫾ 7.41

63.34 ⫾ 4.03 34.91 ⫾ 3.48 57.99 ⫾ 12.13 15.79 ⫾ 0.62 54.49 ⫾ 9.56 120.66 ⫾ 8.85 89.44 ⫾ 24.61 45.12 ⫾ 3.34 70.15 ⫾ 5.20

1 2 3 4 5 6 7 8 9

C

PRE (%)

PRE (mM)

Patient

85.98 ⫾ 2.65 89.66 ⫾ 3.73 110.99 ⫾ 8.14 87.28 ⫾ 2.85 79.70 ⫾ 10.85 97.72 ⫾ 6.23 82.71 ⫾ 6.71 79.38 ⫾ 7.82 75.70 ⫾ 10.57 87.68 ⫾ 10.91

INTRA (%)

NOx continuous flow CPB

100.00 ⫾ 4.85 100.00 ⫾ 8.89 100.00 ⫾ 2.26 100.00 ⫾ 6.80 100.00 ⫾ 9.00 100.00 ⫾ 4.24 100.00 ⫾ 4.96 100.00 ⫾ 4.75 100.00 ⫾ 3.16 100.00 ⫾ 0.00

0.41 ⫾ 0.02 0.21 ⫾ 0.02 0.55 ⫾ 0.01 0.12 ⫾ 0.01 8.08 ⫾ 0.73 2.39 ⫾ 0.10 0.26 ⫾ 0.01 1.47 ⫾ 0.07 5.58 ⫾ 0.18 2.12 ⫾ 2.84

1 2 3 4 5 6 7 8 9 Mean

B

PRE (%)

PRE (mM)

NO2- continuous flow CPB

Patient

A

90.85 ⫾ 6.95 97.95 ⫾ 15.32 108.55 ⫾ 8.50 86.31 ⫾ 12.63 113.35 ⫾ 15.35 97.65 ⫾ 7.39 142.83 ⫾ 11.72 107.38 ⫾ 16.50 125.64 ⫾ 10.48 107.84 ⫾ 17.77

INTRA (%)

95.17 ⫾ 20.26

109.99 ⫾ 24.35 86.13 ⫾ 8.25 141.41 ⫾ 7.12 76.90 ⫾ 6.95 97.67 ⫾ 18.55 93.94 ⫾ 9.35 84.50 ⫾ 2.43 76.15 ⫾ 7.05 89.83 ⫾ 10.93

INTRA (%)

100.00 ⫾ 0.00

100.00 ⫾ 0.14 100.00 ⫾ 53.85 100.00 ⫾ 8.23 100.00 ⫾ 34.31 100.00 ⫾ 4.82 100.00 ⫾ 62.77 100.00 ⫾ 2.39 100.00 ⫾ 45.39 100.00 ⫾ 47.35

PRE (%)

128.28 ⫾ 75.47

54.67 ⫾ 46.70 165.38 ⫾ 73.08 47.19 ⫾ 0.71 219.07 ⫾ 162.88 229.82 ⫾ 46.05 93.51 ⫾ 71.00 90.19 ⫾ 58.36 53.24 ⫾ 4.78 201.46 ⫾ 56.22

INTRA (%)

eNOS activity pulsatile flow CPB

100.00 ⫾ 0.00

100.00 ⫾ 9.17 100.00 ⫾ 1.14 100.00 ⫾ 4.94 100.00 ⫾ 7.17 100.00 ⫾ 20.54 100.00 ⫾ 13.81 100.00 ⫾ 8.51 100.00 ⫾ 21.07 100.00 ⫾ 28.26

PRE (%)

NOx pulsatile flow CPB

100.00 ⫾ 7.57 100.00 ⫾ 13.96 100.00 ⫾ 3.74 100.00 ⫾ 6.59 100.00 ⫾ 20.81 100.00 ⫾ 6.10 100.00 ⫾ 12.19 100.00 ⫾ 5.62 100.00 ⫾ 5.33 100.00 ⫾ 0.00

PRE (%)

NO2- pulsatile flow CPB

101.12 ⫾ 60.47

9.89 ⫾ 9.89 184.62 ⫾ 15.38 63.34 ⫾ 0.70 101.42 ⫾ 5.58 155.15 ⫾ 103.62 114.29 ⫾ 67.53 154.51 ⫾ 70.29 20.14 ⫾ 1.71 106.75 ⫾ 28.97

POST (%)

88.44 ⫾ 16.12

84.01 ⫾ 11.33 120.03 ⫾ 10.32 104.21 ⫾ 9.12 92.78 ⫾ 5.36 70.76 ⫾ 12.72 85.14 ⫾ 19.74 82.03 ⫾ 2.02 67.73 ⫾ 2.51 89.25 ⫾ 2.47

POST (%)

113.03 ⫾ 11.02 95.05 ⫾ 14.91 105.64 ⫾ 1.81 114.42 ⫾ 4.27 96.64 ⫾ 2.82 110.64 ⫾ 6.59 112.47 ⫾ 11.21 83.47 ⫾ 7.05 112.43 ⫾ 2.89 104.87 ⫾ 10.77

POST (%)

TABLE 2. NO2- (A), NOx (B), and citrulline (C) concentration in PRE operative period and percent concentrations in PRE, INTRA, and POST operative periods for the two populations

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E. LANZARONE ET AL. B Pulsatile flow CPB

A Continuous flow CPB

160

NO2 [% of PRE]

NO2 [% of PRE]

160 140 120 100

140 120 100 80

80 60

60

PRE

INTRA

POST

PRE

INTRA

POST

PRE

INTRA

POST

PRE

INTRA

POST

160

NOx [% of PRE]

NOx [% of PRE]

160 140 120 100 80

120 100 80

60

60

40

40 20

20

INTRA

POST

pmol of citrulline/mg/min [% of PRE]

PRE pmol of citrulline/mg/min [% of PRE]

140

450 400 350 300 250 200 150 100 50

450 400 350 300 250 200 150 100 50 0

0

PRE

INTRA

POST

FIG. 1. Percent NO2-, NOx and citrulline concentration in PRE, INTRA, and POST operative periods for continuous (A) and pulsatile (B) flow cardiopulmonary bypass (CPB). Mean values with standard deviations for the two populations (C).

and the beating-heart surgery were not significantly different (Table 3B) and showed a similar behavior. In the postoperative period, only the comparison of NO2- concentration between continuous flow CPB and beating-heart surgery were significant, whereas the different CPB treatments did not differ (Table 3B). Finally, no differences were pointed out concerning NOx concentrations (Table 3B). DISCUSSION Although the literature underlines the importance of accurately reproducing the left ventricle waveform to bring out the beneficial effects of pulsatile perfusion (1,2), pulsatile devices approved for clinical use do not reproduce the physiological waveform provided by the left ventricle. Therefore the pulsatile perfusion was analyzed in the presence of both a physiological flow waveform or the CPB circuit, and accurate analysis of NO metabolites in blood was performed. In our parallel study, a significant reduction of endothelial NO release during continuous flow CBP was found, whereas no differences were observed under beating-heart surgery (unpublished results). Artif Organs, Vol. 33, No. 11, 2009

Nevertheless, the presence of an external CPB circuit implies hemodilution and influences the systemic inflammatory response and the complement activation (interacting with other regulatory mechanisms such as the endothelial NO release [11–13]). Therefore, it was important to also compare continuous with pulsatile flow CPB, to have the same conditions in both the groups, even if the flow waveform supplied in the pulsatile group differs from the physiological one. This work, concerning comparison between continuous and pulsatile flow CPB, revealed the same differences as in the comparison with beating-heart surgery (unpublished results). The overall analysis of the data from both this study and the parallel one confirmed the different concentration of plasmatic NO metabolites between continuous flow CPB and pulsatile perfusion (beating-heart surgery or pulsatile flow CPB). Moreover, this work also validated the results of our parallel study, where hemodilution and CPB circuit were present only in one group. In fact, the results of this work suggested that the differences between beating-heart surgery and continuous flow

NITRIC OXIDE PRESERVATION UNDER PULSATILE FLOW CPB

NO2 [% of PRE]

C — Pulsatile — Continuous 160 140 120 100 80 60

PRE

INTRA

POST

PRE

INTRA

POST

NOx [% of PRE]

140 120 100 80 60

pmol of citrulline/mg/min [% of PRE]

40 300 250 200

933

different plasmatic NO2- and NOx concentrations to the alteration of endothelium release rather than to the erythrocyte contribution. Such a result confirmed the hypothesis of a different endothelial release in the presence of the analysis conducted considering NO metabolites in blood, where other release factors can be present. It also is of crucial importance to validate the literature, in which blood NO metabolites were analyzed without any study of the other factors affecting them (6–10). No differences were also observed concerning the estimation of the systemic inflammatory response. An increase in the patient numbers could bring significant results or confirm an absence of significant difference. The numbers should be increased also to consider the possible significant difference in plasmatic NOx concentration. Finally, other blood samples were prepared for further analysis of NO metabolites in plasma (particularly plasma detection of 3-nitrothyrosine).

150

CONCLUSIONS

100 50 0

PRE

INTRA

POST

FIG. 1. Continued

CPB derived from perfusion modality, as similar results were obtained even considering pulsatile flow CPB. The analysis of NOS activity in erythrocytes revealed no differences between continuous and pulsatile flow CPB. This allows us to mainly ascribe the

This article showed how in clinical practice the presence of a CPB circuit does not reduce NO release, as similar values were found between patients undergoing pulsatile flow CPB or beatingheart surgery (unpublished results). Also, anesthesiologic and pharmacologic treatments did not appear to reduce it. However, the presence of a continuous perfusion heavily induces a reduction in endothelium NO release, even under short-term treatments of a few hours. Results also showed a significant difference in INTRA surgery pressure (Table 1), which is lower

TABLE 3. P values of the performed t-tests for both NO2- and NOx concentrations (A) and analysis of variance pairwise comparisons between groups for both NO2- and NOx concentrations (B) A

NO2-

NOx

INTRA continuous CPB vs. INTRA pulsatile CPB POST continuous CPB vs. POST pulsatile CPB INTRA continuous CPB vs. PRE continuous CPB INTRA beating-heart vs. PRE pulsatile CPB POST continuous CPB vs. PRE pulsatile CPB POST beating-heart vs. PRE pulsatile CPB

0.012 0.058 0.010 0.223 0.156 0.213

0.107 0.495 0.016 0.495 0.032 0.064

B Continuous CPB (Table 2A,B) (unpublished results) vs. beating-heart (unpublished results) Continuous CPB (Table 2A,B) (unpublished results) vs. pulsatile CPB (Table 2A,B) Pulsatile CPB (Table 2A,B) vs. beating-heart (unpublished results)

NO2- INTRA

NO2- POST

NOx INTRA

NOx POST

0.023

0.060

0.821

0.984

0.001

0.106

0.182

0.695

0.589

0.948

0.578

0.850

In Table 3B data concerning both this work and the comparison between continuous flow cardiopulmonary bypass (CPB) and beatingheart surgery (unpublished results) are considered, in which the two continuous groups were put together as no significant differences were assessed in terms of NO2- and NOx plasma concentrations. Artif Organs, Vol. 33, No. 11, 2009

934

E. LANZARONE ET AL.

under pulsatile perfusion. This difference highlighted the strong endothelium mediated vasodilative effect of pulsatile perfusion, especially in the presence of a low number of patients. Therefore, continuous perfusion appears not to be beneficial for CPB patients, according to the recent literature (1,2). Moreover, the reduction in NO release, which already appears under short-term treatments, could bring to heavier effects under prolonged treatments, such as medium- and long-term cardiac assistance. Acknowledgments: The authors are grateful to Rocco Tabbì for his precious collaboration in blood sample acquiring, and to Alessandra Carcereri De Prati and Elisabetta Cavalieri for erythrocyte eNOS analysis. REFERENCES 1. Ündar A, Rosenberg G, Myers JL. Major factors in the controversy of pulsatile versus nonpulsatile flow during acute and chronic cardiac support. ASAIO J 2005;51:173–5. 2. Ji B, Ündar A. An evaluation of the benefits of pulsatile flow versus nonpulsatile perfusion during cardiopulmonary bypass procedures in pediatric and adult cardiac patients. ASAIO J 2006;52:357–61. 3. Fumero R, Montevecchi FM, Scuri S, Carrara B, Gamba A, Parenzan L. Clinical experience with a new pulsatile pump for infant and pediatric cardiopulmonary bypass. Int J Artif Organs 1989;12:314–20. 4. Ignarro LJ. Biosythesis and metabolism of endotheliumderived nitric oxide. Annu Rev Pharmacol Toxicol 1990;30: 535–60. 5. Griffith TM, Edwards DH, Lewis MJ, Newby AC, Henderson AH. The nature of endothelium-derived vascular relaxant factor. Nature 1984;308:645–7. 6. Ogasa T, Nakano H, Ide H, et al. Flow-mediated release of nitric oxide in isolated, perfused rabbit lungs. J Appl Physiol 2001;91:363–70. 7. Dai G, Tsukurov O, Chen M, Gertoler JP, Kamm RD. Endothelial nitric oxide production during in vitro simulation of external limb compression. Am J Physiol—Heart C 2002;282: 2066–75.

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