Open Access

Plasma proteomic changes during hypothermic and normothermic cardiopulmonary bypass in aortic surgeries

  • Authors:
    • Teiji Oda
    • Akane Yamaguchi
    • Masao Yokoyama
    • Koji Shimizu
    • Kosaku Toyota
    • Tetsuro Nikai
    • Ken‑Ichi Matsumoto
  • View Affiliations

  • Published online on: July 15, 2014     https://doi.org/10.3892/ijmm.2014.1855
  • Pages: 947-956
  • Copyright: © Oda et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

Deep hypothermic circulatory arrest (DHCA) is a protective method against brain ischemia in aortic surgery. However, the possible effects of DHCA on the plasma proteins remain to be determined. In the present study, we used novel high‑throughput technology to compare the plasma proteomes during DHCA (22˚C) with selective cerebral perfusion (SCP, n=7) to those during normothermic cardiopulmonary bypass (CPB, n=7). Three plasma samples per patient were obtained during CPB: T1, prior to cooling; T2, during hypothermia; T3, after rewarming for the DHCA group and three corresponding points for the normothermic group. A proteomic analysis was performed using isobaric tag for relative and absolute quantification (iTRAQ) labeling tandem mass spectrometry to assess quantitative protein changes. In total, the analysis identified 262 proteins. The bioinformatics analysis revealed a significant upregulation of complement activation at T2 in normothermic CPB, which was suppressed in DHCA. These findings were confirmed by the changes of the terminal complement complex (SC5b‑9) levels. At T3, however, the level of SC5b‑9 showed a greater increase in DHCA compared to normothermic CPB, while 48 proteins were significantly downregulated in DHCA. The results demonstrated that DHCA and rewarming potentially exert a significant effect on the plasma proteome in patients undergoing aortic surgery.

Introduction

Hypothermia <32–34°C has been clinically utilized only in cardiovascular surgery with cardiopulmonary bypass (CPB) as cardiac function becomes unstable with the decrease in temperature. Profound (<20°C) to deep (<25°C) hypothermic circulatory arrest (HCA) combined with antegrade selective cerebral perfusion (SCP) is a protective strategy against brain ischemia in complex aortic surgery (1). Although there is relatively little evidence of adverse effects of profound/deep hypothermia, there is some concern that HCA may have some drawbacks, such as coagulation disorders (2,3). As a consequence, moderate HCA (25–28°C) combined with increased SCP flow rates for aortic surgery has been utilized as the mainstay of treatment (4).

The development of isobaric tag-based quantitative mass spectrometry has provided a reliable method for high-throughput analysis of proteomes (5). Proteomic changes in human plasma samples have been successfully analyzed through the use of the isobaric tag for relative and absolute quantification (iTRAQ) labeling strategy (6,7). In the present study, a comparison was made of the plasma proteomes of patients undergoing aortic surgery using deep hypothermic circulatory arrest (DHCA) and SCP (DHCA + SCP) to those undergoing aortic surgery using normothermic CPB.

Patients and methods

Patient selection

The present prospective cohort study received approval from the Ethics Committee of Shimane University Faculty of Medicine and was conducted at Shimane University Hospital. The patients provided written informed consent. Patients with annulo-aortic ectasia, and ascending or aortic arch aneurysms were included in this study.

Anesthesia

The same anesthesia and CPB protocols were used for all patients: anesthesia was induced and maintained with fentanyl (15–20 μg/kg, administered intravenously prior to initiation of CPB) and propofol (3–6 mg/kg/h). Catecholamines, vasodilators and antiarrhythmic drugs were administered in a similar manner in the two groups of patients.

CPB

The cardiopulmonary circuit was the same for the two groups, consisting of a hollow-fiber polymer-coated membrane oxygenator (QUADROX-I, HMO70000; Maquet, Rastatt, Germany), heparin-coated venous reservoir (HVR-4WF; Mera, Tokyo, Japan) and polymer-coating cardiotomy reservoir (only used in hypothermic CPB; BO-HC2821; Maquet), centrifugal pump (ROTAFLOW, BO-RF32; Maquet), arterial filter (40 μm Pall arterial line filter, AL6; Pall, Port Washington, NY, USA) and heparin-coated CPB circuit (Mera or JMS, Tokyo, Japan). The pump was primed with bicarbonated Ringer’s solution, mannitol and methylprednisolone (500 mg). After systemic anticoagulation with 3 mg/kg heparin, CPB was instituted at a flow rate of 2.0–2.8 l/min/M2. Blood gases, electrolytes, glucose and the hemoglobin (Hb) concentration were continuously monitored by CDI 500 (Terumo, Tokyo, Japan) and blood gas analysis was performed every 30 min (ABL800 FLEX Blood Gas Analyzer; Radiometer, Copenhagen, Denmark). Each parameter was maintained as follows: PaO2: 300–500 mmHg, PaCO2: 35–45 mmHg (α-stat management), blood glucose: 100–300 mg/dl, Hb: 7–9 g/dl in the two groups. Rectal and urinary bladder temperatures were monitored. Dilutional ultrafiltration was continuously performed during CPB for the patients in the two groups to correct hyperkalemia and fluid imbalance (Blood concentrator PLUS, BC-140 plus; Maquet). In DHCA + SCP, the body was cooled to 22°C with topical head cooling. After cardioplegic cardiac arrest and initiation of HCA, SCP was performed following cannulation into the three arch vessels and maintained by monitoring pressures (both bilateral radial artery and SCP cannula tip) and using an INVOS Oximeter (INVOS 5100C; Somanetics, Troy, MI, USA). Rewarming was initiated following completion of open distal anastomosis and initiation of distal perfusion.

Blood sampling

Three blood samples were collected from an arterial line into ethylenediaminetetraacetic acid (EDTA) tubes as follows: T1 (before cooling, 5 min after pump initiation), T2 (before aortic declamping, 30–160 min after pump initiation in normothermic CPB or during deep hypothermia, 30–124 min after pump initiation in DHCA + SCP), T3 (just before termination of CPB, 90–193 min after pump initiation in normothermic CPB or 121–314 min after pump initiation in DHCA + SCP). Blood samples were centrifuged at 1,400 × g for 5 min, and the plasma layers were stored at −80°C.

Immunodepletion of abundant proteins

The two most abundant plasma proteins, albumin and immunoglobulin (Ig) G, were removed using an immunodepletion column (Albumin & IgG Depletion SpinTrap; GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions and as previously described (7).

iTRAQ labeling and strong cation exchange (SCX) chromatography

Samples were prepared according to the manual published by AB Sciex (Foster city, CA, USA) and as described previously (7). In brief, equal amounts of immunodepleted T1, T2, and T3 samples from each patient were denaturated and reduced, the cysteines were alkylated, and then digested with trypsin (AB Sciex). Each digest was labeled with a different iTRAQ tag using the iTRAQ reagent 4-plex kit (AB Sciex). iTRAQ label 114 was used for the T1 sample, and iTRAQ labels 115, 116 or 117 were randomly selected for the T2 and T3 samples, after which the three samples of each patient were combined. The combined samples were then fractionated into six fractions by SCX chromatography according to the manufacturer’s instructions (AB Sciex) and each fraction was desalted according to the manufacturer’s instructions (Waters, Milford, MA, USA).

NanoLC and MALDI-TOF/TOF MS/MS analysis

One fraction from the SCX chromatography (see above) was fractionated to 171 spots using a DiNa nanoLC system (KYA Technologies, Tokyo, Japan) and collected onto an Opti-TOF LC/MALDI 384 target plate (AB Sciex) according to the manufacturer’s instructions and as previously described (7). Spotted peptide samples were analyzed by a 5800 MALDI-TOF/TOF MS/MS Analyzer with TOF/TOF Series software (version 4.0; AB Sciex). MS/MS data were analyzed using ProteinPilot™ software (version 3.0) and the Paragon™ protein database (AB Sciex). Quantitative changes of proteins at T2 or T3 were calculated using the iTRAQ ratios T2:T1 or T3:T1, respectively.

iTRAQ data analysis and bioinformatic analysis

Proteins identified as showing expression changes were tested for conformity to the following conditions: i) a false discovery rate (FDR) <5% (FDR was estimated by ‘decoy database searching’ using the ProteinPilot Software); and ii) protein confidence >99% (‘unused ProtScore’ >2). Unused ProtScore is defined as −log (1-% confidence/100). Proteins fulfilling these criteria were considered to have ‘statistical significance’ (7,8). PANTHER software (version 8.1, http://www.pantherdb.org) was used to test for statistical overrepresentation of Gene Ontology (GO, http://www.geneontology.org) terms as described in detail elsewhere (9,10). If the number of identified genes in a GO term was significantly larger than that in the whole genome classified by the same GO term, i.e., the number of observed genes in a GO term is significantly larger than the number of expected genes in the same GO term by the binomial test, the GO term was described here as ‘overrepresented’ with statistical significance after a Bonferroni correction for multiple testing. The annotations of identified proteins were obtained from the Uniprot database (http://www.uniprot.org/).

Western blot analysis and enzyme-linked immunosorbent assay (ELISA)

Western blot analyses were performed as described previously (7). In brief, plasma samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotted using rabbit monoclonal carbonic anhydrase 1 (CA1) antibody (Abcam, Tokyo, Japan) and anti-rabbit IR dye 680-conjugated IgG (LI-COR, Lincoln, NE, USA). Protein bands were visualized using an Odyssey (LI-COR) infrared imaging system and their intensities measured for densitometric analyses of CA1. Plasma levels of the complement proteins C5a and SC5b-9 were determined using a commercially available solid-phase sandwich ELISA kit (Quidel Corp., San Diego, CA, USA) following the manufacturer’s recommendations.

Statistical analysis

Continuous variables, such as blood gas analysis and hemodynamic variables were expressed as means ± standard deviation (SD). The variables were tested for statistically significant differences between patient groups using the Student’s t-test. Categorical variables were compared using Fisher’s exact test. For analysis of iTRAQ ratios, i.e. T2:T1 or T3:T1 ratios for each significantly identified protein, P-values were calculated by the one sample t-test of averaged protein ratio against 1 to assess the validity of the protein expression change (11). To assess ELISA and western blot analysis data, statistical comparisons were performed by two-way analysis of variance (ANOVA) (general linear model) with repeated measures followed by a post-hoc Bonferroni test to detect individual differences. The relationships between complement activation (SC5b-9) and the iTRAQ ratios of each protein including CA1 values (western blot analysis) were calculated by single and stepwise multiple regression analyses (forward selection method) (StatFlex version 6; Artech, Osaka, Japan). P<0.05 was considered to indicate statistical significance.

Results

Patient characteristics and CPB data

Baseline characteristics were similar in both groups except for minimal core temperature and CPB time (Table I). There were several small but significant differences in blood gas analysis, Hb and lactate levels in both groups (Table II). Pump flow rate, perfusion pressure, and core temperatures were similar between the two groups at T1 and T3. At T2, there was a significant difference in core temperature between the two groups: 23.0±1.2°C vs. 36.0±1.0°C (P<0.0001). The timing of T1 and T2 sampling from the initiation of CPB did not differ between the two groups (P=1.000 for T1, P=0.250 for T2); however, sampling at T3 was performed significantly later in the DHCA + SCP patient group than in the normothermic CPB group (Table I). The HCA time of the lower body averaged 59±41 min in DHCA + SCP patients.

Table I

Baseline characteristics, type of surgery and intraoperative variables.

Table I

Baseline characteristics, type of surgery and intraoperative variables.

VariablesNormothermic CPB (n=7)DHCA + SCP (n=7)P-value
Age66.0±8.869.9±7.00.3841
Gender (male)341.0000
β-blocker140.2657
ACEIs/ARBs260.1026
Calcium channel blockers350.5921
Oral antihypoglycemic agent101.0000
Statins420.5921
Minimal core temperature (°C)34.6±0.921.8±1.1<0.0001
Aortic cross-clamping time (min)107.0±31.7117.7±68.60.7245
CPB time (min)142.9±34.2218.7±75.70.0457

[i] ACEI, angiotension converting enzyme inhibitor; ARB, angiotensin II receptor blocker; CPB, cardiopulmonary bypass; DHCA + SCP, deep hypothermic circulatory arrest with selective cerebral perfusion.

Table II

Arterial blood gas analysis at three sampling points during CPB for aortic surgery.

Table II

Arterial blood gas analysis at three sampling points during CPB for aortic surgery.

Treatment methodsSampling timepHPaO2 (mmHg)PaCO2 (mmHg)B.E.Glucose (mg/dl)Lactate (mmol/l)Hb (g/dl)
T17.38±0.05387±4941.5±4.5−1.1±2.099±2010.0±4.77.4±0.9
Normothermic CPBT27.35±0.03399±3345.0±2.0a−1.1±2.4125±1810.0±5.98.7±1.3b
T37.37±0.04373±35c43.0±1.8−0.6±1.6d124±2310.7±7.3e8.3±1.4
T17.38±0.03390±3841.4±2.6−0.8±1.9114±2112.0±4.97.1±0.6
DHCA + SCPT27.32±0.04424±4349.7±3.9a−0.9±3.296±3315.1±5.96.7±0.6b
T37.35±0.03321±28c43.0±3.6−2.3±0.8d140±4724.9±12.9e7.6±0.6

{ label (or @symbol) needed for fn[@id='tfn2-ijmm-34-04-0947'] } Statistically significant differences between the two groups were:

a P=0.0157,

b P=0.0058,

c P=0.0102,

d P=0.0297,

e P=0.0270.

{ label (or @symbol) needed for fn[@id='tfn8-ijmm-34-04-0947'] } B.E., base excess; CPB, cardiopulmonary bypass; DHCA, deep hypothermic circulatory arrest; Hb, hemoglobin concentration; SCP, selective cerebral perfusion; T1, 5 min after initiation of CPB; T2, during deep hypothermia or declamping; T3, just before termination of CPB.

iTRAQ data analysis and bioinformatics

Mass spectrometry identified 322 proteins, of which 262 were found to be statistically significant, i.e., the proteins fulfilled both the FDR <5% and protein confidence >99% criteria. Of the 262 proteins, those that increased or decreased (>1.2-fold or <0.833-fold) in at least four of the seven samples in each treatment group were identified to obtain an adequate sample size and statistical power (5,12), subsequently analyzed by PANTHER software. The statistical overrepresentation test identified six GO categories (biological processes) with five proteins significantly increased in the normothermic CPB group at T2 (Tables III and V). By contrast, only one GO category showed significant overrepresentation in the DHCA + SCP group at T2 (Table III). At T3 in the latter treatment group, 11 GO categories were significantly overrepresented with 48 proteins significantly decreased, whereas only one GO category was significantly overrepresented in the normothermic CPB group (Tables IV and VI). In the normothermic CPB group, the iTRAQ ratio of CA1 increased significantly to 1.50±0.23 at T2 and to 1.58±0.30 at T3 in comparison to T1 (P=0.0241 at T2, and P=0.0299 at T3, Table V). The ratio also increased in the DHCA + SCP group to 1.52±0.45 at T2 and 2.65±1.65 at T3, although without statistical significance due to missing data. This increase in the CA1 ratio at T3 is a noteworthy finding as all other proteins decreased significantly in the DHCA + SCP patients, with the exception of HBA1 and HBB (Table VI).

Table III

GO categories showing significant overrepresentation for plasma proteins at T2.

Table III

GO categories showing significant overrepresentation for plasma proteins at T2.

Homo sapiensPlasma sample


GO termDescriptionNo. of genesaNo. of observed genesbNo. of expected genescP-valueGenes
A, Normothermic CPB

0006956Complement activation9960.096.46E-08C8B C1S C1R C6 C4B C5
0006508Proteolysis1,131101.073.29E-06C8B SERPING1 ITIH3 C1S C1R C6 C4B C5 SERPINA4 AGT
0006955Immune response72570.694.82E-04C8B AZGP1 C1S C1R C6 C4B C5
0007596Blood coagulation27140.261.90E-02C8B C1S C1R C6
0009605Response to external stimulus27140.261.90E-02C8B C1S C1R C6
0019538Protein metabolic process3,178103.024.02E-02C8B SERPING1 ITIH3 C1S C1R C6 C4B C5 SERPINA4 AGT

B, DHCA + SCP

0006869Lipid transport22030.123.52E-02APOD APOC3 APOA2

{ label (or @symbol) needed for fn[@id='tfn9-ijmm-34-04-0947'] } The test for statistical overrepresentation of each Gene Ontology (GO) term (biological process) was perfomed using PANTHER software (version 8.1). Upregulated genes are shown in bold.

a The total number of genes in the whole genomes (Homo sapiens, n=20,000) classified by the GO term;

b the number of genes from the input list [n=20 (mapped genes, 19; unmapped gene, 1) in the normothermic cardiopulmonary bypass (CPB) group, n=12 (mapped genes, 11; unmapped gene, 1)’ in the deep hypothermic circulatory arrest (DHCA) with selective cerebral perfusion (SCP) group (DHCA + SCP)] classified by the GO term;

c the number of genes that would be detected in the input list for a particular GO category on the basis of the reference list (Homo sapiens).

{ label (or @symbol) needed for fn[@id='tfn13-ijmm-34-04-0947'] } T2, during deep hypothermia or before declamping.

Table V

Plasma proteins showing significantly changed iTRAQ ratio at T2 or T3 of normothermic CPB.

Table V

Plasma proteins showing significantly changed iTRAQ ratio at T2 or T3 of normothermic CPB.

At T2At T3


Unused ProtScoreCoverage %Peptides (95%)Uniprot No.GeneProteinT2/T1bP-valueT3/T1bP-valueBiological process (GO)
33.3396.639P68871HBBHb subunit β4.070.01388.700.1023Oxygen transport
19.6673.230P69905HBA1Hb subunit α3.790.02167.150.0536Oxygen transport
12.0040.27P00915CA1CA1a1.500.02411.580.0299Bicarbonate transport
23.2935.012P05155SERPING1Plasma protease C1 inhibitor1.290.00881.130.2306Complement activation, blood coagulation
8.0114.34P00736C1RComplement C1r subcomponent1.250.02271.130.3394Complement activation
31.7032.419P04004VTNVTNa1.030.55830.800.0026Innate immune response
12.0138.19P02743APCSSerum amyloid P-component1.070.59550.770.0006Acute-phase response
95.6585.793P00738HPHaptoglobin0.800.07160.620.0035Acute-phase response, defense response

{ label (or @symbol) needed for fn[@id='tfn19-ijmm-34-04-0947'] } Bold, increased ratio (≥1.2-fold) with statistical significance and italics, decreased ratio (≤0.83-fold) with statistical significance. Plasma samplings were performed at T1 [5 min after initiation of cardiopulmonary bypass (CPB)], T2 (before declamping) and T3 (just before termination of CPB). Unused ProtScore indicates protein confidence as expressed by the formula: ProtScore = - log (1 - % confidence/100). Coverage % indicates the percentage of matching amino acids from identified peptides. Peptides (95%) indicates the number of peptides having at least 95% confidence.

a Statistical significance of single regression analysis of relationships between isobaric tag for relative and absolute quantification (iTRAQ) ratios of proteins and SC5b-9 measured by enzyme-linked immunosorbent assay (ELISA);

b iTRAQ average ratio.

{ label (or @symbol) needed for fn[@id='tfn22-ijmm-34-04-0947'] } CA1, carbonic anhydrase 1; GO, Gene Ontology; Hb, hemoglobin; VTN, vitronectin.

Table IV

GO categories showing significant overrepresentation for plasma proteins at T3.

Table IV

GO categories showing significant overrepresentation for plasma proteins at T3.

Homo sapiensPlasma sample


GO termDescriptionNo. of genesaNo. of observed genesbNo. of expected genescP-valueGenes
A, Normothermic CPB

0008015Blood circulation19230.112.36E-02HP HBA1 HBB

B, DHCA + SCP

0006508Proteolysis1131222.943.13E-12SERPING1 PLG SERPINC1 APOH C3 ITIH1 F2 CP A2M C4BPA C4B CFB HP SERINF2 CFH ITIH2 C5 AHSG SERPINA3 ITIH4 AMBP SERPINA1
0006956Complement activation99100.261.98E-11APOH C3 PGLYRP2 A2M C4BPA C4B CFB CFH HP C5
0007596Blood coagulation271100.73.36E-07APOH F2 CP C4BPA CFB CFH HP TF HRG AMBP
0009605Response to external stimulus271100.73.36E-07APOH F2 CP C4BPA CFB CFH HP TF HRG AMBP
0006955Immune response725121.884.68E-05A1BG APOH C3 PGLYRP2 A2M C4BPA C4B CFB CFH HP TF C5
0050896Response to stimulus1767174.592.20E-04A1BG APOH C3 PGLYRP2 F2 CP A2M C4BPA CFB C4B HP CFH TF C5 HRG AMBP
0002376Immune system process2480206.452.92E-04A1BG APOH C3 PGLYRP2 F2 CP A2M C4BPA CFB C4B HP CFH TF C5 SAA4 AHSG HRG AMBP
0019538Protein metabolic process3178228.269.02E-04SERPING1 PLG SERPINC1 APOH C3 ITIH1 F2 CP A2M C4BPA C4B CFB HP SERINF2 CFH ITIH2 C5 AHSG SERPINA3 ITIH4 AMBP SERPINA1
0006869Lipid transport22070.572.89E-04APOB APOD APOA1 APOE APOC3 APOA2 APOL1
0016337Cell-cell adhesion72491.881.63E-02APOH FGB CP C4BPA FGG CFB CFH FGA VTN
0006810Vitamin transport9040.231.60E-02TTR CP HPX RBP4

{ label (or @symbol) needed for fn[@id='tfn14-ijmm-34-04-0947'] } The test for statistical overrepresentation of each Gene Ontology (GO) term (biological process) was perfomed using PANTHER software (version 8.1). Upregulated genes are shown in bold.

a The total number of genes in the whole genomes (Homo sapiens, n=20,000) classified by the GO term;

b the number of genes from the input list [n=11 (mapped genes, 11) in the normothermic cardiopulmonary bypass (CPB) group, n=57 (mapped genes, 52; unmapped gene, 5) in the deep hypothermic circulatory arrest (DHCA) with selective cerebral perfusion (SCP) group (DHCA + SCP)] classified by the GO term;

c the number of genes that would be detected in the input list for a particular GO category on the basis of the reference list (Homo sapiens).

{ label (or @symbol) needed for fn[@id='tfn18-ijmm-34-04-0947'] } T3, just before termination of CPB.

Table VI

Plasma proteins showing significantly changed iTRAQ ratio at T2 or T3 in patients undergoing DHCA and SCP.

Table VI

Plasma proteins showing significantly changed iTRAQ ratio at T2 or T3 in patients undergoing DHCA and SCP.

At T2At T3


Unused ProtScoreCoverage %Peptides (95%)Uniprot No.GeneProteinT2/T1cP-value process (GO)T3/T1cP-valueBiological
19.6673.230P69905HBA1Hb subunit α4.540.00117.740.0002Oxygen transport
33.3396.639P68871HBBHb subunit β4.230.00097.140.0004Oxygen transport
35.5040.118P19827ITIH1Inter-α-trypsin inhibitor heavy chain H11.010.87410.830.0136Hyaluronan metabolic process
35.5649.619P01008SERPINC1 Antithrombin-III0.920.50190.830.0439Blood coagulation
28.5540.518P10909CLUClusterin0.980.89810.820.0036Innate immunity
23.2935.012P05155SERPING1Plasma protease C1 inhibitor0.940.51230.820.0310Complement pathway, blood coagulation
34.5549.220P01011SERPINA3 α-1-antichymotrypsin0.990.89050.810.0056Acute-phase response
28.7661.720P02763ORM1α-1-acid glycoprotein 10.970.78940.810.0008Acute-phase response
38.0741.420Q14624ITIH4Inter-α-trypsin inhibitor heavy chain H40.930.42390.810.0116Acute-phase response
4.0116.82O14791APOL1Apolipoprotein L10.990.90450.800.0181Cholesterol metabolism
45.1133.627P19823ITIH2Inter-α-trypsin inhibitor heavy chain H20.940.43300.800.0046Hyaluronan metabolic process
20.1045.212P02760AMBPProtein AMBP0.930.39310.800.0031Negative regulation of immune response
18.8144.212P27169PON1Serum paraoxonase/arylesterase 10.870.08010.800.0306Response to external stimulus
116.1063.065P00450CPCPa,b0.970.74470.790.0012Cellular iron ion homeostasis
100.6674.492P01009SERPINA1 α-1-antitrypsin0.960.71210.780.0170Acute-phase response
29.2235.116P04196HRGHistidine-rich glycoprotein0.950.54980.780.0225Blood coagulation, chemotaxis
26.3058.015P02749APOH β-2-glycoprotein10.850.05560.780.0222Negative regulation of blood coagulation
12.1356.26P35542SAA4Serum amyloid A-4 protein0.820.03070.780.0176Acute-phase response
83.6774.955P02790HPXHemopexina0.960.63440.770.0013Hb metabolic process, positive regulation of immune response
222.9274.4155P01023A2M α-2-macroglobulin0.930.43390.770.0027Blood coagulation, negative regulation of complement activation
18.9651.211P02753RBP4Retinol-binding protein 40.860.08610.770.0398Positive regulation of Ig secretion
116.3789.986P0C0L5C4BComplement C4-B0.940.40090.760.0015Complement activation
222.4778.5138P01024C3Complement C30.930.50760.760.0048Complement activation
57.4059.537P01871IGHMIg μ chain C region0.930.37550.760.0047Immune response
39.2761.623P00734F2Prothrombina0.910.38170.750.0014Acute-phase response, blood coagulation
98.9580.090P02675FGBFibrinogen β chain0.940.64860.740.0431Blood coagulation
36.1147.722P04217A1BG α-1B-glycoproteina0.940.46310.740.0003No description
35.3443.122P02765AHSG α-2-HS-glycoprotein0.890.21030.740.0013Acute-phase response, regulation of inflammatory response
84.8051.551P08603CFHComplement factor H0.880.11870.740.0005Complement activation
15.2631.08P08697SERPINF2 α-2-antiplasmin0.860.11920.740.0163Acute-phase response, negative regulation of fibrinolysis
31.7032.419P04004VTNVTNa0.940.44420.730.0014Innate immune response, negative regulation of blood coagulation
20.0129.610P43652AFMAfamin0.840.18780.730.0032Vitamin transport
41.0072.838P01042KNG1Kininogen-10.900.13800.720.0003Blood coagulation, inflammatory response
68.1154.07P02774GCVitamin D-binding proteina0.880.27490.720.0020Vitamin D metabolic process
24.0156.26P01842IGLC1Ig λ chain C regions0.870.23960.720.0041Complement activation
63.3074.852P02679FGGFibrinogen γ chain0.920.53110.710.0275Blood coagulation
32.4875.228P02649APOEApolipoprotein E0.870.06060.710.0053Cholesterol metabolism
45.3625.53P00751CFBComplement factor B0.860.15270.710.0013Complement activation
182.8885.793P02787TF Serotransferrin0.880.22970.700.0001Cellular iron ion homeostasis
30.4197.232P01834IGKCIg κ chain C region0.890.33370.690.0002Complement activation
87.7043.624P02647APOA1Apolipoprotein A-I0.840.26540.690.0239Cholesterol metabolism
25.1932.313P04003C4BPAC4b-binding protein α chain0.910.47220.680.0005Complement activation
47.9854.424P00747PLGPlasminogen0.820.01790.670.0002Fibrinolysis
273.7345.0144P04114APOBApolipoprotein B-1000.860.09330.660.0032Cholesterol metabolic process
8.0036.49P02656APOC3Apolipoprotein C-III0.820.04140.660.0169Lipid transport
7.4552.45P02766TTRTransthyretin0.860.19240.650.0218Retinoid metabolic process
7.3125.53Q96PD5PGLYRP2 N-acetylmuramoyl-L-alanine amidase0.750.03950.640.0001Innate immune response
72.1273.772P01876IGHA1Ig α-1 chain C region0.790.11760.600.0003Immune response
17.0253.411P05090APODApolipoprotein D0.740.05140.590.0002Negative regulation of cytokine production
23.4893.014P02652APOA2Apolipoprotein A-II0.720.03130.580.0273Acute inflammatory response, negative regulation of cytokine secretion

{ label (or @symbol) needed for fn[@id='tfn23-ijmm-34-04-0947'] } Bold, increased ratio (≥1.2-fold) with statistical significance and italics, decreased ratio (≤0.833-fold) with statistical significance. Plasma samples were obtained at T1 (5 min after pump initiation), T2 (during deep hypothermia or before declamping), T3 [just before termination of cardiopulmonary bypass (CPB)]. Unused ProtScore indicates protein confidence as derived from the formula: ProtScore = −log (1-% confidence/100). Coverage % indicates the percentage of matching amino acids from identified peptides. Peptides (95%) indicate the number of peptides having at least 95% confidence.

a Statistical significance of single regression analysis of relationships between isobaric tag for relative and absolute quantification (iTRAQ) ratios of proteins and SC5b-9 measured by enzyme-linked immunosorbent assay (ELISA);

b statistical significance of stepwise multiple regression analysis of relationships between iTRAQ ratios of seven proteins identified by single regression analysis and SC5b-9;

c iTRAQ average ratio.

{ label (or @symbol) needed for fn[@id='tfn27-ijmm-34-04-0947'] } CP, ceruloplasmin; GO, Gene Ontology; Hb, hemoglobin; Ig, immunoglobulin; VTN, vitronectin.

Western blot analysis and ELISA

To validate the iTRAQ ratios and complement activation identified by the PANTHER analysis, the protein CA1 was analyzed by western blot analysis and the complement proteins C5a and SC5b-9 were measured by ELISA. In both patient groups, the levels of CA1 continuously and significantly increased during CPB (P<0.0001), although the differences between the groups were not significant (Fig. 1). The level of complement C5a increased, albeit non-significantly, at T3 in the DHCA + SCP group compared to the normothermic group (Fig 2A). The level of SC5b-9 increased significantly at T2 in the normothermic group, and the differences between T1 and T2 and between T1 and T3 were both highly significant (P<0.0001, Fig 2B). In the DHCA + SCP group, the level of SC5b-9 increased slightly from T1 to T2, and then increased rapidly and significantly from T2 to T3. There were significant differences in SC5b-9 values between T3 and T1 and between T3 and T2 in the DHCA + SCP group (P<0.0001), and between both groups at T2, respectively (P=0.008). Since the innate immunity response is regulated by complex protein-protein interactions (13), it is difficult to clarify the causative relationship between the SC5b-9 level and iTRAQ ratios of identified proteins. Therefore, single regression analyses were performed to search for the relationships between the SC5–9 values and the iTRAQ ratios of the 42 proteins identified in all samples including the CA1 value (western blot analysis). Seven proteins (CP, HPX, VTN, F2, A1BG, GC, and CA1) showed significant correlations with SC5b-9 levels. A stepwise multiple regression analysis identified a significant coefficient (R=0.486) and ceruloplasmin as an independent variable (standardized β=−0.468, P=0.012).

Discussion

Exposure of blood components to a CPB circuit activates various blood cell types, endothelial cells and proteins. This activation can improperly stimulate biological processes that may contribute to the development of postoperative complications resulting from the dysfunction of blood cells, coagulation, fibrinolysis, and the kinin-kallikrein and immune systems (1416). Various modifications to the inner surface of the CPB circuit, including heparin- and polymer-coating, reduce adverse effects on complement activation, blood coagulation, proinflammatory cytokines and improve clinical outcomes (1720). The C5 complement inhibitor pexelizumab and a complement factor 1 esterase inhibitor have been tested clinically (21) and in an animal study (22) for their abilities to reduce complement activation during CPB. Although a recent proteomic study demonstrated the possibility of identifying predictive plasma biomarkers in patients undergoing CPB (23), this approach has not been used to investigate the effect of hypothermia/rewarming on the biocompatibility of a CPB circuit.

The proteins that were identified as showing significant protein confidence in this experiment were all included in the list of proteins reported previously for the human plasma proteome (24). Our analysis using iTRAQ labeling mass spectrometry demonstrated that several GO categories related to the complement system and proteolysis were significantly overrepresented at T2 in normothermic CPB in spite of using a heparin-coated CPB circuit and polymer-coated oxygenator. Of note, these changes were not observed at T2 in DHCA + SCP patients. To confirm these findings, the levels of plasma C5a and SC5b-9 were measured by ELISA, which showed a significantly larger increase in SC5b-9 at T2 in patients undergoing normothermic CPB compared to DHCA + SCP. This reduction in the complement activation level in the DHCA + SCP group is similar to the findings from investigations of a randomized CPB study or therapeutic hypothermia for encephalopathy, which showed that hypothermia can suppress the immune response (2527). In this context, our previous proteomic analysis of rat liver samples after a short duration (3 h) of deep hypothermia (23°C) demonstrated that 264 proteins changed significantly with 163 showing a decrease (28). Consequently, the deep hypothermia (22°C) and circulatory arrest of the lower body that occurs in DHCA + SCP might further decrease complement components (activators and regulators) that are mainly produced in the liver. Any such decrease may have a significant effect on the activity of the complement system at T2 in the DHCA + SCP patients.

Complement activation in the DHCA +SCP group was indicated by the rapid increase in the level of SC5b-9 from T2 to T3. To the best of our knowledge, this is the first study to demonstrate that CPB-induced complement activation can be initially suppressed at T2 and later upregulated at T3 in the DHCA + SCP group. The latter response may in part have occurred because complement activation can develop after rewarming. This explanation is supported by a study on mild hypothermia after cardiac arrest which demonstrated that complement activation decreased after the induction of hypothermia (32–34°C), followed by an increase during rewarming (27). Another possible cause of complement activation at T3 may be reperfusion injury of the lower part of the body that developed with the lactate production at T3 (Table II), which may indicate that the metabolism of lower body had been conducted anaerobically with the production of oxygen debt during 59±41 min of DHCA in the DHCA + SCP group (29). Moreover, rewarming has been shown to aggravate cold-induced (4°C) oxidative hepatocyte injury caused by chelatable iron via Fenton chemistry and, thereby, to accelerate complement activation (30). On the other hand, both reperfusion injury and the impact of cooling/rewarming on liver metabolism may underlie the downregulation of some of the identified proteins at T3. A number of proteins were found to be downregulated at T3 in the DHCA + SCP patients (Tables IV and VI). This downregulation seems inconsistent with the complement activation identified by the ELISA analysis. Possibly, this inconsistency might be explained by the tight control of complement activation by its regulators, suggesting that the downregulation of regulators such as vitronectin (VTN) or C1-inhibitor (SERPING1) contribute to complement activation (Table VI) (29). CA1 had a significant positive correlation with SC5b-9 levels by a single regression analysis. It has been demonstrated that CA1 causes kallikrein-kinin system activation (31). There is evidence of an interaction among the complement, coagulation and kinin systems (29,32) in which kallikrein is an initiator of the complement cascade by digestion of factor XIIa into factor XIIf (33) and possibly through the cleavage of C3 or C5 into active forms (32). By contrast, ceruloplasmin, which had a significant negative correlation with the SC5b-9 level by stepwise multiple regression analysis, was downregulated at T3 in the DHCA + SCP. This protein has ferroxidase activity to oxidize Fe2+ to Fe3+, contributing to the antioxidant capacity because of the reduction of oxidation capacity via the Fenton reaction (34). Ceruloplasmin is also reported to be an endogenous inhibitor of myeloperoxidase (35), but is downregulated by reactive oxygen species (34). Taken together, CA1, an upregulated protein at T3, may be a positive regulator of the complement system via kallikrein-kinin system activation. By contrast, ceruloplasmin, a downregulated protein at T3, may be a major negative regulator of the complement system via its antioxidant capacity. Thus, both the increased complement activator and several decreased complement regulators could result in the rapid rise of the SC5b-9 level at T3 in the DHCA + SCP patients. During normothermic CPB, the levels of C5a and SC5b-9 did not change significantly from T2 to T3 after an initial rise at T2, a finding consistent with the relatively unchanged iTRAQ ratio and with reports from previous studies (14,16).

Although we used state-of-the-art iTRAQ labeling technology mass spectroscopy, a considerable number of proteins expressed at low levels may not have been detected because of the large dynamic range in plasma proteins (24). Nevertheless, this technology may become a useful analytical tool in the field of research of biocompatibility of CPB in the future. The comparisons here involved two groups in which the two sets of patients underwent similar thoracic aortic surgery, i.e., DHCA + SCP and normothermic CPB. However, significant differences between the two groups occurred in the CPB time, which in turn affected T3 sampling time, implementation of circulatory arrest and SCP (hypothermic ischemia of lower body), and levels of PaCO2 and Hb at T2. These differences may affect plasma proteomic profiles. To elucidate the effects of hypothermia (and rewarming) per se on the biocompatibility of CPB, it may be necessary to perform a study using an animal model in the future.

In conclusion, results of the comparative iTRAQ proteomic analysis demonstrated that CPB-induced complement activation (and proteolysis) was initially suppressed during DHCA and later upregulated at the end of CPB after rewarming. Single and multiple regression analyses showed that this complement activation/suppression was associated with changes in expression of ceruloplasmin and six other proteins including CA1.

Acknowledgements

We thank Yasuko Sonoyama for the sample collection and technical assistance. This study was supported by a Grant-in Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (nos. 21390389 and 23659845).

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October 2014
Volume 34 Issue 4

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Oda T, Yamaguchi A, Yokoyama M, Shimizu K, Toyota K, Nikai T and Matsumoto KI: Plasma proteomic changes during hypothermic and normothermic cardiopulmonary bypass in aortic surgeries. Int J Mol Med 34: 947-956, 2014
APA
Oda, T., Yamaguchi, A., Yokoyama, M., Shimizu, K., Toyota, K., Nikai, T., & Matsumoto, K. (2014). Plasma proteomic changes during hypothermic and normothermic cardiopulmonary bypass in aortic surgeries. International Journal of Molecular Medicine, 34, 947-956. https://doi.org/10.3892/ijmm.2014.1855
MLA
Oda, T., Yamaguchi, A., Yokoyama, M., Shimizu, K., Toyota, K., Nikai, T., Matsumoto, K."Plasma proteomic changes during hypothermic and normothermic cardiopulmonary bypass in aortic surgeries". International Journal of Molecular Medicine 34.4 (2014): 947-956.
Chicago
Oda, T., Yamaguchi, A., Yokoyama, M., Shimizu, K., Toyota, K., Nikai, T., Matsumoto, K."Plasma proteomic changes during hypothermic and normothermic cardiopulmonary bypass in aortic surgeries". International Journal of Molecular Medicine 34, no. 4 (2014): 947-956. https://doi.org/10.3892/ijmm.2014.1855