Effect of Partial Oxygen Supply on Mitochondrial Electron Transport System During Complete Cardiac Ischemia

424

Effect of Partial Oxygen Supply on Mitochondria1 Electron Transport System During Complete Cardiac Ischemia Cuneyt Konuralp, M.D., F.I.C.S.," Saadettin Guner, Ph.D.,+ Ufuk Cakatay, Ph.D.,* Zeynep Konuralp, M.Sc.,§ Nihan Yapici, M.D.," Huseyin MaGika, M.D.," Hakki Aydogan, M.D.,* Serap Aykut-Aka, M.D.,* Cem Alhan, M.D.,* Mustafa Gultepe, Ph.D.,l and Ergin E. Eren, M.D."

ABSTRACT During complete ischemia w e assessed myocardial utilization of the small amount of oxygen available. We also determined whether blood cardioplegia has any ad- vantage over crystalloid cardioplegia in this setting.

Patients with preserved left ventricular myocardial function and wi thout anterolateral wal l infarct or aneurysm were included t o the study. Intermittent cold blood and crystalloid cardioplegia were used in 10 patients (group BC) and 9 patients (group CC), respectively. From myocardial biopsies, obtained before and after ischemia, complete electron transport system (ETS) enzyme activities (NDH, SDH, NCCR, SCCR, and COX) and lactate content were analyzed. Biochemical and hemodynamic analyses also were done. Myocardial and blood temperatures were monitored.

Ischemic t ime was longer in group CC (p < 0.05). There were no important differences in biochemical and hemodynamic variables between the t w o groups. In addition, there was no difference in NDH and SDH activities as wel l as COX/SCCR and COX/RS-NCCR ratios between the t w o groups before and after ischemia. After Ischemia, RS-NCCR in group CC and SCCR and COX activities in both groups were lower than the control. For all enzymes, ac- t iv i ty change ratios were no t different between groups. Myocardial lactate content was increased in both groups after ischemia. However, the increase in group BC was less (p < 0.01).

Based on our findings, w e believe that the superiority of b lood cardioplegia over crystal- lo id cardioplegia does no t depend on oxygen content, but on other factors such as buffering and free oxygen radical scavenger effects among others. However, with the warm and continuous blood cardioplegia technique, oxygen content might be more important. (J Card Surg 1999; 14:424-434)

"Istanbul Prof. Dr. Siyami Ersek Thoracic and Cardiovascular Surgery Center and Research Hospital, Department of Car- diovascular Surgery, Istanbul, +Karadeniz Technical University, Department of Chemistry, Trabzon; *University of Istanbul, Istanbul Faculty of Medicine, Biochemistry Central Laboratory, Istanbul, $University of Marmara, Department of Biochem- istry. Istanbul, lllstanbul Prof. Dr Siyami Ersek Thoracic and Cardiovascular Surgery Center and Research Hospital, Depart- ment of Anesthesiology and Reanimation, Istanbul, VHaydarpasa Gulhane Military Medical Academy, Department of Bio- chemistry, Istanbul, Turkey Address for correspondence: Cuneyt Konuralp, M D., F 1.C.S . Ayse Cavus Sokak, No: 7J6, Hurl Apt., Suadiye 81070 Istan- bul, Turkey Fax. 90 (2161 363 3642; e-mail: [email protected]

J CARD SURG 1 999; 14:424-434

KONURALP, ET AL. 425 ETS DURING CARDIAC ISCHEMIA

Oxidative phosphorylation is performed in the mitochondria and, in the presence of oxygen, the electrons and protons of the NADH + H+ and FADH, produced from the destruction of energy precursors are transferred to molecular oxygen. Electrochemical gradient energy, which is produced at that moment, is stored as high-energy phosphate compounds, and ATP is synthesized from ADP. This phenomenon is mediated through respiratory chain enzymes (RCE) or electron transport system (ETS) enzymes localized in the inner mitochondrial membrane (Table 1). This reaction chain is coupled with oxygen, and without oxygen ETS enzymes cannot show any activity.'

While the first four enzyme complexes ensure the transfer of hydrogen ions to oxygen, C-V ensures the synthesis of ATP from ADP by using electromechanical energy, which is produced in different stages of this process.

Transferring electrons to oxygen from NADH is performed in nine stages. However, the loss of free energy, which is enough for ATP synthesis, exists in only three stages. As a result, 3 mol ATP are synthesized from 1 mol NADH + H+.

We examined the activities of ETS enzymes in a complete ischemia environment in patients under- going coronary bypass. In this study, we investigated the effect of intermittent oxygen supply. For the complete ischemic condition, we used the patients who were given crystalloid cardioplegia (CC) for myocardial protection, and as the partial oxygen supply environment, we used the patients who were given blood cardioplegia (BC) for myocardial protection. We investigated mitochondrial ETS enzyme activities as markers of aerobic energy me-

~

TABLE 1 Electron Transport System Enzyme

Complexes*

Complex I (C-I): NADH ubiquinone reductase (NADH

Complex I1 (C-11): succinate ubiqurnone reductase

Complex 111 (C-Ill). ubiquinone cytochrome c reductase

Complex IV (C-IV): cytochrome c oxydase (cytochrome

Comolex V (C-V): ATP synthetase (F, F, ATP synthetase)

dehydrogenase; NDH)

(succinate dehydrogenase; SDH)

(cytochrome b-c, complex)

Oxydase, COX)

'NADH Cytochrome c Reductase (NCCR) and Succinate Cytochrome c Reductase (SCCR) enzyme complexes used in our study stand for C-l+C-Ill and C-ll+ C-Ill activities, respectively. '2

tabolism and myocardial lactate contents as a marker of anaerobic energy metabolism.

MATERIAL AND METHODS

Patients

Nineteen patients who underwent coronary artery bypass graft (CABG) surgery were included in this prospective randomized study. Patients who were < 70 years old and had no prior anteroseptal myocardial infarction, ventricular aneurysm, or hypertrophy and > 95% stenosis in the coronary artery system and diffuse lesions in the LAD coronary artery were included. Because mitochondrial ETS enzyme defects have been reported in patients with cardiomyopathy and congenital heart defects,," we excluded the heart valve and congenital heart disease cases. We, also, excluded patient who had < 40% left ventricular ejection fraction, acute MI, and emer- gency PTCA or angiographic complications.

Anesthesia and myocardial protection

Nineteen patients were placed in two groups. Myocardium was protected with intermittent cold blood cardioplegia in the first group (Group BC; n = 10) and with intermittent cold crystalloid cardioplegia in the second group (group CC; n = 9). Cardioplegia was given antegradly in both groups. The content of cardioplegia was analyzed (with al- pha-stat method) and recorded with Stat Profile 5 (Nova Biomedical, serial no: G=5C90100, Waltham, MA, USA) as ten parameters (Na+, K+, Caf2, CI-, HC0,-, Glucose, PO,, Oxygen content, hematocrit, and osmolarity). Temperature, administration pressure, administration rate, and total amount of cardioplegia also were recorded.

Premedication and anesthesia were the same in two groups. Anesthesia was induced with fentanyl (30-50 pg/kg) and diazepam (0.5 mg/kg). Succinyl cholin was given first and the Pancuro- nium as a myorelaxant. Anesthesia was maintained with fentanyl infusion and as needed with isoflurane inhalation.

Surgical techniques were the same in two groups. Nonpulsatile cardiopulmonary bypass at 29" C with a membrane oxygenator (Ultrox 1 Auecor, Plymouth, MA, USA) was used. Mannitol 200 mL, NaHCO, 60 mL, 1 mVkg heparin, and 100 mL 20% human albumin were added to the

426 KONURALP, ET AL. ETS DURING CARDIAC ISCHEMIA

J CARD SURG 1999; 14:424-434

prime solution of Ringer's lactate. Cardioplegia was given antegradely.

In group CC, only cold crystalloid cardioplegia (Plegisol, Abbott, Chicago, IL, USA) and in group BC, cold blood and crystalloid cardioplegia mix- ture (3/4 crystalloid + 1/4 oxygenated patient's blood) was used. In group BC 40 mmol KCI, and in group CC 10 mL NaHCO, and 100 mg (2%) li- docaine were added to the Plegisol.

We did not lower the cardiopulmonary bypass (CPB) flow rate below 2.5 Vmin. After completing each distal anastomosis, 25-30 mL cardioplegia was infused via each vein graft. After release of the cross-clamp, proximal anastomoses were performed, with partial aortic clamping. Rectal, esophageal (Shertan 700 probe, Boston, MA, USA), myocardial (Malinckrodt temperature sen- sor probe, 226 X 18 mm, no: 503-0310, St. Louis, MO, USA) and blood temperatures were monitored during the operation. Perioperative parameters recorded were operation time, CPB time, crossclamp time, graft number, fibrillation before crossclamp, defibrillation requirement after declamping, blood usage, and necessity of inotropic or mechanical support.

Hemodynamic and biochemical studies

Before anesthesia a five-lumen EF Thermoduli- tion catheter (Swan Ganz. Right ventricular ejection fraction/volumetric oximetry TD catheter, Bax- ter, Cath. No: 93A-754H, 7.5F, Irvine, CA, USA) was inserted via the internal jugular vein, and hemodynamic measurements were obtained for preoperative (before giving anesthesia), perioperative (before CPB and 15 minutes after terminating CPB), and postoperative (1 st, Znd, 12th, and 24th hours) periods by using Explorer homodynamic analyzer (Baxter, Edwards Critical Care, serial no: 17914, Irvine, CA, USA) Heart rate (HR), mean ar- terial pressure (MAP), central venous pressure (CVP), mean pulmonary artery pressure (MPAP), pulmonary capillary wedge pressure (PcWP), dou- ble product (DP), cardiac index (CI), systemic vascular resistance index (SVRI), pulmonary vascular resistance index (PVRI), left ventricular stroke work index (LVSWI), right ventricular stroke work index (RVSWI), left cardiac work index (LCWI), right cardiac work index (RCWI), right ventricular ejection fraction (RVEF), mixed venous oxygen sat- uration (SMVO,) and arteriovenous oxygen satura- tion difference (S,,O,) were recorded.

Serum CPK-MB, LDH, and GPT levels in arter- ial blood samples as preoperative, perioperative (1 0 minutes after declamping), and postoperative (0, 4th, 8th, 16th, 24th hours and Znd, 3rd, 7th day) periods were recorded by using a Hitachi 704 Automatic Analyzer (Boehringer Mannheim, serial no: 6231-1 5, Mannheim, Germany).

Preparation of myocardial tissue samples

We obtained myocardial tissue samples from the anterior aspect of the left ventricular septum from each patient twice (after the patients's oral and written consent and with the approval of the medical ethics committee of the hospital). The first biopsywas taken at the beginning of CPB to reflect the aerobic medium. The second biopsy was taken just before the release of the aortic clamp to show the endpoint of the ischemia (bulldog clamp on the IMA graft was released after the biopsy). Using 16- guage Sherwood biopsy needles (Sherwood Med- ical, Monoject ABC needle, 186 x 6 ; 1.2 mm x 150 mm Ref: 1 100-224466, Ballymena, Northern Ireland) 1-2 mg biopsy specimens were obtained. The specimen was placed in a sterile polypropy- lene vial tube immediately (within 10-1 5 seconds). After closing with parafilm, the tube placed into a Devar cup (- 180 C" environment) ensure cessa- tion of enzyme activity.

Analysis of mitochondrial RCE activities in myocardial tissue extracts

Four mitochondrial ETS enzyme activities (SDH, COX, SCCR, and Total [TI and Rotenone sensitive [RSI NCCR) and lactate contents were quantitavely analyzed in biopsy specimens. Also RS-NCCRI SCCR (reflecting NDH activity), COWSCCR, and COWRS-NCCR rates were ~alculated.~

Three measurements of enzyme and lactate content were performed for each specimen and averaged. The total amount of biopsied myocardium was insufficient for myocardial phosphate compounds determination.

The quantity of the substrate (as micromole) changed by enzyme in 1 minute was determined as one enzyme activity unit (1 unit = 1 pmol substrate/min). However, to calculate the specific enzyme activity (enzyme activity unit for each 1 mg protein of myocardium), this value was divided by the amount of biopsy protein. We used that parameter in the statistical analyzes.

J CARD SURG 1999;14:424-434


Tissue homogenates were prepared from each biopsy specimen as described pre~iously.~,6-8 The collected supernatants were pooled and used for the assay of the enzyme activities and lactate content. Protein concentrations of the myocardial extracts were determined by the Lowry methodg (using Shimadz, UV-2100, UV-Visible Recording Spectrophotometer, Tokyo, Japan).

Mitochondria1 respiratory chain enzyme activities were spectrophotometrically measured in mitochondrial homogenates with PU 8740 UV, VIS Scanning Spectrophotometer (Philips, serial no: RS 232, Taipei, Taiwan). SDH,l0*l1 NCCR,5,10*12-14 SCCR.7.10.12.15 and COX"J12-14 activities were determined as previously described.

For NDH enzyme activity, RS-NCCR/SCCR (C- l+C-lll)/(C-ll+C-lll) was used. This ratio is accepted for evaluation for NDH activity qualitatively.12

Analysis of myocardial lactate content

Tissue homogenates from prepared biopsy specimens were placed into tubes prepared with special fluor and nitrite. Lactate content was measured with Analox Lactate Analyzer (Mac Lennon Supplies Ltd., Model no: LM5, serial no: 030, Lon- don, UK). Machine values shown as mmol/L were converted to nmol/kg tissue.

Postoperative period

Postoperative parameters evaluated were du- ration of intubation time in ICU, need for inotropic or mechanical support, reoperation for bleeding, rhythm and/or conduction problem, amount of transfused blood, perioperative MI, postoperative MI, and postoperative mortality.

Statistical analysis

Descriptive statistics values are expressed as mean 5 standard deviation (SD). The geometric mean (GM) is reported for the values analyzed from geometric scale. Student t-test (if the variances are equal) and alternative t-test (if the variances are not equal) were used for normally distributed data. Mann-Whitney U-test was used for categorial data and data that do not show a normal distribution. The one sample t-test was used for blood differences from zero. Data obtained for the mitochondria1 enzyme activities were converted to a logarithmic (1 0- base) scale to provide a normal distribution. For in-

group comparison (before and after ischemia), paired t-test (if the variances are equal), or alternative t-test (if the variances are not equal) was used. For intergroup comparison (chance ratios for both groups), unpaired Student t-test was used. My- ocardial lactate levels were analyzed with paired t- (in-group) and unpaired t- (intergroup) tests in arith- metic scale. For multiple biochemical analyses (non-Gaussian distribution) Friedman repeated measures test (pretest) and Dunn's multiple comparisons test (posttest) were used. For hemodynamic parameters (Gaussian distribution) repeated measured analysis of variance (ANOVA) and Stu- dent-Newman-Keuls Multiple Comparisons test (if the variances are equal) or Tukey-Kramer Multiple Comparisons test (if the variances are not equal) were used. A difference < 0.05 p value (two-tailed) was considered significant.

RESULTS

Preoperative patient characteristics were similar in both groups (Table 2). Perioperative findings are summarized in Table 3. Operative time, CPB time, number of grafts, development of ventricular fibrillation before cross-clamp, defibrillation after cross-clamp, and blood use were not different in the two groups. But cross-clamp time was higher in group CC (61.6% 13.4 min vs. 52.1 2 10.5 min; p < 0.05).

IMA was used in all patients. In one patient in group BC and two patients in group CC, LAD could not be bypassed (small diameter) and IMA was anastomosed to the diagonal artery. Also, diagonal artery (two patients), circumflex artery (one patient), and right coronary artery (one patient) were not suitable for revascularization in group BC. Among minimum rectal, esophageal, blood, and myocardial temperature compared for the two groups, only esophageal temperature was different (27.2 2 0.87 C" in BC vs. 28.49 5 0.97 C" in CC; p < 0.01 1. There was no difference in myocardial temperatures (Table 4). Cardiople- gia characteristics are summarized in Table 5. PO, (p < O.Ol), 0, content (p < 0.0001), and osmolarity (p < 0,05) were higher in BC. Na+ (p < 0.05), K+ (p < 0.05) and HC03- (p < 0.001) were also higher in BC. pH, Ca+,, CI- were not different in two groups. Administration features of cardioplegia are shown in Table 6. Temperature of BC was higher than CC (9.0 -+ 1.2 c" vs. 7.2 -+ 0.8 C"; p < 0.05).


J CARD SURG 1999:14:424-434

~~~

TABLE 2 Preoperative Patient Characteristics

Group BC (n = 10) Group CC In = 9) p Value

Age (year) (range)

Female gender (96) Body surface area (m*) Number of diseased vessels Left main stenosis (YO) Hypertension (%) Diabetes (%) Unstable angina (%) Preexisting MI (YO) NYHA Class (l/ll/lll) Left ventricular ejection fraction (96)"

53.1 t- 9.0 (42-69) 1 (10.0)

1.86 2 0.20 3.4 2 0 5 1 (10.0) 3 (30.0) 4 (40.0) 3 (30.0) 6 (60 0) 31512

54.0 t 6.6

59.8 2 10.1 (44-69) l( 1 1 . 1 )

1.93 2 0.19 3.4 t 0.5 l(l l . l ) 6 (66.7) l( 1 1 . 1 ) 3 (33 3) 6 (66.7) 01811

55.3 t 7.4

NS NS NS NS NS NS NS NS NS NS NS

"By angiography.

TABLE 3 Perioperative Patient Characteristics

Group BC (n = 10) Group CC (n = 9) p Value

Operation time (mid CPB time (min) Cross-clamp time (min) Number of grafts VF before aortic clamp (%) Defibrillation after declamping (%) Blood transfusion (YO) lnotrotx or mechanic sumort (%)

238 5 2 27 9 96.2 t 19.4 52.1 t- 10.5 2 9 2 0.4 3 (30.0) 4 (40.0) 0 (0.0) 0 (0 0)

276.1 2 47.5 137.6 2 54 5 61 6 2 13.4 3.3 t 0.5 2 (22.2) 3 (33.3) 3 (33.3) 1 1 1 1 . 1 )

NS NS

< 0.05 NS NS NS NS NS

TABLE 4 Biopsy Temperature Conditions

p value+

Preischemic Postischemic Group BC Group CC

Temperature (C") Preischemic Postischemic Preischemic Postischemic (BC vs. CC) (BC vs. CC)

Myocardium 34 40 t- 0 34 26 34 t- 0.99 34.99 2 1.31 26.10 t 4.34 NS NS Blood 31.55 t'1.14 33.61 2 1.96 3442 t- 134 33.16 -C 2.62 < 0.05 NS

'Pre- versus postischemic changes in temperature reduction were not significantly different between group BB and CC (for myocardium and blood).

Postoperative patient characteristics did not differ between the two groups (Table 7). But shown). postoperative blood using was lower in group BC

tween any hemodynamic parameter (data not

Myocardial mitochondria1 ETS enzyme activities

(0.4 2 0.7 units vs. 1.6 ? 1.1 units; p < 0.05). Serum CPK-MB, LDH, and GPT levels were not

different globally in pre-, peri-, and postoperative periods and increased over time in both groups (data not shown). There was no difference be-

Because of the large variations of the RCEs in the populations and to minimize possible devia-

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TABLE 5 Properties of Cardioplegic Solutions

Group BC Group CC p Value

Hematocrit (YO) PO, (mmHg) c0, (mUdL) PH Na+ (mmol/L) K+ (mmol/L) Ca+2 (mmol/L) CI- (mmol/L) HC0,- (mmol/L) Glucose (mmol/L) Osmolarity (mOsm/L)

9.80 t 7.24 289 43 2 51.98

4.86 t 3.55 7.538 2 0.059

130.24 t 8.76 26.46 t 7.66 2.10 f 0.51

174.05 t 25.37 14.51 t 3.50 1 1 . 4 3 t 9 11

252.40 t 16.76

0 221.82 t 38.02

0.70 2 0 11 7.621 t 0.080

118.70 t 3.98 17.21 t 1 36

1.84 2 0.57 177.86 t 31.53

8.73 t 2.20 0

231 33 t 6.00

<o 01 CO.01 <0.0001 C0.05

<.001 C0.05

NS NS

<0.001 <o 001 <0.001

TABLE 6 Cardioplegia Administration Characteristics

~

Group BC Group CC p value

Temperature (C") 9.0 t 1.2 7.2 ? 0.8 <o 01 Pressure (mmHg) 137.0 t 26 0 121.1 f 21 5 NS Rate of infusion (mUmin) 212.4 f 10.9 215.6 f 7 9 NS Total volume (mL) 560.0 t 31.6 594.4 t 76.8 NS

TABLE 7 Postoperative Patient Characteristics

Group BC Group CC p Value

Time to extubation (h) 14.4 t 3.2 16.2 t 9 9 NS ICU time (day) 2.0 t 0.0 2 8 5 2 3 NS Blood transfusion (u) Penoperative MI (%) 1 (10.0) 0 (0.0) NS Ventricular arrhythmias (yo) 1 (10.0) 0 (0.0) NS

<0.05 0.4 t 0.7 1 6 2 1.1

tions in different conditions for each patient, we chose not to use arithmetical differences to compare pre- and postischemic changes between groups. Rather, we used changing ratios (percent activity compared to the preischemic period) for statistical analysis.

All enzyme activities decreased numerically at the end of ischemia in both groups (Table 8). How- ever for BC, only. COX decreased significantly (1 10.31 2 82.32 units/mg [GM: 86.15 units/mgl vs. 57.64 t 45.64 units/mg [GM: 43.00 units/mgl; p < 0.05) and for CC, COX (1 71.45 t 169.23 units/mg [GM: 124/18 units/mgl vs. 86.1 5 t 57.09 units/mg [GM: 67.61 unitslmgl; p < 0.051, RS-NCCR (94.68

2 91.89 units/mg [GM: 57.98 units/mgl vs. 35.42 t 40.88 units/mg [GM: 20.38 units/mg]; p < 0.05), and SCCR (1 74.58 I+- 123.64 units/mg [GM: 138.98 units/mg[ vs. 112.14 t 121.68 units/mg [GM: 67.81 units/mgl; p < 0.01) decreased significantly. However, activity change ratios between the groups were not significantly different for these enzymes. COWSCCR and COWRS- NCCR ratios were not significantly changed in both groups. Myocardial lactate content (Table 8) significantly increased in group BC (26.42 ? 16.59 nmol/kg vs. 34.45 2 16.71 nmol/kg; p < 0.05) and in group CC (22.42 t 14.58 nmol/kg vs. 61.45 '-+ 39.74 nmol/kg; p < 0.01). There was a

430 KONURALP. ET AL. ETS DURING CARDIAC ISCHEMIA

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TABLE 8 The Respiratory Chain Enzyme Activities and Lactate Contents of Myocardial Tissues in Blood

and Crystalloid Groups Before and After the Complete Ischemia Blood Cardioplegia Group Crystalloid Cardioplegia Group

Parameters Preischemia Postischemia p Value Preischemia Postichemia p Value

SDH

(pmollm in/mg) COX"

(pmol/min/mg) RS-NCCR"

(pmol/min/mg) SCCR"

(pmoi/min/mg) NDHt COWSCCR COWRS-NCCR

Lactate' qmolkg

71.57247.78

(57.38)' 110.31 282.32

(86.15)' 71 10238.92

(34.81 ) 5 112882116.18

(77.83)' 0.7720.46 1.542 1.49 2.7222.79

Lactate' 26.422 16 59

46 83223.35

(41.34)' 57.64245.64

(43.00)' 42.82228.88

(32.49)' 63.28255.55

(47.72)' 0.5720.33 1.1 120.77 1.5720.96

34.45?16.71

NS

<0.05

NS

NS

NS NS NS

<0.05

61.33268.19

(38.57)' 171.452 169.23

(1 24.18)' 94.68291 89

(57.98)' 175.582123.64

(1 38.98)' 0.7220.84 1.4320.30 4.5423.74

22.422 14.58

45.08231.06

(36.1 6)' 86.1 5 2 57.09

(67.61 )' 35.42240.88

(20.38)' 112 142121.68

(67.81 )' 0.5720.67 1.0820.64 3.7422.91

61.45239.74

NS

<0.05

<0.05

<0.01

NS NS NS

<0.01

"activity change ratios between groups are not significant; +was taken as a ratio of RS-NCWSCCR; *change ratios between groups are significant (p < 0 05); 4 express geometric mean (GM)

significant difference in lactate change ratios between groups (p < 0.01). The increase in group CC was more than that of group BC.

DISCUSSION

The aim of this study was to test whether the heart could utilize oxygen for gaining energy in complete ischemia when limited oxygen was sup- plied for short periods. For this purpose, we used as an experimental environment, procedures that are routinely applied in open heart surgery. To sum- marize our results: (1) in group BC, anaerobic path- way was used less than in group CC, (2) although COX activity decreased after ischemia in group BC, and COX, SCCR, and RS-NCCR activities decreased after ischemia in group CC, there was no difference in rate of change between groups.

The analysis of RCE activity implies that the mitochondrial ETS enzyme activities is impaired or electron transport is blocked at the same points and electrons coming from substrate cannot reach the molecular oxygen in both groups. This implies that cardiomyocytes were not able to use oxygen as an energy source; but for some other reason, total energy gain or maintenance is more satisfactory in the BC group. It has been fact noted in previous reports that the myocardial ATP

and creatine phosphate (CP) stores were maintained better with BC.lgZ1

While the number of diseased vessels was similar in two groups preoperatively, the number of grafts was higher in group CC. Therefore, the ischemic time for this group was longer. This factor can affect the ETS enzyme activities and lactate content. However, even with less ischemia time, we could not demonstrate better aerobic metabolic performance in group BC. This is an important paradox. Therefore, it is important to analyze the relative contribution of each parameter to the inefficient use of oxygen during the total cross-clamp period.

Oxygen content

Silverman22 had proposed that the erythrocytes in BC were required for optimal myocardial protection, but the majority of the protective effect was related to the buffering and free radical scavenger capacity of the blood rather than oxygen-carrying capacity.

de Wit23 showed that excessive oxygenation of cardioplegia did not improve metabolic or functional recovery. We believe there was adequate oxygen in our BC solution. The reason for inefficient oxygen utilization must lie elsewhere.

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KONURALP. ET AL. 431 ETS DURING CARDIAC ISCHEMIA

The majority of studies that compare nonoxygenated crystalloid to blood (oxygenated) cardioplegia have showed that BC is more advantageous than CC for protecting m y ~ c a r d i u m . ~ ~ ~ ~ , ~ ~ ~ ~ How- ever, other articles report no difference between BC and CC.2S29The advocates of BC generally report greater myocardial oxygen consumption in BC than CC. They also have reported better preservation of myocardial energy stores. How- ever, there is no proof that the oxygen was used for gaining energy. We believe these investiga- tors have missed the myoglobin component in their discussion. In fact, most oxygen that can reach the mitochondria is bound to myoglobin (Mb) rather than the c y t o ~ o l e . ~ ~ As opposed to Hb, Mb has a stronger affinity for oxygen and re- leases it less easily; and that affinity increases with lower temperatures. Thus, in these conditions, mitochondria probably do not use all the oxygen for oxidative phosphorylation and thus high oxygen consumption does not reflect oxidative phosphorylation.

Other cellular and acellular components of the blood

We believe other components of the blood are responsible for the superiority of the BC. llies et al.31 used cardioplegia with high hematocrit, low hematocrit, and plasma (without erythrocyte) in dogs. Their study revealed that the availability of

oxygen was not different among the groups de- spite different oxygen content of the solutions. They suggested that the advantageous effects of BC did not depend on the oxygen-carrying capacity of the erythrocytes. Possibly, it may not even depend on the buffer effect. Although the buffering capacity of whole blood is greater than plasma, myocardial protection was the same in three groups. Therefore, some other factors in the plasma may be more important. That study also showed that, contrary to the general belief, the leukocyte and thrombocyte components of the blood may not be harmful to the myocardium.

Temperature

Whether the myocarial temperature influenced the analysis for enzyme activities must be clari- fied. As a rule, A 1 c" drop of tissue temperature results in about 10% decrease of enzyme activity. If we consider that the difference between the two biopsy conditions was approximately 8 c" (Table 41, we estimate that the enzyme activities would be different. However, because there was no statistical difference in myocardial temperature change between groups, it is suggested that enzyme activities were affected from temperature drop to the same degree. Therefore, the statistical relationship of the enzyme activities must not be due to chance. Does the temperature of the BC affect oxygen utilization? Other clinical in-

TABLE 9 Comparative Studies with Different Cardioplegia for

Myocardial Protection and Energy Metabolism

Author Comparison Result

DeBoer, et aL4' 95% oxygenated and nonoxygenated perfusates at different (37 c", 30 c", 20 c". and 13 C") temperatures (rat heart) maintained longer

30Y0, 6OY0, and 90% oxygenated CC (rat heart)

With higher oxygen ratio and lower temperature aerobic metabolism is

Increasing oxygenation to 60% associated de Wit, et aLZ3 with higher myocardial ATP and CP content However, increasing to 9090 did not make any difference

Higher myocardial ATP and CP levels with BC after ischemia and during reperfusion

No difference in myocardial ATP content. Myocardial oxygen consumption is higher in group 4

Lower perop MI rate, myocardial lactate content and better myocardial ATP store in BC group

with higher volume BC)

Engelman, et aL4*

Heitmiller, et

Fremes, et aL1*

Engelman, et a1 43

CC, oxygenated CC, and BC (pig heart)

CC with low Calcium (I), high Calcium (2), 1090 Hct (3). and 30% Hct (4) (canine heart)

BC (1/2 ratio) and CC (human trial)

Normal volume BC and CC vs. higher volume BC Higher volume shows better protection (best and CC (human trial)


J CARD SURG 1999;14:424-434

vestigators report similar32 or better33-35 results with warm BC. It is known that the advantages of the BC are'less with colder temperature^,^^,^^ as the Hb dissociation curve shifts to the left and enzymes do not exhibit optimal activity.

Therefore, it has been ~ t a t e d ~ * , ~ ~ that fluosol cardioplegia is more advantageous than BC (fluosol has a low viscosity and can bind and release the oxygen linearly at all temperatures). We believe that with the release of impaired oxygen and its others components, warm (normothermic) BC should be more beneficial for myocardial protection.

Administration pressure and rate of infusion

In general practice, the typical administration pressure must be between 130-140 mmHg for the antegrade method (BC was delivered on 137 2 26.3 mmHg pressure in our study). However, Buckberg40 suggests > 250 mmHg initial pressure for BCs (at least until electromechanic activity stops). It can be logical to assume that oxygen utilization would be better with higher perfusion pressure.

Timing of delivery+

In our study cardioplegia was given at the beginning of cardiac arrest and just after each distal anastomosis was completed. Cardioplegia solution remained in the coronary vascular system without immediate washout or support. Continu- ous cardioplegia ensures a different physiologic environment and perhaps with this technique, oxygen utilization could be more satisfactory.

CONCLUSION

Due to a number of factors in BC, known or un- known, BC or the cellular environment that is prepared by this perfusate, ATP synthesis is possibly superior to CC. These factors could be the buffering system or free oxygen radical substrates or other at the plasma enzymes. Perhaps an alternative energy-producing system that does not use oxygen is mobilized. However, this system will be limited by the time of the ischemia also,

*Other pertinent comparative studies are summarized at Table 9

and extended time factor will lead to irreversible cell damage.

For that reason, optimization of BC to ensure better oxygen utilization is the crucial p ~ i n t . ~ ~ , ~ ~ Some substrates (glucose, glutamate, aspartate, co-enzyme Q,o) can be added to increase the benefit. However, in the future, ETS systems can be analyzed on warm versus cold and continuous versus intermittent BC groups to understand the mechanism. ATP synthetase (C-V) enzyme and myocardial oxi-myoglobin could be other important factors to examine.

Acknowledgment: We wish to thank our consultant statisti- cian Dr. Yild/rAtakurt (Assatant professor of Biostatistics, Uni- versity of Ankara, Faculty of Medicine, Ankara, Turkey) for valuable contributions to the analysis of the results.

APPENDIX: SELECTED ABBREVIATIONS AND ACRONYMS

BC = blood cardioplegia

CC = crystalloid cardioplegia COX = cytochrome c oxidase CPB = cardiopulmonary bypass ETS = electron transport system IMA = internal mammary artery

NDH = NADH dehydrogenase RCE = respiratory chain enzymes

CABG = coronary artery bypass graft surgery

NCCR = NADH cytochrome c reductase

RS-NCCR = rotenone sensitive NADH cytochrome c reductase

SCCR = succinate cytochrome c reductase

T-NCCR = total NADH cytochrome c reductase SDH = succinate dehydrogenase

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