Prevention of Increased Hemoglobin-Oxygen Affinity in Open-Heart Operations with Inosine-Phosphate* Pyruvate Solution Stanley Giannelli, Jr., M.D., J. Patrick McKenna, M.D., Joseph M. Bordiuk, M.D., Leonard D. Miller, M.D., and Carl R. Jerome, Ph.D.
ABSTRACT In a control group of 32 patients under-
going open-heart operation, erythrocyte 2,3diphosphoglycerate (2,3-DPG) declined progressively during the course of perfusion from a prebypass mean of 17.00 to 11.29 p M per gram of hemoglobin at the end of bypass. The decrease was greater than that attributable merely to dilution of the patients’ cells with the 2,3-DPG-deficient donor cells used to prime the pump oxygenator circuit. Administration of 300 mg of allopurinol, to prevent the conversion of inosine to uric acid, every 8 hours during the 24 hours prior to operation in 11patients did not prevent the 2,3-DPG decrease during heart-lung bypass: prebypass, 18.31; postbypass, 13.56 pMlgm Hgb. The mean Ps0 for both these groups combined decreased from a prebypass mean of 25.7 to a postbypass level of 21.9 torr. A solution of 0.1 M inosine, 0.1 M pyruvate, and 0.066 M phosphate (IPP) in a dosage of 7.5 ml per kilogram of body weight prevented the 2,3-DPG decrease: prebypass, 15.74; postbypass, 14.85. Administration of 15 ml per kilogram of IPP in 15 patients preserved 2,3-DPG: prebypass, 18.09; postbypass, 18.52. The Pj0 remained unchanged in this last group. The method of providing for myocardial oxygen requirements during bypass was not standardized, and therefore the protective effect of IPP against ischemic damage in patients undergoing aortic valve replacement or myocardial revascularization could not be evaluated. No deleterious effects of IPP were noted. From the Departments of Surgery, Pathology, and Pediatrics, St. Vincent’s Hospital and Medical Center, New York, NY, and the Department of Surgery, University of Pennsylvania Medical School and Hospital of the University of Pennsylvania, Philadelphia, PA. Supported in part by Grant no. 5-R01-HL-10781-08from the Department of Health, Education, and Welfare. Accepted for publication Nov 6 , 1975. Address reprint requests to Dr. Giannelli, Division of Cardiac Surgery, St. Vincent’s Hospital and Medical Center of New York, 153 W 11th St, New York, NY 10011.
386
We initially reported, in 1971, that red cell 2,3diphosphoglycerate (2,3-DPG) progressively declines during heart-lung bypass [7]. At the conclusion of perfusion 2,3-DPG levels were about half the prebypassvalues; the extent of the decrease was greater than could be ascribed merely to dilution of the patients’ cells with the 2,3-DPG-deficient blood bank donor cells in the pump prime. The patients in this initial series were perfused with either a rotating disc or a membrane oxygenator [ll], and 2,3-DPG phosphorus was determined by a column chromatographic extraction method [20]. We then studied another series of patients, perfused with a bubble oxygenator, in whom 2,3-DPG was determined by an enzymatic method [15]. During bypass 2,3-DPG progressively decreased [161, and while the amount of the decline was less than we had previously reported [7], it was still greater than could be attributed to dilution by the donor red cells. There was an associated increase in the hemoglobin affinity for oxygen: the average Pjo in 10 patients prior to bypass was 28.7 torr, and at the conclusion of bypass it was 24.4 torr. These latter data were similar to those recently published by Jesch and associates [14], who showed that 2,3DPG declined from a preoperative mean of 13.6 p M per gram of hemoglobin to 10.9 at the completion of operation. The respective Pso values were 27.0 and 24.8 torr. An increased hemoglobin affinity for oxygen theoretically should reduce the maximum tissue oxygen delivery by decreasing the oxygen diffusion pressure gradient between the erythrocytes in the capillaries and the cellular mitochondria. The myocardium would be expected to be particularly vulnerable to decreased Pso, because at rest that organ already extracts oxygen from the blood at a nearly maximal rate. Unlike skeletal
387 Giannelli et al: Prevention of Increased Hemoglobin-Oxygen Affinity
muscle, which obtains additional oxygen during exercise by a combination of increased blood flow and greater oxygen extraction, myocardium is almost solely dependent upon higher blood flow during a period of increased oxygen demand. There are two groups of cardiac surgical patients in whom the balance between oxygen supply and demand is compromised: those with coronary artery disease undergoing myocardial revascularization and those with left ventricular hypertrophy who require aortic valve replacement. Postoperative myocardial ischemic damage has been reported to occur in a significant percentage of patients in each of these groups [lo, 211. If a method were available to prevent P50 from decreasing in patients having open-heart operations, the frequency of myocardial damage possibly could be reduced. This decrease in ischemic damage would provide quantitative documentation of the clinical importance of P50 to myocardial oxygen delivery. Akerblom and associates [l]reported in 1968 that the addition of inosine to 2,3-DPG-deficient blood partially restores the 2,3-DPG level. Fig-
ure 1presents those chemical reactions that are probably responsible for 2,3-DPG synthesis when inosine is added to blood. Oski and coworkers [181 found that the administration of phosphate and pyruvate in addition to inosine (IPP) causes a greater elevation of 2,3-DPG when added to blood in vitro than does inosine alone. Phosphate is required for the formation of ribose-1-phosphate, and the pyruvate serves to replenish nicotinamide-adenine dinucleotide from its reduced form. IPP was safely administered to monkeys and, as was true for the in vitro studies, this combination of substances effected a greater rise in 2,3-DPG than did inosine alone [24]. The solution then was administered to normal human volunteers, as reported by Miller and the other members of Oski’s group [17]. In humans, IPP was also more effective than inosine alone in raising 2,3-DPG. Optimum 2,3DPG elevations were obtained by two separate administrations of 7.5 mllkg of 0.1 M inosine, 0.05 M phosphate in buffer solution, and 0.1 M pyruvate, given rapidly 1 hour apart. The average rise in 2,3-DPG content was 1,600 p M per milliliter of red cells; the mean Pso increase was
Fig I. The route by which inosine enters into the erythrocyte metabolic pathzvays to produce
2,3-DPG.
INOSINE + P i 4
(Purine nucleoside phosphorylase)
(Phosphoribmutase)
-I
HYPOXANTH INE.&WR
RIBOSE-5-P (Transketol ase + transaldolase)
ATP ADP
(Aldolase)
1
G-3-P-OIHYDROXYACETONE
t
NAD NAOH
(G-3-PD)
ATP ADPAPGK
/
I
ALLOPUR INOL
&RIBOSE-I -P
(DPGP)
IC ACID
388 The Annals of Thoracic Surgery Vol 21 No 5 May 1976
3.5 torr. The peak effect was reached 3 to 6 hours after completion of the infusion, and 75% of the elevation was present 24 hours after administration. There are several possible deleterious effects of IPP administration, the most obvious being the formation of uric acid. Miller’s patients were given 300 mg of allopurinol daily for two days prior to IPP administration, and significant rises in uric acid did not occur [17]. Hypoxanthine and inosine are vasodilators, and 2 of the 16 human subjects who received the two 7.5 mllkg doses of IPP experienced transient mild blood pressure depression. Decreased peripheral vascular resistance is common during the early stages of heart-lung bypass, and therefore these vasodilator effects might be of greater significance than in the intact individual. Caen and associates [81 showed that both inosine and hypoxanthine inhibit platelet aggregation induced by adenosine diphosphate (ADP). They proposed that these substances compete with adenosine for a receptor site on the platelet membrane. Platelet counts regularly decrease during the course of heart-lung bypass [91, and those platelets that remain in the circulation have been found to have decreased agglutination in response to ADP [6]. Thus, IPP administration could further impair platelet function and might contribute to clinically significant bleeding after perfusion.
Clinical Material and Methods Serial 2,3-DPG measurements were made during and after heart-lung bypass in four groups of patients operated upon because of congenital or rheumatic heart disease or coronary arteriosclerosis. Only those patients who were perfused longer than 60 minutes are included in this paper. Group 1, serving as controls, was composed of 29 adults and 3 children each 7 years old. The 11adults in Group 2 received 300 mg of allopurinol at 8-hour intervals beginning the day prior to operation; the last dose was administered with a sip of water 1hour prior to going to the operating room. The 13 adults in Group 3 received allopurinol plus 7.5 mllkg of IPP solution. The IPP was infused over approximately 20 minutes beginning about 15 minutes
after the onset of heart-lung bypass. The 15 adults in Group 4 were managed similarly to those in Group 3 but received 15 mllkg of IPP; administration was begun at the same time and continued for 30 to 40 minutes. Evaluation of the incidence of postoperative ischemic damage in the four groups was not considered worthwhile for two reasons: the method of protecting the myocardium from ischemic damage during the period of heart-lung bypass was not constant in those patients operated upon for coronary artery disease and aortic valve disease, and myocardial-specific creatine phosphokinase isoenzyme determinations were not available to us during most of the period covered by this study.
Schedule of PS0 and 2,3-DPG Determinations In all groups, serial 2,3-DPG measurements were made during bypass. Prebypass and priming volume 2,3-DPG levels were obtained in all patients, and subsequent measurements usually were made at 1 hour and at the conclusion of bypass. In a smaller number of these patients, hourly determinations were made for up to 3 hours following bypass. In a total of 19 patients-5 in Group 1, 5 in Group 2, and 9 in Group 4-2,3-DPG measurements were also made on the day prior to operation. For all but 3 patients, in whom hematocrit values were not available, a 2,3-DPG ”dilution” value was calculated; this was an estimate of the 2,3-DPG level that would have resulted from simple admixture of the patients’ cells and the 2,3-DPG-deficient cells in the priming volume. The volume of the patients’ red cells was determined from their hematocrit and their estimated blood volume. It was assumed in all patients that blood volume represented 8% of body weight. Determination of PjOwas done prior to bypass and at the end of bypass in 5 patients in Group 1, 10 patients in Group 2 , l patient in Group 3, and all 15 patients in Group 4. In 5 patients in Group 1, 5 in Group 2, and 9 in Group 4, PjO measurements were also made on the day prior to bypass. All the data for 2,3-DPG and Pjo in the four groups are given in Tables 1 through 4. Variable amounts of additional bank blood were administered during the first 3 hours following bypass, usually 2 to 3 units. Correlation
389 Giannelli et al: Prevention of Increased Hemoglobin-Oxygen Affinity
Table 1 . Red Blood Cell 2,3-Diphosphoglycerate (pMlgrn Hgb) and Ps0 (tory) in the Control Group (Group 1 ) Day Prior to Operation Patient No.
2,3DPG
PjO
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
14.48 13.32 17.73 12.24 13.18
24.3 22.0 24.7 15.2 22.0
Mean SD
Prebypass 2,3DPG
PS0
18.15 13.69 17.46 14.04 14.37 18.20 13.40 18.50 14.80 25.60 17.50 14.78 16.16 15.96 19.44 22.30 17.48 20.26 15.11 15.48 23.22 16.58 11.80 18.78 16.55 16.94 17.40 12.70 20.67 20.84 12.52 18.38
24.7 24.9 27.7 19.3 22.7
17.00 3.20
23.9 2.8
Dilution 2,3DPG 14.92 11.68 12.69 10.86 11.19 10.45 11.75 14.13 12.16 17.60 11.50 10.41 12.15 9.33 14.87 17.08 16.09 12.61 13.40 19.52 14.86 10.79 12.01 14.78 14.47 13.29 11.39 18.05 17.16 11.78 13.90 13.45 2.52
2,3-DPG during Bypass 1Hr
End
at End of Bypass
14.57 11.34 13.60 12.22 10.11 7.50 10.40 9.05 9.10 7.78 7.50 10.54
13.93 10.52 11.69 8.68 12.37
21.2 19.9 21.1 20.0 18.7
2,3-DPG in Postbypass Period
p50
2Hr
3Hr
13.64 10.74 14.05 9.13
11.65 13.95
12.11
6.60
14.62 10.18 6.33 13.83 13.09 12.90 10.62 13.20 17.42 10.36 11.67
12.10 10.43 10.50 9.88 10.30 10.12 13.48 8.82 11.98 11.02 11.30 10.90 12.35 11.28 15.26 14.34 10.71 7.38 11.32 12.14 12.42 9.01 13.67 14.49 10.83 12.59
11.26 2.52
11.9 2.05
11.02 12.82 10.90
1Hr
10.50 9.55 5.30
7.86 8.30
4.20 7.12
8.90 9.42 10.66
17.12 17.12 8.72 11.29 12.11 13.77
13.68 11.30 8.50
8.54
14.57 8.72
9.93
9.37
20.2 0.9
8.40 13.30 14.61 11.65
11.92 12.66
10.87 2.50
10.98 1.93
12.48
Duration of Bypass 2'29" 1'29" 1'30" 2'16" 2'30" 1'58" 1'35" 1'40" 3 '12" 3'29" 2'29" 1'40" 1'40 1'39" 1'39" 1' 3'24" 1' 1'11" 1'46" 1'53" 3'52" 3'7" 1'22" 2'16" 1'35" 1'19" 2'21" 1'23" 2'57" 1'35" 2'21"
10.17 3.54
Conduct of the Operations Nitrous oxide and morphine anesthesia and a Q-100 Temptrol bubble oxygenator* were employed for each operation. Silastic rubber tubing was used throughout the extracorporeal circuit. Prior to introduction of the priming volume into the extracorporeal circuit, 200 units of heparin was added to each of the 4 units of blood and to each 500 ml of the 2,000 ml of saline that
comprised the prime. The blood had been drawn 3 to 10 days prior to use and was stored in either acid-citrate-dextrose or citratephosphate-dextrose solution. The blood was passed through a Swank Dacron-wool CA 100 microfiltert before introduction into the pump oxygenator. The citrate in the blood was neutralized with 2.0 gm of calcium gluconate. Heparin, 200 units per kilogram, was administered intravenously between 5 and 10 minutes prior to bypass, and the initial patient dose was repeated each hour during bypass. When necessary, the perfusate volume was augmented dur-
'Bentley Laboratories, Inc, Irvine, CA
tPioneer Filters, Inc, Beaverton, OR
was not made between 2,3-DPG levels in the hours after bypass and the amount of transfused bank blood.
390 The Annals of Thoracic Surgery Vol 21 No 5 May 1976
Table 2. Red Blood Cell 2,3-Diphosphoglycerate (pMlgm Hgb) and Allopurinol (Group 2) Day Prior to Operation Patient
No.
2,3DPG
2,3DPG
P,,
1 2 3 4 5 6 7 8 9 10 11
19.33 24.59 12.40
23.2 30.7 25.3
20.58 14.82
28.1 23.8
Mean SD
2,3-DPG during Bypass
Prebypass
Pso
(torr) in the Patients Receiving
P5, at
Dilution 2,3DPG
1 Hr
End
15.31 18.39 15.89 12.83 16.70 16.19 14.79 10.87 14.32 13.86 11.05
14.68 18.04 15.36 12.71 14.40 16.10 11.43 10.29 15.57 10.57 10.00
23.1 25.3 25.3 24.8 21.7 21.6 25.3 23.5
14.56 2.21
13.56 2.58
22.7 2.8
18.02 18.55 19.59 16.97 19.92 21.06 23.38 13.24 18.43 17.17 15.08
26.6 28.4 28.9 30.7 22.5 22.8 32.8 26.0 24.3 23.0
14.57 18.85 15.94 13.98 17.68 17.13 17.59 11.43 15.23 14.47 9.57
18.31 2.64
26.6 3.4
15.13 2.66
ing perfusion with blood and saline in approximately 2 : l volume ratio. In only 4 of the 43 patients in Groups 1and 2 was more than 2 units of blood added during the course of bypass. The Dacron-wool filter in the coronary suction line provided the only filtration in the entire extracorporeal circuit [25]. No attempt was made to control Paq; during bypass it was usually in the 200 to 300 torr range. Arterial blood was analyzed every 30 to 40
End of Bypass
15.8 20.9
2,3-DPG in Postbypass Period 1Hr
2Hr
3Hr
14.04 14.45 12.16 13.20 13.75 9.87 7.31 11.88 12.10 11.71
12.10 14.20 10.00 10.00 14.40
11.40
5.95 11.27 10.94
12.00 2.09
11.11 2.51
12.31 10.22
10.89 9.37
Duration of Bypass 1' 1'40" 2'6" 1'18" 2'27" 3'7" 3'35" 1' 1'32" 3'16"
10.84
1.00
minutes for pH, Pcq, and bicarbonate. These measurements were also made with each P50and 2,3-DPG determination.
Chemical Determinations The 2,3-DPG level was measured by the enzymatic method of Krimsky [15]. In order to determine the normal values and the reproducibility of the measurements, two assays were made in blood drawn from 20 randomly selected
Table 3. Red Blood Cell 2,3-Diphosphoglycerate (pMlgrn Hgb) and P5,, (torr) in the Patients Receiving 7.5 mllkg of lnosine (Group 3)a
Patient
No.
Prebypass 2,3-DPG
2,3-DPG during Bypass
pJ0 at End of Bypass
Pjo
Dilution 2,3-DPG
1 Hr
End
15.35 16.12 14.44 11.80 15.60 13.37 16.41 10.00 14.69 22.50 14.49 14.32 13.00
16.63 15.00 14.18 12.52 14.85 12.84 18.49 12.45 13.55 20.95 15.47 14.08 12.08
25.00
14.78 2.81
14.85 2.48
25.00 0.0
2,3-DFG in Postbypass Period 1 Hr
2 Hr
3 Hr
17.17 14.50 15.68 12.86 16.52 14.08 17.09 12.52 12.22 20.40 17.32 14.59
16.49
17.20 14.89
15.41 2.31
14.84 2.93
Duration of Bypass
~
1 2 3 4 5 6 7 8 9 10 11 12 13
18.17 15.34 15.45 13.47 14.49 14.74 16.48 12.07 16.80 25.53 14.13 14.72 15.29
27.6
14.24 13.95 13.41 12.47 12.87 13.13 14.54 11.18 12.10 20.02 13.47 12.83 11.78
Mean SD
15.74 2.68
27.6 0.0
13.54 2.09
PNeithervalue was determined on the day prior to operation
11.42 13.22 11.87 11.68 19.24 17.73 17.05
14.37 10.53 10.63
10.99
16.75 13.21 13.57 2.51
1'11" 1' 1'56" 1'56"
1'11" 2'47" 3'24" 2'30" 2'57" 3'2" 1'25" 2'4"
391 Giannelli et al: Prevention of Increased Hemoglobin-Oxygen Affinity
Table 4. Red Blood Cell 2,3-Diphosphoglycerate (pMlgm Hgb) and Ps0 (torr) in Patients Receiving 15 mllkg of Inosine (Group 4 ) Day Prior to Operation Patient No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Mean SD
2,3DPG
Pa
25.21 15.95 16.12 14.93 17.74 22.22 19.29
26.6 23.0 26.6 26.3 24.6 30.7 26.8
20.99 14.37
25.0 23.3
2,3-DPG during Bypass
Prebypass 2,3DPG
PW
18.17 13.82 23.67 24.68 14.79 16.64 14.25 15.05 21.39 19.85 17.76 21.26 14.29 19.45 16.33
27.8 22.7 28.4
21.5 23.4 27.0 21.2 24.9 19.4
18.09 3.40
24.8 3.0
30.0 22.2 26.8 23.5 24.9
28.0
Dilution 2,3DPG 1 Hr
End
PSOat End of Bypass
16.14 11.44 20.09 21.51 12.33 15.87 10.64 12.05 18.60 14.97 14.52 16.42 12.56
18.58 15.93 27.28 24.63 15.00 18.70 18.02 12.36 18.89 17.22 17.39 17.33 16.25
19.54 15.81 26.67 26.94 16.07 18.31 16.11 16.29 21.76 13.75 16.16 15.74 18.28 19.69 16.74
29.7 21.6 28.7 30.4 24.4 25.3 22.9 24.2 26.6 23.2 23.4 23.0 26.4 23.3 19.8
15.16 3.27
18.27 3.71
18.52 3.77
24.9 2.9
donors to the hospital blood bank. The average difference between the two determinations in each donor was 1.5 pMlgm Hgb (SE k 1.8). Whole blood and red cell pH were measured in duplicate with a pHlblood gas analyzer.* Generally, measurements on the same sample were identical. Red blood cell pH was measured in packed, hemolyzed cells prepared by Bartels’ method [12]. Serum blood urea nitrogen and creatinine measurements were made preoperatively and on each of the first five postoperative days in those patients who received allopurinol alone or allopurinol and IPP. Pso DETERMINATIONS. The Pso was determined by measuring the oxyhemoglobin saturation of the blood sample after tonometry with a gas mixture of 5.04% COz, 4.27% 02,and 90.69% N2. Saturation was obtained from Van Slyke analyses [26], and the oxygen percentage of the oxygenating mixture was determined by averaging fifteen Scholander analyses [221 that ranged from 4.23 to 4.33%. If the Van Slyke measurements on the same sample differed by more than 0.3 ml per 100 ml ‘Model 113, Instrumentation Laboratory Inc, Boston, MA.
2,3-DPG in Postbypass Period 1 Hr
2Hr
3 Hr
20.58 9.57 27.92 28.15 16.33 19.40 12.62 10.46 9.40
19.23 13.27 24.32 23.48 16.25 21.62 11.18 10.55 8.21
19.00 14.00 25.00 19.00
14.64 11.89 15.18
14.79
12.50
16.35 6.24
16.29 5.38
16.58 4.99
10.00
Duration of Bypass 1’24” 2’4” 1’23” 2’25” 1’14” 5’20 1’31” 3’17” 2‘49” 5’37” 2’20” 2‘49 2’34“
of blood, additional measurements were made until agreement was obtained. In forty Van Slyke measurements performed by the same personnel who made the measurements reported in this paper, the average difference between measurements was 0.09 ml of oxygen per 100 ml of blood (range, 0.00 to 0.22). After correction for pH [231, the P q was plotted against saturation, which usually was slightly above 50%. A line parallel to the normal oxyhemoglobin dissociation curve was drawn through this point to intercept the 50% saturation P q value. In order to evaluate the reproducibility of our Pso measurements, duplicate determinations were made upon fourteen blood samples from our laboratory personnel.
Preparation of IPP For the IPP solution (0.1M inosine, 0.1 M pyruvate, 0.066 M phosphate buffer), the following were added to each 1,000 ml of sterile water for injection USP: 26.8 gm of inosine, 10.3 gm of pyruvate, 1.8 gm of KH2P04, and 7.65 gm of NaZPHO4.The resulting solution was buffered with Na2PH04to a pH of 7.35 to 7.40. Then the solution was rendered sterile by filtration
392 The Annals of Thoracic Surgery Vol 21 No 5 May 1976
through a 0.22 (u filter* and delivered directly into 1,000 ml plastic intravenous dispensing bags. An aliquot of each bag was cultured for 72 hours. The IPP was stored at 4°C and warmed to room temperature prior to administration.
Results There was no evidence that allopurinol or allopurinol plus IPP was deleterious. BUN and creatinine levels in those patients who received IPP were similar to the values in patients in the control and allopurinol groups. Arterial blood pressure during the period of IPP administration was thought by the anesthesiologist to be lower than in the other groups. However, the pressure was easily maintained in the desired range by administration of vasoconstrictors. The total amount of blood given during the first 24 hours after bypass was similar for each group. Arterial blood pH, PCQ, and bicarbonate levels tended to remain in the normal range during perfusion. There was considerable variation but no systematic trend for any of these values. 2,3-DPG and Ps0 during and after Heart-Lung Bypass A stability coefficient of 0.997 was obtained from the fourteen pairs of replicated P5,, mea‘7.03 Filter, Falcon Plastics, Los Angeles, CA.
Fig2. Mean2,3-DPGlez~els(+ 2 SD)duringand after bypass for the fourgroups.
surements. Using a chi-square (14 df) statistic, a 99% upper bound of confidence of 0.799 was obtained for the standard deviation of the difference between replications of Pjo measurements. Tables 1 through 4 contain the 2,3-DPG and Pj0 data obtained from the surgical patients. Figure 2 presents the serial mean 2,3-DPG data for each of the four groups. These data were evaluated for statistical significance by a nonparametric one-way analysis of variance with regard to four variables [131. A concatenated method of multiple comparisons was used, eg, the three groups with the lowest (and highest) mean ranks were tested for significant differences if, and only if, the four groups together had shown significant differences. For each of the four variables, those groups not found to be significantly different were then pooled, and the hypothesis that their median (ie, the median of their underlying population) was zero was tested with the Fisher sign test. The 2,3-DPG levels of the four groups were compared by evaluating the changes in 2,3-DPG at 1hour of bypass, at the conclusion of bypass, and 1hour after bypass from the prebypass dilution levels. Both the control and the allopurinol group showed significant decreases in 2,3-DPG level at 1hour of bypass from the initial dilution level ( p < 0.001 and < 0.01, respectively). The allopurinol group had a significantly smaller decrease than did the control group, however. IPP, by contrast, caused significant rises in 2,3-DPG levels at both the 7.5 mllkg and 15 mllkg dosages
393 Giannelli et al: Prevention of Increased Hemoglobin-Oxygen Affinity
(p < 0.05 and 0.005, respectively). In terms of differences in 2,3-DPG level at end of bypass from the initial dilution level, Groups 1 and 2 were similar and showed a significant decrease in 2,3-DPG ( p < 0.0001). Groups 3 and 4 had significant rises of 2,3-DPG from the dilution level, with p values of < 0.005 and < 0.025, respectively. The larger IPP dose resulted in a significantly higher 2,3-DPG increase than did Fig3. (A)Relationships between 2,3-DPG and Ps0 prior to and at the conclusion of bypass for the 5 patients in Group 2 and 10 patients in Group 2 (control and allopurinol)f o r w h o m these data are available. T h e P,,, and 2,3-DPG behavior was similar f o r the twogroups. ( B ) Same data f o r the 25 patients in Group 4 (25 rnllkg IPP).
c
a
.w-
I
I
9ic. w
15
!lo-
rn ".
N
the 7.5 mllkg dose (p < 0.001). Groups 1 and 2 (control and allopurinol) behaved similarly to one another, as did Groups 3 and 4, with regard to changes in the 2,3-DPG level at 1hour after bypass from the initial dilution level. The pooled sample of Groups 1and 2 showed a significant fall from the dilution levels (p < O.OOOl), while Groups 3 and 4 had a significant increase ( p < 0.0025). Figure 3 presents the and 2,3-DPG data for the combined control and allopurinol and the 15 mllkg IPP group before and after bypass; the single patient with data in Group 3 (7.5 ml/kg IPP) is not included in the figure or in the analysis. The behavior of Groups 1 and 2 was similar, and together they showed a significant
394 The Annals of Thoracic Surgery Vol 21 No 5 May 1976
decline in P50: prebypass mean P50, 25.7 torr; postbypass, 21.9 torr ( p < 0.001). In Group 4 the prebypass and postbypass levels of 24.8 and 24.9 torr, respectively, were not significantly different. Thus IPP prevented the fall in P50 observed in the control and allopurinol groups. These data show that the usual open-heart surgical procedure is accompanied by drops in 2,3-DPG and P50 levels. Apparently the only alteration in this tendency produced by the administration of allopurinol is a mild delay in the drop of the 2,3-DPG level. From these data it is not clear how much of the drop in P50 was attributable to dilution effect and how much to bypass itself. Indirectly from the 2,3-DPG results, however, coupled with the theoretical and actually observed correlation between 2,3-DPG and P50, a major portion of the drop probably resulted from bypass. The data present strong evidence that IPP not only prevents a drop in 2,3-DPG and P50 levels but actually raises 2,3DPG during bypass. The higher dose of IPP tends to elevate 2,3-DPG more than the lower dose during bypass, while 1 hour after bypass their effect is about the same. 2,3-DPG LEVELS DURING AND AFTER HEARTLUNG BYPASS. These data do not provide additional information regarding the etiology of the progressive decline in 2,3-DPG during bypass. They confirm our earlier studies demonstrating that 2,3-DPG falls below levels attributable to dilution by the bank blood erythrocytes placed in the pump oxygenator priming volume. Neither was the further dilution provided by the 1 or 2 additional units of bank blood frequently added during bypass sufficient to explain the decrease in 2,3-DPG that occurred during the course of perfusion. Our earlier attempts to determine the effect of simple in vitro recirculation of fresh human blood through the extracorporeal circulation were inconclusive. We plan to do studies that will explore further the effects of simple in vitro recirculation, different priming solutions, the introduction of microfilters into the extracorporeal circuit, steroid administration, and the influence of hypothermia.
Relationships between 2,3-DPG and P50 Two methods of approach were employed to determine whether the operative procedure or
Table 5. Correlations between
P50
and 2,3-DPG
Variables
Control & Allo15 M1 purinol IPP (n = 15) (n = 15)
Prebypass Pso,
0.484
0.713
0.541
Postbypass Pjo,
0.485
0.780
0.725
0.379
0.375
0.658
2,3-DPG
2,3- DPG APw, A2,3-DPG
Whole Sample (n = 30)
the administration of IPP affectsthe relationship between 2,3-DPG and P50. The first was to determine the correlation between 2,3-DPG and P50 values at the beginning and end of bypass; this was done for all the patients for whom these determinations were available and then separately for the 15 mllkg IPP group and for the combined control and allopurinol groups. The second approach was to determine the correlation between P50 and 2,3-DPG at the beginning and end of bypass in the control plus allopurinol group, the 15 mllkg IPP group, and for all patients combined. (The correlation between AP50 and A2,3-DPG evaluated the variability of the slopes of the arrows in Figure 3.) Table 5 presents these data. Neither the IPP group nor the control plus allopurinol group showed a significant change in 2,3-DPG and Pso correlation before and after bypass; nor does IPP influence the prebypass to postbypass AP50A2,3-DPG correlation. Thus there is no evidence that either heart-lung bypass or IPP administration affects the relationship of 2,3-DPG to P50.
Clinical Significance of These Studies The data demonstrate that IPP can be administered safely during extracorporeal circulation, thus preventing the decreases in 2,3-DPG and P50 that normally would occur. Because the method of providing for myocardial oxygen requirements during bypass was not standardized, conclusions are not possible regarding the protection against myocardial ischemic damage afforded by IPP in patients with aortic valve disease and those with coronary atherosclerosis. A randomized study has been initiated in these two groups of patients, with standardized methods of handling the problem
395 Giannelli et al: Prevention of Increased Hemoglobin-Oxygen Affinity
of myocardial oxygenation, to evaluate whether IPP protects against ischemic damage. Decreased Pso has relatively little effect u p o n maximal overall bodily oxygen consumption. Two clinical studies have compared reduction i n oxygen supply by hypoxia a n d carbon monoxide [19,27]. The oxygen content of arterial blood w a s reduced 10% by either inspiration of low oxygen mixtures or saturation of 10% of the hemoglobin with carbon monoxide; i n the latter case, the P50 of the remaining hemoglobin was reduced by about 6 torr. I n each of these studies maximal oxygen uptake w a s equally impaired by t h e two interventions. Similarly, i n rats, a fall i n Pj0 from 36 torr (normal for that species) to 30 torr d i d not reduce maximal oxygen uptake [281. A further decrease t o 23 torr produced t h e same work reduction as a 10% diminution i n hemoglobin concentration. Oski's group studied 2 persons w i t h similar degrees of anemia whose oxyhemoglobin dissociation curves were markedly shifted i n opposite directions: Pso of 19 a n d 38 torr, respectively. The patient with the left-shifted curve showed a s h a r p initial fall i n central venous oxygen tension with mild exercise a n d then a marked increase i n cardiac o u t p u t with further exertion. The other patient accommodated to progressive exercise w i t h a gradually accelerating increase i n oxygen extraction so that oxygen requirements were met by a smaller increase in cardiac output. deIn contrast to the minor influence of a crease of several torr i n normal subjects, some studies demonstrate that moderately elevated hemoglobin affinity for oxygen significantly impairs tissue oxygen delivery w h e n arterial disease limits the capacity for increased organ blood flow. Aronow a n d colleagues [4] showed that a 3% hemoglobin carbon monoxide level, induced b y breathing carbon monoxide, adversely affected muscle oxygenation i n 10 calves with lower extremity arterial occlusion. While they performed light exercise, t h e average time to t h e development of intermittent claudication w a s reduced by 20% after carbon monoxide inhalation. Aronow's g r o u p performed similar studies on patients with coronary artery disease and showed that angina w a s induced more quickly d u r i n g controlled exercise testing w h e n 3 t o 4% hemoglobin carbon monoxide levels
were induced by any of several methods: cigarette smoking [2], carbon monoxide inhalation [3], a n d exposure to Los Angeles freeway traffic
[51. These semiquantitative data suggest that increased hemoglobin affinity for oxygen probably is a n important determinant of t h e sufficiency of myocardial oxygenation in patients w i t h coronary artery disease.
References 1. Akerblom 0, DeVerdier CH, Garby L, et al: Restoration of defective oxygen-transport function of stored red blood cells by addition of inosine. Scand J Clin Lab Invest 21:235, 1968 2. Aronow WS, Dedinger J, Rokow SN: Heart rate and carbon monoxide level after smoking high, low and non-nicotine cigarettes: a study in the male patients with angina pectoris. Ann Intern Med 74:697, 1971 3. Aronow WS, Isbell MW: Carbon monoxide effect on exercise-induced angina pectoris. Ann Intern Med 79:392, 1973 4. Aronow WS, Stemmer EA, Isbell MW: Effect of carbon monoxide exposure on intermittent claudication. Circulation 49:415, 1974 5. Aronow WS, Harris CN, Isbell MW, et al: Effect of heavy freeway traffic on cardiopulmonary function in angina (abstract). Chest 62:358, 1972 6. Berman IR, Acinapura A, Zucker MB, et al: Alteration of platelet reactivity during cardio-bypass in man (abstract). Circulation 43,44:Suppl2: 11,1971 7. Bordiuk JM, McKenna PJ, Giannelli S Jr, et al: Alterations in 2,3 diphosphoglycerate and O2 hemoglobin affinity in patients undergoing open heart surgery. Circulation 43:Suppl 1:141, 1971 8. Caen JP, Jenkins CSP, Michel H, et al: Adenosine metabolism in platelets and plasma. Ser Haematol 6:317, 1973 9. DeLevine M, Hill JD, Mielke CH, et al: Blood platelets and coagulation mechanisms during extracorporeal circulation (abstract). Presented at the 45th Scientific Session of the American Heart Association, Dallas, TX, Nov 1972 10. Dixon SH Jr, Limbird BS, Roe CR, et al: Recognition of post-operative acute myocardial infarction: application of isoenzyme techniques. Circulation 47,48:Suppl 3:137, 1973 11. Giannelli S Jr, Conklin EF, Ayres SM, et al: A practical method of transmembrane gas exchange for open heart surgery. Am J Surg 119:519, 1970 12. Hilpert P, Fleischmann RG, Kempe D, et al: The Bohr effect related to blood and erythrocyte pH. Am J Physiol 205:337, 1963 13. Hollander M, Wolfe DA: Non Parametric Methods. New York, Wiley, 1973, p 114
396 The Annals of Thoracic Surgery Vol 21 No 5 May 1976
14. Jesch F, Weber LM, Carey JS: Oxygen affinity of hemoglobin: influence of blood replacement and hemodilution after cardiac surgery, Advances in Experimental Medicine and Biology: Volume 37A, Oxygen Transport to Tissue. Edited by Bmley DF, Bicher HI. New York, Plenum, 1973 15. Krimsky IL: D-2-3 diphosphoglycerate, Methods of Enzymatic Analysis. Edited by Bergmeyer HU. New York, Academic, 1963 16. McKenna PJ, Giannelli S Jr, Bordiuk JM: Increased hemoglobin affinity for oxygen in open heart patients. Crit Care Med 2:73, 1974 17. Miller ID, Sugerman HJ, Cromie WJ, et al: Administration of inosine to man, Proceedings of Conference on preservation of red blood cells. National Academy of Science, Washington DC, 1973 18. Oski FA, Travis SF, Miller LD, et al: The in vitro restoration of red cell 2-3 diphosphoglycerate levels in banked blood. Blood 37:52, 1971 19. Pirnay F, Dujardin J, Deroanne R, et al: Muscular exercise during intoxication by carbon monoxide. J Appl Physiol 31:573, 1971 20. Robinson MA, Louder PB, DeGrachy GC: Red cell metabolism in nonspherocytic congenital haemolytic anemia. Br J Haematol 7:327, 1961 21. Sapsford RN, Blackstone EH, Kirklin JW, et al: Coronary perfusion versus cold ischemic arrest
22. 23. 24.
25. 26.
27. 28.
during aortic valve surgery. Circulation 49: 1190, 1974 Scholander PF: Analyzer for accurate estimation of respiratory gases in one-half cubic centimeter samples. J Biol Chem 167:235, 1947 Severinghaus JW, Bradley AF: Electrodes for blood Po2 and PcoL determinations. J Appl Physiol 13:515, 1958 Sugarman HJ, Pollock TW, Rosato EF, et al: Experimentally induced alterations in affinity of hemoglobin for oxygen: 11. In vivo effect of inosine, pyruvate, and phosphate on oxygenhemoglobin affinity in Rhesus monkey. Blood 39:525, 1972 Swank RL, Hirsch H, Bruer M, et al: Effect of glass wool filtration on blood during extracorporeal circulation. Surg Gynecol Obstet 117:547, 1963 Van Slyke DD, McNeill JM: The determination of gases in blood and other solutions by vacuum extraction and manometric measurement. J Biol Chem 61:523, 1924 Vogel JA, Gleser MA, Wheeler RC, et al: Carbon monoxide and physical work capacity. Arch Environ Health 24:198, 1972 Woodson RD, Wranne 8, Detter JC: Effect of increased blood oxygen affinity on work performance of rats. J Clin Invest 52:2717, 1973
ABSTRACT In a control group of 32 patients under-
going open-heart operation, erythrocyte 2,3diphosphoglycerate (2,3-DPG) declined progressively during the course of perfusion from a prebypass mean of 17.00 to 11.29 p M per gram of hemoglobin at the end of bypass. The decrease was greater than that attributable merely to dilution of the patients’ cells with the 2,3-DPG-deficient donor cells used to prime the pump oxygenator circuit. Administration of 300 mg of allopurinol, to prevent the conversion of inosine to uric acid, every 8 hours during the 24 hours prior to operation in 11patients did not prevent the 2,3-DPG decrease during heart-lung bypass: prebypass, 18.31; postbypass, 13.56 pMlgm Hgb. The mean Ps0 for both these groups combined decreased from a prebypass mean of 25.7 to a postbypass level of 21.9 torr. A solution of 0.1 M inosine, 0.1 M pyruvate, and 0.066 M phosphate (IPP) in a dosage of 7.5 ml per kilogram of body weight prevented the 2,3-DPG decrease: prebypass, 15.74; postbypass, 14.85. Administration of 15 ml per kilogram of IPP in 15 patients preserved 2,3-DPG: prebypass, 18.09; postbypass, 18.52. The Pj0 remained unchanged in this last group. The method of providing for myocardial oxygen requirements during bypass was not standardized, and therefore the protective effect of IPP against ischemic damage in patients undergoing aortic valve replacement or myocardial revascularization could not be evaluated. No deleterious effects of IPP were noted. From the Departments of Surgery, Pathology, and Pediatrics, St. Vincent’s Hospital and Medical Center, New York, NY, and the Department of Surgery, University of Pennsylvania Medical School and Hospital of the University of Pennsylvania, Philadelphia, PA. Supported in part by Grant no. 5-R01-HL-10781-08from the Department of Health, Education, and Welfare. Accepted for publication Nov 6 , 1975. Address reprint requests to Dr. Giannelli, Division of Cardiac Surgery, St. Vincent’s Hospital and Medical Center of New York, 153 W 11th St, New York, NY 10011.
386
We initially reported, in 1971, that red cell 2,3diphosphoglycerate (2,3-DPG) progressively declines during heart-lung bypass [7]. At the conclusion of perfusion 2,3-DPG levels were about half the prebypassvalues; the extent of the decrease was greater than could be ascribed merely to dilution of the patients’ cells with the 2,3-DPG-deficient blood bank donor cells in the pump prime. The patients in this initial series were perfused with either a rotating disc or a membrane oxygenator [ll], and 2,3-DPG phosphorus was determined by a column chromatographic extraction method [20]. We then studied another series of patients, perfused with a bubble oxygenator, in whom 2,3-DPG was determined by an enzymatic method [15]. During bypass 2,3-DPG progressively decreased [161, and while the amount of the decline was less than we had previously reported [7], it was still greater than could be attributed to dilution by the donor red cells. There was an associated increase in the hemoglobin affinity for oxygen: the average Pjo in 10 patients prior to bypass was 28.7 torr, and at the conclusion of bypass it was 24.4 torr. These latter data were similar to those recently published by Jesch and associates [14], who showed that 2,3DPG declined from a preoperative mean of 13.6 p M per gram of hemoglobin to 10.9 at the completion of operation. The respective Pso values were 27.0 and 24.8 torr. An increased hemoglobin affinity for oxygen theoretically should reduce the maximum tissue oxygen delivery by decreasing the oxygen diffusion pressure gradient between the erythrocytes in the capillaries and the cellular mitochondria. The myocardium would be expected to be particularly vulnerable to decreased Pso, because at rest that organ already extracts oxygen from the blood at a nearly maximal rate. Unlike skeletal
387 Giannelli et al: Prevention of Increased Hemoglobin-Oxygen Affinity
muscle, which obtains additional oxygen during exercise by a combination of increased blood flow and greater oxygen extraction, myocardium is almost solely dependent upon higher blood flow during a period of increased oxygen demand. There are two groups of cardiac surgical patients in whom the balance between oxygen supply and demand is compromised: those with coronary artery disease undergoing myocardial revascularization and those with left ventricular hypertrophy who require aortic valve replacement. Postoperative myocardial ischemic damage has been reported to occur in a significant percentage of patients in each of these groups [lo, 211. If a method were available to prevent P50 from decreasing in patients having open-heart operations, the frequency of myocardial damage possibly could be reduced. This decrease in ischemic damage would provide quantitative documentation of the clinical importance of P50 to myocardial oxygen delivery. Akerblom and associates [l]reported in 1968 that the addition of inosine to 2,3-DPG-deficient blood partially restores the 2,3-DPG level. Fig-
ure 1presents those chemical reactions that are probably responsible for 2,3-DPG synthesis when inosine is added to blood. Oski and coworkers [181 found that the administration of phosphate and pyruvate in addition to inosine (IPP) causes a greater elevation of 2,3-DPG when added to blood in vitro than does inosine alone. Phosphate is required for the formation of ribose-1-phosphate, and the pyruvate serves to replenish nicotinamide-adenine dinucleotide from its reduced form. IPP was safely administered to monkeys and, as was true for the in vitro studies, this combination of substances effected a greater rise in 2,3-DPG than did inosine alone [24]. The solution then was administered to normal human volunteers, as reported by Miller and the other members of Oski’s group [17]. In humans, IPP was also more effective than inosine alone in raising 2,3-DPG. Optimum 2,3DPG elevations were obtained by two separate administrations of 7.5 mllkg of 0.1 M inosine, 0.05 M phosphate in buffer solution, and 0.1 M pyruvate, given rapidly 1 hour apart. The average rise in 2,3-DPG content was 1,600 p M per milliliter of red cells; the mean Pso increase was
Fig I. The route by which inosine enters into the erythrocyte metabolic pathzvays to produce
2,3-DPG.
INOSINE + P i 4
(Purine nucleoside phosphorylase)
(Phosphoribmutase)
-I
HYPOXANTH INE.&WR
RIBOSE-5-P (Transketol ase + transaldolase)
ATP ADP
(Aldolase)
1
G-3-P-OIHYDROXYACETONE
t
NAD NAOH
(G-3-PD)
ATP ADPAPGK
/
I
ALLOPUR INOL
&RIBOSE-I -P
(DPGP)
IC ACID
388 The Annals of Thoracic Surgery Vol 21 No 5 May 1976
3.5 torr. The peak effect was reached 3 to 6 hours after completion of the infusion, and 75% of the elevation was present 24 hours after administration. There are several possible deleterious effects of IPP administration, the most obvious being the formation of uric acid. Miller’s patients were given 300 mg of allopurinol daily for two days prior to IPP administration, and significant rises in uric acid did not occur [17]. Hypoxanthine and inosine are vasodilators, and 2 of the 16 human subjects who received the two 7.5 mllkg doses of IPP experienced transient mild blood pressure depression. Decreased peripheral vascular resistance is common during the early stages of heart-lung bypass, and therefore these vasodilator effects might be of greater significance than in the intact individual. Caen and associates [81 showed that both inosine and hypoxanthine inhibit platelet aggregation induced by adenosine diphosphate (ADP). They proposed that these substances compete with adenosine for a receptor site on the platelet membrane. Platelet counts regularly decrease during the course of heart-lung bypass [91, and those platelets that remain in the circulation have been found to have decreased agglutination in response to ADP [6]. Thus, IPP administration could further impair platelet function and might contribute to clinically significant bleeding after perfusion.
Clinical Material and Methods Serial 2,3-DPG measurements were made during and after heart-lung bypass in four groups of patients operated upon because of congenital or rheumatic heart disease or coronary arteriosclerosis. Only those patients who were perfused longer than 60 minutes are included in this paper. Group 1, serving as controls, was composed of 29 adults and 3 children each 7 years old. The 11adults in Group 2 received 300 mg of allopurinol at 8-hour intervals beginning the day prior to operation; the last dose was administered with a sip of water 1hour prior to going to the operating room. The 13 adults in Group 3 received allopurinol plus 7.5 mllkg of IPP solution. The IPP was infused over approximately 20 minutes beginning about 15 minutes
after the onset of heart-lung bypass. The 15 adults in Group 4 were managed similarly to those in Group 3 but received 15 mllkg of IPP; administration was begun at the same time and continued for 30 to 40 minutes. Evaluation of the incidence of postoperative ischemic damage in the four groups was not considered worthwhile for two reasons: the method of protecting the myocardium from ischemic damage during the period of heart-lung bypass was not constant in those patients operated upon for coronary artery disease and aortic valve disease, and myocardial-specific creatine phosphokinase isoenzyme determinations were not available to us during most of the period covered by this study.
Schedule of PS0 and 2,3-DPG Determinations In all groups, serial 2,3-DPG measurements were made during bypass. Prebypass and priming volume 2,3-DPG levels were obtained in all patients, and subsequent measurements usually were made at 1 hour and at the conclusion of bypass. In a smaller number of these patients, hourly determinations were made for up to 3 hours following bypass. In a total of 19 patients-5 in Group 1, 5 in Group 2, and 9 in Group 4-2,3-DPG measurements were also made on the day prior to operation. For all but 3 patients, in whom hematocrit values were not available, a 2,3-DPG ”dilution” value was calculated; this was an estimate of the 2,3-DPG level that would have resulted from simple admixture of the patients’ cells and the 2,3-DPG-deficient cells in the priming volume. The volume of the patients’ red cells was determined from their hematocrit and their estimated blood volume. It was assumed in all patients that blood volume represented 8% of body weight. Determination of PjOwas done prior to bypass and at the end of bypass in 5 patients in Group 1, 10 patients in Group 2 , l patient in Group 3, and all 15 patients in Group 4. In 5 patients in Group 1, 5 in Group 2, and 9 in Group 4, PjO measurements were also made on the day prior to bypass. All the data for 2,3-DPG and Pjo in the four groups are given in Tables 1 through 4. Variable amounts of additional bank blood were administered during the first 3 hours following bypass, usually 2 to 3 units. Correlation
389 Giannelli et al: Prevention of Increased Hemoglobin-Oxygen Affinity
Table 1 . Red Blood Cell 2,3-Diphosphoglycerate (pMlgrn Hgb) and Ps0 (tory) in the Control Group (Group 1 ) Day Prior to Operation Patient No.
2,3DPG
PjO
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
14.48 13.32 17.73 12.24 13.18
24.3 22.0 24.7 15.2 22.0
Mean SD
Prebypass 2,3DPG
PS0
18.15 13.69 17.46 14.04 14.37 18.20 13.40 18.50 14.80 25.60 17.50 14.78 16.16 15.96 19.44 22.30 17.48 20.26 15.11 15.48 23.22 16.58 11.80 18.78 16.55 16.94 17.40 12.70 20.67 20.84 12.52 18.38
24.7 24.9 27.7 19.3 22.7
17.00 3.20
23.9 2.8
Dilution 2,3DPG 14.92 11.68 12.69 10.86 11.19 10.45 11.75 14.13 12.16 17.60 11.50 10.41 12.15 9.33 14.87 17.08 16.09 12.61 13.40 19.52 14.86 10.79 12.01 14.78 14.47 13.29 11.39 18.05 17.16 11.78 13.90 13.45 2.52
2,3-DPG during Bypass 1Hr
End
at End of Bypass
14.57 11.34 13.60 12.22 10.11 7.50 10.40 9.05 9.10 7.78 7.50 10.54
13.93 10.52 11.69 8.68 12.37
21.2 19.9 21.1 20.0 18.7
2,3-DPG in Postbypass Period
p50
2Hr
3Hr
13.64 10.74 14.05 9.13
11.65 13.95
12.11
6.60
14.62 10.18 6.33 13.83 13.09 12.90 10.62 13.20 17.42 10.36 11.67
12.10 10.43 10.50 9.88 10.30 10.12 13.48 8.82 11.98 11.02 11.30 10.90 12.35 11.28 15.26 14.34 10.71 7.38 11.32 12.14 12.42 9.01 13.67 14.49 10.83 12.59
11.26 2.52
11.9 2.05
11.02 12.82 10.90
1Hr
10.50 9.55 5.30
7.86 8.30
4.20 7.12
8.90 9.42 10.66
17.12 17.12 8.72 11.29 12.11 13.77
13.68 11.30 8.50
8.54
14.57 8.72
9.93
9.37
20.2 0.9
8.40 13.30 14.61 11.65
11.92 12.66
10.87 2.50
10.98 1.93
12.48
Duration of Bypass 2'29" 1'29" 1'30" 2'16" 2'30" 1'58" 1'35" 1'40" 3 '12" 3'29" 2'29" 1'40" 1'40 1'39" 1'39" 1' 3'24" 1' 1'11" 1'46" 1'53" 3'52" 3'7" 1'22" 2'16" 1'35" 1'19" 2'21" 1'23" 2'57" 1'35" 2'21"
10.17 3.54
Conduct of the Operations Nitrous oxide and morphine anesthesia and a Q-100 Temptrol bubble oxygenator* were employed for each operation. Silastic rubber tubing was used throughout the extracorporeal circuit. Prior to introduction of the priming volume into the extracorporeal circuit, 200 units of heparin was added to each of the 4 units of blood and to each 500 ml of the 2,000 ml of saline that
comprised the prime. The blood had been drawn 3 to 10 days prior to use and was stored in either acid-citrate-dextrose or citratephosphate-dextrose solution. The blood was passed through a Swank Dacron-wool CA 100 microfiltert before introduction into the pump oxygenator. The citrate in the blood was neutralized with 2.0 gm of calcium gluconate. Heparin, 200 units per kilogram, was administered intravenously between 5 and 10 minutes prior to bypass, and the initial patient dose was repeated each hour during bypass. When necessary, the perfusate volume was augmented dur-
'Bentley Laboratories, Inc, Irvine, CA
tPioneer Filters, Inc, Beaverton, OR
was not made between 2,3-DPG levels in the hours after bypass and the amount of transfused bank blood.
390 The Annals of Thoracic Surgery Vol 21 No 5 May 1976
Table 2. Red Blood Cell 2,3-Diphosphoglycerate (pMlgm Hgb) and Allopurinol (Group 2) Day Prior to Operation Patient
No.
2,3DPG
2,3DPG
P,,
1 2 3 4 5 6 7 8 9 10 11
19.33 24.59 12.40
23.2 30.7 25.3
20.58 14.82
28.1 23.8
Mean SD
2,3-DPG during Bypass
Prebypass
Pso
(torr) in the Patients Receiving
P5, at
Dilution 2,3DPG
1 Hr
End
15.31 18.39 15.89 12.83 16.70 16.19 14.79 10.87 14.32 13.86 11.05
14.68 18.04 15.36 12.71 14.40 16.10 11.43 10.29 15.57 10.57 10.00
23.1 25.3 25.3 24.8 21.7 21.6 25.3 23.5
14.56 2.21
13.56 2.58
22.7 2.8
18.02 18.55 19.59 16.97 19.92 21.06 23.38 13.24 18.43 17.17 15.08
26.6 28.4 28.9 30.7 22.5 22.8 32.8 26.0 24.3 23.0
14.57 18.85 15.94 13.98 17.68 17.13 17.59 11.43 15.23 14.47 9.57
18.31 2.64
26.6 3.4
15.13 2.66
ing perfusion with blood and saline in approximately 2 : l volume ratio. In only 4 of the 43 patients in Groups 1and 2 was more than 2 units of blood added during the course of bypass. The Dacron-wool filter in the coronary suction line provided the only filtration in the entire extracorporeal circuit [25]. No attempt was made to control Paq; during bypass it was usually in the 200 to 300 torr range. Arterial blood was analyzed every 30 to 40
End of Bypass
15.8 20.9
2,3-DPG in Postbypass Period 1Hr
2Hr
3Hr
14.04 14.45 12.16 13.20 13.75 9.87 7.31 11.88 12.10 11.71
12.10 14.20 10.00 10.00 14.40
11.40
5.95 11.27 10.94
12.00 2.09
11.11 2.51
12.31 10.22
10.89 9.37
Duration of Bypass 1' 1'40" 2'6" 1'18" 2'27" 3'7" 3'35" 1' 1'32" 3'16"
10.84
1.00
minutes for pH, Pcq, and bicarbonate. These measurements were also made with each P50and 2,3-DPG determination.
Chemical Determinations The 2,3-DPG level was measured by the enzymatic method of Krimsky [15]. In order to determine the normal values and the reproducibility of the measurements, two assays were made in blood drawn from 20 randomly selected
Table 3. Red Blood Cell 2,3-Diphosphoglycerate (pMlgrn Hgb) and P5,, (torr) in the Patients Receiving 7.5 mllkg of lnosine (Group 3)a
Patient
No.
Prebypass 2,3-DPG
2,3-DPG during Bypass
pJ0 at End of Bypass
Pjo
Dilution 2,3-DPG
1 Hr
End
15.35 16.12 14.44 11.80 15.60 13.37 16.41 10.00 14.69 22.50 14.49 14.32 13.00
16.63 15.00 14.18 12.52 14.85 12.84 18.49 12.45 13.55 20.95 15.47 14.08 12.08
25.00
14.78 2.81
14.85 2.48
25.00 0.0
2,3-DFG in Postbypass Period 1 Hr
2 Hr
3 Hr
17.17 14.50 15.68 12.86 16.52 14.08 17.09 12.52 12.22 20.40 17.32 14.59
16.49
17.20 14.89
15.41 2.31
14.84 2.93
Duration of Bypass
~
1 2 3 4 5 6 7 8 9 10 11 12 13
18.17 15.34 15.45 13.47 14.49 14.74 16.48 12.07 16.80 25.53 14.13 14.72 15.29
27.6
14.24 13.95 13.41 12.47 12.87 13.13 14.54 11.18 12.10 20.02 13.47 12.83 11.78
Mean SD
15.74 2.68
27.6 0.0
13.54 2.09
PNeithervalue was determined on the day prior to operation
11.42 13.22 11.87 11.68 19.24 17.73 17.05
14.37 10.53 10.63
10.99
16.75 13.21 13.57 2.51
1'11" 1' 1'56" 1'56"
1'11" 2'47" 3'24" 2'30" 2'57" 3'2" 1'25" 2'4"
391 Giannelli et al: Prevention of Increased Hemoglobin-Oxygen Affinity
Table 4. Red Blood Cell 2,3-Diphosphoglycerate (pMlgm Hgb) and Ps0 (torr) in Patients Receiving 15 mllkg of Inosine (Group 4 ) Day Prior to Operation Patient No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Mean SD
2,3DPG
Pa
25.21 15.95 16.12 14.93 17.74 22.22 19.29
26.6 23.0 26.6 26.3 24.6 30.7 26.8
20.99 14.37
25.0 23.3
2,3-DPG during Bypass
Prebypass 2,3DPG
PW
18.17 13.82 23.67 24.68 14.79 16.64 14.25 15.05 21.39 19.85 17.76 21.26 14.29 19.45 16.33
27.8 22.7 28.4
21.5 23.4 27.0 21.2 24.9 19.4
18.09 3.40
24.8 3.0
30.0 22.2 26.8 23.5 24.9
28.0
Dilution 2,3DPG 1 Hr
End
PSOat End of Bypass
16.14 11.44 20.09 21.51 12.33 15.87 10.64 12.05 18.60 14.97 14.52 16.42 12.56
18.58 15.93 27.28 24.63 15.00 18.70 18.02 12.36 18.89 17.22 17.39 17.33 16.25
19.54 15.81 26.67 26.94 16.07 18.31 16.11 16.29 21.76 13.75 16.16 15.74 18.28 19.69 16.74
29.7 21.6 28.7 30.4 24.4 25.3 22.9 24.2 26.6 23.2 23.4 23.0 26.4 23.3 19.8
15.16 3.27
18.27 3.71
18.52 3.77
24.9 2.9
donors to the hospital blood bank. The average difference between the two determinations in each donor was 1.5 pMlgm Hgb (SE k 1.8). Whole blood and red cell pH were measured in duplicate with a pHlblood gas analyzer.* Generally, measurements on the same sample were identical. Red blood cell pH was measured in packed, hemolyzed cells prepared by Bartels’ method [12]. Serum blood urea nitrogen and creatinine measurements were made preoperatively and on each of the first five postoperative days in those patients who received allopurinol alone or allopurinol and IPP. Pso DETERMINATIONS. The Pso was determined by measuring the oxyhemoglobin saturation of the blood sample after tonometry with a gas mixture of 5.04% COz, 4.27% 02,and 90.69% N2. Saturation was obtained from Van Slyke analyses [26], and the oxygen percentage of the oxygenating mixture was determined by averaging fifteen Scholander analyses [221 that ranged from 4.23 to 4.33%. If the Van Slyke measurements on the same sample differed by more than 0.3 ml per 100 ml ‘Model 113, Instrumentation Laboratory Inc, Boston, MA.
2,3-DPG in Postbypass Period 1 Hr
2Hr
3 Hr
20.58 9.57 27.92 28.15 16.33 19.40 12.62 10.46 9.40
19.23 13.27 24.32 23.48 16.25 21.62 11.18 10.55 8.21
19.00 14.00 25.00 19.00
14.64 11.89 15.18
14.79
12.50
16.35 6.24
16.29 5.38
16.58 4.99
10.00
Duration of Bypass 1’24” 2’4” 1’23” 2’25” 1’14” 5’20 1’31” 3’17” 2‘49” 5’37” 2’20” 2‘49 2’34“
of blood, additional measurements were made until agreement was obtained. In forty Van Slyke measurements performed by the same personnel who made the measurements reported in this paper, the average difference between measurements was 0.09 ml of oxygen per 100 ml of blood (range, 0.00 to 0.22). After correction for pH [231, the P q was plotted against saturation, which usually was slightly above 50%. A line parallel to the normal oxyhemoglobin dissociation curve was drawn through this point to intercept the 50% saturation P q value. In order to evaluate the reproducibility of our Pso measurements, duplicate determinations were made upon fourteen blood samples from our laboratory personnel.
Preparation of IPP For the IPP solution (0.1M inosine, 0.1 M pyruvate, 0.066 M phosphate buffer), the following were added to each 1,000 ml of sterile water for injection USP: 26.8 gm of inosine, 10.3 gm of pyruvate, 1.8 gm of KH2P04, and 7.65 gm of NaZPHO4.The resulting solution was buffered with Na2PH04to a pH of 7.35 to 7.40. Then the solution was rendered sterile by filtration
392 The Annals of Thoracic Surgery Vol 21 No 5 May 1976
through a 0.22 (u filter* and delivered directly into 1,000 ml plastic intravenous dispensing bags. An aliquot of each bag was cultured for 72 hours. The IPP was stored at 4°C and warmed to room temperature prior to administration.
Results There was no evidence that allopurinol or allopurinol plus IPP was deleterious. BUN and creatinine levels in those patients who received IPP were similar to the values in patients in the control and allopurinol groups. Arterial blood pressure during the period of IPP administration was thought by the anesthesiologist to be lower than in the other groups. However, the pressure was easily maintained in the desired range by administration of vasoconstrictors. The total amount of blood given during the first 24 hours after bypass was similar for each group. Arterial blood pH, PCQ, and bicarbonate levels tended to remain in the normal range during perfusion. There was considerable variation but no systematic trend for any of these values. 2,3-DPG and Ps0 during and after Heart-Lung Bypass A stability coefficient of 0.997 was obtained from the fourteen pairs of replicated P5,, mea‘7.03 Filter, Falcon Plastics, Los Angeles, CA.
Fig2. Mean2,3-DPGlez~els(+ 2 SD)duringand after bypass for the fourgroups.
surements. Using a chi-square (14 df) statistic, a 99% upper bound of confidence of 0.799 was obtained for the standard deviation of the difference between replications of Pjo measurements. Tables 1 through 4 contain the 2,3-DPG and Pj0 data obtained from the surgical patients. Figure 2 presents the serial mean 2,3-DPG data for each of the four groups. These data were evaluated for statistical significance by a nonparametric one-way analysis of variance with regard to four variables [131. A concatenated method of multiple comparisons was used, eg, the three groups with the lowest (and highest) mean ranks were tested for significant differences if, and only if, the four groups together had shown significant differences. For each of the four variables, those groups not found to be significantly different were then pooled, and the hypothesis that their median (ie, the median of their underlying population) was zero was tested with the Fisher sign test. The 2,3-DPG levels of the four groups were compared by evaluating the changes in 2,3-DPG at 1hour of bypass, at the conclusion of bypass, and 1hour after bypass from the prebypass dilution levels. Both the control and the allopurinol group showed significant decreases in 2,3-DPG level at 1hour of bypass from the initial dilution level ( p < 0.001 and < 0.01, respectively). The allopurinol group had a significantly smaller decrease than did the control group, however. IPP, by contrast, caused significant rises in 2,3-DPG levels at both the 7.5 mllkg and 15 mllkg dosages
393 Giannelli et al: Prevention of Increased Hemoglobin-Oxygen Affinity
(p < 0.05 and 0.005, respectively). In terms of differences in 2,3-DPG level at end of bypass from the initial dilution level, Groups 1 and 2 were similar and showed a significant decrease in 2,3-DPG ( p < 0.0001). Groups 3 and 4 had significant rises of 2,3-DPG from the dilution level, with p values of < 0.005 and < 0.025, respectively. The larger IPP dose resulted in a significantly higher 2,3-DPG increase than did Fig3. (A)Relationships between 2,3-DPG and Ps0 prior to and at the conclusion of bypass for the 5 patients in Group 2 and 10 patients in Group 2 (control and allopurinol)f o r w h o m these data are available. T h e P,,, and 2,3-DPG behavior was similar f o r the twogroups. ( B ) Same data f o r the 25 patients in Group 4 (25 rnllkg IPP).
c
a
.w-
I
I
9ic. w
15
!lo-
rn ".
N
the 7.5 mllkg dose (p < 0.001). Groups 1 and 2 (control and allopurinol) behaved similarly to one another, as did Groups 3 and 4, with regard to changes in the 2,3-DPG level at 1hour after bypass from the initial dilution level. The pooled sample of Groups 1and 2 showed a significant fall from the dilution levels (p < O.OOOl), while Groups 3 and 4 had a significant increase ( p < 0.0025). Figure 3 presents the and 2,3-DPG data for the combined control and allopurinol and the 15 mllkg IPP group before and after bypass; the single patient with data in Group 3 (7.5 ml/kg IPP) is not included in the figure or in the analysis. The behavior of Groups 1 and 2 was similar, and together they showed a significant
394 The Annals of Thoracic Surgery Vol 21 No 5 May 1976
decline in P50: prebypass mean P50, 25.7 torr; postbypass, 21.9 torr ( p < 0.001). In Group 4 the prebypass and postbypass levels of 24.8 and 24.9 torr, respectively, were not significantly different. Thus IPP prevented the fall in P50 observed in the control and allopurinol groups. These data show that the usual open-heart surgical procedure is accompanied by drops in 2,3-DPG and P50 levels. Apparently the only alteration in this tendency produced by the administration of allopurinol is a mild delay in the drop of the 2,3-DPG level. From these data it is not clear how much of the drop in P50 was attributable to dilution effect and how much to bypass itself. Indirectly from the 2,3-DPG results, however, coupled with the theoretical and actually observed correlation between 2,3-DPG and P50, a major portion of the drop probably resulted from bypass. The data present strong evidence that IPP not only prevents a drop in 2,3-DPG and P50 levels but actually raises 2,3DPG during bypass. The higher dose of IPP tends to elevate 2,3-DPG more than the lower dose during bypass, while 1 hour after bypass their effect is about the same. 2,3-DPG LEVELS DURING AND AFTER HEARTLUNG BYPASS. These data do not provide additional information regarding the etiology of the progressive decline in 2,3-DPG during bypass. They confirm our earlier studies demonstrating that 2,3-DPG falls below levels attributable to dilution by the bank blood erythrocytes placed in the pump oxygenator priming volume. Neither was the further dilution provided by the 1 or 2 additional units of bank blood frequently added during bypass sufficient to explain the decrease in 2,3-DPG that occurred during the course of perfusion. Our earlier attempts to determine the effect of simple in vitro recirculation of fresh human blood through the extracorporeal circulation were inconclusive. We plan to do studies that will explore further the effects of simple in vitro recirculation, different priming solutions, the introduction of microfilters into the extracorporeal circuit, steroid administration, and the influence of hypothermia.
Relationships between 2,3-DPG and P50 Two methods of approach were employed to determine whether the operative procedure or
Table 5. Correlations between
P50
and 2,3-DPG
Variables
Control & Allo15 M1 purinol IPP (n = 15) (n = 15)
Prebypass Pso,
0.484
0.713
0.541
Postbypass Pjo,
0.485
0.780
0.725
0.379
0.375
0.658
2,3-DPG
2,3- DPG APw, A2,3-DPG
Whole Sample (n = 30)
the administration of IPP affectsthe relationship between 2,3-DPG and P50. The first was to determine the correlation between 2,3-DPG and P50 values at the beginning and end of bypass; this was done for all the patients for whom these determinations were available and then separately for the 15 mllkg IPP group and for the combined control and allopurinol groups. The second approach was to determine the correlation between P50 and 2,3-DPG at the beginning and end of bypass in the control plus allopurinol group, the 15 mllkg IPP group, and for all patients combined. (The correlation between AP50 and A2,3-DPG evaluated the variability of the slopes of the arrows in Figure 3.) Table 5 presents these data. Neither the IPP group nor the control plus allopurinol group showed a significant change in 2,3-DPG and Pso correlation before and after bypass; nor does IPP influence the prebypass to postbypass AP50A2,3-DPG correlation. Thus there is no evidence that either heart-lung bypass or IPP administration affects the relationship of 2,3-DPG to P50.
Clinical Significance of These Studies The data demonstrate that IPP can be administered safely during extracorporeal circulation, thus preventing the decreases in 2,3-DPG and P50 that normally would occur. Because the method of providing for myocardial oxygen requirements during bypass was not standardized, conclusions are not possible regarding the protection against myocardial ischemic damage afforded by IPP in patients with aortic valve disease and those with coronary atherosclerosis. A randomized study has been initiated in these two groups of patients, with standardized methods of handling the problem
395 Giannelli et al: Prevention of Increased Hemoglobin-Oxygen Affinity
of myocardial oxygenation, to evaluate whether IPP protects against ischemic damage. Decreased Pso has relatively little effect u p o n maximal overall bodily oxygen consumption. Two clinical studies have compared reduction i n oxygen supply by hypoxia a n d carbon monoxide [19,27]. The oxygen content of arterial blood w a s reduced 10% by either inspiration of low oxygen mixtures or saturation of 10% of the hemoglobin with carbon monoxide; i n the latter case, the P50 of the remaining hemoglobin was reduced by about 6 torr. I n each of these studies maximal oxygen uptake w a s equally impaired by t h e two interventions. Similarly, i n rats, a fall i n Pj0 from 36 torr (normal for that species) to 30 torr d i d not reduce maximal oxygen uptake [281. A further decrease t o 23 torr produced t h e same work reduction as a 10% diminution i n hemoglobin concentration. Oski's group studied 2 persons w i t h similar degrees of anemia whose oxyhemoglobin dissociation curves were markedly shifted i n opposite directions: Pso of 19 a n d 38 torr, respectively. The patient with the left-shifted curve showed a s h a r p initial fall i n central venous oxygen tension with mild exercise a n d then a marked increase i n cardiac o u t p u t with further exertion. The other patient accommodated to progressive exercise w i t h a gradually accelerating increase i n oxygen extraction so that oxygen requirements were met by a smaller increase in cardiac output. deIn contrast to the minor influence of a crease of several torr i n normal subjects, some studies demonstrate that moderately elevated hemoglobin affinity for oxygen significantly impairs tissue oxygen delivery w h e n arterial disease limits the capacity for increased organ blood flow. Aronow a n d colleagues [4] showed that a 3% hemoglobin carbon monoxide level, induced b y breathing carbon monoxide, adversely affected muscle oxygenation i n 10 calves with lower extremity arterial occlusion. While they performed light exercise, t h e average time to t h e development of intermittent claudication w a s reduced by 20% after carbon monoxide inhalation. Aronow's g r o u p performed similar studies on patients with coronary artery disease and showed that angina w a s induced more quickly d u r i n g controlled exercise testing w h e n 3 t o 4% hemoglobin carbon monoxide levels
were induced by any of several methods: cigarette smoking [2], carbon monoxide inhalation [3], a n d exposure to Los Angeles freeway traffic
[51. These semiquantitative data suggest that increased hemoglobin affinity for oxygen probably is a n important determinant of t h e sufficiency of myocardial oxygenation in patients w i t h coronary artery disease.
References 1. Akerblom 0, DeVerdier CH, Garby L, et al: Restoration of defective oxygen-transport function of stored red blood cells by addition of inosine. Scand J Clin Lab Invest 21:235, 1968 2. Aronow WS, Dedinger J, Rokow SN: Heart rate and carbon monoxide level after smoking high, low and non-nicotine cigarettes: a study in the male patients with angina pectoris. Ann Intern Med 74:697, 1971 3. Aronow WS, Isbell MW: Carbon monoxide effect on exercise-induced angina pectoris. Ann Intern Med 79:392, 1973 4. Aronow WS, Stemmer EA, Isbell MW: Effect of carbon monoxide exposure on intermittent claudication. Circulation 49:415, 1974 5. Aronow WS, Harris CN, Isbell MW, et al: Effect of heavy freeway traffic on cardiopulmonary function in angina (abstract). Chest 62:358, 1972 6. Berman IR, Acinapura A, Zucker MB, et al: Alteration of platelet reactivity during cardio-bypass in man (abstract). Circulation 43,44:Suppl2: 11,1971 7. Bordiuk JM, McKenna PJ, Giannelli S Jr, et al: Alterations in 2,3 diphosphoglycerate and O2 hemoglobin affinity in patients undergoing open heart surgery. Circulation 43:Suppl 1:141, 1971 8. Caen JP, Jenkins CSP, Michel H, et al: Adenosine metabolism in platelets and plasma. Ser Haematol 6:317, 1973 9. DeLevine M, Hill JD, Mielke CH, et al: Blood platelets and coagulation mechanisms during extracorporeal circulation (abstract). Presented at the 45th Scientific Session of the American Heart Association, Dallas, TX, Nov 1972 10. Dixon SH Jr, Limbird BS, Roe CR, et al: Recognition of post-operative acute myocardial infarction: application of isoenzyme techniques. Circulation 47,48:Suppl 3:137, 1973 11. Giannelli S Jr, Conklin EF, Ayres SM, et al: A practical method of transmembrane gas exchange for open heart surgery. Am J Surg 119:519, 1970 12. Hilpert P, Fleischmann RG, Kempe D, et al: The Bohr effect related to blood and erythrocyte pH. Am J Physiol 205:337, 1963 13. Hollander M, Wolfe DA: Non Parametric Methods. New York, Wiley, 1973, p 114
396 The Annals of Thoracic Surgery Vol 21 No 5 May 1976
14. Jesch F, Weber LM, Carey JS: Oxygen affinity of hemoglobin: influence of blood replacement and hemodilution after cardiac surgery, Advances in Experimental Medicine and Biology: Volume 37A, Oxygen Transport to Tissue. Edited by Bmley DF, Bicher HI. New York, Plenum, 1973 15. Krimsky IL: D-2-3 diphosphoglycerate, Methods of Enzymatic Analysis. Edited by Bergmeyer HU. New York, Academic, 1963 16. McKenna PJ, Giannelli S Jr, Bordiuk JM: Increased hemoglobin affinity for oxygen in open heart patients. Crit Care Med 2:73, 1974 17. Miller ID, Sugerman HJ, Cromie WJ, et al: Administration of inosine to man, Proceedings of Conference on preservation of red blood cells. National Academy of Science, Washington DC, 1973 18. Oski FA, Travis SF, Miller LD, et al: The in vitro restoration of red cell 2-3 diphosphoglycerate levels in banked blood. Blood 37:52, 1971 19. Pirnay F, Dujardin J, Deroanne R, et al: Muscular exercise during intoxication by carbon monoxide. J Appl Physiol 31:573, 1971 20. Robinson MA, Louder PB, DeGrachy GC: Red cell metabolism in nonspherocytic congenital haemolytic anemia. Br J Haematol 7:327, 1961 21. Sapsford RN, Blackstone EH, Kirklin JW, et al: Coronary perfusion versus cold ischemic arrest
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