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The metabolic effect of fluoride inhibition of anaerobic glycolysis during hemorrhagic shock
THE
METABOLIC ANAEROBIC
EFFECT GLYCOLYSIS
J*
KENT LESTER
OFFLUORIDE DURING SHOCK
TRINKLE, R.
M.D., BRYANT,
AFTER CLINICAL and experimental hemdeclines and orrhage, oxygen consumption blood lactate concentration rises, indicating a decrease in oxidative metabolism and an increase in anaerobic glycolvsis. It is assumed that anaerobic glycolysis -is then a major metabolic pathway providing energy for cellular function [3, 4, 71. Fluoride, an enzvme inhibitor which blocks an essential step in anaerobic glycolysis [l], was administered to cats prior to bleeding in order to czarnine the significance of anaerobic glycolIsiS as an energy source during hemorrhagic shock. Cats receiving fluoride were compared to control animals in which anaerobic pathwavs were intact.
METHODS Thirty cats were lightly anesthetized with intraperitoneal phenobarbital, 10 mg./lb., and ventilated via an endotracheal tube, respirator, and spirometer with 100% oxygen. The femoral vessels were cannulated and 2 mg.,, lb. was given. intravenous heparin, Each experiment lasted 2 hours: 30-minute From the Division of Cardiothoracic Surgery, Department of Surgery, University of Kentucky hledical Center, Lexington, Kentucky 40506. Submitted for publication Feb. 20, 1969.
INHIBITION HEMORRHAGIC
F.A.C.S., M.D.,
OF
AND F.A.C.S.
control period with anesthesia and mechanical ventilation, 30-minute shock period after rapidlv bleeding &c/lb., and two 30minute observation periods after giving lactated Ringers solution (LRS) 45 ct./lb. Arterial and venous pressure, pH, POZ, and hematocrit, arterial L-lactate, and oxygen consumption were recorded at the conclusion of each 30-minute interval. Half of the animals received sodium fluoride (NaF), 10 mg./lb. 30 minutes prior to bleeding. The animals were divided into four groups by random selection of numbered cards after induction of anesthesia: I. Unbled control, anesthesia and ventilation only (5 cats) II. NaF 10 mg./lb. and unbled control (5 cats ) HIBled 1.5 cc. ,lb. and LRS 45 cc/lb. after 30 minutes of shock (10 cats) I\‘. NaF 10 mg. ‘lb., bled 15 cc. lb., and LRS 45cc. lb. after 30 minutes of shock. (10 cats 1
RESULTS Tables l-6 summarize the in each of the four groups of I was compared with II, and significant differences bv The
averaged data animals. Group III with IV for Wilcoxon Rank $3
JOURSAL
OF
Table
1.
SURGICAL
RESEARCH
Averaged
Arterial
9
VOL.
SO.
and Venous
10,
1969
OCTOBER
pff for Each
Group
of Anin&
at 3O-Minr&
Arterial Groups I II III IV
Control
Shock
7.37 7.37 7.38 7.36
7.36 7.34 7.26 7.27
Control NaF and Control Bled and LRS NaF, bled, and LRS
Table
2.
Averaged
Arterial
and Venous
Venous Observation 7.34 7.35 7.29 7.24
7.36 7.37 7.31 7.30
POz for Each
I
Control
Control NaF and control Bled and LRS NaF. bled. and LRS
II III IV
Table
3.
Averaged
Shock
129 127 123 131
Group
Arterial
and Venous
135 132 130 134
Shock
7.34 7.33 7.36
7.32 7.31 i.21 7.23
of Animals
137 129 133 141
PCOa for Each Group
I II III IV
Control
Control NaF and control Bled and LRS NaF, bled, and LRS
Table
34 35 27 30
4.
Averaged
of Animals
I II III IV
Control
Control NaF and control Bled and LRS NaF, bled, and LRS
Table
-. I II III IV 584
37 32 35 34
5.
Groups Control NaF and control Bled and LRS NaF, bled, and LRS
31 28 28 25
Averaged
Control 88 85 91 92
Lactate
7.31 7.31 7.26 7.24
Intervals
Venous
51 54 74 53
51 60 38 37
Observation 52 56 43 ijl
at 30-Minute
51 53 36 41
Intervals
Venous Observation 33 24 27 27
31 26 32 28
Arterial Lactate, Hematocrit, Weight, for Each Group of Animals
Arterial Groups
Shock
7.30 7.30 7.23 7.20
Shock
Arterial G-.oups
Observation
at 30-Minute
~ Control
Observation
129 130 124 141
Control
i.33
Arterial Groups
Intervals
(mg./lOO
ml.)
Shock
Observation
35 30 100 63
29 33 95 50
Hematocrit Control
Control
Shock
40 38 33 35
33 32 38 30
and A4ortality
(s ) Final
36 34 37 40
33 30 27 29
Observation 35 26 46 33
33 28 37 36
Rate
Weight (lb.1 5.4 5.6 5.5 5.6
Mortality (74) 0 0 30 20
Mean Arterial and Venous Pressure for Each Group of Animals at 30-Minute Intervals Arterial (mm. Hg) Shock Observation 87 89 61 57
92 86 69 64
Control 94 90 81 78
2.2 2.4 1.9 2.1
Venous (mm. Hg) Shock Observation 2.0 2.6 1.0 0.6
2.6 2.6 2.8 2.1
3.0 2.6 2.7 2.2
TRINKLE
Table
6.
Averaged
Oxygen
AND
Consumption
BRYANT:
for
INHIBITION
Each
Group
I
II III
I’i’
Control
4.11
NaF and control Bled and LRS NaF, bled, and LRS
3.96
4.27 4.10 3.32 3.28
4.21 4.16
Sum Test [lo]. There were no apparent or statistical differences in any data except those for arterial lactate. The lactate concentrations :30 minutes after bleeding and 60 minutes after LRS administration were significantly lower in group IV compared to group III (P = .Ol )
DISCUSSION After hemorrhage the decline in oxygen consumption indicates a decrease in aerobic glycolysis, and the simultaneous rise in serum lactate suggests that anaerobic glvcolysis increases. The level to which lactate concentration rises is a rough index of the prognosis and severitv of shock-the higher the lactate the lower the survival rate [2], Lactate accumulates as a metabolic waste due to inadequate oxygen molecules to act as the hydrogen receptor needed to convert lactate to pvruvate [4]. Prior work in this laborator! indicates that lactate per se is not detrimental, and that an exogenous lactate load can be metabolized after circulating volume is restored [9]. Therefore, lactate is merely a svmptom of cellular hypoxia. The next question is whether anaerobic glycolysis yields a significant energy contribution to the cell while the normal aerobic pathwav is suppressed. Animals in group IV, which received NaF before being bled, had lower serum lactate concentrations than group III animals which did not receive NaF. Fluoride blocks anaerobic glycolysis by specifically inhibiting enolase, an enzyme which catalvzes an essential step in anaerobic glycdlysis-the conversion of 2-phosphoglyceric acid to phospho-enol-pyruvic acid plus an energy-rich phosphate bond [ 11. The lower lactate concentration (P = .Ol) in animals
of Animals
Shock
Control
OF
AXAEROBIC
GLYCOLYSIS
at 30-Minute
__ 4.21 4.23 4.47 4.!5?
Intervals
Observation .4.:% 4.21 4.31 3.39
receiving NaF prior to bleeding indicates that the dose of fluoride utilized was sufficient to inhibit enolasc and thus suppress anaerobic glycolysis. In spite of this, there was no significant difference in survival or anv other parameters measured. This indicates t’hat the metabolic contribution of annerobic glycolvsis is insignificant and suggests that cndogenous stores of ATP maintain cellular function during the period when aerobic glycolvsis is suppressed. This conclusion is supported b\r studies demonstrating decreases in tissue ATP concentration during shock [ 5-81.
Sodium fluoride, a metabolic inhibit01 which blocks an essential step in anaerobic glycolysis, was administered to cats prior to hemorrhage. Lower serum lactate (P = .Ol) in the animals receiving NaF indicates that anaerobic glycolysis was effectively suppressed. No significant differences in survival, pH, PO?, PCOZ, or arterial and venous pressures were noted in a similar group of animals which did not receive NaF. These data suggest that anaerobic glycolysis accompanying hemorrhage merclv reflects the presence of cellular hypoxia &d is in itself of little or no metabolic significance.
REFERENCES 1.
2.
3.
Baldwin, E. Dynamic A.Ypects of Biochemistry. London and New York: Cambridge Univ. Press, 1953. Broder, G., and Weil, M. H. Excess lactate: Index of reversibility of shock in hulnan patients. Science 143: 1457, 1964. Bruce, H. A., Jones, J. W., and Strait, G. B. Anaerobic lnetabolic responses to acute maximal
JOURNAL
4.
5. 6.
7.
OF
SURGICAL
RESEARCH
VOL.
9
h-0.
exercise in male athletes, Amer. Heart I. 67:643, 1964. Huckabee, W. E. Relationship of pyruvate and lactate during anaerobic metabolism. III. Effect of breathing low-oxygen gases. C&n. Incest. 37: 264, 1958. Kovach, A. G. B. The Biochemical Response to Injury. New York: Academic Press, Inc., 1960. Lepage, G. A. Biological energy transformations during shock as shown by tissue analysis. Amer. J. Physiol. 146:267, 1946. McShan, W. H., Potter, V. It., Goldman, A., Shipley, E. G., and Meyer, R. K. Biological
586
10,
OCTOBER
8.
1969
energy transfomrations during shock as shown by blood chemistry. Amer. 1. Physiol. 145:93, 1945. Talaat, S. M., Masson, W. H., and Schilling, J. A. Effects of adenosine triphosphate administration in irreversible hemorrhagic shock. Sal-
gery 55:813, 1964. 9.
10.
Trinkle, J. K., Rush, B. F., and Eiseman, B. Metabolism of lactate following major blood loss. Surgery 63:782, 1968. Wilcoxon, F., and Wilcox, R. A. Some rapid approximate statistical procedures. Pearl River, N.Y.: Lederle Laboratories, 1964.
METABOLIC ANAEROBIC
EFFECT GLYCOLYSIS
J*
KENT LESTER
OFFLUORIDE DURING SHOCK
TRINKLE, R.
M.D., BRYANT,
AFTER CLINICAL and experimental hemdeclines and orrhage, oxygen consumption blood lactate concentration rises, indicating a decrease in oxidative metabolism and an increase in anaerobic glycolvsis. It is assumed that anaerobic glycolysis -is then a major metabolic pathway providing energy for cellular function [3, 4, 71. Fluoride, an enzvme inhibitor which blocks an essential step in anaerobic glycolysis [l], was administered to cats prior to bleeding in order to czarnine the significance of anaerobic glycolIsiS as an energy source during hemorrhagic shock. Cats receiving fluoride were compared to control animals in which anaerobic pathwavs were intact.
METHODS Thirty cats were lightly anesthetized with intraperitoneal phenobarbital, 10 mg./lb., and ventilated via an endotracheal tube, respirator, and spirometer with 100% oxygen. The femoral vessels were cannulated and 2 mg.,, lb. was given. intravenous heparin, Each experiment lasted 2 hours: 30-minute From the Division of Cardiothoracic Surgery, Department of Surgery, University of Kentucky hledical Center, Lexington, Kentucky 40506. Submitted for publication Feb. 20, 1969.
INHIBITION HEMORRHAGIC
F.A.C.S., M.D.,
OF
AND F.A.C.S.
control period with anesthesia and mechanical ventilation, 30-minute shock period after rapidlv bleeding &c/lb., and two 30minute observation periods after giving lactated Ringers solution (LRS) 45 ct./lb. Arterial and venous pressure, pH, POZ, and hematocrit, arterial L-lactate, and oxygen consumption were recorded at the conclusion of each 30-minute interval. Half of the animals received sodium fluoride (NaF), 10 mg./lb. 30 minutes prior to bleeding. The animals were divided into four groups by random selection of numbered cards after induction of anesthesia: I. Unbled control, anesthesia and ventilation only (5 cats) II. NaF 10 mg./lb. and unbled control (5 cats ) HIBled 1.5 cc. ,lb. and LRS 45 cc/lb. after 30 minutes of shock (10 cats) I\‘. NaF 10 mg. ‘lb., bled 15 cc. lb., and LRS 45cc. lb. after 30 minutes of shock. (10 cats 1
RESULTS Tables l-6 summarize the in each of the four groups of I was compared with II, and significant differences bv The
averaged data animals. Group III with IV for Wilcoxon Rank $3
JOURSAL
OF
Table
1.
SURGICAL
RESEARCH
Averaged
Arterial
9
VOL.
SO.
and Venous
10,
1969
OCTOBER
pff for Each
Group
of Anin&
at 3O-Minr&
Arterial Groups I II III IV
Control
Shock
7.37 7.37 7.38 7.36
7.36 7.34 7.26 7.27
Control NaF and Control Bled and LRS NaF, bled, and LRS
Table
2.
Averaged
Arterial
and Venous
Venous Observation 7.34 7.35 7.29 7.24
7.36 7.37 7.31 7.30
POz for Each
I
Control
Control NaF and control Bled and LRS NaF. bled. and LRS
II III IV
Table
3.
Averaged
Shock
129 127 123 131
Group
Arterial
and Venous
135 132 130 134
Shock
7.34 7.33 7.36
7.32 7.31 i.21 7.23
of Animals
137 129 133 141
PCOa for Each Group
I II III IV
Control
Control NaF and control Bled and LRS NaF, bled, and LRS
Table
34 35 27 30
4.
Averaged
of Animals
I II III IV
Control
Control NaF and control Bled and LRS NaF, bled, and LRS
Table
-. I II III IV 584
37 32 35 34
5.
Groups Control NaF and control Bled and LRS NaF, bled, and LRS
31 28 28 25
Averaged
Control 88 85 91 92
Lactate
7.31 7.31 7.26 7.24
Intervals
Venous
51 54 74 53
51 60 38 37
Observation 52 56 43 ijl
at 30-Minute
51 53 36 41
Intervals
Venous Observation 33 24 27 27
31 26 32 28
Arterial Lactate, Hematocrit, Weight, for Each Group of Animals
Arterial Groups
Shock
7.30 7.30 7.23 7.20
Shock
Arterial G-.oups
Observation
at 30-Minute
~ Control
Observation
129 130 124 141
Control
i.33
Arterial Groups
Intervals
(mg./lOO
ml.)
Shock
Observation
35 30 100 63
29 33 95 50
Hematocrit Control
Control
Shock
40 38 33 35
33 32 38 30
and A4ortality
(s ) Final
36 34 37 40
33 30 27 29
Observation 35 26 46 33
33 28 37 36
Rate
Weight (lb.1 5.4 5.6 5.5 5.6
Mortality (74) 0 0 30 20
Mean Arterial and Venous Pressure for Each Group of Animals at 30-Minute Intervals Arterial (mm. Hg) Shock Observation 87 89 61 57
92 86 69 64
Control 94 90 81 78
2.2 2.4 1.9 2.1
Venous (mm. Hg) Shock Observation 2.0 2.6 1.0 0.6
2.6 2.6 2.8 2.1
3.0 2.6 2.7 2.2
TRINKLE
Table
6.
Averaged
Oxygen
AND
Consumption
BRYANT:
for
INHIBITION
Each
Group
I
II III
I’i’
Control
4.11
NaF and control Bled and LRS NaF, bled, and LRS
3.96
4.27 4.10 3.32 3.28
4.21 4.16
Sum Test [lo]. There were no apparent or statistical differences in any data except those for arterial lactate. The lactate concentrations :30 minutes after bleeding and 60 minutes after LRS administration were significantly lower in group IV compared to group III (P = .Ol )
DISCUSSION After hemorrhage the decline in oxygen consumption indicates a decrease in aerobic glycolysis, and the simultaneous rise in serum lactate suggests that anaerobic glvcolysis increases. The level to which lactate concentration rises is a rough index of the prognosis and severitv of shock-the higher the lactate the lower the survival rate [2], Lactate accumulates as a metabolic waste due to inadequate oxygen molecules to act as the hydrogen receptor needed to convert lactate to pvruvate [4]. Prior work in this laborator! indicates that lactate per se is not detrimental, and that an exogenous lactate load can be metabolized after circulating volume is restored [9]. Therefore, lactate is merely a svmptom of cellular hypoxia. The next question is whether anaerobic glycolysis yields a significant energy contribution to the cell while the normal aerobic pathwav is suppressed. Animals in group IV, which received NaF before being bled, had lower serum lactate concentrations than group III animals which did not receive NaF. Fluoride blocks anaerobic glycolysis by specifically inhibiting enolase, an enzyme which catalvzes an essential step in anaerobic glycdlysis-the conversion of 2-phosphoglyceric acid to phospho-enol-pyruvic acid plus an energy-rich phosphate bond [ 11. The lower lactate concentration (P = .Ol) in animals
of Animals
Shock
Control
OF
AXAEROBIC
GLYCOLYSIS
at 30-Minute
__ 4.21 4.23 4.47 4.!5?
Intervals
Observation .4.:% 4.21 4.31 3.39
receiving NaF prior to bleeding indicates that the dose of fluoride utilized was sufficient to inhibit enolasc and thus suppress anaerobic glycolysis. In spite of this, there was no significant difference in survival or anv other parameters measured. This indicates t’hat the metabolic contribution of annerobic glycolvsis is insignificant and suggests that cndogenous stores of ATP maintain cellular function during the period when aerobic glycolvsis is suppressed. This conclusion is supported b\r studies demonstrating decreases in tissue ATP concentration during shock [ 5-81.
Sodium fluoride, a metabolic inhibit01 which blocks an essential step in anaerobic glycolysis, was administered to cats prior to hemorrhage. Lower serum lactate (P = .Ol) in the animals receiving NaF indicates that anaerobic glycolysis was effectively suppressed. No significant differences in survival, pH, PO?, PCOZ, or arterial and venous pressures were noted in a similar group of animals which did not receive NaF. These data suggest that anaerobic glycolysis accompanying hemorrhage merclv reflects the presence of cellular hypoxia &d is in itself of little or no metabolic significance.
REFERENCES 1.
2.
3.
Baldwin, E. Dynamic A.Ypects of Biochemistry. London and New York: Cambridge Univ. Press, 1953. Broder, G., and Weil, M. H. Excess lactate: Index of reversibility of shock in hulnan patients. Science 143: 1457, 1964. Bruce, H. A., Jones, J. W., and Strait, G. B. Anaerobic lnetabolic responses to acute maximal
JOURNAL
4.
5. 6.
7.
OF
SURGICAL
RESEARCH
VOL.
9
h-0.
exercise in male athletes, Amer. Heart I. 67:643, 1964. Huckabee, W. E. Relationship of pyruvate and lactate during anaerobic metabolism. III. Effect of breathing low-oxygen gases. C&n. Incest. 37: 264, 1958. Kovach, A. G. B. The Biochemical Response to Injury. New York: Academic Press, Inc., 1960. Lepage, G. A. Biological energy transformations during shock as shown by tissue analysis. Amer. J. Physiol. 146:267, 1946. McShan, W. H., Potter, V. It., Goldman, A., Shipley, E. G., and Meyer, R. K. Biological
586
10,
OCTOBER
8.
1969
energy transfomrations during shock as shown by blood chemistry. Amer. 1. Physiol. 145:93, 1945. Talaat, S. M., Masson, W. H., and Schilling, J. A. Effects of adenosine triphosphate administration in irreversible hemorrhagic shock. Sal-
gery 55:813, 1964. 9.
10.
Trinkle, J. K., Rush, B. F., and Eiseman, B. Metabolism of lactate following major blood loss. Surgery 63:782, 1968. Wilcoxon, F., and Wilcox, R. A. Some rapid approximate statistical procedures. Pearl River, N.Y.: Lederle Laboratories, 1964.