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Meeting Abstracts  |   July 2005
Pyruvate Modulates Hepatic Mitochondrial Functions and Reduces Apoptosis Indicators during Hemorrhagic Shock in Rats
Author Affiliations & Notes
  • Pushpa Sharma, Ph.D.
    *
  • Kane T. Walsh, M.D.
  • Kimberly A. Kerr-Knott, M.D.
  • John E. Karaian, B.S.
  • Paul D. Mongan, M.D.
    §
  • * Assistant Professor, † Staff Anesthesiologist, ‡ Research Assistant, § Staff Anesthesiologist and Associate Professor.
Article Information
Meeting Abstracts   |   July 2005
Pyruvate Modulates Hepatic Mitochondrial Functions and Reduces Apoptosis Indicators during Hemorrhagic Shock in Rats
Anesthesiology 7 2005, Vol.103, 65-73. doi:
Anesthesiology 7 2005, Vol.103, 65-73. doi:
MULTIPLE organ failure in hemorrhagic shock (HS) has been commonly related to metabolic failure due to dysfunctional mitochondria. The liver is known to be the first organ to display the signs of injury during HS due to its high mitochondrial content, increased metabolic rate, lactic acid clearance, and increased production of oxygen and nitrogen species in comparison with other organs.1–3 A relation between mitochondrial dysfunction and the irreversibility of pathologic damage in liver has been reported.4–6 Therefore, it is logical to hypothesize that the pharmacologic agents that can reduce reactive oxygen and nitrogen species and prevent mitochondrial damage may offer a promising treatment for HS.
To test our hypothesis, we examined and compared the effects of pyruvate with another pharmacologic agent, β-hydroxybutyrate (β-HOB), in resuscitative fluids of rats subjected to HS. The rationale for the use of pyruvate and β-HOB is that they are antioxidants, decrease membrane permeability, and produce acetyl coenzyme A as the immediate substrate of the Krebs cycle to generate mitochondrial matrix reduced adenine dinucleotide for oxidative phosphorylation.7 The uptake of β-HOB by mitochondria is adenosine triphosphate (ATP) dependent, whereas pyruvate requires the activity of the mitochondrial enzyme pyruvate dehydrogenase, which is regulated by the content of its E-1α subunit.8 However, it is not known whether pharmacologic activation of pyruvate dehydrogenase complex (PDH) and up-regulation of its E-1α subunit could be linked to the improved hepatococellular ATP, mitochondrial functions, and decreased apoptosis. In addition, the use of β-HOB in the treatment of HS has not been extensively studied in HS. On the other hand, recent work from our laboratory and those of others have shown the use of pyruvate administration in restoring the cellular metabolic functions in the brain and heart and also in enhancing the survival rates during HS or ischemia by preventing the loss of ATP.7,9–12 Despite this, the mechanisms for these responses are not fully understood, and the role of pyruvate in restoring the mitochondrial energetic functions in one of the most important organs, the liver, is still unresolved. The use of pyruvate and β-HOB as alternate adjuvants to metabolic resuscitative fluids may therefore provide a rational approach for the development of an effective metabolic therapy for HS by maintaining the hepatocellular functions.
Materials and Methods
The protocol and experimental procedures were reviewed and approved by the Animal Care and Use Committee of the Uniformed Services University of the Health Sciences at Bethesda, Maryland, with adherence to the Guide for Care and Use of Laboratory Animals  .13 
Animal and Surgical Preparation
Male Sprague-Dawley rats (Taconic Farms, German Town, NY) weighing 225–250 g were used in this study. Rats were not fasted at the time of experiment. Animals were anesthetized by spontaneously breathing 1.6% halothane in room air through a nose cone. The femoral artery and vein were surgically cannulated with PE-50 polyethylene tubing for fluid administration, blood sampling, and continuous monitoring of blood pressure. The incision was closed with interrupted sutures. Core temperature (rectal) was maintained at 37°± 0.2°C with a heat lamp. The rats were continuously monitored for mean arterial blood pressure (MAP) by connecting the arterial catheter to a pressure transducer and computerized physiograph (LABVIEW 5; National Instruments, Austin, TX). The readings were recorded every 10 s and averaged per minute over the course of the experiment. The baseline MAP was stabilized by infusing normal saline. At this point, the concentration of halothane was reduced to 1% throughout the experiment.
Experimental Protocol
Induction of HS.
Computer-controlled arterial hemorrhage to a target of MAP of 40 mmHg was achieved over 15 min by the withdrawal of blood from femoral arterial catheter using an Instech P720 peristaltic pump (Instech Laboratories Inc., Plymouth Meeting, PA). This MAP of 40 mmHg was maintained by either removal or infusion of blood for a 60-min (T60) shock period.
Resuscitation Groups.
The animals were randomly assigned to the four treatment groups (n = 6 animals/group). In group 1, instrumented time control rats were anesthetized and instrumented in the same manner as rats in the other group but did not undergo arterial hemorrhage except the withdrawal of blood for laboratory investigation. Vital signs were recorded for 120 min by infusing normal saline three times the volume of blood lost. Groups 2, 3, and 4 were subjected to hemorrhage and resuscitation, but these three groups differed in the composition of resuscitative fluids: Group 2 received 7.5% hypertonic saline (HTS) to match the osmolality effects of sodium pyruvate, group 3 received 256 mg/ml hypertonic β-HOB, and group 4 received 225 mg/ml hypertonic sodium pyruvate infused at a rate of 5 ml/kg for 1 h.
Analytical Sampling Procedures.
Immediately before hemorrhage (T0) and every 60 min thereafter, arterial blood was collected for the measurement of pH, base excess, and blood gases (IL 1610 Blood Gas Analyzer; Instrumentation Laboratories, Lexington, KY). Serum lactate and pyruvate from arterial blood samples were also measured with a CMA 600 Analyzer (CMA/Microdialysis, Acton, MA).
Tissue Sampling.
Before death at T120, the rats’ livers were excised immediately. The left lobe was placed in ice-cold isotonic sucrose Tris EDTA buffer, pH 7.4, for isolation of mitochondrial and cytosolic fraction,10 while the remaining tissue was quickly frozen in liquid nitrogen, pulverized, and stored in cryovials at −80°C for analysis of lipid peroxidation, energy metabolites, and Western blotting for the semiquantitation of nitric oxide synthase and poly–adenosine diphosphate (ADP) ribose polymerase (PARP) proteins. Animals were then killed by exsanguination.
Isolation of Mitochondrial and Cytosolic Fractions.
The duration from harvesting of liver to homogenization was kept as short as possible (< 5 min). Tissue was minced, washed, and homogenized in ice-cold mitochondrial isolation buffer consisting of 0.25 m sucrose, 10 mm HEPES buffer, pH 7.4, and 1 mm EGTA in a ratio of 1:10 (vol/vol). Mitochondria were isolated by differential centrifugation at −4°C. In brief, cell membranes and nuclei were separated by centrifugation of tissue homogenate at 1,000g  . The first supernatant was centrifuged at 10,000g  for 15 min to get a mitochondrial pellet (S1). The supernatant thus obtained was centrifuged at high speed, 100,000g  , for 60 min in a Beckman Ultracentrifuge-TL100 (Scientific Surplus, LLC, Hills Borough, NJ) to obtain the cytosolic pellet. The mitochondrial pellet was washed three times in mitochondrial isolation buffer without EGTA to obtain relatively pure mitochondria for determining the respiratory rates, activation state of PDH, level of its E-1α subunit, and citrate synthase activities. The cytosolic fraction was used for Western analysis of cytochrome c. All measurements were performed in duplicate, and the average of the two from each of the six animals in each group was used for data analysis.
Analytical Procedures
Measurement of Liver Enzymes.
The serum contents of liver enzymes alanine aminotransferase and aspartate aminotransferase were determined in our clinical chemistry laboratory at the Uniformed Services University of the Health Sciences.
Measurement of Tissue Lipid Peroxidation Level.
Malondialdehyde, a degradation product of peroxidized phospholipids, was quantified as an indirect marker of oxidative stress using a colorimetric thiobarbituric acid assay kit from Calbiochem (La Jolla, CA). The Malondialdehyde concentrations were normalized to protein content and expressed as nmol/mg tissue protein.
Protein Assay.
Protein contents in tissue, mitochondrial, or cytosolic samples were determined according to Bradford assay using bovine serum albumin as a standard.14 
Determination of Adenine Nucleotides and Energy Charge in Liver.
To determine the contents of cellular nucleotides, pulverized frozen T120 liver tissue was deproteinated by incubation in 1.5 m perchloric acid (3 ml/mg protein) for 10 min and then neutralized with 2 m KOH. Nucleotides ATP, ADP, and adenosine monophosphate (AMP) were determined as a measure of reduced adenine dinucleotide formation at 340 nm using hexokinase and glucose 6-phosphate dehydrogenase as substrates.15 Energy charge was calculated as an indicator of cellular metabolic status in liver by using the equation Energy Charge =[ATP]+ 0.5[ADP]/([ATP]+[ADP]+[AMP])
Determination of Pyruvate Dehydrogenase Complex Activity.
The total PDH (PDHt) and the native PDH (PDHa) in their activated or dephosphorylated form at T120 were determined in mitochondrial samples. For the measurement of PDHtactivity, the inactive (phosphorylated) form of the enzyme was converted to the “active” (dephosphorylated) form by incubating 25 mg mitochondrial protein in 1.0 ml of 10 mm MgCl2and 10 mm CaCl2for 5 min at 25°C. The activities of PDHtand PDHawere determined spectrophotometrically after reduction of p  -iodonitrotetrazolium violet by PDH-dependent reduced adenine dinucleotide production at 500 nm.16 The PDH activity was calculated using the molar extinction coefficient of p  -iodonitrotetrazolium violet (5.4 × 103cm−1) and expressed as μmol · min−1· mg−1protein. The fractional activity of PDHain PDHtwas calculated as (PDHa/PDHt) × 100. To examine the relation between the fraction of active PDH and its E-1α component, Western blot analysis was conducted using the same mitochondrial preparations.
Citrate Synthase Assay.
As a standard marker of mitochondrial content in the samples, citrate synthase activity was measured in the mitochondrial pellet. The assay buffer included 50 mm Tris (pH 8.0), 0.5 mm DTNB, 0.1 mm acetyl coenzyme A, 0.05% Triton X-100, and 20 μg mitochondrial protein. The reaction was started by the addition of 0.5 mm oxaloacetate, and absorbance at 412 nm followed for several minutes at 30°C. Citrate synthase activity was determined according to the method of Srere and Matsuoka,17 which couples coenzyme A to DTNB.
Measurement of Mitochondrial Respiratory Activity.
The rate of oxygen consumption in freshly isolated liver mitochondria was measured at room temperature using three-channel titration-injection respirometers (StrathKelvin oxygen microelectrodes with Oxygraph [Glasgow, United Kingdom] for data acquisition and analysis). One hundred microliters of the respiratory buffer (250 mm sucrose, 10 mm HEPES, pH 7.2, 20 mm KCl, 5 mm potassium phosphate, and 2 mm MgCl2) was added to each electrode, stirred, and equilibrated for 30 min with air in the Oxygraph chambers until a stable baseline was obtained. The mitochondrial suspension was added to give a final concentration of 1 mg/ml protein in 100 μl incubation buffer consisting of respiratory buffer plus substrates/inhibitors. Oxygen flux at the resting state or state 4 respiration through various segments of the electron transport chain (ETC) was determined using (1) flux of electrons from complex I to the end of the ETC (10 mm glutamate and 5 mm malate); (2) flux of electrons from complex II to the end of the ETC (after inhibition of complex I with 5 μm rotenone; state 4 respiration was restored by adding 10 mm succinate as complex II substrate); (3) flux of electrons from complex III to the end of the ETC (inhibition of complex III with 5 mm antimycin A was followed by adding 0.5 mm TMPD and 2 mm ascorbate substrates for complex IV [cytochrome c oxidase, cyclooxygenase]). Oxygen consumption measured in the presence of added ADP (active) was defined as state 3 respiration, and that measured after consumption of ADP (resting) was defined as state 4 respiration.18 Respiratory control ratio was calculated as ratio of state 3 respiration to state 4 respiration and was used as a marker of mitochondrial respiratory activity. Mitochondrial respiration was calculated as the nanomoles of oxygen per minute per milligram of protein.
Western Blot Analysis for E-1α Subunit of PDH, Nitric Oxide Synthase, and Indicators of Apoptosis.
Mitochondria were used for the immunodetection of E-1α protein, tissue homogenates for nitric oxide synthase and PARP, and cytosol was used for the detection of cytochrome c. The samples for Western blotting were prepared in ice-cold lysing buffer (1:10 [wt/vol]) consisting of phosphate-buffered saline at a pH of 7.4, 230 mg/ml PMSF, 1 μg/ml leupeptin, and 1 μg/ml aprotinin in 1% Triton X-100. After brief sonication and centrifugation (1,000g  × 10 min), these samples were denatured in Laemmli sample buffer. Approximately 15 μg protein was resolved on 10% sodium dodecyl sulfate gel and transferred to the nitrocellulose membrane of 0.45-μm pore size. The membranes were probed with primary mouse monoclonal antibody to either anti E-1α (dilution, 1:1,000; Molecular Probes, Eugene, OR), anti–nitric oxide synthase (dilution, 1:500; BD Transduction Laboratories, Lexington, KY), anti–cytochrome c (dilution, 1:500; Trevigen, Gaithersburg, MD), or anti PARP (dilution, 1:2,000; Biomol Research Laboratories Inc., Plymouth Meeting, PA). Detection was accomplished using horseradish peroxidase–conjugated antibody and enhanced chemiluminescence reagents (Pierce, Rockford, IL). Ponceau staining of the membranes with transferred proteins showed an equal amount of protein loaded in each lane (data not shown). Negative controls were performed by omitting the primary antibodies. The regulation of nitric oxide synthase, PARP, and cytochrome c was also standardized against antibodies to antiactin as a housekeeping gene. Human heart mitochondria from Mito-sciences (Eugene, OR) were used as control/marker for E-1α protein. Quantitative determinations of signal intensity of the protein bands were performed by measuring the integrated optical density using the Scion image program from Scion Corporation (Frederick, MD).
Statistical Analysis
All data are presented as mean ± SE or median and range between 25 and 75% distribution. Statistical inference was performed using Sigma-Stat 3.1 (Aspire Software International, Leesburg, VA). Differences between groups for nonrecurring measurements were assessed using one-way analysis of variance. Differences between and within groups for multiple independent samples were performed by Kruskal-Wallis one-way analysis of variance on ranks followed by the Student-Newman-Keuls method. The level of statistical significance was accepted at P  < 0.05 after correction for multiple comparisons.
Results
Hemodynamic and Physiologic Response to HS and Resuscitation
Hemodynamic parameters in the control group (group 1) were stable throughout the experiment (table 1). The volume of shed blood for the induction of shock (40 mmHg) did not differ significantly among the various shock groups (groups 2, 3, and 4; table 1; P  > 0.05) and was in the range previously reported for the same level (40 mmHg) of HS.19 Because this was a computer-controlled shock model, the volumes of resuscitative fluids infused in each shock group were also similar (table 1). In comparison to baseline values, all animals in the hemorrhaged group had a significant increase in arterial partial pressure of oxygen (Po2). All treatments were equally effective in decreasing Po2and increasing partial pressure of carbon dioxide (Pco2) (P  > 0.05).
Table 1. Hemodynamic and Metabolic Parameters during the Experiments 
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Table 1. Hemodynamic and Metabolic Parameters during the Experiments 
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Osmolality, Acid–Base Parameters, and Markers of Ischemic Injury
Serum osmolality values were similar in all resuscitated groups. The arterial base excess, a reliable indicator of the severity of HS, oxygen debt, and volume deficit, was negative at the end of the 60-min shock period. Pyruvate administration prevented the magnitude of changes in acid–base parameters that occurred in other treatment groups (P  < 0.05). Also, in comparison to controls, hemorrhaged animals showed signs of ischemic conditions characterized by significant lactic acidosis, a low lactate/pyruvate ratio, and a decrease in pH (P  < 0.05) at T60. However, pyruvate treatment was able to reduce these ischemic events when compared with HTS or β-HOB.
Pyruvate Effects the Release of Hepatic Enzymes and Lipid Peroxidation
The data presented in table 2indicate that after HS at T120, plasma concentrations of aspartate aminotransferase and alanine aminotransferase were significantly increased in the HTS and β-HOB groups, and infusion of pyruvate reduced their concentrations, which were similar to those of control operated animals. Also, in these animals, HTS or β-HOB treatment showed a significant increase in the accumulation of malondialdehyde concentrations in liver tissue when compared with pyruvate or control animals (P  < 0.05).
Table 2. Effect of Various Treatments on Liver Dysfunction, Lipid Peroxidation, Adenine Nucleotides, and Energy Charge 
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Table 2. Effect of Various Treatments on Liver Dysfunction, Lipid Peroxidation, Adenine Nucleotides, and Energy Charge 
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Pyruvate Effects on Activation State of Mitochondrial Pyruvate Dehydrogenase and Up-regulation of the E-1α Subunit
The data depicted in figure 1show that in comparison to control operated animals, the activities of both PDHtand PDHawere significantly reduced in HTS and β-HOB groups and not in pyruvate-treated animals (P  > 0.05). The protective effect of pyruvate on the recovery of PDH activities was also substantiated by the observations that PDHawas 57 ± 5.9, 23 ± 4.5, 42 ± 1.8, and 61 ± 2.9% of PDHtin control, HTS, β-HOB, and pyruvate groups, respectively (P  < 0.05 between HTS and β-HOB). The citrate synthase activity in hepatic mitochondria (nmol · min−1· mg−1mitochondrial protein) was approximately similar in all treatment groups (mean ± SE: control, 121 ± 30.7; HTS, 129 ± 25.8; β-HOB, 123 ± 27.5; pyruvate, 133 ± 22.3). The Western blot analysis of 40-kd E-1α protein in figure 2also shows significantly darker protein bands in control and pyruvate groups when compared with HTS– or β-HOB–treated animals. The quantitative densitometric analysis of this protein also indicates that it was increased in pyruvate-treated animals by factors of approximately 5 and 3 in comparison with the HTS and β-HOB groups, respectively (P  < 0.05).
Fig. 1. Effect of treatments on the activity of total and active pyruvate dehydrogenase complex in rat liver mitochondria. Data are presented as mean ± SEM of four to six experiments. The resuscitative fluids containing hypertonic saline (HTS), β-hydroxybutyrate (β-HOB), and pyruvate were started 60 min after the beginning of the hemorrhage procedure. The control group of animals (instrumented time control) received only normal saline. The liver was harvested at 120 min. The activities of native or total pyruvate dehydrogenase complex (total PDH) and the active form of pyruvate dehydrogenase complex (active PDH) were determined spectrophotometrically. UNHANDLED_ENTITY  P  < 0.05 comparing the pyruvate group with HTS or β-HOB for the total pyruvate dehydrogenase complex activity. *  P  < 0.05 comparing the pyruvate group with HTS and β-HOB for the active form of pyruvate dehydrogenase complex. 
Fig. 1. Effect of treatments on the activity of total and active pyruvate dehydrogenase complex in rat liver mitochondria. Data are presented as mean ± SEM of four to six experiments. The resuscitative fluids containing hypertonic saline (HTS), β-hydroxybutyrate (β-HOB), and pyruvate were started 60 min after the beginning of the hemorrhage procedure. The control group of animals (instrumented time control) received only normal saline. The liver was harvested at 120 min. The activities of native or total pyruvate dehydrogenase complex (total PDH) and the active form of pyruvate dehydrogenase complex (active PDH) were determined spectrophotometrically. UNHANDLED_ENTITY  P  < 0.05 comparing the pyruvate group with HTS or β-HOB for the total pyruvate dehydrogenase complex activity. *  P  < 0.05 comparing the pyruvate group with HTS and β-HOB for the active form of pyruvate dehydrogenase complex. 
Fig. 1. Effect of treatments on the activity of total and active pyruvate dehydrogenase complex in rat liver mitochondria. Data are presented as mean ± SEM of four to six experiments. The resuscitative fluids containing hypertonic saline (HTS), β-hydroxybutyrate (β-HOB), and pyruvate were started 60 min after the beginning of the hemorrhage procedure. The control group of animals (instrumented time control) received only normal saline. The liver was harvested at 120 min. The activities of native or total pyruvate dehydrogenase complex (total PDH) and the active form of pyruvate dehydrogenase complex (active PDH) were determined spectrophotometrically. UNHANDLED_ENTITY  P  < 0.05 comparing the pyruvate group with HTS or β-HOB for the total pyruvate dehydrogenase complex activity. *  P  < 0.05 comparing the pyruvate group with HTS and β-HOB for the active form of pyruvate dehydrogenase complex. 
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Fig. 2. A representative Western blot of rat liver mitochondria showing the expression of E-1α subunit of pyruvate dehydrogenase complex in various treatment groups. Mitochondrial samples obtained from liver tissues at T120 were subjected to electrophoresis and immunostaining with antibodies to anti–E-1α protein. The considerably darker protein band with significantly higher integrated optical density (IOD) in the pyruvate-treated group when compared with others indicates that pyruvate prevented the loss of pyruvate dehydrogenase E-1α subunit. 
Fig. 2. A representative Western blot of rat liver mitochondria showing the expression of E-1α subunit of pyruvate dehydrogenase complex in various treatment groups. Mitochondrial samples obtained from liver tissues at T120 were subjected to electrophoresis and immunostaining with antibodies to anti–E-1α protein. The considerably darker protein band with significantly higher integrated optical density (IOD) in the pyruvate-treated group when compared with others indicates that pyruvate prevented the loss of pyruvate dehydrogenase E-1α subunit. 
Fig. 2. A representative Western blot of rat liver mitochondria showing the expression of E-1α subunit of pyruvate dehydrogenase complex in various treatment groups. Mitochondrial samples obtained from liver tissues at T120 were subjected to electrophoresis and immunostaining with antibodies to anti–E-1α protein. The considerably darker protein band with significantly higher integrated optical density (IOD) in the pyruvate-treated group when compared with others indicates that pyruvate prevented the loss of pyruvate dehydrogenase E-1α subunit. 
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Pyruvate Effects on Hepatic Adenine Nucleotides Contents and Energy Charge
The changes in adenine nucleotides and purine catabolites reflecting energy metabolites in hepatic tissue after resuscitation are shown in table 2. Data suggests that pyruvate was significantly effective in preventing the loss of hepatocellular ATP in comparison with HTS or β-HOB treatments (percent of controls ATP in HTS, 17%; β-HOB, 33%; pyruvate, 72%, P  < 0.05). A concomitant increase in ADP and decrease in AMP concentrations after pyruvate treatment in comparison with other groups resulted in a significantly higher ATP/ADP ratio and a higher energy charge, which was similar to that of the control group (P  > 0.05). However, total adenine nucleotide content (ATP plus ADP plus AMP) remained unchanged in all treatment groups.
Pyruvate Effects on Hepatic Mitochondrial Respiration in HS
The capacity of the ETC to oxidize substrates at its various segments was examined by measuring the respiratory control ratio with complex specific substrates. Data presented in table 3indicate that in comparison to control animals, liver mitochondria from HTS– and β-HOB–treated animals showed significantly increased state 4 (resting) respiration. Treatment with pyruvate attenuated these mitochondrial respiratory dysfunctions after HS. Rotenone-sensitive decrease in respiratory control ratio with substrates for complex II and I was significantly low in the HTS and β-HOB groups when compared with pyruvate. In addition, the oxygen consumption was still decreased after complex III inhibition with antimycin (antimycin A plus substrates of complex IV). Our results also demonstrate that respiratory control ratios with complex IV substrates were significantly lower in comparison with the substrates for complex I and II in HTS and β-HOB treatment groups when compared with pyruvate.
Table 3. Effect of Resuscitation Medium on Hepatic Mitochondrial Respiration in Hemorrhagic Shock 
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Table 3. Effect of Resuscitation Medium on Hepatic Mitochondrial Respiration in Hemorrhagic Shock 
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Evidence of Decreased Nitric Oxide Synthase, Low Cytochrome c Release, and Decreased Poly-ADP Ribose Polymerase Fragmentation in Liver by Pyruvate in HS
To gain insight into the molecular mechanisms of the action of HTS, β-HOB, and pyruvate on mitochondrial dysfunctions and cell death, inducible nitric oxide synthase, cytosolic cytochrome c, and PARP fragmentation expression were examined in liver from each treatment group. The Western blotting data in figure 3show significantly lighter protein bands from the pyruvate treatment in comparison with HTS and β-HOB. The integrated optical density of inducible nitric oxide synthase (table 4) in pyruvate-treated animals was not significantly different from that of the control group (P  > 0.05), but it was 25% and 43% of that of the HTS and β-HOB groups, respectively (P  < 0.05). The release of cytochrome c from mitochondria into the cytosol is an initial step to trigger the apoptotic events leading to DNA damage and cell death. The middle panel of figure 3shows that cytochrome c release into the cytosol was significantly lower in the pyruvate group than the remaining other groups except the control group, confirmed by integrated optical density data. The bottom panel of figure 3shows that the intact PARP was abundant in all liver samples as a 116-kd protein. Examining the PARP cleavage pattern in these samples shows that a background apoptosis signal was detectable as the faint 85-kd fragments of PARP in the liver of pyruvate-treated animals, but 85-kd and 50-kd bands with higher integrated optical density were noticed in the HTS– and β-HOB–treated HS rats (table 4; P  < 0.05, n = 3). The percentage of PARP fragmentation was calculated by using the formula (optical density of 85-kd fragmented PARP)/optical density of total PARP (116-kd intact PARP + 85-kd fragmented PARP) × 100. The percentage of total PARP fragmentation was 21 ± 5% in the control group, 73 ± 5% for HTS, 68 ± 6% for β-HOB, and 27 ± 4% for pyruvate (table 4; P  < 0.05, n = 3). In addition, the total PARP was significantly increased in all HS groups when compared with controls (P  < 0.05), indicating a trigger of DNA repair mechanism by the activation of PARP.
Fig. 3. Representative Western blot of rat liver showing the expression of inducible nitric oxide synthase (iNOS), poly–adenosine diphosphate ribose polymerase (PARP), cytochrome c, and actin in various treatment groups. β-HOB =β-hydroxybutyrate; HTS = hypertonic saline. 
Fig. 3. Representative Western blot of rat liver showing the expression of inducible nitric oxide synthase (iNOS), poly–adenosine diphosphate ribose polymerase (PARP), cytochrome c, and actin in various treatment groups. β-HOB =β-hydroxybutyrate; HTS = hypertonic saline. 
Fig. 3. Representative Western blot of rat liver showing the expression of inducible nitric oxide synthase (iNOS), poly–adenosine diphosphate ribose polymerase (PARP), cytochrome c, and actin in various treatment groups. β-HOB =β-hydroxybutyrate; HTS = hypertonic saline. 
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Table 4. Intensity of Nitric Oxide Synthase, Cytochrome c, and PARP Protein Bands in Response to Various Resuscitative Fluids in Hemorrhagic Shock Rat Liver 
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Table 4. Intensity of Nitric Oxide Synthase, Cytochrome c, and PARP Protein Bands in Response to Various Resuscitative Fluids in Hemorrhagic Shock Rat Liver 
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Discussion
This study demonstrates the beneficial effects of pyruvate on liver function, hepatocellular injury, mitochondrial functions, and indicators of apoptosis when compared with another ketone body, β-HOB, or saline solutions. In terms of MAP (40 mmHg), amount of blood shed, base excess, pH, Po2, Pco2, and lactate, there were no differences among the various shock groups, indicating that variation in the intensity of HS was not likely the cause of differences in the outcome of various treatments.
Our data strongly suggest a role for pyruvate in preventing liver damage in HS as indicated by a significant decrease in plasma concentrations of liver enzymes aspartate aminotransferase and alanine aminotransferase in comparison to hypertonic saline or β-HOB treatments. This effect of pyruvate was not related to the osmotic effects of the resuscitative fluids because serum osmolality values at the end of the resuscitation period were similar in all animal groups. However, the protective effect of pyruvate in reducing liver injury could be attributed to its antioxidant properties, because free radical scavengers have shown beneficial effects on liver function after ischemia–reperfusion.20 In addition, a significant decrease in lipid peroxidation (accumulation of malondialdehyde in liver) with pyruvate treatment reported in the current study raises the possibility that HS-mediated liver injury is associated with oxidative stress, which can be either a cause or a consequence of mitochondrial dysfunctions.
At low pyruvate concentrations, the availability of pyruvate controls its own decarboxylation, which is not affected by any component of PDH. However, at high pyruvate concentrations, the reaction is strictly controlled by all of the PDH components, especially E-1α.21 The amount of pyruvate we have used in the perfusate is of supramaximal level, and it seems that PDH activity in HS is regulated by the content of E-1α protein. We have observed a threefold higher level of E-1α protein in the pyruvate-treated animals when compared with HTS and β-HOB treatment. These observations show either an increased expression or attenuation in loss of the E-1α protein. The activities of PDHtand percentage activation of PDHareported in the control group are comparatively lower than the values reported for controls in the literature. The underlying mechanisms may be the nutritional state of the animal, techniques of the PDH measurement, and the fact that in our study control group, animals were not totally controlled because they were subjected to the trauma of surgery and anesthesia. Although the results of these experiments cannot account for why these differences occurred, they do contribute toward our understanding of pyruvate utilization via  regulation of PDH activity in the liver during HS.
In this study, we have reported a significant decrease of PDHtin HTS– and β-HOB–treated animals when compared with pyruvate. This difference in mitochondrial PDHtmay indicate oxidative damage of PDH components because citrate synthase (mitochondrial matrix protein), which is more resistant to oxidative stress, was not affected by control or shock treatments. Our in vivo  studies have confirmed a positive relation between the decreased mitochondrial PDHtand the percentage of PARP fragmentation, an indicator of apoptosis, which can be due to increased oxidative stress during HS because pyruvate treatment was able to reverse these changes. In this study, we have provided evidence supporting the notion that increases in PDHaby pyruvate can enhance hepatic energy charge. Our results are in accord with those of Jope and Blass,22 who reported that the proportion of PDH in the active form in both brain and liver mitochondria changed inversely with changes in mitochondrial energy charge. We found that inclusion of pyruvate into the resuscitation fluid can stimulate hepatic metabolism by increasing the pool of high-energy adenine nucleotides, and these pyruvate-mediated changes may have subtle effects on mitochondrial dysfunctions leading to cellular energy depletion. These observations can also explain why pyruvate is an effective protective agent and β-HOB is not, because the entry of β-HOB into the cell is controlled by the availability of cellular ATP, which is depleted in hemorrhagic shock, whereas pyruvate can enhance its own oxidation by stimulating PDH activity.23–25 
Availability of mitochondrial substrate, oxygen, and an intact ETC are the key components for adequate ATP production. Our results suggest that in the liver, HS can cause poor flux of electrons from the segments after complex I and II, and also, the defect lies in the transfer of electrons at the terminal end of the ETC (complex IV or cytochrome oxidase). The precise mechanism of respiratory control ratio inhibition at complex IV is not known, but it has been suggested (1) that nitric oxide could inhibit the enzyme cytochrome oxidase by competing with oxygen that can result in the formation of harmful molecules ONOO− (nitrates) 26,27 and (2) that the loss of cytochrome c is an important substrate for enzyme cytochrome c oxidase (complex IV).
Stimulation of nitric oxide by inducible nitric oxide synthase can also be viewed as a defense mechanism to abate oxidative stress28 in HS because, in our control study, although nitric oxide synthase was slightly up-regulated, it did not trigger the apoptotic events such as release of cytochrome c and PARP fragmentation. The release of cytochrome c out of the intramitochondrial space into the cytosol is an important trigger for caspase activation, which leads to the events characteristic of apoptosis. Our data show that pyruvate treatment of HS inhibits cytochrome c release from the mitochondria, thereby preventing up-regulation of indicators of cell death such as PARP fragmentation. Rapid activation of this enzyme depletes the intracellular concentration of its substrate, nicotinamide adenine dinucleotide, thus slowing the rate of glycolysis, electron transport, and subsequently ATP formation. This process can result in cell dysfunction and cell death.29 Because of the conflicting role of PARP in apoptosis and necrosis,30,31 it is difficult to define the HS-mediated cell death by apoptosis or necrosis, but it is clear from our study that inhibition of PARP fragmentation attenuates liver injury and prevents loss of cellular ATP. Thus, pyruvate also mediates its pharmacologic effects, in part by preventing the cleavage of PARP.
In conclusion, the beneficial effects of pyruvate as an alternate substrate in preventing HS-mediated liver injury can be attributed to increases in cellular energetic levels through its antioxidant and antinitrosyl properties and prevention of mitochondrial damage.
The authors thank Mulchand S. Patel, Ph.D. (Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York), for discussion with pyruvate dehydrogenase complex activity, Michael Graham Espey, Ph.D. (Radiation Biology Branch, National Institutes of Health/National Cancer Institute, Bethesda, Maryland), for discussion with inducible nitric oxide synthase study, and Michael J. Dymond, B.S. (Department of Anesthesiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland), for technical help.
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Fig. 1. Effect of treatments on the activity of total and active pyruvate dehydrogenase complex in rat liver mitochondria. Data are presented as mean ± SEM of four to six experiments. The resuscitative fluids containing hypertonic saline (HTS), β-hydroxybutyrate (β-HOB), and pyruvate were started 60 min after the beginning of the hemorrhage procedure. The control group of animals (instrumented time control) received only normal saline. The liver was harvested at 120 min. The activities of native or total pyruvate dehydrogenase complex (total PDH) and the active form of pyruvate dehydrogenase complex (active PDH) were determined spectrophotometrically. UNHANDLED_ENTITY  P  < 0.05 comparing the pyruvate group with HTS or β-HOB for the total pyruvate dehydrogenase complex activity. *  P  < 0.05 comparing the pyruvate group with HTS and β-HOB for the active form of pyruvate dehydrogenase complex. 
Fig. 1. Effect of treatments on the activity of total and active pyruvate dehydrogenase complex in rat liver mitochondria. Data are presented as mean ± SEM of four to six experiments. The resuscitative fluids containing hypertonic saline (HTS), β-hydroxybutyrate (β-HOB), and pyruvate were started 60 min after the beginning of the hemorrhage procedure. The control group of animals (instrumented time control) received only normal saline. The liver was harvested at 120 min. The activities of native or total pyruvate dehydrogenase complex (total PDH) and the active form of pyruvate dehydrogenase complex (active PDH) were determined spectrophotometrically. UNHANDLED_ENTITY  P  < 0.05 comparing the pyruvate group with HTS or β-HOB for the total pyruvate dehydrogenase complex activity. *  P  < 0.05 comparing the pyruvate group with HTS and β-HOB for the active form of pyruvate dehydrogenase complex. 
Fig. 1. Effect of treatments on the activity of total and active pyruvate dehydrogenase complex in rat liver mitochondria. Data are presented as mean ± SEM of four to six experiments. The resuscitative fluids containing hypertonic saline (HTS), β-hydroxybutyrate (β-HOB), and pyruvate were started 60 min after the beginning of the hemorrhage procedure. The control group of animals (instrumented time control) received only normal saline. The liver was harvested at 120 min. The activities of native or total pyruvate dehydrogenase complex (total PDH) and the active form of pyruvate dehydrogenase complex (active PDH) were determined spectrophotometrically. UNHANDLED_ENTITY  P  < 0.05 comparing the pyruvate group with HTS or β-HOB for the total pyruvate dehydrogenase complex activity. *  P  < 0.05 comparing the pyruvate group with HTS and β-HOB for the active form of pyruvate dehydrogenase complex. 
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Fig. 2. A representative Western blot of rat liver mitochondria showing the expression of E-1α subunit of pyruvate dehydrogenase complex in various treatment groups. Mitochondrial samples obtained from liver tissues at T120 were subjected to electrophoresis and immunostaining with antibodies to anti–E-1α protein. The considerably darker protein band with significantly higher integrated optical density (IOD) in the pyruvate-treated group when compared with others indicates that pyruvate prevented the loss of pyruvate dehydrogenase E-1α subunit. 
Fig. 2. A representative Western blot of rat liver mitochondria showing the expression of E-1α subunit of pyruvate dehydrogenase complex in various treatment groups. Mitochondrial samples obtained from liver tissues at T120 were subjected to electrophoresis and immunostaining with antibodies to anti–E-1α protein. The considerably darker protein band with significantly higher integrated optical density (IOD) in the pyruvate-treated group when compared with others indicates that pyruvate prevented the loss of pyruvate dehydrogenase E-1α subunit. 
Fig. 2. A representative Western blot of rat liver mitochondria showing the expression of E-1α subunit of pyruvate dehydrogenase complex in various treatment groups. Mitochondrial samples obtained from liver tissues at T120 were subjected to electrophoresis and immunostaining with antibodies to anti–E-1α protein. The considerably darker protein band with significantly higher integrated optical density (IOD) in the pyruvate-treated group when compared with others indicates that pyruvate prevented the loss of pyruvate dehydrogenase E-1α subunit. 
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Fig. 3. Representative Western blot of rat liver showing the expression of inducible nitric oxide synthase (iNOS), poly–adenosine diphosphate ribose polymerase (PARP), cytochrome c, and actin in various treatment groups. β-HOB =β-hydroxybutyrate; HTS = hypertonic saline. 
Fig. 3. Representative Western blot of rat liver showing the expression of inducible nitric oxide synthase (iNOS), poly–adenosine diphosphate ribose polymerase (PARP), cytochrome c, and actin in various treatment groups. β-HOB =β-hydroxybutyrate; HTS = hypertonic saline. 
Fig. 3. Representative Western blot of rat liver showing the expression of inducible nitric oxide synthase (iNOS), poly–adenosine diphosphate ribose polymerase (PARP), cytochrome c, and actin in various treatment groups. β-HOB =β-hydroxybutyrate; HTS = hypertonic saline. 
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Table 1. Hemodynamic and Metabolic Parameters during the Experiments 
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Table 1. Hemodynamic and Metabolic Parameters during the Experiments 
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Table 2. Effect of Various Treatments on Liver Dysfunction, Lipid Peroxidation, Adenine Nucleotides, and Energy Charge 
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Table 2. Effect of Various Treatments on Liver Dysfunction, Lipid Peroxidation, Adenine Nucleotides, and Energy Charge 
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Table 3. Effect of Resuscitation Medium on Hepatic Mitochondrial Respiration in Hemorrhagic Shock 
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Table 3. Effect of Resuscitation Medium on Hepatic Mitochondrial Respiration in Hemorrhagic Shock 
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Table 4. Intensity of Nitric Oxide Synthase, Cytochrome c, and PARP Protein Bands in Response to Various Resuscitative Fluids in Hemorrhagic Shock Rat Liver 
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Table 4. Intensity of Nitric Oxide Synthase, Cytochrome c, and PARP Protein Bands in Response to Various Resuscitative Fluids in Hemorrhagic Shock Rat Liver 
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