SHOCK, Vol. 41, No. 1, pp. 67Y71, 2014
DIFFERENTIAL CHANGES IN HEPATIC SYNTHESIS OF ALBUMIN AND FIBRINOGEN AFTER SEVERE HEMORRHAGIC SHOCK IN PIGS Wenjun Z. Martini, Kevin K. Chung, and Michael A. Dubick US Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas Received 10 Sep 2013; first review completed 24 Sep 2013; accepted in final form 07 Oct 2013 ABSTRACT—Introduction: Changes of plasma albumin and fibrinogen after various insults have been described as acute phase responses. This study investigated the acute changes of hepatic synthesis of albumin and fibrinogen after hemorrhage and resuscitation with lactated Ringer’s (LR) solution or normal saline (NS) in pigs. Methods: Twenty anesthetized pigs were randomized into control (n = 6), LR solution (n = 7), and NS (n = 7) groups. Hemorrhage of 60% estimated blood volume was induced in the LR and NS groups by removing blood from the left femoral artery with a computer-controlled pump, followed by resuscitation with either LR solution at three times the bled volume, or NS to reach the same mean arterial pressure as in the LR group. Stable isotope 1-13C-phenylalanine was infused for 6 h with hourly blood sampling and subsequent gas chromatography and mass spectrometry analysis to quantify hepatic protein synthesis. Results: Hemorrhage decreased mean arterial pressure and increased heart rate. Resuscitation with LR solution or NS corrected these changes. Compared with baseline, hemorrhage and resuscitation decreased albumin levels to 49% T 2% and 44% T 3% and fibrinogen levels to 50% T 2% and 53% T 2% in LR solution and NS (all P G 0.05), respectively. Albumin synthesis was impaired from 8.8 T 1.4 mg/kg per hour (control) to 5.3 T 0.8 mg/kg per hour in LR solution and 3.9 T 0.6 mg/kg per hour in NS (both P G 0.05). No changes were observed in fibrinogen synthesis after hemorrhage and resuscitation with LR solution (4.4 T 0.7 mg/kg per hour) or NS (3.3 T 0.4 mg/kg per hour), compared with the control (3.5 T 0.3mg/kg per hour). Conclusions: Hemorrhage and resuscitation compromised albumin synthesis, but not fibrinogen synthesis. There were no differences in hepatic synthesis of albumin or fibrinogen between LR solution and NS resuscitation. KEY
WORDS—Acute
phase proteins, stable isotope infusion, protein synthesis, and gas chromatography and mass spectrometry
INTRODUCTION
synthesis, fluctuating plasma volume, and/or increased degradation. At present, it is unclear whether albumin synthesis is impaired after hemorrhagic shock. Together with albumin, fibrinogen has been identified as an acute phase protein for decades (15, 16). The general concept of the acute phase response has been described as increased concentrations of positive acute phase proteins (e.g., fibrinogen) and decreased concentrations of negative acute phase proteins (e.g., albumin) (15Y17). As the precursor for clot formation, fibrinogen plays an essential role in coagulation function. Fibrinogen has been reported to be the first coagulation protein to drop below critical levels in acutely injured trauma patients (3, 18). To reveal the underlying mechanisms, our previous study showed that following a moderate hemorrhage (35%), hepatic synthesis of fibrinogen was acutely maintained (19), followed by a sustained increase for 5 days after hemorrhage (20). It is unclear whether the acute change of albumin is similar to fibrinogen, or the acute change of fibrinogen synthesis under a severe hemorrhage is different from a moderate hemorrhage. Although damage control resuscitation was introduced recently to initiate early use of blood products for severely injured hypotensive trauma patients, blood products are not generally available at prehospital and far-forward military settings. Crystalloids remain to be used as resuscitation fluids under these conditions with survival benefit (21). Normal saline (NS) and lactated Ringer’s (LR) solution have been used as crystalloid fluids for decades, but controversies continue as to which crystalloid is best. The effects of these crystalloids on hepatic synthesis of albumin and fibrinogen are unclear. Recognizing the important roles of albumin in homeostasis and fibrinogen in hemostasis, we designed the current study to investigate mechanisms related to acute changes of albumin and fibrinogen in a swine model with severe hemorrhagic shock.
Hemorrhage remains a major cause of death in civilian trauma and on conventional battlefields (1). The immediate effects of hemorrhage include reducing blood pressure and compromising tissue perfusion. Hemorrhage resulting in decreased perfusion also impairs functions of liver, lung, kidney, and gastrointestinal systems and coagulation (2Y5). Although hemodynamics and tissue perfusion can be improved with fluid resuscitation, the impact of hemorrhage on metabolism and coagulation appears to last days after the insults (3, 6). The effects of resuscitation fluids, such as crystalloids and colloids, on restoration of hemodynamics and hemostasis after hemorrhage have been well described (7Y11), but very limited information is available about the effects of hemorrhage on acute phase responses, specifically hepatic protein synthesis. Albumin is the most abundant protein in plasma and plays essential physiological functions in generating colloid-oncotic pressure, transporting lipid and fatty acids, scavenging free radicals, and maintaining capillary permeability (12). Decreased albumin levels have been reported in patients following infection, surgery, or trauma (13, 14). Various factors may contribute to the depletion of albumin levels, such as compromised hepatic Address reprint requests to Wenjun Z. Martini, PhD, The US Army Institute of Surgical Research, 3698 Chambers Pass, JBSA Fort Sam Houston, TX 78234. E-mail: [email protected]. Support was received from the Veterinary Support Branch and the Laboratory Support Section at the US Army Institute of Surgical Research. This study was also supported by the US Army Medical Research and Materiel Command. No funding was received from the National Institutes of Health, Wellcome Trust, or Howard Hughes Medical Institute. The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. DOI: 10.1097/SHK.0000000000000071 Copyright Ó 2013 by the Shock Society
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Copyright © 2013 by the Shock Society. Unauthorized reproduction of this article is prohibited.
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We hypothesized that hepatic synthesis of albumin and fibrinogen is compromised after a severe hemorrhagic shock. In addition, we compared the effects of resuscitation fluids, LR solution and NS, on hepatic protein synthesis. MATERIALS AND METHODS Experimental design This study was approved by the Institutional Animal Care and Use Committee of the US Army Institute of Surgical Research and has been conducted in compliance with the Animal Welfare Act and the implementing Animal Welfare Regulations and in accordance with the principles of the Guide for the Care and Use of Laboratory Animals. A total of 20 sexually immature female pigs (34.6 T 1.2 kg) were randomly allocated into three experimental groups: sham control (C, n = 6), hemorrhage and LR resuscitation (LR, n = 7), and hemorrhage and NS resuscitation (NS, n = 7). After an overnight fast, the pigs were preanesthetized with glycopyrrolate (0.1 mg/kg) and tiletamine (Telazol) (6 mg/kg). The pigs were then intubated, and anesthesia was maintained by 1.5% to 2.5% isoflurane in 100% oxygen for the surgical procedures. Polyvinyl chloride catheters were inserted into the thoracic aorta via the carotid artery to measure mean arterial pressure (MAP), systolic and diastolic blood pressure, and heart rate. A Swan-Ganz thermodilution catheter was inserted in the pulmonary artery via the left jugular vein to measure cardiac output and temperature. The right femoral artery was cannulated for arterial blood sampling and induction of bleeding. The right jugular vein was cannulated for venous blood sampling. The left femoral vein was cannulated for resuscitation. The right femoral vein was cannulated for intravenous (i.v.) anesthesia of ketamine during the study. No splenectomy was performed in this study. Upon completion of surgical procedures, anesthesia was switched to a combination of isoflurane (0.5%) and continuous i.v. drip of ketamine (0.15 mL/kg per hour of 100 mg/mL) in all pigs throughout the entire study period. After a 10-min stabilization period, blood samples were taken from the femoral artery for baseline measurements. Hemorrhage was then induced by bleeding from the femoral artery into sterile empty blood bags containing standard anticoagulant. The pigs were hemorrhaged 60% of their estimated blood volume over 60 min using a computer-controlled pump as previously described (22). Pigs were randomized into LR or NS groups 15 min after the completion of hemorrhage. Pigs in the LR group were resuscitated with LR solution at three times the bled volume over a 45-min period. Pigs in the NS group were resuscitated with NS to reach the same MAP as those in the LR group. Pigs in the control group were not hemorrhaged or resuscitated. Pigs in all three groups were given the same amount of anesthesia and maintenance fluid (NS, 0.04 mL/kg per minute). No heparin was used in this study.
Stable isotope infusion for hepatic synthesis Upon the completion of resuscitation, a 6-h isotope infusion was performed to quantify hepatic synthesis of albumin and fibrinogen. Briefly, the sterile stable isotope solution of 1-13C-phenylalanine (100 Hmol/mL) was made in 0.45% saline. After a priming dose of 1-13C-phenylalanine (18 2mol/kg), 1-13C-phenylalanine (0.3 2mol/kg per minute) was infused for 6 h with hourly blood sampling during the infusion. To assess changes of plasma volume following
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hemorrhage and resuscitation, plasma volume was measured at baseline; after hemorrhage; and 15 min, 3 h, and 6 h after resuscitation. At each of these time points, a bolus of sterile indocyanine green (Akorn Inc, Buffalo Grove, Ill) dye solution (10 mL of 2.5 mg/mL) was injected via the femoral vein, and blood samples (2 mL each) were collected at 5, 10, and 15 min postYindocyanine green injection to measure changes of plasma volume. Because plasma volume reached steady state within 3 h after resuscitation (Fig. 1), the average plasma volumes at 3 and 6 h after hemorrhage and resuscitation were used for synthesis calculation. All pigs were monitored during the 6-h isotope infusion after the completion of resuscitation. Mean arterial pressure and heart rate were recorded continuously during the study. Blood samples were taken at baseline; after hemorrhage; and at 15 min, 3 h, and 6 h after resuscitation for measurements of blood gases and blood chemistry. At the end of the 6-h isotope infusion and blood sampling, the pigs were killed by i.v. injection of a veterinary euthanasia solution of sodium pentobarbital (100 mg/kg; Fatal Plus, Fort Dodge, Iowa).
Calculations for synthesis of albumin and fibrinogen Quantification of albumin synthesis was based on the changes of isotope tracer labeling in albumin-bound 1-13C-phenylalanine during the 6-h infusion of 1-13C-phenylalanine. Similarly, quantification of fibrinogen synthesis was based on the changes of isotope tracer labeling in fibrinogen-bound 1-13Cphenylalanine during the 6-h infusion of 1-13C-phenylalanine. The fractional synthesis rates (FSRs) were calculated by using the formula, as described previously (23): FSR ¼ ½ EBðt2Þ j EBðt1Þ =ð EF tÞ; where EB(t) is the isotope labeling of albumin (or fibrinogen)Ybound 1-13Cphenylalanine; EF is the isotope labeling of plasma-free 1-13C-phenylalanine; t1 and t2 are the time points when infusion started and stopped, respectively; and t is the duration of the infusion. The absolute synthesis rate of albumin was calculated by multiplying albumin FSR with plasma volume and albumin concentration. Similarly, the absolute synthesis rate of fibrinogen was calculated by multiplying fibrinogen FSR with plasma volume and fibrinogen concentration.
Analytical methods Measurements of blood gases (pH, base excess [BE], lactate, hematocrit [Hct], etc.) and blood chemistry (total protein, albumin, etc.) were determined by standard clinical laboratory analysis. Plasma fibrinogen concentration was measured using the BCS Coagulation System (Dade Behring, Deerfield, Ill). Plasma-free amino acid enrichments from the infusion of 1-13C-phenylalnine were determined following procedures described previously (19, 23). Briefly, 0.5 mL of acidified plasma was loaded on a cation exchange column (AG 50W-X8 resin, 200 to 400 mesh, H+ form; Bio-Rad Laboratory, Hercules, Calif). Amino acids were separated after elution with ammonic hydroxide. The extracts were dried under speed vacuum and derivatized by N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide at 100-C for 1 h. Plasma albumin was isolated by ethanol extraction from trichloroacetic acidYprecipitated plasma protein, following procedures described by Debro and Korner (24). The isolated albumin was dried and hydrolyzed in 6N hydrochloric acid at 110-C for 24 h. The released amino acids after hydrolysis were isolated, dried, and derivatized in the same manner as for plasma-free amino acids. The enrichments of phenylalanine from plasma-free amino acid pool and from
FIG. 1. Changes in plasma volume after hemorrhage and resuscitation with LR solution or NS in pigs. *P G 0.05 compared with the corresponding baseline values and *P G 0.05 compared with corresponding control values.
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SHOCK JANUARY 2014
HEPATIC SYNTHESIS AND HEMORRHAGIC SHOCK
albumin protein were determined by gas chromatographyYmass spectrometry (model 5973; Hewlett-Packard, Palo Alto, Calif) in the electron impact ionization mode. A selective ion-monitoring method was used at nominal mass-tocharge ratio (m/z) of 336 (m + 0) and 337 (m + 1) for phenylalanine. Plasma fibrinogen was isolated as described previously (19, 23). Specifically, fibrinogen was isolated following the procedures described by Stein et al. (25). The clot was then washed, hydrolyzed, and dried. The enrichments of phenylalanine from plasma-free amino acids and from fibrinogen protein were determined by gas chromatographyYmass spectrometry (model 5973; Hewlett-Packard). A selective ion-monitoring method was used at nominal mass-to-charge ratio (m/z) of 336 (m + 0) and 337 (m + 1) for phenylalanine.
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TABLE 1. Changes of plasma protein levels after hemorrhage and resuscitation with LR solution or NS in pigs Group
Baseline
After hemorrhage and resuscitation
6h
Hct, % Control
28.4 T 0.8
28.9 T 0.8
26.7 T 0.8
LR
30.2 T 1.0
13.1 T 0.9*†
14.8 T 0.6*†
NS
29.0 T 1.0
11.6 T 0.8*†
12.6 T 0.8*†
Total protein, g/dL
Statistical analysis
Control
5.2 T 0.2
5.5 T 0.3
4.9 T 0.1
Data were expressed as means T SEM and analyzed using SAS statistical software (Cary, NC). A two-way analysis of variance with repeated measures using a Tukey adjustment was used to compare the changes over time between the groups. A one-way analysis of variance with repeated measures using a Dunnett adjustment was used to compare the changes to the baseline within the group. The statistically significant level was set at P G 0.05.
LR
5.6 T 0.2
2.7 T 0.1*†
3.3 T 0.1*†
NS
5.6 T 0.3
3.0 T 0.2*†
3.8 T 0.3*†
RESULTS
Albumin, g/dL Control
2.4 T 0.1
2.3 T 0.1
2.1 T 0.2
LR
2.6 T 0.1
1.3 T 0.2*†
1.8 T 0.2*†
NS
2.5 T 0.1
1.0 T 0.3*†
1.7 T 0.1*†
170 T 7
Fibrinogen, mg/dL
Hemodynamics and acid-base status
Control
180 T 6
All pigs survived to the end of the 6-h infusion study. No significant changes in the hemodynamics were observed in the control group over the experimental period. A 60% hemorrhage (42 mL/kg) dropped MAP from the baseline value of 79 T 6 mm Hg to 31 T 3 mm Hg (P G 0.05). Resuscitation with LR solution at three times the bled volume (119 T 7 mL/kg) returned the MAP to prehemorrhage levels in the LR group. In the NS group, NS of 183 T 9 mL/kg returned the MAP to a level similar to that of the LR group. Heart rate was increased by hemorrhage, followed by a return to near baseline levels (101 T 8 beats/min [bpm]) in the NS group (118 T 9 bpm) but remained higher over the baseline in the LR group (151 T 11 bpm, P G 0.05). Cardiac output returned to prehemorrhage levels (3.7 T 0.2 L/min) within 15 min after LR resuscitation (3.9 T 0.3 L/min) and remained higher than prehemorrhage levels for 6 h after NS resuscitation (4.5 T 0.2 L/min, P G 0.05). Base excess in the LR group fell from a prehemorrhage value of 6.8 T 0.6 mM to j2.7 T 1.7 after hemorrhage and returned to prehemorrhage levels with LR resuscitation by 3 h. In the NS group, BE fell from a prehemorrhage value of 6.5 T 0.9 mM to j0.8 T 1.4 mM after hemorrhage, with no return to a prehemorrhage level during the remainder of the study with NS resuscitation. Arterial pH was decreased from a prehemorrhage value of 7.41 T 0.02 to 7.35 T 0.01 after NS resuscitation (P G 0.05) but returned to the prehemorrhage value at the 3-h time point. No significant changes in arterial pH occurred in the LR group during the study.
LR
199 T 12
99 T 7*†
132 T 15*†
NS
189 T 9
101 T 6*†
129 T 7*†
186 T 12
Animal groups include control (C, n = 6), hemorrhage with LR resuscitation (LR, n = 6), and hemorrhage with NS resuscitation (NS, n = 6). Data are expressed as means T SE. *P G 0.05 compared with corresponding baseline values within the group. † P G 0.05 compared with corresponding control values.
resuscitation with LR solution or NS (Table 1) and remained below prehemorrhage levels afterward (Table 1). Hepatic synthesis of albumin
Albumin synthesis was quantified based on changes of isotope enrichments of precursor (free amino acids) and isotope enrichment of product (albumin) from the 6-h isotope infusion. Following 1-h infusion of 1-13C-phenylalanine, the isotope labeling of plasma 1-13C-phenylalanine reached plateau values in all three animal groups (19.8% T 0.6% in control, 20.0% T 1.4% in LR solution, and 20.2% T 1.4% in NS). Plasma albumin-bound 1-13C- phenylalanine labeling increased linearly during the infusion of 1-13C-phenylalanine. Albumin FSRs, calculated from the increased slope of albumin-bound phenylalanine labeling, were 0.43% T 0.02%/h in control, 0.29% T 0.04%/h in LR solution, and 0.50% T 0.08%/h in NS. The absolute synthesis rate, calculated by multiplying FSR with plasma volume and albumin concentration from the corresponding group, was similarly decreased after hemorrhage and resuscitation with LR solution or NS, as compared with control value (Fig. 2).
Hemodilution, plasma volume, and plasma protein
Hepatic synthesis of fibrinogen
Hemorrhage and resuscitation caused significant changes in Hct and plasma volume. Hematocrit dropped by 50% after hemorrhage and resuscitation with LR solution or NS and remained at the lower levels for the remaining 6-h study period (Table 1). Similar reductions in plasma volume were observed after hemorrhage (Fig. 1). Resuscitation with LR solution and NS immediately elevated plasma volume above prehemorrhage levels, followed by returning to prehemorrhage levels within 3 h after resuscitation (Fig. 1). Levels of total protein, albumin, and fibrinogen were also reduced by 50% after hemorrhage and
Fibrinogen synthesis was quantified based on changes of plasma-free phenylalanine isotopic enrichments and fibrinogenbound phenylalanine isotopic enrichment from 1-13C-phenyalanine infusion. Plasma fibrinogen-bound 1-13C-phenylalanine labeling increased linearly during the infusion of 1-13C-phenylalanine in all three animal groups. Fibrinogen FSRs, calculated from the increased slope of fibrinogen-bound 1-13C-phenylalanine labeling, were 3.38% T 0.23%/h in control, 3.35% T 0.30%/h in LR solution, and 4.25% T 0.36%/h in NS. The absolute synthesis rates of fibrinogen, calculated by multiplying FSR with plasma volume
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FIG. 2. Changes in albumin and fibrinogen synthesis after 6-h isotope infusion following hemorrhage and resuscitation with LR solution or NS in pigs. *P G 0.05 compared with control values.
and fibrinogen concentration from the corresponding group, were similar among the three groups (Fig. 2). DISCUSSION We previously reported the acute (6 h) and long-term (5 days) effects of moderate hemorrhage on fibrinogen synthesis in pigs (19, 20). In the current study, we investigated the effects of severe hemorrhagic shock on hepatic synthesis. To our knowledge, this is the first study to reveal the effects of severe hemorrhagic shock on hepatic synthesis of albumin and fibrinogen. Using a large-animal model with severe hemorrhage and resuscitation, our data showed that albumin synthesis was impaired but that fibrinogen synthesis was maintained. Furthermore, there were no differences in hepatic synthesis of albumin or fibrinogen between LR and NS resuscitation. Metabolic responses to trauma, infection, inflammation, or sepsis are generalized by an increase in whole-body protein turnover rate (26). Amino acid release from muscle protein breakdown into circulation and amino acid uptake by the splanchnic bed are increased (26), shifting amino acid sources from the muscle to the liver. This shift is considered to be beneficial because it may facilitate hepatic synthesis of proteins that are critical for survival (15, 27Y29). Clinical data have shown that after trauma and sepsis, synthesis of proteins that are important to immune response, coagulation, and organ preservation must be maintained (16, 27), and failure to have this response is associated with adverse outcomes (30). In this study, we observed that fibrinogen synthesis was maintained, but albumin synthesis decreased after severe hemorrhage. These differential changes in synthesis might also reflect a shift of hepatic synthesis toward producing proteins necessary to survive from severe hemorrhagic shock, although much of the underlying mechanism remains to be explored. Nevertheless, prioritizing hepatic synthesis after insults appears to be a generalized response, occurring at whole-body level as well as within an individual organ. It should be emphasized that hepatic synthesis is a dynamic process. Changes in albumin and fibrinogen synthesis at different time points may differ from what was observed in this study. Although fibrinogen synthesis was maintained after severe hemorrhagic shock, it is important to point out that quantitatively fibrinogen synthesis rate was very slow, i.e., 4% of the entire
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body pool per hour. Thus, a 60% blood loss (60% fibrinogen loss) would require at least 15 h to synthesize enough new fibrinogen to compensate the lost fibrinogen, assuming there is no degradation. Thus, data from this study support the approach of fibrinogen supplementation to restore hemostasis in acutely bleeding patients after hemorrhagic shock. Fibrinogen is an acute phase reactant, and albumin is a constitutive protein. After various stresses, including hemorrhagic shock, the levels of constitutive proteins decrease, and acute phase reactants increases. In this study, fibrinogen level did not increase, although fibrinogen synthesis was maintained after hemorrhage. Our previous study showed that after a moderate hemorrhage, fibrinogen level was doubled at 24 h after a moderate hemorrhage. Thus, the lack of increase in fibrinogen level in this study is likely due to the short duration of the study (6 h). However, changes of fibrinogen to changes in C-reactive protein are unclear. In this study, resuscitation with LR solution at three times the bled volume returned the MAP to the prehemorrhage level. To reach the same goal, about 50% more volume of NS was used in the NS group. Despite similar restoration of hemodynamics, there was a significant separation of acid-base status between the two resuscitation groups: LR resuscitation returned BE and bicarbonate to prehemorrhage levels within 3 h, but no return of BE or bicarbonate was observed with NS resuscitation during the study. As an index of shock, acidosis is associated with well-known detrimental effects on coagulation (31, 32). However, changes in synthesis of fibrinogen and albumin were similar between the LR and the NS groups in this study, suggesting that hepatic synthesis was not affected by changes in acid-base status. Consistently, when acidosis of 7.1 was induced in pigs, we previously reported that fibrinogen synthesis in those acidotic pigs was similar to that of control animals (pH 7.4) (33). On the other hand, when hypothermia of 32-C was induced in pigs, fibrinogen synthesis in those pigs was inhibited to 50% of that of control pigs (34). Thus, it appears that hepatic synthesis is sensitive to changes in body temperature but resistant to changes in acid-base status. We observed similar changes of albumin synthesis and fibrinogen synthesis in the LR and NS groups in this study, suggesting that the effects of LR solution and NS on hepatic protein synthesis were similar. Previously in a swine model with a moderate hemorrhage (35%), we found that fibrinogen synthesis was similar in hemorrhaged pigs with and without LR resuscitation in a swine model with a moderate (35%) hemorrhage, indicating that LR resuscitation does not affect synthesis (19). Put together, these data suggest that changes of hepatic protein synthesis reflect the responses of hemorrhage and that crystalloid resuscitation has minimal effects on hepatic synthesis. However, it remains to be confirmed whether this conclusion holds under colloid resuscitation, because more dramatic changes in hemodilution and coagulation are observed with colloid resuscitation (32). Although hemorrhage and resuscitation decreased albumin synthesis, we did not observe a corresponding decrease in albumin concentration during 6 h after resuscitation in the study, except changes due to hemodilution from resuscitation. This apparent discrepancy is likely due to fluctuations of plasma volume after resuscitation. With direct measurements of plasma volumes at different time points after resuscitation,
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SHOCK JANUARY 2014 we found that plasma volume was elevated above prehemorrhage value at 15 min after resuscitation but reached equilibrium within 3 h after resuscitation. Thus, the reduction of plasma volume a few hours after resuscitation might cause a Bpseudo[ increase in albumin concentration, which offset the effect of decreased synthesis of albumin. In addition, albumin transport from different pools is likely to be another confounding factor. As a transport protein, albumin has a molecular weight of 65,000, and about 50% of its content is confined in the vascular pool (12). Albumin transport, assessed as the transvascular escape rate, in normal subjects is found to be about 4% to 5%/h (12), which is greater than 20 times higher than that of normal albumin synthesis. Thus, changes in albumin transport can have a significant impact on plasma albumin levels, although this was not quantified in the present study. Nevertheless, because hepatic synthesis is the only source for new albumin production, it is assumed that changes in albumin synthesis would be reflected in albumin concentration eventually. Our study had some limitations. First, a volume-controlled hemorrhage model was used in this study to evaluate the effects of hemorrhage alone on hepatic synthesis of albumin and fibrinogen. Although allowing better standardization and clarification, this model was not able to fully represent clinical bleeding situation. Second, tissue injury was not included in this study, which may have significant impact on the acute phase response, including hepatic synthesis. Thus, changes of hepatic synthesis of albumin and fibrinogen may be different in bleeding patients with traumatic injury. In conclusion, we investigated the responses of acute hepatic protein synthesis to hemorrhage in a swine model. Severe hemorrhagic shock caused differential changes in hepatic protein synthesis: albumin synthesis was inhibited, but fibrinogen synthesis remained unchanged. Resuscitation with LR solution and NS to comparable blood pressure had equivalent effects on hepatic synthesis of albumin and fibrinogen, despite better acid-base status with LR solution. With growing interests in fibrinogen supplementation in trauma patients to restore coagulation, future effort is warranted to investigate hepatic synthesis of acute phase proteins following albumin therapy and fibrinogen supplementation. ACKNOWLEDGMENTS
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The authors thank Douglas Cortez, Shavaughn Colvin, and Nahir Miranda for their technical assistance in animal study and sample analysis; Mr. John Jones for statistical analysis; and Otilia Sa´nchez for editing the article.
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molecular weight hydroxyethyl starch) versus 5% albumin for plasma volume expansion after cardiac operations. J Thorac Cardiovasc Surg 97(5):785Y797, 1989. Alderson P, Schierhout G, Roberts I, Bunn F: Colloids versus crystalloids for fluid resuscitation in critically ill patients. Cochrane Database Syst Rev (2):CD000567, 2000. Moggio RA, Rha CC, Somberg ED, Praeger PI, Pooley RW, Reed GE: Hemodynamic comparison of albumin and hydroxyethyl starch in postoperative cardiac surgery patients. Crit Care Med 11(12):943Y945, 1983. Gan TJ, Bennett-Guerrero E, Phillips-Bute B, Wakeling H, Moskowitz DM, Olufolabi Y, Konstadt SN, Bradford C, Glass PS, Machin SJ, et al.: Hextend, a physiologically balanced plasma expander for large volume use in major surgery: a randomized phase III clinical trial. Hextend Study Group. Anesth Analg 88(5):992Y998, 1999. Innerhofer P, Fries D, Margreiter J, Klingler A, Kuhbacher G, Wachter B, Oswald E, Salner E, Frischhut B, Schobersberger W: The effects of perioperatively administered colloids and crystalloids on primary platelet-mediated hemostasis and clot formation. Anesth Analg 95(4):858Y865, table of contents, 2002. Mendez CM, McClain CJ, Marsano LS: Albumin therapy in clinical practice. Nutr Clin Pract 20(3):314Y320, 2005. Fleck A, Raines G, Hawker F, Trotter J, Wallace PI, Ledingham IM, Calman KC: Increased vascular permeability: a major cause of hypoalbuminaemia in disease and injury. Lancet 1(8432):781Y784, 1985. Bonate PL: Pathophysiology and pharmacokinetics following burn injury. Clin Pharmacokinet 18(2):118Y130, 1990. Fleck A: Clinical and nutritional aspects of changes in acute-phase proteins during inflammation. Proc Nutr Soc 48(3):347Y354, 1989. Kushner I: The phenomenon of the acute phase response. Ann N Y Acad Sci 389:39Y48, 1982. Koj A: Cytokines regulating acute inflammation and synthesis of acute phase proteins. Blut 51(4):267Y274, 1985. Armand R, Hess JR: Treating coagulopathy in trauma patients. Transfus Med Rev 17(3):223Y231, 2003. Martini WZ, Chinkes DL, Sondeen J, Dubick MA: Effects of hemorrhage and lactated Ringer’s resuscitation on coagulation and fibrinogen metabolism in swine. Shock 26(4):396Y401, 2006. Martini WZ, Chung KK, Dubick MA, Blackbourne LH: Daily profiles of fibrinogen metabolism for 5 days following hemorrhage and lactated Ringer’s resuscitation in pigs. Shock 37(6):605Y610, 2012. Spoerke N, Michalek J, Schreiber M, Brasel KJ, Vercruysse G, MacLeod J, Dutton RP, Duchesne JC, McSwain NE, Muskat P, et al.: Crystalloid resuscitation improves survival in trauma patients receiving low ratios of fresh frozen plasma to packed red blood cells. J Trauma 71(2 Suppl 3):S380YS383, 2011. Burns JW, Baer LA, Darlington DN, Dubick MA, Wade CE: Screening of potential small volume resuscitation products using a severe hemorrhage sedated swine model. Int J Burns Trauma 2(1):59Y67, 2012. Martini WZ, Chinkes DL, Pusateri AE, Holcomb JB, Yu YM, Zhang XJ, WolfeRR: Acute changes in fibrinogen metabolism and coagulation after hemorrhage in pigs. Am J Physiol Endocrinol Metab 289(5):E930YE934, 2005. Debro JR, Korner A: Solubility of albumin in alcohol after precipitation by trichloroacetic acid: a simplified procedure for separation of albumin. Nature 178(4541):1067, 1956. Stein TP, Leskiw MJ, Wallace HW: Measurement of half-life human plasma fibrinogen. Am J Physiol 234(5):D504YD510, 1978. Clowes GH Jr, Randall HT, Cha CJ: Amino acid and energy metabolism in septic and traumatized patients. JPEN J Parenter Enteral Nutr 4(2):195Y205, 1980. Sganga G, Siegel JH, Brown G, Coleman B, Wiles CE 3rd, Belzberg H, Wedel S, Placko R: Reprioritization of hepatic plasma protein release in trauma and sepsis. Arch Surg 120(2):187Y199, 1985. Beisel WR: Magnitude of the host nutritional responses to infection. Am J Clin Nutr 30(8):1236Y1247, 1977. Clowes GH Jr, Hirsch E, George BC, Bigatello LM, Mazuski JE, Villee CA Jr: Survival from sepsis. The significance of altered protein metabolism regulated by proteolysis inducing factor, the circulating cleavage product of interleukin-1. Ann Surg 202(4):446Y458, 1985. McMenamy RH, Birkhahn R, Oswald G, Reed R, Rumph C, Vaidyanath N, Yu L, Cerra FB, Sorkness R, Border JR: Multiple systems organ failure: I. The basal state. J Trauma 21(2):99Y114, 1981. Martini WZ, Dubick MA, Pusateri AE, Park MS, Ryan KL, Holcomb JB: Does bicarbonate correct coagulation function impaired by acidosis in swine? J Trauma 61(1):99Y106, 2006. Martini WZ, Dubick MA, Blackbourne LH: Comparisons of lactated Ringer’s and Hextend resuscitation on hemodynamics and coagulation following femur injury and severe hemorrhage in pigs. J Trauma Acute Care Surg 74(3):732Y740, 2013. Martini WZ, Holcomb JB: Acidosis and coagulopathy: the differential effects on fibrinogen synthesis and breakdown in pigs. Ann Surg 246(5):831Y835, 2007. Martini WZ: The effects of hypothermia on fibrinogen metabolism and coagulation function in swine. Metabolism 56(2):214Y221, 2007.
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DIFFERENTIAL CHANGES IN HEPATIC SYNTHESIS OF ALBUMIN AND FIBRINOGEN AFTER SEVERE HEMORRHAGIC SHOCK IN PIGS Wenjun Z. Martini, Kevin K. Chung, and Michael A. Dubick US Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas Received 10 Sep 2013; first review completed 24 Sep 2013; accepted in final form 07 Oct 2013 ABSTRACT—Introduction: Changes of plasma albumin and fibrinogen after various insults have been described as acute phase responses. This study investigated the acute changes of hepatic synthesis of albumin and fibrinogen after hemorrhage and resuscitation with lactated Ringer’s (LR) solution or normal saline (NS) in pigs. Methods: Twenty anesthetized pigs were randomized into control (n = 6), LR solution (n = 7), and NS (n = 7) groups. Hemorrhage of 60% estimated blood volume was induced in the LR and NS groups by removing blood from the left femoral artery with a computer-controlled pump, followed by resuscitation with either LR solution at three times the bled volume, or NS to reach the same mean arterial pressure as in the LR group. Stable isotope 1-13C-phenylalanine was infused for 6 h with hourly blood sampling and subsequent gas chromatography and mass spectrometry analysis to quantify hepatic protein synthesis. Results: Hemorrhage decreased mean arterial pressure and increased heart rate. Resuscitation with LR solution or NS corrected these changes. Compared with baseline, hemorrhage and resuscitation decreased albumin levels to 49% T 2% and 44% T 3% and fibrinogen levels to 50% T 2% and 53% T 2% in LR solution and NS (all P G 0.05), respectively. Albumin synthesis was impaired from 8.8 T 1.4 mg/kg per hour (control) to 5.3 T 0.8 mg/kg per hour in LR solution and 3.9 T 0.6 mg/kg per hour in NS (both P G 0.05). No changes were observed in fibrinogen synthesis after hemorrhage and resuscitation with LR solution (4.4 T 0.7 mg/kg per hour) or NS (3.3 T 0.4 mg/kg per hour), compared with the control (3.5 T 0.3mg/kg per hour). Conclusions: Hemorrhage and resuscitation compromised albumin synthesis, but not fibrinogen synthesis. There were no differences in hepatic synthesis of albumin or fibrinogen between LR solution and NS resuscitation. KEY
WORDS—Acute
phase proteins, stable isotope infusion, protein synthesis, and gas chromatography and mass spectrometry
INTRODUCTION
synthesis, fluctuating plasma volume, and/or increased degradation. At present, it is unclear whether albumin synthesis is impaired after hemorrhagic shock. Together with albumin, fibrinogen has been identified as an acute phase protein for decades (15, 16). The general concept of the acute phase response has been described as increased concentrations of positive acute phase proteins (e.g., fibrinogen) and decreased concentrations of negative acute phase proteins (e.g., albumin) (15Y17). As the precursor for clot formation, fibrinogen plays an essential role in coagulation function. Fibrinogen has been reported to be the first coagulation protein to drop below critical levels in acutely injured trauma patients (3, 18). To reveal the underlying mechanisms, our previous study showed that following a moderate hemorrhage (35%), hepatic synthesis of fibrinogen was acutely maintained (19), followed by a sustained increase for 5 days after hemorrhage (20). It is unclear whether the acute change of albumin is similar to fibrinogen, or the acute change of fibrinogen synthesis under a severe hemorrhage is different from a moderate hemorrhage. Although damage control resuscitation was introduced recently to initiate early use of blood products for severely injured hypotensive trauma patients, blood products are not generally available at prehospital and far-forward military settings. Crystalloids remain to be used as resuscitation fluids under these conditions with survival benefit (21). Normal saline (NS) and lactated Ringer’s (LR) solution have been used as crystalloid fluids for decades, but controversies continue as to which crystalloid is best. The effects of these crystalloids on hepatic synthesis of albumin and fibrinogen are unclear. Recognizing the important roles of albumin in homeostasis and fibrinogen in hemostasis, we designed the current study to investigate mechanisms related to acute changes of albumin and fibrinogen in a swine model with severe hemorrhagic shock.
Hemorrhage remains a major cause of death in civilian trauma and on conventional battlefields (1). The immediate effects of hemorrhage include reducing blood pressure and compromising tissue perfusion. Hemorrhage resulting in decreased perfusion also impairs functions of liver, lung, kidney, and gastrointestinal systems and coagulation (2Y5). Although hemodynamics and tissue perfusion can be improved with fluid resuscitation, the impact of hemorrhage on metabolism and coagulation appears to last days after the insults (3, 6). The effects of resuscitation fluids, such as crystalloids and colloids, on restoration of hemodynamics and hemostasis after hemorrhage have been well described (7Y11), but very limited information is available about the effects of hemorrhage on acute phase responses, specifically hepatic protein synthesis. Albumin is the most abundant protein in plasma and plays essential physiological functions in generating colloid-oncotic pressure, transporting lipid and fatty acids, scavenging free radicals, and maintaining capillary permeability (12). Decreased albumin levels have been reported in patients following infection, surgery, or trauma (13, 14). Various factors may contribute to the depletion of albumin levels, such as compromised hepatic Address reprint requests to Wenjun Z. Martini, PhD, The US Army Institute of Surgical Research, 3698 Chambers Pass, JBSA Fort Sam Houston, TX 78234. E-mail: [email protected]. Support was received from the Veterinary Support Branch and the Laboratory Support Section at the US Army Institute of Surgical Research. This study was also supported by the US Army Medical Research and Materiel Command. No funding was received from the National Institutes of Health, Wellcome Trust, or Howard Hughes Medical Institute. The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. DOI: 10.1097/SHK.0000000000000071 Copyright Ó 2013 by the Shock Society
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Copyright © 2013 by the Shock Society. Unauthorized reproduction of this article is prohibited.
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We hypothesized that hepatic synthesis of albumin and fibrinogen is compromised after a severe hemorrhagic shock. In addition, we compared the effects of resuscitation fluids, LR solution and NS, on hepatic protein synthesis. MATERIALS AND METHODS Experimental design This study was approved by the Institutional Animal Care and Use Committee of the US Army Institute of Surgical Research and has been conducted in compliance with the Animal Welfare Act and the implementing Animal Welfare Regulations and in accordance with the principles of the Guide for the Care and Use of Laboratory Animals. A total of 20 sexually immature female pigs (34.6 T 1.2 kg) were randomly allocated into three experimental groups: sham control (C, n = 6), hemorrhage and LR resuscitation (LR, n = 7), and hemorrhage and NS resuscitation (NS, n = 7). After an overnight fast, the pigs were preanesthetized with glycopyrrolate (0.1 mg/kg) and tiletamine (Telazol) (6 mg/kg). The pigs were then intubated, and anesthesia was maintained by 1.5% to 2.5% isoflurane in 100% oxygen for the surgical procedures. Polyvinyl chloride catheters were inserted into the thoracic aorta via the carotid artery to measure mean arterial pressure (MAP), systolic and diastolic blood pressure, and heart rate. A Swan-Ganz thermodilution catheter was inserted in the pulmonary artery via the left jugular vein to measure cardiac output and temperature. The right femoral artery was cannulated for arterial blood sampling and induction of bleeding. The right jugular vein was cannulated for venous blood sampling. The left femoral vein was cannulated for resuscitation. The right femoral vein was cannulated for intravenous (i.v.) anesthesia of ketamine during the study. No splenectomy was performed in this study. Upon completion of surgical procedures, anesthesia was switched to a combination of isoflurane (0.5%) and continuous i.v. drip of ketamine (0.15 mL/kg per hour of 100 mg/mL) in all pigs throughout the entire study period. After a 10-min stabilization period, blood samples were taken from the femoral artery for baseline measurements. Hemorrhage was then induced by bleeding from the femoral artery into sterile empty blood bags containing standard anticoagulant. The pigs were hemorrhaged 60% of their estimated blood volume over 60 min using a computer-controlled pump as previously described (22). Pigs were randomized into LR or NS groups 15 min after the completion of hemorrhage. Pigs in the LR group were resuscitated with LR solution at three times the bled volume over a 45-min period. Pigs in the NS group were resuscitated with NS to reach the same MAP as those in the LR group. Pigs in the control group were not hemorrhaged or resuscitated. Pigs in all three groups were given the same amount of anesthesia and maintenance fluid (NS, 0.04 mL/kg per minute). No heparin was used in this study.
Stable isotope infusion for hepatic synthesis Upon the completion of resuscitation, a 6-h isotope infusion was performed to quantify hepatic synthesis of albumin and fibrinogen. Briefly, the sterile stable isotope solution of 1-13C-phenylalanine (100 Hmol/mL) was made in 0.45% saline. After a priming dose of 1-13C-phenylalanine (18 2mol/kg), 1-13C-phenylalanine (0.3 2mol/kg per minute) was infused for 6 h with hourly blood sampling during the infusion. To assess changes of plasma volume following
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hemorrhage and resuscitation, plasma volume was measured at baseline; after hemorrhage; and 15 min, 3 h, and 6 h after resuscitation. At each of these time points, a bolus of sterile indocyanine green (Akorn Inc, Buffalo Grove, Ill) dye solution (10 mL of 2.5 mg/mL) was injected via the femoral vein, and blood samples (2 mL each) were collected at 5, 10, and 15 min postYindocyanine green injection to measure changes of plasma volume. Because plasma volume reached steady state within 3 h after resuscitation (Fig. 1), the average plasma volumes at 3 and 6 h after hemorrhage and resuscitation were used for synthesis calculation. All pigs were monitored during the 6-h isotope infusion after the completion of resuscitation. Mean arterial pressure and heart rate were recorded continuously during the study. Blood samples were taken at baseline; after hemorrhage; and at 15 min, 3 h, and 6 h after resuscitation for measurements of blood gases and blood chemistry. At the end of the 6-h isotope infusion and blood sampling, the pigs were killed by i.v. injection of a veterinary euthanasia solution of sodium pentobarbital (100 mg/kg; Fatal Plus, Fort Dodge, Iowa).
Calculations for synthesis of albumin and fibrinogen Quantification of albumin synthesis was based on the changes of isotope tracer labeling in albumin-bound 1-13C-phenylalanine during the 6-h infusion of 1-13C-phenylalanine. Similarly, quantification of fibrinogen synthesis was based on the changes of isotope tracer labeling in fibrinogen-bound 1-13Cphenylalanine during the 6-h infusion of 1-13C-phenylalanine. The fractional synthesis rates (FSRs) were calculated by using the formula, as described previously (23): FSR ¼ ½ EBðt2Þ j EBðt1Þ =ð EF tÞ; where EB(t) is the isotope labeling of albumin (or fibrinogen)Ybound 1-13Cphenylalanine; EF is the isotope labeling of plasma-free 1-13C-phenylalanine; t1 and t2 are the time points when infusion started and stopped, respectively; and t is the duration of the infusion. The absolute synthesis rate of albumin was calculated by multiplying albumin FSR with plasma volume and albumin concentration. Similarly, the absolute synthesis rate of fibrinogen was calculated by multiplying fibrinogen FSR with plasma volume and fibrinogen concentration.
Analytical methods Measurements of blood gases (pH, base excess [BE], lactate, hematocrit [Hct], etc.) and blood chemistry (total protein, albumin, etc.) were determined by standard clinical laboratory analysis. Plasma fibrinogen concentration was measured using the BCS Coagulation System (Dade Behring, Deerfield, Ill). Plasma-free amino acid enrichments from the infusion of 1-13C-phenylalnine were determined following procedures described previously (19, 23). Briefly, 0.5 mL of acidified plasma was loaded on a cation exchange column (AG 50W-X8 resin, 200 to 400 mesh, H+ form; Bio-Rad Laboratory, Hercules, Calif). Amino acids were separated after elution with ammonic hydroxide. The extracts were dried under speed vacuum and derivatized by N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide at 100-C for 1 h. Plasma albumin was isolated by ethanol extraction from trichloroacetic acidYprecipitated plasma protein, following procedures described by Debro and Korner (24). The isolated albumin was dried and hydrolyzed in 6N hydrochloric acid at 110-C for 24 h. The released amino acids after hydrolysis were isolated, dried, and derivatized in the same manner as for plasma-free amino acids. The enrichments of phenylalanine from plasma-free amino acid pool and from
FIG. 1. Changes in plasma volume after hemorrhage and resuscitation with LR solution or NS in pigs. *P G 0.05 compared with the corresponding baseline values and *P G 0.05 compared with corresponding control values.
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HEPATIC SYNTHESIS AND HEMORRHAGIC SHOCK
albumin protein were determined by gas chromatographyYmass spectrometry (model 5973; Hewlett-Packard, Palo Alto, Calif) in the electron impact ionization mode. A selective ion-monitoring method was used at nominal mass-tocharge ratio (m/z) of 336 (m + 0) and 337 (m + 1) for phenylalanine. Plasma fibrinogen was isolated as described previously (19, 23). Specifically, fibrinogen was isolated following the procedures described by Stein et al. (25). The clot was then washed, hydrolyzed, and dried. The enrichments of phenylalanine from plasma-free amino acids and from fibrinogen protein were determined by gas chromatographyYmass spectrometry (model 5973; Hewlett-Packard). A selective ion-monitoring method was used at nominal mass-to-charge ratio (m/z) of 336 (m + 0) and 337 (m + 1) for phenylalanine.
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TABLE 1. Changes of plasma protein levels after hemorrhage and resuscitation with LR solution or NS in pigs Group
Baseline
After hemorrhage and resuscitation
6h
Hct, % Control
28.4 T 0.8
28.9 T 0.8
26.7 T 0.8
LR
30.2 T 1.0
13.1 T 0.9*†
14.8 T 0.6*†
NS
29.0 T 1.0
11.6 T 0.8*†
12.6 T 0.8*†
Total protein, g/dL
Statistical analysis
Control
5.2 T 0.2
5.5 T 0.3
4.9 T 0.1
Data were expressed as means T SEM and analyzed using SAS statistical software (Cary, NC). A two-way analysis of variance with repeated measures using a Tukey adjustment was used to compare the changes over time between the groups. A one-way analysis of variance with repeated measures using a Dunnett adjustment was used to compare the changes to the baseline within the group. The statistically significant level was set at P G 0.05.
LR
5.6 T 0.2
2.7 T 0.1*†
3.3 T 0.1*†
NS
5.6 T 0.3
3.0 T 0.2*†
3.8 T 0.3*†
RESULTS
Albumin, g/dL Control
2.4 T 0.1
2.3 T 0.1
2.1 T 0.2
LR
2.6 T 0.1
1.3 T 0.2*†
1.8 T 0.2*†
NS
2.5 T 0.1
1.0 T 0.3*†
1.7 T 0.1*†
170 T 7
Fibrinogen, mg/dL
Hemodynamics and acid-base status
Control
180 T 6
All pigs survived to the end of the 6-h infusion study. No significant changes in the hemodynamics were observed in the control group over the experimental period. A 60% hemorrhage (42 mL/kg) dropped MAP from the baseline value of 79 T 6 mm Hg to 31 T 3 mm Hg (P G 0.05). Resuscitation with LR solution at three times the bled volume (119 T 7 mL/kg) returned the MAP to prehemorrhage levels in the LR group. In the NS group, NS of 183 T 9 mL/kg returned the MAP to a level similar to that of the LR group. Heart rate was increased by hemorrhage, followed by a return to near baseline levels (101 T 8 beats/min [bpm]) in the NS group (118 T 9 bpm) but remained higher over the baseline in the LR group (151 T 11 bpm, P G 0.05). Cardiac output returned to prehemorrhage levels (3.7 T 0.2 L/min) within 15 min after LR resuscitation (3.9 T 0.3 L/min) and remained higher than prehemorrhage levels for 6 h after NS resuscitation (4.5 T 0.2 L/min, P G 0.05). Base excess in the LR group fell from a prehemorrhage value of 6.8 T 0.6 mM to j2.7 T 1.7 after hemorrhage and returned to prehemorrhage levels with LR resuscitation by 3 h. In the NS group, BE fell from a prehemorrhage value of 6.5 T 0.9 mM to j0.8 T 1.4 mM after hemorrhage, with no return to a prehemorrhage level during the remainder of the study with NS resuscitation. Arterial pH was decreased from a prehemorrhage value of 7.41 T 0.02 to 7.35 T 0.01 after NS resuscitation (P G 0.05) but returned to the prehemorrhage value at the 3-h time point. No significant changes in arterial pH occurred in the LR group during the study.
LR
199 T 12
99 T 7*†
132 T 15*†
NS
189 T 9
101 T 6*†
129 T 7*†
186 T 12
Animal groups include control (C, n = 6), hemorrhage with LR resuscitation (LR, n = 6), and hemorrhage with NS resuscitation (NS, n = 6). Data are expressed as means T SE. *P G 0.05 compared with corresponding baseline values within the group. † P G 0.05 compared with corresponding control values.
resuscitation with LR solution or NS (Table 1) and remained below prehemorrhage levels afterward (Table 1). Hepatic synthesis of albumin
Albumin synthesis was quantified based on changes of isotope enrichments of precursor (free amino acids) and isotope enrichment of product (albumin) from the 6-h isotope infusion. Following 1-h infusion of 1-13C-phenylalanine, the isotope labeling of plasma 1-13C-phenylalanine reached plateau values in all three animal groups (19.8% T 0.6% in control, 20.0% T 1.4% in LR solution, and 20.2% T 1.4% in NS). Plasma albumin-bound 1-13C- phenylalanine labeling increased linearly during the infusion of 1-13C-phenylalanine. Albumin FSRs, calculated from the increased slope of albumin-bound phenylalanine labeling, were 0.43% T 0.02%/h in control, 0.29% T 0.04%/h in LR solution, and 0.50% T 0.08%/h in NS. The absolute synthesis rate, calculated by multiplying FSR with plasma volume and albumin concentration from the corresponding group, was similarly decreased after hemorrhage and resuscitation with LR solution or NS, as compared with control value (Fig. 2).
Hemodilution, plasma volume, and plasma protein
Hepatic synthesis of fibrinogen
Hemorrhage and resuscitation caused significant changes in Hct and plasma volume. Hematocrit dropped by 50% after hemorrhage and resuscitation with LR solution or NS and remained at the lower levels for the remaining 6-h study period (Table 1). Similar reductions in plasma volume were observed after hemorrhage (Fig. 1). Resuscitation with LR solution and NS immediately elevated plasma volume above prehemorrhage levels, followed by returning to prehemorrhage levels within 3 h after resuscitation (Fig. 1). Levels of total protein, albumin, and fibrinogen were also reduced by 50% after hemorrhage and
Fibrinogen synthesis was quantified based on changes of plasma-free phenylalanine isotopic enrichments and fibrinogenbound phenylalanine isotopic enrichment from 1-13C-phenyalanine infusion. Plasma fibrinogen-bound 1-13C-phenylalanine labeling increased linearly during the infusion of 1-13C-phenylalanine in all three animal groups. Fibrinogen FSRs, calculated from the increased slope of fibrinogen-bound 1-13C-phenylalanine labeling, were 3.38% T 0.23%/h in control, 3.35% T 0.30%/h in LR solution, and 4.25% T 0.36%/h in NS. The absolute synthesis rates of fibrinogen, calculated by multiplying FSR with plasma volume
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FIG. 2. Changes in albumin and fibrinogen synthesis after 6-h isotope infusion following hemorrhage and resuscitation with LR solution or NS in pigs. *P G 0.05 compared with control values.
and fibrinogen concentration from the corresponding group, were similar among the three groups (Fig. 2). DISCUSSION We previously reported the acute (6 h) and long-term (5 days) effects of moderate hemorrhage on fibrinogen synthesis in pigs (19, 20). In the current study, we investigated the effects of severe hemorrhagic shock on hepatic synthesis. To our knowledge, this is the first study to reveal the effects of severe hemorrhagic shock on hepatic synthesis of albumin and fibrinogen. Using a large-animal model with severe hemorrhage and resuscitation, our data showed that albumin synthesis was impaired but that fibrinogen synthesis was maintained. Furthermore, there were no differences in hepatic synthesis of albumin or fibrinogen between LR and NS resuscitation. Metabolic responses to trauma, infection, inflammation, or sepsis are generalized by an increase in whole-body protein turnover rate (26). Amino acid release from muscle protein breakdown into circulation and amino acid uptake by the splanchnic bed are increased (26), shifting amino acid sources from the muscle to the liver. This shift is considered to be beneficial because it may facilitate hepatic synthesis of proteins that are critical for survival (15, 27Y29). Clinical data have shown that after trauma and sepsis, synthesis of proteins that are important to immune response, coagulation, and organ preservation must be maintained (16, 27), and failure to have this response is associated with adverse outcomes (30). In this study, we observed that fibrinogen synthesis was maintained, but albumin synthesis decreased after severe hemorrhage. These differential changes in synthesis might also reflect a shift of hepatic synthesis toward producing proteins necessary to survive from severe hemorrhagic shock, although much of the underlying mechanism remains to be explored. Nevertheless, prioritizing hepatic synthesis after insults appears to be a generalized response, occurring at whole-body level as well as within an individual organ. It should be emphasized that hepatic synthesis is a dynamic process. Changes in albumin and fibrinogen synthesis at different time points may differ from what was observed in this study. Although fibrinogen synthesis was maintained after severe hemorrhagic shock, it is important to point out that quantitatively fibrinogen synthesis rate was very slow, i.e., 4% of the entire
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body pool per hour. Thus, a 60% blood loss (60% fibrinogen loss) would require at least 15 h to synthesize enough new fibrinogen to compensate the lost fibrinogen, assuming there is no degradation. Thus, data from this study support the approach of fibrinogen supplementation to restore hemostasis in acutely bleeding patients after hemorrhagic shock. Fibrinogen is an acute phase reactant, and albumin is a constitutive protein. After various stresses, including hemorrhagic shock, the levels of constitutive proteins decrease, and acute phase reactants increases. In this study, fibrinogen level did not increase, although fibrinogen synthesis was maintained after hemorrhage. Our previous study showed that after a moderate hemorrhage, fibrinogen level was doubled at 24 h after a moderate hemorrhage. Thus, the lack of increase in fibrinogen level in this study is likely due to the short duration of the study (6 h). However, changes of fibrinogen to changes in C-reactive protein are unclear. In this study, resuscitation with LR solution at three times the bled volume returned the MAP to the prehemorrhage level. To reach the same goal, about 50% more volume of NS was used in the NS group. Despite similar restoration of hemodynamics, there was a significant separation of acid-base status between the two resuscitation groups: LR resuscitation returned BE and bicarbonate to prehemorrhage levels within 3 h, but no return of BE or bicarbonate was observed with NS resuscitation during the study. As an index of shock, acidosis is associated with well-known detrimental effects on coagulation (31, 32). However, changes in synthesis of fibrinogen and albumin were similar between the LR and the NS groups in this study, suggesting that hepatic synthesis was not affected by changes in acid-base status. Consistently, when acidosis of 7.1 was induced in pigs, we previously reported that fibrinogen synthesis in those acidotic pigs was similar to that of control animals (pH 7.4) (33). On the other hand, when hypothermia of 32-C was induced in pigs, fibrinogen synthesis in those pigs was inhibited to 50% of that of control pigs (34). Thus, it appears that hepatic synthesis is sensitive to changes in body temperature but resistant to changes in acid-base status. We observed similar changes of albumin synthesis and fibrinogen synthesis in the LR and NS groups in this study, suggesting that the effects of LR solution and NS on hepatic protein synthesis were similar. Previously in a swine model with a moderate hemorrhage (35%), we found that fibrinogen synthesis was similar in hemorrhaged pigs with and without LR resuscitation in a swine model with a moderate (35%) hemorrhage, indicating that LR resuscitation does not affect synthesis (19). Put together, these data suggest that changes of hepatic protein synthesis reflect the responses of hemorrhage and that crystalloid resuscitation has minimal effects on hepatic synthesis. However, it remains to be confirmed whether this conclusion holds under colloid resuscitation, because more dramatic changes in hemodilution and coagulation are observed with colloid resuscitation (32). Although hemorrhage and resuscitation decreased albumin synthesis, we did not observe a corresponding decrease in albumin concentration during 6 h after resuscitation in the study, except changes due to hemodilution from resuscitation. This apparent discrepancy is likely due to fluctuations of plasma volume after resuscitation. With direct measurements of plasma volumes at different time points after resuscitation,
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SHOCK JANUARY 2014 we found that plasma volume was elevated above prehemorrhage value at 15 min after resuscitation but reached equilibrium within 3 h after resuscitation. Thus, the reduction of plasma volume a few hours after resuscitation might cause a Bpseudo[ increase in albumin concentration, which offset the effect of decreased synthesis of albumin. In addition, albumin transport from different pools is likely to be another confounding factor. As a transport protein, albumin has a molecular weight of 65,000, and about 50% of its content is confined in the vascular pool (12). Albumin transport, assessed as the transvascular escape rate, in normal subjects is found to be about 4% to 5%/h (12), which is greater than 20 times higher than that of normal albumin synthesis. Thus, changes in albumin transport can have a significant impact on plasma albumin levels, although this was not quantified in the present study. Nevertheless, because hepatic synthesis is the only source for new albumin production, it is assumed that changes in albumin synthesis would be reflected in albumin concentration eventually. Our study had some limitations. First, a volume-controlled hemorrhage model was used in this study to evaluate the effects of hemorrhage alone on hepatic synthesis of albumin and fibrinogen. Although allowing better standardization and clarification, this model was not able to fully represent clinical bleeding situation. Second, tissue injury was not included in this study, which may have significant impact on the acute phase response, including hepatic synthesis. Thus, changes of hepatic synthesis of albumin and fibrinogen may be different in bleeding patients with traumatic injury. In conclusion, we investigated the responses of acute hepatic protein synthesis to hemorrhage in a swine model. Severe hemorrhagic shock caused differential changes in hepatic protein synthesis: albumin synthesis was inhibited, but fibrinogen synthesis remained unchanged. Resuscitation with LR solution and NS to comparable blood pressure had equivalent effects on hepatic synthesis of albumin and fibrinogen, despite better acid-base status with LR solution. With growing interests in fibrinogen supplementation in trauma patients to restore coagulation, future effort is warranted to investigate hepatic synthesis of acute phase proteins following albumin therapy and fibrinogen supplementation. ACKNOWLEDGMENTS
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The authors thank Douglas Cortez, Shavaughn Colvin, and Nahir Miranda for their technical assistance in animal study and sample analysis; Mr. John Jones for statistical analysis; and Otilia Sa´nchez for editing the article.
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