Research Note
Blood biochemical changes in pigs after infusion with acetate-buffered or lactatebuffered crystalloid solutions
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© 2015 Nature America, Inc. All rights reserved.
Claudia Keibl, DVM1, Wolfgang Sipos, DVM, PhD, Dipl. ECPHM2, Martin Ponschab, MD1 & Christoph J. Schlimp, MD1
Perioperative fluid therapy is an important component of many medical procedures with animals. Buffered crystalloid solutions avoid inducing metabolic acidosis, but lactated solutions can elevate blood lactate concentrations and acetated solutions have not been thoroughly investigated using large animals. Here, the authors compare blood biochemical parameters in 20 juvenile pigs after perioperative fluid administration of an acetate-buffered solution (Elo-Mel isoton, EMI) or a lactate-buffered solution (lactated Ringer’s solution, LRS). The authors measured blood lactate, glucose and electrolyte concentrations before and after administering the test fluid during surgery. Blood lactate concentration after administration was significantly higher in pigs that received LRS than in pigs that received EMI, but glucose and electrolyte concentrations did not differ significantly between treatment groups before or after administration. These findings suggest that EMI might be a preferable option for perioperative fluid therapy in pigs. Perioperative fluid therapy is an important component of various veterinary medical procedures. Nevertheless, clear guidelines for perioperative fluid replacement are lacking and choice of fluid generally depends on hospital policy and the personal preferences of individual physicians. Crystalloid solutions are mostly used in routine clinical infusion regimens and various types are available for perioperative fluid replacement. Crystalloid solutions are designed to be physiologically compatible, consisting of 0.9% saline containing only sodium (154 mmol/l) and chloride (154 mmol/l). Plain Ringer’s solution is one crystalloid fluid that is nearly isotonic and contains sodium (147.2 mmol/l), chloride (155.7 mmol/l), potassium (4.0 mmol/l) and calcium (2.25 mmol/l). The anion content of these solutions is higher than that of blood plasma, and they can therefore induce a hyperchloremic overload and cause acidosis during or after infusion1. For this reason,
chemically buffered solutions are used more frequently in human medicine2. Buffered solutions are designed to maintain acid–base equilibrium during and after infusion. Lactate has been used to buffer crystalloid solutions for decades, and more recently acetate has also been used to maintain acid–base equilibrium in perioperative fluids3. Lactated Ringer’s solution (LRS) is a lactate-buffered electrolyte solution with a hypotonic ion concentration of 277 mOsmol/l. LRS contains sodium (131.0 mmol/l), chloride (112.0 mmol/l), potassium (5.4 mmol/l), calcium (1.8 mmol/l) and lactate (28.0 mmol/l) at a pH of 5.0–7.0. The anionic sodium lactate component of LRS functions as a buffer and can be metabolized to bicarbonate to prevent dilutional acidosis. Infusion of LRS also increases blood lactate, which normally forms endogenously from lactic acid that is produced during glycolysis under anaerobic conditions in skin, muscle, erythrocytes, brain and intestinal mucosa. Anaerobic
1Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Austrian Cluster for Tissue Regeneration, Vienna, Austria. 2University of Veterinary Medicine Vienna, Department for Farm Animals and Veterinary Public Health, Vienna, Austria. Correspondence should be addressed to C.K. ([email protected]).
268 Volume 44, No. 7 | JULY 2015
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Research Note
npg
© 2015 Nature America, Inc. All rights reserved.
metabolism is a consequence of inadequate oxygen supply to tissues, and excess lactate is often used as a prognostic indicator for critically ill patients4. Lactate is an important indicator for the intensity of sepsis or shock, for tissue hypoxia after hypoperfusion and for general physical performance and exercise. Clinicians must therefore avoid using lactated fluids in many clinical scenarios to prevent misinterpretations from a therapeutically altered blood lactate concentration5,6. Elo-Mel isoton solution (EMI) is an acetate-buffered crystalloid solution with an isotonic ion concentration of 302 mOsmol/l. EMI contains sodium (140.0 mmol/l), chloride (108.0 mmol/l), potassium (5.0 mmol/l), calcium (2.5 mmol/l) and acetate (45.0 mmol/l) at a pH of 6.0–7.5. This composition imitates the electrolyte composition of plasma more closely than LRS does and does not include lactate5. Acetated solutions alkalize plasma as quickly as solutions that contain bicarbonate7, and acetate is also metabolized more quickly than lactate8. One study of normal humans showed that less than 10% of total administrated acetate was excreted renally9. Vasodilation can be a side effect of acetate10, and blood pressure can briefly fluctuate during fluid therapy with acetated solutions3,11. Acetated solutions are used to alkalize biochemical systems during many medical treatments that include management of diabetic lactic acidosis, alkalization of urine and reduction of calcium excretion during hemorrhagic shock and hepatectomy12–14. Many studies have also used LRS to replace fluids in pigs modeling hemorrhage, resuscitation, trauma and combinations thereof15–19, and more recent research has used EMI while investigating inhalational anesthetics in goats and pigs and hypothermia in pigs20,21. Juvenile pigs are frequently used in preclinical studies for a wide range of conditions, including cardiovascular and resuscitation medicine, osteology and bone repair, trauma, shock, hernia repair, skin grafting, allergic skin diseases and burns22–29. This study compares serum biochemical and hematological effects of LRS and EMI in juvenile pigs after perioperative fluid administration during the preparatory phase of an experimental hemorrhagic shock and coagulation therapy model. We primarily focused on blood lactate concentration, predicting that EMI, unlike LRS, would not alter perioperative blood lactate concentration. We also measured whole blood glucose concentration, electrolyte concentrations and acid–base balance, although we did not expect these parameters to differ between treatments. METHODS Pigs and husbandry This experiment took place during the surgical preparation phase for a large hemorrhagic shock study designed to test a novel coagulation therapy. The goal of this surgical LAB ANIMAL
preparation is to place one central venous access at the external jugular vein for intraoperative drug administration, one arterial access at the internal carotid artery for blood pressure monitoring and a urinary catheter through a laparotomy. The local Animal Investigation Committee of Vienna, Austria, approved all experiments. All materials and methods have been previously described30, except the infusion therapy of this study. This study used 20 juvenile male Landrace pigs, 12–16 weeks old and weighing 30.9 ± 4.4 kg purchased from a government-approved farmer in Münichsthal, Austria. We housed pigs in groups of two on straw bedding in a 5.04-m2 open-air stable within our research facilities. Animal care was carried out and documented by qualified personnel and supervised by veterinarians. All pigs were fed ad libitum with shredded diet consisting of products from domestic farms (40% maize, 19% barley, 17% soya, 10% wheat bran, 5% peas, 5% triticale) and nutritional supplement (4% porkovit-F, Garant-Tiernahrung, Pöchlarn, Austria). Tap water was available ad libitum. The stable was kept on a 12-h:12-h light:dark cycle with dimmable light, at an average temperature within 19–23 °C and at relative humidity of approximately 55 ± 10%. Pigs were housed in this environment for 2 d to acclimatize before surgery. Pigs were fasted during the night before surgery to prevent vomiting during premedication but had free access to water until administration of anesthesia on the day of surgery. Surgical procedure Surgery consisted of the insertion of a perfusor catheter into the external jugular vein for intraoperative drug administration and fluid therapy30. We premedicated the pigs using an intramuscular injection of butorphanol (0.1 mg/kg body weight; Richter Pharma, Wels, Austria), medetomidine (0.03 mg/kg body weight; Eurovet Animal Health, Bladel, the Netherlands) and midazolam (0.5 mg/kg body weight; Erwo Pharma, Brunn, Austria). We established auricular vein access by inserting an intravenous cannula (Venflon 20-gauge, Becton Dickinson Infusion Therapy, Helsingborg, Sweden) into the lateral auricular vein of each pig. We then sedated the pigs by administering intravenous (i.v.) ketamine (0.07 mg/kg body weight; AniMedica, Wels, Austria) through the auricular vein cannula and placed the pigs in a supine position on a vacuum mattress for the remainder of the procedure. About 15 min after premedication and before intubation, we collected 1 ml of venous blood from the auricular vein of each pig using a 1-ml heparin-treated syringe (Safe PICO 70, Radiometer, Copenhagen, Denmark). Immediately after collecting the first blood sample, we intubated pigs with a 6.5-mm tracheal tube (Rüsch, Vienna, Austria) and maintained volume-controlled ventilation (Primus, Dräger, Lübeck, Germany) with Volume 44, No. 7 | JULY 2015 2 69
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© 2015 Nature America, Inc. All rights reserved.
Research Note
a tidal volume of 10 ml per kg body weight, a rate of 20 breaths per min and a positive end-expiratory pressure of 3 mbar for normocapnia with an end-tidal carbon dioxide concentration of 4.5–5.5%. We randomly assigned each pig to receive either LRS (Braun, Melsungen, Germany) or EMI (Fresenius Kabi, Graz, Austria) to replace fluids during the surgical procedure (n = 10 per group) and began administering a continuous i.v. infusion of test fluid with rocuronium bromide (5 mg/kg body weight/h; Fresenius Kabi, Graz, Austria) and sufentanil (0.008 mg/kg body weight/h; Janssen, Vienna, Austria) to each pig. We administered this infusion through the auricular vein until we catheterized the external jugular vein, whereupon we administered drugs and test fluids only through the external jugular vein. We maintained oxygen saturation and monitored the depth of anesthesia throughout the procedure for each pig, using an Infinity Patient Monitoring System (Draeger, Danvers, MA) with a pulse oximeter on the tail, electrocardiogram electrodes clipped to the chest and an oral temperature sensor. After tracheal intubation and until the catheter had been placed in the external jugular vein, all pigs inhaled 1% isoflurane (AbbVie, Vienna, Austria) and an inspiratory oxygen concentration of 30%. After we placed the catheter in the external jugular vein, we maintained anesthesia solely with i.v. drug administration. After tracheal intubation we created an incision at the neck through skin, fat and muscle tissue. We incised the external jugular vein and intraluminally inserted a perfusion catheter (Perfusor Line, Braun, Melsungen, Germany) filled with test fluid, as previously described30. We secured the catheter with two ligatures around the vein (Dagrofil green 2/0, Braun, Tuttlingen, Germany), ceased i.v. infusion through the auricular vein and resumed i.v. infusion of test fluid with rocuronium bromide (5 mg/kg body weight/h; Fresenius Kabi, Graz, Austria), sufentanil (0.008 mg/kg body weight/h; Janssen, Vienna, Austria) and midazolam (0.8 mg/kg body weight/h; Erwo Pharma, Brunn, Austria) through the external jugular vein. We then located and incised the internal carotid artery on the same side of the neck and intraluminally inserted a catheter by which we monitored intra-arterial blood pressure using the Infinity Patient Monitoring System (Draeger, Danvers, MA). We secured this catheter in the same fashion, with two ligatures around the artery. We also carried out a laparotomy of about 5 cm on each pig to place a urinary catheter. We made a small incision into the ventral apical bladder pole and inserted the urinary catheter (Ureofix 500 classic, Braun, Melsungen, Germany; balloon catheter brilliant size 20, Rüsch, Vienna, Austria), which we secured with a purse-string suture. We closed all neck and laparotomy wounds with sutures. 270 Volume 44, No. 7 | JULY 2015
We administered 1 l of test fluid over a total period of 1 h, first through the auricular vein access with rocuronium and sufentanil, then through the external jugular vein access with rocuronium, sufentanil and midazolam. We recorded the amount of time needed to infuse the test fluid for each pig. After infusion of the test fluid, we sampled 1 ml of arterial blood from each pig from the internal carotid artery using a 1-ml heparin-treated syringe. After fluid administration and blood sampling, all pigs were used in a subsequent hemorrhagic shock study. At the end of this second study, all pigs were euthanized in deep anesthesia with an i.v. overdose of a barbiturate (Sandoz, Kundl, Austria) and T61 (Intervet, Vienna, Austria). Blood sample analysis We analyzed all blood samples immediately after blood collection using a blood gas analyzer (ABL 870 Flex, Radiometer, Copenhagen, Denmark) to measure concentrations of lactate, glucose, sodium, chloride, potassium, calcium and bicarbonate (HCO3); pH; and base excess. We created the reference range for lactate using previously reported values from untreated pigs31,32 and obtained other reference ranges from ref. 33 (Table 1). We calculated a reference range for calcium using 50% of reported values for total calcium to account for calcium bound in proteins and ion complexes. Statistical analysis All data are expressed as mean ± standard deviation (s.d.). We tested the data for normality using the Kolmogorov–Smirnov test (Table 1). We compared data between treatment groups using independent samples t-tests with parametric data or the Mann–Whitney U-test with non-parametric data, and we compared data within treatment groups using paired samples t-tests with parametric data or Wilcoxon signed-rank tests with non-parametric data. Venous and arterial blood naturally differ in bicarbonate concentration, pH and base excess, owing to their different concentrations of oxygen and carbon dioxide; we therefore did not compare these parameters within each group, as one sample was collected from the jugular vein and the other sample was collected from the carotid artery. P values
Blood biochemical changes in pigs after infusion with acetate-buffered or lactatebuffered crystalloid solutions
npg
© 2015 Nature America, Inc. All rights reserved.
Claudia Keibl, DVM1, Wolfgang Sipos, DVM, PhD, Dipl. ECPHM2, Martin Ponschab, MD1 & Christoph J. Schlimp, MD1
Perioperative fluid therapy is an important component of many medical procedures with animals. Buffered crystalloid solutions avoid inducing metabolic acidosis, but lactated solutions can elevate blood lactate concentrations and acetated solutions have not been thoroughly investigated using large animals. Here, the authors compare blood biochemical parameters in 20 juvenile pigs after perioperative fluid administration of an acetate-buffered solution (Elo-Mel isoton, EMI) or a lactate-buffered solution (lactated Ringer’s solution, LRS). The authors measured blood lactate, glucose and electrolyte concentrations before and after administering the test fluid during surgery. Blood lactate concentration after administration was significantly higher in pigs that received LRS than in pigs that received EMI, but glucose and electrolyte concentrations did not differ significantly between treatment groups before or after administration. These findings suggest that EMI might be a preferable option for perioperative fluid therapy in pigs. Perioperative fluid therapy is an important component of various veterinary medical procedures. Nevertheless, clear guidelines for perioperative fluid replacement are lacking and choice of fluid generally depends on hospital policy and the personal preferences of individual physicians. Crystalloid solutions are mostly used in routine clinical infusion regimens and various types are available for perioperative fluid replacement. Crystalloid solutions are designed to be physiologically compatible, consisting of 0.9% saline containing only sodium (154 mmol/l) and chloride (154 mmol/l). Plain Ringer’s solution is one crystalloid fluid that is nearly isotonic and contains sodium (147.2 mmol/l), chloride (155.7 mmol/l), potassium (4.0 mmol/l) and calcium (2.25 mmol/l). The anion content of these solutions is higher than that of blood plasma, and they can therefore induce a hyperchloremic overload and cause acidosis during or after infusion1. For this reason,
chemically buffered solutions are used more frequently in human medicine2. Buffered solutions are designed to maintain acid–base equilibrium during and after infusion. Lactate has been used to buffer crystalloid solutions for decades, and more recently acetate has also been used to maintain acid–base equilibrium in perioperative fluids3. Lactated Ringer’s solution (LRS) is a lactate-buffered electrolyte solution with a hypotonic ion concentration of 277 mOsmol/l. LRS contains sodium (131.0 mmol/l), chloride (112.0 mmol/l), potassium (5.4 mmol/l), calcium (1.8 mmol/l) and lactate (28.0 mmol/l) at a pH of 5.0–7.0. The anionic sodium lactate component of LRS functions as a buffer and can be metabolized to bicarbonate to prevent dilutional acidosis. Infusion of LRS also increases blood lactate, which normally forms endogenously from lactic acid that is produced during glycolysis under anaerobic conditions in skin, muscle, erythrocytes, brain and intestinal mucosa. Anaerobic
1Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Austrian Cluster for Tissue Regeneration, Vienna, Austria. 2University of Veterinary Medicine Vienna, Department for Farm Animals and Veterinary Public Health, Vienna, Austria. Correspondence should be addressed to C.K. ([email protected]).
268 Volume 44, No. 7 | JULY 2015
www.labanimal.com
Research Note
npg
© 2015 Nature America, Inc. All rights reserved.
metabolism is a consequence of inadequate oxygen supply to tissues, and excess lactate is often used as a prognostic indicator for critically ill patients4. Lactate is an important indicator for the intensity of sepsis or shock, for tissue hypoxia after hypoperfusion and for general physical performance and exercise. Clinicians must therefore avoid using lactated fluids in many clinical scenarios to prevent misinterpretations from a therapeutically altered blood lactate concentration5,6. Elo-Mel isoton solution (EMI) is an acetate-buffered crystalloid solution with an isotonic ion concentration of 302 mOsmol/l. EMI contains sodium (140.0 mmol/l), chloride (108.0 mmol/l), potassium (5.0 mmol/l), calcium (2.5 mmol/l) and acetate (45.0 mmol/l) at a pH of 6.0–7.5. This composition imitates the electrolyte composition of plasma more closely than LRS does and does not include lactate5. Acetated solutions alkalize plasma as quickly as solutions that contain bicarbonate7, and acetate is also metabolized more quickly than lactate8. One study of normal humans showed that less than 10% of total administrated acetate was excreted renally9. Vasodilation can be a side effect of acetate10, and blood pressure can briefly fluctuate during fluid therapy with acetated solutions3,11. Acetated solutions are used to alkalize biochemical systems during many medical treatments that include management of diabetic lactic acidosis, alkalization of urine and reduction of calcium excretion during hemorrhagic shock and hepatectomy12–14. Many studies have also used LRS to replace fluids in pigs modeling hemorrhage, resuscitation, trauma and combinations thereof15–19, and more recent research has used EMI while investigating inhalational anesthetics in goats and pigs and hypothermia in pigs20,21. Juvenile pigs are frequently used in preclinical studies for a wide range of conditions, including cardiovascular and resuscitation medicine, osteology and bone repair, trauma, shock, hernia repair, skin grafting, allergic skin diseases and burns22–29. This study compares serum biochemical and hematological effects of LRS and EMI in juvenile pigs after perioperative fluid administration during the preparatory phase of an experimental hemorrhagic shock and coagulation therapy model. We primarily focused on blood lactate concentration, predicting that EMI, unlike LRS, would not alter perioperative blood lactate concentration. We also measured whole blood glucose concentration, electrolyte concentrations and acid–base balance, although we did not expect these parameters to differ between treatments. METHODS Pigs and husbandry This experiment took place during the surgical preparation phase for a large hemorrhagic shock study designed to test a novel coagulation therapy. The goal of this surgical LAB ANIMAL
preparation is to place one central venous access at the external jugular vein for intraoperative drug administration, one arterial access at the internal carotid artery for blood pressure monitoring and a urinary catheter through a laparotomy. The local Animal Investigation Committee of Vienna, Austria, approved all experiments. All materials and methods have been previously described30, except the infusion therapy of this study. This study used 20 juvenile male Landrace pigs, 12–16 weeks old and weighing 30.9 ± 4.4 kg purchased from a government-approved farmer in Münichsthal, Austria. We housed pigs in groups of two on straw bedding in a 5.04-m2 open-air stable within our research facilities. Animal care was carried out and documented by qualified personnel and supervised by veterinarians. All pigs were fed ad libitum with shredded diet consisting of products from domestic farms (40% maize, 19% barley, 17% soya, 10% wheat bran, 5% peas, 5% triticale) and nutritional supplement (4% porkovit-F, Garant-Tiernahrung, Pöchlarn, Austria). Tap water was available ad libitum. The stable was kept on a 12-h:12-h light:dark cycle with dimmable light, at an average temperature within 19–23 °C and at relative humidity of approximately 55 ± 10%. Pigs were housed in this environment for 2 d to acclimatize before surgery. Pigs were fasted during the night before surgery to prevent vomiting during premedication but had free access to water until administration of anesthesia on the day of surgery. Surgical procedure Surgery consisted of the insertion of a perfusor catheter into the external jugular vein for intraoperative drug administration and fluid therapy30. We premedicated the pigs using an intramuscular injection of butorphanol (0.1 mg/kg body weight; Richter Pharma, Wels, Austria), medetomidine (0.03 mg/kg body weight; Eurovet Animal Health, Bladel, the Netherlands) and midazolam (0.5 mg/kg body weight; Erwo Pharma, Brunn, Austria). We established auricular vein access by inserting an intravenous cannula (Venflon 20-gauge, Becton Dickinson Infusion Therapy, Helsingborg, Sweden) into the lateral auricular vein of each pig. We then sedated the pigs by administering intravenous (i.v.) ketamine (0.07 mg/kg body weight; AniMedica, Wels, Austria) through the auricular vein cannula and placed the pigs in a supine position on a vacuum mattress for the remainder of the procedure. About 15 min after premedication and before intubation, we collected 1 ml of venous blood from the auricular vein of each pig using a 1-ml heparin-treated syringe (Safe PICO 70, Radiometer, Copenhagen, Denmark). Immediately after collecting the first blood sample, we intubated pigs with a 6.5-mm tracheal tube (Rüsch, Vienna, Austria) and maintained volume-controlled ventilation (Primus, Dräger, Lübeck, Germany) with Volume 44, No. 7 | JULY 2015 2 69
npg
© 2015 Nature America, Inc. All rights reserved.
Research Note
a tidal volume of 10 ml per kg body weight, a rate of 20 breaths per min and a positive end-expiratory pressure of 3 mbar for normocapnia with an end-tidal carbon dioxide concentration of 4.5–5.5%. We randomly assigned each pig to receive either LRS (Braun, Melsungen, Germany) or EMI (Fresenius Kabi, Graz, Austria) to replace fluids during the surgical procedure (n = 10 per group) and began administering a continuous i.v. infusion of test fluid with rocuronium bromide (5 mg/kg body weight/h; Fresenius Kabi, Graz, Austria) and sufentanil (0.008 mg/kg body weight/h; Janssen, Vienna, Austria) to each pig. We administered this infusion through the auricular vein until we catheterized the external jugular vein, whereupon we administered drugs and test fluids only through the external jugular vein. We maintained oxygen saturation and monitored the depth of anesthesia throughout the procedure for each pig, using an Infinity Patient Monitoring System (Draeger, Danvers, MA) with a pulse oximeter on the tail, electrocardiogram electrodes clipped to the chest and an oral temperature sensor. After tracheal intubation and until the catheter had been placed in the external jugular vein, all pigs inhaled 1% isoflurane (AbbVie, Vienna, Austria) and an inspiratory oxygen concentration of 30%. After we placed the catheter in the external jugular vein, we maintained anesthesia solely with i.v. drug administration. After tracheal intubation we created an incision at the neck through skin, fat and muscle tissue. We incised the external jugular vein and intraluminally inserted a perfusion catheter (Perfusor Line, Braun, Melsungen, Germany) filled with test fluid, as previously described30. We secured the catheter with two ligatures around the vein (Dagrofil green 2/0, Braun, Tuttlingen, Germany), ceased i.v. infusion through the auricular vein and resumed i.v. infusion of test fluid with rocuronium bromide (5 mg/kg body weight/h; Fresenius Kabi, Graz, Austria), sufentanil (0.008 mg/kg body weight/h; Janssen, Vienna, Austria) and midazolam (0.8 mg/kg body weight/h; Erwo Pharma, Brunn, Austria) through the external jugular vein. We then located and incised the internal carotid artery on the same side of the neck and intraluminally inserted a catheter by which we monitored intra-arterial blood pressure using the Infinity Patient Monitoring System (Draeger, Danvers, MA). We secured this catheter in the same fashion, with two ligatures around the artery. We also carried out a laparotomy of about 5 cm on each pig to place a urinary catheter. We made a small incision into the ventral apical bladder pole and inserted the urinary catheter (Ureofix 500 classic, Braun, Melsungen, Germany; balloon catheter brilliant size 20, Rüsch, Vienna, Austria), which we secured with a purse-string suture. We closed all neck and laparotomy wounds with sutures. 270 Volume 44, No. 7 | JULY 2015
We administered 1 l of test fluid over a total period of 1 h, first through the auricular vein access with rocuronium and sufentanil, then through the external jugular vein access with rocuronium, sufentanil and midazolam. We recorded the amount of time needed to infuse the test fluid for each pig. After infusion of the test fluid, we sampled 1 ml of arterial blood from each pig from the internal carotid artery using a 1-ml heparin-treated syringe. After fluid administration and blood sampling, all pigs were used in a subsequent hemorrhagic shock study. At the end of this second study, all pigs were euthanized in deep anesthesia with an i.v. overdose of a barbiturate (Sandoz, Kundl, Austria) and T61 (Intervet, Vienna, Austria). Blood sample analysis We analyzed all blood samples immediately after blood collection using a blood gas analyzer (ABL 870 Flex, Radiometer, Copenhagen, Denmark) to measure concentrations of lactate, glucose, sodium, chloride, potassium, calcium and bicarbonate (HCO3); pH; and base excess. We created the reference range for lactate using previously reported values from untreated pigs31,32 and obtained other reference ranges from ref. 33 (Table 1). We calculated a reference range for calcium using 50% of reported values for total calcium to account for calcium bound in proteins and ion complexes. Statistical analysis All data are expressed as mean ± standard deviation (s.d.). We tested the data for normality using the Kolmogorov–Smirnov test (Table 1). We compared data between treatment groups using independent samples t-tests with parametric data or the Mann–Whitney U-test with non-parametric data, and we compared data within treatment groups using paired samples t-tests with parametric data or Wilcoxon signed-rank tests with non-parametric data. Venous and arterial blood naturally differ in bicarbonate concentration, pH and base excess, owing to their different concentrations of oxygen and carbon dioxide; we therefore did not compare these parameters within each group, as one sample was collected from the jugular vein and the other sample was collected from the carotid artery. P values
