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Neonatal endotoxemia affects heart but not kidney bioenergetics
Neonatal Endotoxemia Affects Heart But Not Kidney Bioenergetics By Koji Fukumoto, Agostino Pierro, Lewis Spitz, and Simon Eaton London, England
Purpose: The aim was to determine the effects of early and late endotoxemia on neonatal cardiac and renal mitochondrial energetics. Methods: Suckling rats received intraperitoneal 300 g/kg lipopolysaccharide; controls received saline. Heart and kidney mitochondria were isolated after 2 hours (early) or 6 hours (late sepsis). State 3 (maximum mitochondrial flux) and 4 O2 consumption and complex I activity were measured. Results, expressed as mean ⫾ SEM normalized to citrate synthase (CS), were compared using paired t tests. Results: Mortality rate was zero within 2 hours, 2.7% between 2 and 6 hours of endotoxemia, and 100% 6 to 8 hours; therefore, we consider that 2 hours and 6 hours represent early and late endotoxemia, respectively. Endotoxic heart mitochondria had unaltered O2 consumption at 2 hours but
S
EPSIS REMAINS an important cause of morbidity and mortality in infants and children.1 It is associated with many metabolic and pathophysiologic alterations.2 However, the metabolic response to sepsis in infants and children is little known. A proportion of infants and children with sepsis progress to multiple system organ failure including cardiac and renal failure, the pathogenesis of which remains unclear.3-5 The early events that follow the host response to sepsis are represented by cytokine production. This process, which is essential for the antibiotic activity of phagocytes, produces significant quantities of reactive oxygen species including hydrogen peroxide (H2O2) and nitric oxide (NO). However, in addition to an involvement in the bacterial killing process, reactive oxygen species can contribute also to organ damage and to multiple organ failure. We have shown that H2O2 inhibits hepatic mitochondrial oxygen consumption6,7 and that hepatocyte mitochondrial function is similarly impaired in neonatal From the Department of Paediatric Surgery, Institute of Child Health, London, England. Presented at the 34th Annual Meeting of the Canadian Association of Paediatric Surgeons, Vancouver, British Columbia, Canada, September 19-22, 2002. Address reprint requests to Simon Eaton, PhD, Department of Paediatric Surgery, Institute of Child Health, 30 Guilford St, London WC1N 1EH, England. © 2003 Elsevier Inc. All rights reserved. 0022-3468/03/3805-0008$30.00/0 10.1016/S0022-3468(03)00028-9 690
significantly decreased state 3 after 6 hours, resulting in significantly decreased respiratory control ratio. Complex I activity, which could affect O2 consumption, was decreased significantly at 6 hours (9.8 ⫾ 0.6 mU/U CS; n ⫽ 15) versus controls (11.3 ⫾ 0.8, n ⫽ 15; P ⫽ .04), but not at 2 hours. There were no differences in these measurements at either 2 hours or 6 hours in kidney mitochondria. Conclusions: The respiratory chain is affected late in endotoxemia. Neither early nor late endotoxemia affects oxidative function of kidney mitochondria. J Pediatr Surg 38:690-693. © 2003 Elsevier Inc. All rights reserved. INDEX WORDS: Endotoxin, cardiac mitochondrial activity, renal mitochondrial activity, oxygen consumption.
endotoxaemia.8 Other groups have similarly shown decreased respiratory chain function in septic liver mitochondria.9 However, knowledge about the effects of sepsis on neonatal heart and kidney function is limited. Both organs have a high demand for adenosine triphosphate (ATP), and utilize fatty acid substrates during the neonatal period.10 We have shown that endotoxemia inhibits carnitine palmitoyl transferase I (a rate-controlling enzyme of fatty acid -oxidation) activity in the heart but not in the kidney,11 and that heart and kidney mitochondria respond differently to H2O2.12 However, its is not known whether endotoxemia affects respiratory chain function of neonatal heart or kidney mitochondria, which could contribute to bioenergetic failure. MATERIALS AND METHODS
Model of Neonatal Endotoxemia Endotoxemia was modelled in suckling Wistar rats (11 to 13 days) by intraperitoneal injection of 300 g/kg of 12.5 mg/L lipopolysaccharide (LPS; Escherichia coli 055:B5, Sigma, Poole, Dorset, England) as previously described.8,11 Controls were injected with isovolemic normal saline. Animals were killed and organs extracted after either 2 or 6 hours. All experimental protocols were in accordance with local and national rules for the proper care and use of experimental animals.
Isolation of Mitochondria Cardiac and renal mitochondria were isolated from the heart and kidney of 11 to 13-day-old (peak suckling) Wistar rats as previously described.11 Journal of Pediatric Surgery, Vol 38, No 5 (May), 2003: pp 690-693
NEONATAL ENDOTOXEMIA
691
Mitochondrial Oxygen Consumption Oxygen consumption was measured polarographically in mitochondria incubated in 0.75 mL medium containing 120 mmol/L KCl, 10 mmol/L HEPES, 10 mmol/L phosphate, 1 mmol/L EDTA, 0.2 mg/mL cytochrome c, and 2 mg/mL BSA, pH 7.2 for heart13 and 110 mmol/L KCl, 10 mmol/L HEPES, 2.5 mmol/L phosphate, 1 mmol/L EDTA, 5 mmol/L MgCl2, 0.2 mg/mL cytochrome c, and 2 mg/mL BSA, pH 7.2 for kidney.14 Glutamate 10 mmol/L plus 1 mmol/L malate was used as respiratory substrate; state 3 respiration (representing maximum oxidative flux) was measured after addition of 0.18 mol/L ADP (adenosine diphosphate). State 4, representing leakage of extruded protons, was measured after all the ADP was converted to ATP. The respiratory control ratio was calculated as the ratio of the state 3 to the state 4 rates.
Complex I Assay Complex I activity was measured spectrophotometrically as rotenone-sensitive NADH-dehydrogenase activity, as described by BirchMachin et al15: mitochondria were diluted to 1 mg/mL in 25 mmol/L KH2PO4/5 mmol/L MgCl2, pH 7.2 and freeze-thawed 3 times in liquid nitrogen. NADH-dehydrogenase then was assayed in 1 mL of a medium containing 25 mmol/L PO4/5 mmol/L MgCl2/ 2.5 mg/mL BSA/ 2 mmol/L KCN, 2 g/mL antimycin, 65 mol/L CoQ1, pH 7.2. Reactions were initiated with 0.13 mmol/L NADH and consumption of NADH followed spectrophotometrically at 340 nm and 25°C. After a linear rate was obtained, 2 g/mL rotenone was added and the rotenone-insensitive rate recorded. Complex I is completely rotenone sensitive, so complex I activity was calculated after subtracting the rotenone-insensitive rate, using an extinction coefficient of 6.22 mmol/ L⫺1 cm⫺1 for NADH at 340 nm.
Citrate Synthase Assay Oxygen consumption and complex I activity were normalized to citrate synthase activity, measured spectrophotometrically as described by Shepherd and Garland.16 Results were normalized also to mitochondrial protein and were qualitatively very similar.
Statistical Analyses Data are expressed as means ⫾ SEM. State 3 and state 4 oxygen consumption data were normalized to controls from the same litter of rats and analyzed by one-sample t test. Comparisons of the results were performed using paired or unpaired Student’s t test as indicated. Results showing probability levels of less than .05 were considered significant.
RESULTS
Mortality No rats died up to 2 hours of endotoxemia, and only 2.7% of rats died between 2 and 6 hours of endotoxemia. The remaining 96.3% of rats died between 6 and 8 hours so that we consider that 2 hours and 6 hours represent early and late endotoxemia, respectively. Heart Oxygen Consumption State 3 oxygen consumption, which is measured in the presence of ADP and represents maximum mitochondrial oxidative flux, was unaltered after 2 hours of endotoxemia in heart mitochondria but was decreased significantly after 6 hours of endotoxemia (Fig 1A). State 4 oxygen consumption, which is measured after all the ADP is utilized and represents oxygen consumption that
Fig 1. Heart mitochondrial oxygen consumption. Mitochondria were isolated from control or endotoxemic rat hearts after 2 or 6 hours and state 3 and state 4 oxygen consumption measured. (A) Light shading, state 3 oxygen consumption normalized to control; darker shading, state 4 oxygen consumption normalized to control, n ⴝ 15 per group, *P < .005. (B) Respiratory control ratio of control (C) and endotoxic hearts (E). **P ⴝ .0005 versus control.
is wasted and not used for ATP generation, was increased compared with control after 6 hours of endotoxemia, but this difference did not reach significance (P ⫽ .070; Fig 1A). Hence, the respiratory control ratio, which is the ratio of state 3 to state 4 and reflects the efficiency of oxidative metabolism to produce ATP, was decreased in heart mitochondria only after 6 hours of endotoxemia (Fig 1B). Kidney Oxygen Consumption There were no significant effects of endotoxemia on state 3 or state 4 oxygen consumption of kidney mitochondria at either 2 hours or 6 hours (Fig 2A). Hence, the respiratory control ratio of kidney mitochondria was unaltered at either 2 or 6 hours of endotoxemia (Fig 2B). Complex I Activity of Heart Mitochondria Activity of complex I of the respiratory chain was unaltered in heart mitochondria isolated after 2 hours of endotoxemia but was significantly decreased after 6 hours of endotoxemia (Fig 3).
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Fig 2. Kidney mitochondrial oxygen consumption. Mitochondria were isolated from control or endotoxemic rat kidneys after 2 or 6 hours and state 3 and state 4 oxygen consumption measured. (A) Light shading, state 3 oxygen consumption normalized to control, darker shading, state 4 oxygen consumption normalized to control, n ⴝ 11 to 15 per group. (B) Respiratory control ratio of control (C) and endotoxic kidney (E).
DISCUSSION
This study shows that neonatal endotoxemia has different effects on heart and kidney mitochondria. Detrimental effects on the heart respiratory chain are observed after 6 hours of endotoxemia, a phenomenon that closely precedes the death of the animals. Mitochondrial dysfunction resulting from endotoxemia/sepsis has been implicated in the pathogenesis of organ failure in the liver,9,8 heart,17,18 and kidney.19,20 However, it is not known whether this mitochondrial dysfunction occurs in neonates, because mitochondrial metabolism alters greatly during the transition from newborn to adult.21,22,10 We have previously shown that carnitine palmitoyl transferase I activity, which is crucial for the oxidation of fatty acids, is impaired in the heart but not the kidney of endotoxemic suckling rats.11 However, whether endotoxemia directly affects the activity of the mitochondrial respiratory chain of heart or kidney in neonates is not known. In the current study we undertook measurements of oxygen consumption in mitochondria
isolated from heart and kidney at 2 different times after endotoxin injection. Because suckling rats given endotoxin die after 6 to 8 hours, we consider mitochondria isolated after 6 hours of endotoxemia to represent the later stages of sepsis/organ failure. Biochemical effects at 2 or 6 hours were not caused by lack of fluid resuscitation because animals did not become significantly dehydrated during the course of the experiment. Despite our previous findings, using the same model, that cardiac fatty acid oxidation is already altered after 2 hours endotoxaemia,11 we did not find any alteration in mitochondrial oxygen consumption after 2 hours of endotoxemia. However, after 6 hours of endotoxemia, we measured a significant decrease in mitochondrial oxygen consumption. This was associated with a decrease in the activity of complex I of the respiratory chain, which has been suggested previously to be sensitive to damage caused by sepsis17 and other pathologic situations involving increased free radical production.23,24 These findings suggest that a switch from fat oxidation to carbohydrate oxidation in the heart precedes bioenergetic failure. However, in keeping with the findings of others,25 we could detect no evidence for alteration of kidney mitochondrial function at either time-point. Because neonatal kidney mitochondria are sensitive to the effects of H2O2,12 it is possible that in vivo during endotoxemia, kidney mitochondria are either exposed to lower levels of free radicals than the heart or are better protected from the effects of free radicals. Because cardiac mitochondrial respiratory chain metabolism is only altered in later stages of sepsis, this could provide a window of opportunity in which nutritional support (eg, by glutamine as a precursor of the antioxidant glutathione) or pharmacologic support (eg, by free-radical scavengers) could be beneficial.
Fig 3. Heart mitochondrial complex I activity. Mitochondria were isolated from control (C) or endotoxaemic (E) rat heart after 6 hours and complex I activity measured spectrophotometrically. Results, expressed as mU complex I activity per U citrate synthase activity, were compared by paired t test *P ⴝ .040.
NEONATAL ENDOTOXEMIA
693
REFERENCES 1. Ford HR, Rowe MI: Sepsis and related considerations, in O’Neill JA, Rowe MI, Grosfeld JL (eds): Pediatric Surgery. St Louis, MO Mosby-Year Book, 1998, pp 135-155 2. Vlessis AA, Goldman RK, Trunkey DD: New concepts in the pathophysiology of oxygen-metabolism during sepsis. Br J Surg 82: 870-876, 1995 3. Smith SD, Tagge EP, Hannakan C, et al: Characterization of neonatal multisystem organ failure in the surgical newborn. J Pediatr Surg 26:494-499, 1991 4. Morecroft JA, Spitz L, Hamilton PA, et al: Necrotizing enterocolitis—Multisystem organ failure of the newborn. Acta Paediatr 83: 21-23, 1994 5. Avanoglu A, Ergun O, Bakirtas E, et al: Characteristics of multisystem organ failure in neonates. Eur J Pediatr Surg 7:263-266, 1997 6. Romeo C, Eaton S, Quant PA, et al: Neonatal oxidative liver metabolism: Effects of hydrogen peroxide, a putative mediator of septic damage. J Pediatr Surg 34:1107-1111, 1999 7. Romeo C, Eaton S, Spitz L, et al: Nitric oxide inhibits neonatal hepatocyte oxidative metabolism. J Pediatr Surg 35:44-48, 2000 8. Markley MA, Pierro A, Eaton S: Hepatocyte mitochondrial metabolism is inhibited in neonatal rat endotoxaemia: Effects of glutamine. Clin Sci 102:337-344, 2002 9. Kantrow SP, Taylor DE, Carraway MS, et al: Oxidative metabolism in rat hepatocytes and mitochondria during sepsis. Arch Biochem Biophys 345:278-288, 1997 10. Girard J, Ferre´ P, Pe´ gorier J-P, et al: Adaptations of glucose and fatty-acid metabolism during perinatal-period and suckling-weaning transition. Physiol Rev 72:507-562, 1992 11. Fukumoto K, Pierro A, Spitz L, et al: Differential effects of neonatal endotoxemia on heart and kidney carnitine palmitoyl transferase I. J Pediatr Surg 37:723-726, 2002 12. Fukumoto K, Eaton S, Spitz L, et al: Cardiac and renal mitochondria respond differently to mediators of sepsis in neonates. Abstracts of the XLVII meeting of the British Association of Paediatric Surgeons. Italy, Sorrento, P23-P23, 2000 13. Sherratt HSA, Watmough NJ, Johnson MA, et al: Methods for study of normal and abnormal skeletal muscle mitochondria. Meth Biochem Anal 33:243-335, 1988
14. Turnbull DM, Bartlett K, Younan SI, et al: The effects of 2,5-(4-chlorophenyl)pentyloxirane-2-carbonyl-Co-A on mitochondrial oxidations. Biochem Pharmacol 33:475-481, 1984 15. Birch-Machin MA, Briggs HL, Saborido AA, et al: An evaluation of the measurement of the activities of complexes I-IV in the respiratory chain of human skeletal muscle mitochondria. Biochem Med Metab Biol 51:35-42, 1994 16. Shepherd D, Garland PB: Citrate synthase from rat liver. Meth Enzymol 13:11-16, 1969 17. Gellerich FN, Trumbeckaite S, Hertel K, et al: Impaired energy metabolism in hearts of septic baboons: Diminished activities of Complex I and Complex II of the mitochondrial respiratory chain. Shock 11:336-341, 1999 18. Trumbeckaite S, Opalka JR, Neuhof C, et al: Different sensitivity of rabbit heart and skeletal muscle to endotoxin-induced impairment of mitochondrial function. Eur J Biochem 268:1422-1429, 2001 19. Messner UK, Briner VA, Pfeilschifter J: Tumor necrosis factoralpha and lipopolysaccharide induce apoptotic cell death in bovine glomerular endothelial cells. Kidney Int 55:2322-2337, 1999 20. Kang YH, Falk MC, Bentley TB, et al: Distribution and role of lipopolysaccharide in the pathogenesis of acute renal proximal tubule injury. Shock 4:441-449, 1995 21. Valcarce C, Navarrete R, Encabo P, et al: Postnatal-development of rat-liver mitochondrial functions—the roles of protein-synthesis and of adenine-nucleotides. J Biol Chem 263:7767-7775, 1988 22. Lionetti L, Iossa S, Liverini G, et al: Changes in the hepatic mitochondrial respiratory system in the transition from weaning to adulthood in rats. Arch Biochem Biophys 352:240-246, 1998 23. Borutaite V, Budriunaite A, Brown GC: Reversal of nitric oxide-, peroxynitrite- and S-nitrosothiol-induced inhibition of mitochondrial respiration or complex I activity by light and thiols. Biochim Biophys Acta 1459:405-412, 2000 24. Hardy L, Clark JB, Darley-Usmar VM, et al: Reoxygenationdependent decrease in mitochondrial NADH:CoQ reductase (Complex I) activity in the hypoxic/reoxygenated rat heart. Biochem J 274:133137, 1991 25. Millar CGM, Brand MP, Heales SJR, et al: Preservation of renal mitochondrial function in endotoxemic acute renal failure. J Am Soc Nephrol 8:A2755, 1997
Purpose: The aim was to determine the effects of early and late endotoxemia on neonatal cardiac and renal mitochondrial energetics. Methods: Suckling rats received intraperitoneal 300 g/kg lipopolysaccharide; controls received saline. Heart and kidney mitochondria were isolated after 2 hours (early) or 6 hours (late sepsis). State 3 (maximum mitochondrial flux) and 4 O2 consumption and complex I activity were measured. Results, expressed as mean ⫾ SEM normalized to citrate synthase (CS), were compared using paired t tests. Results: Mortality rate was zero within 2 hours, 2.7% between 2 and 6 hours of endotoxemia, and 100% 6 to 8 hours; therefore, we consider that 2 hours and 6 hours represent early and late endotoxemia, respectively. Endotoxic heart mitochondria had unaltered O2 consumption at 2 hours but
S
EPSIS REMAINS an important cause of morbidity and mortality in infants and children.1 It is associated with many metabolic and pathophysiologic alterations.2 However, the metabolic response to sepsis in infants and children is little known. A proportion of infants and children with sepsis progress to multiple system organ failure including cardiac and renal failure, the pathogenesis of which remains unclear.3-5 The early events that follow the host response to sepsis are represented by cytokine production. This process, which is essential for the antibiotic activity of phagocytes, produces significant quantities of reactive oxygen species including hydrogen peroxide (H2O2) and nitric oxide (NO). However, in addition to an involvement in the bacterial killing process, reactive oxygen species can contribute also to organ damage and to multiple organ failure. We have shown that H2O2 inhibits hepatic mitochondrial oxygen consumption6,7 and that hepatocyte mitochondrial function is similarly impaired in neonatal From the Department of Paediatric Surgery, Institute of Child Health, London, England. Presented at the 34th Annual Meeting of the Canadian Association of Paediatric Surgeons, Vancouver, British Columbia, Canada, September 19-22, 2002. Address reprint requests to Simon Eaton, PhD, Department of Paediatric Surgery, Institute of Child Health, 30 Guilford St, London WC1N 1EH, England. © 2003 Elsevier Inc. All rights reserved. 0022-3468/03/3805-0008$30.00/0 10.1016/S0022-3468(03)00028-9 690
significantly decreased state 3 after 6 hours, resulting in significantly decreased respiratory control ratio. Complex I activity, which could affect O2 consumption, was decreased significantly at 6 hours (9.8 ⫾ 0.6 mU/U CS; n ⫽ 15) versus controls (11.3 ⫾ 0.8, n ⫽ 15; P ⫽ .04), but not at 2 hours. There were no differences in these measurements at either 2 hours or 6 hours in kidney mitochondria. Conclusions: The respiratory chain is affected late in endotoxemia. Neither early nor late endotoxemia affects oxidative function of kidney mitochondria. J Pediatr Surg 38:690-693. © 2003 Elsevier Inc. All rights reserved. INDEX WORDS: Endotoxin, cardiac mitochondrial activity, renal mitochondrial activity, oxygen consumption.
endotoxaemia.8 Other groups have similarly shown decreased respiratory chain function in septic liver mitochondria.9 However, knowledge about the effects of sepsis on neonatal heart and kidney function is limited. Both organs have a high demand for adenosine triphosphate (ATP), and utilize fatty acid substrates during the neonatal period.10 We have shown that endotoxemia inhibits carnitine palmitoyl transferase I (a rate-controlling enzyme of fatty acid -oxidation) activity in the heart but not in the kidney,11 and that heart and kidney mitochondria respond differently to H2O2.12 However, its is not known whether endotoxemia affects respiratory chain function of neonatal heart or kidney mitochondria, which could contribute to bioenergetic failure. MATERIALS AND METHODS
Model of Neonatal Endotoxemia Endotoxemia was modelled in suckling Wistar rats (11 to 13 days) by intraperitoneal injection of 300 g/kg of 12.5 mg/L lipopolysaccharide (LPS; Escherichia coli 055:B5, Sigma, Poole, Dorset, England) as previously described.8,11 Controls were injected with isovolemic normal saline. Animals were killed and organs extracted after either 2 or 6 hours. All experimental protocols were in accordance with local and national rules for the proper care and use of experimental animals.
Isolation of Mitochondria Cardiac and renal mitochondria were isolated from the heart and kidney of 11 to 13-day-old (peak suckling) Wistar rats as previously described.11 Journal of Pediatric Surgery, Vol 38, No 5 (May), 2003: pp 690-693
NEONATAL ENDOTOXEMIA
691
Mitochondrial Oxygen Consumption Oxygen consumption was measured polarographically in mitochondria incubated in 0.75 mL medium containing 120 mmol/L KCl, 10 mmol/L HEPES, 10 mmol/L phosphate, 1 mmol/L EDTA, 0.2 mg/mL cytochrome c, and 2 mg/mL BSA, pH 7.2 for heart13 and 110 mmol/L KCl, 10 mmol/L HEPES, 2.5 mmol/L phosphate, 1 mmol/L EDTA, 5 mmol/L MgCl2, 0.2 mg/mL cytochrome c, and 2 mg/mL BSA, pH 7.2 for kidney.14 Glutamate 10 mmol/L plus 1 mmol/L malate was used as respiratory substrate; state 3 respiration (representing maximum oxidative flux) was measured after addition of 0.18 mol/L ADP (adenosine diphosphate). State 4, representing leakage of extruded protons, was measured after all the ADP was converted to ATP. The respiratory control ratio was calculated as the ratio of the state 3 to the state 4 rates.
Complex I Assay Complex I activity was measured spectrophotometrically as rotenone-sensitive NADH-dehydrogenase activity, as described by BirchMachin et al15: mitochondria were diluted to 1 mg/mL in 25 mmol/L KH2PO4/5 mmol/L MgCl2, pH 7.2 and freeze-thawed 3 times in liquid nitrogen. NADH-dehydrogenase then was assayed in 1 mL of a medium containing 25 mmol/L PO4/5 mmol/L MgCl2/ 2.5 mg/mL BSA/ 2 mmol/L KCN, 2 g/mL antimycin, 65 mol/L CoQ1, pH 7.2. Reactions were initiated with 0.13 mmol/L NADH and consumption of NADH followed spectrophotometrically at 340 nm and 25°C. After a linear rate was obtained, 2 g/mL rotenone was added and the rotenone-insensitive rate recorded. Complex I is completely rotenone sensitive, so complex I activity was calculated after subtracting the rotenone-insensitive rate, using an extinction coefficient of 6.22 mmol/ L⫺1 cm⫺1 for NADH at 340 nm.
Citrate Synthase Assay Oxygen consumption and complex I activity were normalized to citrate synthase activity, measured spectrophotometrically as described by Shepherd and Garland.16 Results were normalized also to mitochondrial protein and were qualitatively very similar.
Statistical Analyses Data are expressed as means ⫾ SEM. State 3 and state 4 oxygen consumption data were normalized to controls from the same litter of rats and analyzed by one-sample t test. Comparisons of the results were performed using paired or unpaired Student’s t test as indicated. Results showing probability levels of less than .05 were considered significant.
RESULTS
Mortality No rats died up to 2 hours of endotoxemia, and only 2.7% of rats died between 2 and 6 hours of endotoxemia. The remaining 96.3% of rats died between 6 and 8 hours so that we consider that 2 hours and 6 hours represent early and late endotoxemia, respectively. Heart Oxygen Consumption State 3 oxygen consumption, which is measured in the presence of ADP and represents maximum mitochondrial oxidative flux, was unaltered after 2 hours of endotoxemia in heart mitochondria but was decreased significantly after 6 hours of endotoxemia (Fig 1A). State 4 oxygen consumption, which is measured after all the ADP is utilized and represents oxygen consumption that
Fig 1. Heart mitochondrial oxygen consumption. Mitochondria were isolated from control or endotoxemic rat hearts after 2 or 6 hours and state 3 and state 4 oxygen consumption measured. (A) Light shading, state 3 oxygen consumption normalized to control; darker shading, state 4 oxygen consumption normalized to control, n ⴝ 15 per group, *P < .005. (B) Respiratory control ratio of control (C) and endotoxic hearts (E). **P ⴝ .0005 versus control.
is wasted and not used for ATP generation, was increased compared with control after 6 hours of endotoxemia, but this difference did not reach significance (P ⫽ .070; Fig 1A). Hence, the respiratory control ratio, which is the ratio of state 3 to state 4 and reflects the efficiency of oxidative metabolism to produce ATP, was decreased in heart mitochondria only after 6 hours of endotoxemia (Fig 1B). Kidney Oxygen Consumption There were no significant effects of endotoxemia on state 3 or state 4 oxygen consumption of kidney mitochondria at either 2 hours or 6 hours (Fig 2A). Hence, the respiratory control ratio of kidney mitochondria was unaltered at either 2 or 6 hours of endotoxemia (Fig 2B). Complex I Activity of Heart Mitochondria Activity of complex I of the respiratory chain was unaltered in heart mitochondria isolated after 2 hours of endotoxemia but was significantly decreased after 6 hours of endotoxemia (Fig 3).
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FUKUMOTO ET AL
Fig 2. Kidney mitochondrial oxygen consumption. Mitochondria were isolated from control or endotoxemic rat kidneys after 2 or 6 hours and state 3 and state 4 oxygen consumption measured. (A) Light shading, state 3 oxygen consumption normalized to control, darker shading, state 4 oxygen consumption normalized to control, n ⴝ 11 to 15 per group. (B) Respiratory control ratio of control (C) and endotoxic kidney (E).
DISCUSSION
This study shows that neonatal endotoxemia has different effects on heart and kidney mitochondria. Detrimental effects on the heart respiratory chain are observed after 6 hours of endotoxemia, a phenomenon that closely precedes the death of the animals. Mitochondrial dysfunction resulting from endotoxemia/sepsis has been implicated in the pathogenesis of organ failure in the liver,9,8 heart,17,18 and kidney.19,20 However, it is not known whether this mitochondrial dysfunction occurs in neonates, because mitochondrial metabolism alters greatly during the transition from newborn to adult.21,22,10 We have previously shown that carnitine palmitoyl transferase I activity, which is crucial for the oxidation of fatty acids, is impaired in the heart but not the kidney of endotoxemic suckling rats.11 However, whether endotoxemia directly affects the activity of the mitochondrial respiratory chain of heart or kidney in neonates is not known. In the current study we undertook measurements of oxygen consumption in mitochondria
isolated from heart and kidney at 2 different times after endotoxin injection. Because suckling rats given endotoxin die after 6 to 8 hours, we consider mitochondria isolated after 6 hours of endotoxemia to represent the later stages of sepsis/organ failure. Biochemical effects at 2 or 6 hours were not caused by lack of fluid resuscitation because animals did not become significantly dehydrated during the course of the experiment. Despite our previous findings, using the same model, that cardiac fatty acid oxidation is already altered after 2 hours endotoxaemia,11 we did not find any alteration in mitochondrial oxygen consumption after 2 hours of endotoxemia. However, after 6 hours of endotoxemia, we measured a significant decrease in mitochondrial oxygen consumption. This was associated with a decrease in the activity of complex I of the respiratory chain, which has been suggested previously to be sensitive to damage caused by sepsis17 and other pathologic situations involving increased free radical production.23,24 These findings suggest that a switch from fat oxidation to carbohydrate oxidation in the heart precedes bioenergetic failure. However, in keeping with the findings of others,25 we could detect no evidence for alteration of kidney mitochondrial function at either time-point. Because neonatal kidney mitochondria are sensitive to the effects of H2O2,12 it is possible that in vivo during endotoxemia, kidney mitochondria are either exposed to lower levels of free radicals than the heart or are better protected from the effects of free radicals. Because cardiac mitochondrial respiratory chain metabolism is only altered in later stages of sepsis, this could provide a window of opportunity in which nutritional support (eg, by glutamine as a precursor of the antioxidant glutathione) or pharmacologic support (eg, by free-radical scavengers) could be beneficial.
Fig 3. Heart mitochondrial complex I activity. Mitochondria were isolated from control (C) or endotoxaemic (E) rat heart after 6 hours and complex I activity measured spectrophotometrically. Results, expressed as mU complex I activity per U citrate synthase activity, were compared by paired t test *P ⴝ .040.
NEONATAL ENDOTOXEMIA
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REFERENCES 1. Ford HR, Rowe MI: Sepsis and related considerations, in O’Neill JA, Rowe MI, Grosfeld JL (eds): Pediatric Surgery. St Louis, MO Mosby-Year Book, 1998, pp 135-155 2. Vlessis AA, Goldman RK, Trunkey DD: New concepts in the pathophysiology of oxygen-metabolism during sepsis. Br J Surg 82: 870-876, 1995 3. Smith SD, Tagge EP, Hannakan C, et al: Characterization of neonatal multisystem organ failure in the surgical newborn. J Pediatr Surg 26:494-499, 1991 4. Morecroft JA, Spitz L, Hamilton PA, et al: Necrotizing enterocolitis—Multisystem organ failure of the newborn. Acta Paediatr 83: 21-23, 1994 5. Avanoglu A, Ergun O, Bakirtas E, et al: Characteristics of multisystem organ failure in neonates. Eur J Pediatr Surg 7:263-266, 1997 6. Romeo C, Eaton S, Quant PA, et al: Neonatal oxidative liver metabolism: Effects of hydrogen peroxide, a putative mediator of septic damage. J Pediatr Surg 34:1107-1111, 1999 7. Romeo C, Eaton S, Spitz L, et al: Nitric oxide inhibits neonatal hepatocyte oxidative metabolism. J Pediatr Surg 35:44-48, 2000 8. Markley MA, Pierro A, Eaton S: Hepatocyte mitochondrial metabolism is inhibited in neonatal rat endotoxaemia: Effects of glutamine. Clin Sci 102:337-344, 2002 9. Kantrow SP, Taylor DE, Carraway MS, et al: Oxidative metabolism in rat hepatocytes and mitochondria during sepsis. Arch Biochem Biophys 345:278-288, 1997 10. Girard J, Ferre´ P, Pe´ gorier J-P, et al: Adaptations of glucose and fatty-acid metabolism during perinatal-period and suckling-weaning transition. Physiol Rev 72:507-562, 1992 11. Fukumoto K, Pierro A, Spitz L, et al: Differential effects of neonatal endotoxemia on heart and kidney carnitine palmitoyl transferase I. J Pediatr Surg 37:723-726, 2002 12. Fukumoto K, Eaton S, Spitz L, et al: Cardiac and renal mitochondria respond differently to mediators of sepsis in neonates. Abstracts of the XLVII meeting of the British Association of Paediatric Surgeons. Italy, Sorrento, P23-P23, 2000 13. Sherratt HSA, Watmough NJ, Johnson MA, et al: Methods for study of normal and abnormal skeletal muscle mitochondria. Meth Biochem Anal 33:243-335, 1988
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