Adenosine triphosphate--magnesium chloride ameliorates reperfusion injury following ischemia as determined by phosphorus nuclear magnetic resonance

We used high-resolution phosphate 31(31P)--nuclear magnetic resonance spectroscopy to study the effects of ischemia and reperfusion on intracellular adenosine triphosphate (ATP) and pH changes in isolated perfused rat kidneys. With renal ischemia, ATP levels fell rapidly and the inorganic phosphate (Pi) peak shifted, indicating acidosis. On reperfusion after 45 minutes of warm ischemia, there was a 56% rise in tissue ATP levels within ten minutes that then slowly declined; by 75 minutes the levels were only 33% of normal. Perfusate flow decreased from 21.2 +/- 0.9 mL/min (mean +/- SE) to 16.5 +/- 1.1 mL/min and the Pi peak did not shift during reperfusion. When 0.3mM ATP complexed to magnesium chloride (ATP-MgCl2) was added to the perfusate after ischemia, renal ATP levels increased to 69% of normal within ten minutes of reperfusion and by 75 minutes they were normal. Perfusate flow was also normal during reperfusion. The Pi peak shifted back to the normal frequency, indicating correction of the intracellular acidosis. Thus, intracellular acidosis, ATP depletion, and decreased flow during reperfusion injury were rapidly reversed and sustained by the postischemic administration of ATP-MgCl2.     

In vivo phosphorus 31 magnetic resonance spectroscopy of rat hind limb skeletal muscle during sepsis

High-energy phosphate metabolism in skeletal muscle is altered during sepsis, although the chronology of events is uncertain. Phosphorus 31 magnetic resonance spectroscopy was used to measure changes in muscle energy stores of the left hind limb musculature of adult male rats during sepsis. Following control scans, cecal ligation and puncture were performed and scanning was repeated 6, 24, and 48 hours after surgery. The ratios of phosphocreatine (PCr) to inorganic phosphate (Pi), a measure of energy stores, and adenosine triphosphate (ATP) to Pi ratio, a measure of the energy available for immediate use, were determined from peak heights. Intracellular pH was calculated using the distance between Pi and PCr peaks. In surviving animals, a 40% decrease in PCr/Pi ratio (+/- SEM) was observed by 24 hours (22.3 +/- 3.0 at time 0 vs 13.3 +/- 2.8 at 24 hours), whereas energy availability (beta-ATP/Pi) was statistically unchanged (18.2 +/- 2.2 at time 0 vs 15.2 +/- 1.2 at 48 hours). Intracellular pH did not change. Both PCr/Pi and ATP/Pi ratios were inversely correlated with time. In this model of documented peritonitis, skeletal muscle energy metabolism is rapidly altered following severe infection, and these changes can be detected using 31P magnetic resonance spectroscopy.     

Real-time noninvasive assessment of pancreatic ATP levels during cold preservation

31P-NMR spectroscopy was utilized to investigate rat and porcine pancreatic ATP:P(i) ratios to assess the efficacy of existing protocols for cold preservation (CP) in maintaining organ quality. Following sacrifice, rat pancreata were immediately excised or left enclosed in the body for 15 minutes of warm ischemia (WI). After excision, rat pancreata were stored at 6 degrees C to 8 degrees C using histidine-tryptophan-ketoglutarate solution (HTK) presaturated with air (S1), HTK presaturated with O2 (S2), or the HTK/perfluorodecalin two-layer method (TLM) with both liquids presaturated with O2 (S3). 31P-NMR spectra were sequentially collected at 3, 6, 9, 12, and 24 hours of CP from pancreata stored with each of the three protocols examined. The ATP:Pi ratio for rat pancreata exposed to 15 minutes of WI and stored with S3 increased during the first 9 hours of CP, approaching values observed for organs procured with no WI. A marked reduction in the ATP:Pi ratio was observed beyond 12 hours of CP with S3. After 6 hours of CP, the ATP:Pi ratio was highest for S3, substantially decreased for S2, and below detection for S1. In sharp contrast to the rat model, ATP was barely detectable in porcine pancreata exposed to minimal warm ischemia (<15 minutes) stored with the TLM regardless of CP time. We conclude that 31P-NMR spectroscopy is a powerful tool that can be used to (1) noninvasively evaluate pancreata prior to islet isolation, (2) assess the efficacy of different preservation protocols, (3) precisely define the timing of reversible versus irreversible damage, and (4) assess whether intervention will extend this timing.     

Pancreatitis-induced ascitic fluid and hepatocellular dysfunction in severe acute pancreatitis

BACKGROUND. Multiple organ failure (MOF) is the most serious complication in severe acute pancreatitis, contributing to its high mortality. It has been suggested that changes of high-energy phosphates, intracellular pH, and intracellular cation homeostasis are closely related to hepatocellular injury associated with MOF. METHODS. Phosphorus metabolites, intracellular pH (pHi), and intracellular Na+ concentration ([Na+]i) were measured in rat livers in vivo using 31P and 23Na NMR spectroscopy after deoxycholic acid (DCA)-induced pancreatitis or intraperitoneal injection (ip) of pancreatitis-induced ascitic fluid (PAF). RESULTS. Two hours after induction of DCA-pancreatitis, the liver experienced significant intracellular acidosis (pHi = 6.99 +/- 0.16) and sodium loading (75 +/- 9 mM) and a reduction in its energy state (beta-ATP/Pi = 0.2 +/- 0.03 and Pi = 164 +/- 12). Although ip injection of PAF into healthy rats did not induce systemic hypotension, the livers under these conditions also developed severe disturbances in hepatocellular ion homeostasis and depletion of its bioenergetics. The longer the abdomen was exposed to the PAF, the worse the changes were. At 3 h after ip injection of PAF, hepatic [Na+]i significantly increased (42 +/- 3 mM) along with a significant decrease in pHi (7.30 +/- 0. 03). At 6 h after ip injection of PAF, the hepatic beta-ATP/Pi ratio decreased to 0.34 +/- 0.05 and Pi increased to 97 +/- 27. CONCLUSIONS. PAF induced severe hepatocellular acidosis, rapid accumulation of hepatic intracellular sodium, impaired hepatic cytosolic phosphorylation potential, and increased hepatic utilization of ATP. These effects may account for the eventual development of liver dysfunction associated with necrotizing pancreatitis.     

Sequential in vivo measurement of cerebral intracellular metabolites with phosphorus-31 magnetic resonance spectroscopy during global cerebral ischemia and reperfusion in rats

Phosphorus-31 magnetic resonance (31P MR) spectroscopy was used to obtain serial in vivo measurements of cerebral adenosine triphosphate (ATP), phosphocreatine (PCr), inorganic phosphate (Pi), and intracellular pH levels in rats during temporary global cerebral ischemia and reperfusion. Three groups of 4 rats each that recovered from permanent bilateral vertebral artery occlusion were placed in a MR spectrometer and subjected to remotely controlled bilateral carotid artery occlusion lasting 6, 15, or 30 minutes followed by 1 hour of reperfusion. Four additional rats that developed systemic hypotension (2 during a 6-minute occlusion and 2 during a 15-minute occlusion) were also studied. 31P MR spectra were obtained in each rat before, during, and after ischemia. Rats in which MR spectra showed metabolic recovery underwent a second occlusion followed by reperfusion and sacrifice. In the 12 normotensive rats, metabolic alterations began within 3 minutes after the onset of global ischemia. By the end of the occlusion period, cerebral ATP had decreased by 20 to 100% in 10 rats and PCr had decreased by 15 to 75% in all 12; Pi increased by 25 to 240%. The mean intracellular pH decreased from 7.33 to 6.9 +/- 0.6. The degree of metabolic deterioration during ischemia was not related to the duration of occlusion. During reperfusion, ATP, PCr, Pi, and intracellular pH returned to normal in 4 rats; 5 rats had partial metabolic recovery, and 3 had minimal or transient metabolic recovery followed by progressive deterioration. All rats that developed systemic hypotension had a decrease in ATP, PCr, and intracellular pH and an increase in Pi during the initial occlusion. Each had transient partial recovery in ATP during reperfusion, and 2 had slight recovery of PCr. The onset of hypotension was followed by depletion of these metabolites, progressive increase in Pi, and progressive intracellular acidosis. All rats that deteriorated metabolically after reversal of carotid occlusion died by the end of the reperfusion period or soon after. The 8 rats that recovered from the first occlusion were subjected to a second period of ischemia, during which each rat showed severe depletion of metabolites. During the second reperfusion, only 1 rat showed significant metabolic recovery, which lasted only 30 minutes and was followed by progressive deterioration. Severe global cerebral ischemia was associated with a progressive decline in both ATP and PCr, whereas less complete ischemia seemed to be characterized by stabilization or recovery of ATP and continued depression of PCr.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lower liver transplant success in females: gender differences in metabolic response to global ischemia

BACKGROUND: Research has shown that gender plays a significant role in the metabolic processes of different organs and that transplanting livers of females into male or female recipients has significantly higher failure rates. To understand why, this study examined whether gender differences exist in various metabolic responses of livers to ischemia. METHODS: The following metabolic liver parameters in Sprague-Dawley rats (male, n = 14; and female, n = 18) were examined; adenosine triphosphate (ATP) and lactate expressed as micromoles/g dry weight, and hydrogen ion content [H+] expressed as 10(-8) mol/L. In vivo liver biopsy specimens were compared with ischemic biopsy specimens at 3, 10, 15, 30, and 45 minutes (37 degrees C). RESULTS: In vivo female ATP values (9.9 +/- 0.8) were similar to males (9.8 +/- 0.9) and both had early, rapid decline during ischemia reaching 20% of baseline by 10 minutes of ischemia. In contrast, male liver lactate accumulation peaked by 3 minutes and at much lower levels (35 +/- 13), whereas female liver lactate peaked by 10 minutes at 71 +/- 11. For the rest of the ischemic period, female livers exhibited significantly (P < .05) greater lactate accumulation. Female liver H+ levels also increased to higher levels (55 +/- 10) than the male livers (37 +/- 7) and this pattern was significantly (P < .05) different from 10 minutes onward. CONCLUSIONS: Although livers of females ultimately have similar ATP profiles to livers of males, they experienced more rapid and greater degree of tissue lactate and H+ accumulation during ischemia. Therefore, female livers have increased acidosis during ischemia, which could adversely affect transplant outcome.     

Subnormothermic machine perfusion protects against rat liver preservation injury: a comparative evaluation with conventional cold storage

Hypothermic machine perfusion (MP) of the liver has been reported to improve graft function reclaiming marginal livers, such as those from non-heart-beating donors. Livers from obese donors often have fatty infiltrates and are more susceptible to hypothermic conditions. No data exist about MP at temperatures >4 degrees C. This study evaluated liver function after organ preservation by comparing MP at 20 degrees C with conventional cold storage. METHODS: For MP, rat livers were perfused for 6 hours using an oxygenated Krebs-Henseleit (KH) solution at 20 degrees C (pH 7.4). For cold storage, livers were perfused in situ and preserved with Celsior solution at 4 degrees C for 6 hours. The reperfusion period with KH (2 hours at 37 degrees C) was performed under the same conditions both among livers preserved by MP or cold storage. Hepatic enzyme release (aspartate aminotransferase [AST], alanine aminotransferase [ALT], lactate dehydrogenase [LDH], and gamma-glutamyl transferase [GGT]), bile production, and ATP levels were measured during MP and reperfusion. RESULTS: At the end of reperfusion, livers preserved by MP showed significantly decreased liver damage compared with cold storage: AST, 18 +/- 4 vs. 45 +/- 6 mU/mL (P < .01); ALT, 1.5 +/- .07 vs. 6 +/- 0.5 mU/mL (P < .01); and LDH, 82 +/- 2 vs. 135 +/- 29 mU/mL (P < .05). No difference was observed between bile production between MP and cold storage. High levels of biliary GGT and LDH were found in cold preserved livers. ATP levels were higher in livers preserved with MP compared with those preserved by cold storage. CONCLUSIONS: MP at 20 degrees C resulted in a better quality of liver preservation, improving hepatocyte survival, compared with conventional cold storage. This may provide a new method for successful utilization of marginal livers, in particular fatty livers.     

Renal corticomedullary metabolite gradients during graded arterial occlusion: a localized 31P magnetic resonance spectroscopy study

In order to investigate the role of the outer medulla in acute ischemic renal failure (Epstein FH, Balaban RS, Ross BD: Redox state of cytochrome aa3 in isolated perfused rat kidney. Am J Physiol 1982;243: F356-F363), the distribution of ATP in the in vivo porcine kidney and its relationship to Na transport and to ischemia was examined by using localized 31P magnetic resonance spectroscopy. Renal cortex (ATP) was higher than medulla. Reduction in Na transport produced by partial renal arterial occlusion ("hypofiltration"), resulted in a 13% increase in the ATP/Pi ratio of the whole kidney (from 2.61 +/- 0.26 to 2.96 +/- 0.27; P less than 0.03). This increase was accounted for by a statistically significant increase in (ATP) in the cortex, with medulla contributing to an insignificant extent. Further occlusion of the renal artery to reduce GFR to zero ("hypoperfusion") resulted in a 70% fall in ATP/Pi ratio. (ATP) was reduced most in the cortex, but pH fell equally in cortex and medulla. After release of arterial occlusion, cortical ATP recovered less completely than medulla ATP. Intracellular pH and Pi were restored in both cortex and medulla. It was concluded that cortex and medulla contribute equally to the pattern of disordered energy metabolism in acute renal failure. Sparing of ATP during hypofiltration may reflect the reduced energy requirements of active Na transport.     

Effects of regenerating liver cytosol on drug-induced hepatic failure

Despite various interventions, the mortality rate after acute hepatic failure remains extremely high. Although it has been shown that administration of regenerating liver cytosol (RLC) after hepatic failure improves the survival rate of animals, the sequence in which various hepatic functions are altered after acute hepatic failure and their improvement by RLC remain unknown. To study this, fulminant hepatic failure in rats was produced by intraperitoneal injection of 1.5 gm/kg D-galactosamine (GAL). Twenty-four hours after 68% hepatectomy in normal rats, RLC was prepared and 4 ml of this solution (40 to 50 mg protein) was injected intraperitoneally in other animals at 6 or 24 hours after GAL administration. The long-term survival rate was 19.4% in the GAL-vehicle (control) group, 26.7% in the GAL-treated rats given liver cytosol from normal rats, and 72.2% (p less than 0.01 compared with both groups) in rats given RLC even 24 hours after GAL administration. Reticuloendothelial function was depressed at 24 and 48 hours after GAL administration; however, treatment with RLC but not vehicle or normal liver cytosol at 6 or even 24 hours after GAL administration significantly improved reticuloendothelial function 24 hours after GAL administration. DNA synthesis (an indicator of cell proliferation) did not increase in any group 24 hours after GAL administration; however, it increased strikingly at 48 hours in the GAL-RLC groups. Serum bilirubin levels were also lower in the RLC group 48 hours after GAL administration. Thus RLC administration improved survival, reticuloendothelial function, DNA synthesis, and hepatocyte function after GAL-induced acute hepatic failure. Improvement of reticuloendothelial function by RLC occurred before stimulation of DNA synthesis and appears to play an important role in improving survival after hepatic failure.     

Warm ischemia-reperfusion injury is decreased by tacrolimus in steatotic rat liver.

Ischemia-reperfusion (I-R) injury is poorly tolerated by fatty livers, most probably secondary to reduced cellular adenosine triphosphate (ATP) levels. We investigated the effectiveness of tacrolimus pretreatment on fatty liver I-R injury in obese Zucker rats. Tacrolimus (0.3 mg/kg, intravenously) was injected 24 hours before a 75-minute ischemic period and rats were sacrificed 6 hours later. Tacrolimus modified the response to I-R observed in obese Zucker rats, when compared to nontreated obese rats: a significant reduction in hepatocyte necrosis was associated with a significant increase in hepatocyte apoptosis. In addition, cell necrosis and apoptosis were significantly and inversely correlated in lean nontreated and treated obese Zucker rats following I-R. Tacrolimus also significantly increased the hepatic ATP levels, reduced in nontreated obese rats, toward values found in lean Zucker rat livers. This protective effect of tacrolimus was further confirmed in vivo by a significantly improved survival following pretreatment with tacrolimus, 24 hours prior to ischemia. In conclusion, in obese Zucker rat livers, tacrolimus pretreatment reversed the I-R injury toward the one found in lean Zucker rats. The correlations between ATP levels and the opposite changes in necrosis and apoptotic pathways strongly suggest a cause-effect relationship between tacrolimus and changes in ATP levels.     

Brain bioenergetics during cardiopulmonary resuscitation in dogs.

Cardiac arrest causes a rapid loss of cerebral adenosine triphosphate [corrected] (ATP) and a decrease in cerebral intracellular pH (pHi). Depending on the efficacy of cardiopulmonary resuscitation (CPR), cerebral blood flow levels (CBF) ranging from near zero to near normal have been reported experimentally. Using 31P magnetic resonance spectroscopy, the authors tested whether experimental CPR with normal levels of cerebral blood flow can rapidly restore cerebral ATP and pHi despite the progressive systemic acidemia associated with CPR. After 6 min of ventricular fibrillation in six dogs anesthetized with fentanyl and pentobarbital, ATP was reduced to undetectable concentrations and pHi decreased from 7.11 +/- 0.02 to 6.28 +/- 0.09 (+/- SE) as measured by 31P magnetic resonance spectroscopy. Application of cyclic chest compression by an inflatable vest placed around the thorax and infusion of epinephrine (40 micrograms/kg bolus plus 8 micrograms/kg/min, intravenously) maintained cerebral perfusion pressure greater than 70 mmHg for 50 min with the dog remaining in the magnet. Prearrest cerebral blood flows were generated. Cerebral pHi recovered to 7.03 +/- 0.03 by 35 min of CPR, whereas arterial pH decreased from 7.41 +/- 0.4 to 7.08 +/- 0.04 and cerebral venous pH decreased from 7.29 +/- 0.03 to 7.01 +/- 0.04. Cerebral ATP levels recovered to 86 +/- 7% (+/- SE) of prearrest concentration by 6 min of CPR. There was no further recovery of ATP, which remained significantly less than control. Therefore, in contrast to hyperemic reperfusion with spontaneous circulation and full ATP recovery, experimental CPR may not be able to restore ATP completely after 6 min of global ischemia despite restoration of CBF and brain pHi to prearrest levels.     

In vivo 31P-NMR studies on energy metabolism and the effect of methotrexate in murine implanted osteosarcoma

The energy metabolism of Dunn osteosarcoma subcutaneously implanted in C3H/He mice was studied in vivo by a 31P-NMR spectrometer with surface-coils. The spectra of Dunn osteosarcoma showed peaks of sugar phosphate, inorganic phosphate, phosphocreatine, phosphomonoester, and ATPs. In the early stage of the tumor growth phosphocreatine and ATP showed large signal intensities and the tissue pH was 7.23 +/- 0.08. Following the tumor growth phosphocreatine and ATP decreased and the tissue pH fell to 6.82 +/- 0.08. Immediately after a small dose of MTX (2 mg/kg) was administered, an increase of inorganic phosphate and a decrease of phosphocreatine were temporarily observed when MTX concentrations of the tumor tissues were maximum. High energy metabolites were apparently consumed with the active transport of MTX. After twelve hours of a high dose of MTX (500 mg/kg) was administered, disappearance of phosphocreatine and ATP with an increase of inorganic phosphate was observed previous to the histological change. In vivo 31P-NMR spectroscopy may be useful in the evaluation of chemotherapy.     

热缺血损伤对大鼠移植肝组织能量代谢及存活期的影响

目的：探讨不同热缺血时间下大鼠肝组织能量代谢变化规律,预测供肝耐受热缺血的安全时限。方法：实验动物按供肝热缺血时间分别为0、10、15、20、30、45和60min,随机分为7组。采用反相高效液相色谱法测定单纯热缺血后肝组织能量代谢指标并进行超微结构的观察。然后按各组条件分别作原位肝移植,观察移植后24、48h各组肝组织能量代谢指标的恢复性变化,并统计生存时间。结果：供肝经受热缺血损伤后,肝组织ATP含量和EC水平远逐渐下降,其中前30min下降比较急剧,以后趋向平缓。热缺血30min内肝组织ATP含量和EC水平在肝移植再复流24h后基本得到恢复,术后大鼠仍可以获长期存活。45min组,移植肝在48h后能量代谢的功能也基本恢复,虽不足以影响术后的1周存活率,但对大鼠肝移植术后的3个月存活率影响显著。60min组,肝脏能量代谢储备功能难以恢复,大鼠术后生存天数显著降低。结论：供肝组织三磷酸腺苷（ATP）含量和能荷（EC）水平以及其移植术后恢复的潜能是衡量供肝质量的重要标准。供肝热缺血损伤的时间与肝组织能量代谢功能的恢复及术后动物生存情况密切相关。     

High energy compound stability during experimental brain death

The aim of this study was to examine the effect of sudden brain death (BD) on myocardial function and high energy phosphate (HEP) stores. BD was induced by cerebral vessel ligation in six swine (BD group) that were compared to six control swine. At the end of the BD period (3 hours), harvested hearts were stored at 4 degrees C. Myocardial tissue HEP were assessed by: (i) (31)P-NMR spectroscopy of left ventricle for phosphocreatine (PCr), adenosine triphosphate (ATP), inorganic phosphate (Pi) and intracellular pH (pHi), and by (ii) HPLC for ATP, ADP, and AMP levels in left ventricle biopsies. Brain death resulted in a instantaneous major increase in catecholamines (>50-fold, P < .001) and paradoxically a significant progressive decrease in the regional contractility of the left ventricle. After cardioplegia, no significant differences on HEP compounds (ATP/Pi, PCr/Pi, ATP, energetic index) or in pHi were observed between BD and control groups. These data suggest that early heart injury occurring during BD does not seem to be an ischemic phenomenon.     

Assessment of renal preservation by phosphorus-31 magnetic resonance spectroscopy: in vivo normothermic blood perfusion

To evaluate the usefulness of the monophosphate/inorganic phosphate ratio (MP/Pi) in assessing renal viability in a renal transplantation setting, we monitored intracellular phosphorous metabolites of 33 canine kidneys by phosphorus-31 magnetic resonance spectroscopy (MRS) after various amounts of ischemia. Renal viability (adequate perfusion and function) was directly assessed by the presence of intraoperative urine production in each kidney. Twelve normal, well-perfused kidneys (Group 1) exhibited high control MP/Pi ratios, with a mean of 0.87 +/- 0.12. Six in situ kidneys (Group 2), subjected to 45 minutes' warm ischemia followed by reperfusion, had a mean MP/Pi ratio of 0.50 +/- 0.12 after warm ischemia, which increased by a mean of 0.50 +/- 0.11 (to 1.0 +/- 0.07) after two to four hours of reperfusion. Fifteen kidneys (Group 3) were removed, cold-flushed and transplanted after 24 hours of hypothermic storage. In eight (Group 3A), reperfusion was excellent; in seven (Group 3B), reperfusion was inadequate secondary to hypotension in two, hemorrhage in two, and renal vein thrombosis in three. Group 3A kidneys had a mean MP/Pi ratio after cold-storage ischemia of 0.54 +/- 0.08. After successful transplantation and two to four hours of reperfusion, this increased by a mean of 0.23 +/- 0.12 to 0.77 +/- 0.15. Group 3B kidneys all showed a continuous decline of MP/Pi, with a mean loss of 0.26 +/- 0.09 from baseline values (mean 0.56 +/- 0.08) to nonviable levels of 0.28 +/- 0.12 within four hours of transplantation. We conclude that MP/Pi ratios enable assessment of renal viability and ischemic damage and can predict the efficacy of renal preservation maneuvers in the dog kidney. These preliminary data support the theory that MRS can be applied to the noninvasive assessment of viability in ex vivo, cold-stored cadaveric human kidneys awaiting renal transplantation.     

Prolonged effect of leukocytosis on reperfusion injury of rat intestine: real-time ATP change studied using (31)P MRS

BACKGROUND: The intestine is one of the most sensitive tissues to ischemia and reperfusion (I/R). Polymorphonuclear neutrophils (PMN) may play an important role in ischemic injury. (31)P magnetic resonance spectroscopy (MRS) has been used to continuously monitor the energy metabolism of an animal in situ. We have applied MRS to study the effect of PMN on the I/R injury of rat intestine. MATERIAL AND METHODS: In a rat model of 30 min of intestinal ischemia and reperfusion, the number of PMNs was manipulated: group A, control; group B, leukopenia induced by cyclophosphamide; group C, leukocytosis induced by granulocyte colony-stimulating factor (G-CSF). MRS was employed to measure the level of real-time intestinal ATP and pH in vivo. RESULTS: In group A, ATP rapidly recovered on reperfusion to 61.0 +/- 11.0% of the preischemia level and maintained that level during reperfusion. The other two groups showed similar recovery of ATP at the initial phase of the reperfusion (<10 min). ATP in group B continued to recover, reaching 74.0 +/- 10.0% of the preischemia level. After the initial recovery, ATP in group C deteriorated reaching 46.0 +/- 4.4% of the preischemic level at 150 min of reperfusion. In group A and group B tissue pH decreased on ischemia and recovered on reperfusion in a similar manner. In group C, tissue pH was significantly lower than in other groups during I/R. CONCLUSION: Leukocytosis induced by G-CSF exerts a prolonged effect on ATP during I/R and leukocyte depletion helps protect against the I/R injury.     

加氧低温灌流下31磷-磁共振频谱与肝酶测定在判断大鼠供肝活力中的意义

目的　探讨判断经受热缺血的肝脏活力的方法。方法　将 18只SD大鼠随机分成 3组 :A组肝脏热缺血时间 (WI)为 0min ;B组WI为 30min ;C组WI为 6 0min。各组肝脏冷保存后 2～2 .5h ,采用31磷 磁共振频谱 (31P MRS)测定各组加氧低温灌流 30min时的离体肝脏频谱 ,同时测定保存液和灌流液中肝酶的变化。结果　β 三磷酸核酸 (β NTP)与亚甲基二磷酸 (MDP)的比值 ,B组与A组比较 ,P >0 .0 5 ;C组与A、B组比较 ,P <0 .0 1。肝酶测定结果 :随着热缺血时间的延长 ,保存液中的肝酶逐渐升高 ,每组之间比较 ,差异均有显著性 (P <0 .0 5 ) ;灌流液中的肝酶 ,A组与B组比较 ,差异无显著性 (P >0 .0 5 ) ,C组与A、B两组比较 ,差异均有极显著性 (P <0 .0 1)。结论　测定加氧低温灌流下大鼠离体肝脏的ATP再生能力能较好判断其活力 ,该方法在临床热缺血供肝活力的判断上具有一定参考价值 ;同时测定保存液和灌流液中的肝酶含量 ,能配合31P MRS判断肝脏的活力。     

In vivo 31P NMR spectroscopic changes during liver regeneration

Liver regeneration following partial hepatectomy involves rapid cell division 24 to 72 hr postresection. This cell division would necessarily involve changes in intracellular energy stores and cell membrane phospholipid precursors. In tumor models 31P nuclear magnetic resonance (NMR) has been shown to identify intracellular substrate changes associated with cell growth. The ability to monitor early changes in adenosine triphosphate (ATP), inorganic orthophosphate (Pi), phosphomonoesters (PME), or phosphodiesters (PDE) after liver resection could indicate the intracellular changes necessary for hepatocellular regeneration. In vivo 31P NMR scans of the liver were performed in both normal rats and in rats at 24, 48, 72, and 120 hr after 70% hepatectomy. At 48 hr, total ATP fell to 18.9% (P less than 0.05) and both Pi/beta-ATP and PME/beta-ATP were significantly elevated (P less than 0.01) from controls. These changes correlate with the known mitotic peak in the rat following hepatectomy. We conclude that in vivo 31P NMR is a potentially valuable tool for studying hepatic regeneration. The data also suggest that hepatocellular regeneration may be critically dependent on cellular ATP stores.     

Hindlimb ischemia-reperfusion increases complement deposition and glycolysis.

BACKGROUND: Hindlimb ischemia-reperfusion (HIR) impairs cellular energy metabolism and causes local muscle injury possibly through free radical or complement-mediated mechanisms. MATERIALS AND METHODS: To determine the relationship among myocellular energetics, histopathological injury, and mediator activity, male Wistar rats underwent 4 h of Sham (n = 8), Unilateral (n = 8), or Bilateral (n = 8) hindlimb ischemia followed by 4 h of reperfusion. All rats underwent 31P magnetic resonance spectroscopy of their right gastrocnemius muscle to determine various high-energy phosphate ratios including ATP to Pi (ATP/Pi, a measure of energy status) and phosphocreatine to Pi (PCr/Pi, a measure of thermodynamic capacity). Gastrocnemius muscles were then harvested to determine muscle damage and complement membrane attack complex (MAC) deposition by immunohistochemical staining [grade 0 (none) to 3 (very severe)] and to measure glutathione (GSH), DNA, and enzyme activities: beta-hydroxyacyl-CoA dehydrogenase, phosphofructokinase, and citrate synthetase. RESULTS: HIR was associated with significant declines in ATP/Pi and PCr/Pi (P < 0.001). Progressively more severe HIR (Sham, Unilateral, Bilateral) was associated with greater MAC deposition (0. 0 +/- 0.0, 1.0 +/- 0.3, 1.5 +/- 0.4, P = 0.06, mean +/- SEM) and histological damage (0.0 +/- 0.0, 0.9 +/- 0.3, 1.3 +/- 0.4, P < 0. 05). GSH levels, beta-hydroxyacyl-CoA dehydrogenase, and citrate synthetase activities were not affected by HIR, but phosphofructokinase activity increased (24.09 +/- 2.42, 35.16 +/- 5. 26, 59.29 +/- 9.82 mmol/mg of DNA/min, P < 0.05). Although GSH levels were not significantly altered, complement deposition was closely associated with skeletal muscle injury and compensatory changes in glycolysis. Alterations in myocellular bioenergetics after HIR closely paralleled complement deposition rather than GSH depletion. CONCLUSIONS: Therapeutic strategies aimed at controlling complement activity and assessment techniques based on bioenergetics may allow more precise determinations of the effects of HIR injury.     

肌红蛋白的表达对肝缺血再灌注损伤的保护作用

目的　探讨以腺病毒介导的肌红蛋白的表达对肝脏的ATP水平的影响及其在肝脏缺血再灌注 (I R)的保护作用。方法　将携带有人体肌红蛋白基因的腺病毒转染至白鼠的肝脏 ,然后检测肌红蛋白的表达 ,同时检测肝脏的ATP水平和肝功能。对肌红蛋白对肝脏I R损伤动物模型的保护效应进行评价。结果　腺病毒介导的肌红蛋白可转染至白鼠肝脏。转染 72h后白鼠肝脏的ATP水平较对照组明显升高。肌红蛋白的表达能减轻肝脏的I R损伤。结论　肌红蛋白在体肝脏的表达可提高肝脏的ATP水平 ,并可能成为减轻I R损伤的新的治疗方法。