Bioenergetic Heterogeneity in Mycobacterium tuberculosis Residing in Different Subcellular Niches.

ATP/ADP depicts the bioenergetic state of Mycobacterium tuberculosis (Mtb). However, the metabolic state of Mtb during infection remains poorly defined due to the absence of appropriate tools. Perceval HR (PHR) was recently developed to measure intracellular ATP/ADP levels, but it cannot be employed in mycobacterial cells due to mycobacterial autofluorescence. Here, we reengineered the ATP/ADP sensor Perceval HR into PHR-mCherry to analyze ATP/ADP in fast- and slow-growing mycobacteria. ATP/ADP reporter strains were generated through the expression of PHR-mCherry. Using the Mtb reporter strain, we analyzed the changes in ATP/ADP levels in response to antimycobacterial agents. As expected, bedaquiline induced a decrease in ATP/ADP. Interestingly, the transcriptional inhibitor rifampicin led to the depletion of ATP/ADP levels, while the cell wall synthesis inhibitor isoniazid did not affect the ATP/ADP levels in Mtb. The usage of this probe revealed that Mtb faces depletion of ATP/ADP levels upon phagocytosis. Furthermore, we observed that the activation of macrophages with interferon gamma and lipopolysaccharides leads to metabolic stress in intracellular Mtb. Examination of the bioenergetics of mycobacteria residing in subvacuolar compartments of macrophages revealed that the bacilli residing in phagolysosomes and autophagosomes have significantly less ATP/ADP than the bacilli residing in phagosomes. These observations indicate that phagosomes represent a niche for metabolically active Mtb, while autophagosomes and phagolysosomes harbor metabolically quiescent bacilli. Interestingly, even in activated macrophages, Mtb residing in phagosomes remains metabolically active. We further observed that macrophage activation affects the metabolic state of intracellular Mtb through the trafficking of Mtb from phagosomes to autophagosomes and phagolysosomes. IMPORTANCE ATP/ADP levels guide bacterial cells, whether to replicate or to enter nonreplicating persistence. However, tools for measuring ATP/ADP levels with spatiotemporal resolution are lacking. Here, we describe a method for tracking ATP/ADP levels at the single-cell and population levels. Using this tool, we have demonstrated that the transcription inhibitor rifampicin induces metabolic stress. In contrast, the cell wall synthesis inhibitor isoniazid does not alter the metabolic state of the bacilli, suggesting that transcription is tightly intertwined with metabolism, while cell wall synthesis is not. Furthermore, we analyzed the metabolic state of mycobacteria residing in different compartments of macrophages. We observed that Mtb cells residing inside phagosomes have healthy ATP/ADP levels. In contrast, the bacteria residing inside phagolysosomes and autophagosomes face depletion of ATP. Interestingly, the activation of macrophages facilitates the trafficking of mycobacterial cells from metabolism-conducive phagosomes to metabolism-averse phagolysosomes and autophagosomes. We believe that this tool holds the key to the identification of inhibitors of mycobacterial metabolism.

Metabolomic and Imaging Mass Spectrometric Assays of Labile Brain Metabolites: Critical Importance of Brain Harvest Procedures.

Metabolomic technologies including imaging mass spectrometry (IMS; also called mass spectrometry imaging, MSI, or matrix-assisted laser desorption/ionization-mass spectrometry imaging, MALDI MSI) are important methods to evaluate levels of many compounds in brain with high spatial resolution, characterize metabolic phenotypes of brain disorders, and identify disease biomarkers. ATP is central to brain energetics, and reports of its heterogeneous distribution in brain and regional differences in ATP/ADP ratios reported in IMS studies conflict with earlier studies. These discordant data were, therefore, analyzed and compared with biochemical literature that used rigorous methods to preserve labile metabolites. Unequal, very low regional ATP levels and low ATP/ADP ratios are explained by rapid metabolism during postmortem ischemia. A critical aspect of any analysis of brain components is their stability during and after tissue harvest so measured concentrations closely approximate their physiological levels in vivo. Unfortunately, the requirement for inactivation of brain enzymes by freezing or heating is not widely recognized outside the neurochemistry discipline, and procedures that do not prevent postmortem autolysis, including decapitation, brain removal/dissection, and 'snap freezing' are commonly used. Strong emphasis is placed on use of supplementary approaches to calibrate metabolite abundance in units of concentration in IMS studies and comparison of IMS results with biochemical data obtained by different methods to help identify potential artifacts.

The dynamics of cellular energetics during continuous yeast culture.

A plethora of data is accumulating from high throughput methods on metabolites, coenzymes, proteins, and nucleic acids and their interactions as well as the signalling and regulatory functions and pathways of the cellular network. The frozen moment viewed in a single discrete time sample requires frequent repetition and updating before any appreciation of the dynamics of component interaction becomes possible. Even then in a sample derived from a cell population, time-averaging of processes and events that occur in out-of-phase individuals blur the detailed complexity of single cell organization. Continuously-grown cultures of yeast can become spontaneously self-synchronized, thereby enabling resolution of far more detailed temporal structure. Continuous on-line monitoring by rapidly responding sensors (O2 electrode and membrane-inlet mass spectrometry for O2, CO2 and H2S; direct fluorimetry for NAD(P)H and flavins) gives dynamic information from time-scales of minutes to hours. Supplemented with capillary electophoresis and gas chromatography mass spectrometry and transcriptomics the predominantly oscillatory behaviour of network components becomes evident, with a 40 min cycle between a phase of increased respiration (oxidative phase) and decreased respiration (reductive phase). Highly pervasive, this ultradian clock provides a coordinating function that links mitochondrial energetics and redox balance to transcriptional regulation, mitochondrial structure and organelle remodelling, DNA duplication and cell division events. Ultimately, this leads to a global partitioning of anabolism and catabolism and the enzymes involved, mediated by a relatively simple ATP feedback loop on chromatin architecture.

Selective detection of ATP and ADP in aqueous solution by using a fluorescent zinc receptor.

We report on the successful use of a new zinc complex for the selective fluorescent detection of ADP and ATP in water. This is achieved by the complementary coordination of the phosphate groups to the metal centre and hydrogen bonding of the adenosine with the coordinated ligand.

Altered seed oil and glucosinolate levels in transgenic plants overexpressing the Brassica napus SHOOTMERISTEMLESS gene.

SHOOTMERISTEMLESS (STM) is a homeobox gene conserved among plant species which is required for the formation and maintenance of the shoot meristem by suppressing differentiation and maintaining an undetermined cell fate within the apical pole. To assess further the role of this gene during seed storage accumulation, transgenic Brassica napus (Bn) plants overexpressing or down-regulating BnSTM under the control of the 35S promoter were generated. Overexpression of BnSTM increased seed oil content without affecting the protein and sucrose level. These changes were accompanied by the induction of genes encoding several transcription factors promoting fatty acid (FA) synthesis: LEAFY COTYLEDON1 (BnLEC1), BnLEC2, and WRINKLE1 (BnWRI1). In addition, expression of key representative enzymes involved in sucrose metabolism, glycolysis, and FA biosynthesis was up-regulated in developing seeds ectopically expressing BnSTM. These distinctive expression patterns support the view of an increased carbon flux to the FA biosynthetic pathway in developing transformed seeds. The overexpression of BnSTM also resulted in a desirable reduction of seed glucosinolate (GLS) levels ascribed to a transcriptional repression of key enzymes participating in the GLS biosynthetic pathway, and possibly to the differential utilization of common precursors for GLS and indole-3-acetic acid synthesis. No changes in oil and GLS levels were observed in lines down-regulating BnSTM. Taken together, these findings provide evidence for a novel function for BnSTM in promoting desirable changes in seed oil and GLS levels when overexpressed in B. napus plants, and demonstrate that this gene can be used as a target for genetic improvement of oilseed species.

Value of dynamic ³¹P magnetic resonance spectroscopy technique in in vivo assessment of the skeletal muscle mitochondrial function in type 2 diabetes.

BACKGROUND: Phosphorous magnetic resonance spectroscopy ((31)P-MRS) has been successfully applied to study intracellular membrane compounds and high-energy phosphate metabolism. This study aimed to evaluate the capability of dynamic (31)P-MRS for assessing energy metabolism and mitochondrial function in skeletal muscle from type 2 diabetic patients. METHODS: Dynamic (31)P-MRS was performed on 22 patients with type 2 diabetes and 26 healthy volunteers. Spectra were acquired from quadriceps muscle while subjects were in a state of rest, at exercise and during recovery. The peak areas of inorganic phosphate (Pi), phosphocreatine (PCr), and adenosine triphosphate (ATP) were measured. The concentration of adenosine diphosphate (ADP) and the intracellular pH value were calculated from the biochemistry reaction equilibrium. The time constant and recovery rates of Pi, PCr, and ADP were analyzed using exponential curve fitting. RESULTS: As compared to healthy controls, type 2 diabetes patients had significantly lower skeletal muscle concentrations of Pi, PCr and ß-ATP, and higher levels of ADP and Pi/PCr. During exercise, diabetics experienced a significant Pi peak increase and PCr peak decrease, and once the exercise was completed both Pi and PCr peaks returned to resting levels. Quantitatively, the mean recovery rates of Pi and PCr in diabetes patients were (10.74 ± 1.26) mmol/s and (4.74 ± 2.36) mmol/s, respectively, which was significantly higher than in controls. CONCLUSIONS: Non-invasive quantitative (31)P-MRS is able to detect energy metabolism inefficiency and mitochondrial function impairment in skeletal muscle of type 2 diabetics.

High-throughput, homogeneous, fluorescence intensity-based measurement of adenosine diphosphate and other ribonucleoside diphosphates with nanomolar sensitivity.

A new, homogeneous, high-throughput-compatible assay method is described for the fluorescence-based quantitation of nanomolar concentrations of ribonucleoside diphosphates (rNDPs). The principle of the method is the conversion of the rNDPs to RNA by the enzyme polynucleotide phosphorylase (EC 2.7.7.8) and detection of the RNA by the increased fluorescence of a commercial nucleic acid detection dye. A commercial RNA homopolymer complementary to the RNA product is included to increase the sensitivity for ADP and UDP. Standard curves for nanomolar concentrations of ADP, UDP, GDP, and CDP are shown. The assay detected 75 nM ADP produced by the pyruvate kinase-catalyzed phosphorylation of pyruvate with a signal-to-baseline ratio of 2.8. The assay may be used in either a continuous or a discontinuous mode.

Development and validation of a high-density fluorescence polarization-based assay for the trypanosoma RNA triphosphatase TbCet1.

RNA triphosphatases are attractive and mostly unexplored therapeutic targets for the development of broad spectrum antiprotozoal, antiviral and antifungal agents. The use of malachite green as a readout for phosphatases is well characterized and widely employed. However, the reaction depends on high quantities of inorganic phosphate to be generated, which makes this assay not easily amenable to screening in 1536-well format. The overly long reading times required also prohibit its use to screen large chemical libraries. To overcome these limitations, we sought to develop a fluorescence polarization (FP) -based assay for triphosphatases, compatible with miniaturization and fast readouts. For this purpose, we took advantage of the nucleoside triphosphatase activity of this class of enzyme to successfully adapt the Transcreener ADP assay based on the detection of generated ADP by immunocompetition fluorescence polarization to the RNA triphosphatase TbCet1 in 1536-well format. We also tested the performance of this newly developed assay in a pilot screen of 3,000 compounds and we confirmed the activity of the obtained hits. We present and discuss our findings and their importance for the discovery of novel drugs by high throughput screening.

Hexokinase inhibitor screening based on adenosine 5'-diphosphate determination by electrophoretically mediated microanalysis.

A CE-based method for hexokinase inhibitor screening was developed in the present paper. In this method, hexokinase activity was assayed via electrophoretically mediated microanalysis (EMMA), which combines on-column hexokinase-mediated reaction and measurement of produced adenosine 5'-diphosphate (ADP) via electrophoretical separation and UV detection. Enzyme inhibition can be read out directly from the reduced peak area of ADP in comparison with a reference electropherogram obtained in the absence of any inhibitor. Conditions for on-column enzyme reaction and separation of adenosine 5'-triphosphate (ATP) and ADP were optimized. The optimal buffer composition for enzymatic reaction was 25 mM HEPES buffer (pH 7.5) containing 5 mM MgCl(2), whereas the optimal buffer composition for separation was 100 mM Tris-phosphate buffer (pH 5.5) containing 0.02% (m/v) hexadimethrine bromide (HDB). Fortunately, discontinuous buffer system can be adapted easily in the EMMA method. The time for separation was reduced dramatically to less than 3 min by reversing the direction of EOF via dynamically coating the capillary wall with the cationic polyelectrolyte HDB. Moreover, the peak tailing of ATP was also reduced by HDB coating. The Z' factor as high as 0.98 was obtained, indicating a high quality of the screening data. The present method is simple, robust and cost-effective.

In vivo evidence for cerebral depletion in high-energy phosphates in progressive supranuclear palsy.

Indirect evidence from laboratory studies suggests that mitochondrial energy metabolism is impaired in progressive supranuclear palsy (PSP), but brain energy metabolism has not yet been studied directly in vivo in a comprehensive manner in patients. We have used combined phosphorus and proton magnetic resonance spectroscopy to measure adenosine-triphosphate (ATP), adenosine-diphosphate (ADP), phosphorylated creatine, unphosphorylated creatine, inorganic phosphate and lactate in the basal ganglia and the frontal and occipital lobes of clinically probable patients (N=21; PSP stages II to III) and healthy controls (N=9). In the basal ganglia, which are severely affected creatine in PSP patients, the concentrations of high-energy phosphates (=ATP+phosphorylated creatine) and inorganic phosphate, but not low-energy phosphates (=ADP+unphosphorylated creatine), were decreased. The decrease probably does not reflect neuronal death, as the neuronal marker N-acetylaspartate was not yet significantly reduced in the early-stage patients examined. The frontal lobe, also prone to neurodegeneration in PSP, showed similar alterations, whereas the occipital lobe, typically unaffected, showed less pronounced alterations. The levels of lactate, a product of anaerobic glycolysis, were elevated in 35% of the patients. The observed changes in the levels of cerebral energy metabolites in PSP are consistent with a functionally relevant impairment of oxidative phosphorylation.

Discovery of acetyl-coenzyme A carboxylase 2 inhibitors: comparison of a fluorescence intensity-based phosphate assay and a fluorescence polarization-based ADP Assay for high-throughput screening.

Acetyl-coenzyme A carboxylase (ACC) enzymes exist as two isoforms, ACC1 and ACC2, which play critical roles in fatty acid biosynthesis and oxidation. Though each isoform differs in tissue and subcellular localization, both catalyze the biotin- and ATP-dependent carboxylation of acetyl-coenzyme A to generate malonyl-coenzyme A, a key metabolite in the control of fatty acid synthesis and oxidation. The cytosolic ACC1 is expressed primarily in liver and adipose tissue, and uses malonyl-coenzyme A as a key building block in fatty acid biosynthesis. The mitochondrial ACC2 is primarily expressed in heart and skeletal muscle, where it is involved in the regulation of fatty acid oxidation. Inhibitors of ACC enzymes may therefore be useful therapeutics for diabetes, obesity, and metabolic syndrome. Two assay formats for these ATP-utilizing enzymes amenable to high-throughput screening are compared: a fluorescence intensity-based assay to detect inorganic phosphate and a fluorescence polarization-based assay to detect ADP. Acetyl-coenzyme A carboxylase inhibitors were identified by these high-throughput screening methods and were confirmed in a radiometric high performance liquid chromatography assay of malonyl-coenzyme A production.

Selective loss of glucose-induced amplification of insulin secretion in mouse pancreatic islets pretreated with sulfonylurea in the absence of fuels.

AIMS/HYPOTHESIS: The beta cell metabolism of glucose, and some other fuels, initiates insulin secretion by closure of ATP-sensitive K+ channels and amplifies the secretory response via unknown metabolic intermediates. The aim of this study was to further characterise the mechanism responsible for the metabolic amplification of insulin secretion. MATERIALS AND METHODS: Pancreatic islets were isolated from albino mice by collagenase digestion. Insulin secretion in perifused islets was determined by ELISA. Bioluminometry was used to determine the ATP and ADP content of the incubated islets. RESULTS: After perifusing islets for 60 min with 2.7 micromol/l glipizide (closing all ATP-sensitive K+ channels) in the absence of any fuel, perifusion with a test medium containing 2.7 micromol/l glipizide plus 30 mmol/l glucose did not enhance insulin secretion. However, test media supplemented with 2.7 micromol/l glipizide plus either 10 mmol/l alpha-ketoisocaproate or 10 mmol/l 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid amplified the glipizide-induced insulin secretion. In pancreatic islets preincubated for 60 min with 2.7 micromol/l glipizide in the absence of any fuel, 40 min incubations in the presence of 2.7 micromol/l glipizide plus 30 mmol/l glucose or plus 10 mmol/l alpha-ketoisocaproate produced an increase in the ATP content, no change in the ADP content and a rather small increase in the ATP:ADP ratio. The corresponding effects of glucose and alpha-ketoisocaproate were similar. CONCLUSIONS/INTERPRETATION: These results suggest that metabolic amplification of fuel-induced insulin secretion is not mediated by changes in the beta cell content of ATP and ADP, but might be due to export of citrate cycle intermediates to the beta cell cytosol.

Uniformly oriented bacterial F0F1-ATPase immobilized on a semi-permeable membrane: a step towards biotechnological energy transduction.

The immobilization of F(0)F(1)-ATPase in uniform orientation is reported. The biotinylated and histidine-tagged subunits of the bacterial F(0)F(1)-ATPase complex were used for immobilization of the complex on artificial semi-permeable membranes resulting in 88+/-7.8 and 72+/-5.2% coupling of the enzymes. The immobilized enzymes retained over 90% activity. The immobilized ATPase/synthase was used for generation of ATP from ADP and P(i) at the expense of electrochemical potential energy. The re-usability, ratio of amount of enzyme immobilized to enzymic activity conferred on the membranes, ATP synthesized by assembled system and suitability of ATP generated for use in coupled enzymic reactions were determined.

Single dose of glutamine enhances myocardial tissue metabolism, glutathione content, and improves myocardial function after ischemia-reperfusion injury.

BACKGROUND: Myocardial ischemia and reperfusion (I/R) injury causes significant morbidity and mortality. Protection against I/R injury may occur via preservation of tissue metabolism and ATP content, preservation of reduced glutathione, and stimulation of heat shock protein (HSP) synthesis. Supplementation with glutamine (GLN) has been reported to have beneficial effects on all of these protective pathways. Thus, we hypothesized that GLN pretreatment given to the rat in vivo would protect the myocardium against I/R-induced dysfunction. METHODS: GLN (0.52 g/kg, intraperitoneally, given as alanine-glutamine dipeptide), alanine alone (0.23 g/kg), or a Ringer's lactate solution (control) was administered to Sprague-Dawley rats 18 hours before heart excision, perfusion, exposure to global ischemia (15 minutes) and reperfusion (1 hour). Tissue metabolites were analyzed via magnetic resonance spectroscopy. RESULTS: In control and alanine-treated animals, I/R injury resulted in cardiac dysfunction, indicated by a decrease in cardiac output. Administration of GLN 18 hours before I/R injury preserved cardiac output after reperfusion. Metabolic analysis of the myocardial tissue revealed that [/R injury led to significant diminution of myocardial tissue glutamate, ATP content, accumulation of myocardial lactate, and a reduction in reduced glutathione content in control animals. GLN significantly reduced the deleterious changes in myocardial metabolism and improved reduced glutathione content. No changes in pre- or post-I/R injury HSP expression were observed after GLN administration. CONCLUSIONS: These observations demonstrate that remote in vivo administration of GLN before cardiac I/R injury can improve post-I/R cardiac function. This effect may be mediated via improved myocardial metabolism and enhanced reduced glutathione content.

Glutamine administration during total parenteral nutrition protects liver adenosine nucleotides during and after subsequent hemorrhagic shock.

BACKGROUND: Glutamine supplementation of total parenteral nutrition (TPN) in stressed patients has been advocated. To determine whether glutamine supplementation affects the host response to conditions of stress, animals were given TPN with or without glutamine for 7 days. They were then subjected to the acute stress of hemorrhagic shock, which results in marked loss of hepatic adenosine triphosphate (ATP) and adenosine diphosphate (ADP), with accumulation of adenosine monophosphate (AMP) and the metabolites adenosine, inosine, hypoxanthine, and xanthine. This loss of ATP and accumulation of metabolites contributes to subsequent tissue damage. The hypothesis of the study was that glutamine supplementation would significantly improve restoration of hepatic adenosine nucleotides before and after hemorrhagic shock. METHODS: Sprague-Dawley rats were given TPN for 7 days. One half of the animals (n = 8) received TPN supplemented with glutamine, while one half received TPN with an isonitrogenous mixture of alanine and glycine. Animals were subjected to hemorrhagic shock for 30 minutes and then resuscitated using only heparinized shed blood. Liver biopsies were taken pre- and post-shock, and at 30 and 60 minutes after resuscitation. ATP, ADP, AMP, and their metabolites were measured using gradient high-performance liquid chromatography. RESULTS: After 7 days of TPN, baseline values of ATP, ADP, AMP, and metabolites were similar between the 2 groups before the initiation of shock. Glutamine-treated animals manifested a 40% decrease in ATP level immediately after shock and recovered to 90% of baseline within 60 minutes. By contrast, the control animals manifested a 66% decrease in ATP level after the shock period and recovered only to 60% of baseline at 1 hour postresuscitation. Similar changes were observed in ADP levels and were accompanied by corresponding changes in AMP and adenosine metabolites, all of which rose during shock and fell after resuscitation. CONCLUSIONS: Glutamine supplementation significantly protected the liver from tissue damage caused by hemorrhagic shock. ATP levels remained higher during shock and recovered more rapidly after resuscitation. Glutamine supplementation may help to protect cellular energy stores in the stressed organism and may offer opportunities for therapeutic intervention during and after stress.

Effects of epinephrine and norepinephrine on hemodynamics, oxidative metabolism, and organ energetics in endotoxemic rats.

OBJECTIVE: To determine whether epinephrine increases lactate concentration in sepsis through hypoxia or through a particular thermogenic or metabolic pathway. DESIGN: Prospective, controlled experimental study in rats. SETTING: Experimental laboratory in a university teaching hospital. INTERVENTIONS: Three groups of anesthetized, mechanically ventilated male Wistar rats received an intravenous infusion of 15 mg/kg Escherichia coli O127:B8 endotoxin. Rats were treated after 90 min by epinephrine ( n=14), norepinephrine ( n=14), or hydroxyethyl starch ( n=14). Three groups of six rats served as time-matched control groups and received saline, epinephrine, or norepinephrine from 90 to 180 degrees min. Mean arterial pressure, aortic, renal, mesenteric and femoral blood flow, arterial blood gases, lactate, pyruvate, and nitrate were measured at baseline and 90 and 180 min after endotoxin challenge. At the end of experiments biopsy samples were taken from the liver, heart, muscle, kidney, and small intestine for tissue adenine nucleotide and lactate/pyruvate measurements. MEASUREMENTS AND RESULTS: Endotoxin induced a decrease in mean arterial pressure and in aortic, mesenteric, and renal blood flow. Plasmatic and tissue lactate increased with a high lactate/pyruvate (L/P) ratio. ATP decreased in liver, kidney, and heart. The ATP/ADP ratio did not change, and phosphocreatinine decreased in all organs. Epinephrine and norepinephrine increased mean arterial pressure to baseline values. Epinephrine increased aortic blood flow while renal blood low decreased with both drugs. Plasmatic lactate increased with a stable L/P ratio with epinephrine and did not change with norepinephrine compared to endotoxin values. Nevertheless epinephrine and norepinephrine when compared to endotoxin values did not change tissue L/P ratios or ATP concentration in muscle, heart, gut, or liver. In kidney both drugs decreased ATP concentration. CONCLUSIONS: Our data demonstrate in a rat model of endotoxemia that epinephrine-induced hyperlactatemia is not related to cellular hypoxia.

gamma-glutamyltranspeptidase-deficient knockout mice as a model to study the relationship between glutathione status, mitochondrial function, and cellular function.

gamma-Glutamyltranspeptidase (GGT)-deficient mice (GGT(-/-)) display chronic glutathione (GSH) deficiency, growth retardation, and die at a young age (<20 weeks). Using livers from these mice, we investigated the relationship between GSH content, especially mitochondrial, and mitochondrial and cellular function. We found that the GSH content of isolated liver mitochondria was diminished by >/=50% in GGT(-/-) mice when compared with wild-type mice. Respiratory control ratios (RCRs) of GGT(-/-) mice liver mitochondria were /=40% in mitochondria obtained from GGT(-/-) mice. We observed a strong correlation between mitochondrial GSH content and RCRs. Even moderate decreases (<50%) correlated with adverse effects with respect to respiration. Electron microscopy revealed that livers from GGT(-/-) knockout mice were deprived of fat and glycogen, and swollen mitochondria were observed in animals that were severely deprived of GSH. Thus, GGT(-/-) mice exhibit a loss of GSH homeostasis and impaired oxidative phosphorylation, which may be related to the rate of adenosine triphosphate (ATP) formation and subsequently leads to progressive liver injury, which characterizes the diseased state. We also found that supplementation of GGT(-/-) mice with N-acetylcysteine (NAC) partially restored liver GSH, but fully restored mitochondrial GSH and respiratory function. Electron microscopy revealed that the livers of NAC-supplemented GGT(-/-) mice contained fat and glycogen; however, slightly enlarged mitochondria were found in some livers. NAC supplementation did not have any beneficial effect on the parameters examined in wild-type mice.

Influence of vitamin E succinate on retinal cell survival.

In this study, we analyzed the influence of vitamin E succinate (5-80 microM), supplemented in the culture medium, on the survival of cultured retinal cells. The release of lactate dehydrogenase (LDH) was decreased in the presence of low concentrations (10-20 microM) of vitamin E succinate, whereas high concentrations (80 microM) induced a significant increase (about 2-fold) in the release of LDH, indicating a reduction of plasma membrane integrity. Supplementing with vitamin E succinate (80 microM) greatly enhanced its cellular content, as compared to vitamin E acetate (80 microM), and the membrane order of the retinal cells, as evaluated by the fluorescence anisotropy (r) of TMA-DPH (1-(4-(trimethylammonium)-phenyl)-6-phenylhexa-1,3,5-triene), was not altered. Furthermore, vitamin E succinate was more potent than vitamin E acetate in reducing thiobarbituric acid reactive substances (TBARS) formation upon ascorbate-Fe2+-induced oxidative stress (TBARS formation after cell oxidation decreased by about 15-fold or 1.6 fold, respectively, in the presence of 20 microM vitamin E succinate or 20 microM vitamin E acetate). A decrease in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction induced by supplementing with vitamin E succinate (80 microM), to 35.99 +/- 1.96% as compared to the control, but not by vitamin E acetate (80 microM), suggests that vitamin E succinate may affect the mitochondrial activity. Vitamin E succinate also reduced significantly the ATP:ADP ratio in a dose-dependent manner, indicating that vitamin E succinate-mediated cytotoxic effects involve a decrement of mitochondrial function.

Serial experimental and clinical studies on the pathogenesis of multiple organ dysfunction syndrome (MODS) in severe burns.

These serial clinical and experimental studies were designed to clarify the pathogenesis of postburn MODS. Both animal and clinical studies were performed. In animal experiments, 46 male cross-bred dogs were cannulated with Swan-Ganz catheters and 39 of them were inflicted with 50% TBSA third degree burns (7 were used as controls). The burned dogs were randomly divided into 4 groups: immediate infusion, delayed infusion, delayed fast infusion and delayed fast infusion combined with ginsenosides. All dogs were kept under constant barbiturate sedation during the whole study period. Hemodynamics, visceral MDA, mitochondrial respiratory control rate (RCR) and ADP/O ratio, ATP, succinic dehydrogenase (SDH), organ water content as well as light and electron microscopy of visceral tissues were determined. In the clinical study, 61 patients with extensive deep burns were chosen, of which 16 sustained MODS. Plasma TXB2/6-keto-PGF1alpha ratio, TNF, SOD, MDA, circulatory platelet aggregate ratio (CPAR), PGE2, interleukin-1, total organ water content and pathological observations of visceral tissues from patients who died of MODS were carried out. Results demonstrated that ischemic-reperfusion damage due to severe shock, sepsis and inhalation injury are three main causes of postburn death. All inflammatory mediators increased markedly in both animals and patients who sustained organ damage or MODS. SDH, RCR, ADP/O and ATP decreased significantly. These findings suggested that ischemic damage and systemic inflammatory response syndrome (SIRS) initiated by mediators or cytokines might be important in the pathogenesis of postburn MODS.

The effects of halothane and isoflurane on the phosphoenergetic state of the liver during hemorrhagic shock in rats: an in vivo 31P nuclear magnetic resonance spectroscopic study.

We studied the effects of halothane versus isoflurane on the phosphoenergetic state and intracellular pH (pHi) of the rat liver using in vivo 31P nuclear magnetic resonance (NMR) spectroscopy during and after hemorrhagic shock. Seventeen rats were anesthetized with 1 minimum alveolar anesthetic concentration of halothane or isoflurane. The mean arterial blood pressure was reduced to 40 mm Hg and maintained at this level for 45 min by withdrawing blood from the common carotid artery. The shed blood was then returned slowly. In vivo 31P NMR spectra were consecutively collected throughout the study. The phosphoenergetic state of the liver was evaluated from the changes in adenosine triphosphate (ATP) and inorganic phosphate (P(i)) levels. pHi was calculated from the chemical shifts of P(i) and alpha-ATP peaks. During hemorrhagic shock, beta-ATP decreased to 35% and 45%, and P(i) increased to 300% and 230% of their initial values in the halothane and isoflurane groups, respectively. Intracellular acidosis was more severe in the halothane group. The recoveries of beta-ATP and P(i) were better in the isoflurane group. Halothane showed a more detrimental effect than isoflurane on the hepatic phosphoenergetic level during and after hemorrhagic shock.