Exercise-induced oxidative stress in elderly subjects: the effect of red orange supplementation on the biochemical and cellular response to a single bout of intense physical activity.

Aging is characterized by an impaired capacity to maintain the redox balance both in physiological and pathological situations associated with an increased production of reactive oxygen species. Since the extent of this phenomenon may be influenced by an antioxidants-rich diet, we investigated the effect of supplementation with fresh red orange juice (ROJ) on biochemical and cellular biomarkers of oxidative stress in healthy, trained elderly women after a single bout of exhaustive exercise (EE). To this purpose, a sample of 22 females, 15 (69.0 ± 5.1 years) taking the ROJ supplementation and 7 (68.1 ± 2.7 years) as Control group, was constituted. Blood samples were collected immediately before, 30 minutes, and 24 hr after a single bout of EE, at baseline and after 4 weeks. Our results demonstrate that markers of DNA damage or apoptosis were not affected by EE both in Control and ROJ group, and by ROJ, whereas, exercise temporarily affected the redox balance in both groups. Controls didn't change their response to EE after the experimental period, but experimental group after ROJ supplementation had lower EE-induced MDA, consumed less ascorbic acid, and had less activation of the hypoxanthine/xanthine system, i.e., they seemed to be protected from hypoxia/reoxygenation mechanisms.

Cerebrospinal fluid ATP metabolites in multiple sclerosis.

Increased axonal energy demand and mitochondrial failure have been suggested as possible causes for axonal degeneration and disability in multiple sclerosis. Our objective was to test whether ATP depletion precedes clinical, imaging and biomarker evidence for axonal degeneration in multiple sclerosis. The method consisted of a longitudinal study which included 21 patients with multiple sclerosis. High performance liquid chromatography was used to quantify biomarkers of the ATP metabolism (oxypurines and purines) from the cerebrospinal fluid at baseline. The Expanded Disability Status Scale, MRI brain imaging measures for brain atrophy (ventricular and parenchymal fractions), and cerebrospinal fluid biomarkers for axonal damage (phosphorylated and hyperphosphorylated neurofilaments) were quantified at baseline and 3-year follow-up. Central ATP depletion (sum of ATP metabolites >19.7 micromol/litre) was followed by more severe progression of disability if compared to normal ATP metabolites (median 1.5 versus 0, p< 0.05). Baseline ATP metabolite levels correlated with change of Expanded Disability Status Scale in the pooled cohort (r= 0.66, p= 0.001) and subgroups (relapsing-remitting patients: r= 0.79, p< 0.05 and secondary progressive/primary progressive patients: r= 0.69, p< 0.01). There was no relationship between central ATP metabolites and either biomarker or MRI evidence for axonal degeneration. The data suggests that an increased energy demand in multiple sclerosis may cause a quantifiable degree of central ATP depletion. We speculate that the observed clinical disability may be related to depolarisation associated conduction block.

Myocardial metabolism of exogenous FDP is consistent with transport by a dicarboxylate transporter.

The extent to and the mechanism by which fructose-1,6-bisphosphate (FDP) crosses cell membranes are unknown. We hypothesized that its transport is either via band 3 or a dicarboxylate transporter. The question was addressed in isolated Langendorff rat hearts perfused under normoxic conditions. Groups of hearts received the following metabolic substrates (in mM): 5 FDP; 5 FDP + either 5, 10, or 20 fumarate; 10 FDP and either 5, 10, or 20 fumarate; or 5 FDP + 2 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), a band 3 inhibitor. FDP uptake and metabolism were measured as production of [(13)C]lactate from [(13)C]FDP or (14)CO(2) and [(14)C]lactate from uniformly labeled [(14)C]FDP in sample perfusates. During 30 min of perfusion, FDP metabolism was 12.4 +/- 2.6 and 31.2 +/- 3.0 micromol for 5 and 10 mM FDP, respectively. Addition of 20 mM fumarate reduced FDP metabolism over a 30-min perfusion period to 3.1 +/- 0.6 and 6.3 +/- 0.5 micromol for 5 and 10 mM FDP groups, respectively. DNDS did not affect FDP utilization. These data are consistent with transport of FDP by a dicarboxylate transport system.

N-Acetylaspartate reduction as a measure of injury severity and mitochondrial dysfunction following diffuse traumatic brain injury.

N-Acetylaspartate (NAA) is considered a neuron-specific metabolite and its reduction a marker of neuronal loss. The objective of this study was to evaluate the time course of NAA changes in varying grades of traumatic brain injury (TBI), in concert with the disturbance of energy metabolites (ATP). Since NAA is synthesized by the mitochondria, it was hypothesized that changes in NAA would follow ATP. The impact acceleration model was used to produce three grades of TBI. Sprague-Dawley rats were divided into the following four groups: sham control (n = 12); moderate TBI (n = 36); severe TBI (n = 36); and severe TBI coupled with hypoxia-hypotension (n = 16). Animals were sacrificed at different time points ranging from 1 min to 120 h postinjury, and the brain was processed for high-performance liquid chromatography (HPLC) analysis of NAA and ATP. After moderate TBI, NAA reduced gradually by 35% at 6 h and 46% at 15 h, accompanied by a 57% and 45% reduction in ATP. A spontaneous recovery of NAA to 86% of baseline at 120 h was paralleled by a restoration in ATP. In severe TBI, NAA fell suddenly and did not recover, showing critical reduction (60%) at 48 h. ATP was reduced by 70% and also did not recover. Maximum NAA and ATP decrease occurred with secondary insult (80% and 90%, respectively, at 48 h). These data show that, at 48 h post diffuse TBI, reduction of NAA is graded according to the severity of insult. NAA recovers if the degree of injury is moderate and not accompanied by secondary insult. The highly similar time course and correlation between NAA and ATP supports the notion that NAA reduction is related to energetic impairment.

Oxidative stress induces impairment of human erythrocyte energy metabolism through the oxygen radical-mediated direct activation of AMP-deaminase.

The effect of oxidative stress on human red blood cell AMP-deaminase activity was studied by incubating either fresh erythrocytes or hemolysates with H(2)O(2) (0.5, 1, 2, 4, 6, 8, and 10 mm) or NaNO(2) (1, 5, 10, 20, and 50 mm), for 15 min at 37 degrees C. AMP-deaminase tremendously increased by increasing H(2)O(2) or NaNO(2) at up to 4 and 20 mm, respectively (maximal effect for both oxidants was 9.5 and 6.5 times higher enzymatic activity than control erythrocytes or hemolysates, respectively). The incubation of hemolysates with iodoacetate (5-100 mm), N-ethylmaleimide (0.1-10 mm), or p-hydroxymercuribenzoate (0.1-5 mm) mimicked the effect of oxidative stress on AMP-deaminase, indicating that sulfhydryl group modification is involved in the enzyme activation. In comparison with control hemolysates, changes of the kinetic properties of AMP-deaminase (decrease of AMP concentration necessary for half-maximal activation, increase of V(max), modification of the curve shape of V(o) versus [S], Hill plots, and coefficients) were recorded with 4 mm H(2)O(2)- and 1 mm N-ethylmaleimide-treated hemolysates. Data obtained using 90% purified enzyme, incubated with Fenton reagents (Fe(2+) + H(2)O(2)) or -SH-modifying compounds, demonstrated that (i) reactive oxygen species are directly responsible for AMP-deaminase activation; (ii) this phenomenon occurs through sulfhydryl group modification; and (iii) the activation does not involve the loss of the tetrameric protein structure. Results of experiments conducted with glucose-6-phosphate dehydrogenase-deficient erythrocytes, challenged with increasing doses of the anti-malarial drug quinine hydrochloride and showing dramatic AMP-deaminase activation, suggest relevant physiopathological implications of this enzymatic activation in conditions of increased oxidative stress. To the best of our knowledge, this is the first example of an enzyme, fundamental for the maintenance of the correct red blood cell energy metabolism, that is activated (rather than inhibited) by the interaction with reactive oxygen species.

Early onset of lipid peroxidation after human traumatic brain injury: a fatal limitation for the free radical scavenger pharmacological therapy?

BACKGROUND: On the basis of the contradiction between data on experimental head trauma showing oxidative stress-mediated cerebral tissue damage and failure of the majority of clinical trials using free radical scavenger drugs, we monitored the time-course changes of malondialdehyde (MDA, an index of cell lipid peroxidation), ascorbate, and dephosphorylated ATP catabolites in cerebrospinal fluid (CSF) of traumatic brain-injured patients. METHODS: CSF samples were obtained from 20 consecutive patients suffering from severe brain injury. All patients were comatose, with a Glasgow Coma Scale on admission of 6 +/- 1. The first CSF sample for each patient was collected within a mean value of 2.95 hours from trauma (SD=1.98), after the insertion of a ventriculostomy catheter for the continuous monitoring of intracranial pressure. During the next 48 hours, CSF was withdrawn from each patient once every 6 hours. All samples were analyzed by an ion-pairing high-performance liquid chromatographic method for the simultaneous determination of MDA, ascorbic acid, hypoxanthine, xanthine, uric acid, inosine, and adenosine. RESULTS: In comparison with values recorded in 10 herniated-lumbar-disk, noncerebral control patients, data showed that all CSF samples of brain-injured patients had high values (0.226 micromol/L; SD=0.196) of MDA (undetectable in samples of control patients) and decreased ascorbate levels (96.25 micromol/L; SD=31.74), already at the time of first withdrawal at the time of hospital admission. MDA was almost constant in the next two withdrawals and tended to decrease thereafter, although 48 hours after hospital admission, a mean level of 0.072 micromol/L CSF (SD=0.026) was still recorded. The ascorbate level was normalized 42 hours after hospital admission. Changes in the CSF values of ATP degradation products (oxypurines and nucleosides) suggested a dramatic alteration of neuronal energy metabolism after traumatic brain injury. CONCLUSIONS: On the whole, these data demonstrate the early onset of oxygen radical-mediated oxidative stress, proposing a valid explanation for the failure of clinical trials based on the administration of oxygen free radical scavenger drugs and suggesting a possible rationale for testing the efficacy of lipid peroxidation "chain breakers" in future clinical trials.

The potency of acyclovir can be markedly different in different cell types.

Acyclovir is an acyclic guanine analog with a considerable activity against herpes simplex viruses. We studied the antiherpetic activity of acyclovir in macrophages and fibroblast cell lines. Utilising a plaque reduction assay we found that acyclovir potently inhibited the HSV-1 replication in macrophages (EC50) = 0.0025 microM) compared to Vero (EC50 = 8.5 microM) and MRC-5 (EC50 = 3.3 microM) cells. The cytotoxicity of acyclovir was not detected at concentrations < or = 20 microM, thus the selective index in macrophages was >8000. This marked difference in antiherpetic activity between macrophages and fibroblasts was not observed with Foscarnet and PMEA. We suggest that this potent antiviral effect of acyclovir is mainly due to a proficient phosphorylation of the drug and/or a favourable dGTP/acyclovir triphosphate ratio in macrophage cells.

Effectiveness of a new device to retain carcinogenic compounds of tar from mainstream cigarette smoke for the prevention of smoking-associated tumors.

PURPOSE: Effectiveness of a new device inserted within the common cellulose acetate cigarette filter (named hypobaric chamber tar-removing system, HCTRS) to remove tar and its carcinogenic compounds from mainstream cigarette smoke (MCS). METHODS: Eighty HCTRS prototypes were mounted inside the cellulose acetate filter of commercial-brand cigarettes (13 mg tar) and smoked by eighty smoker volunteers. Tar retained by HCTRS prototypes was determined by weighing them before and after smoking. Subsequently, an aliquot (3-5 mg) of the tar retained by twenty randomly chosen HCTRS prototypes was analysed by high-performance liquid chromatography (HPLC) for the detection of polycyclic aromatic hydrocarbons (PAH). RESULTS: The mean value of tar retained was 12.80 mg/HCTRS prototype (S.D. = 5.31), thus showing that this simple device is capable of removing 98.5% of tar present in MCS. Minimal and maximal amounts of retained tar were 4.15 and 31.47 mg/HCTRS prototype, respectively. Moreover, these tar samples contained, although in differing amounts, each of the 16 priority pollutant PAH. A mean value of 259.42 ng/mg of tar (S.E.M. = 44.37) of the 16 main PAH was found in the tar of the 20 HCTRS prototypes examined. These data cogently demonstrate that the use of the HCTRS prototype can effectively eliminate about 100% of tar from MCS, thus reducing the inhalation of PAH (considered the most obvious carcinogenic tar components). CONCLUSIONS: The application of this device could be a suitable tool for effectively improving human health through the prevention of smoking-associated cancer.

Activity and mechanism of the antioxidant properties of cyanidin-3-O-beta-glucopyranoside.

In the present study, the antioxidant activity, the interaction with reactive oxygen species and the redox potential of cyanidin-3-O-beta-glucopyranoside (C-3-G), the main anthocyanin present in juice of pigmented oranges, were evaluated in detail. C-3-G effects on low density lipoproteins (LDL) oxidation induced by 40 microM Cu at a pH of 7.4 were compared with those of resveratrol and ascorbic acid, two other natural antioxidants. All cyanidin-3-O-beta-glucopyranoside concentrations used (1, 2, 5, 10, 20, 50, 100 and 200 microM) inhibited malondialdehyde (MDA) generation (an index of lipid peroxidation), the inhibition being significantly higher than that obtained with equal concentrations of resveratrol and ascorbic acid (IC50 = 6.5 microM for C-3-G, 34 microM for resveratrol and 212 microM for ascorbic acid). Experiments of LDL oxidation performed at a pH of 5.0 or 6.0 showed that C-3-G antioxidant activity is not influenced by pH variations between 5.0 and 7.4. This suggests that metal chelation, exerted by C-3-G through the eventual dissociation of its phenolic groups, plays a minor role in its protective mechanism. The presence of C-3-G produced significantly higher protective effects of pigmented orange juice (obtained from Moro cultivar) with respect to blond orange juice, when tested on copper-induced LDL oxidation. The evaluation of the direct interaction with reactive oxygen species (H2O2, -O2, OH*), demonstrated that C-3-G is quickly oxidized by these compounds and it is, therefore, a highly efficient oxygen free radical scavenger. The powerful C-3-G antioxidant activity is in excellent agreement with the very negative redox potential (-405 mV), determined through direct current cyclic voltammetry measurements. On the basis of these results, C-3-G should be considered as one of the most effective antioxidants that can be assumed with dietary plants; therefore, pigmented oranges represent a very relevant C-3-G source because of the high content of this anthocyanin in their juice.

Modifications of mitochondria in human tumor cells during anthracycline-induced apoptosis.

Adriamycin (ADR), one of the major antitumor agents used for the clinical treatment of a wide variety of human cancers and its glutathione(GSH)-conjugated adduct, ADRIGLU, induced apoptosis in K562 erythroleukemia and TVM-A12 clone 2 melanoma human cell lines. We have previously reported that ADR has nuclear localization and that ADRIGLU localizes exclusively in the cytoplasm. During ADR or ADRIGLU treatment, significant depletion of the cell energy state, demonstrated by a reduction in high-energy phosphates (ATP and GTP) and a decrease in energy charge potential (ECP), were recorded between 2 hours and 24 hours, by HPLC analysis. Transmission electron microscopy also revealed that between 2 hours and 24 hours of ADR or ADRIGLU treatment, mitochondria underwent evident morphological changes, from an initial "high amplitude swelling state" to a "shrinkage state" and finally, in early apoptotic cells, to an "abnormal shrinkage state", in which a marked accumulation of pycnotic mitochondria was also observed. Confocal microscopic analysis, using the potential-sensitive dye JC-1, showed that inhibition of cell energy metabolism was preceded by a rapid decrease in mitochondrial transmembrane potential (delta psi m). With the progression of exposure time, the early depolarization of the mitochondrial membrane was followed by a transient reversion to normal delta psi m until, in apoptotic cells, almost all mitochondrial subpopulations appeared to be hyperpolarized. Our results indicated that mitochondria are actively involved in anthracycline-induced programmed cell death, suggesting a novel mechanism that may be common to all forms of apoptosis.

Determination of boronophenylalanine in biological samples using precolumn o-phthalaldehyde derivatization and reversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method for the detection of boronophenylalanine is described. Determination was obtained by precolumn reaction of o-phthalaldehyde with a mixture of standard amino acids containing boronophenylalanine and separating the corresponding o-phthalaldehyde derivatives, using a Kromasil C-18, 250 x 4.6 mm, 5-microm particle size column, a step gradient with two buffers, a flow rate of 1.2 ml/min, a column temperature of 23 degrees C, and fluorimetric detection (excitation and emission wavelengths of 330 and 430 nm, respectively). The use of such a method for assaying boronophenylalanine in biological samples was tested in neutralized perchloric acid blood and cerebral tissue extracts of rats treated with intracarotid administration of 300 mg/kg of body weight boronophenylalanine. Results of these experiments showed that the present HPLC method represents a valid alternative to currently available analytical techniques for assaying boronophenylalanine based on boron determination in terms of reproducibility, recovery, or sensitivity. Therefore, it is suggested that the present method may routinely be used in all preclinical and clinical studies in which quantification of circulating and tissue concentrations of boronophenylalanine is critical for the application of boron neutron capture therapy.

Direct NAD(P)H hydrolysis into ADP-ribose(P) and nicotinamide induced by reactive oxygen species: a new mechanism of oxygen radical toxicity.

The effect of different oxygen radical-generating systems on NAD(P)H was determined by incubating the reduced forms of the pyridine coenzymes with either Fe2+-H2O2 or Fe3+-ascorbate and by analyzing the reaction mixtures using a HPLC separation of adenine nucleotide derivatives. The effect of the azo-initiator 2,2'-azobis(2-methylpropionamidine)dihydrochloride was also tested. Results showed that, whilst all the three free radical-producing systems induced, with different extent, the oxidation of NAD(P)H to NAD(P)+, only Fe2+-H2O2 also caused the formation of equimolar amounts of ADP-ribose(P) and nicotinamide. Dose-dependent experiments, with increasing Fe2+ iron (concentration range 3-180 microM) or H2O2 (concentration range 50-1000 microM), were carried out at pH 6.5 in 50 mM ammonium acetate. NAD(P)+, ADP-ribose(P) and nicotinamide formation increased by increasing the amount of hydroxyl radicals produced in the medium. Under such incubation conditions NAD(P)+/ADP-ribose(P) ratio was about 4 at any Fe2+ or H2O2 concentration. By varying pH to 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0 and 7.4, NAD(P)+/ADP-ribose(P) ratio changed to 5.5, 3.2, 1.8, 1.6, 2.0, 2.5, 3.0, 5.4 and 6.5, respectively. Kinetic experiments indicated that 90-95% of all compounds were generated within 5s from the beginning of the Fenton reaction. Inhibition of ADP-ribose(P), nicotinamide and NAD(P)+ production of Fe2+-H2O2-treated NAD(P)H samples, was achieved by adding mannitol (10-50 mM) to the reaction mixture. Differently, selective and total inhibition of ADP-ribose(P) and nicotinamide formation was obtained by performing the Fenton reaction in an almost completely anhydrous medium, i.e. in HPLC-grade methanol. Experiments carried out in isolated postischemic rat hearts perfused with 50 mM mannitol, showed that, with respect to values of control hearts, this hydroxyl radical scavenger prevented reperfusion-associated pyridine coenzyme depletion and ADP-ribose formation. On the basis of these results, a possible mechanism of action of ADP-ribose(P) and nicotinamide generation through the interaction between NAD(P)H and hydroxyl radical (which does not involve the C-center where "conventional" oxidation occurs) is presented. The implication of this phenomenon in the pyridine coenzyme depletion observed in postischemic tissues is also discussed.

Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress.

To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5-10 mM) of hydrogen peroxide for 1 h at 37 degrees C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mM H2O2), oxidative stress produced, starting from 1 mM H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mM). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mM H2O2-treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mM H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress.

Ion-pairing high-performance liquid chromatographic method for the detection of N-acetylaspartate and N-acetylglutamate in cerebral tissue extracts.

An ion-pairing high-performance liquid chromatographic method for the determination of N-acetylaspartate and N-acetylglutamate using a C-18 column and a UV detection at 210 nm wavelength, by means of a diode array detector, is presented. A buffer containing 2.8 mM tetrabutylammonium hydroxide, 25 mM KH(2)PO(4), 1.25% methanol, pH 7. 00, is utilized for the isocratic separation of these N-acetylated amino acids, at a flow rate of 1 ml/min and a column temperature of 23 degrees C. The suitability of this chromatographic separation (without additional chromatographic steps prior to HPLC assay) to monitor variations both of N-acetylaspartate and of N-acetylglutamate in perchloric acid brain extracts from rats subjected to the impact acceleration model of diffuse brain injury is also reported. According to the data presented, this HPLC method allows the separation of the two N-acetylated amino acids considered from the many possible interfering compounds, commonly present in extracts of cerebral tissue, which have high extinction coefficients at 210 nm wavelength. Values of N-acetylaspartate and N-acetylglutamate determined by this method showed that cerebral trauma negatively affects both compounds, according to the severity of trauma itself.

Comparison of NAA measures by MRS and HPLC.

This work investigates the accuracy of an in vivo estimation of absolute N-acetyl aspartate (NAA) concentrations by magnetic resonance spectroscopy (MRS) using cerebral water as an internal reference standard. Single-voxel, proton spectroscopy was carried out in two groups of rats (normal and diffuse head injury), using a PRESS sequence with TR = 3 s, TE = 135 ms. Fully relaxed water spectra and water-suppressed proton spectra were obtained from a 7 x 5 x 5 mm3 volume of tissue. MRI-based brain water content measurements were also performed. Following MRS, HPLC determinations of NAA were carried out. In the normal rats the MRS yielded 10.98 +/- 0.83 mmol/kg w.w. vs 10.76 +/- 0.76 for HPLC with a mean absolute difference of 0.8. In the injured rats the corresponding results were 9.41 +/- 1.78 (MRS) and 8.16 +/- 0.77 (HPLC) with a mean absolute difference of 1.66. The in vivo absolute method accurately documented the temporal NAA changes compared to the NAA/Cr approach.

Changes of cerebral energy metabolism and lipid peroxidation in rats leading to mitochondrial dysfunction after diffuse brain injury.

The effect of mild closed head trauma, induced by the weight-drop method (450 g from a 1-m height), on lipid peroxidation and energy metabolism of brain tissue was determined at various times after cerebral injury in spontaneously breathing rats (1, 10, 30 minutes and 2, 6, 15, 24, 48, and 120 hours). Animals were continuously monitored for the evaluation of blood pressure, blood gases, heart rate, and intracranial pressure. Analysis of malondialdehyde (MDA) as an index of lipid peroxidation, ascorbic acid, high-energy phosphates, nicotinic coenzymes, oxypurines, and nucleosides was performed by high-performance liquid chromatography (HPLC) on neutralized perchloric acid extract of the whole brain. Data showed that MDA, undetectable in control, sham-operated rats, was already present within 1 minute of trauma (1.77 nmol/g wet weight; SD = 0.29) and reached maximal values by 2 hours (72.26 nmol/g w.w.; SD = 11.26), showing a progressive slow decrease thereafter. In contrast, ATP, GTP, and nicotinic coenzyme (NAD and NADP) concentrations showed significant reduction only by the second hour postinjury. Maximal decrease of the ATP and GTP concentrations were seen at 6 hours postinjury, whereas NAD and NADP concentrations showed maximum decline by 15 hours. Values recorded in mechanically ventilated rats did not differ significantly from those obtained in spontaneously breathing animals. These findings, supported by the absence of blood gas and blood pressure changes in the spontaneously breathing rats, strongly support the premise that biochemical changes (primarily lipid peroxidation) are not caused by secondary ischemic-hypoxic phenomena but rather are triggered by these forces acting on the brain at the time of impact. In addition, these results suggest that depression of energy metabolism might be caused by peroxidation of the mitochondrial membrane with a consequent alteration of the main mitochondrial function-that is, the energy supply.

Differential effects of acute morphine administrations on polymorphonuclear cell metabolism in various mouse strains.

This paper shows that an acute morphine treatment dose-dependently alters the energetic and oxidative metabolism of polymorphonuclear leukocytes obtained from BALB/c and DBA/2 mice, while phagocytic cells from C57BL/6 were not affected. In sensitive mouse strains, i.e. BALB/c and DBA/2, morphine decreased both ATP concentration and energy charge potential. At the same time, ATP catabolic products, i.e. nucleosides (inosine+adenosine) and oxypurines (hypoxanthine+xanthine+uric acid), significantly increased, indicating an imbalance between energy production and consumption. Morphine treatment also induced malondialdehyde and superoxide anions production in leukocyte cells from sensitive mice. The opiate antagonist naloxone blocked morphine-induced modifications by the lower morphine dose. The same parameters in cells from C57BL/6 mice were not affected. These findings confirm that: i) the phagocytic cells are an important target for the in vivo effects of morphine, and ii) the genotype-dependent variation influences the immunological responsiveness to opiates.

Clinical implications of HIV dynamics and drug resistance in macrophages.

Macrophages are widely recognized as the second major target of HIV in the body. The cellular characteristics of such resting cells markedly affect the dynamics of virus lifecycle, that is slower but far more prolonged that in lymphocytes. In addition, the limited concentrations of endogenous nucleotide pools in macrophages downregulate the enzymatic activity of reverse transcriptase. As a consequence, both the anti-HIV activity and the development of resistance to antiviral drugs in macrophages are substantially different than those found in activated lymphocytes. These peculiar characteristics of virus replication and efficacy of antiviral drugs in macrophages have a natural in vivo counterpart in extralymphoid tissues, where macrophages account for the majority of cells infected by HIV. Furthermore, the replication of HIV in macrophages of testis and central nervous system is far less affected by antiviral drugs than in lymph nodes, because of the presence of natural barriers that markedly diminish the concentration of such drugs. For all these reasons, HIV infection of macrophages should be taken into account in therapeutic strategies aimed to achieve an optimal therapeutic effect in all tissue compartments where the virus hides and replicates.

Lipid peroxidation, tissue necrosis, and metabolic and mechanical recovery of isolated reperfused rat heart as a function of increasing ischemia.

Isolated Langendorff-perfused rat hearts, after 30 min of preperfusion, were submitted to increasing times of global normothermic ischemia (1, 2, 5, 10, 20 and 30 min) or to the same times of ischemia followed by 30 min of reperfusion. Analysis of malondialdehyde, ascorbic acid, oxypurines, nucleosides, nicotinic coenzymes and high-energy phosphates was carried out by HPLC on neutralized perchloric acid extracts of freeze-clamped tissues. In addition, maximum rate of intraventricular pressure development and cardiac output of malondialdehyde, lactate dehydrogenase, oxypurines and nucleosides were monitored during both preperfusion and reperfusion. Besides decreasing energy metabolites and nicotinic coenzyme pool, prolonged ischemia produced oxidation of significant amounts of hypoxanthine and xanthine to uric acid and generation of detectable levels of malondialdehyde (0.002 micromol/g dry weight). After oxygen and substrate readmission, tissue and perfusate malondialdehyde increased only if previous ischemia was longer than 5 min, while lactate dehydrogenase was detected in perfusate of reperfused hearts following 10, 20, and 30 min of ischemia. Highest values of tissue malondialdehyde and total malondialdehyde output were recorded in reperfused hearts subjected to 30 min of ischemia (0.043 micromol/g dry weight and 0.069 micromol/30 min/g dry weight, respectively). Since tissue malondialdehyde was observed without detectable lactate dehydrogenase release in perfusate, it might be stated that malondialdehyde generation (i.e., lipid peroxidation) temporally preceded lactate dehydrogenase release (i.e., tissue necrosis). In reperfused hearts, evaluation of myocardial energy state and of mechanical recovery allowed us to determine times of ischemia beyond which reperfusion did not positively affect these metabolic and functional parameters. Main findings are that, under these experimental conditions, lipid peroxidation might be the cause and not the consequence of tissue necrosis and that duration of ischemia might be the factor deciding effectiveness of reperfusion.