Plumbagin can potently enhance the activity of xanthine oxidase: in vitro, in vivo and in silico studies.

BACKGROUND: Abnormally elevated xanthine oxidase (XO) activity has been verified to cause various pathological processes, such as gout, oxidative stress injury and metabolic syndrome. Thus, XO activators may exhibit above potential toxicological properties. Plumbagin (PLB) is an important active compound in traditional Chinese medicine (TCM), while its obvious toxic effects have been reported, including diarrhea, skin rashes and hepatic toxicity. However, the potential toxicity associated with enhancement of XO activity has not been fully illuminated so far. METHODS: The present study investigated the effect of PLB on XO activity by culturing mouse liver S9 (MLS9), human liver S9 (HLS9), XO monoenzyme system with PLB and xanthine. Then, the molecular docking and biolayer interferometry analysis were adopted to study the binding properties between PLB and XO. Finally, the in vivo acceleration effect also investigated by injected intraperitoneally PLB to KM mice for 3 days. RESULTS: PLB could obviously accelerate xanthine oxidation in the above three incubation systems. Both the Vmax values and intrinsic clearance values (CLint, Vmax/Km) of XO in the three incubation systems increased along with elevated PLB concentration. In addition, the molecular docking study and label-free biolayer interferometry assay displayed that PLB was well bound to XO. In addition, the in vivo results showed that PLB (2 and 10 mg/kg) significantly increased serum uric acid levels and enhanced serum XO activity in mice. CONCLUSION: In summary, this study outlines a potential source of toxicity for PLB due to the powerful enhancement of XO activity, which may provide the crucial reminding for the PLB-containing preparation development and clinical application.

Stored RBC metabolism as a function of caffeine levels.

BACKGROUND: Coffee consumption is extremely common in the United States. Coffee is rich with caffeine, a psychoactive, purinergic antagonist of adenosine receptors, which regulate red blood cell energy and redox metabolism. Since red blood cell (purine) metabolism is a critical component to the red cell storage lesion, here we set out to investigate whether caffeine levels correlated with alterations of energy and redox metabolism in stored red blood cells. STUDY DESIGN AND METHODS: We measured the levels of caffeine and its main metabolites in 599 samples from the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study via ultra-high-pressure-liquid chromatography coupled to high-resolution mass spectrometry and correlated them to global metabolomic and lipidomic analyses of RBCs stored for 10, 23, and 42 days. RESULTS: Caffeine levels positively correlated with increased levels of the main red cell antioxidant, glutathione, and its metabolic intermediates in glutathione-dependent detoxification pathways of oxidized lipids and sugar aldehydes. Caffeine levels were positively correlated with transamination products and substrates, tryptophan, and indole metabolites. Expectedly, since caffeine and its metabolites belong to the family of xanthine purines, all xanthine metabolites were significantly increased in the subjects with the highest levels of caffeine. However, high-energy phosphate compounds ATP and DPG were not affected by caffeine levels, despite decreases in glucose oxidation products-both via glycolysis and the pentose phosphate pathway. CONCLUSION: Though preliminary, this study is suggestive of a beneficial correlation between the caffeine levels and improved antioxidant capacity of stored red cells.

Caffeine metabolism during cultivation of oyster mushroom (Pleurotus ostreatus) with spent coffee grounds.

In coffee-producing countries, waste products from coffee production are useful substrates for cultivation of Pleurotus ostreatus. This species is relatively easy to grow, coffee waste substrates are readily available and the mushroom fruiting bodies are a valuable source of nutrition and income. In developed countries, cultivation of P. ostreatus on spent coffee grounds (SCG) from coffee consumption is a novel way to recycle this urban waste product. Here, we studied the effect of SCG and caffeine on growth of a commercial strain of P. ostreatus in liquid and solid cultures, and on a commercial scale. The presence of caffeine inhibited mycelial growth on agar and in liquid culture in the laboratory. Increased levels of SCG in an SCG/sawdust substrate also delayed mycelial growth and delayed or prevented fruiting during commercial cultivation. Despite growth inhibition, partial degradation of caffeine to xanthine by P. ostreatus mycelium was observed in all SCG-containing substrate mixtures. Degradation of caffeine proceeded mainly via sequential N-demethylation to theophylline (1,3-dimethylxanthine) and 3-methylxanthine, although both paraxanthine and theobromine also accumulated in the substrate. Caffeine and its demethylated metabolites were also detected in fruiting bodies, but it was not clear whether caffeine metabolism occurred in the fruiting bodies themselves or whether caffeine metabolites were translocated there from the mycelium. Based on the caffeine concentrations measured in fruiting bodies after growth with SCG, it would be necessary to consume ~ 250 kg of fresh oyster mushrooms to obtain the amount of caffeine equivalent to one cup of espresso coffee, suggesting that the health impact of caffeine in these mushrooms is low. However, the ability of P. ostreatus to degrade caffeine indicates that this and other species in this genus may have potential applications in detoxification of coffee production wastes.

Multi-omics identify xanthine as a pro-survival metabolite for nematodes with mitochondrial dysfunction.

Aberrant mitochondrial function contributes to the pathogenesis of various metabolic and chronic disorders. Inhibition of insulin/IGF-1 signaling (IIS) represents a promising avenue for the treatment of mitochondrial diseases, although many of the molecular mechanisms underlying this beneficial effect remain elusive. Using an unbiased multi-omics approach, we report here that IIS inhibition reduces protein synthesis and favors catabolism in mitochondrial deficient Caenorhabditis elegans We unveil that the lifespan extension does not occur through the restoration of mitochondrial respiration, but as a consequence of an ATP-saving metabolic rewiring that is associated with an evolutionarily conserved phosphoproteome landscape. Furthermore, we identify xanthine accumulation as a prominent downstream metabolic output of IIS inhibition. We provide evidence that supplementation of FDA-approved xanthine derivatives is sufficient to promote fitness and survival of nematodes carrying mitochondrial lesions. Together, our data describe previously unknown molecular components of a metabolic network that can extend the lifespan of short-lived mitochondrial mutant animals.

Different Catabolism Pathways Triggered by Various Methylxanthines in Caffeine-Tolerant Bacterium Pseudomonas putida CT25 Isolated from Tea Garden Soil.

The degradation efficiency and catabolism pathways of the different methylxanthines (MXs) in isolated caffeine-tolerant strain Pseudomonas putida CT25 were comprehensively studied. The results showed that the degradation efficiency of various MXs varied with the number and position of the methyl groups on the molecule (i.e., xanthine > 7-methylxanthine ≈ theobromine > caffeine > theophylline > 1-methylxanthine). Multiple MX catabolism pathways coexisted in strain CT25, and a different pathway would be triggered by various MXs. Demethylation dominated in the degradation of N-7-methylated MXs (such as 7-methylxanthine, theobromine, and caffeine), where C-8 oxidation was the major pathway in the catabolism of 1-methylxanthine, whereas demethylation and C-8 oxidation are likely both involved in the degradation of theophylline. Enzymes responsible for MX degradation were located inside the cell. Both cell culture and cell-free enzyme assays revealed that N-1 demethylation might be a rate-limiting step for the catabolism of the MXs. Surprisingly, accumulation of uric acid was observed in a cell-free reaction system, which might be attributed to the lack of activity of uricase, a cytochrome c-coupled membrane integral enzyme.

Metabolomic response to coffee consumption: application to a three-stage clinical trial.

BACKGROUND: Coffee is widely consumed and contains many bioactive compounds, any of which may impact pathways related to disease development. OBJECTIVE: To identify individual metabolite changes in response to coffee. METHODS: We profiled the metabolome of fasting serum samples collected from a previously reported single-blinded, three-stage clinical trial. Forty-seven habitual coffee consumers refrained from drinking coffee for 1 month, consumed four cups of coffee/day in the second month and eight cups/day in the third month. Samples collected after each coffee stage were subject to nontargeted metabolomic profiling using UPLC-ESI-MS/MS. A total of 733 metabolites were included for univariate and multivariate analyses. RESULTS: A total of 115 metabolites were significantly associated with coffee intake (P < 0.05 and Q < 0.05). Eighty-two were of known identity and mapped to one of 33 predefined biological pathways. We observed a significant enrichment of metabolite members of five pathways (P < 0.05): (i) xanthine metabolism: includes caffeine metabolites, (ii) benzoate metabolism: reflects polyphenol metabolite products of gut microbiota metabolism, (iii) steroid: novel but may reflect phytosterol content of coffee, (iv) fatty acid metabolism (acylcholine): novel link to coffee and (v) endocannabinoid: novel link to coffee. CONCLUSIONS: The novel metabolites and candidate pathways we have identified may provide new insight into the mechanisms by which coffee may be exerting its health effects.

Total glycosides of Paeony shows Neuroprotective effects against Semen Strychni-induced neurotoxicity by recovering secretion of hormones and improving brain energy metabolism.

In this study, we investigated the protective effect of total glycosides of paeony against Semen Strychni-induced neurotoxicity and discussed some probably mechanisms. Levels of estrone, estradiol, estriol and growth hormone in male rats' serum were determined by ELISA, levels of ATP and substances associated with energy metabolism in rats' brain were determined by HPLC and levels of progesterone was determined by a UPLC-MS/MS method. The results showed that neurotoxicity induced by Semen Strychni could cause a significant decrease (p < 0.05, compare to the blank group) in secretion of estrogens and GH and disorder brain energy metabolism at the same time. While, rats with total glycosides of paeony pre-protection (orally administrated with total glycosides of paeony for 15 days before administrating Semen Strychni extract) showed a much better condition in the secretion of hormones and brain energy metabolism, and showed no significant changes in most of those associated substances when comparing to the blank group. Our study indicated that total glycosides of paeony have neuroprotective effects on Semen Strychni-induced neurotoxicity. It could recover the disordered hormone secretion and improve the brain energy metabolism. Total glycosides of paeony is potential to be further used in clinic to protect against neurotoxicity induced by other reasons.

Protective Effects of Tinospora cordifolia on Hepatic and Gastrointestinal Toxicity Induced by Chronic and Moderate Alcoholism.

AIMS: Heavy alcohol intake depletes the plasma vitamins due to hepatotoxicity and decreased intestinal absorption. However, moderate alcohol intake is often thought to be healthy. Therefore, effects of chronic moderate alcohol intake on liver and intestine were studied using urinary vitamin levels. Furthermore, effects of Tinospora cordifolia water extract (TCE) (hepatoprotective) on vitamin excretion and intestinal absorption were also studied. METHODS: In the study, asymptomatic moderate alcoholics (n = 12) without chronic liver disease and healthy volunteers (n = 14) of mean age 39 ± 2.2 (mean ± SD) were selected and divided into three groups. TCE treatment was performed for 14 days. The blood and urine samples were collected on Day 0 and 14 after treatment with TCE and analyzed. RESULTS: In alcoholics samples, a significant increase in the levels of gamma-glutamyl transferase, aspartate transaminase, alanine transaminase, Triglyceride, Cholesterol, HDL and LDL (P < 0.05) was observed but their level get downregulated after TCE intervention. Multivariate analysis of metabolites without missing values showed an increased excretion of 7-dehydrocholesterol, orotic acid, pyridoxine, lipoamide and niacin and TCE intervention depleted their levels (P < 0.05). In contrast, excretion of biotin, xanthine, vitamin D2 and 2-O-p-coumaroyltartronic acid (CA, an internal marker of intestinal absorption) were observed to be decreased in alcoholic samples; however, TCE intervention restored the CA and biotin levels. Vitamin metabolism biomarkers, i.e. homocysteine and xanthurenic acid, were also normalized after TCE intervention. CONCLUSION: Overall data depict that moderate alcohol intake is also hepatotoxic and decreases intestinal absorption. However, TCE treatment effectively increased the intestinal absorption and retaining power of liver that regulated alcohol-induced multivitamin deficiency.

Metabolism of purine alkaloids and xanthine in leaves of maté (Ilex paraguariensis).

Accumulation and metabolism of purine alkaloids in leaves of maté (Ilex paraguariensis) were investigated. In winter, leaves accumulated caffeine but not theobromine, indicating that caffeine is the end product of purine alkaloid synthesis in maté. To elucidate the purine alkaloid metabolism in maté leaves, the metabolic fate of [8-(14)C]theobromine, [8-(14)C]theophylline, [8-(14)C]caffeine and [8-(14)C] xanthine was investigated in the leaf disks of young and mature leaves. In young maté leaves, significant amounts of theobromine and theophylline were utilized for caffeine biosynthesis, but the conversion was not observed in mature leaves. A small amount of theophylline was converted to theobromine. Practically no caffeine catabolism was detected in maté leaves during a 24 h-incubation. Catabolism of theobromine and theophylline via 3-methylxanthine was observed mainly in mature leaves. Xanthine was catabolised extensively via ureides in both young and mature leaves, but limited amounts are also utilized for the synthesis of theobromine, theophylline and caffeine. Possible pathways for the metabolism of purine alkaloids in maté leaves are discussed.

Expression of a xanthine permease and phosphate transporter in cultures and field populations of the harmful alga Aureococcus anophagefferens: tracking nutritional deficiency during brown tides.

Targeted gene expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to track patterns in the expression of genes indicative of nitrogen or phosphorus deficiency in the brown tide-forming alga Aureococcus anophagefferens. During culture experiments, a xanthine/uracil/vitamin C permease (XUV) was upregulated ∼20-fold under nitrogen-deficient conditions relative to a nitrogen-replete control and rapidly returned to nitrogen-replete levels after nitrogen-deficient cells were resupplied with nitrate or ammonium. It was not responsive to phosphorus deficiency. Expression of an inorganic phosphate transporter (PTA3) was enriched ∼10-fold under phosphorus-deficient conditions relative to a phosphorus-replete control, and this signal was rapidly lost upon phosphate resupply. PTA3 was not upregulated by nitrogen deficiency. Natural A. anophagefferens populations from a dense brown tide that occurred in Long Island, NY, in 2009 were assayed for XUV and PTA3 expression and compared with nutrient concentrations over the peak of a bloom. Patterns in XUV expression were consistent with nitrogen-replete growth, never reaching the values observed in N-deficient cultures. PTA3 expression was highest prior to peak bloom stages, reaching expression levels within the range of P-deficient cultures. These data highlight the value of molecular-level assessments of nutrient deficiency and suggest that phosphorus deficiency could play a role in the dynamics of destructive A. anophagefferens blooms.

Xanthine oxidase inhibition attenuates skeletal muscle signaling following acute exercise but does not impair mitochondrial adaptations to endurance training.

The aim of this research was to examine the impact of the xanthine oxidase (XO) inhibitor allopurinol on the skeletal muscle activation of cell signaling kinases' and adaptations to mitochondrial proteins and antioxidant enzymes following acute endurance exercise and endurance training. Male Sprague-Dawley rats performed either acute exercise (60 min of treadmill running, 27 m/min, 5% incline) or 6 wk of endurance training (5 days/wk) while receiving allopurinol or vehicle. Allopurinol treatment reduced XO activity to 5% of the basal levels (P < 0.05), with skeletal muscle uric acid levels being almost undetectable. Following acute exercise, skeletal muscle oxidized glutathione (GSSG) significantly increased in allopurinol- and vehicle-treated groups despite XO activity and uric acid levels being unaltered by acute exercise (P < 0.05). This suggests that the source of ROS was not from XO. Surprisingly, muscle GSSG levels were significantly increased following allopurinol treatment. Following acute exercise, allopurinol treatment prevented the increase in p38 MAPK and ERK phosphorylation and attenuated the increase in mitochondrial transcription factor A (mtTFA) mRNA (P < 0.05) but had no effect on the increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor-2, GLUT4, or superoxide dismutase mRNA. Allopurinol also had no impact on the endurance training-induced increases in PGC-1α, mtTFA, and mitochondrial proteins including cytochrome c, citrate synthase, and ß-hydroxyacyl-CoA dehydrogenase. In conclusion, although allopurinol inhibits cell signaling pathways in response to acute exercise, the inhibitory effects of allopurinol appear unrelated to exercise-induced ROS production by XO. Allopurinol also has little effect on increases in mitochondrial proteins following endurance training.

Cellular protection during oxidative stress: a potential role for D-ribose and antioxidants.

A healthy cellular system involves the maintenance of an intracellular metabolic balance. Reactive oxygen species (ROS) are constantly produced as a normal product of cellular metabolism; however, during situations of cellular stress, these levels can increase dramatically with the potential to cause deleterious cellular structural and/or functional consequences. There is a significant elevation in these ROS following stressful situations, such as ischemia, hypoxia, high-intensity exercise, and in many diseases. To combat these ROS, neutralizing endogenous enzymes, as well as exogenous antioxidants, can aid in minimizing their potential untoward cellular effects. Exogenous reducing antioxidant agents, such as vitamin C and/or E, play a role in addressing these formed species; however, recent research has suggested that fruit seed extracts may provide additional cellular benefits beyond their antioxidant features. Furthermore, supplemental D-ribose enhances the recovery of high-energy phosphates following stress and appears to potentially offer additional benefits by reducing radical formation. Specifically, during periods of hypoxia/ischemia, supplemental D-ribose may play an inhibitory role in the breakdown of adenine nucleotides, influencing the subsequent formation of xanthine and uric acid compounds; and thereby affecting the release of superoxide anion radicals. The combination of D-ribose with reducing antioxidants may provide a more optimal state of cellular protection during and following times of oxidative stress.

Polar extracts from (Tunisian) Acacia salicina Lindl. Study of the antimicrobial and antigenotoxic activities.

BACKGROUND: Methanolic, aqueous and Total Oligomer Flavonoids (TOF)-enriched extracts obtained from the leaves of Acacia salicina 'Lindl.' were investigated for antibacterial, antimutagenic and antioxidant activities. METHODS: The antimicrobial activity was tested on the Gram positive and Gram negative reference bacterial strains. The Mutagenic and antimutagenic activities against direct acting mutagens, methylmethane sulfonate (MMS) and 4-nitro-o-phenylenediamine (NOPD), and indirect acting mutagens, 2-aminoanthracene (2-AA) and benzo[a]pyrene (B(a)P) were performed with S. typhimurium TA102 and TA98 assay systems. In addition, the enzymatic and nonenzymatic methods were employed to evaluate the anti-oxidative effects of the tested extracts. RESULTS: A significant effect against the Gram positive and Gram negative reference bacterial strains was observed with all the extracts. The mutagenic and antimutagenic studies revealed that all the extracts decreased the mutagenicity induced by B(a)P (7.5 µg/plate), 2-AA (5 µg/plate), MMS (1.3 mg/plate) and NOPD (10 µg/plate). Likewise, all the extracts showed an important free radical scavenging activity towards the superoxide anion generated by the xanthine/xanthine oxidase assay system, as well as high Trolox Equivalent Antioxidant Capacity (TEAC), against the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)⁺• radical. TOF-enriched extract exhibited the highest protective effect against free radicals, direct acting-mutagen and metabolically activated S9-dependent mutagens. CONCLUSIONS: The present study indicates that the extracts from A. salicina leaves are a significant source of compounds with the antimutagenic and antioxidant activities, and this may be useful for developing potential chemopreventive substances.

Novel high-throughput assay for antioxidant capacity against superoxide anion.

A novel high-throughput assay for measuring antioxidant capacity against superoxide anion has been developed and validated. In this assay, hydroethidine (HE), a fluorescent probe, is oxidized by superoxide anion generated by xanthine and xanthine oxidase and increases its fluorescence intensity. Therefore, the inhibition of loss of HE's fluorescence intensity in the presence of antioxidant is an index of antioxidant capacity. The result is expressed as superoxide dismutase (SOD) equivalent. Unlike other probes, such as tetrazolium and lucigenin, one major advantage of this assay is that the use of HE is not prone to artifact. The method was rigorously validated through linearity, precision, accuracy, and ruggedness. The linear range, limit of quantitation (LOQ), and limit of detection (LOD) are 0.22-3.75 units/mL, 0.30 unit/mL, and 0.10 unit/mL, respectively. A wide variety of phenolic compounds and fruit extracts were analyzed.

Effect of Indian herbal hypoglycemic agents on antioxidant capacity and trace elements content in diabetic rats.

In the present investigation we report the protective potential of some herbal hypoglycemic agents on antioxidant status and levels of metal ions in streptozotocin-induced diabetic rats. Furthermore, in vitro antioxidant activity of the herbs was also evaluated. Induction of diabetes mellitus in rats caused an increase in blood lipid peroxide levels that was associated with the reduced activity of red blood cell (RBC) antioxidant enzymes--namely, superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase--along with depletion of plasma reduced glutathione (GSH) and copper, zinc, iron, magnesium, and selenium levels. Oral treatment of diabetic rats with Allium sativum, Azadirachta indica, Momordica charantia, and Ocimum sanctum extracts (500 mg/kg of body weight) not only lowered the blood glucose level but also inhibited the formation of lipid peroxides, reactivated the antioxidant enzymes, and restored levels of GSH and metals in the above-mentioned model. The herbal extracts (50-500 microg) inhibited the generation of superoxide anions (O(2)(-.)) in both enzymatic and nonenzymatic in vitro systems. These preparations also inhibited the ferrous-sodium ascorbate-induced formation of lipid peroxides in RBCs. The in vivo and in vitro protective effects of the above-mentioned herbal drugs were also compared with that of glibenclamide. On the basis of our results, we conclude that the above-mentioned herbal plants not only possess hypoglycemic properties, but they also decrease oxidative load in diabetes mellitus. Therefore, we propose that long-term use of such agents might help in the prevention of diabetes-associated complications. However, the extrapolation of these results to humans needs further in-depth study.

Renierol from marine sponge Haliclona.SP.: a natural inhibitor of xanthine oxidase with hypouricemic effects.

The purpose of this study was to evaluate the inhibitory effect of renierol, extracted from marine sponge Halicdona.SP., on xanthine oxidase (XO) and its hypouricemic effect in vivo. Renierol and a positive control, allopurinol, were tested for their effects on XO activity by measuring the formation of uric acid and superoxide radical from xanthine. Renierol inhibited XO in a concentration-dependent and competitive manner. IC(50) value was 1.85 microg.ml(-1) through the measuring of uric acid and was 1.36 microg.ml(- 1) through the measuring of superoxide radical. Renierol was found to have an in vivo hypouricemic activity against potassium oxonate-induced hyperuricaemia in mice. After oral administration of renierol at doses of 10, 20 and 30 mg.kg(- 1), there was a significant decrease in the serum urate level (4.08 +/- 0.09 mg.dl(- 1), P < 0.01), (3.47 +/- 0.11 mg.dl(- 1), P < 0.01) and (3.12 +/- 0.08 mg.dl(- 1), P < 0.01), when compared to the hyperuricaemic control (6.74 +/- 0.23 mg.dl(- 1)). Renierol was a potent XO inhibitor with hypouricemic activity in mice.

EPR studies on the superoxide-scavenging capacity of the nutraceutical resveratrol.

Resveratrol (3,4',5-trihydroxystilbene), a polyphenolic compound found in mulberries, grapes, and red wine, has received considerable attention because of its apparent protective effects against various degenerative diseases due to its potential antioxidant activities. However, direct evidence for the superoxide-scavenging capacity of resveratrol is lacking in literature. In this study, electron paramagnetic resonance spectroscopy in combination with 5-(diethoxyphosphoryl)-5-methylpyrroline-N-oxide (DEPMPO)-spin trapping technique was utilized to determine the ability of resveratrol in scavenging superoxide anions generated from both potassium superoxide and the xanthine oxidase/xanthine system. We have demonstrated here for the first time that the presence of resveratrol resulted in decreased formation of DEPMPO-superoxide adduct (DEPMPO-OOH) in both the potassium superoxide and xanthine oxidase/xanthine systems, indicating that resveratrol could directly scavenge superoxide anions. The inhibition of DEPMPO-OOH in the xanthine oxidase/xanthine system, however, was found to be much potent as compared to that observed in potassium superoxide system. It was further shown that resveratrol could also directly inhibit xanthine oxidase activity as assessed by oxygen consumption and formation of uric acid. Taken together, the dual role of resveratrol in directly scavenging superoxide and inhibiting its generation via xanthine oxidase reported in this study may explain, at least in part, the protective role of this compound against oxidative injury in various disease processes.

Administration of glutamine after hemorrhagic shock restores cellular energy, reduces cell apoptosis and damage, and increases survival.

BACKGROUND: Hemorrhagic shock causes a rapid depletion of adenosine triphosphate (ATP) and an increase of the terminal metabolite xanthine. Free radicals generated from xanthine oxidase play a major role in cell injury. Programmed cell death, apoptosis, is a major pathway causing reperfusion injury. During apoptosis, cytosolic cytochrome-c is released from damaged mitochondria, and it further initiates activation of apoptosis as evidenced by the appearance of caspase-3. The bcl-2 protein serves as an antiapoptosis found on the mitochondrial membrane. Glutamine has been known as a conditionally essential nutrient and seems to have beneficial effects in critically ill patients. The hypothesis of the present study is that glutamine administered during resuscitation following hemorrhagic shock would restore the depletion of hepatic ATP, reduce cellular apoptosis, and increase survival. METHODS: Male Sprague-Dawley rats were randomly assigned to 3 groups for resuscitation after the same pattern of hemorrhagic shock: Ringer's lactate (LR 21 ml/kg); Alanine-glycine (LR with alanine 0.15 gm/kg and glycine 0.18 gm/kg); and glutamine (LR with glutamine 0.3 gm/kg). Hepatic ATP and xanthine was measured at different time periods. Hepatic apoptosis was measured and the levels of cytosolic cytochrome-c, caspase-3 and bcl-2 were analyzed. Another group of rats were used for survival study. RESULTS: Glutamine administered during resuscitation following hemorrhagic shock partially restored the depletion of hepatic ATP, reduced cellular apoptosis, and increased survival. CONCLUSIONS: Glutamine administration during resuscitation significantly protected the liver from tissue damage caused by hemorrhagic shock. Glutamine supplementation may offer opportunities for therapeutic intervention during and after shock.

Profiles of purine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers.

To find general metabolic profiles of purine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, we looked at the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers. The activities of key enzymes in potato tuber extracts were also studied. Of the precursors for the intermediates in de novo purine biosynthesis, [(14)C]formate, [2-(14)C]glycine and [2-(14)C]5-aminoimidazole-4-carboxyamide ribonucleoside were metabolised to purine nucleotides and were incorporated into nucleic acids. The rates of uptake of purine ribo- and deoxyribonucleosides by the disks were in the following order: deoxyadenosine > adenosine > adenine > guanine > guanosine > deoxyguanosine > inosine > hypoxanthine > xanthine > xanthosine. The purine ribonucleosides, adenosine and guanosine, were salvaged exclusively to nucleotides, by adenosine kinase (EC 2.7.1.20) and inosine/guanosine kinase (EC 2.7.1.73) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Inosine was also salvaged by inosine/guanosine kinase, but to a lesser extent. In contrast, no xanthosine was salvaged. Deoxyadenosine and deoxyguanosine, was efficiently salvaged by deoxyadenosine kinase (EC 2.7.1.76) and deoxyguanosine kinase (EC 2.7.1.113) and/or non-specific nucleoside phosphotransferase (EC 2.7.1.77). Of the purine bases, adenine, guanine and hypoxanthine but not xanthine were salvaged for nucleotide synthesis. Since purine nucleoside phosphorylase (EC 2.4.2.1) activity was not detected, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) seem to play the major role in salvage of adenine, guanine and hypoxanthine. Xanthine was catabolised by the oxidative purine degradation pathway via allantoin. Activity of the purine-metabolising enzymes observed in other organisms, such as purine nucleoside phosphorylase (EC 2.4.2.1), xanthine phosphoribosyltransferase (EC 2.4.2.22), adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4) and guanine deaminase (EC 3.5.4.3), were not detected in potato tuber extracts. These results suggest that the major catabolic pathways of adenine and guanine nucleotides are AMP --> IMP --> inosine --> hypoxanthine --> xanthine and GMP --> guanosine --> xanthosine --> xanthine pathways, respectively. Catabolites before xanthosine and xanthine can be utilised in salvage pathways for nucleotide biosynthesis.

Prevention of cellular oxidative damage by an aqueous extract of Anoectochilus formosanus.

Anoectochilus formosanus (AF) is a popular folk medicine in Taiwan whose pharmacological effects have been characterized. In this work we investigated the antioxidant properties of an aqueous extract prepared from AF. The AF extract was capable of scavenging H2O2 in a dose-dependent manner. We induced oxidative stress in HL-60 cells, either by the addition of hydrogen peroxide (H2O2) or by the xanthine/xanthine oxidase reaction. Apoptosis caused by oxidative damage was displayed by DNA fragmentation on gel electrophoresis, and the apoptotic fraction was quantified with flow cytometry. The cell damage induced by oxidative stress was prevented by the plant extract in a concentration-dependent manner. Furthermore, the proteolytic cleavage of poly(ADP-ribose) polymerase during the apoptotic process was also inhibited by AF extract. Our results provide the basis for determining an AF extract to be an antioxidant.