Expressional regulation of key hepatic enzymes of intermediary metabolism in European seabass (Dicentrarchus labrax) during food deprivation and refeeding

We hypothesized that the analysis of mRNA level and activity of key enzymes in amino acid and carbohydrate metabolism in a feeding/fasting/refeeding setting could improve our understanding of how a carnivorous fish, like the European seabass (Dicentrarchus labrax), responds to changes in dietary intake at the hepatic level. To this end cDNA fragments encoding genes for cytosolic and mitochondrial alanine aminotransferase (cALT; mALT), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were cloned and sequenced. Measurement of mRNA levels through quantitative real-time PCR performed in livers of fasted seabass revealed a significant increase in cALT (8.5-fold induction)while promoting a drastic 45-fold down-regulation of PK in relation to the levels found in fed seabass. These observations were corroborated by enzyme activity meaning that during food deprivation an increase in the capacity of pyruvate generation happened via alanine to offset the reduction in pyruvate derived via glycolysis. After a 3-day refeeding period cALT returned to control levels while PK was not able to rebound. No alterations were detected in the expression levels of G6PDH while 6PGDH was revealed to be more sensitive specially to fasting, as confirmed by a significant 5.7-fold decrease in mRNA levels with no recovery after refeeding. Our results indicate that in early stages of refeeding, the liver prioritized the restoration of systemic normoglycemia and replenishment of hepatic glycogen. In a later stage, once regular feeding is re-established, dietary fuel may then be channeled to glycolysis and de novo lipogenesis.

Acid-base Balance and Oxidative Metabolism in Calcified Tissues

The calcified tissues, comprising bone and cartilage, are metabolically active tissues that bind and release calcium, bicarbonate and other substances according to systemic needs. Understanding the regulation of cellular metabolism in bone and cartilage is an important issue, since a link between the metabolism and diseases of these tissues is clear. An essential element in the function of bone-resorbing osteoclasts, namely regulation of bicarbonate transport, has not yet been thoroughly studied. Another example of an important but at the same time fairly unexplored subject of interest in this field is cartilage degeneration, an important determinant for development of osteoarthritis. The link between this and oxidative metabolism has rarely been studied. In this study, we have investigated the significance of bicarbonate transport in osteoclasts. We found that osteoclasts possess several potential proteins for bicarbonate transport, including carbonic anhydrase IV and XIV, and an electroneutral bicarbonate co-transporter NBCn1. We have also shown that inhibiting the function of these proteins has a significant impact on bone resorption and osteoclast morphology. Furthermore, we have explored oxidative metabolism in chondrocytes and found that carbonic anhydrase III (CA III), a protein linked to the prevention of protein oxidation in muscle cells, is also present in mouse chondrocytes, where its expression correlates with the presence of reactive oxygen species. Thus, our study provides novel information on the regulation of cellular metabolism in calcified tissues.

The Role of Cytochrome P450 3A Inducers and Inhibitors in the Metabolism and the Effects of Oxycodone

Oxycodone is an opioid used in the treatment of moderate or severe pain. It is principally metabolized in the liver by cytochrome P450 3A (CYP3A) enzymes whereas approximately 10% is metabolized by CYP2D6. Little is known about the interactions between oxycodone and other drugs, herbals and nutritional substances. In this work the effects of CYP3A inducers rifampicin and St. John’s wort and CYP3A inhibitors voriconazole, grapefruit juice, ritonavir and lopinavir/ritonavir were investigated on the pharmacokinetics and pharmacodynamics of oxycodone. All studies were randomized, balanced, placebo-controlled crossover clinical studies in healthy volunteers. The plasma concentrations of oxycodone and its metabolites were determined for 48 hours and pharmacodynamic parameters were recorded for 12 hours in each study. Pharmacokinetic parameters were calculated by noncompartmental methods. Rifampicin decreased the plasma concentrations, analgesic effects, and oral bioavailability of oral oxycodone. St. John’s wort reduced the concentrations of oxycodone and diminished the self-reported drug effect. Voriconazole increased the exposure to oral oxycodone by 3.6-fold whereas grapefruit juice, which inhibits predominantly the intestinal CYP3A, elevated the mean concentrations of oxycodone by 1.7-fold. Ritonavir and lopinavir/ritonavir increased the mean AUC of oxycodone by 3.0- and 2.6-fold, respectively, and prolonged its elimination half-life. In spite of increased oxycodone plasma concentrations during concomitant administration of CYP3A inhibitors, the analgesic effects were not increased. These studies show that the induction or inhibition of CYP3A alters the pharmacokinetics and pharmacologic effects of oxycodone. The exposure to oxycodone decreased after induction and increased after inhibition of CYP3A. As a conclusion, the clinicians should avoid concomitant administration of CYP3A inducers or inhibitors and oral oxycodone. If this is not possible, they should be prepared to interactions leading to impaired analgesia after CYP3A inducers or increased adverse effects after CYP3A inhibitors and oral oxycodone.

Berry polyphenol absorption and the effect of northern berries on metabolism, ectopic fat accumulation, and associated diseases

The prevalence of obesity and type 2 diabetes has increased at an alarming rate in developed countries. It seems in the light of current knowledge that metabolic syndrome may not develop at all without NAFLD, and NAFLD is estimated to be as common as metabolic syndrome in western population (23 % occurrence). Fat in the liver is called ectopic fat, which is triacylglycerols within the cells of non-adipose tissue. Serum alanine aminotransferase (ALT) values correlate positively with liver fat proportions, and increased activity of ALT predicts type 2 diabetes independently from obesity. Berries, high in natural bioactive compounds, have indicated the potential to reduce the risk of obesity-related diseases. Ectopic fat induces common endocrine excretion of adipose tissue resulting in the overproduction of inflammatory markers, which further induce insulin resistance by multiple mechanisms. Insulin resistance inducing hyperinsulinemia and lipolysis in adipocytes increases the concentration of free fatty acids and consequently causes further fat accumulation in hepatocytes. Polyphenolic fractions of berries have been shown to reverse inflammatory reaction cascades in in vitro and animal studies, and moreover to decrease ectopic fat accumulation. The aim of this thesis was to explore the role of northern berries in obesity-related diseases. The absorption and metabolism of selected berry polyphenols, flavonol glycosides and anthocyanins, was investigated in humans, and metabolites of the studied compounds were identified in plasma and urine samples (I, II). Further, the effects of berries on the risk factors of metabolic syndrome were studied in clinical intervention trials (III, IV), and the different fractions of sea buckthorn berry were tested for their ability to reduce postprandial glycemia and insulinemia after high-glucose meal in a postprandial study with humans (V). The marked impact of mixed berries on plasma ALT values (III), as well as indications of the positive effects of sea buckthorn, its fractions and bilberry on omental adiposity and adhesion molecules (IV) were observed. In study V, sea buckthorn and its polyphenol fractions had a promising effect on potprandial metabolism after high-glucose meal. In the literature review, the possible mechanisms behind the observed effects have been discussed with a special emphasis on ectopic fat accumulation. The literature review indicated that especially tannins and flavonoids have shown potential in suppressing diverse reaction cascades related to systemic inflammation, ectopic fat accumulation and insulin resistance development.

Physiological and biochemical characterization of the assai palm (Euterpe oleracea Mart.) during seed germination and seedling growth under aerobic and anaerobic conditions

Physiological and biochemical aspects of assai palm during seed germination and early seedling growth were investigated. Seeds collected from plants growing in flooded and upland forests were used to determine the influence of normoxic (aerobic) and anoxic (anaerobic) conditions in germination and the initial and average time of development in the roots and shoots. After 75 days, seedlings germinated under normoxia were transferred to trays and submitted to flooding. Seed reserves (lipids, proteins, soluble sugars and starch) were monitored for quiescent and germinated seeds maintained under normoxic and anoxic conditions, as well as after 5, 10 and 20 days of seedling growth. Alcohol dehydrogenase (ADH) activity was quantified in roots and leaves of seedlings without or with flooding (partial and total). Seeds were not able to germinate under anoxia. Different strategies of storage mobilization of lipids, proteins, soluble sugars and starch were observed in seeds of each environment. ADH activity was induced by anoxia, with the highest level observed in the leaves. This study showed that, under normoxic conditions, the best developmental performance of assai palm seeds, from flooded or upland forest areas, during germination was associated with primary metabolites mobilization and seedling flooding tolerance with increased ADH activity. We conclude that the assai palm is well adapted to the anoxic conditions provoked by flooding.

Anaerobic-aerobic treatment of swine wastewater in uasb and batch reactors in series

In this work it was evaluated the performance of two systems of swine wastewater treatment consisting of two-stage upflow anaerobic sludge blanket (UASB) reactors, with and without post-treatment in sequencing batch reactor (SBR), fed continuously, with aerobic phase. The UASB reactors in the first stage had 908 L in the sets I and II, and in the second stage 350 and 188 L, respectively. In the set II the post-treatment was performed in a SBR of 3,000 L. The hydraulic detention times in the anaerobic treatment systems were 100, 75 and 58 h in the set I; 87, 65 and 51 h in the set II; and 240 and 180 h in the SBR. The volumetric organic load applied in the first stage UASB reactors ranged from 6.9 to 12.6 g total COD (L d)-1 in the set I and 7.5 to 9.8 g total COD (L d)-1 in the set II. The average removal efficiencies of total COD, total phosphorus (Ptotal), and Kjeldahl and organic nitrogen (KN and Norg) in the anaerobic treatment systems were similar and reached maximum values of 97%, 64%, 68%, and 98%. In the SBR, the removal efficiencies of total COD and thermotolerant coliforms were up to 62 and 92% resulting, respectively, in effluent concentrations of 135 mg L-1 and 2x10(4)MPN (100 mL)-1. For Ptotal, total nitrogen (TN) and Norg, the average removal efficiencies in the SBR were up to 58, 25 and 73%, respectively.

Performance of an anaerobic-aerobic reactor and kinetic study of organic matter removal of cattle slaughterhouse effluent

This paper sought to evaluate the behavior of an upflow Anaerobic-Aerobic Fixed Bed Reactor (AAFBR) in the treatment of cattle slaughterhouse effluent and determine apparent kinetic constants of the organic matter removal. The AAFBR was operated with no recirculation (Phase I) and with 50% of effluent recirculation (Phase II), with θ of 11h and 8h. In terms of pH, bicarbonate alkalinity and volatile acids, the results indicated the reactor ability to maintain favorable conditions for the biological processes involved in the organic matter removal in both operational phases. The average removal efficiencies of organic matter along the reactor height, expressed in terms of raw COD, were 49% and 68% in Phase I and 54% and 86% in Phase II for θ of 11h and 8h, respectively. The results of the filtered COD indicated removal efficiency of 52% and k = 0.0857h-1 to θ of 11h and 42% and k = 0.0880h-1 to θ of 8h in the Phase I. In Phase II, the removal efficiencies were 59% and 51% to θ of 11h and 8h, with k = 0.1238h-1 and k = 0.1075 h-1, respectively. The first order kinetic model showed good adjustment and described adequately the kinetics of organic matter removal for θ of 11h, with r² equal to 0.9734 and 0.9591 to the Phases I and II, respectively.

Metabolism and gene polymorphisms of the folate pathway in Brazilian women with history of recurrent abortion

PURPOSE: To investigate the association between polymorphisms in genes that encode enzymes involved in folate- and vitamin B12-dependent homocysteine metabolism and recurrent spontaneous abortion (RSA).METHODS: We investigated the C677T and A1298C polymorphisms of the methylenetetrahydrofalate reductase gene (MTHFR), the A2756G polymorphism of the methionine synthase gene (MS) and the 844ins68 insertion of the cystathionine beta synthetase gene (CBS). The PCR technique followed by RFLP was used to assess the polymorphisms; the serum levels of homocysteine, vitamin B12 and folate were investigated by chemiluminescence. The EPI Info Software version 6.04 was used for statistical analysis. Parametric variables were compared by Student's t-test and nonparametric variables by the Wilcoxon rank sum test.RESULTS: The frequencies of gene polymorphisms in 89 women with a history of idiopathic recurrent miscarriage and 150 controls were 19.1 and 19.6% for the C677T, insertion, 20.8 and 26% for the A1298C insertion, 14.2 and 21.9% for the A2756G insertion, and 16.4 and 18% for the 844ins68 insertion, respectively. There were no significant differences between case and control groups in any of the gene polymorphisms investigated. However, the frequency of the 844ins68 insertion in the CBS gene was higher among women with a history of loss during the third trimester of pregnancy (p=0.003). Serum homocysteine, vitamin B12 and folate levels id not differ between the polymorphisms studied in the case and control groups. However, linear regression analysis showed a dependence of serum folate levels on the maintenance of tHcy levels.CONCLUSION: The investigated gene polymorphisms and serum homocysteine, vitamin B12 and folate levels were not associated with idiopathic recurrent miscarriage in the present study. Further investigations are needed in order to confirm the role of the CBS 844ins68 insertion in recurrent miscarriage.

Parenteral administration of vitamins A, D and E on the oxidative metabolism and function of polymorphonuclear leukocytes in swine

The weaning period of piglets is characterized by physiological alterations, such as decreased weight gain, increased reactive oxygen species (ROS) and increased serum cortisol levels with possible effects on the immune response. The effect of parenteral administration of vitamins A, D and E on production performance, oxidative metabolism, and the function of polymorphonuclear leukocytes (PMNLs) was assessed in piglets during the weaning period. The sample was comprised of 20 male piglets that were given an injectable ADE vitamin combination (135,000 IU vitamin A, 40,000 IU vitamin D and 40mg vitamin E/ animal) at 20 and 40 days of age. Weight gain, concentration of reduced glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD) and the microbicidal and phagocytic activity of PMNLs were assessed. No difference was observed in the average piglet weight during the study; however, a greater percentage of weight gain was observed after weaning in the treated group. The concentrations of GSH and SOD did not differ between groups, although lipid peroxidation was greater in the control group at 60 days of age. The investigated variables of oxidative metabolism were correlated as follows: -0.41 for GSH and MDA, -0.54 for GSH and SOD and 0.34 for MDA and SOD. The intensity of intracellular ROS production, the percentage of ROS-producing PMNLs and the intensity of phagocytosis by PMNLs did not differ between treatment groups. Administration of the injectable ADE combination improved the percentage of weight gain between 20 and 40 days of age, decreased oxidative stress at 60 days of age and did not influence the function of PMNLs in piglets.

On Glucose Metabolism in Patients with the m.3243A>G Mutation

Background: The m.3243A>G mutation in mitochondrial DNA is the most common cause for mitochondrial diabetes. In addition, unexpected deaths related to the m.3243A>G associate with encephalopathy and cardiomyopathy. Failing mitochondrial respiratory chain in neurons, myocytes and beta cells is considered to underlie the multiorgan manifestations of the m.3243A>G. Aims: The primary aim of the study was to characterize the organ-specific glucose metabolism in patients with m.3243A>G and secondly, to study patients with or without signs of diabetes, cardiomyopathy or encephalopathy. The insulin-stimulated glucose metabolism in brain, heart, skeletal muscle, adipose tissue and liver were measured with 2-deoxy-2-[18F]fluoro-α-D-glucose in 15 patients and 14 controls. Brain oxygen metabolism was assessed with [15O]oxygen and insulin secretion was modelled based on oral glucose tolerance test. Results: The glucose oxidation in brain was globally decreased in patients with or without clinical encephalopathy. The insulin-stimulated glucose influx to skeletal muscle and adipose tissue was decreased in patients with or without diabetes as the hepatic glucose metabolism was normal. Impaired beta cell function and myocardial glucose uptake were associated with the high m.3243A>G heteroplasmy. Conclusions: This cross-sectional study suggests that: 1) The ability of insulin to stimulate glucose metabolism in skeletal muscle and adipose tissue is weakened before the beta cell failure results in mitochondrial diabetes. 2) Glucose oxidation defect is detected in otherwise unaffected cerebral regions in patients with the m.3243A>G, thus it likely precedes the clinical encephalopathy. 3) Uneconomical glucose hypometabolism during hyperinsulinemia contributes to the cardiac vulnerability in patients with high m.3243A>G heteroplasmy

Effects of cytochrome P450 enzyme inhibitors and inducers on the metabolism of S-ketamine

The human body eliminates foreign compounds primarily by metabolizing them to hydrophilic forms to facilitate effective excretion through the kidneys. Cytochrome P450 (CYP) enzymes in the liver and intestine contribute to the metabolism of many drugs. Pharmacokinetic drugdrug interactions occur if the activity of CYPs are inhibited or induced by another drug. Prescribing multiple drugs to the improve effectiveness of therapy or to treat coexisting diseases is a common practice in clinical medicine. Polypharmacy predisposes patients to adverse effects because of the profound unpredictability in CYP enzymatic-mediated drug metabolism. S-ketamine is a phencyclidine derivative which functions as an antagonist of the N-methyl-Daspartate (NMDA) receptor in the central nervous system. It is a unique anaesthetic producing “dissociative anaesthesia” in high doses and analgesia in low doses. Studies with human liver microsomes suggest that ketamine is metabolized primarily via CYP3A4 and CYP2B6 enzymes. In this thesis, in healthy volunteers, randomized and controlled cross-over studies were conducted to investigate the effects of different CYP inducers and inhibitors on the pharmacokinetics and pharmacodynamics of oral and intravenous S-ketamine. The plasma concentrations of ketamine and its metabolite, norketamine, were determined at different timepoints over a 24 hour period. Other pharmacodynamic variables were examined for 12 hours. Results of these studies showed that the inhibition of the CYP3A4 pathway by clarithromycin or grapefruit juice increased the exposure to oral S-ketamine by 2.6- and 3.0-fold. Unexpectedly, CYP3A4 inhibition by itraconazole caused no significant alterations in the plasma concentrations of oral S-ketamine. CYP3A4 induction by St. John´s wort or rifampicin decreased profoundly the concentrations of oral S-ketamine. However, after rifampicin, there were no significant differences in the plasma concentrations of S-ketamine when it was administered intravenously. This demonstrated that rifampicin inhibited the metabolism of Sketamine at the intestinal level. When CYP2B6 was inhibited by ticlopidine, there was a 2.4- fold increase in the exposure of S-ketamine. These studies demonstrated that low dose oral Sketamine is metabolized both via CYP3A4 and CYP2B6 pathways. The concomitant use of drugs that affect CYP3A4 or CYP2B6, during oral S-ketamine treatment, may cause clinically significant drug-drug interactions.

Effect of exposure to sublethal concentrations of sodium cyanide on the carbohydrate metabolism of the Indian Major Carp Labeo rohita (Hamilton, 1822)

Experiments were designed to study in-vivo effects of sodium cyanide on biochemical endpoints in the freshwater fish Labeo rohita. Fish were exposed to two sublethal concentrations (0.106 and 0.064mg/L) for a period of 15 days. Levels of glycogen, pyruvate, lactate and the enzymatic activities of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH), phosphorylase, alkaline phosphatase (ALP), acid phosphatase (AcP) were assessed in different tissues (liver, muscle and gills). Result indicated a steady decrease in glycogen, pyruvate, SDH, ALP and AcP activity with a concomitant increase in the lactate, phosphorylase, LDH and G6PD activity in all selected tissues. The alterations in all the above biochemical parameters were significantly (p<0.05) time and dose dependent. In all the above parameters, liver pointing out the intensity of cyanide intoxication compare to muscle and gills. Study revealed change in the metabolic energy by means of altered metabolic profile of the fish. Further, these observations indicated that even sublethal concentrations of sodium cyanide might not be fully devoid of deleterious influence on metabolism in L. rohita.

Comparative uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid in callus cultures of monocot (Dioscorea spp.) and dicot (Nicotiana tabacum L.) plants

(Comparative uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid in callus cultures of monocot (Dioscorea spp.) and dicot (Nicotiana tabacum L.) plants). The uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid (2,4-D) were investigated in leaf calluses of Nicotiana tabacum, tuber calluses of Dioscorea opposita and calluses derived from zygotic embryos, leaves and petioles of Dioscorea composita. Striking similarities were evident in the patterns of 2,4-D metabolites and their chemical characteristics in the three callus types of D. composita compared, but significant differences were detected among the patterns of rnetabolites in the three species studied. Preliminary investigations on the stability of various metabolites (separated using TLC) by hydrolysis showed that sugar esters appeared to be the major metabolites in tobacco whilst in yams (D. opposita) glycosides were shown to be the main ones, which indicated a similarity between plants of Gramineae and Dioscoreaceae in terms of 2,4-D metabolism. Release of 2,4-D from tobacco callus cells upon their transfer to 2,4-D-free medium was detected and the implications of this are discussed in relation to the cultural conditions necessary to induce morphogenesis in vitro.