Similar Metabolic Patterns Research Articles

Glucose metabolic brain patterns to discriminate amyotrophic lateral sclerosis from Parkinson plus syndromes

Background18F-FDG brain PET measures metabolic changes in neurodegenerative disorders and may discriminate between different diseases even at an early stage. The objective of this study was to classify patients with amyotrophic lateral sclerosis (ALS) and Parkinson plus syndromes (PP). To this end, different approaches were evaluated using generalized linear models and corresponding glucose metabolic brain patterns. Besides direct classification, healthy controls were also included to generate disease-specific metabolic brain patterns and to perform a classification using disease expression scores.MethodsALS patients (n = 70) and PP patients (n = 33: 20 PSP, 3 CBD, and 10 MSA) were available from an existing database of patients with neuromuscular and movement disorders while age-matched healthy controls (n = 29) were selected from a prospective study. To generate both disease-discriminative (direct classification) and disease-specific (classification versus controls) metabolic brain patterns, data were spatially normalized and a principal component analysis (PCA) was performed prior to classification using either logistic regression (PCA-LR) or a support vector machine (PCA-SVM). Furthermore, a direct SVM approach was considered. To compare the three different approaches, Pearson correlations (r) between pattern expression scores and metabolic brain patterns were evaluated, while pairs of ALS- and PP-specific pattern expression scores were compared using the RV coefficient.ResultsClassification between ALS and PP resulted in a sensitivity and specificity ≥ 0.82 for both direct classification and classification according to disease-specific pattern expression scores. PCA-LR, PCA-SVM, and SVM generated very similar metabolic brain patterns with voxelwise correlations ≥ 0.66, while all patterns allowed straightforward identification of ALS- and PP-specific brain regions of hyper- and hypometabolism. Moreover, pattern expression scores were highly correlated among different classifiers with a mean r of 0.94 while a RV coefficient ≥ 0.91 was found between pairs of ALS- and PP-specific pattern expression scores.ConclusionWe demonstrated that a classification between ALS and PP using expression scores of an ALS and PP metabolic brain pattern leads to a similar and high prediction accuracy as direct classification between ALS and PP. Classification performance and disease-specific metabolic patterns, which could support visual reading and improve insight in brain pathology, were very related for different classifiers.

Gender-specific metabolic responses in gonad of mussel Perna viridis to triazophos

Triazophos, as a lipophilic organophosphate pesticide, displays higher bioaccumulation in the gonads of shellfish. To study the reproductive toxicity of triazophos, we applied metabolomics to characterize the gender-specific metabolic responses in mussel Perna viridis exposed to triazophos. Metabolites were differently altered by triazophos in ovaries of mussel at different concentrations and time intervals, while basically similar metabolic response patterns were observed in male mussels at the two tested concentrations after exposure for 24 and 48h. The significant changes of metabolites in ovaries of mussel exhibited the disturbances in energy metabolism and osmotic regulation, while in male samples triazophos only affected the energy metabolism. Moreover, glycine, sn-glycero-3-phosphocholine, ethanol, aspartate, etc. exhibited consistent variation tendency in both male and female individuals. While the changes of homarine, betaine, taurine, hypotaurine, malonate, β-alanine, succinate, and choline showed obviously gender-specific responses. Overall, this study confirmed the gender-specific responses in gonad of P. viridis to triazophos exposure.

Characteristics of cerebral glucose metabolism on 18F-FDG PET imaging in patients with Parkinson′s disease

Objective To validate the reproducibility of abnormal cerebral metabolic characteristics in PD patients from different medical centers using 18F-FDG PET imaging. Methods A total of 108 subjects who were referred for resting-state brain 18F-FDG PET imaging were retrospectively reviewed. Thirty-three PD patients (15 males, 18 females, age: 38-79 years) and 33 age-matched healthy controls (15 males, 18 females, age: 40-77 years) underwent evaluation at Shanghai Huashan Hospital Affiliated to Fudan University. Seventeen PD patients (10 males, 7 females, age: 44-74 years) and 17 age-matched healthy controls (6 males, 11 females, age: 42-67 years) underwent evaluation at the First Affiliated Hospital of Sun Yat-sen University. SPM was used to investigate the cerebral metabolic characteristics of the patients with two-sample t test. Statistically significant voxels were obtained by using familywise error rate (FWE; P<0.05). Two sets of PD patients with abnormal glucose metabolism of brain regions were obtained and the cerebral metabolic characteristics were compared. Results Regarding the PD patients from Shanghai Huashan Hospital, the features of cerebral glucose metabolism by SPM analysis were demonstrated as follows: increased metabolism was found in the region of pons, cerebellum, thalamus, putamen and pallidum, while decreased metabolism was displayed in the region of parietal lobe and occipital lobe. The increased regions referred to 8 110 voxels and decreased regions referred to 2 810 voxels (P<0.05). The similar metabolic pattern was found in PD patients from the First Affiliated Hospital of Sun Yat-sen University. The increased metabolism was shown in the regions of pons, cerebellum, thalamus, putamen and pallidum, and referred to 15 573 voxels. The metabolism-decreased regions included parietal lobe, occipital lobe and frontal lobe, and referred to 3 945 voxels (P<0.05). Conclusions 18F-FDG PET/CT imaging results demonstrate similar metabolic pattern in PD patients from different medical centers, in whom the metabolism-increased regions are found in the pons, cerebellum, thalamus and pallidum and decreased regions were demonstrated in the parietal lobe and occipital lobe. The reproducibility from 18F-FDG PET/CT imaging provides reliable evidence for the multi-center study in the differential diagnosis of PD. Key words: Parkinson disease; Brain; Metabolism; Positron-emission tomography; Deoxyglucose

P04.16 Multiparametric magnetic resonance imaging revealed tumor niches in glioblastoma patients

Abstract Heterogeneity poses challenge to personalised treatment of glioblastoma (GBM). Different habitats within GBM may have diverse treatment response. Magnetic resonance imaging (MRI) is crucial for targeted surgery. However, conventional imaging methods fail to target intratumor habitats. Finding validated imaging method to address GBM regional heterogeneity is crucial. Methods: Patients 112 patients (mean age 59.41 years, range 22–76, 84 males) with newly diagnosed supratentorial GBM were recruited. All patients received surgery and diagnosis was confirmed by pathology. Survival data of 79 patients were available for study. Exclusion criteria include history of cranial surgery/radiotherapy/chemotherapy. The study was approved by local Institutional Review Board. Anatomical, spectroscopy (MRS), dynamic susceptibility contrast (DSC) and diffusion tensor imaging (DTI) were acquired preoperatively with a 3T MRI scanner. All images were co-registered to T2W images. DTI, DSC and MRS data were processed as previously described. Imaging markers, i.e., apparent diffusion coefficient (ADC), relative cerebral blood volume (rCBV) and a series of metabolites were calculated. Region of interest (ROI) was manually delineated on the contrast-enhanced (CE) T1W and FLAIR images. ADC and rCBV values were obtained from each pixel within CE. Thresholds of 0.25 and 0.75 were used for both rCBV and ADC maps to generate four overlapping ADC-rCBV ROIs, which were low-ADC-high-rCBV (LH), low-ADC-low-rCBV (LL), high-ADC-high-rCBV (HH) and high-ADC-low-rCBV (HL) ROIs. The volumetric and metabolic analysis of all ROIs were performed. Results of volume and metabolites were correlated with patient survival. LH, HH, LL, and HL all displayed higher Choline/N-acetylaspartate ratio than normal appearing white matter (<0.001, <0.001, <0.001, <0.01). HL, mainly locating in the necrosis on T1 contrast images, showed higher lactate level than LH, HH, LL (<0.001, <0.001, <0.001) and higher macromolecule/lipid level than LH and HH (<0.05, <0.05). LL showed similar metabolic pattern with HL but may display enhancement on T1 contrast images. The volumes of LL and LH were inversely correlated (<0.001). The proportional volume of LH in CE were positively correlated with non-enhancing tumor volume in FLAIR (<0.01). Kaplan-Meier and multivariate survival analysis showed an increased volume ratio of LH/LL in CE was associated with worse progress free survival (p<0.05). Difference in cellularity and vascularity can create diverse tumor microenvironment. The LH, LL, HL and HH regions may represent the proliferative, hypoxic, necrotic and invasive tumor niches respectively. This approach shows potential in targeting intratumor niches. In next step we will take multiple biopsies and validate this approach at histological and genomic levels.

Metabolic Patterns of Fentanyl, Meperidine, Methylphenidate, Tapentadol and Tramadol Observed in Urine, Serum or Plasma

Drug testing is a useful tool to identify drug use or monitor adherence to prescription drugs. The interpretation of drug results can be complicated based on the pattern and proportional concentrations of drugs and/or drug metabolite(s). The purpose of this retrospective study was to detect the positivity rates and metabolic patterns of five prescription drugs, including fentanyl, meperidine, methylphenidate, tapentadol and tramadol. Retrospective data were retrieved from the laboratory information system in a national reference laboratory. Drug testing was performed using four mass spectrometry methods that were validated for clinical use. For urine specimens, the positivity rate was the highest for methylphenidate (62.3%, n = 2,489), followed by tramadol (43.7%, n = 3,483), fentanyl (41.9%, n = 4,657), tapentadol (37.9%, n = 736) and meperidine (8.3%, n = 138). Among positive samples, both parent drug and metabolite(s) was detectable in 94.9% of meperidine samples, 94.5% of tramadol samples, 93.8% of fentanyl samples, 89.9% of methylphenidate and 86.6% of tapentadol samples. For serum or plasma specimens, the positivity rate was the highest for tapentadol (75.0%, n = 39), followed by methylphenidate (74.2%, n = 569), fentanyl (53.6%, n = 113), meperidine (41.9%, n = 18) and tramadol (28.9%, n = 213). Similar metabolic patterns were found in serum or plasma. Of positive results, both parent drug and metabolite(s) were found in 94.7% of fentanyl samples, 83.3% of meperidine samples, 79.6% of methylphenidate samples, 53.8% of tapentadol samples and 44.1% of tramadol samples. Our data demonstrates the metabolic patterns of five drugs from a random urine or serum/plasma collection in patients that have been prescribed these medications. The data presented can be used to guide clinicians in determining drug adherence by assessing the positivity rates of the parent drug and corresponding metabolite(s).

Metabolic profiling during ex vivo machine perfusion of the human liver.

As donor organ shortages persist, functional machine perfusion is under investigation to improve preservation of the donor liver. The transplantation of donation after circulatory death (DCD) livers is limited by poor outcomes, but its application may be expanded by ex vivo repair and assessment of the organ before transplantation. Here we employed subnormothermic (21 °C) machine perfusion of discarded human livers combined with metabolomics to gain insight into metabolic recovery during machine perfusion. Improvements in energetic cofactors and redox shifts were observed, as well as reversal of ischemia-induced alterations in selected pathways, including lactate metabolism and increased TCA cycle intermediates. We next evaluated whether DCD livers with steatotic and severe ischemic injury could be discriminated from ‘transplantable’ DCD livers. Metabolomic profiling was able to cluster livers with similar metabolic patterns based on the degree of injury. Moreover, perfusion parameters combined with differences in metabolic factors suggest variable mechanisms that result in poor energy recovery in injured livers. We conclude that machine perfusion combined with metabolomics has significant potential as a clinical instrument for the assessment of preserved livers.

Rapid comparison of metabolic equivalence of standard medicinal parts from medicinal plants and their in vitro-generated adventitious roots using FT-IR spectroscopy

Myung Suk Ahn, Sung Ran Min, Eun Yee Jie, Eun Jin So, So Yeon Choi, Byeong Cheol Moon, Young Min Kang, So-Young Park, and Suk Weon Kim. J Plant Biotechnol 2015;42:257-64. https://doi.org/10.5010/JPB.2015.42.3.257

Lower brain 18F-fluorodeoxyglucose uptake but normal 11C-acetoacetate metabolism in mild Alzheimer's disease dementia.

The cerebral metabolic rate of glucose (CMRg) is lower in specific brain regions in Alzheimer's disease (AD). The ketones, acetoacetate and β-hydroxybutyrate, are the brain's main alternative energy substrates to glucose. To gain insight into brain fuel metabolism in mild AD dementia by determining whether the regional CMR and the rate constant of acetoacetate (CMRa and Ka, respectively) reflect the same metabolic deficit reported for cerebral glucose uptake (CMRg and Kg). Mild AD dementia (Mild AD; n = 10, age 76 y) patients were compared with gender- and age-matched cognitively normal older adults (Controls; n = 29, age 75 y) using a PET/MRI protocol and analyzed with both ROI- and voxel-based methods. ROI-based analysis showed 13% lower global CMRg in the gray matter of mild AD dementia versus Controls (34.2 ± 5.0 versus 38.3 ± 4.7 μmol/100 g/min, respectively; p = 0.015), with CMRg and Kg in the parietal cortex, posterior cingulate, and thalamus being the most affected (p ≤ 0.022). Neither global nor regional CMRa or Ka differed between the two groups (all p ≥ 0.188). Voxel-based analysis showed a similar metabolic pattern to ROI-based analysis with seven clusters of significantly lower CMRg in the mild AD dementia group (uncorrected p ≤ 0.005) but with no difference in CMRa. Regional brain energy substrate hypometabolism in mild AD dementia may be specific to impaired glucose uptake and/or utilization. This suggests a potential avenue for compensating brain energy deficit in AD dementia with ketones.

Functional pattern of brain FDG-PET in amyotrophic lateral sclerosis.

We investigated a large sample of patients with amyotrophic lateral sclerosis (ALS) at rest in order to assess the value of (18)F-2-fluoro-2-deoxy-d-glucose ((18)F-FDG) PET as a biomarker to discriminate patients from controls. A total of 195 patients with ALS and 40 controls underwent brain (18)F-FDG-PET, most within 5 months of diagnosis. Spinal and bulbar subgroups of ALS were also investigated. Twenty-five bilateral cortical and subcortical volumes of interest and cerebellum were taken into account, and (18)F-FDG uptakes were individually normalized by whole-brain values. Group analyses investigated the ALS-related metabolic changes. Discriminant analysis investigating sensitivity and specificity was performed using the 51 volumes of interest as well as age and sex. Metabolic connectivity was explored by voxel-wise interregional correlation analysis. Hypometabolism was found in frontal, motor, and occipital cortex and hypermetabolism in midbrain, temporal pole, and hippocampus in patients with ALS compared to controls. A similar metabolic pattern was also found in the 2 subgroups. Discriminant analysis showed a sensitivity of 95% and a specificity of 83% in separating patients from controls. Connectivity analysis found a highly significant positive correlation between midbrain and white matter in corticospinal tracts in patients with ALS. (18)F-FDG distribution changes in ALS showed a clear pattern of hypometabolism in frontal and occipital cortex and hypermetabolism in midbrain. The latter might be interpreted as the neurobiological correlate of diffuse subcortical gliosis. Discriminant analysis resulted in high sensitivity and specificity in differentiating patients with ALS from controls. Once validated by diseased-control studies, the present methodology might represent a potentially useful biomarker for ALS diagnosis. This study provides Class III evidence that (18)F-FDG-PET accurately distinguishes patients with ALS from normal controls (sensitivity 95.4%, specificity 82.5%).

Changes of microbial substrate metabolic patterns through a wastewater reuse process, including WWTP and SAT concerning depth

In this study, changes of microbial substrate metabolic patterns by BIOLOG assay were discussed through a sequential wastewater reuse process, which includes activated sludge and treated effluent in wastewater treatment plant and soil aquifer treatment (SAT), especially focussing on the surface sand layer in conjunction with the vadose zone, concerning sand depth. A SAT pilot-scale reactor, in which the height of packed sand was 237 cm (vadose zone: 17 cm and saturated zone 220 cm), was operated and fed continuously by discharged anaerobic–anoxic–oxic (A2O) treated water. Continuous water quality measurements over a period of 10 months indicated that the treatment performance of the reactor, such as 83.2% dissolved organic carbon removal, appeared to be stable. Core sampling was conducted for the surface sand to a 30 cm depth, and the sample was divided into six 5 cm sections. Microbial activities, as evaluated by fluorescein diacetate, sharply decreased with increasing distance from the surface of the 30 cm core sample, which included significant decreases only 5 cm from the top surface. A similar microbial metabolic pattern containing a high degree of carbohydrates was obtained among the activated sludge, A2O treated water (influent to the SAT reactor) and the 0–5 cm layer of sand. Meanwhile, the 10–30 cm sand core layers showed dramatically different metabolic patterns containing a high degree of carboxylic acid and esters, and it is possible that the metabolic pattern exhibited by the 5–10 cm layer is at a midpoint of the changing pattern. This suggests that the removal of different organic compounds by biodegradation would be expected to occur in the activated sludge and in the SAT sand layers immediately below 5 cm from the top surface. It is possible that changes in the composition of the organic matter and/or transit of the limiting factor for microbial activities from carbon to phosphorus might have contributed to the observed dramatic changes in SAT metabolic patterns.

Similar patterns of myocardial metabolism and perfusion in patients with type 2 diabetes and heart disease of ischaemic and non-ischaemic origin

Type 2 diabetes and insulin resistance are often associated with the co-occurrence of coronary atherosclerosis and cardiac dysfunction. The aim of this study was to define the independent relationships between left ventricular dysfunction or ischaemia and patterns of myocardial perfusion and metabolism in type 2 diabetes. Twenty-four type 2 diabetic patients--12 with coronary artery disease (CAD) and preserved left ventricular function and 12 with non-ischaemic heart failure (HF)--were enrolled in a cross-sectional study. Positron emission tomography (PET) was used to assess myocardial blood flow (MBF) at rest, after pharmacological stress and under euglycaemic hyperinsulinaemia. Insulin-mediated myocardial glucose disposal was determined with 2-deoxy-2-[(18)F]fluoroglucose PET. There was no difference in myocardial glucose uptake (MGU) between the healthy myocardium of CAD patients and the dysfunctional myocardium of HF patients. MGU was strongly influenced by levels of systemic insulin resistance in both groups (CAD, r = 0.85, p = 0.005; HF, r = 0.77, p = 0.01). In HF patients, there was an inverse association between MGU and the coronary flow reserve (r = -0.434, p = 0.0115). A similar relationship was observed in non-ischaemic segments of CAD patients. Hyperinsulinaemia increased MBF to a similar extent in the non-ischaemic myocardial of CAD and HF patients. In type 2 diabetes, similar metabolic and perfusion patterns can be detected in the non-ischaemic regions of CAD patients with normal cardiac function and in the dysfunctional non-ischaemic myocardium of HF patients. This suggests that insulin resistance, rather than diagnosis of ischaemia or left ventricular dysfunction, affects the metabolism and perfusion features of patients with type 2 diabetes.

Determination of Absolute Counts of Circulating Regulatory T Cells in Cynomolgus Macaques Using an Optimized Flow Cytometric Method

Regulatory T cells (Tregs) are a rare subset of lymphocytes that inhibit the activation and effector functions of T cells and are important regulators of immune responses. Although Tregs are well characterized in humans and rodents, little is known about their immunophenotyping (IP) profile in cynomolgus macaques (Macaca fascicularis), which is an important species for pharmacological and toxicological evaluation of potential immune modulators because of their similar physiologic, genetic, and metabolic response patterns to humans. The authors have developed an immunophenotyping panel using a high-throughput 96-well microtiter plate-based assay to detect circulating Tregs (CD3(+)CD4(+)CD25(hi)FoxP3(+)) and have determined the normal range for the number of Tregs in naive healthy cynomolgus macaques to be 56.4 to 179.7 cells/µL (mean ± SEM = 113.6 ± 5.1 cells/µL; n = 25). Furthermore, the authors compared the resulting FoxP3(+) Treg profiles with a CD127(lo) cell-surface panel (CD3(+)CD4(+)CD25(hi) CD127(lo)) and found a close correlation between the absolute numbers of CD3(+)CD4(+)CD25(hi)FoxP3(+) and CD3(+)CD4(+)CD25(hi)CD127(lo) cells (mean ± SD = 120 ± 8.0 cells/µL). Quantification of circulating Tregs in cynomolgus macaques in this high-throughput assay may help to identify drug candidates that affect this rare, but critical, immunoregulatory cell population.

Metabolomic phenotype of gastric cancer and precancerous stages based on gas chromatography time‐of‐flight mass spectrometry

To study the low-molecular-weight metabolites in blood plasma of patients with the progressive disease, gastric cancer, and to characterize different stages from chronic superficial gastritis (CSG) to chronic atrophic gastritis (CAG), intestinal metaplasia (IM), gastric dysplasia (DYS) and finally gastric cancer (GC). We applied gas chromatography time-of-flight mass spectrometry (GC/TOF-MS) to determine metabolites levels in plasma obtained from 80 patients including 19 with CSG, 13 with CAG, 10 with IM, 15 with DYS and 22 with GC (nine preoperation and 13 postoperation). Principal component analysis (PCA) and statistics were used to differentiate the stages and to identify the markers of gastric cancer. Totally, 223 peaks were detected in GC/TOF-MS and 72 compounds were authentically identified. CSG showed distinct difference from the other groups of CAG, IM, DYS and GC, whose plots clustered closely. IM clustered closely to GC, suggesting similar metabolic patterns of them. Fifteen identified metabolites contributed most to the differentiating between CSG and GC, and characterized different stages of GC. Statistics revealed elevated levels of 2-Hydroxybutyrate, pyroglutamate, glutamate, asparagine, azelaic acid, ornithine, urate, 11-eicosenoic acid, 1-monohexadecanoylglycerol and γ-tocopherol, while downregulation of creatinine, threonate in GC group, indicating that GC patients were obviously involved in oxidative stress, and perturbed metabolism of amino acids and fatty acids. The metabolic phenotype of CSG is significantly different from GC, while that of IM is similar to it. The discriminatory metabolites characterizing progressive stages from CSG to GC might be the potential markers to indicate a risk of GC.

Tissue Distribution and Characterization of Drug-Related Material in Rats and Dogs after Repeated Oral Administration of Casopitant

Casopitant [1-piperidinecarboxamide,4-(4-acetyl-1-piperazinyl)-N-((1R)-1-(3,5-bis(trifluoromethyl)phenyl)-ethyl)-2-(4-fluoro-2-methylphenyl)-N-methyl-(2R,4S)] has been shown to be a potent and selective antagonist of the human neurokinin 1 receptor, the primary receptor for substance P. During long-term toxicity studies conducted in rat and dog, evidence of cardiomyopathy and increased cardiac weight were observed. The distribution and metabolism of casopitant were studied in both species evaluating the accumulation of drug-related material (DRM) after repeat dosing and its potential relationship with pathological findings observed in myocardium. After repeat oral administration of [(14)C]casopitant to rats (20 days) and dogs (14 days), DRM was quantifiable in all of the tissues examined with lung and liver containing the highest level of radioactivity. The concentration of radioactivity was significantly higher in tissues than in plasma, declining slowly and still quantifiable after a recovery period of 20 days. The principal circulating components identified in both species were casopitant, M12 (oxidized deacetylated), M13 (hydroxylated piperazine), and M31 and M134 (two N-dealkylated piperazines). In tissues, a similar metabolic pattern was observed, in which casopitant, M31, M134, M76 (N-deacetylated), and M200 (N-deacetylated N,N-deethylated) were the major components quantified. After a 26-week repeat dose study in dog, casopitant and M13 were the major circulating components, whereas in myocardium, M200 and M134 were the major ones and their levels increased over time, reaching considerable concentrations (millimolar magnitude). After a washout period, all circulating derivatives decreased to undetectable levels, whereas M200 was still the major component in myocardium. Overall DRM in plasma did not correlate with the respective concentrations in tissues.

Selective enumeration strategies for Brevundimonas diminuta from drinking water

Brevundimonas diminuta is used as a control organism for validating the efficiency of water filtration systems. Since these protocols use nonselective growth media, heterotrophic plate count bacteria (HPCs) indigenous to the water distribution system may interfere with B. diminuta enumeration, thus leading to inaccurate assessment of the filter's microbial reduction capability. This could negatively impact public health as unsafe drinking water may be produced. This study was conducted to evaluate different potential routes for selective enumeration of B. diminuta in drinking water. B. diminuta's biochemical and molecular relationships to HPCs recovered from a laboratory drinking-water system were investigated. Of the 24 HPC morphotypes recovered, members of the Alpha- and Betaproteobacteria were most commonly identified. Based on comparisons of catabolic profiles (generated by the Biolog system) using principal component analysis, B. diminuta possessed similar metabolic patterns to several of the Alphaproteobacteria (Sphingomonas and Caulobacter), indicating that development of a selective medium based solely on carbon source was not feasible. Antibiotic susceptibility profiles revealed that the HPCs were least resistant to kanamycin, making it a candidate for future selective applications. Sequence comparisons of partial 16S rRNA sequences did not reveal any distinct similarities. However, basic local alignment search tool (BLAST) alignments of the gyrB and rpoD sequences for B. diminuta did show uniqueness, with the next closest match being to Caulobacter (88% and 79% similarity, respectively). Future investigation will focus on applying molecular assays, such as fluorescent in situ hybridization and quantitative real-time polymerase chain reaction (PCR), and incorporating an antibiotic marker or expressed fluorescent protein into the wild-type strain of B. diminuta for selective enumeration of B. diminuta.

Preclinical pharmacokinetics and interspecies scaling of ragaglitazar, a novel biliary excreted PPAR dual activator

Allometric scaling has been used as an effective tool for the prediction of human pharmacokinetic parameters. Allometry has been a useful approach for the analysis of compounds that are eliminated unchanged in the urine and/or exhibit similar metabolic patterns across species. However, it has been a challenging issue to correctly predict human pharmacokinetic parameters for drugs that are eliminated intact and/or as conjugates in the bile. Ragaglitazar is a novel, non-thiazolidinedione peroxisome proliferator-activated receptor (PPAR) alpha- and gamma-agonist. In our investigation, preclinical pharmacokinetic data on ragaglitazar were gathered for several animal species (mice, rats, rabbits and dogs). Ragaglitazar when administered orally has shown a low clearance rate (Cl/F; < 5% of hepatic blood flow) in mice, rats and rabbits and a moderately high Cl/F in dogs (> 15% of hepatic blood flow). A qualitative estimation of rat bile has unequivocally confirmed the elimination of ragaglitazar in the bile. The human pharmacokinetic data are also indicative of the involvement of enterohepatic biliary recycling. In order to predict key parameters such as Cl/F and volume of distribution (V/F), simple allometry was the approach adopted at the onset. Although V/F scaled adequately, it failed to accurately predict human Cl/F. Therefore, standard correction factors such as maximum life span potential (MLP) and brain weight were also included. Although such modifications improved the linearity (r2 > 0.9), they failed to predict the investigated values. Further incorporation of correction factors particularly relevant to biliary excreted drugs improved the prediction of these values. Interestingly, the exclusion of dog data from the interspecies scaling considerably improved the prediction of both Cl/F and V/F.

Treatment of rats with the Pelargonium sidoides extract EPs® 7630 has no effect on blood coagulation parameters or on the pharmacokinetics of warfarin

Umckaloabo is a herbal drug for the treatment of respiratory tract infections. It contains an aqueous ethanolic extract from roots of Pelargonium sidoides DC (EPs) 7630) as the active ingredient. Polymeric polyphenols and coumarins have been identified as the principal ingredients of EPs 7630. In view of the coumarin content, it has been suggested that the administration of Umckaloabo could possibly be associated with an increased risk of bleeding. This study, therefore, investigated whether a change in blood coagulation parameters or an interaction with coumarin-type anticoagulants occurred after administration of EPs 7630 to rats. No effect on thromboplastin time (TPT), partial TPT (PTPT) or thrombin time (TT) was observed after oral administration of EPs 7630 (10, 75, 500 mg/kg) for 2 weeks, while treatment with warfarin (0.05 mg/kg) for the same period resulted in significant changes in TPT and PTPT. If EPs 7630 (500 mg/kg) and warfarin (0.05 mg/kg) were given concomitantly, the anticoagulant action of warfarin was not influenced. Similarly, the pharmacokinetics of warfarin were unchanged after pre-treatment with EPs 7630 for 2 weeks. Coumarin-type anticoagulants inhibit the synthesis of vitamin K-dependent coagulation factors via an identical mechanism in rat and man, and have a similar pattern of metabolism in both species. Moreover, as the coumarins so far identified in EPs 7630 do not posses the structural characteristics needed for anticoagulant activity, it appears unlikely that an increased tendency to haemorrhage arises in patients after intake of Umckaloabo.

Metabolic activation of zebularine, a novel DNA methylation inhibitor, in human bladder carcinoma cells: Ben-Kasus T, Ben-Zvi Z, Marquez VE, Kelley JA, Agbaria R, Department of Clinical Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Zebularine (2(1H)-pyrimidinone riboside, Zeb), a synthetic analogue of cytidine that is a potent inhibitor of cytidine deaminase, has been recently identified as a general inhibitor of DNA methylation. This inhibition of DNA methyltransferase (DNMT) is hypothesized to be mechanism-based and result from formation of a covalent complex between the enzyme and zebularine-substituted DNA. Metabolic activation of Zeb thus requires that it be phosphorylated and incorporated into DNA. We have quantitatively assessed the phosphorylation and DNA incorporation of Zeb in T24 cells using 2-[(14)C]-Zeb in conjunction with gradient anion-exchange HPLC and selected enzymatic and spectroscopic analyses. The corresponding 5′-mono-, di- and triphosphates of Zeb were readily formed in a dose- and time-dependent manner. Two additional Zeb-containing metabolites were tentatively identified as diphosphocholine (Zeb-DP-Chol) and diphosphoethanolamine adducts. Intracellular concentrations of Zeb-TP and Zeb-DP-Chol were similar and greatly exceeded those of other metabolites. DNA incorporation occurred but was surpassed by that of RNA by at least seven-fold. Equivalent levels and similar intracellular metabolic patterns were also observed in the Molt-4 (human T-lymphoblasts) and MC38 (murine colon carcinoma) cell lines. For male BALB/c nu/nu mice implanted s.c. with the EJ6 variant of T24 bladder carcinoma and treated i.p. with 500mg/kg 2-[(14)C]-Zeb, the in vivo phosphorylation pattern of Zeb in tumor tissue examined 24h after drug administration was similar to that observed in vitro. The complex metabolism of Zeb and its limited DNA incorporation suggest that these are the reasons why it is less potent than either 5-azacytidine or 5-aza-2′-deoxycytidine and requires higher doses for equivalent inhibition of DNMT.

Effects of Sodium Valproate on Renal Functions in Rats

In recent years, it has been reported that sodium valproate occasionally can cause renal tubular impairment. This study was designed to demonstrate the renal tubular and glomerular functions in rats given sodium valproate as monotherapy, as well as to determine any reversibility of dysfunctions. Female rats were randomly allocated to three groups: group 1 received sodium valproate 500 mg/kg/d intraperitoneal for six weeks; after the same injection period, group 2 was housed for another six weeks, after which laboratory investigations were completed; and group 3 served as a control group made up of 20 healthy rats living in same condition without any treatment. Serum ALT, total protein, uric acid, ALP, phosphorus, sodium levels, and urine Ca/cr ratio were significantly different between groups 1 and 3 (p < 0.025), but this difference was not seen between groups 2 and 3. On the other hand, other parameters such as TRP, Ccr, NAG, and MDA were not significantly different among the three groups ( p > 0.025) These results suggest that SV does not have a significant dose- or time-related side effect on renal functions. Minor biochemical dysfunctions related to long-term sodium valproate therapy is reversible, and the minimal renal fibrosis that showed histopathologically is not clinically important. The renal tissues of rats are known to show similar metabolic and histological patterns with human renal tissues. No renal dysfunction was expected in humans because there were no clinically statistically significant renal side effects in this study.

Metabolic activation of zebularine, a novel DNA methylation inhibitor, in human bladder carcinoma cells

Zebularine (2(1H)-pyrimidinone riboside, Zeb), a synthetic analogue of cytidine that is a potent inhibitor of cytidine deaminase, has been recently identified as a general inhibitor of DNA methylation. This inhibition of DNA methyltransferase (DNMT) is hypothesized to be mechanism-based and result from formation of a covalent complex between the enzyme and zebularine-substituted DNA. Metabolic activation of Zeb thus requires that it be phosphorylated and incorporated into DNA. We have quantitatively assessed the phosphorylation and DNA incorporation of Zeb in T24 cells using 2-[ 14C]-Zeb in conjunction with gradient anion-exchange HPLC and selected enzymatic and spectroscopic analyses. The corresponding 5′-mono-, di- and triphosphates of Zeb were readily formed in a dose- and time-dependent manner. Two additional Zeb-containing metabolites were tentatively identified as diphosphocholine (Zeb-DP-Chol) and diphosphoethanolamine adducts. Intracellular concentrations of Zeb-TP and Zeb-DP-Chol were similar and greatly exceeded those of other metabolites. DNA incorporation occurred but was surpassed by that of RNA by at least seven-fold. Equivalent levels and similar intracellular metabolic patterns were also observed in the Molt-4 (human T-lymphoblasts) and MC38 (murine colon carcinoma) cell lines. For male BALB/c nu/ nu mice implanted s.c. with the EJ6 variant of T24 bladder carcinoma and treated i.p. with 500 mg/kg 2-[ 14C]-Zeb, the in vivo phosphorylation pattern of Zeb in tumor tissue examined 24 h after drug administration was similar to that observed in vitro. The complex metabolism of Zeb and its limited DNA incorporation suggest that these are the reasons why it is less potent than either 5-azacytidine or 5-aza-2′-deoxycytidine and requires higher doses for equivalent inhibition of DNMT.