The 13C-bicarbonate technique as a tool for measurement of energy expenditure in overweight dogs undergoing body weight reduction and the effect of different dietary composition.

Changes in body size and composition, i.e., body weight (BW) gain or loss, affect the daily energy expenditure (EE). To ensure an appropriate BW reduction and to find an efficient strategy to reduce and maintain a target BW, regular evaluations and adjustments of energy allowance are important. This study aimed to provide a detailed knowledge about the possible changes in resting EE using the oral 13C-bicarbonate technique (o13CBT) as a research tool in 16 overweight pet dogs undergoing BW reduction. Dietary composition (i.e., in % of dry matter [DM] being a high protein [33.3], low fat [9.6], and high crude fiber [18.0] diet [LFHFibre], and a high protein [37.9], high fat [52.0], carbohydrate-free diet [HFat]) during 16 wk of energy restriction were evaluated regarding effects on resting EE, rate of BW reduction, body composition, and plasma concentrations of metabolic hormones involved in energy metabolism and appetite regulation. The mean BW loss was higher (P < 0.05) for the dogs fed the LFHFibre diet (1.1%/wk) than that for dogs fed the HFat diet (0.8%/wk), but the total BW reduction of 14.6% and 12.0% of initial BW did not differ significantly (P > 0.05). Resting EE was lower (P < 0.02) after the BW reduction; 414 kJ (99 kcal)/kg BW0.75/d at the start (week 0) and 326 kJ (78 kcal)/kg BW0.75/d at the end (week 16) of the study. The BW reduction in both groups (P > 0.05) consisted of both fat mass (FM) and fat-free mass (FFM). Energy expenditure, calculated in relation to amount of FFM, was not significantly (P > 0.05) affected by BW reduction. Dietary composition did not significantly affect (P > 0.05) plasma concentrations of insulin, leptin, and ghrelin, and no effect (P > 0.05) of BW reduction was observed on hormone concentrations. In conclusion, the o13CBT proved to be a useful research method for studying short-term EE in overweight dogs. Even though all dogs lost BW, most dogs were still overweight at the end of the study. Due to a high individual variation among dogs, a longer experimental period with a larger sample size would be desirable.

The most common nutritional disorder in dogs is overweight, and knowledge about dogs' energy requirement is therefore important to adjust daily feed allowance. Changes in body weight may affect energy expenditure (EE) and, thereby, energy requirement. This study aimed to measure such potential changes under resting conditions in overweight dogs. It was found that the minimally invasive 13C-bicarbonate technique was a useful research method for studies regarding EE during weight loss (WL) in dogs. EE decreased when the dogs lost weight, and energy allowance needed to be reduced to maintain WL. The second objective of this study was to evaluate the effects of feeding diets with different macronutrient compositions on EE, rate of WL, body composition, and plasma concentrations of hormones involved in energy metabolism and appetite regulation. The mean WL rate was slightly higher for dogs fed a diet with high protein, low fat, and high crude fiber contents than those fed a carbohydrate-free diet with a high protein and fat contents. However, diet did not affect the resting EE, measured plasma hormone concentrations, or the total WL at the end of the study.

Metabolic turnover and dynamics of modified ribonucleosides by 13C labeling.

Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications are poorly understood. Here, we developed a 13C labeling approach, called 13C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleoside samples and showed the distinct kinetics of the N6-methyladenosine (m6A) versus 7-methylguanosine (m7G) modification in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m62A) exhibits distinct turnover in small RNAs and free ribonucleosides when compared to known m62A-modified large rRNAs. Finally, combined measurements of turnover and abundance of these modifications informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, 13C-dynamods enables studies of the origin of modified RNAs at steady-state and subsequent dynamics under nonstationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs.

Source of 12C in Calvin-Benson cycle intermediates and isoprene emitted from plant leaves fed with 13CO2.

Feeding 14CO2 was crucial to uncovering the path of carbon in photosynthesis. Feeding 13CO2 to photosynthesizing leaves emitting isoprene has been used to develop hypotheses about the sources of carbon for the methylerythritol 4-phosphate pathway, which makes the precursors for terpene synthesis in chloroplasts and bacteria. Both photosynthesis and isoprene studies found that products label very quickly (<10 min) up to 80-90% but the last 10-20% of labeling requires hours indicating a source of 12C during photosynthesis and isoprene emission. Furthermore, studies with isoprene showed that the proportion of slow label could vary significantly. This was interpreted as a variable contribution of carbon from sources other than the Calvin-Benson cycle (CBC) feeding the methylerythritol 4-phosphate pathway. Here, we measured the degree of label in isoprene and photosynthetic metabolites 20 min after beginning to feed 13CO2. Isoprene labeling was the same as labeling of photosynthesis intermediates. High temperature reduced the label in isoprene and photosynthesis intermediates by the same amount indicating no role for alternative carbon sources for isoprene. A model assuming glucose, fructose, and/or sucrose reenters the CBC as ribulose 5-phosphate through a cytosolic shunt involving glucose 6-phosphate dehydrogenase was consistent with the observations.

Mitochondrial division inhibitor (mdivi-1) decreases oxidative metabolism in cancer.

BACKGROUND: Previous studies suggested that mdivi-1 (mitochondrial division inhibitor), a putative inhibitor of dynamin-related protein (DRP1), decreased cancer cell proliferation through inducing mitochondrial fusion and altering oxygen consumption. However, the metabolic reprogramming underlying the DRP1 inhibition is still unclear in cancer cells. METHODS: To better understand the metabolic effect of DRP1 inhibition, [U-13C]glucose isotope tracing was employed to assess mdivi-1 effects in several cancer cell lines, DRP1-WT (wild-type) and DRP1-KO (knockout) H460 lung cancer cells and mouse embryonic fibroblasts (MEFs). RESULTS: Mitochondrial staining confirmed that mdivi-1 treatment and DRP1 deficiency induced mitochondrial fusion. Surprisingly, metabolic isotope tracing found that mdivi-1 decreased mitochondrial oxidative metabolism in the lung cancer cell lines H460, A549 and the colon cancer cell line HCT116. [U-13C]glucose tracing studies also showed that the TCA cycle intermediates had significantly lower enrichment in mdivi-1-treated cells. In comparison, DRP1-WT and DRP1-KO H460 cells had similar oxidative metabolism, which was decreased by mdivi-1 treatment. Furthermore, mdivi-1-mediated effects on oxidative metabolism were independent of mitochondrial fusion. CONCLUSIONS: Our data suggest that, in cancer cells, mdivi-1, a putative inhibitor of DRP1, decreases oxidative metabolism to impair cell proliferation.

Simultaneous tracers and a unified model of positional and mass isotopomers for quantification of metabolic flux in liver.

Computational models based on the metabolism of stable isotope tracers can yield valuable insight into the metabolic basis of disease. The complexity of these models is limited by the number of tracers and the ability to characterize tracer labeling in downstream metabolites. NMR spectroscopy is ideal for multiple tracer experiments since it precisely detects the position of tracer nuclei in molecules, but it lacks sensitivity for detecting low-concentration metabolites. GC-MS detects stable isotope mass enrichment in low-concentration metabolites, but lacks nuclei and positional specificity. We performed liver perfusions and in vivo infusions of 2H and 13C tracers, yielding complex glucose isotopomers that were assigned by NMR and fit to a newly developed metabolic model. Fluxes regressed from 2H and 13C NMR positional isotopomer enrichments served to validate GC-MS-based flux estimates obtained from the same experimental samples. NMR-derived fluxes were largely recapitulated by modeling the mass isotopomer distributions of six glucose fragment ions measured by GC-MS. Modest differences related to limited fragmentation coverage of glucose C1-C3 were identified, but fluxes such as gluconeogenesis, glycogenolysis, cataplerosis and TCA cycle flux were tightly correlated between the methods. Most importantly, modeling of GC-MS data could assign fluxes in primary mouse hepatocytes, an experiment that is impractical by 2H or 13C NMR.

A framework for tracer-based metabolism in mammalian cells by NMR.

Metabolism changes extensively during the normal proliferation and differentiation of mammalian cells, and in cancer and inflammatory diseases. Since changes in the metabolic network reflect interactions between genetic, epigenetic and environmental changes, it is helpful to study the flow of label from isotopically labelled precursors into other metabolites rather than static metabolite levels. For this Nuclear Magnetic Resonance (NMR) spectroscopy is an attractive technique as it can quantify site-specific label incorporation. However, for applications using human cells and cell lines, the challenge is to optimize the process to maximize sensitivity and reproducibility. Here we present a new framework to analyze metabolism in mammalian cell lines and primary cells, covering the workflow from the preparation of cells to the acquisition and analysis of NMR spectra. We have applied this new approach in hematological and liver cancer cell lines and confirm the feasibility of tracer-based metabolism in primary liver cells.

Can stable isotope markers be used to distinguish wild and mass-reared Anastrepha fraterculus flies?

The availability of accurate techniques to discriminate between marked laboratory-reared flies and unmarked wild flies captured in monitoring traps is essential for programs that integrate the Sterile Insect Technique (SIT) to manage fruit flies. In this study, the feasibility of using a stable isotope marking technique for the South American fruit fly, Anastrepha fraterculus (Wiedemann), was assessed. Wild flies were collected from apple orchards, which are a target of a SIT project in southern Brazil. To verify if adult flies could be labelled by the stable isotopes from larval diets, larvae were reared on two different C4-based diets and fruits in laboratory. To evaluate the influence of the two most common attractants applied to capture A. fraterculus (grape juice and CeraTrapTM) and the most common preservation method in fruit fly collections (ethanol), laboratory-reared flies were immersed in McPhail traps containing the respective treatments for two periods of time. Samples were analyzed in an elemental analyzer coupled to a Continuous Flow Isotope Ratio Mass Spectrometer (CF-IRMS) at CENA/USP. The δ13C signatures of flies reared on artificial diets differed significantly from the δ13C of flies whose larvae were reared on fruits and from wild flies. In contrast, the δ15N values were less conclusive and the technique could not rely solely on them. In all cases considered, the δ13C and δ15N signatures from males did not differ from females. Despite the alterations caused by the attractants tested and ethanol, laboratory-flies could be distinguished from the wild ones based on δ13C signatures. This is the first comprehensive study to demonstrate that it is possible to distinguish wild A. fraterculus from flies reared on larval diets containing C4 sugar. The first experimentally derived trophic discrimination factors were also obtained for this species. Thus, intrinsic isotope labelling can serve as a backup to conventional dye marking.

Hyperpolarized 13C MR metabolic imaging can detect neuroinflammation in vivo in a multiple sclerosis murine model.

Proinflammatory mononuclear phagocytes (MPs) play a crucial role in the progression of multiple sclerosis (MS) and other neurodegenerative diseases. Despite advances in neuroimaging, there are currently limited available methods enabling noninvasive detection of MPs in vivo. Interestingly, upon activation and subsequent differentiation toward a proinflammatory phenotype MPs undergo metabolic reprogramming that results in increased glycolysis and production of lactate. Hyperpolarized (HP) 13C magnetic resonance spectroscopic imaging (MRSI) is a clinically translatable imaging method that allows noninvasive monitoring of metabolic pathways in real time. This method has proven highly useful to monitor the Warburg effect in cancer, through MR detection of increased HP [1-13C]pyruvate-to-lactate conversion. However, to date, this method has never been applied to the study of neuroinflammation. Here, we questioned the potential of 13C MRSI of HP [1-13C]pyruvate to monitor the presence of neuroinflammatory lesions in vivo in the cuprizone mouse model of MS. First, we demonstrated that 13C MRSI could detect a significant increase in HP [1-13C]pyruvate-to-lactate conversion, which was associated with a high density of proinflammatory MPs. We further demonstrated that the increase in HP [1-13C]lactate was likely mediated by pyruvate dehydrogenase kinase 1 up-regulation in activated MPs, resulting in regional pyruvate dehydrogenase inhibition. Altogether, our results demonstrate a potential for 13C MRSI of HP [1-13C]pyruvate as a neuroimaging method for assessment of inflammatory lesions. This approach could prove useful not only in MS but also in other neurological diseases presenting inflammatory components.

Soil anammox community structure in different land use soils treatment with 13C urea as determined by analysis of phospholipid fatty acids.

The anaerobic ammonium oxidation (anammox) process is globally an important nitrogen-cycling process mediated by specialized microbes. However, still little information is documented about anammox microbial community structure under agricultural soils. The anaerobic incubation experiment was conducted to study the impacts of different land use soils fertilized by 13C-urea on the activity and diversity of anammox bacteria using stable isotope to probe the phospholipid fatty acid (PLFA-SIP). The 13C was preferentially incorporated in ratios PLFAs 16:1ω7c, 16:1ω5c, and 16:0. The results revealed that the abundance of the anammox bacteria (both hzs-ß and hzo) were observed in vegetable soil V1 and paddy soils (R1 and R2) means that they were positively correlated with 13C-urea but were negatively correlated with NO3--N and NH4+-N concentrations. Thus, 13C-PLFAs 16:1ω7c, 16:1ω5c, and 16:0 could be the biomarker as soil anammox. The anaerobic microbial community composition of soils under different land use systems was diverse, and V1, R1, and R2 had similar microbial diversity and higher microbial biomass. The principal component analysis between soil properties and gene abundance suggested that not only pH but also soil organic matter, available P, and available K were important factors for the anammox process. This study suggested that 13C-Urea-PLFA for anaerobic incubation was a simple method to study anammox microbial community structure through affecting the soil nutrients, and the different land use systems played important roles in determining the microbial composition of soils.

Mentally stimulating activities associate with better cognitive performance in Parkinson disease.

Subjects at risk of dementia benefit from participation in mentally stimulating activities, but no prior studies have investigated similar associations in Parkinson disease (PD). The aim of this study was to investigate the relationship between times spent engaging in mentally stimulating activities and cognitive functions in PD while accounting for the degree of primary neurodegenerations. PD patients (N = 41, 33 males; age 68.5 ± 7.2; Hoehn and Yahr stage 2.6 ± 0.6) completed the Community Health Activities Model Program for Seniors questionnaire, mini-mental state examination (MMSE), and [11C]dihydrotetrabenazine dopaminergic and [11C]piperidinyl propionate acetylcholinesterase PET imaging. The subset of mentally stimulating activity items of the Community Health Activities Model Program for Seniors questionnaire was used to develop a rating scale as primary outcome variable in this study. Findings showed that mean rating scale score of time spent in mentally stimulating activities over a 4-week timespan was 20.0 ± 8.3 h and mean MMSE score was 28.4 ± 1.9. Regression analysis showed that duration of participation in mentally stimulating activities was a significant predictor of MMSE scores (standardized ß = 0.39, t = 2.8, p = 0.009; total model: F (6,34) = 3.5, p = 0.005) independent from significant effects for cortical cholinergic activity (ß = 0.35, t = 2.4, p = 0.024). Caudate nucleus dopaminergic activity, age, education, or duration of disease were not significant regressors. Post hoc analysis did not show significant effects of motor disease severity or level of physical activities. We conclude that engagement in mentally stimulating activities is associated with better cognitive abilities in PD, independent of education, severity of motor disease, nigrostriatal dopaminergic and cortical cholinergic degenerations.

The use of stable isotopes in the study of human pathophysiology.

The growing prevalence of metabolic diseases including fatty liver disease and Type 2 diabetes has increased the emphasis on understanding metabolism at the mechanistic level and how it is perturbed in disease. Metabolomics is a continually expanding field that seeks to measure metabolites in biological systems during a physiological stimulus or a genetic alteration. Typically, metabolomics studies provide total pool sizes of metabolites rather than dynamic flux measurements. More recently there has been a resurgence in approaches that use stable isotopes (e.g. 2H and 13C) for the unambiguous tracking of individual atoms through compartmentalised metabolic networks in humans to determine underlying mechanisms. This is known as metabolic flux analysis and enables the capture of a dynamic picture of the metabolome and its interactions with the genome and proteome. In this review, we describe current approaches using stable isotope labelling in the field of metabolomics and provide examples of studies that led to an improved understanding of glucose, fatty acid and amino acid metabolism in humans, particularly in relation to metabolic disease. Examples include the use of stable isotopes of glucose to study tumour bioenergetics as well as brain metabolism during traumatic brain injury. Lipid tracers have also been used to measure non-esterified fatty acid production whilst amino acid tracers have been used to study the rate of protein digestion on whole body postprandial protein metabolism. In addition, we illustrate the use of stable isotopes for measuring flux in human physiology by providing examples of breath tests to measure insulin resistance and gastric emptying rates.

13C-assisted metabolomics analysis reveals the positive correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size in Saccharopolyspora erythraea.

Metabolomics analysis is extremely essential to explore the metabolism characteristics of Saccharopolyspora erythraea. The lack of suitable methods for the determination of intracellular metabolites, however, hinders the application of metabolomics analysis for S. erythraea. Acyl-CoAs are important precursors of erythromycin; phosphorylated sugars are intermediate metabolites in EMP pathway or PPP pathway; organic acids are intermediate metabolites in TCA cycle. Reliable determination methods for intracellular acyl-CoAs, phosphorylated sugars, and organic acids of S. erythraea were designed and validated in this study. Using the optimized determination methods, the pool sizes of intracellular metabolites during an erythromycin fermentation process were precisely quantified by isotope dilution mass spectroscopy method. The quantification results showed that the specific erythromycin production rate was positively correlated with the pool sizes of propionyl-CoA as well as many other intracellular metabolites. The experiment under the condition without propanol, which is a precursor of propionyl-CoA and an important substrate in industrial erythromycin production process, also corroborated the correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size. As far as we know, this is the first paper to conduct the metabolomics analysis of S. erythraea, which makes the metabolomics analysis of S. erythraea in the industrial erythromycin production process possible.

Rerouting of carbon flux in a glycogen mutant of cyanobacteria assessed via isotopically non-stationary 13 C metabolic flux analysis.

Cyanobacteria, which constitute a quantitatively dominant phylum, have attracted attention in biofuel applications due to favorable physiological characteristics, high photosynthetic efficiency and amenability to genetic manipulations. However, quantitative aspects of cyanobacterial metabolism have received limited attention. In the present study, we have performed isotopically non-stationary 13 C metabolic flux analysis (INST-13 C-MFA) to analyze rerouting of carbon in a glycogen synthase deficient mutant strain (glgA-I glgA-II) of the model cyanobacterium Synechococcus sp. PCC 7002. During balanced photoautotrophic growth, 10-20% of the fixed carbon is stored in the form of glycogen via a pathway that is conserved across the cyanobacterial phylum. Our results show that deletion of glycogen synthase gene orchestrates cascading effects on carbon distribution in various parts of the metabolic network. Carbon that was originally destined to be incorporated into glycogen gets partially diverted toward alternate storage molecules such as glucosylglycerol and sucrose. The rest is partitioned within the metabolic network, primarily via glycolysis and tricarboxylic acid cycle. A lowered flux toward carbohydrate synthesis and an altered distribution at the glucose-1-phosphate node indicate flexibility in the network. Further, reversibility of glycogen biosynthesis reactions points toward the presence of futile cycles. Similar redistribution of carbon was also predicted by Flux Balance Analysis. The results are significant to metabolic engineering efforts with cyanobacteria where fixed carbon needs to be re-routed to products of interest. Biotechnol. Bioeng. 2017;114: 2298-2308. © 2017 Wiley Periodicals, Inc.

Specificity of exogenous acetate and glutamate as astrocyte substrates examined in acute brain slices from female mice using methionine sulfoximine (MSO) to inhibit glutamine synthesis.

Removal of endogenously released glutamate is mediated primarily by astrocytes and exogenous 13 C-labeled glutamate has been applied to study glutamate metabolism in astrocytes. Likewise, studies have clearly established the relevance of 13 C-labeled acetate as an astrocyte specific metabolic substrate. Recent studies have, however, challenged the arguments used to anchor this astrocyte specificity of acetate and glutamate. The aim of the current study was to evaluate the specificity of acetate and glutamate as astrocyte substrates in brain slices. Acutely isolated hippocampal and cerebral cortical slices from female NMRI mice were incubated in media containing [1,2-13 C]acetate or [U-13 C]glutamate, with or without methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Tissue extracts were analyzed by gas chromatography-mass spectrometry. Blocking GS abolished the majority of glutamine 13 C-labeling from [1,2-13 C]acetate as intended. However, 13 C-labeling of GABA was only 40-50% reduced by MSO, suggesting considerable neuronal uptake of acetate. Moreover, labeling of glutamate from [1,2-13 C]acetate in the presence of MSO exceeded the level probable from exclusive labeling of the astrocytic pool, which likewise suggests neuronal acetate metabolism. Approximately 50% of glutamate was uniformly labeled in slices incubated with [U-13 C]glutamate in the presence of MSO, suggesting that neurons exhibit substantial uptake of exogenously provided glutamate. © 2017 Wiley Periodicals, Inc.

Monitoring creatine and phosphocreatine by (13)C MR spectroscopic imaging during and after (13)C4 creatine loading: a feasibility study.

Creatine (Cr) supplementation to enhance muscle performance shows variable responses among individuals and different muscles. Direct monitoring of the supplied Cr in muscles would address these differences. In this feasibility study, we introduce in vivo 3D (13)C MR spectroscopic imaging (MRSI) of the leg with oral ingestion of (13)C4-creatine to observe simultaneously Cr and phosphocreatine (PCr) for assessing Cr uptake, turnover, and the ratio PCr over total Cr (TCr) in individual muscles. (13)C MRSI was performed of five muscles in the posterior thigh in seven subjects (two males and two females of ~20 years, one 82-year-old male, and two neuromuscular patients) with a (1)H/(13)C coil in a 3T MR system before, during and after intake of 15 % (13)C4-enriched Cr. Subjects ingested 20 g Cr/day for 4 days in four 5 g doses at equal time intervals. The PCr/TCr did not vary significantly during supplementation and was similar for all subjects and investigated muscles (average 0.71 ± 0.07), except for the adductor magnus (0.64 ± 0.03). The average Cr turnover rate, assessed in male muscles, was 2.1 ± 0.7 %/day. The linear uptake rates of Cr were variable between muscles, although not significantly different. This assessment was possible in all investigated muscles of young male volunteers, but less so in muscles of the other subjects due to lower signal-to-noise ratio. Improvements for future studies are discussed. In vivo (13)C MRSI after (13)C-Cr ingestion is demonstrated for longitudinal studies of Cr uptake, turnover, and PCr/TCr ratios of individual muscles in one exam.

Isotopic Discrimination in the Double-Crested Cormorant (Phalacrocorax auritus).

The diet-tissue discrimination factor is the amount by which a consumer's tissue varies isotopically from its diet, and is therefore a key element in models that use stable isotopes to estimate diet composition. In this study we measured discrimination factors in blood (whole blood, red blood cells and plasma), liver, muscle and feathers of Double-crested Cormorants (Phalacrocorax auritus) for stable isotope ratios of carbon, nitrogen and sulfur. Cormorants exhibited discrimination factors that differed significantly among tissue types (for carbon and nitrogen), and differed substantially (in the context of the isotopic variation among relevant prey species) from those observed in congeneric species. The Double-crested Cormorant has undergone rapid population expansion throughout much of its historic range over the past three decades, leading to both real and perceived conflicts with fisheries throughout North America, and this study provides an essential link for the use of stable isotope analysis in researching foraging ecology, diet, and resource use of this widespread and controversial species.

In Vivo Metabolic Trapping Radiotracers for Imaging Monoamine Oxidase-A and -B Enzymatic Activity.

The isozymes of monoamine oxidase (MAO-A and MAO-B) are important enzymes involved in the metabolism of numerous biogenic amines, including the neurotransmitters serotonin, dopamine, and norepinephrine. Recently, changes in concentrations of MAO-B have been proposed to be an in vivo marker of neuroinflammation associated with Alzheimer's disease. Previous developments of in vivo radiotracers for imaging changes in MAO enzyme expression or activity have utilized the irreversible propargylamine-based suicide inhibitors or high-affinity reversibly binding inhibitors. As an alternative approach, we have investigated 1-[(11)C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines as metabolic trapping agents for the monoamine oxidases. MAO-mediated oxidation and spontaneous hydrolysis yield 1-[(11)C]methyl-2,3-dihydro-4-pyridinone as a hydrophilic metabolite that is trapped within brain tissues. Radiotracers with phenyl, biphenyl, and 7-coumarinyl ethers were evaluated using microPET imaging in rat and primate brains. No isozyme selectivity for radiotracer trapping was observed in the rat brain for any compound, but in the monkey brain, the phenyl ether demonstrated MAO-A selectivity and the coumarinyl ether showed MAO-B selectivity. These are lead compounds for further development of 1-[(11)C]methyl-4-aryloxy-1,2,3,6-tetrahydropyridines with optimized brain pharmacokinetics and isozyme selectivity.

Impact of kinetic isotope effects in isotopic studies of metabolic systems.

BACKGROUND: Isotope labeling experiments (ILEs) are increasingly used to investigate the functioning of metabolic systems. Some enzymes are subject to kinetic isotope effects (KIEs) which modulate reaction rates depending on the isotopic composition of their substrate(s). KIEs may therefore affect both the propagation of isotopes through metabolic networks and their operation, and ultimately jeopardize the biological value of ILEs. However, the actual impact of KIEs on metabolism has never been investigated at the system level. RESULTS: First, we developed a framework which integrates KIEs into kinetic and isotopic models of metabolism, thereby accounting for their system-wide effects on metabolite concentrations, metabolic fluxes, and isotopic patterns. Then, we applied this framework to assess the impact of KIEs on the central carbon metabolism of Escherichia coli in the context of (13)C-ILEs, under different situations commonly encountered in laboratories. Results showed that the impact of KIEs strongly depends on the label input and on the variable considered but is significantly lower than expected intuitively from measurements on isolated enzymes. The global robustness of both the metabolic operation and isotopic patterns largely emerge from intrinsic properties of metabolic networks, such as the distribution of control across the network and bidirectional isotope exchange. CONCLUSIONS: These results demonstrate the necessity of investigating the impact of KIEs at the level of the entire system, contradict previous hypotheses that KIEs would have a strong effect on isotopic distributions and on flux determination, and strengthen the biological value of (13)C-ILEs. The proposed modeling framework is generic and can be used to investigate the impact of all the isotopic tracers ((2)H, (13)C, (15)N, (18)O, etc.) on different isotopic datasets and metabolic systems. By allowing the integration of isotopic and metabolomics data collected under stationary and/or non-stationary conditions, it may also assist interpretations of ILEs and facilitate the development of more accurate kinetic models with improved explicative and predictive capabilities.

Noninvasive estimation of disease activity and liver fibrosis in nonalcoholic fatty liver disease using anthropometric and biochemical characteristics, including insulin, insulin resistance, and 13C-methionine breath test.

OBJECTIVE: The purpose of this study was to noninvasively estimate disease activity and liver fibrosis in patients with nonalcoholic fatty liver disease (NAFLD) using anthropometric and biochemical characteristics and the C-methionine breath test (MeBT). METHODS: A total of 164 patients with histologically proven NAFLD and 56 healthy controls were included in the study. Anthropometric and biochemical analyses and the MeBT were performed on all patients and controls. RESULTS: BMI; waist circumference; waist-hip ratio; transaminase, lipid, γ-glutamyl transpeptidase (GGT), glucose, and insulin levels; and insulin resistance were significantly higher in patients with NAFLD than in controls. The GGT level and the MeBT were independent predictors of nonalcoholic steatohepatitis (NASH). Fibrosis was correlated with GGT, bilirubin, cholesterol, and insulin levels, and the MeBT, but the test was the only independent predictor of significant fibrosis. Patients with simple steatosis had similar MeBT values as controls. The MeBT values were significantly lower in NASH and NASH-cirrhosis patients (P<0.001) compared with simple steatosis patients and controls. Patients with advanced fibrosis (F2-3) had significantly lower MeBT values than patients with mild fibrosis (F0-1; P<0.001). The area under the receiving operating characteristic curve for NASH and advanced fibrosis was estimated to be 0.95 in the total cohort. CONCLUSION: This study indicates that anthropometric and biochemical parameters are insufficient for estimating the presence of NASH or the fibrosis stage. However, the MeBT is a suitable noninvasive method for accurately predicting which patients suffer from simple steatosis, NASH, or NASH-cirrhosis.

[11C]PBR28 PET imaging is sensitive to neuroinflammation in the aged rat.

Neuroinflammation in the aging rat brain was investigated using [(11)C]PBR28 microPET (positron emission tomography) imaging. Normal rats were studied alongside LRRK2 p.G2019S transgenic rats; this mutation increases the risk of Parkinson's disease in humans. Seventy [(11)C]PBR28 PET scans were acquired. Arterial blood sampling enabled tracer kinetic modeling and estimation of VT. In vitro autoradiography was also performed. PBR28 uptake increased with age, without differences between nontransgenic and transgenic rats. In 12 months of aging (4 to 16 months), standard uptake value (SUV) increased by 56% from 0.44 to 0.69 g/mL, whereas VT increased by 91% from 30 to 57 mL/cm(3). Standard uptake value and VT were strongly correlated (r = 0.52, 95% confidence interval (CI) = 0.31 to 0.69, n = 37). The plasma free fraction, fp, was 0.21 ± 0.03 (mean ± standard deviation, n = 53). In vitro binding increased by 19% in 16 months of aging (4 to 20 months). The SUV was less variable across rats than VT; coefficients of variation were 13% (n = 27) and 29% (n = 12). The intraclass correlation coefficient for SUV was 0.53, but was effectively zero for VT. These data show that [(11)C]PBR28 brain uptake increases with age, implying increased microglial activation in the aged brain.