Human Metabolism Studies Research Articles

Prediction of Human Disproportionate and Biliary Excreted Metabolites Using Chimeric Mice with Humanized Liver.

The PXB-mouse is potentially a useful in vivo model to predict human hepatic metabolism and clearance. Four model compounds, [14C]desloratadine, [3H]mianserin, cyproheptadine, and [3H]carbazeran, all reported with disproportionate human metabolites, were orally administered to PXB- or control SCID mice to elucidate the biotransformation of each of them. For [14C]desloratadine in PXB-mice, O-glucuronide of 3-hydroxydesloratadine was observed as the predominant metabolite in both the plasma and urine. Both 3-hydroxydesloratadine and its O-glucuronide were detected as major drug-related materials in the bile, whereas only 3-hydroxydesloratadine was detected in the feces, suggesting that a fraction of 3-hydroxydesloratadine in feces was derived from deconjugation of its O-glucuronide by gut microflora. This information can help understand the biliary clearance mechanism of a drug and may fill the gap in a human absorption, distribution, metabolism, and excretion study, in which the bile samples are typically not available. The metabolic profiles in PXB-mice were qualitatively similar to those reported in humans in a clinical study in which 3-hydroxydesloratadine and its O-glucuronide were major and disproportionate metabolites compared with rat, mouse, and monkey. In the control SCID mice, neither of the metabolites was detected in any matrix. Similarly, for the other three compounds, all human specific or disproportionate metabolites were detected at a high level in PXB-mice, but they were either minimally observed or not observed in the control mice. Data from these four compounds indicate that studies in PXB-mice can help predict the potential for the presence of human disproportionate metabolites (relative to preclinical species) prior to conducting clinical studies and understand the biliary clearance mechanism of a drug. SIGNIFICANCE STATEMENT: Studies in PXB-mice have successfully predicted the human major and disproportionate metabolites compared with preclinical safety species for desloratadine, mianserin, cyproheptadine, and carbazeran. In addition, biliary excretion data from PXB-mice can help illustrate the human biliary clearance mechanism of a drug.

Contemporary stable isotope tracer approaches: Insights into skeletal muscle metabolism in health and disease.

What is the topic of this review? This review discusses the application of new stable isotope tracer techniques in understanding the control of skeletal muscle mass. What advances does it highlight? This review highlights current advances in stable isotope tracer techniques through their combination with high-throughput proteomics technologies. Beyond its primary locomotory and key structural functions, skeletal muscle provides additional vital roles for maintenance of metabolic health, acting as a storage point for glucose and intramuscular lipids for energy production, alongside being the largest reservoir for amino acids in the body. Therefore, maintenance of muscle mass is key to the promotion of health and well-being across the lifespan and in several disease states. As such, when skeletal muscle is lost, in either clinical (cancer, organ failure etc.) or non-clinical (ageing, inactivity) situations, there are potentially devastating consequences attached, with robust links existing between muscle mass loss and mortality. Great efforts are being made to reverse or slow muscle mass declines in health and disease, through combinations of lifestyle changes and nutritional and/or pharmaceutical intervention. However, despite this comprehensive research effort, the underlying metabolic and molecular mechanisms have yet to be defined properly. However, with the rapid acceleration of analytical developments over recent years, the application of stable isotope tracers to the study of human muscle metabolism is providing unique insights into the mechanisms controlling skeletal muscle loss and allowing more targeted therapeutic strategies to be developed. The aim of this review is to highlight the technical breakthroughs in our understanding of muscle wasting in health and disease and how future directions and developments incorporating 'omics' with stable isotope tracers will allow for a more personalized and stratified therapeutic approach.

Mitochondrial Dysfunction, Insulin Resistance, and Potential Genetic Implications.

Insulin resistance (IR) is fundamental to the development of type 2 diabetes (T2D) and is present in most prediabetic (preDM) individuals. Insulin resistance has both heritable and environmental determinants centered on energy storage and metabolism. Recent insights from human genetic studies, coupled with comprehensive in vivo and ex vivo metabolic studies in humans and rodents, have highlighted the critical role of reduced mitochondrial function as a predisposing condition for ectopic lipid deposition and IR. These studies support the hypothesis that reduced mitochondrial function, particularly in insulin-responsive tissues such as skeletal muscle, white adipose tissue, and the liver, is inextricably linked to tissue and whole body IR through the effects on cellular energy balance. Here we discuss these findings as well as address potential mechanisms that serve as the nexus between mitochondrial malfunction and IR.

Determination of di-n-butyl adipate (DnBA) metabolites as possible biomarkers of exposure in human urine by online-SPE-LC-MS/MS

Di-n-butyl adipate (DnBA) is an alternative to the anti-androgenic and strictly regulated di-n-butyl phthalate (DnBP) used as a cosmetic ingredient, plasticizer, and in various articles of everyday life. Hence, exposures of the general population have to be expected. Currently, biomarkers of DnBA exposure and methods for their determination are not available. Here, we describe a sensitive, rugged and precise analytical method for the determination of the DnBA monoester metabolite mono-n-butyl adipate (MnBA), as well as its potential downstream metabolites 3-hydroxy-mono-n-butyl adipate (3OH-MnBA) and 3-carboxy-mono-n-propyl adipate (3cx-MnPrA) in human urine. Glucuronic acid conjugates present in urine were deconjugated using a pure β-glucuronidase. The metabolites were then analyzed by liquid chromatography on a C18 column with superficially porous particles coupled to electrospray ionization-triple quadrupole-tandem mass spectrometry, applying online turbulent flow chromatography for analyte enrichment and matrix depletion (online-SPE-LC-MS/MS). The metabolites were quantified using stable isotope dilution analysis with limits of quantification of 0.05 µg/L (MnBA), 0.1 µg/L (3OH-MnBA), and 0.5 µg/L (3cx-MnPrA). Method imprecision in urinary matrix was below 7% (coefficient of variation) for all analytes. Mean relative recoveries were between 93% and 107%. The suitability of the DnBA metabolites as biomarkers of exposure was demonstrated after dermal application of a commercially available sunscreen containing DnBA. Maximum concentrations were reached 6.5 h after dose (219 µg/L 3cx-MnPrA, 91 µg/L MnBA, and 3.9 µg/L 3OH-MnBA). Elimination kinetics were similar for all three metabolites. We were able to quantify 3cx-MnPrA and MnBA until 4 d after sunscreen application. In a sample set of 35 urine samples from the general German population, 3cx-MnPrA was quantified in 94% (median 2.54 µg/L, maximum 78.3 µg/L) and MnBA in 3% (median < LOQ, maximum 0.18 µg/L) of the samples. The method will be applied in future human metabolism and human biomonitoring population studies.

Biotransformation Pathways and Metabolite Profiles of Oral [14C]Alisertib (MLN8237), an Investigational Aurora A Kinase Inhibitor, in Patients with Advanced Solid Tumors.

Alisertib (MLN8237) is an investigational, orally available, selective aurora A kinase inhibitor in clinical development for the treatment of solid tumors and hematologic malignancies. This metabolic profiling analysis was conducted as part of a broader phase 1 study evaluating mass balance, pharmacokinetics, metabolism, and routes of excretion of alisertib following a single 35-mg dose of [14C]alisertib oral solution (∼80 μCi) in three patients with advanced malignancies. On average, 87.8% and 2.7% of the administered dose was recovered in feces and urine, respectively, for a total recovery of 90.5% by 14 days postdose. Unchanged [14C]alisertib was the predominant drug-related component in plasma, followed by O-desmethyl alisertib (M2), and alisertib acyl glucuronide (M1), which were present at 47.8%, 34.6%, and 12.0% of total plasma radioactivity. In urine, of the 2.7% of the dose excreted, unchanged [14C]alisertib was a negligible component (trace), with M1 (0.84% of dose) and glucuronide conjugate of hydroxy alisertib (M9; 0.66% of dose) representing the primary drug-related components in urine. Hydroxy alisertib (M3; 20.8% of the dose administered) and unchanged [14C]alisertib (26.3% of the dose administered) were the major drug-related components in feces. In vitro, oxidative metabolism of alisertib was primarily mediated by CYP3A. The acyl glucuronidation of alisertib was primarily mediated by uridine 5'-diphospho-glucuronosyltransferase 1A1, 1A3, and 1A8 and was stable in 0.1 M phosphate buffer and in plasma and urine. Further in vitro evaluation of alisertib and its metabolites M1 and M2 for cytochrome P450-based drug-drug interaction (DDI) showed minimal potential for perpetrating DDI with coadministered drugs. Overall, renal elimination played an insignificant role in the disposition of alisertib, and metabolites resulting from phase 1 oxidative pathways contributed to >58% of the alisertib dose recovered in urine and feces over 192 hours postdose. SIGNIFICANCE STATEMENT: This study describes the primary clearance pathways of alisertib and illustrates the value of timely conduct of human absorption, distribution, metabolism, and excretion studies in providing guidance to the clinical pharmacology development program for oncology drugs, for which a careful understanding of sources of exposure variability is crucial to inform risk management for drug-drug interactions given the generally limited therapeutic window for anticancer drugs and polypharmacy that is common in cancer patients.

In-Vitro assessment of the inhibitory effects of dietary flavones on the regulated metabolism of CYP 450: Potential for Herb-drug interactions

Background and objective: Dietary flavones like chrysin are found in foods and can contribute to human health through prevention of cardiovascular and cancer supplements. Recently, as herbal medicines and traditional allopathic medicines are used together, drug food or herbal drug interactions have become the target of our body and can lead to potential herbal-drug interactions. The present study aims to determine the metabolic stability and CYP inhibition liabilities in human liver microsomes in order to rule out the association between herb drugs and the dietary flavone chrysin. Methods: Standard procedures were used for metabolic stability study in human liver microsomes (Phase I) and metabolic stability study in cryopreserved human hepatocytes (Phase II). Human liver microsomes (HLMs) were added to each selective probe substrates of cytochrome P450 enzymes with or without of chrysin and IC50 was calculated. Results: Metabolic stability data reveals that Chrysin underwent phase II metabolism predominantly as it was highly stable in HLM and comparatively faster metabolism in hepatocytes. Chrysin was found to be a potent inhibitor of CYP1A2 and CYP2C9 with IC50 values of 0.6 μM and 1.6μM, respectively. Chrysin has weak inhibitory effect on other CYP isoforms such as CYP2C9, CY2D6 (~25μM) and no inhibition of CYP3A4 up to 100μM. Conclusion: These results could be useful in clinical practice for safely and effectively use of flavone chrysin. Our report showed that in vivo interactions of drugs and pharmaceuticals with dietary flavones must be examined for CYP1A2 and CYP2C9 substrates.

Simultaneous quantitative determination of seven novel tyrosine kinase inhibitors in plasma by a validated UPLC-MS/MS method and its application to human microsomal metabolic stability study

A rapid, sensitive and reproducible ultra-high performance liquid chromatography mass spectrometry method was developed and validated for simultaneous determination of seven tyrosine kinase inhibitors (dasatinib, foretinib, osimertinib, gefitinib, ibrutinib, linifanib and motesanib) in human plasma samples using quizartinib as internal standard (IS). The sample preparation was performed by liquid-liquid extraction method, using a mixture of ethyl acetate and tert butyl methyl ether (50:50, v/v) as extracting solvents. Chromatographic separation was achieved using acquity UPLC BEH C18, 1.7 µm 2.1 × 100 mm column and a mobile phase consisting of a mixture of acetonitrile (0.1% formic acid) and 20 mM ammonium acetate (95:5) at a flow rate of 0.25 mL/min. All the analytes and IS were eluted within 2 min, with total run time of 3 min only. The electrospray ionization in positive mode was used and all analytes were monitored using multiple reaction monitoring (MRM) mode. The method was linear and reproducible for all the compounds in the range of 5–1000 ng/mL. Between- and within-run accuracy ranged from 86.7% to 92.5%, and the precision was less than 11.9% for all seven compounds. The developed method was successfully applied to in-vitro human microsomal metabolic stability study. This method could be useful in clinical application for therapeutic drug monitoring (TDM) of these analytes and for individualization of therapeutic regimens.

First hyperpolarized [2-13C]pyruvate MR studies of human brain metabolism.

We developed methods for the preparation of hyperpolarized (HP) sterile [2-13C]pyruvate to test its feasibility in first-ever human NMR studies following FDA-IND & IRB approval. Spectral results using this MR stable-isotope imaging approach demonstrated the feasibility of investigating human cerebral energy metabolism by measuring the dynamic conversion of HP [2-13C]pyruvate to [2-13C]lactate and [5-13C]glutamate in the brain of four healthy volunteers. Metabolite kinetics, signal-to-noise (SNR) and area-under-curve (AUC) ratios, and calculated [2-13C]pyruvate to [2-13C]lactate conversion rates (kPL) were measured and showed similar but not identical inter-subject values. The kPL measurements were equivalent with prior human HP [1-13C]pyruvate measurements.

Metabolic modulation predicts heart failure tests performance.

The metabolic changes that accompany changes in Cardiopulmonary testing (CPET) and heart failure biomarkers (HFbio) are not well known. We undertook metabolomic and lipidomic phenotyping of a cohort of heart failure (HF) patients and utilized Multiple Regression Analysis (MRA) to identify associations to CPET and HFBio test performance (peak oxygen consumption (Peak VO2), oxygen uptake efficiency slope (OUES), exercise duration, and minute ventilation-carbon dioxide production slope (VE/VCO2 slope), as well as the established HF biomarkers of inflammation C-reactive protein (CRP), beta-galactoside-binding protein (galectin-3), and N-terminal prohormone of brain natriuretic peptide (NT-proBNP)). A cohort of 49 patients with a left ventricular ejection fraction < 50%, predominantly males African American, presenting a high frequency of diabetes, hyperlipidemia, and hypertension were used in the study. MRA revealed that metabolic models for VE/VCO2 and Peak VO2 were the most fitted models, and the highest predictors’ coefficients were from Acylcarnitine C18:2, palmitic acid, citric acid, asparagine, and 3-hydroxybutiric acid. Metabolic Pathway Analysis (MetPA) used predictors to identify the most relevant metabolic pathways associated to the study, aminoacyl-tRNA and amino acid biosynthesis, amino acid metabolism, nitrogen metabolism, pantothenate and CoA biosynthesis, sphingolipid and glycerolipid metabolism, fatty acid biosynthesis, glutathione metabolism, and pentose phosphate pathway (PPP). Metabolite Set Enrichment Analysis (MSEA) found associations of our findings with pre-existing biological knowledge from studies of human plasma metabolism as brain dysfunction and enzyme deficiencies associated with lactic acidosis. Our results indicate a profile of oxidative stress, lactic acidosis, and metabolic syndrome coupled with mitochondria dysfunction in patients with HF tests poor performance. The insights resulting from this study coincides with what has previously been discussed in existing literature thereby supporting the validity of our findings while at the same time characterizing the metabolic underpinning of CPET and HFBio.

NGP 555, a γ-secretase modulator, shows a beneficial shift in the ratio of amyloid biomarkers in human cerebrospinal fluid at safe doses

IntroductionCurrently, there is no cure for Alzheimer's disease (AD), and it is widely accepted that AD is a complex disease with multiple approaches necessary to prevent and treat the disease. MethodsUsing amyloid biomarkers in human cerebrospinal fluid, pharmacokinetic, safety, and metabolism studies, we investigate the properties of NGP 555, γ-secretase modulator, for the first time in human clinical trials. ResultsOur preclinical and clinical studies combined show beneficial effects with NGP 555 on synaptic response and amyloid cerebrospinal fluid biomarkers while avoiding negative side effects. Importantly, pharmacokinetic and pharmacodynamic parameters combined with safety outcomes indicate that NGP 555 penetrates the blood-brain barrier and increases the ratio of amyloid-β peptide Aβ37 and Aβ38 compared with that of Aβ42, establishing a proof of target engagement in humans in a 14 day, once-daily oral dosing trial. DiscussionIn humans, NGP 555 has demonstrated a beneficial shift in the production of Aβ37 and Aβ38 versus Aβ42 biomarker levels in the cerebrospinal fluid while maintaining an adequate safety profile. The overall clinical goal is to achieve an optimal balance of efficacy for altering amyloid-β peptide (Aβ) biomarkers while maintaining a safe profile so that NGP 555 can be given early in AD to prevent production of Aβ42 and accumulation of amyloid plaques, in an effort to prevent aggregation of tau and destruction of neurons and synapses resulting in cognitive decline.

Increased Cardiac Uptake of Ketone Bodies and Free Fatty Acids in Human Heart Failure and Hypertrophic Left Ventricular Remodeling.

Deranged energy metabolism contributes to the pathophysiology of heart failure (HF). Recent studies showed diminished free fatty acid (FFA) oxidation in experimental HF models with a shift towards oxidation of ketone bodies. However, conflicting clinical data on FFA metabolism and limited knowledge on ketone body metabolism in human HF mandate additional metabolic profiling studies. We, therefore, investigated cardiac uptake of FFAs and ketone bodies (β-hydroxybutyrate and acetoacetate) in patients with HF with reduced ejection fraction (HFrEF) or with aortic stenosis (AS)-induced left ventricular hypertrophy. We hypothesized that FFA oxidation is impaired in HFrEF and in AS and results in decreased concentrations of free carnitine, the necessary carrier for mitochondrial entry of activated FFAs, and in accumulation of metabolic intermediates. We collected arterial and coronary sinus blood samples in patients with HFrEF (n=15), in AS patients with preserved systolic function (n=15), and in control patients (n=15). Plasma concentration gradients across the heart show significantly greater uptake of ketone bodies in patients with HFrEF than in controls. Patients with AS show significantly increased uptake of β-hydroxybutyrate and FFAs. Free carnitine concentration and concentration gradients of intermediates of FFA oxidation were comparable between groups. In conclusion, our results show significantly increased cardiac uptake of ketone bodies in patients with stable HFrEF and AS and increased uptake of FFAs in AS compared with control patients. The lack of myocardial release of acyl-carnitine species or change in free carnitine uptake suggests no impairment of FFA oxidation.

Genomic and Transcriptomic Analysis of Hypercholesterolemic Rabbits: Progress and Perspectives.

Rabbits (Oryctolagus cuniculus) are one of the most widely used animal models for the study of human lipid metabolism and atherosclerosis because they are more sensitive to a cholesterol diet than other experimental animals such as rodents. Currently, two hypercholesterolemic rabbit models are frequently used for atherosclerosis studies. One is a cholesterol-fed wild-type rabbit and the other is the Watanabe heritable hyperlipidemic (WHHL) rabbit, which is genetically deficient in low density lipoprotein (LDL) receptor function. Wild-type rabbits can be easily induced to develop severe hypercholesterolemia with a cholesterol-rich diet due to the marked increase in hepatically and intestinally derived remnant lipoproteins, called β-very low density lipoproteins (VLDL), which are rich in cholesteryl esters. WHHL rabbits are characterized by elevated plasma LDL levels on a standard chow diet, which resembles human familial hypercholesterolemia. Therefore, both rabbit models develop aortic and coronary atherosclerosis, but the elevated plasma cholesterol levels are caused by completely different mechanisms. In addition, cholesterol-fed rabbits but not WHHL rabbits exhibit different degrees of hepatosteatosis. Recently, we along with others have shown that there are many differentially expressed genes in the atherosclerotic lesions and livers of cholesterol-fed rabbits that are either significantly up- or down-regulated, compared with those in normal rabbits, including genes involved in the regulation of inflammation and lipid metabolism. Therefore, dietary cholesterol plays an important role not only in hypercholesterolemia and atherosclerosis but also in hepatosteatosis. In this review, we make an overview of the recent progress in genomic and transcriptomic analyses of hypercholesterolemic rabbits. These transcriptomic profiling data should provide novel insight into the relationship between hypercholesterolemia and atherosclerosis or hepatic dysfunction caused by dietary cholesterol.

Editorial introductions

Editorial introductions

Measuring Human Lipid Metabolism Using Deuterium Labeling: In Vivo and In Vitro Protocols.

Stable isotopes are powerful tools for tracing the metabolic fate of molecules in the human body. In this chapter, we focus on the use of deuterium (2H), a stable isotope of hydrogen, in the study of human lipid metabolism within the liver in vivo in humans and in vitro using hepatocyte cellular models. The measurement of de novo lipogenesis (DNL) will be focussed on, as the synthesis of fatty acids, specifically palmitate, has been gathering momentum as being implicated in cellular dysfunction, which may be involved in the development of non-alcoholic fatty liver disease (NAFLD). Therefore, this chapter focusses specifically on the use of 2H2O (heavy water) to measure hepatic DNL.

German Environmental Specimen Bank: Urine Samples from 1999-2017 Document Rapid Increase in Exposure to the Para-Phthalate Plasticizer DEHTP

Di(2-ethylhexyl) terephthalate (DEHTP) is increasingly used as an alternative to reprotoxic ortho-phthalate plasticizers, e.g. di(2-ethylhexyl) phthalate (DEHP), in everyday products such as food contact materials, toys and medical devices. In Western Europe, DEHTP consumption rose from 2,000 t in 2002, to 100,000 t in 2014 and a predicted 135,000 t in 2019.To investigate for possible increases of DEHTP exposure in the German population over time, we analyzed 600 24-hour urine samples of the German Environmental Specimen Bank for specific DEHTP metabolites as exposure biomarkers. Samples were collected in the years 1999-2017 (ten sampling years, 30 male and 30 female volunteers each, aged 20-29). The samples were blinded and randomized before analysis. Four urinary DEHTP metabolites (side-chain oxidized monoesters) were determined by online SPE LC-MS/MS with isotope dilution quantification. The limits of quantification were 0.2 µg/L (5cx-MEPTP and 5oxo-MEHTP), 0.3 µg/L (5OH-MEHTP) and 0.4 µg/L (2cx-MMHTP).From 1999 until 2009 the main oxidized DEHTP metabolite 5cx-MEPTP was rarely detected. Afterwards, detection rates increased significantly (8% in 2009, 42% in 2011, 95% in 2013, 98% in 2015, and 100% in 2017). The median concentration for 5cx-MEPTP increased from &amp;lt;LOQ in 1999 to 3.35 µg/L in 2017. Detection rates and concentrations for the other metabolites were lower, as expected from human metabolism studies, but strongly correlated with 5cx-MEPTP. Overall, urinary 5cx-MEPTP concentrations were well below the health based guidance value (HBM-I) of 2800 µg/L, and calculated daily intakes were well below the tolerable daily intake of 1 mg/kg bw*d.For the first time, we document the increasing exposure to DEHTP of the German population in a robust set of 600 24-hour urine samples collected between 1999 and 2017. Although considered uncritical for now, the steep increase in DEHTP exposure calls for a continued exposure monitoring.

Spontaneous severe hypercholesterolemia and atherosclerosis lesions in rabbits with deficiency of low-density lipoprotein receptor (LDLR) on exon 7

Rabbits (Oryctolagus cuniculus) have been the very frequently used as animal models in the study of human lipid metabolism and atherosclerosis, because they have similar lipoprotein metabolism to humans. Most of hyperlipidemia and atherosclerosis rabbit models are produced by feeding rabbits a high-cholesterol diet. Gene editing or knockout (KO) offered another means of producing rabbit models for study of the metabolism of lipids and lipoproteins. Even so, apolipoprotein (Apo)E KO rabbits must be fed a high-cholesterol diet to induce hyperlipidemia.In this study, we used the CRISPR/Cas9 system anchored exon 7 of low-density lipoprotein receptor (LDLR) in an attempt to generate KO rabbits. We designed two sgRNA sequences located in E7:g.7055–7074 and E7:g.7102–7124 of rabbit LDLR gene, respectively. Seven LDLR-KO founder rabbits were generated, and all of them contained biallelic modifications. Various mutational LDLR amino acid sequences of the 7 founder rabbits were subjected to tertiary structure modeling with SWISS-MODEL, and results showed that the structure of EGF-A domain of each protein differs from the wild-type. All the founder rabbits spontaneously developed hypercholesterolemia and atherosclerosis on a normal chow (NC) diet. Analysis of their plasma lipids and lipoproteins at the age of 12 weeks revealed that all these KO rabbits exhibited markedly increased levels of plasma TC (the highest of which was 1013.15 mg/dl, 20-fold higher than wild-type rabbits), LDL-C (the highest of which was 730.00 mg/dl, 35-fold higher than wild-type rabbits) and TG accompanied by reduced HDL-C levels. Pathological examinations of a founder rabbit showed prominent aortic atherosclerosis lesions and coronary artery atherosclerosis.In conclusion, we have reported the generation LDLR-KO rabbit model for the study of spontaneous hypercholesterolemia and atherosclerosis on a NC diet. The LDLR-KO rabbits should be a useful rabbit model of human familial hypercholesterolemia (FH) for the simulations of human primary hypercholesterolemia and such models would allow more exact research into cardio-cerebrovascular disease.

My family and other animals: comparative requirements for human and animal metabolism studies

Insects used to treat organic waste streams and produce valuable protein products are increasingly exposed to plastic contaminated source material assimilating plastic carbon into organic biomass, which is pervasive and hazardous to organisms. Our understanding of this increased insect-plastic interaction remains limited and needs urgent scientific attention if plastic biodegradation and production rates of quality protein are to be improved. Herein, we investigated the biochemical impact of various plastics using three insect models. Black Soldier Fly (BSF), Mealworm (MW), and Wax Moth (WM) larva were each exposed to a plastic substrate (PET, PE, PS, Expanded PE, PP, and PLA) as the primary carbon source for five days to explore any positive metabolic benefits in terms of insect performance and plastic degradation potential. Central carbon metabolism (CCM) metabolites were analyzed via a targeted tMRM liquid chromatography triple quadrupole mass spectrometry (LC-QqQ-MS) method. Unique expressed pathways were observed for each insect model. When reared on PET, BSF larvae were found to have an elevated pyrimidine metabolism, while the purine metabolism pathway was strongly expressed on other plastics. BSF also exhibited a downregulated Vitamin B6 metabolism across all plastics, indicating a likely gut-symbiont breakdown. The MW and WM model insects were metabolically more active on PLA and expanded foam plastics. Further, WM exhibited an elevation in Vitamin B6 metabolism. This data suggests a positive insect-specific interaction towards certain plastic types that warrants further investigation. It is anticipated that through deeper insight into the metabolic impact and benefits afforded from certain plastics, an insect biotransformation pipeline can be established that links fit-for-purpose insect models to individual plastic types that address our growing plastic waste issue.

Cytochrome P450 1A2 Messenger RNA is a More Reliable Marker than Cytochrome P450 1A2 Activity, Phenacetin O-Deethylation, for Assessment of Induction Potential of Drug-Metabolizing Enzymes Using HepaRG Cells.

The HepaRG cells have key drug metabolism functionalities comparable to those of primary human hepatocytes. Many studies have reported that this cell line can be used as a reliable in vitro model for human drug metabolism studies, including the assessment of cytochrome P450 (CYP) induction. The objective of this study is to determine whether CYP mRNA level measurement is superior to the CYP enzyme activity measurement as a convenient high-throughput method for evaluating CYP induction potential using HepaRG cells. QuantiGene Plex 2.0 Assay and LC/MS/MS. mRNA expression levels and enzyme activities of CYP1A2, CYP2B6, and CYP3A in HepaRG cells treated with prototypical inducers of each CYP isoform [omeprazole (OME) for CYP1A2, phenobarbital (PB) for CYP2B6, and rifampicin (RIF) for CYP3A] were evaluated. Although the activities of CYP2B6 and CYP3A were induced by treatment with PB and RIF, we found that the activity of phenacetin O-deethylase (PHOD), which is known as a marker of the activity of CYP1A2, was also enhanced by treatment with these non-CYP1A2 inducers in HepaRG cells. Based on previously published reports, we hypothesized that the expression ratio of CYP3A to CYP1A2 is much higher in HepaRG cells than in human hepatocytes; this may result in a nonnegligible contribution of CYP3A to the PHOD reaction in HepaRG cells. Studies using CYP3A inhibitor and pregnane X receptor-knockout HepaRG cells supported this hypothesis. The measurement of mRNA serves as a higher reliable indicator for the evaluation of CYP induction potential when using HepaRG cells.

Unlocking Human Brain Metabolism by Genome-Scale and Multiomics Metabolic Models: Relevance for Neurology Research, Health, and Disease.

Neurology research and clinical practice are transforming toward postgenomics integrative biology. One such example is the study of human brain metabolism that is highly sophisticated due to reactions occurring in and between the astrocytes and neurons. Because of the inherent difficulty of performing experimental studies in human brain, metabolic network modeling has grown in importance to decipher the contribution of brain metabolite kinetics to human health and disease. Multiomics system science-driven metabolic models, using genome-scale and transcriptomics Big Data, offer the promise of new insights on metabolic networks in human brain. Added to this, the availability of omics technologies in both developed and developing world, neurology research, and clinical practice ought to be repositioned with a view to systems medicine. In this expert analysis, we present a critical and in-depth overview of the basic tenets of human brain metabolism, together with the most recent metabolic modeling strategies and computational studies of brain in health and neurological diseases. Human genome-scale metabolic models developed in a both global and brain-specific manner and multiomics synthesis of knowledge are highlighted in particular. We conclude by underscoring the value of multiomics modeling for metabolic diseases and computational investigations of the brain networks, with a view to unlocking the pathophysiology of Alzheimer's disease, Parkinson's disease, migraine, stroke, epilepsy, and multiple sclerosis, among other neurological disorders of importance for global health.

HEPATOKIN1 is a biochemistry-based model of liver metabolism for applications in medicine and pharmacology

The epidemic increase of non-alcoholic fatty liver diseases (NAFLD) requires a deeper understanding of the regulatory circuits controlling the response of liver metabolism to nutritional challenges, medical drugs, and genetic enzyme variants. As in vivo studies of human liver metabolism are encumbered with serious ethical and technical issues, we developed a comprehensive biochemistry-based kinetic model of the central liver metabolism including the regulation of enzyme activities by their reactants, allosteric effectors, and hormone-dependent phosphorylation. The utility of the model for basic research and applications in medicine and pharmacology is illustrated by simulating diurnal variations of the metabolic state of the liver at various perturbations caused by nutritional challenges (alcohol), drugs (valproate), and inherited enzyme disorders (galactosemia). Using proteomics data to scale maximal enzyme activities, the model is used to highlight differences in the metabolic functions of normal hepatocytes and malignant liver cells (adenoma and hepatocellular carcinoma).