Review on in vivo profiling of drug metabolites with LC-MS/MS in the past decade

Metabolite profiling is an indispensable part of drug discovery and development, enabling a comprehensive understanding of the drug's metabolic behavior. Liquid chromatography-mass spectrometry facilitates metabolite profiling by reducing sample complexity and providing high sensitivity. This review discusses the in vivo metabolite profiling involving LC-MS/MS and the utilization of QTOF, QQQ mass analyzers with a particular emphasis on a mass filter. Further, a summary of sample extraction procedures in biological matrices such as plasma, urine, feces, serum and hair as in vivo samples are outlined. toward the end, we present 15 case studies in biological matrices and their LC-MS/MS conditions to understand the metabolic disposition.

The Effect of a Host on the Primary Metabolic Profiling of Cuscuta Campestris’ Main Organs, Haustoria, Stem and Flower

Cuscuta campestris (dodder) is a stem holoparasitic plant without leaves or roots that parasitizes various types of host plants and causes damage to certain crops worldwide. This study aimed at gaining more knowledge about the effect of the hosts on the parasite’s levels of primary metabolites. To this end, metabolic profiling analyses were performed on the parasite’s three main organs, haustoria, stem and flowers, which developed on three hosts, Heliotropium hirsutissimum, Polygonum equisetiforme and Amaranthus viridis. The results showed significant differences in the metabolic profiles of C. campestris that developed on the different hosts, suggesting that the parasites rely highly on the host’s metabolites. However, changes in the metabolites’ contents between the organs that developed on the same host suggest that the parasite can also self-regulate its metabolites. Flowers, for example, have significantly higher levels of most of the amino acids and sugar acids, while haustoria and stem have higher levels of several sugars and polyols. Determination of total soluble proteins and phenolic compounds showed that the same pattern is detected in the organs unrelated to the hosts. This study contributes to our knowledge about the metabolic behavior of this parasite.

Hedgehog signaling can enhance glycolytic ATP production in the Drosophila wing disc

ABSTRACTEnergy production and utilization is critically important for animal development and growth. How it is regulated in space and time during tissue growth remains largely unclear. Toward this end, we used a FRET-based adenosine triphosphate (ATP) sensor to dynamically monitor ATP levels across a growing tissue, using the Drosophila wing disc. We discovered that steady-state levels of ATP are spatially uniform across the wing pouch. Pharmacologically inhibiting oxidative phosphorylation, however, reveals spatial heterogeneities in metabolic behavior, whereby signaling centers at compartment boundaries produce more ATP from glycolysis than the rest of the tissue. Genetic perturbations indicate that the conserved Hedgehog (Hh) signaling pathway can enhance ATP production by glycolysis. Collectively, our work reveals a positive feedback loop between Hh signaling and energy metabolism, advancing our understanding of the connection between conserved developmental patterning genes and energy production during animal tissue development.

Biotransformation of Timosaponin BII into Seven Characteristic Metabolites by the Gut Microbiota

Timosaponin BII is one of the most abundant Anemarrhena saponins and is in a phase II clinical trial for the treatment of dementia. However, the pharmacological activity of timosaponin BII does not match its low bioavailability. In this study, we aimed to determine the effects of gut microbiota on timosaponin BII metabolism. We found that intestinal flora had a strong metabolic effect on timosaponin BII by HPLC-MS/MS. At the same time, seven potential metabolites (M1-M7) produced by rat intestinal flora were identified using HPLC/MS-Q-TOF. Among them, three structures identified are reported in gut microbiota for the first time. A comparison of rat liver homogenate and a rat liver microsome incubation system revealed that the metabolic behavior of timosaponin BII was unique to the gut microbiota system. Finally, a quantitative method for the three representative metabolites was established by HPLC-MS/MS, and the temporal relationship among the metabolites was initially clarified. In summary, it is suggested that the metabolic characteristics of gut microbiota may be an important indicator of the pharmacological activity of timosaponin BII, which can be applied to guide its application and clinical use in the future.

Metabolic Profiling of VOCs Emitted by Bacteria Isolated from Pressure Ulcers and Treated with Different Concentrations of Bio-AgNPs

Considering the advent of antibiotic resistance, the study of bacterial metabolic behavior stimulated by novel antimicrobial agents becomes a relevant tool to elucidate involved adaptive pathways. Profiling of volatile metabolites was performed to monitor alterations of bacterial metabolism induced by biosynthesized silver nanoparticles (bio-AgNPs). Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae and Proteus mirabilis were isolated from pressure ulcers, and their cultures were prepared in the presence/absence of bio-AgNPs at 12.5, 25 and 50 µg mL−1. Headspace solid phase microextraction associated to gas chromatography–mass spectrometry was the employed analytical platform. At the lower concentration level, the agent promoted positive modulation of products of fermentation routes and bioactive volatiles, indicating an attempt of bacteria to adapt to an ongoing suppression of cellular respiration. Augmented response of aldehydes and other possible products of lipid oxidative cleavage was noticed for increasing levels of bio-AgNPs. The greatest concentration of agent caused a reduction of 44 to 80% in the variety of compounds found in the control samples. Pathway analysis indicated overall inhibition of amino acids and fatty acids routes. The present assessment may provide a deeper understanding of molecular mechanisms of bio-AgNPs and how the metabolic response of bacteria is untangled.

Understanding FBA Solutions under Multiple Nutrient Limitations

Genome-scale stoichiometric modeling methods, in particular Flux Balance Analysis (FBA) and variations thereof, are widely used to investigate cell metabolism and to optimize biotechnological processes. Given (1) a metabolic network, which can be reconstructed from an organism’s genome sequence, and (2) constraints on reaction rates, which may be based on measured nutrient uptake rates, FBA predicts which reactions maximize an objective flux, usually the production of cell components. Although FBA solutions may accurately predict the metabolic behavior of a cell, the actual flux predictions are often hard to interpret. This is especially the case for conditions with many constraints, such as for organisms growing in rich nutrient environments: it remains unclear why a certain solution was optimal. Here, we rationalize FBA solutions by explaining for which properties the optimal combination of metabolic strategies is selected. We provide a graphical formalism in which the selection of solutions can be visualized; we illustrate how this perspective provides a glimpse of the logic that underlies genome-scale modeling by applying our formalism to models of various sizes.

Mitochondrial metabolism supports resistance to IDH mutant inhibitors in acute myeloid leukemia

Mutations in IDH induce epigenetic and transcriptional reprogramming, differentiation bias, and susceptibility to mitochondrial inhibitors in cancer cells. Here, we first show that cell lines, PDXs, and patients with acute myeloid leukemia (AML) harboring an IDH mutation displayed an enhanced mitochondrial oxidative metabolism. Along with an increase in TCA cycle intermediates, this AML-specific metabolic behavior mechanistically occurred through the increase in electron transport chain complex I activity, mitochondrial respiration, and methylation-driven CEBPα-induced fatty acid β-oxidation of IDH1 mutant cells. While IDH1 mutant inhibitor reduced 2-HG oncometabolite and CEBPα methylation, it failed to reverse FAO and OxPHOS. These mitochondrial activities were maintained through the inhibition of Akt and enhanced activation of peroxisome proliferator-activated receptor-γ coactivator-1 PGC1α upon IDH1 mutant inhibitor. Accordingly, OxPHOS inhibitors improved anti-AML efficacy of IDH mutant inhibitors in vivo. This work provides a scientific rationale for combinatory mitochondrial-targeted therapies to treat IDH mutant AML patients, especially those unresponsive to or relapsing from IDH mutant inhibitors.

Antioxidant activity and cytotoxic effect of Chilean Buddleja globosa (Matico) and Ribes magellanicum (Zarzaparrilla) flower extracts

The native Chilean shrubs Buddleja globosa (Matico) and Ribes magellanicum (Zarzaparrilla) are used widely at a rural level, due to their medicinal properties. Nevertheless, little is known about their secondary metabolites and cytotoxic effect. The aim of this study was to evaluate the content of different compounds like catechin, epicatechin, p-coumaric acid and the antioxidant capacity by ABTS, ORAC, FRAP and DPPH methods. In addition, the cytotoxic activity of both extracts was evaluated against Chinese hamster ovary (CHO-K1) cell lines by MTT and neutral red assays. The results suggest that the most abundant constituent in Budleja globosa and Ribes magellanicum were catechin (682.43 mg/100 g DW) and epicatechin (3362.08 mg/100 g DW) respectively; while the ORAC methodology showed an elevated antioxidant activity for Matico (134147.31 μmol Trolox Eq/100 g DW). On the other hand, both extracts at the assayed concentrations affect the membrane stability and cellular metabolic capacity of the CHO-K1 cell lines. These finding provide a direction for further researches, and suggest the Matico and Zarzaparrilla flower extracts as promising sources of antioxidants, and as research objects through the analyze of their metabolic behavior and antitumoral potential.