Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing

Meissen, John K.; Pirman, David; Wan, Min; Miller, Emily; Jatkar, Aditi; Miller, Russell A.; Steenwyk, Rick C.; Blatnik, Matthew

doi:10.1016/j.ab.2016.06.019

Cited by 9 publications

(3 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore, the link between the urea and TCA cycles indirectly compensates for the three ATP molecules required for ureagenesis. Furthermore, oxaloacetate is transaminated with glutamate to yield α‐ketoglutarate and aspartate, which can then be reused in the urea cycle via the aspartate–argininosuccinate shunt, providing metabolic links between the urea cycle and TCA cycle (Meissen et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

Simultaneous activation of genes encoding urea cycle enzymes and gluconeogenetic enzymes coincides with a corticosterone surge period before metamorphosis in Xenopus laevis

Konno

2022

Dev Growth Differ

View full text Add to dashboard Cite

Amphibian tadpoles are postulated to excrete ammonia as nitrogen metabolites but to shift from ammonotelism to ureotelism during metamorphosis. However, it is unknown whether ureagenesis occurs or plays a functional role before metamorphosis. Here, the mRNA-expression levels of two urea cycle enzymes (carbamoyl phosphate synthetase I [CPSI] and ornithine transcarbamylase [OTC]) were measured beginning with stage-47 Xenopus tadpoles at 5 days post-fertilization (dpf), between the onset of feeding (stage 45, 4 dpf) and metamorphosis (stage 55, 32 dpf). CPSI and OTC expression levels increased significantly from stage 49 (12 dpf). Urea excretion was also detected at stage 47. A transient corticosterone surge peaking at stage 48 was previously reported, supporting the hypothesis that corticosterone can induce CPSI expression in tadpoles, as found in adult frogs and mammals. Stage-46 tadpoles were exposed to a synthetic glucocorticoid, dexamethasone (Dex, 10-500 nM) for 3 days. CPSI mRNA expression was significantly higher in tadpoles exposed to Dex than in tadpoles exposed to the vehicle control. Furthermore, glucocorticoid receptor mRNA expression increased during the pre-metamorphic period. In addition to CPSI and OTC mRNA upregulation, the expression levels of three gluconeogenic enzyme genes (glucose 6-phosphatase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase 1) increased with the onset of urea synthesis and excretion. These results suggest that simultaneous induction of the urea cycle and gluconeogenic enzymes coincided with a corticosterone surge occurring prior to metamorphosis. These metabolic changes preceding metamorphosis may be closely related to the onset of feeding and nutrient accumulation required for metamorphosis.

show abstract

Section: Discussionmentioning

confidence: 99%

Simultaneous activation of genes encoding urea cycle enzymes and gluconeogenetic enzymes coincides with a corticosterone surge period before metamorphosis in Xenopus laevis

Konno

2022

Dev Growth Differ

View full text Add to dashboard Cite

show abstract

“…25−27 For many compounds including sugars and hydroxyl acids, 28−31 GC−MS offers an easier route to chromatographic separation than liquid chromatography (LC)−MS unless specific procedures like ion-pairing LC are used. 32 to increase volatility, selectivity, and sensitivity, especially for metabolites bearing acidic protons. 30,33 A typical silylation reagent used for derivatization in stable isotope tracing is Nmethyl-N-tert-butyldimethylsilyltrifl uoroacetamide (MTBSTFA), which replaces nonsterically hindered active hydrogens (e.g., on −COOH, −OH, NH 2 , −HN, −SH groups) with tert-butyldimethylsilyl (TBDMS).…”

Section: ■ Introductionmentioning

confidence: 99%

“…Mass spectrometry (MS) has played a central role in metabolomics and stable isotope tracing studies . Gas chromatography with mass spectrometry (GC–MS) has been used for a long time for stable isotope labeling studies because many target compounds are directly amenable to GC–MS, including metabolites in the oxidative part of glycolysis, the tricarboxylic acid (TCA) cycle, and adjacent amino acids. − For many compounds including sugars and hydroxyl acids, − GC–MS offers an easier route to chromatographic separation than liquid chromatography (LC)–MS unless specific procedures like ion-pairing LC are used . Small molecules analyzed by GC–MS require derivatization to decrease their polarity and to increase volatility, selectivity, and sensitivity, especially for metabolites bearing acidic protons. , A typical silylation reagent used for derivatization in stable isotope tracing is N -methyl- N-tert -butyldimethylsilyltrifluoroacetamide (MTBSTFA), which replaces nonsterically hindered active hydrogens (e.g., on −COOH, −OH, NH 2 , −HN, −SH groups) with tert -butyldimethylsilyl (TBDMS).…”

Section: Introductionmentioning

confidence: 99%

Comparing Stable Isotope Enrichment by Gas Chromatography with Time-of-Flight, Quadrupole Time-of-Flight, and Quadrupole Mass Spectrometry

et al. 2021

View full text Add to dashboard Cite

Stable isotope tracers are applied for in vivo and in vitro studies to reveal the activity of enzymes and intracellular metabolic pathways. Most often, such tracers are used with gas chromatography coupled to mass spectrometry (GC−MS) owing to its ease of operation and reproducible mass spectral databases. Differences in isotope tracer performance of the classic GC− quadrupole MS instrument and newer time-of-flight instruments are not well studied. Here, we used three commercially available instruments for the analysis of identical samples from a stable isotope labeling study that used [U-13 C 6 ] D-glucose to investigate the metabolism of the bacterium Rothia mucilaginosa with respect to 29 amino acids and hydroxyl acids involved in primary metabolism. The prokaryote R. mucilaginosa belongs to the family of Micrococcaceae and is present and metabolically active in the airways and sputum of cystic fibrosis patients. Overall, all three GC−MS instruments (low-resolution GC-SQ MS, low-resolution GC-TOF MS, and high-resolution GC-QTOF MS) can be used to perform stable isotope tracing studies for glycolytic intermediates, tricarboxylic acid (TCA) metabolites, and amino acids, yielding similar biological results, with high-resolution GC-QTOF MS offering additional capabilities to identify the chemical structures of unknown compounds that might show significant isotope enrichments in biological studies.

show abstract

Deglucosylation of zearalenone-14-glucoside in animals and human liver leads to underestimation of exposure to zearalenone in humans

Yang

Zhang

Zhang³

et al. 2018

Arch Toxicol

View full text Add to dashboard Cite

Zearalenone-14-glucoside (ZEN-14G), the modified mycotoxin of zearalenone (ZEN), has attracted considerable attention due to its high potential to be hydrolyzed into ZEN, which would exert toxicity. It has been confirmed that the microflora could metabolize ZEN-14G to ZEN. However, the metabolic profile of ZEN-14G and whether it could be deglucosidated in the liver are unknown. To thoroughly investigate the metabolism of ZEN-14G, in vitro metabolism including phase I and phase II metabolism was studied using liquid chromatography coupled to high-resolution mass spectrometry. Additionally, in vivo metabolism of ZEN-14G was conducted in model animals, rats, by oral administration. As a result, 29 phase I metabolites and 6 phase II metabolites were identified and significant inter-species metabolic differences were observed as well. What is more, ZEN-14G could be considerably deglucosidated into its free form of ZEN after the incubation with animals and human liver microsomes in the absence of NADPH, which was mainly metabolized by human carboxylesterase CES-I and II. Furthermore, results showed that the major metabolic pathways of ZEN-14G were deglucosylation, hydroxylation, hydrogenation and glucuronidation. Although interspecies differences in the biotransformation of ZEN-14G were observed, ZEN, α-ZEL-14G, β-ZEL-14G, α-ZEL, ZEN-14G-16GlcA and ZEN-14GlcA were the major metabolites of ZEN-14G. Additionally, a larger yield of 6-OH-ZEN-14G and 8-OH-ZEN-14G was also observed in human liver microsomes. The obtained data would be of great importance for the safety assessment of modified mycotoxin, ZEN-14G, and provide another perspective for risk assessment of mycotoxin.

show abstract

Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing

Cited by 9 publications

References 33 publications

Simultaneous activation of genes encoding urea cycle enzymes and gluconeogenetic enzymes coincides with a corticosterone surge period before metamorphosis in Xenopus laevis

Simultaneous activation of genes encoding urea cycle enzymes and gluconeogenetic enzymes coincides with a corticosterone surge period before metamorphosis in Xenopus laevis

Comparing Stable Isotope Enrichment by Gas Chromatography with Time-of-Flight, Quadrupole Time-of-Flight, and Quadrupole Mass Spectrometry

Deglucosylation of zearalenone-14-glucoside in animals and human liver leads to underestimation of exposure to zearalenone in humans

Contact Info

Product

Resources

About