Bayesian 13C-Metabolic Flux Analysis of Parallel Tracer Experiments in Granulocytes: A Directional Shift within the Non-Oxidative Pentose Phosphate Pathway Supports Phagocytosis

Abstract

The pentose phosphate pathway (PPP) plays a key role in the cellular regulation of immune function; however, little is known about the interplay of metabolic adjustments in granulocytes, especially regarding the non-oxidative PPP. For the determination of metabolic mechanisms within glucose metabolism, we propose a novel set of measures for 13C-metabolic flux analysis based on ex vivo parallel tracer experiments ([1,2-13C]glucose, [U-13C]glucose, [4,5,6-13C]glucose) and gas chromatography–mass spectrometry labeling measurements of intracellular metabolites, such as sugar phosphates and their fragments. A detailed constraint analysis showed that the permission range for net and irreversible fluxes was limited to a three-dimensional space. The overall workflow, including its Bayesian flux estimation, resulted in precise flux distributions and pairwise confidence intervals, some of which could be represented as a line due to the strength of their correlation. The principal component analysis that was enabled by these behaviors comprised three components that explained 99.6% of the data variance. It showed that phagocytic stimulation reversed the direction of non-oxidative PPP net fluxes from ribose-5-phosphate biosynthesis toward glycolytic pathways. This process was closely associated with the up-regulation of the oxidative PPP to promote the oxidative burst.

13C-Metabolic flux analysis detected a hyperoxemia-induced reduction of tricarboxylic acid cycle metabolism in granulocytes during two models of porcine acute subdural hematoma and hemorrhagic shock

“…SIM methods, device settings, and oven programs are mentioned in the Supplementary File . Peak area integration was performed with our in-house program ( 28 ). Mass isotopomer distributions (MIDs) were converted into carbon mass distributions (CMDs) by deducting isotopic interferences with a correction matrix approach ( 20 , 29 ).…”

Ex Vivo 13C-Metabolic Flux Analysis of Porcine Circulating Immune Cells Reveals Cell Type-Specific Metabolic Patterns and Sex Differences in the Pentose Phosphate Pathway

“…The water/methanol-acetonitrile extraction was performed as recently described in Hogg et al [33]. Briefly, the frozen pellet was extracted with 100 µL ice-cold Ampuwa water, vortexed vigorously, and sonicated in an ice bath for 10 min.…”

“…The ethoxime-trimethylsilyl derivatization (EtOx-TMS) and following GC-MS analysis were performed as recently described [33]. Briefly, the dried cell extracts and at least five staggered levels of reference standard mix containing all the monitored metabolites (∑19; list is available in the Supplementary Material) were dissolved in 50 µL derivatization reagent (ethoxyamine hydrochloride in pyridine (2%, w/v) (Sigma-Aldrich, St. Louis, MO, USA) and sonicated for 10 min at room temperature.…”

“…Recently, we proposed a novel GC-MS-based Bayesian 13 C-MFA for precise determination of glucose metabolism that enables the simultaneous quantification of glycolytic, PPP, and TCA cycle fluxes ex vivo. The Bayesian approach additionally provides pairwise correlations of fluxes with their joint confidence regions, which provide valuable information regarding cellular mechanisms [33].…”

“…In this study, we applied our Bayesian 13 C-MFA to unstimulated PBMCs and granulocytes from healthy pigs of either sex for characterization of their immunometabolism [33,34]. Furthermore, we included the total oxygen consumption and the O 2…”