Crym-positive striatal astrocytes gate perseverative behaviour.

Astrocytes are heterogeneous glial cells of the central nervous system1-3. However, the physiological relevance of astrocyte diversity for neural circuits and behaviour remains unclear. Here we show that a specific population of astrocytes in the central striatum expresses µ-crystallin (encoded by Crym in mice and CRYM in humans) that is associated with several human diseases, including neuropsychiatric disorders4-7. In adult mice, reducing the levels of µ-crystallin in striatal astrocytes through CRISPR-Cas9-mediated knockout of Crym resulted in perseverative behaviours, increased fast synaptic excitation in medium spiny neurons and dysfunctional excitatory-inhibitory synaptic balance. Increased perseveration stemmed from the loss of astrocyte-gated control of neurotransmitter release from presynaptic terminals of orbitofrontal cortex-striatum projections. We found that perseveration could be remedied using presynaptic inhibitory chemogenetics8, and that this treatment also corrected the synaptic deficits. Together, our findings reveal converging molecular, synaptic, circuit and behavioural mechanisms by which a molecularly defined and allocated population of striatal astrocytes gates perseveration phenotypes that accompany neuropsychiatric disorders9-12. Our data show that Crym-positive striatal astrocytes have key biological functions within the central nervous system, and uncover astrocyte-neuron interaction mechanisms that could be targeted in treatments for perseveration.

Mitochondrial morphology controls fatty acid utilization by changing CPT1 sensitivity to malonyl-CoA.

Changes in mitochondrial morphology are associated with nutrient utilization, but the precise causalities and the underlying mechanisms remain unknown. Here, using cellular models representing a wide variety of mitochondrial shapes, we show a strong linear correlation between mitochondrial fragmentation and increased fatty acid oxidation (FAO) rates. Forced mitochondrial elongation following MFN2 over-expression or DRP1 depletion diminishes FAO, while forced fragmentation upon knockdown or knockout of MFN2 augments FAO as evident from respirometry and metabolic tracing. Remarkably, the genetic induction of fragmentation phenocopies distinct cell type-specific biological functions of enhanced FAO. These include stimulation of gluconeogenesis in hepatocytes, induction of insulin secretion in islet ß-cells exposed to fatty acids, and survival of FAO-dependent lymphoma subtypes. We find that fragmentation increases long-chain but not short-chain FAO, identifying carnitine O-palmitoyltransferase 1 (CPT1) as the downstream effector of mitochondrial morphology in regulation of FAO. Mechanistically, we determined that fragmentation reduces malonyl-CoA inhibition of CPT1, while elongation increases CPT1 sensitivity to malonyl-CoA inhibition. Overall, these findings underscore a physiologic role for fragmentation as a mechanism whereby cellular fuel preference and FAO capacity are determined.

Extreme shifts in pyrite sulfur isotope compositions reveal the path to bonanza gold.

Pyrite is the most common sulfide mineral in hydrothermal ore-forming systems. The ubiquity and abundance of pyrite, combined with its ability to record and preserve a history of fluid evolution in crustal environments, make it an ideal mineral for studying the genesis of hydrothermal ore deposits, including those that host critical metals. However, with the exception of boiling, few studies have been able to directly link changes in pyrite chemistry to the processes responsible for bonanza-style gold mineralization. Here, we report the results of high-resolution secondary-ion mass spectrometry and electron microprobe analyses conducted on pyrite from the Brucejack epithermal gold deposit, British Columbia. Our δ34S and trace element results reveal that the Brucejack hydrothermal system experienced abrupt fluctuations in fluid chemistry, which preceded and ultimately coincided with the onset of ultra-high-grade mineralization. We argue that these fluctuations, which include the occurrence of extraordinarily negative δ34S values (e.g., -36.1‰) in zones of auriferous, arsenian pyrite, followed by sharp increases of δ34S values in syn-electrum zones of nonarsenian pyrite, were caused by vigorous, fault valve-induced episodic boiling (flashing) and subsequent inundation of the hydrothermal system by seawater. We conclude that the influx of seawater was the essential step to forming bonanza-grade electrum mineralization by triggering, through the addition of cationic flocculants and cooling, the aggregation of colloidal gold suspensions. Moreover, our study demonstrates the efficacy of employing high-resolution, in situ analytical techniques to map out individual ore-forming events in a hydrothermal system.

The BCKDK inhibitor BT2 is a chemical uncoupler that lowers mitochondrial ROS production and de novo lipogenesis.

Elevated levels of branched chain amino acids (BCAAs) and branched-chain α-ketoacids are associated with cardiovascular and metabolic disease, but the molecular mechanisms underlying a putative causal relationship remain unclear. The branched-chain ketoacid dehydrogenase kinase (BCKDK) inhibitor BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid) is often used in preclinical models to increase BCAA oxidation and restore steady-state BCAA and branched-chain α-ketoacid levels. BT2 administration is protective in various rodent models of heart failure and metabolic disease, but confoundingly, targeted ablation of Bckdk in specific tissues does not reproduce the beneficial effects conferred by pharmacologic inhibition. Here, we demonstrate that BT2, a lipophilic weak acid, can act as a mitochondrial uncoupler. Measurements of oxygen consumption, mitochondrial membrane potential, and patch-clamp electrophysiology show that BT2 increases proton conductance across the mitochondrial inner membrane independently of its inhibitory effect on BCKDK. BT2 is roughly sixfold less potent than the prototypical uncoupler 2,4-dinitrophenol and phenocopies 2,4-dinitrophenol in lowering de novo lipogenesis and mitochondrial superoxide production. The data suggest that the therapeutic efficacy of BT2 may be attributable to the well-documented effects of mitochondrial uncoupling in alleviating cardiovascular and metabolic disease.

Calculation of ATP production rates using the Seahorse XF Analyzer.

Oxidative phosphorylation and glycolysis are the dominant ATP-generating pathways in mammalian metabolism. The balance between these two pathways is often shifted to execute cell-specific functions in response to stimuli that promote activation, proliferation, or differentiation. However, measurement of these metabolic switches has remained mostly qualitative, making it difficult to discriminate between healthy, physiological changes in energy transduction or compensatory responses due to metabolic dysfunction. We therefore present a broadly applicable method to calculate ATP production rates from oxidative phosphorylation and glycolysis using Seahorse XF Analyzer data and empirical conversion factors. We quantify the bioenergetic changes observed during macrophage polarization as well as cancer cell adaptation to in vitro culture conditions. Additionally, we detect substantive changes in ATP utilization upon neuronal depolarization and T cell receptor activation that are not evident from steady-state ATP measurements. This method generates a single readout that allows the direct comparison of ATP produced from oxidative phosphorylation and glycolysis in live cells. Additionally, the manuscript provides a framework for tailoring the calculations to specific cell systems or experimental conditions.

Colloidal transport and flocculation are the cause of the hyperenrichment of gold in nature.

Aqueous complexation has long been considered the only viable means of transporting gold to depositional sites in hydrothermal ore-forming systems. A major weakness of this hypothesis is that it cannot readily explain the formation of ultrahigh-grade gold veins. This is a consequence of the relatively low gold concentrations typical of ore fluids (tens of parts per billion [ppb]) and the fact that these "bonanza" veins can contain weight-percent levels of gold in some epithermal and orogenic deposits. Here, we present direct evidence for a hypothesis that could explain these veins, namely, the transport of the gold as colloidal particles and their flocculation in nanoscale calcite veinlets. These gold-bearing nanoveinlets bear a remarkable resemblance to centimeter-scale ore veins in many hydrothermal gold deposits and give unique insight into the scale invariability of colloidal flocculation in forming hyperenriched gold deposits. Using this evidence, we propose a model for the development of bonanza gold veins in high-grade deposits. We argue that gold transport in these systems is largely mechanical and is the result of exceptionally high degrees of supersaturation that preclude precipitation of gold crystals and instead lead to the formation of colloidal particles, which flocculate and form much larger masses. These flocculated masses aggregate locally, where they are seismically pumped into fractures to locally form veins composed largely of gold. This model explains how bonanza veins may form from fluids containing ppb concentrations of gold and does not require prior encapsulation of colloidal gold particles in silica gel, as proposed by previous studies.

Blocking mitochondrial pyruvate import in brown adipocytes induces energy wasting via lipid cycling.

Combined fatty acid esterification and lipolysis, termed lipid cycling, is an ATP-consuming process that contributes to energy expenditure. Therefore, interventions that stimulate energy expenditure through lipid cycling are of great interest. Here we find that pharmacological and genetic inhibition of the mitochondrial pyruvate carrier (MPC) in brown adipocytes activates lipid cycling and energy expenditure, even in the absence of adrenergic stimulation. We show that the resulting increase in ATP demand elevates mitochondrial respiration coupled to ATP synthesis and fueled by lipid oxidation. We identify that glutamine consumption and the Malate-Aspartate Shuttle are required for the increase in Energy Expenditure induced by MPC inhibition in Brown Adipocytes (MAShEEBA). We thus demonstrate that energy expenditure through enhanced lipid cycling can be activated in brown adipocytes by decreasing mitochondrial pyruvate availability. We present a new mechanism to increase energy expenditure and fat oxidation in brown adipocytes, which does not require adrenergic stimulation of mitochondrial uncoupling.

Forces, fluxes, and fuels: tracking mitochondrial metabolism by integrating measurements of membrane potential, respiration, and metabolites.

Assessing mitochondrial function in cell-based systems is a central component of metabolism research. However, the selection of an initial measurement technique may be complicated given the range of parameters that can be studied and the need to define the mitochondrial (dys)function of interest. This methods-focused review compares and contrasts the use of mitochondrial membrane potential measurements, plate-based respirometry, and metabolomics and stable isotope tracing. We demonstrate how measurements of 1) cellular substrate preference, 2) respiratory chain activity, 3) cell activation, and 4) mitochondrial biogenesis are enriched by integrating information from multiple methods. This manuscript is meant to serve as a perspective to help choose which technique might be an appropriate initial method to answer a given question, as well as provide a broad "roadmap" for designing follow-up assays to enrich datasets or resolve ambiguous results.

ACE overexpression in myeloid cells increases oxidative metabolism and cellular ATP.

Angiotensin-converting enzyme (ACE) affects blood pressure. In addition, ACE overexpression in myeloid cells increases their immune function. Using MS and chemical analysis, we identified marked changes of intermediate metabolites in ACE-overexpressing macrophages and neutrophils, with increased cellular ATP (1.7-3.0-fold) and Krebs cycle intermediates, including citrate, isocitrate, succinate, and malate (1.4-3.9-fold). Increased ATP is due to ACE C-domain catalytic activity; it is reversed by an ACE inhibitor but not by an angiotensin II AT1 receptor antagonist. In contrast, macrophages from ACE knockout (null) mice averaged only 28% of the ATP levels found in WT mice. ACE overexpression does not change cell or mitochondrial size or number. However, expression levels of the electron transport chain proteins NDUFB8 (complex I), ATP5A, and ATP5ß (complex V) are significantly increased in macrophages and neutrophils, and COX1 and COX2 (complex IV) are increased in macrophages overexpressing ACE. Macrophages overexpressing ACE have increased mitochondrial membrane potential (24% higher), ATP production rates (29% higher), and maximal respiratory rates (37% higher) compared with WT cells. Increased cellular ATP underpins increased myeloid cell superoxide production and phagocytosis associated with increased ACE expression. Myeloid cells overexpressing ACE indicate the existence of a novel pathway in which myeloid cell function can be enhanced, with a key feature being increased cellular ATP.

Steel slag carbonation in a flow-through reactor system: the role of fluid-flux.

Steel production is currently the largest industrial source of atmospheric CO2. As annual steel production continues to grow, the need for effective methods of reducing its carbon footprint increases correspondingly. The carbonation of the calcium-bearing phases in steel slag generated during basic oxygen furnace (BOF) steel production, in particular its major constituent, larnite {Ca2SiO4}, which is a structural analogue of olivine {(MgFe)2SiO4}, the main mineral subjected to natural carbonation in peridotites, offers the potential to offset some of these emissions. However, the controls on the nature and efficiency of steel slag carbonation are yet to be completely understood. Experiments were conducted exposing steel slag grains to a CO2-H2O mixture in both batch and flow-through reactors to investigate the impact of temperature, fluid flux, and reaction gradient on the dissolution and carbonation of steel slag. The results of these experiments show that dissolution and carbonation of BOF steel slag are more efficient in a flow-through reactor than in the batch reactors used in most previous studies. Moreover, they show that fluid flux needs to be optimized in addition to grain size, pressure, and temperature, in order to maximize the efficiency of carbonation. Based on these results, a two-stage reactor consisting of a high and a low fluid-flux chamber is proposed for CO2 sequestration by steel slag carbonation, allowing dissolution of the slag and precipitation of calcium carbonate to occur within a single flow-through system.

The metabolic cofactor Coenzyme A enhances alternative macrophage activation via MyD88-linked signaling.

Metabolites and metabolic co-factors can shape the innate immune response, though the pathways by which these molecules adjust inflammation remain incompletely understood. Here we show that the metabolic cofactor Coenzyme A (CoA) enhances IL-4 driven alternative macrophage activation [m(IL-4)] in vitro and in vivo. Unexpectedly, we found that perturbations in intracellular CoA metabolism did not influence m(IL-4) differentiation. Rather, we discovered that exogenous CoA provides a weak TLR4 signal which primes macrophages for increased receptivity to IL-4 signals and resolution of inflammation via MyD88. Mechanistic studies revealed MyD88-linked signals prime for IL-4 responsiveness, in part, by reshaping chromatin accessibility to enhance transcription of IL-4-linked genes. The results identify CoA as a host metabolic co-factor that influences macrophage function through an extrinsic TLR4-dependent mechanism, and suggests that damage-associated molecular patterns (DAMPs) can prime macrophages for alternative activation and resolution of inflammation.

Peroxisome proliferator-activated receptor alpha is essential factor in enhanced macrophage immune function induced by angiotensin converting enzyme.

An upregulation of angiotensin-converting enzyme (ACE) expression strengthens the immune activity of myeloid lineage cells as a natural functional regulation mechanism in our immunity. ACE10/10 mice, possessing increased ACE expression in macrophages, exhibit enhanced anti-tumor immunity and anti-bactericidal effects compared to those of wild type (WT) mice, while the detailed molecular mechanism has not been elucidated yet. In this report, we demonstrate that peroxisome proliferator-activated receptor alpha (PPARα) is a key molecule in the functional upregulation of macrophages induced by ACE. The expression of PPARα, a transcription factor regulating fatty acid metabolism-associated gene expressions, was upregulated in ACE-overexpressing macrophages. To pinpoint the role of PPARα in the enhanced immune function of ACE-overexpressing macrophages, we established a line with myeloid lineage-selective PPARα depletion employing the Lysozyme 2 (LysM)-Cre system based on ACE 10/10 mice (named A10-PPARα-Cre). Interestingly, A10-PPARα-Cre mice exhibited larger B16-F10-originated tumors than original ACE 10/10 mice. PPARα depletion impaired cytokine production and antigen-presenting activity in ACE-overexpressing macrophages, resulting in reduced tumor antigen-specific CD8+ T cell activity. Additionally, the anti-bactericidal effect was also impaired in A10-PPARα-Cre mice, resulting in similar bacterial colonization to WT mice in Methicillin-Resistant Staphylococcus aureus (MRSA) infection. PPARα depletion downregulated phagocytic activity and bacteria killing in ACE-overexpressing macrophages. Moreover, THP-1-ACE-derived macrophages, as a human model, expressing upregulated PPARα exhibited enhanced cytotoxicity against B16-F10 cells and MRSA killing. These activities were further enhanced by the PPARα agonist, WY 14643, while abolished by the antagonist, GW6471, in THP-1-ACE cells. Thus, PPARα is an indispensable molecule in ACE-dependent functional upregulation of macrophages in both mice and humans.

Pro-inflammatory macrophage activation does not require inhibition of mitochondrial respiration.

Pro-inflammatory macrophage activation is a hallmark example of how mitochondria serve as signaling organelles. Upon classical macrophage activation, oxidative phosphorylation sharply decreases and mitochondria are repurposed to accumulate signals that amplify effector function. However, evidence is conflicting as to whether this collapse in respiration is essential or largely dispensable. Here we systematically examine this question and show that reduced oxidative phosphorylation is not required for pro-inflammatory macrophage activation. Only stimuli that engage both MyD88- and TRIF-linked pathways decrease mitochondrial respiration, and different pro-inflammatory stimuli have varying effects on other bioenergetic parameters. Additionally, pharmacologic and genetic models of electron transport chain inhibition show no direct link between respiration and pro-inflammatory activation. Studies in mouse and human macrophages also reveal accumulation of the signaling metabolites succinate and itaconate can occur independently of characteristic breaks in the TCA cycle. Finally, in vivo activation of peritoneal macrophages further demonstrates that a pro-inflammatory response can be elicited without reductions to oxidative phosphorylation. Taken together, the results suggest the conventional model of mitochondrial reprogramming upon macrophage activation is incomplete.

The role of an oxidized lithospheric mantle in gold mobilization.

Phanerozoic orogenic gold mineralization at craton margins is related to the metasomatism of the lithospheric mantle by crustal material. Slab subduction transfers Au from the crust to the metasomatized mantle and oxidizes the latter to facilitate the mobilization of Au into mantle melts. The role of volatiles in the mobilization of Au in the mantle is unclear because of the absence of direct geochemical evidence relating the mantle source of Au to Au mineralization in the overlying crust. This study uses lithium isotopes from a large suite of lamprophyres to characterize the mantle beneath the eastern North China Craton, which hosts giant Mesozoic gold deposits. Our results indicate a strong genetic link between carbonate metasomatism in the mantle and Au mineralization in the overlying crust. Although pre-enrichment of Au in the mantle is critical for forming giant Au provinces, the oxidation of the lithospheric mantle controls the mobilization of Au.

Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome.

Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxICAT, Biotin Switch, and SP3-Rox, these methods typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. Here we establish the local cysteine capture (Cys-LoC) and local cysteine oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole-cell proteomic analysis. Application of the Cys-LOx method to LPS-stimulated immortalized murine bone marrow-derived macrophages (iBMDM), revealed previously unidentified, mitochondrially localized cysteine oxidative modifications upon pro-inflammatory activation, including those associated with oxidative mitochondrial metabolism.

Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome.

Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxiCat, Biotin Switch, and SP3-Rox, they typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. To obviate requirements for laborious biochemical fractionation, here, we develop and apply an unprecedented two step cysteine capture method to establish the Local Cysteine Capture (Cys-LoC), and Local Cysteine Oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole cell proteomic analysis. Application of the Cys-LOx method to LPS stimulated murine immortalized bone marrow-derived macrophages (iBMDM), revealed previously unidentified mitochondria-specific inflammation-induced cysteine oxidative modifications including those associated with oxidative phosphorylation. These findings shed light on post-translational mechanisms regulating mitochondrial function during the cellular innate immune response.

The BCKDK inhibitor BT2 is a chemical uncoupler that lowers mitochondrial ROS production and de novo lipogenesis.

Elevated levels of branched chain amino acids (BCAAs) and branched-chain α-ketoacids (BCKAs) are associated with cardiovascular and metabolic disease, but the molecular mechanisms underlying a putative causal relationship remain unclear. The branched-chain ketoacid dehydrogenase kinase (BCKDK) inhibitor BT2 is often used in preclinical models to increase BCAA oxidation and restore steady-state BCAA and BCKA levels. BT2 administration is protective in various rodent models of heart failure and metabolic disease, but confoundingly, targeted ablation of Bckdk in specific tissues does not reproduce the beneficial effects conferred by pharmacologic inhibition. Here we demonstrate that BT2, a lipophilic weak acid, can act as a mitochondrial uncoupler. Measurements of oxygen consumption, mitochondrial membrane potential, and patch-clamp electrophysiology show BT2 increases proton conductance across the mitochondrial inner membrane independently of its inhibitory effect on BCKDK. BT2 is roughly five-fold less potent than the prototypical uncoupler 2,4-dinitrophenol (DNP), and phenocopies DNP in lowering de novo lipogenesis and mitochondrial superoxide production. The data suggest the therapeutic efficacy of BT2 may be attributable to the well-documented effects of mitochondrial uncoupling in alleviating cardiovascular and metabolic disease.

MYC is a regulator of androgen receptor inhibition-induced metabolic requirements in prostate cancer.

Advanced prostate cancers are treated with therapies targeting the androgen receptor (AR) signaling pathway. While many tumors initially respond to AR inhibition, nearly all develop resistance. It is critical to understand how prostate tumor cells respond to AR inhibition in order to exploit therapy-induced phenotypes prior to the outgrowth of treatment-resistant disease. Here, we comprehensively characterize the effects of AR blockade on prostate cancer metabolism using transcriptomics, metabolomics, and bioenergetics approaches. The metabolic response to AR inhibition is defined by reduced glycolysis, robust elongation of mitochondria, and increased reliance on mitochondrial oxidative metabolism. We establish DRP1 activity and MYC signaling as mediators of AR-blockade-induced metabolic phenotypes. Rescuing DRP1 phosphorylation after AR inhibition restores mitochondrial fission, while rescuing MYC restores glycolytic activity and prevents sensitivity to complex I inhibition. Our study provides insight into the regulation of treatment-induced metabolic phenotypes and vulnerabilities in prostate cancer.

Macrophage COX2 Mediates Efferocytosis, Resolution Reprogramming, and Intestinal Epithelial Repair.

BACKGROUND AND AIMS: Phagocytosis (efferocytosis) of apoptotic neutrophils by macrophages anchors the resolution of intestinal inflammation. Efferocytosis prevents secondary necrosis and inhibits further inflammation, and also reprograms macrophages to facilitate tissue repair and promote resolution function. Macrophage efferocytosis and efferocytosis-dependent reprogramming are implicated in the pathogenesis of inflammatory bowel disease. We previously reported that absence of macrophage cyclooxygenase 2 (COX2) exacerbates inflammatory bowel disease-like intestinal inflammation. To elucidate the underlying pathogenic mechanism, we investigated here whether COX2 mediates macrophage efferocytosis and efferocytosis-dependent reprogramming, including intestinal epithelial repair capacity. METHODS: Using apoptotic neutrophils and synthetic apoptotic targets, we determined the effects of macrophage specific Cox2 knockout and pharmacological COX2 inhibition on the efferocytosis capacity of mouse primary macrophages. COX2-mediated efferocytosis-dependent eicosanoid lipidomics was determined by liquid chromatography tandem mass spectrometry. Small intestinal epithelial organoids were employed to assay the effects of COX2 on efferocytosis-dependent intestinal epithelial repair. RESULTS: Loss of COX2 impaired efferocytosis in mouse primary macrophages, in part, by affecting the binding capacity of macrophages for apoptotic cells. This effect was comparable to that of high-dose lipopolysaccharide and was accompanied by both dysregulation of macrophage polarization and the inhibited expression of genes involved in apoptotic cell binding. COX2 modulated the production of efferocytosis-dependent lipid inflammatory mediators that include the eicosanoids prostaglandin I2, prostaglandin E2, lipoxin A4, and 15d-PGJ2; and further affected secondary efferocytosis. Finally, macrophage efferocytosis induced, in a macrophage COX2-dependent manner, a tissue restitution and repair phenotype in intestinal epithelial organoids. CONCLUSIONS: Macrophage COX2 potentiates efferocytosis capacity and efferocytosis-dependent reprogramming, facilitating macrophage intestinal epithelial repair capacity.

A Single LC-MS/MS Analysis to Quantify CoA Biosynthetic Intermediates and Short-Chain Acyl CoAs.

Coenzyme A (CoA) is an essential cofactor for dozens of reactions in intermediary metabolism. Dysregulation of CoA synthesis or acyl CoA metabolism can result in metabolic or neurodegenerative disease. Although several methods use liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS) to quantify acyl CoA levels in biological samples, few allow for simultaneous measurement of intermediates in the CoA biosynthetic pathway. Here we describe a simple sample preparation and LC-MS/MS method that can measure both short-chain acyl CoAs and biosynthetic precursors of CoA. The method does not require use of a solid phase extraction column during sample preparation and exhibits high sensitivity, precision, and accuracy. It reproduces expected changes from known effectors of cellular CoA homeostasis and helps clarify the mechanism by which excess concentrations of etomoxir reduce intracellular CoA levels.