Proximity proteomic analysis of the NRF family reveals the Parkinson's disease protein ZNF746/PARIS as a co-complexed repressor of NRF2.

The nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor activates cytoprotective and metabolic gene expression in response to various electrophilic stressors. Constitutive NRF2 activity promotes cancer progression, whereas decreased NRF2 function contributes to neurodegenerative diseases. We used proximity proteomic analysis to define protein networks for NRF2 and its family members NRF1, NRF3, and the NRF2 heterodimer MAFG. A functional screen of co-complexed proteins revealed previously uncharacterized regulators of NRF2 transcriptional activity. We found that ZNF746 (also known as PARIS), a zinc finger transcription factor implicated in Parkinson's disease, physically associated with NRF2 and MAFG, resulting in suppression of NRF2-driven transcription. ZNF746 overexpression increased oxidative stress and apoptosis in a neuronal cell model of Parkinson's disease, phenotypes that were reversed by chemical and genetic hyperactivation of NRF2. This study presents a functionally annotated proximity network for NRF2 and suggests a link between ZNF746 overexpression in Parkinson's disease and inhibition of NRF2-driven neuroprotection.

Hepatic palmitoyl-proteomes and acyl-protein thioesterase protein proximity networks link lipid modification and mitochondria.

Acyl-protein thioesterases 1 and 2 (APT1 and APT2) reverse S-acylation, a potential regulator of systemic glucose metabolism in mammals. Palmitoylation proteomics in liver-specific knockout mice shows that APT1 predominates over APT2, primarily depalmitoylating mitochondrial proteins, including proteins linked to glutamine metabolism. miniTurbo-facilitated determination of the protein-protein proximity network of APT1 and APT2 in HepG2 cells reveals APT proximity networks encompassing mitochondrial proteins including the major translocases Tomm20 and Timm44. APT1 also interacts with Slc1a5 (ASCT2), the only glutamine transporter known to localize to mitochondria. High-fat-diet-fed male mice with dual (but not single) hepatic deletion of APT1 and APT2 have insulin resistance, fasting hyperglycemia, increased glutamine-driven gluconeogenesis, and decreased liver mass. These data suggest that APT1 and APT2 regulation of hepatic glucose metabolism and insulin signaling is functionally redundant. Identification of substrates and protein-protein proximity networks for APT1 and APT2 establishes a framework for defining mechanisms underlying metabolic disease.

Protein proximity networks and functional evaluation of the casein kinase 1 gamma family reveal unique roles for CK1γ3 in WNT signaling.

Aberrant activation or suppression of WNT/ß-catenin signaling contributes to cancer initiation and progression, neurodegeneration, and bone disease. However, despite great need and more than 40 years of research, targeted therapies for the WNT pathway have yet to be fully realized. Kinases are considered exceptionally druggable and occupy key nodes within the WNT signaling network, but several pathway-relevant kinases remain understudied and "dark." Here, we studied the function of the casein kinase 1γ (CSNK1γ) subfamily of human kinases and their roles in WNT signaling. miniTurbo-based proximity biotinylation and mass spectrometry analysis of CSNK1γ1, CSNK1γ2, and CSNK1γ3 revealed numerous components of the ß-catenin-dependent and ß-catenin-independent WNT pathways. In gain-of-function experiments, we found that CSNK1γ3 but not CSNK1γ1 or CSNK1γ2 activated ß-catenin-dependent WNT signaling, with minimal effect on other signaling pathways. We also show that within the family, CSNK1γ3 expression uniquely induced low-density lipoprotein receptor-related protein 6 phosphorylation, which mediates downstream WNT signaling transduction. Conversely, siRNA-mediated silencing of CSNK1γ3 alone had no impact on WNT signaling, though cosilencing of all three family members decreased WNT pathway activity. Finally, we characterized two moderately selective and potent small-molecule inhibitors of the CSNK1γ family. We show that these inhibitors and a CSNK1γ3 kinase-dead mutant suppressed but did not eliminate WNT-driven low-density lipoprotein receptor-related protein 6 phosphorylation and ß-catenin stabilization. Our data suggest that while CSNK1γ3 expression uniquely drives pathway activity, potential functional redundancy within the family necessitates loss of all three family members to suppress the WNT signaling pathway.

Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice.

A wide range of protein acyl modifications has been identified on enzymes across various metabolic processes; however, the impact of these modifications remains poorly understood. Protein glutarylation is a recently identified modification that can be nonenzymatically driven by glutaryl-CoA. In mammalian systems, this unique metabolite is only produced in the lysine and tryptophan oxidative pathways. To better understand the biology of protein glutarylation, we studied the relationship between enzymes within the lysine/tryptophan catabolic pathways, protein glutarylation, and regulation by the deglutarylating enzyme sirtuin 5 (SIRT5). Here, we identify glutarylation on the lysine oxidation pathway enzyme glutaryl-CoA dehydrogenase (GCDH) and show increased GCDH glutarylation when glutaryl-CoA production is stimulated by lysine catabolism. Our data reveal that glutarylation of GCDH impacts its function, ultimately decreasing lysine oxidation. We also demonstrate the ability of SIRT5 to deglutarylate GCDH, restoring its enzymatic activity. Finally, metabolomic and bioinformatic analyses indicate an expanded role for SIRT5 in regulating amino acid metabolism. Together, these data support a feedback loop model within the lysine/tryptophan oxidation pathway in which glutaryl-CoA is produced, in turn inhibiting GCDH function via glutaryl modification of GCDH lysine residues and can be relieved by SIRT5 deacylation activity.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

Early-life mitochondrial DNA damage results in lifelong deficits in energy production mediated by redox signaling in Caenorhabditis elegans.

The consequences of damage to the mitochondrial genome (mtDNA) are poorly understood, although mtDNA is more susceptible to damage resulting from some genotoxicants than nuclear DNA (nucDNA), and many environmental toxicants target the mitochondria. Reports from the toxicological literature suggest that exposure to early-life mitochondrial damage could lead to deleterious consequences later in life (the "Developmental Origins of Health and Disease" paradigm), but reports from other fields often report beneficial ("mitohormetic") responses to such damage. Here, we tested the effects of low (causing no change in lifespan) levels of ultraviolet C (UVC)-induced, irreparable mtDNA damage during early development in Caenorhabditis elegans. This exposure led to life-long reductions in mtDNA copy number and steady-state ATP levels, accompanied by increased oxygen consumption and altered metabolite profiles, suggesting inefficient mitochondrial function. Exposed nematodes were also developmentally delayed, reached smaller adult size, and were rendered more susceptible to subsequent exposure to chemical mitotoxicants. Metabolomic and genetic analysis of key signaling and metabolic pathways supported redox and mitochondrial stress-response signaling during early development as a mechanism for establishing these persistent alterations. Our results highlight the importance of early-life exposures to environmental pollutants, especially in the context of exposure to chemicals that target mitochondria.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell.

Established in vitro models for SARS-CoV-2 infection are limited and include cell lines of non-human origin and those engineered to overexpress ACE2, the cognate host cell receptor. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. Infection of H522 cells required the SARS-CoV-2 spike protein, though in contrast to ACE2-dependent models, spike alone was not sufficient for H522 infection. Temporally resolved transcriptomic and proteomic profiling revealed alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. Focused chemical screens point to important roles for clathrin-mediated endocytosis and endosomal cathepsins in SARS-CoV-2 infection of H522 cells. These findings imply the utilization of an alternative SARS-CoV-2 host cell receptor which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

Multiple metabolic changes mediate the response of Caenorhabditis elegans to the complex I inhibitor rotenone.

Rotenone, a mitochondrial complex I inhibitor, has been widely used to study the effects of mitochondrial dysfunction on dopaminergic neurons in the context of Parkinson's disease. Although the deleterious effects of rotenone are well documented, we found that young adult Caenorhabditis elegans showed resistance to 24 and 48 h rotenone exposures. To better understand the response to rotenone in C. elegans, we evaluated mitochondrial bioenergetic parameters after 24 and 48 h exposures to 1 µM or 5 µM rotenone. Results suggested upregulation of mitochondrial complexes II and V following rotenone exposure, without major changes in oxygen consumption or steady-state ATP levels after rotenone treatment at the tested concentrations. We found evidence that the glyoxylate pathway (an alternate pathway not present in higher metazoans) was induced by rotenone exposure; gene expression measurements showed increases in mRNA levels for two complex II subunits and for isocitrate lyase, the key glyoxylate pathway enzyme. Targeted metabolomics analyses showed alterations in the levels of organic acids, amino acids, and acylcarnitines, consistent with the metabolic restructuring of cellular bioenergetic pathways including activation of complex II, the glyoxylate pathway, glycolysis, and fatty acid oxidation. This expanded understanding of how C. elegans responds metabolically to complex I inhibition via multiple bioenergetic adaptations, including the glyoxylate pathway, will be useful in interrogating the effects of mitochondrial and bioenergetic stressors and toxicants.

Sirtuin 5 Is Regulated by the SCFCyclin F Ubiquitin Ligase and Is Involved in Cell Cycle Control.

The ubiquitin-proteasome system is essential for cell cycle progression. Cyclin F is a cell cycle-regulated substrate adapter F-box protein for the Skp1, CUL1, and F-box protein (SCF) family of E3 ubiquitin ligases. Despite its importance in cell cycle progression, identifying cyclin F-bound SCF complex (SCFCyclin F) substrates has remained challenging. Since cyclin F overexpression rescues a yeast mutant in the cdc4 gene, we considered the possibility that other genes that genetically modify cdc4 mutant lethality could also encode cyclin F substrates. We identified the mitochondrial and cytosolic deacylating enzyme sirtuin 5 (SIRT5) as a novel cyclin F substrate. SIRT5 has been implicated in metabolic processes, but its connection to the cell cycle is not known. We show that cyclin F interacts with and controls the ubiquitination, abundance, and stability of SIRT5. We show SIRT5 knockout results in a diminished G1 population and a subsequent increase in both S and G2/M. Global proteomic analyses reveal cyclin-dependent kinase (CDK) signaling changes congruent with the cell cycle changes in SIRT5 knockout cells. Together, these data demonstrate that SIRT5 is regulated by cyclin F and suggest a connection between SIRT5, cell cycle regulation, and metabolism.

daf-16/FoxO promotes gluconeogenesis and trehalose synthesis during starvation to support survival.

daf-16/FoxO is required to survive starvation in Caenorhabditis elegans, but how daf-16IFoxO promotes starvation resistance is unclear. We show that daf-16/FoxO restructures carbohydrate metabolism by driving carbon flux through the glyoxylate shunt and gluconeogenesis and into synthesis of trehalose, a disaccharide of glucose. Trehalose is a well-known stress protectant, capable of preserving membrane organization and protein structure during abiotic stress. Metabolomic, genetic, and pharmacological analyses confirm increased trehalose synthesis and further show that trehalose not only supports survival as a stress protectant but also serves as a glycolytic input. Furthermore, we provide evidence that metabolic cycling between trehalose and glucose is necessary for this dual function of trehalose. This work demonstrates that daf-16/FoxO promotes starvation resistance by shifting carbon metabolism to drive trehalose synthesis, which in turn supports survival by providing an energy source and acting as a stress protectant.

Mechanism-Based Inhibitors of the Human Sirtuin 5 Deacylase: Structure-Activity Relationship, Biostructural, and Kinetic Insight.

The sirtuin enzymes are important regulatory deacylases in a variety of biochemical contexts and may therefore be potential therapeutic targets through either activation or inhibition by small molecules. Here, we describe the discovery of the most potent inhibitor of sirtuin 5 (SIRT5) reported to date. We provide rationalization of the mode of binding by solving co-crystal structures of selected inhibitors in complex with both human and zebrafish SIRT5, which provide insight for future optimization of inhibitors with more "drug-like" properties. Importantly, enzyme kinetic evaluation revealed a slow, tight-binding mechanism of inhibition, which is unprecedented for SIRT5. This is important information when applying inhibitors to probe mechanisms in biology.

Nicotinamide mononucleotide requires SIRT3 to improve cardiac function and bioenergetics in a Friedreich's ataxia cardiomyopathy model.

Increasing NAD+ levels by supplementing with the precursor nicotinamide mononucleotide (NMN) improves cardiac function in multiple mouse models of disease. While NMN influences several aspects of mitochondrial metabolism, the molecular mechanisms by which increased NAD+ enhances cardiac function are poorly understood. A putative mechanism of NAD+ therapeutic action exists via activation of the mitochondrial NAD+-dependent protein deacetylase sirtuin 3 (SIRT3). We assessed the therapeutic efficacy of NMN and the role of SIRT3 in the Friedreich's ataxia cardiomyopathy mouse model (FXN-KO). At baseline, the FXN-KO heart has mitochondrial protein hyperacetylation, reduced Sirt3 mRNA expression, and evidence of increased NAD+ salvage. Remarkably, NMN administered to FXN-KO mice restores cardiac function to near-normal levels. To determine whether SIRT3 is required for NMN therapeutic efficacy, we generated SIRT3-KO and SIRT3-KO/FXN-KO (double KO [dKO]) models. The improvement in cardiac function upon NMN treatment in the FXN-KO is lost in the dKO model, demonstrating that the effects of NMN are dependent upon cardiac SIRT3. Coupled with cardio-protection, SIRT3 mediates NMN-induced improvements in both cardiac and extracardiac metabolic function and energy metabolism. Taken together, these results serve as important preclinical data for NMN supplementation or SIRT3 activator therapy in Friedreich's ataxia patients.

A Class of Reactive Acyl-CoA Species Reveals the Non-enzymatic Origins of Protein Acylation.

The mechanisms underlying the formation of acyl protein modifications remain poorly understood. By investigating the reactivity of endogenous acyl-CoA metabolites, we found a class of acyl-CoAs that undergo intramolecular catalysis to form reactive intermediates that non-enzymatically modify proteins. Based on this mechanism, we predicted, validated, and characterized a protein modification: 3-hydroxy-3-methylglutaryl(HMG)-lysine. In a model of altered HMG-CoA metabolism, we found evidence of two additional protein modifications: 3-methylglutaconyl(MGc)-lysine and 3-methylglutaryl(MG)-lysine. Using quantitative proteomics, we compared the "acylomes" of two reactive acyl-CoA species, namely HMG-CoA and glutaryl-CoA, which are generated in different pathways. We found proteins that are uniquely modified by each reactive metabolite, as well as common proteins and pathways. We identified the tricarboxylic acid cycle as a pathway commonly regulated by acylation and validated malate dehydrogenase as a key target. These data uncover a fundamental relationship between reactive acyl-CoA species and proteins and define a new regulatory paradigm in metabolism.

From the Cover: Arsenite Uncouples Mitochondrial Respiration and Induces a Warburg-like Effect in Caenorhabditis elegans.

Millions of people worldwide are chronically exposed to arsenic through contaminated drinking water. Despite decades of research studying the carcinogenic potential of arsenic, the mechanisms by which arsenic causes cancer and other diseases remain poorly understood. Mitochondria appear to be an important target of arsenic toxicity. The trivalent arsenical, arsenite, can induce mitochondrial reactive oxygen species production, inhibit enzymes involved in energy metabolism, and induce aerobic glycolysis in vitro, suggesting that metabolic dysfunction may be important in arsenic-induced disease. Here, using the model organism Caenorhabditis elegans and a novel metabolic inhibition assay, we report an in vivo induction of aerobic glycolysis following arsenite exposure. Furthermore, arsenite exposure induced severe mitochondrial dysfunction, including altered pyruvate metabolism; reduced steady-state ATP levels, ATP-linked respiration and spare respiratory capacity; and increased proton leak. We also found evidence that induction of autophagy is an important protective response to arsenite exposure. Because these results demonstrate that mitochondria are an important in vivo target of arsenite toxicity, we hypothesized that deficiencies in mitochondrial electron transport chain genes, which cause mitochondrial disease in humans, would sensitize nematodes to arsenite. In agreement with this, nematodes deficient in electron transport chain complexes I, II, and III, but not ATP synthase, were sensitive to arsenite exposure, thus identifying a novel class of gene-environment interactions that warrant further investigation in the human populace.

A pilot study to assess effects of long-term inhalation of airborne particulate matter on early Alzheimer-like changes in the mouse brain.

Exposure to air pollutants, including particulate matter, results in activation of the brain inflammatory response and Alzheimer disease (AD)-like pathology in dogs and humans. However, the length of time required for inhalation of ambient particulate matter to influence brain inflammation and AD pathology is less clear. Here, we studied the effect of 3 and 9 months of air particulate matter (<2.5 µm diameter, PM2.5) exposure on brain inflammatory phenotype and pathological hallmarks of AD in C57BL/6 mice. Using western blot, ELISA, and cytokine array analysis we quantified brain APP, beta-site APP cleaving enzyme (BACE), oligomeric protein, total Aß 1-40 and Aß 1-42 levels, inducible nitric oxide synthase (iNOS), nitrotyrosine-modified proteins, HNE-Michael adducts, vascular cell adhesion molecule 1 (VCAM-1), glial markers (GFAP, Iba-1), pre- and post- synaptic markers (synaptophysin and PSD-95), cyclooxygenase (COX-1, COX-2) levels, and the cytokine profile in PM2.5 exposed and filtered air control mice. Only 9 month PM2.5 exposure increased BACE protein levels, APP processing, and Aß 1-40 levels. This correlated with a concomitant increase in COX-1 and COX-2 protein levels and a modest alteration in the cytokine profile. These data support the hypothesis that prolonged exposure to airborne particulate matter has the potential to alter brain inflammatory phenotype and promote development of early AD-like pathology.

Acetate supplementation modulates brain adenosine metabolizing enzymes and adenosine A2A receptor levels in rats subjected to neuroinflammation.

BACKGROUND: Acetate supplementation reduces neuroglia activation and pro-inflammatory cytokine expression in rat models of neuroinflammation and Lyme neuroborreliosis. Because single-dose glyceryl triacetate (GTA) treatment increases brain phosphocreatine and reduces brain AMP levels, we postulate that GTA modulates adenosine metabolizing enzymes and receptors, which may be a possible mechanism to reduce neuroinflammation. METHODS: To test this hypothesis, we quantified the ability of GTA to alter brain levels of ecto-5'-nucleotidase (CD73), adenosine kinase (AK), and adenosine A2A receptor using western blot analysis and CD73 activity by measuring the rate of AMP hydrolysis. Neuroinflammation was induced by continuous bacterial lipopolysaccharide (LPS) infusion in the fourth ventricle of the brain for 14 and 28 days. Three treatment strategies were employed, one and two where rats received prophylactic GTA through oral gavage with LPS infusion for 14 or 28 days. In the third treatment regimen, an interventional strategy was used where rats were subjected to 28 days of neuroinflammation, and GTA treatment was started on day 14 following the start of the LPS infusion. RESULTS: We found that rats subjected to neuroinflammation for 28 days had a 28% reduction in CD73 levels and a 43% increase in AK levels that was reversed with prophylactic acetate supplementation. CD73 activity in these rats was increased by 46% with the 28-day GTA treatment compared to the water-treated rats. Rats subjected to neuroinflammation for 14 days showed a 50% increase in levels of the adenosine A2A receptor, which was prevented with prophylactic acetate supplementation. Interventional GTA therapy, beginning on day 14 following the induction of neuroinflammation, resulted in a 67% increase in CD73 levels and a 155% increase in adenosine A2A receptor levels. CONCLUSION: These results support the hypothesis that acetate supplementation can modulate brain CD73, AK and adenosine A2A receptor levels, and possibly influence purinergic signaling.

Acetate treatment increases fatty acid content in LPS-stimulated BV2 microglia.

Acetate supplementation increases plasma acetate, brain acetyl-CoA, histone acetylation, phosphocreatine levels, and is anti-inflammatory in models of neuroinflammation and neuroborreliosis. Although radiolabeled acetate is incorporated into the cellular lipid pools, the effect that acetate supplementation has on lipid deposition has not been quantified. To determine the impact acetate-treatment has on cellular lipid content, we investigated the effect of acetate in the presence of bacterial lipopolysaccharide (LPS) on fatty acid, phospholipid, and cholesterol content in BV2 microglia. We found that 1, 5, and 10 mM of acetate in the presence of LPS increased the total fatty acid content in BV2 cells by 23, 34, and 14 % at 2 h, respectively. Significant increases in individual fatty acids were also observed with all acetate concentrations tested with the greatest increases occurring with 5 mM acetate in the presence of LPS. Treatment with 5 mM acetate in the absence of LPS increased total cholesterol levels by 11 %. However, neither treatment in the absence of LPS significantly altered the content of individual phospholipids or total phospholipid content. To determine the minimum effective concentration of acetate we measured the time- and concentration-dependent changes in histone acetylation using western blot analysis. These studies showed that 5 mM acetate was necessary to induce histone acetylation and at 10 mM acetate, the histone acetylation-state increased as early as 0.5 h following the start of treatment. These data suggest that acetate increases fatty acid content in LPS-stimulated BV2 microglia that is reflected by an increase in fatty acids esterified into membrane phospholipids.

Acetate supplementation increases brain phosphocreatine and reduces AMP levels with no effect on mitochondrial biogenesis.

Acetate supplementation in rats increases plasma acetate and brain acetyl-CoA levels. Although acetate is used as a marker to study glial energy metabolism, the effect that acetate supplementation has on normal brain energy stores has not been quantified. To determine the effect(s) that an increase in acetyl-CoA levels has on brain energy metabolism, we measured brain nucleotide, phosphagen and glycogen levels, and quantified cardiolipin content and mitochondrial number in rats subjected to acetate supplementation. Acetate supplementation was induced with glyceryl triacetate (GTA) by oral gavage (6 g/kg body weight). Rats used for biochemical analysis were euthanized using head-focused microwave irradiation at 2, and 4h following treatment to immediately stop metabolism. We found that acetate did not alter brain ATP, ADP, NAD, GTP levels, or the energy charge ratio [ECR, (ATP+½ ADP)/(ATP+ADP+AMP)] when compared to controls. However, after 4h of treatment brain phosphocreatine levels were significantly elevated with a concomitant reduction in AMP levels with no change in glycogen levels. In parallel studies where rats were treated with GTA for 28 days, we found that acetate did not alter brain glycogen and mitochondrial biogenesis as determined by measuring brain cardiolipin content, the fatty acid composition of cardiolipin and using quantitative ultra-structural analysis to determine mitochondrial density/unit area of cytoplasm in hippocampal CA3 neurons. Collectively, these data suggest that an increase in brain acetyl-CoA levels by acetate supplementation does increase brain energy stores however it has no effect on brain glycogen and neuronal mitochondrial biogenesis.

A sensitive HPLC-based method to quantify adenine nucleotides in primary astrocyte cell cultures.

In mono-layered primary cell cultures baseline AMP and ADP levels are found nominally in the mid to low picomolar range and are thus difficult to measure with conventional HPLC methods that often require the pooling of samples or require indirect detection methods using radiotracers or enzyme coupled assays. To address this issue, we developed a highly sensitive and selective ion-pairing HPLC method with fluorescence detection to quantify adenine nucleotides and the adenylate energy charge in primary astrocyte cell cultures. To accomplish this, we optimized the fluorescence derivatization conditions and the HPLC parameters to achieve baseline separation and quantification of all adenine nucleotides. Nucleotides were converted to their respective 1, N(6)-etheno derivatives by incubating with chloroacetaldehyde at pH 4.5 and 60°C for 60 min. Under these conditions, the loss of the adenine nucleotides due to hydrolysis was minimized with a derivatization yield of 94.1% for 1, N(6)-ethenoadenosine. The optimal concentration of tetrabutylammonium phosphate, the ion-pairing reagent, required to achieve a reproducible separation of the adenine nucleotides was found to be 0.8mM. Calibration curves of nucleotide standards were linear within the range of 0.16-10.4 pmol for adenosine, 0.16-20.6 pmol for AMP, 0.15-19.2 pmol for ADP, and 0.15-19.5 pmol for ATP. The limits of detection and quantification for all adenine nucleotides were approximately 0.08 and 0.16 pmol, respectively. The intra- and inter-day variability for this method was less than 5.1 and 3.4%, respectively. This method was successfully used to measure all adenine nucleotides and an adenylate energy charge of 0.92±0.02 in primary astrocyte cell cultures.