Overexpression of Nudt7 decreases bile acid levels and peroxisomal fatty acid oxidation in the liver
2019; Elsevier BV; Volume: 60; Issue: 5 Linguagem: Inglês
10.1194/jlr.m092676
ISSN1539-7262
AutoresStephanie A. Shumar, Evan W. Kerr, Paolo Fagone, Aniello M. Infante, Roberta Leonardi,
Tópico(s)Metabolism and Genetic Disorders
ResumoLipid metabolism requires CoA, an essential cofactor found in multiple subcellular compartments, including the peroxisomes. In the liver, CoA levels are dynamically adjusted between the fed and fasted states. Elevated CoA levels in the fasted state are driven by increased synthesis; however, this also correlates with decreased expression of Nudix hydrolase (Nudt)7, the major CoA-degrading enzyme in the liver. Nudt7 resides in the peroxisomes, and we overexpressed this enzyme in mouse livers to determine its effect on the size and composition of the hepatic CoA pool in the fed and fasted states. Nudt7 overexpression did not change total CoA levels, but decreased the concentration of short-chain acyl-CoAs and choloyl-CoA in fasted livers, when endogenous Nudt7 activity was lowest. The effect on these acyl-CoAs correlated with a significant decrease in the hepatic bile acid content and in the rate of peroxisomal fatty acid oxidation, as estimated by targeted and untargeted metabolomics, combined with the measurement of fatty acid oxidation in intact hepatocytes. Identification of the CoA species and metabolic pathways affected by the overexpression on Nudt7 in vivo supports the conclusion that the nutritionally driven modulation of Nudt7 activity could contribute to the regulation of the peroxisomal CoA pool and peroxisomal lipid metabolism. Lipid metabolism requires CoA, an essential cofactor found in multiple subcellular compartments, including the peroxisomes. In the liver, CoA levels are dynamically adjusted between the fed and fasted states. Elevated CoA levels in the fasted state are driven by increased synthesis; however, this also correlates with decreased expression of Nudix hydrolase (Nudt)7, the major CoA-degrading enzyme in the liver. Nudt7 resides in the peroxisomes, and we overexpressed this enzyme in mouse livers to determine its effect on the size and composition of the hepatic CoA pool in the fed and fasted states. Nudt7 overexpression did not change total CoA levels, but decreased the concentration of short-chain acyl-CoAs and choloyl-CoA in fasted livers, when endogenous Nudt7 activity was lowest. The effect on these acyl-CoAs correlated with a significant decrease in the hepatic bile acid content and in the rate of peroxisomal fatty acid oxidation, as estimated by targeted and untargeted metabolomics, combined with the measurement of fatty acid oxidation in intact hepatocytes. Identification of the CoA species and metabolic pathways affected by the overexpression on Nudt7 in vivo supports the conclusion that the nutritionally driven modulation of Nudt7 activity could contribute to the regulation of the peroxisomal CoA pool and peroxisomal lipid metabolism. The liver is a metabolically flexible organ that plays a key role in the maintenance of whole-body lipid and glucose homeostasis (1.Weickert M.O. Pfeiffer A.F. Signalling mechanisms linking hepatic glucose and lipid metabolism.Diabetologia. 2006; 49: 1732-1741Crossref PubMed Scopus (84) Google Scholar, 2.Bechmann L.P. Hannivoort R.A. Gerken G. Hotamisligil G.S. Trauner M. Canbay A. The interaction of hepatic lipid and glucose metabolism in liver diseases.J. Hepatol. 2012; 56: 952-964Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar, 3.Rui L. Energy metabolism in the liver.Compr. Physiol. 2014; 4: 177-197Crossref PubMed Scopus (1093) Google Scholar). Several pathways, including gluconeogenesis, ketogenesis, and bile acid synthesis, occur primarily in this organ and rely on the availability of the essential cofactor, CoA. CoA is the major acyl group carrier in mammalian cells. 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The nudix hydrolase 7 is an acyl-CoA diphosphatase involved in regulating peroxisomal coenzyme A homeostasis.J. Biochem. 2008; 144: 655-663Crossref PubMed Scopus (45) Google Scholar, 18.Shumar S.A. Kerr E.W. Geldenhuys W.J. Montgomery G.E. Fagone P. Thirawatananond P. Saavedra H. Gabelli S.B. Leonardi R. Nudt19 is a renal CoA diphosphohydrolase with biochemical and regulatory properties that are distinct from the hepatic Nudt7 isoform.J. Biol. Chem. 2018; 293: 4134-4148Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). CoA synthesis requires five enzymatic steps and is regulated through feedback inhibition of the first and rate-limiting enzyme of the pathway, pantothenate kinase, by free CoA and acyl-CoAs (19.Zhang Y.M. Rock C.O. Jackowski S. Biochemical properties of human pantothenate kinase 2 isoforms and mutations linked to pantothenate kinase-associated neurodegeneration.J. Biol. 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J. 2001; 357: 33-38Crossref PubMed Scopus (79) Google Scholar). Both enzymes are members of the Nudt superfamily, specifically hydrolyze CoA species to produce 3′,5′-ADP and (acyl-)phosphopantetheine, and reside in the peroxisomes, organelles that play a key role in lipid metabolism (18.Shumar S.A. Kerr E.W. Geldenhuys W.J. Montgomery G.E. Fagone P. Thirawatananond P. Saavedra H. Gabelli S.B. Leonardi R. Nudt19 is a renal CoA diphosphohydrolase with biochemical and regulatory properties that are distinct from the hepatic Nudt7 isoform.J. Biol. Chem. 2018; 293: 4134-4148Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 22.Ofman R. Speijer D. Leen R. Wanders R.J. Proteomic analysis of mouse kidney peroxisomes: identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity.Biochem. J. 2006; 393: 537-543Crossref PubMed Scopus (70) Google Scholar, 23.Gasmi L. McLennan A.G. The mouse Nudt7 gene encodes a peroxisomal nudix hydrolase specific for coenzyme A and its derivatives.Biochem. J. 2001; 357: 33-38Crossref PubMed Scopus (79) Google Scholar, 24.Shumar S.A. Fagone P. Alfonso-Pecchio A. Gray J.T. Rehg J.E. Jackowski S. Leonardi R. Induction of neuron-specific degradation of coenzyme a models pantothenate kinase-associated neurodegeneration by reducing motor coordination in mice.PLoS One. 2015; 10: e0130013Crossref PubMed Scopus (19) Google Scholar, 25.Van Veldhoven P.P. Biochemistry and genetics of inherited disorders of peroxisomal fatty acid metabolism.J. Lipid Res. 2010; 51: 2863-2895Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 26.Wanders R.J. Vreken P. Ferdinandusse S. Jansen G.A. Waterham H.R. van Roermund C.W. Van Grunsven E.G. Peroxisomal fatty acid alpha- and beta-oxidation in humans: enzymology, peroxisomal metabolite transporters and peroxisomal diseases.Biochem. Soc. Trans. 2001; 29: 250-267Crossref PubMed Scopus (225) Google Scholar). Nudt19 is the major isoform in the kidneys, where it contributes to the regulation of total CoA levels in these organs (18.Shumar S.A. Kerr E.W. Geldenhuys W.J. Montgomery G.E. Fagone P. Thirawatananond P. Saavedra H. Gabelli S.B. Leonardi R. Nudt19 is a renal CoA diphosphohydrolase with biochemical and regulatory properties that are distinct from the hepatic Nudt7 isoform.J. Biol. Chem. 2018; 293: 4134-4148Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Nudt7 is the major CoA-degrading enzyme in the liver. Recombinant Nudt7 hydrolyzes a broad range of CoA substrates, including free CoA, short- and medium-chain acyl-CoAs, and the CoA thioesters of bile acids (17.Reilly S.J. Tillander V. Ofman R. Alexson S.E. Hunt M.C. The nudix hydrolase 7 is an acyl-CoA diphosphatase involved in regulating peroxisomal coenzyme A homeostasis.J. Biochem. 2008; 144: 655-663Crossref PubMed Scopus (45) Google Scholar, 18.Shumar S.A. Kerr E.W. Geldenhuys W.J. Montgomery G.E. Fagone P. Thirawatananond P. Saavedra H. Gabelli S.B. Leonardi R. Nudt19 is a renal CoA diphosphohydrolase with biochemical and regulatory properties that are distinct from the hepatic Nudt7 isoform.J. Biol. Chem. 2018; 293: 4134-4148Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Unlike Nudt19, Nudt7 is not inhibited by bile acids and its expression varies with fasting and feeding. In particular, Nudt7 levels decrease with fasting, correlating with the increase in CoA observed in fasted livers. While these findings have established that Nudt7 can degrade CoA in vitro, it has yet to be determined whether Nudt7 is actively involved in regulating hepatic CoA levels in vivo. Indeed, the peroxisomal CoA pool is estimated to represent only 4–10% of total liver CoA (10.Horie S. Ishii H. Suga T. Changes in peroxisomal fatty acid oxidation in the diabetic rat liver.J. Biochem. 1981; 90: 1691-1696Crossref PubMed Scopus (142) Google Scholar, 11.Van Broekhoven A. Peeters M.C. Debeer L.J. Mannaerts G.P. Subcellular distribution of coenzyme A: evidence for a separate coenzyme A pool in peroxisomes.Biochem. Biophys. Res. Commun. 1981; 100: 305-312Crossref PubMed Scopus (43) Google Scholar), and it is currently unclear to what extent the activity of Nudt7 in liver peroxisomes could affect total CoA levels in the fed and fasted states. Furthermore, the localization, substrate specificity, and nutritional regulation of Nudt7 expression suggest that this enzyme may be involved in the regulation of peroxisomal pathways (27.Hunt M.C. Tillander V. Alexson S.E. Regulation of peroxisomal lipid metabolism: the role of acyl-CoA and coenzyme A metabolizing enzymes.Biochimie. 2014; 98: 45-55Crossref PubMed Scopus (61) Google Scholar), but direct evidence is lacking. To gain insight into the physiological role of Nudt7 and its ability to regulate liver CoA levels in vivo, we overexpressed the enzyme in the liver of C57BL/6J mice. Overexpression of Nudt7 in the fasted state, when endogenous Nudt7 activity was lowest, did not prevent the increase in total hepatic CoA levels that occurs with fasting, but it changed the acyl-CoA composition of the liver by significantly decreasing the concentration of short-chain acyl-CoAs and choloyl-CoA, a peroxisomal intermediate in bile acid synthesis. Untargeted and targeted metabolomics analysis revealed that fasted livers overexpressing Nudt7 contained significantly reduced levels of primary and secondary bile acids, in addition to an accumulation of medium- and long-chain acylcarnitines, which correlated with a lower rate of peroxisomal fatty acid oxidation. Our data show that overexpression of Nudt7 specifically interfered with the output of peroxisomal fatty acid oxidation and the bile acid biosynthetic pathway in the fasted state. Thus, changes in Nudt7 activity between the fed and fasted states could act to regulate the peroxisomal CoA pool and, in turn, peroxisomal lipid metabolism. Reagents were purchased from the following suppliers: HRP-conjugated GFP antibody, Alexa Fluor 647- and HRP-conjugated goat anti-rabbit IgG from Thermo Fisher Scientific; the antibodies against GAPDH and the FLAG epitope (DYKDDDK) from Cell Signaling; Waymouth MB 752/1 medium, medium 199 (M199), and the antibody against ATP-binding cassette subfamily D member 3 (PMP70) from Sigma-Aldrich. The Nudt7 antibody was generated as previously described (15.Leonardi R. Rock C.O. Jackowski S. Pank1 deletion in leptin-deficient mice reduces hyperglycaemia and hyperinsulinaemia and modifies global metabolism without affecting insulin resistance.Diabetologia. 2014; 57: 1466-1475Crossref PubMed Scopus (26) Google Scholar). The fluorescent CoA derivative of monobromobimane (mBB), mBB-CoA, was synthesized as previously described (18.Shumar S.A. Kerr E.W. Geldenhuys W.J. Montgomery G.E. Fagone P. Thirawatananond P. Saavedra H. Gabelli S.B. Leonardi R. Nudt19 is a renal CoA diphosphohydrolase with biochemical and regulatory properties that are distinct from the hepatic Nudt7 isoform.J. Biol. Chem. 2018; 293: 4134-4148Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). All other reagents were of analytical grade or better and were purchased from Sigma-Aldrich or Fisher Scientific, unless otherwise stated. Plasmid pscAAV-LP1-EGFP, carrying the enhanced green fluorescent protein coding sequence (henceforth referred to as GFP), was developed by John T. Gray and provided by St. Jude Children's Research Hospital, Memphis, TN. Plasmid pscAAV-LP1-mNudt7, obtained by replacing the coding sequence of GFP with the sequence of mouse Nudt7 containing a FLAG tag at the N terminus, was a kind gift of Suzanne Jackowski, St. Jude Children's Research Hospital, Memphis, TN. To produce adeno-associated virus (AAV), HEK 293T cells were transiently transfected with a 1:1:2.5 (mass ratio) mixture of plasmids pHGTI-adeno1 (developed by John T. Gray and provided by Harvard College, Cambridge, MA), pLTAAVhelp2-8 (developed by John T. Gray and provided by St. Jude Children's Research Hospital, Memphis, TN), and either pscAAV-LP1-mNudt7 or pscAAV-LP1-EGFP using polyethylenimine. Transfected cells were harvested 72 h post transfection and lysed in 50 mM Tris-HCl, 50 mM NaCl, 1 mM MgCl2, 2% Triton X-100, 50 U/ml Pierce™ Universal Nuclease (pH 8.0), incubating for 20 min at 37°C. The lysate was then supplemented with 0.05 vol of sodium deoxycholate (10% solution in water) and further incubated at 37°C for 10 min. Following the addition of 0.1 vol of 5 M NaCl, the lysate was clarified by centrifugation at 4,000 g for 20 min at 20°C. The cleared lysate was layered on top of a discontinuous sucrose density gradient generated by layers of 60, 40, 25, and 15% sucrose in 20 mM diethanolamine hydrochloride (pH 9.0). Following centrifugation at 104,000 g (Beckman Optima LE-80) for 3 h at 20°C, the virus was recovered from the 40% sucrose layer and the 40–60% sucrose interface region of the gradient. The AAV particles were further purified by ion exchange chromatography on a POROS 50 HQ column (Thermo Fisher Scientific). AAV elution was monitored by measuring the absorbance at both 260 and 280 nm. Fractions containing the AAV particles were buffer exchanged into phosphate-buffered saline containing 0.01% Pluronic F-68, filter-sterilized, and quantified using the agarose gel method previously described (28.Fagone P. Wright J.F. Nathwani A.C. Nienhuis A.W. Davidoff A.M. Gray J.T. Systemic errors in quantitative polymerase chain reaction titration of self-complementary adeno-associated viral vectors and improved alternative methods.Hum. Gene Ther. Methods. 2012; 23: 1-7Crossref PubMed Scopus (65) Google Scholar). Mice were fed a standard chow diet (Tekland 2018S) and maintained at a room temperature of 22.2 ± 0.2°C, room humidity of 40 ± 2%, and a 12 h light/12 h dark cycle, with the dark cycle starting at 6:00 PM. Six-week-old C57BL/6J male mice were purchased from Jackson Laboratory. Following a 2 week acclimation period, these mice were injected with 2.5 × 1011 genome copies of GFP- or Nudt7-AAV in 150 μl of sterile phosphate-buffered saline and, unless otherwise indicated, were euthanized 3–4 weeks after injection. Unless otherwise stated, fasting experiments were started at 7:00 AM, and mice were placed in cages with grids without food for the indicated amount of time. To measure food consumption, mice were individually housed in cages with grids and provided with a preweighed amount of food. Seventy-two hours later, the leftover food was weighed and used to calculate the average food consumption per mouse per 24 h. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using Stanbio kits (EKF Diagnostic USA) and 3-hydroxybutyrate was determined using a 3-hydroxybutyrate enzyme solution and colorimetric detector from Cayman Chemicals, as per the manufacturer's instructions. All studies were approved by the Institutional Animal Care and Use Committees of West Virginia University. The concentration of total CoA (free CoA plus CoA thioesters) in liver homogenates was determined after conversion of the cofactor to the mBB derivative, as previously described (24.Shumar S.A. Fagone P. Alfonso-Pecchio A. Gray J.T. Rehg J.E. Jackowski S. Leonardi R. Induction of neuron-specific degradation of coenzyme a models pantothenate kinase-associated neurodegeneration by reducing motor coordination in mice.PLoS One. 2015; 10: e0130013Crossref PubMed Scopus (19) Google Scholar). For Western blot analysis, flash-frozen tissues were homogenized in ice-cold radioimmunoprecipitation assay buffer supplemented with protease inhibitors (Biotool), and centrifuged at 10,000 g for 10 min at 4°C. Proteins (5 or 20 μg) were fractionated on 4–12% bis-Tris polyacrylamide gels and transferred onto nitrocellulose membranes using an iBlot dry transfer system (Thermo Fisher Scientific). The GAPDH and Nudt7 antibodies were used at a 1:5,000 and 1:3,000 dilution, respectively. Bound primary antibodies were detected by chemiluminescence with HRP-conjugated goat anti-rabbit IgG at a 1:45,000 dilution. The HRP-conjugated GFP antibody was used at a 1:7,000 dilution. RNA was isolated from flash-frozen tissue as previously described (29.Garcia M. Leonardi R. Zhang Y.M. Rehg J.E. Jackowski S. Germline deletion of pantothenate kinases 1 and 2 reveals the key roles for CoA in postnatal metabolism.PLoS One. 2012; 7: e40871Crossref PubMed Scopus (54) Google Scholar). Following the removal of genomic DNA with Turbo DNA-free kit (Thermo Fisher Scientific), mRNA levels were quantified in triplicate by RT-PCR using the Quantitect SYBR Green RT-PCR kit (Qiagen) and the primers previously reported (29.Garcia M. Leonardi R. Zhang Y.M. Rehg J.E. Jackowski S. Germline deletion of pantothenate kinases 1 and 2 reveals the key roles for CoA in postnatal metabolism.PLoS One. 2012; 7: e40871Crossref PubMed Scopus (54) Google Scholar). The relative abundance of each gene was calculated using the CT method, and the amount (2-ΔCT) reported relative to the average of ribosomal protein L22 (Rpl22) and β2-microglobulin (B2m). Primers 5′-GTGCCTTTCTCCAAAAGGTATTT-3′ and 5′-CTCTCTTTGCTGTTGGCGAC-3′ were used as forward and reverse primers, respectively, to amplify Rpl22. Primers 5′-ACTGACCGGCCTGTATGCTA-3′ and 5′-ATGTTCGGCTTCCCATTCTCC-3′ were used as forward and reverse primers, respectively for B2m. Total RNA from mouse liver tissue was isolated as described above and delivered to the West Virginia University Genomics Core Facility for quality control and library preparation. The concentration of extracted total RNA was determined using a Qubit fluorometer (Thermo Fisher Scientific), while RNA quality was ascertained on an Agilent 2100 bioanalyzer using an RNA Nano 6000 chip. All of the samples used for the library preparation had RNA integrity number (RIN) values >8.0. Each library was built using 500 ng of RNA as input material for the stranded mRNA library preparation kit from KAPA Biosystems, following the manufacturer's recommended protocol with nine cycles of PCR. The cDNA libraries were quantified via Qubit, pooled in equimolar ratios, and sent to the Genomics Core Facility at Marshall University where the libraries were sequenced on four lanes generating PE50bp reads with the Illumina HiSeq1500 system (16–36M reads per sample, ∼175M total reads). Read quality was assessed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc) and deemed to require no trimming. The reads were quantified using Salmon (30.Patro R. Duggal G. Love M.I. Irizarry R.A. Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression.Nat. Methods. 2017; 14: 417-419Crossref PubMed Scopus (3607) Google Scholar) with transcripts obtained from Ensembl, version GRCm38.84. Using tximport (31.Soneson C. Love M.I. Robinson M.D. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences.F1000 Res. 2015; 4: 1521Crossref PubMed Google Scholar), the data was imported into R and differential expression was computed using DESeq2 (32.Love M.I. Huber W. Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol. 2014; 15: 550Crossref PubMed Scopus (32370) Google Scholar). We removed all genes that did not have at least a count of 10 in at least three samples. Genes were considered to be significantly changed if the Benjamini-Hockburg adjusted P-value was less than 0.01. WebGestalt (http://www.webgestalt.org) was used to conduct gene ontology and pathway enrichment analyses. The RNA-Seq raw data were deposited and are accessible through NCBI Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) with the GEO Series accession number GSE125237. Total liver CoA diphosphohydrolase activity was measured in liver extracts obtained by homogenizing flash-frozen tissue (∼20 mg) in 750 μl of 20 mM Tris-HCl (pH 8.0). The homogenates were incubated on ice for 10 min before centrifuging at 20,000 g for 10 min. The supernatants were removed and increasing amounts of protein (6.25–50 μg) were incubated with 100 μM of mBB-CoA for 10 min at 37°C. The reactions were then stopped and analyzed as described previously (18.Shumar S.A. Kerr E.W. Geldenhuys W.J. Montgomery G.E. Fagone P. Thirawatananond P. Saavedra H. Gabelli S.B. Leonardi R. Nudt19 is a renal CoA diphosphohydrolase with biochemical and regulatory properties that are distinct from the hepatic Nudt7 isoform.J. Biol. Chem. 2018; 293: 4134-4148Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Serum cholesterol and triglycerides were measured using Stanbio kits (EKF Diagnostics USA), as per the manufacturer's instructions. Liver lipids were extracted from ∼50 mg of liver using a modification of the Bligh and Dyer procedure (33.Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42694) Google Scholar) optimized for lipid quantitation by the LipidMaps group (34.Ivanova P.T. Milne S.B. Byrne M.O. Xiang Y. Brown H.A. Glycerophospholipid identification and quantitation by electrospray ionization mass spectrometry.Methods Enzymol. 2007; 432: 21-57Crossref PubMed Scopus (136) Google Scholar). Following extraction, a portion of the lipids was dried and resuspended in 100 μl of 5% NP-40 for cholesterol analysis using the same Stan-Bio kit utilized for the determination of cholesterol in the serum. A second portion of the total lipid extract was fractionated for triglyceride analysis on HPTLC silica plates developed in hexane:ether:acetic acid (80:20:1, v:v:v). Resolved lipid bands and serial dilutions of a tripalmitin standard were visualized and quantitated by spraying with a 0.002% primulin solution followed by fluorescence analysis using G:BOX Chemi XX9 imaging system (Syngene). Global metabolomics profiling, combined with statistical and pathway enrichment analyses, were conducted by Metabolon, Inc. Raw data and metabolite fold-changes between groups are reported in supplemental Table S1. Principal component analysis, normalization, and scaling of selected metabolites for the generation of heat plots were conducted using MetaboAnalyst 4.0 (https://www.metaboanalyst.ca/) (35.Chong J. Soufan O. Li C. Caraus I. Li S. Bourque G. Wishart D.S. Xia J. MetaboAnalyst 4.0: towards more transparent and integrative metabolomics analysis.Nucleic Acids Res. 2018; 46: W486-W494Crossref PubMed Scopus (2374) Google Scholar). Liver, small intestine, and bile samples were analyzed for their bile acid composition using a targeted assay by Metabolon, Inc. For the analysis of the acyl-CoA pool composition, CoA species were extracted as described by Minkler et al. (36.Minkler P.E. Kerner J. Ingalls S.T. Hoppel C.L. Novel isolation procedure for short-, medium-, and long-chain acyl-coenzyme A esters from tissue.Anal. Biochem. 2008; 376: 275-276Crossref PubMed Scopus (59) Google Scholar). Briefly, flash-frozen livers (∼50 mg) were homogenized in 1.5 ml of a 3:1 (v:v) mixture of acetonitrile:isopropanol containing 0.625 μM of heptanoyl- and heptadecanoyl-CoA standards. Following the addition of 0.5 ml of 0.1 M KH2PO4 (pH 6.7), the homogenates were centrifuged at 16,000 g for 5 min. The supernatants were acidified by the addition of 0.25 vol of glacial acetic acid and applied onto 2-(2-pyridyl)ethyl-functionalized silica columns (100 mg; Sigma-Aldrich) previously conditioned with 1 ml of a 9:3:4:4 (v:v) mixture of acetonitrile:isopropanol:water:ace-tic acid. The columns were washed with 1 ml of the same solvent mixture and 1 ml of water before eluting the bound compounds with 4 ml of 90% methanol containing 15 mM ammonium
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