Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets.

Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [(13)C1]propionate and [(13)C3]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[(2)H2]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer studies confirmed that the new acyl-CoA was trans-2-methyl-2-pentenoyl-CoA. Acyl-CoA profiles following treatment of HepG2 cells with mono-unsaturated six-carbon fatty acids also supported this conclusion. Similar results were obtained with human platelets, mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells. Interestingly, trans-2-methyl-2-pentenoyl-CoA corresponds to a previously described acylcarnitine tentatively described in patients with propionic and methylmalonic acidemia. We have proposed a mechanism for this metabolic route consistent with all of the above findings.

extraction conditions were identical to those used for HepG2 cells.

Human platelet studies
Blood was donated by healthy volunteers under the Children's Hospital of Philadelphia protocol #01-002609. Platelet-rich plasma (PRP) was isolated as previously described ( 11 ). Briefl y, donor blood was collected into acid-citrate-dextrose tubes, gently mixed by inversion then transferred to 15 ml conical tubes. Whole blood was spun at 170 g for 15 min at 25°C with no breaks. The resulting upper layer of PRP was transferred, avoiding the buffy coat and red blood cells, to a 1.5 ml Eppendorf tube. The PRP was centrifuged at 400 g for 10 min to pellet the platelets. The supernatant was aspirated and the platelets were gently resuspended in Tyrode's buffer with or without propionate, [ 13 C 3 ] propionate, [ 13 C 1 ]propionate, or 2,2-[ 2 H 2 ]propionate. The samples were incubated for 1 h at 37°C, and then centrifuged at 10,000 g for 2 min to pellet the platelets. The resulting pellet was then taken for extraction and analysis.

Extraction of acyl-CoAs from cell lines and platelets
The procedures for cell extraction have been described in detail previously ( 11,(14)(15)(16)(17). Briefl y, cells or platelets were gently lifted and transferred to 15 ml conical tubes, centrifuged at 500 g for 5 min and resuspended in 1 ml of ice-cold 10% TCA. They were then pulse-sonicated for 30 s on ice using a sonic dismembrator (Fisher), followed by a 5 min centrifugation at 15,000 g at 4°C. The supernatant was transferred to a fresh tube, and the pellet was discarded. The supernatant was purifi ed by solid-phase extraction as follows: Oasis HLB 1 cm 3 (30 mg) solid-phase extraction columns (Waters) were conditioned with 1 ml of methanol followed by 1 ml of water. The collected supernatant was applied, washed with 1 ml of water, and fi nally eluted using three subsequent applications of 0.5 ml of methanol containing 25 mM ammonium acetate. Eluted compounds were dried down under nitrogen and resuspended in 50 l of 5% 5-sulfosalicylic acid. Injections of 10 l were made for LC-ESI/MS analysis.

LC-MS/MS and LC-SRM/MS analysis
Acyl-CoAs were separated using a reversed-phase HPLC Phenomenex Luna C18 column (2.0 × 150 mm, particle size 5 m) with 5 mM ammonium acetate in water as solvent A, 5 mM ammonium acetate in 95/5 ACN/water (v/v) as solvent B, and 80/20/0.1 ACN/water/formic acid (v/v/v) as solvent C. Gradient conditions were as follows: 2% B for 1.5 min, increased to 25% over 3.5 min, increased to 100% B in 0.5 min and held for 8.5 min, washed with 100% C for 5 min before equilibration to 2% B for 5 min. The fl ow rate was 200 l/min. Samples were analyzed using an API 4000 triple-quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) in the positive ESI mode. Samples (10 l) were injected using a Leap CTC autosampler (CTC Analytics, Switzerland) where they were maintained at 4°C, and data were analyzed with Analyst 1.4.1 software. The column effl uent was diverted to the mass spectrometer from 8 to 23 min and to waste for the remainder of the run. The mass spectrometer operating conditions were as follows: ion spray voltage (5.0 kV), nitrogen as curtain gas (15 units), ion source gas 1 (8 units), gas 2 (15 units), and collision-induced dissociation gas (5 units). The ESI probe temperature was 450°C, the declustering potential was 105 V, the entrance potential was 10 V, the collision energy was 45 eV, and the collision exit potential was 15 V. A constant neutral loss of 507 Da was monitored for each acyl-CoA. LC-SRM/MS analysis was conducted using similar instrument parameters and specifi c selected ions as noted in the text. LC-HRMS was conducted on a LTQ-Orbitrap XL provides orthogonal separations and structural information that are ideal for analysis of complex analytes such as acyl-CoAs that are present in biological matrices ( 10 ). Development of new model systems, such as isolated human platelets, may provide a future platform integrating acyl-CoA analysis into quantitative metabolic studies ( 11 ). Furthermore, coupling of stable isotopic labels, including [ 13 C]-and [ 2 H]labeled precursors, to metabolic analysis can provide new insight into cellular metabolism in humans, as well as in animal models ( 12 ).
When employing LC-MS/MS to examine the utilization of isotopically labeled precursors including [ 13 C 1 ]propionate and [ 13 C 3 ]propionate for acyl-CoA biosynthesis, we discovered a high abundance unknown chromatographic spectral feature which was inconsistent with existing mammalian metabolic models. The present study was designed to test the hypothesis that the unknown feature resulted from propionate metabolism to a previously undescribed acyl-CoA.

Cell line studies
HepG2 cells were grown and maintained in DMEM:F12 with 2% FBS and 2 mM glutamine with 100,000 units/l penicillin and 100 mg/l streptomycin. For all experiments, cells were grown to 80% confl uence prior to treatment. For dose response studies, solutions of sodium propionate in maintenance medium were prepared by serial dilution from the most concentrated stock after fi ltration through a 0.2 m sterile fi lter (Corning Inc., Corning, NY). Spent medium was aspirated and new medium containing 0, 1 M, 100 M, 1 mM, 10 mM, or 100 mM propionate was added. After incubations lasting 1 h, the cells were extracted and analyzed. For time-course studies, cells were grown as above, and spent medium was replaced with 1 mM propionate-containing medium. At indicated time-points, cells were taken for extraction and analysis. For all mass isotopologue distribution (MID) analysis, samples were treated with an equal concentration to the tracer with unlabeled propionate (as a control) and processed in parallel. The untreated samples were then analyzed and used for isotope correction as previously described ( 13 ). Mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells were cultured in similar conditions to the HepG2 cells, except the base medium was RPMI1640 for the H358 cells and F-12 K for the LoVo cells. Treatment and under the same conditions with [ 13 C 1 ]propionate revealed a similar acyl-CoA profi le except that MH + for the new acyl-CoA appeared at m/z 866 ( Fig. 1C ) corresponding to a C6 acyl-CoA isotopologue containing both [ 13 C]labeled carbon atoms. Furthermore, [ 13 C 3 ]propionate treatment revealed a highly abundant ion at m/z 870, which would correspond to MH + of a C6 mono-unsaturated acyl-CoA containing all six [ 13 C]atoms ( Fig. 1D ). Finally, treatment of the cells with trans -2-methyl-2-pentenoic acid resulted in the formation of a C6 acyl-CoA with an MH + at m/z 864 and similar constant neutral loss mass spectrum to that observed with propionate ( Fig. 1E ).
Treatment of HepG2 cells with other mono-unsaturated six-carbon fatty acids for 1 h resulted in the substratespecifi c acyl-CoA profi les as demonstrated by constant neutral loss scans of 507 Da (supplementary Fig. 1). However, the LC-MS chromatograms observed after treatment of cells operated in positive ion mode at a resolution of 100,000 as previously described ( 17 ), except that an ESI source was used and the mass spectrometer was coupled to the LC system described above.

Metabolism of propionate in HepG2 cells
Treatment of HepG2 cells with 10 mM propionate revealed an upregulation of certain acyl-CoA species after analysis by the constant neutral loss 507 Da scans to survey short chain acyl-CoA content ( Fig. 1 ). The expected increase of propionyl-CoA with a protonated molecule (MH + ) at m/z 824 was coupled to an unexpected increase in an intense ion at m/z 864 corresponding to MH + of a new C6 mono-unsaturated acyl-CoA ( Fig. 1B ). Treatment    C 3 ]propionate could only be reproduced by the addition of trans -2-methyl-2-pentenoic acid. Importantly, the ion at m/z 838 corresponding to MH + of butyryl-CoA was only observed after treatment with unbranched hexanoyl fatty acids. This confi rmed the differential metabolism of a 2-methyl-substituted pentanoate when compared with an unbranched hexanoate ( 18 ). Correspondingly, propionate did not increase the formation of butyryl-CoA, confi rming that neither propionyl-CoA nor its metabolites were catabolized via unbranched fatty acid ␤ -oxidation. Treatment of other cell lines with propionate, including mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells, also resulted in generation of the new acyl-CoA with MH + at m/z 864 (supplementary Fig. 2).

Dose-response and time-course experiments with propionate
Formation of the new acyl-CoA increased in a dosedependent manner with increasing propionate treatment over the range of 0-1 mM. Its formation reduced with 10 mM and 100 mM propionate suggesting that this new metabolic pathway could be inhibited at high propionate concentrations or that the new acyl-CoA underwent further propionate-dependent metabolism ( Fig. 2A ). An increase of almost an order of magnitude in concentration of the new acyl-CoA was observed between 1 and 100 M propionate with almost another order of magnitude increase between 100 M and 1 mM. Following treatment with 1 mM propionate, the MH + at m/z 864 increased rapidly to a maximum at 1 h, with a stable plateau of levels from 1 h to 12 h, followed by a decrease until 24 h ( Fig. 2B ). [ 13 C 3 15 N 1 ]pantothenic acid was used to label all CoA species in mouse hepatocellular carcinoma Hepa1c1c7 cells, as previously described ( 14 ).  ( Fig. 3C ).

Stable isotope labeling by essential nutrients in cell culture
LC-HRMS analysis was employed to confi rm that the MH + at m/z 864 arose from a C6 mono-unsaturated acyl-CoA ( Table 1 ). An intense MH + was observed at m/z 864.1776 which corresponded to a molecular formula of C 27

MID analyses
HepG2 cells treated with 1 mM [ 13 C 3 ]propionate revealed the anticipated M3 enrichment into MH + of propionyl-CoA ( Fig. 4A ) 4B ); indicating direct incorporation of six carbon atoms from two molecules of propionyl-CoA without any carbon loss. Deuterium labeling in the new acyl-CoA was almost identical to the labeling in propionyl-CoA, with nearly equal enrichment of the M1 and M2 of MH + and virtually no labeling in M3 or M4 ( Fig. 4C ). M3 labeling, which was present in succinyl-CoA only for the [ 13 C 3 ] propionate treatment, arose from anaplerosis into the Krebs cycle ( Fig. 4D ).

Human platelet studies
Platelets from healthy volunteers were isolated for isotopic tracer studies using a previously described procedure ( 11 ). Studies were conducted using platelets because although they are anuclear, they are rich in mitochondria, and produce much higher levels of mitochondrial metabolites than lymphocytes ( 11 ) or neutrophils (data not shown). Thus, platelets have a much higher basal oxygen consumption rate when compared with lymphocytes or neutrophils ( 19 ). In contrast to platelets, lymphocytes primarily only utilize oxidative phosphorylation under basal conditions and they have a limited capacity to increase glycolytic fl ux ( 19 ). Neutrophils have little or no dependence on oxidative phosphorylation and glycolysis is not increased when mitochondrial ATP synthase is inhibited ( 19 ). Furthermore, platelets are amenable to isolation and purifi cation, and do not require culturing, which could otherwise introduce changes in cellular metabolism. Finally, alterations in platelet mitochondrial function have been demonstrated in a variety of diseases so that they have been proposed to serve as potential markers of systemic mitochondrial dysfunction ( 11,20 ).

Characterization of the new acyl-CoA as 2-methyl-2pentenoyl-CoA and mechanism of formation
The LC-MS/MS properties of the new six carbon monounsaturated acyl-CoA were identical with the acyl-CoA that was formed by the addition of trans -2-methyl-2-pentenoic acid to human cell lines and platelets. This fi nding together with the isotope labeling data provided compelling evidence for the structural assignment of the new acyl-CoA as trans -2-methyl-2-pentenoyl-CoA ( Fig. 6 ). Its mechanism of formation must account for the results of three isotopelabeling experiments: 1 ) conservation of the [ 13 C 1 ]atom from each precursor propionate; 2 ) conservation of all [ 13 C 3 ]atoms from precursor propionate; and 3 ) exchangeable deuterium labeling with a maximum of two total conserved [ 2 H]atoms from precursor propionate ( Fig. 6 ). The proposed mechanism involves initial formation of propionyl-CoA through the action of a short chain CoA synthetase ( Fig. 6 ). The limited protium/deuterium exchange observed in the formation of propionyl-CoA from was converted to the fi nal tran s-2-methyl-2-pentenoyl-CoA molecule by a transacylase ( Fig. 6 ). All deuterium atoms that were originally on C2 from the fi rst propionate molecule were lost during the dehydration step ( Fig. 6 ). The remaining deuterium atoms at C2 from the second propionate molecule were partially exchanged for protium ( Fig. 5B ) in a similar manner to that observed in propionyl-CoA ( Fig. 5A ).

DISCUSSION
Diseases of propionic acid metabolism, most notably, propionic and methylmalonic acidemias, result in a widely dysregulated metabolome (21)(22)(23). This follows from the 2,2-[ 2 H 2 ]propionate ( Fig. 5A ) was consistent with the labiality of the deuterium atoms on the ␣ -carbon atom (C2) during CoA thioester formation. The propionyl-CoA was then converted to propionyl-acyl carrier protein (ACP) and 2-methyl-malonyl-CoA through the action of propionyl-CoA:ACP transacylase or propionyl-CoA carboxylase, respectively ( Fig. 6 ). Subsequently, 3-keto-ACP synthase added an additional propionate moiety from propionyl-ACP with a concomitant loss of CO 2 (containing the carbon added during the formation of 2-methyl-malonyl-CoA) and free ACP . This conserved the [ 13 C]atom from 1-[ 13 C]propionate and partially conserved one of the deuterium atoms at C2 derived from the fi rst molecule of 2,2-[ 2 H 2 ]propionate. Sequential actions of a reductase and dehydratase resulted in the generation of an olefi n, which  patients ( 22 ). This would occur through fatty acid elongation, oxidation, or other metabolism from the precursor metabolite, identifi ed here as trans -2-methyl-2-pentenoyl-CoA. Our study has provided the fi rst identifi cation of this metabolite and so there is currently no information on the physiological relevance of this fi nding. However, previous untargeted metabolomics surveys have most likely observed trans -2-methyl-2-pentenoylcarnitine but lacked suffi cient depth of experimental information to characterize the exact structure or propose a mechanism of formation ( 23 ). Clearly, further research is needed to examine the biochemistry of the newly identifi ed trans -2methyl-2-pentenoyl-CoA, as we did not investigate the enzymes involved in its formation or its potential for further metabolism. Although we did not quantify the levels of propionic acid in the media over the course of the experiments, an understanding of the utilization of the substrate propionate in terms of kinetics and relative incorporation into downstream metabolites would be useful in the future.
Relevant to the present study, the normal physiological range of propionic acid in portal vein blood is reported to range between 0.1 and 0.3 mM in nonfasting humans, and lower in fasting individuals ( 27 ). Peripheral blood concentrations are much lower at 6 M, but patients in ketotic hyperglycemia may have 0.1-0.3 mM propionic acid in peripheral blood ( 28 ). Reported incompatibility with life was described at blood concentrations of 4-6 mM propionic acid in propionic acidemia patients in crisis ( 29 ). Although we observed no immediate cellular toxicity by light microscopy within the time frame of treatment at 10 mM and 100 mM, we cannot discount the seemingly likely conclusion that the decrease in trans -2-methyl-2-pentenoic acid formation at these doses was due to cell death. The lack of detection of the intermediate at basal conditions may relate to the fact that there is no sodium propionate added to DMEM base media. Thus, the doses of propionate used in this study range from below to above physiological relevance.
Understanding of propionate metabolism has been driven not only by human disease but also by the critical nature of propionate for branched chain hydrocarbons and hormones in insects ( 6,30 ). In fact, 2-methyl-2-pentenoic acid is an aggregation pheromone of the grain borer, and is produced by the grain borer when feeding ( 31 ). As sources of propionate are relatively energetically expensive to a developing insect, branched chain products are likely to be biologically important ( 32 ). Such branched chain products include 2-methyl branched alkanes, critical components of the cuticular lipids that compose signifi cant portions of the outer shells of insects.
Absolute quantifi cation of intracellular metabolites serves an indispensable role in identifying and characterizing new metabolic pathways. For this purpose, LC-SRM/ MS is the gold standard for quantifi cation because it affords high sensitivity and specifi city from complex biological matrices, especially when coupled to the isotopically labeled analogs of target analytes as internal standards to adjust for variation in extraction and analysis ( 33 ). diverse metabolic capacity that utilizes propionyl-CoA as an intermediate, such as catabolism of the branched chain amino acids, isoleucine and valine, via propionyl-CoA. Propionyl-CoA can also be formed directly from activation of propionate with free CoASH. Additionally, catabolism of threonine, methionine, cholesterol, odd-chain fatty acids, and C 5 -ketone bodies are precursors in the formation of propionyl-CoA. Importantly, metabolism via propionyl-CoA provides an anaplerotic pathway (through D-malonyl-CoA then L-malonyl-CoA) to generate succinyl-CoA as a precursor to other intermediates in the Krebs cycle ( 24,25 ). The effectiveness of propionyl-CoA as an anaplerotic precursor is driven by a tightly regulated equilibrium, allowing a diverse pool of substrates to enter the Krebs cycle ( 26 ).
Considering this diversity of substrates and products for reactions involving propionyl-CoA, it is not surprising that many studies have implicated aberrant propionate metabolism as leading to disruptions in other metabolic pathways. The existence of a propionyl-CoA to trans -2methyl-2-pentenoyl-CoA metabolic pathway may explain the fi ndings of unique acylcarnitines with branched medium chain acyl groups in the urine of propionic acidemia However, it is also useful in combination with stable isotope labeling for examining alterations that can occur to cellular metabolite pools ( 9,13 ). Monitoring the uptake and conversion of isotopically labeled nutrients into downstream metabolic pathways can shed light on unknown components of metabolism ( 12 ). The utility of stable isotopes for metabolic elucidation is demonstrated by the fi ndings in this report, where [ 13 C 1 ]-, [ 13 C 3 ]-, and 2,2-[ 2 H 2 ]labeled propionate provided complimentary metabolic information in conjunction with LC-SRM/MS and LC-HRMS. For quantitative studies, stable isotope analog internal standards utilizing carbon or nitrogen labels are more desirable than deuterium, which causes small LC retention time shifts and can potentially undergo protium/deuterium exchange. However, deuterated analogs may provide insight into unexpected metabolic pathways, as demonstrated in this study and in the recent elucidation of folate-dependent generation of NADPH ( 34 ). Constantly improving capabilities of LC-SRM/MS and LC-HRMS may warrant reexamination of previously studied metabolic pathways, and may make identifi cation of previously unrecognized pathways possible.
In summary, we have used absolute quantifi cation in combination with MID analysis to show both the propionatedependent metabolism and labeling from upstream metabolic sources of trans -2-methyl-2-pentenoyl-CoA in multiple biological systems. Future work could elucidate the enzymology of this pathway completely, as well as assess the contribution of trans -2-methyl-2-pentenoic acid to metabolic crisis in propionic acidemia patients.
The authors thank Drs. David Lynch and Peisong Ma for technical assistance in isolating platelets. They also thank Barry Slaff and Dr. Aalim Weljie for providing advice on the structural assignments of acyl-CoAs.