Isocitrate

alpha-PBP and alpha-PEP were provided by the Bavarian State Criminal Police Office (Munich, Germany). NADP-Na₂, acetonitrile (LC-MS grade), and methanol (LC-MS grade) were obtained from VWR (Darmstadt, Germany), MgCl₂, K₂HPO₄, K₃PO₄, superoxide dismutase, isocitrate dehydrogenase, isocitrate, ammonium formate, ammonium acetate, and formic acid from Sigma (Taufkirchen, Germany). Water was purified with a Millipore filtration unit (18.2 Ω × cm water resistance). pHLM (pool of 25 donors, 20 mg microsomal protein/mL) were obtained from Corning (Amsterdam, The Netherlands). After delivery, the pHLM were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.

d-2-Hydroxyglutarate (d-2-HG), l-2-hydroxyglutarate (l-2-HG), d-lactate, l-lactate, d-malate, l-malate, d-tartrate, glutarate, 2-ketoglutarate (2-KG), d-glycerate, l-glycerate, succinate, pyruvate, oxaloacetate, cis-aconitate, citrate, isocitrate, dichlorophenol-indophenol (DCPIP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phenazine methosulfate (PMS), NADH, NAD⁺, SYPRO orange, bovine serum albumin (BSA), GSK864, and AGI-6780 used in this study were purchased from Sigma-Aldrich (USA). AlphaScreen donor and acceptor beads were purchased from PerkinElmer (USA). Fumarate and human serum were purchased from Beijing Solarbio Science & Technology Co., Ltd (China). Urine samples were collected from a healthy volunteer; informed consent was provided. d,l-2-Hydroxyglutarate disodium salt (2,3,3-D3) was purchased from Cambridge Isotope Laboratories, Inc. (USA). All other chemicals were of analytical reagent grade.

Compounds of CP-17 and AGI-6780 were purchased from TargetMol (Shanghai, China) and MedChemExpress (Shanghai, China) respectively. The human recombinant C-terminal FLAG-tag IDH enzymes including IDH2/R140Q, IDH2/WT, IDH1/R132H and IDH1/WT homodimer were purchased from BPS Bioscience (San Diego, USA). Antibodies of Hemoglobin γ (HBG) (catalog number: 39386S), H3K9me3 (catalog number: 5327S), H3K27me3 (catalog number: 9733S), H3K4me3 (catalog number: 9725S), and H3 (catalog number: 4499S) were purchased from Cell Signaling Technology (Massachusetts, USA). Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) were obtained from Peprotech (New jersey, USA). NADPH, NADP, α-KG, isocitrate, D-2-HG acid disodium, DMSO, diaphorase and resazurin were purchased from Sigma-Aldrich (Missouri, USA).

Methoxyacetylfentanyl hydrochloride (purity ≥ 98%), U-51754 hydrochloride (purity 98%), and U-47931E were purchased from LGC Standards (Wesel, Germany). Isocitrate, Isocitrate dehydrogenase, superoxide dismutase, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), S-(5′-adenosyl)-L-methionine (SAM), dithiothreitol (DTT), reduced glutathione (GSH), acetyl carnitine, and acetyl coenzyme A (AcCoA) were all purchased from Sigma (Taufkirchen, Germany), NADP⁺ from Biomol (Hamburg, Germany), and acetonitrile (LC-MS grade), ammonium formate (analytical grade), formic acid (LC-MS grade), methanol (LC-MS grade), glucuronidase (EC No. 3.2.1.32)/arylsulfatase (EC No. 3.1.6.1) from Helix pomatia L, and all other chemicals and reagents (analytical grade) from VWR (Darmstadt, Germany). phS9 (20 mg protein/mL, from 30 individual donors), UGT reaction mix solution A (25 mM UDP-glucuronic acid), and UGT reaction mix solution B (250 mM Tris–HCl, 40 mM MgCl₂, and 0.125 mg/mL alamethicin) were obtained from Corning (Amsterdam, The Netherlands). After delivery, the enzyme preparations were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.

A-CHMINACA was provided by the EU project ADEBAR/State Bureau of Criminal Investigation Schleswig-Holstein (Kiel, Germany) for research purpose. The chemical purity and identity of the compound were verified by MS and nuclear magnetic resonance analysis. Ammonium formate, ammonium acetate, formic acid, isocitrate dehydrogenase, isocitrate, dipotassium phosphate, tripotassium phosphate, magnesium chloride, and superoxide dismutase were obtained from Sigma (Taufkirchen, Germany). Acetonitrile (LC-MS grade), methanol (LC-MS grade), and NADP-Na₂ were from VWR (Darmstadt, Germany). pHLM (20 mg microsomal protein mL⁻¹) was obtained from Corning (Amsterdam, The Netherlands). After delivery, pHLM were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.

Ferric ammonium citrate (FAC), citrate, isocitrate, succinate, malate, lactate, pyruvate, deferiprone, and deferoxamine were purchased from Sigma-Aldrich (St. Louis, MO). The antibody for ferroportin was obtained from Abcam, Inc. (Cambridge, MA), which was raised against a peptide in human ferroportin. Radiolabeled citrate (1,5-[¹⁴C]-citrate; specific radioactivity, 116.4 mCi/mmol) and radiolabeled Fe³⁺ (⁵⁹FeCl₃; specific radioactivity, >5 mCi/mg) were obtained from PerkinElmer (Waltham, MA). The sequences of the primers used in mRNA analysis and quantification for different citrate and iron/zinc transporters are listed in Supplemental Table 1.