Alpha-Ketoglutaric Acid

The harvested cells were added to metabolites and antibiotic and incubated at 30°C for 6 h. After incubation, cells were collected and re-suspended in sterile saline to OD₆₀₀ = 1.0. Samples with 1 mL were collected by centrifugation at 8,000 rpm for 5 min. Pellets were re-suspended in PBS and broke down by sonication for 2 min at a 200 W power setting on ice, and then centrifuged at 12,000 rpm for 10 min to remove insoluble material. Supernatants containing 400 µg total proteins were transferred to pyruvate dehydrogenase (PDH) reaction mix (0.5 mM MTT, 1 mM MgCl₂, 6.5 mM PMS, 0.2 mM TPP, 2 mM sodium pyruvate, 50 mM PBS), ketoglutarate dehydrogenase (KGDH) reaction mix (0.5 mM MTT, 1 mM MgCl₂, 6.5 mM PMS, 0.2 mM TPP, 50 mM alpha-ketoglutaric acid potassium salt, 50 mM PBS), or succinate dehydrogenase (SDH) reaction mix (0.5 mM MTT, 13 mM PMS, 5 mM sodium succinate, 50 mM PBS), to a final volume of 200 µL in 96-well plate. Subsequently, the plate was incubated at 37°C for 5 min for SDH/PDH/OGD assays, and then measured at 566 nm for colorimetric readings. The plate was protected from light during the incubation. Experiments were repeated at least in three independent biological replicates.

For measurement of pyruvate dehydrogenase (PDH), alpha-ketoglutarate dehydrogenase (KGDH), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) activities, bacterial cells were suspended in 1× phosphate-buffered saline (PBS; pH 7.4) and adjusted to an OD₆₀₀ of 1.0. An aliquot of 50 ml of cells was collected and transferred to a 1.5-ml centrifuge tube. The cells were resuspended with 1 ml of 1× PBS and disrupted by sonic oscillation for 6 min (200-W total power with 35% output, 2-s pulse, and 3-s pause over ice). Following centrifugation at 12,000 × g for 10 min at 4°C, supernatants were collected. The protein concentration of the supernatants was quantified with a BCA protein concentration determination kit (Beyotime; P0009). Then, 300 μg of proteins was used for determination of enzyme activity. For PDH and KGDH measurement, the reaction mixture contained 0.15 mM 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2.5 mM MgCl₂, 6.5 mM phenazine methosulfate (PMS), 0.2 mM thiamine PP_i (TPP), and 80 mM sodium pyruvate/alpha-ketoglutaric acid potassium salt, with water added to 200 μl. For SDH and MDH measurement, the reaction mixture contained 0.15 mM MTT, 2.5 mM MgCl₂, 13 mM PMS, and 80 mM sodium succinate/sodium malate, with water added to 200 μl. All of the reaction mixtures were incubated at 37°C, for 5 min for MDH/PDH/KGDH and for SDH for 10 min. Finally, they were measured at 562 nm for colorimetric readings. Light was avoided in all reactions. Experiments were repeated in at least three independent biological replicates.

Optima™ LC/MS-grade formic acid and HPLC-grade water were purchased from Fisher Scientific (Ottawa, ON, Canada). HPLC-grade pyridine and HPLC-grade methanol, 3-nitrophenylhydrazine (3-NPH), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and butylated hydroxytoluene (BHT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The pooled urine was purchased from Lee Biosolutions (Maryland Heights, MO, USA). 3-(3-Hydroxyphenyl)-3-hydroxypropionic acid (HPHPA) and ¹³C-labeled HPHPA standards were synthesized and purified in-house. All other organic acid standards including lactic acid, beta-hydroxybutyric acid, alpha-ketoglutaric acid, citric acid, butyric acid, isobutyric acid, propionic acid, p-hydroxyhippuric acid, succinic acid, fumaric acid, pyruvic acid, hippuric acid, methylmalonic acid, homovanillic acid, indole-3-acetic acid, uric acid, and their isotope-labeled standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). All 17 acids along with HPHPA were part of the same assay. Nunc^® 96 DeepWell™ plates were purchased from Sigma-Aldrich (St. Louis, MO, USA). Millex-HA Syringe Filter Units (0.22 µm) were purchased from Fisher Scientific (Ottawa, ON, Canada).
Solid chemical standards for all organic acids mentioned above were carefully weighed using a CPA225D semi-micro electronic balance (Sartorius, Bohemia, NY, USA) with a precision of 0.0001 g. Stock solutions, 40 mM for each analyte, were prepared in water by dissolving the weighed solid into specified volumes. Calibration curve solutions were prepared by mixing and diluting the stock solutions with water to generate solutions with concentration ranges from 5 to 400 µM for HPHPA (details of the concentration ranges are not provided here for all other organic acids mentioned above except HPHPA). Stock solutions, 10 mM each, of the isotope-labeled compounds were prepared by dissolving the accurately weighed solids in 75% aqueous methanol. A working internal standard solution mixture in 75% aqueous methanol was made by mixing and diluting all the isotope-labeled stock solutions, with a final concentration of 160 µM for ¹³C-HPHPA. All standard solutions were then stored at −80 °C until further use.
To measure the concentrations of HPHPA in different human urine samples, we used previously collected human urine from 10 healthy volunteers. The study was conducted in compliance with the ethical standards of, and was approved by, the University of Alberta’s HREB biomedical ethics committee (Pro00074045) on 3 October 2017. The urine samples were thawed on ice and filtered with a 0.22 µm Millex-HA syringe filter unit before any further sample preparation. For urine analysis, 10 μL of the ISTD (isotopic standard) mixture solution and 10 μL of the samples were pipetted directly into each spot in a Nunc^® 96 DeepWell™ plate. Thirty microliters of 75% aqueous methanol was then added to each well, followed by the addition of 25 μL each of the following three solutions: 3-NPH (3-nitrophenylhydrazine, 250 mM in 50% aqueous methanol), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 150 mM in methanol), and pyridine (7.5% in 75% aqueous methanol). The whole plate was then shaken at room temperature to allow the derivatization reagents to react for 2 h. After derivatization, 350 μL of water and 25 μL of BHT solution (2 mg/mL of butylated hydroxytoluene, in methanol) were added to each plate well to dilute and stabilize the final solutions. Ten microliters of each solution was injected for LC-MS/MS analysis.
Online LC-MS/MS was performed using an Agilent 1100 series HPLC system (Agilent, Palo Alto, CA, USA) equipped with an Agilent reversed-phase Zorbax Eclipse XDB C18 column (3.0 mm × 100 mm, 3.5 μm particle size, 80 Å pore size) coupled to a Phenomenex (Torrance, CA, USA) SecurityGuard C18 pre-column (4.0 mm × 3.0 mm) connected with an AB SCIEX QTRAP^® 4000 mass spectrometer (SCIEX, Concord, ON, Canada). The controlling software for the LC-MS system was Analyst^® 1.6.2. For the HPLC chromatography, solvent A was 0.01% (v/v) formic acid in water, and solvent B was 0.01% (v/v) formic acid in methanol. The solvent gradient profile was as follows: t = 0 min, 30% B; t = 1.5 min, 30% B; t = 12.5 min, 85% B; t = 12.51 min, 100% B; t = 13.51 min, 100% B; t = 13.6 min, 30% B and finally maintained at 30% B for 6.4 min. The column oven was set to 40 °C. The flow rate was 300 μL/min, and the sample injection volume was 10 μL. The MS instrument was set to a negative electrospray ionization mode with multiple reaction monitoring. The IonSpray voltage was set to −4500 volts and the temperature was set to 400 °C. The curtain gas (CUR), ion source gas 1 (GAS1), ion source gas 2 (GAS2) and collision gas (CAD) were set at 20, 30, 30, and medium, respectively. The entrance potential (EP) was set to −10 V. Additionally, the declustering potential (DP), collision energy (CE), collision cell exit potential (CXP), MRM Q1 and Q3 were optimized and set individually for each analyte and each corresponding isotopically-labeled internal standard. In particular, quantification was performed using MRM transition m/z 316.2→194.2 (DP = −70 V, CE = −20 V, and CXP = −9 V) for HPHPA and m/z 317.1→195.0 (DP = −69 V, CE = −18 V, and CXP = −9 V) for ¹³C-HPHPA.
Details regarding the method validation for measuring HPHPA, including calibration regression, accuracy and precision, recovery, LOQ, and LOD are given in the Supplementary Materials.

Import of nuclear-encoded proteins into the mitochondrial matrix requires a normal mitochondrial membrane potential (ΔΨ) [69 (link)]. Thus, we used the chimeric fusion protein preCox4-mCherry that includes the inner mitochondrial membrane targeting pre-sequence of Cox4 fused to mCherry as a readout to monitor the mitochondrial membrane potential in vivo [43 (link)]. In our experiments, we designed the strain MCY2080 carrying OM45-GFP as a control mitochondrial marker and pre-COX4-mCherry which was integrated at an intergenic region near the centromere of chromosome IV. While pre-COX4-mCherry requires the ΔΨ for its import into mitochondria, OM45 is an outer membrane protein for which import is not affected by changes in the ΔΨ [70 (link)]. Prior to imaging, cells were grown to mid-log phase at 30 °C in SD medium and supplemented when required with 10 mM citrate (citric acid—Merck, C2404), 10 mM alpha-ketoglutarate (alpha-ketoglutaric acid—Merck,75890), or 10 mM glutamate (L-glutamic acid—Merck, G5889). The pH of the media was adjusted using few drops of KOH 10 M solution upon addition of citrate or alpha-ketoglutarate. Cells were subsequently fixed with 3.7% formaldehyde for at least 20 min at room temperature. Using conventional fluorescent microscopy, we counted cells which exhibited a diffuse cytoplasmic localization of preCox4-mCherry, reflecting defects in pre-COX4-mCherry import and in turn a drop in the ΔΨ, while the mitochondrial localization of OM45-GFP was unaffected. The localization of preCox4-mCherry was assessed in at least 100 cells. Data reported are the mean and SEM (error bars) from 3 independent experiments.

All patients were diagnosed, treated, and received follow-up assessments at Hospital de Especialidades, Centro Médico Nacional Siglo XXI by the non-functioning adenoma clinic as part of the Endocrinology Service. The demographic, clinical, hormonal, and imaging characteristics of the patients are summarized in Table 1.
After tumor extraction, the tissue was transported in the cold organ preservation medium, CUSTODIOL^® (in mM: 15 NaCl, 9 KCl, 1 Alpha monopotassium ketoglutaric acid, 4 MgCl₂ hexahydrate, 18 L-Histidine monohydrochloride monohydrate, 180 L-Histidine, 2 L-Tryptophan, 30 Mannitol, 0.015 CaCl₂ dihydrate, 1000 mL vehicle g.s.). No more than 30 min elapsed between the surgery and the beginning of the experimental protocol. Pituitary tumors were divided into two sections with the aid of a scalpel; the first section was isolated to record the cell activity of intracellular calcium and the association of these cells with the vasculature. The second section was paraffin embedded and taken for routine characterization by immunohistochemistry using specific antibodies for the pituitary hormones (TSH, GH, PRL, FSH, LH, and ACTH) and transcription factors (NR5A1, POU1F1, and TBX19) [21 (link),39 (link)].

For extraction of intracellular metabolites, cells were washed with 1 mL ice-cold 150 mM ammonium acetate (NH₄AcO, pH 7.3). After that, 1 mL of −80 °C cold 80% MeOH was added to the wells, and samples were incubated at −80 °C for 20 min before cells were scraped off and transferred into tubes and centrifuged at 4 °C for 10 min at 21,000 g. The supernatants were transferred into new tubes, and the cell pellets were re-extracted with 200 μl ice-cold 80% MeOH, spun down and the supernatants were combined. Metabolites were dried at room temperature under vacuum and re-suspended in water for LC-MS run. Targeted Metabolomics analyses were performed at Proteomics and Metabolomics Core of Cleveland Clinic Lerner Research Institute.
Isotope tracing experiments were performed as previously described^{74 (link)–76 (link)}. For glycolysis tracing, cells were cultured with medium containing [U-¹³C]-labeled glucose for 15 min. For de novo pyrimidine synthesis activity, cells were cultured with medium containing [Amide-¹⁵N] glutamine for 15 min. Metabolite extraction was then performed.
Samples were randomized and analyzed on a Q-Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer coupled to Vanquish UHPLC system (Thermo Fisher). The mass spectrometer was run in polarity switching mode ( + 3.00 kV/−2.25 kV) with an m/z window ranging from 65 to 975. Mobile phase A was 5 mM NH₄AcO, pH 9.9, and mobile phase B was acetonitrile. Metabolites were separated on a Luna 3 µm NH₂ 100 Å (150 × 2.0 mm) column (Phenomenex). The flow rate was 300 µl/min, and the gradient was from 15% A to 95% A in 18 min, followed by an isocratic step for 9 min and re-equilibration for 7 min. All samples were run in at least three biological replicates.
Metabolites were detected and quantified as area under the curve based on retention time and accurate mass ( ≤ 5 ppm) using TraceFinder 4.1 (Thermo Scientific) software against known external standards. Raw data was corrected for naturally occurring ¹³C and ¹⁵N abundance and tracer impurity using the IsoCorrectoR package^{77 (link)}. The full panel of metabolites were then subjected to cell number normalization and data were presented as normalized peak area. Metabolite levels were further compared using a two-tailed, unpaired Student’s t test.
Full name of the metabolites. PEP: phosphoenolpyruvate; 2-PG: 2-phosphoglyceric acid; 3-PG: 3-phosphoglyceric acid; G-6-P: glucose-6-phosphate; F-6-P: fructose-6-phosphate; F-1,6-BP: fructose-1,6-biphosphate; 1,3-BPG: 1,3-biphosphoglyceric acid; DHAP: dihydroxyacetone phosphate; GADP: glyceraldehyde-3-phosphate; NAD: nicotinamide adenine dinucleotide; NAM: nicotinamide; NMN: nicotinamide mononucleotide; UMP: uridine monophosphate; UDP: uridine diphosphate; IMP: inosine monophosphate; AMP: adenosine monophosphate; Ribulose-1,5-BP: ribulose-1,5-biphosphoate; 6-PG: 6-phosphogluconate; Ribose-5-P: ribose-5-phosphoate; α-KG: alpha-ketoglutaric acid.