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| Targets |
2-Methylcitric acid targets mitochondrial respiration. It markedly inhibits ADP-stimulated and uncoupled respiration in mitochondria supported by glutamate, likely by interfering with the tricarboxylic acid (TCA) cycle or by inhibiting mitochondrial transporters. This mechanism contributes to the neurological and metabolic dysfunction seen in propionic acidemia and methylmalonic acidemia, where 2-methylcitric acid accumulates. The compound is formed via the action of methylcitrate synthase (EC 2.3.3.5) from propionyl-CoA and oxaloacetate, and its accumulation serves as a diagnostic biomarker.
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| ln Vitro |
Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as quantitative tracers while the drugs were being developed. Because deuteration may have an effect on a drug's pharmacokinetics and metabolic properties, it is a cause for concern [1].
In vitro, 2-Methylcitric acid (unlabeled) markedly inhibits ADP-stimulated and uncoupled respiration in mitochondria supported by glutamate at concentrations that accumulate in patients with propionic acidemia and methylmalonic acidemia. This inhibition of mitochondrial respiration contributes to the cellular energy deficit and organ dysfunction observed in these disorders. The compound is also used as a metabolite standard to study the metabolism of propionyl-CoA and the TCA cycle. In hepatocyte cultures, 2-methylcitric acid accumulation can be induced by treatment with propionate or by inhibition of methylmalonyl-CoA mutase. 2-Methylcitric acid-d3 is not used for activity studies; it serves as an analytical internal standard. |
| ln Vivo |
In vivo, 2-Methylcitric acid accumulates in patients with methylmalonic acidemia and propionic acidemia, inborn errors of metabolism caused by deficiencies in methylmalonyl-CoA mutase or propionyl-CoA carboxylase, respectively. Elevated levels of 2-methylcitric acid are detected in urine and plasma and serve as a diagnostic and monitoring biomarker for these disorders. The accumulation contributes to the metabolic acidosis, hyperammonemia, and neurological damage observed in affected patients. The compound is not used as a therapeutic agent. In animal models, administration of propionate or its precursors leads to increased urinary 2-methylcitric acid levels.
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| Enzyme Assay |
For non-cellular assays (analytical quantification), 2-Methylcitric acid-d3 is prepared as a stock solution in water or methanol (1 mg/mL). For LC-MS/MS analysis, a calibration curve for 2-methylcitric acid is prepared in human urine or plasma (0.1-1000 ng/mL) with a fixed concentration of the deuterated internal standard (e.g., 50 ng/mL). Sample preparation: 100 uL urine or plasma + 20 uL internal standard + 880 uL acetonitrile. After centrifugation, the supernatant is diluted with water (1:1) and injected onto a C18 column or a HILIC column with a mobile phase of 5 mM ammonium acetate in water and acetonitrile (gradient elution). MRM transitions: 2-methylcitric acid 205→143 (loss of two H2O) and 205→99; 2-methylcitric acid-d3 208→146 and 208→102. For non-cellular assays of mitochondrial respiration, isolated mitochondria (from rat liver) are used.
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| Cell Assay |
For cell-based assays, primary hepatocytes or fibroblasts from patients with propionic acidemia or methylmalonic acidemia are cultured in DMEM with 10% FBS. Cells are seeded in 6-well plates (1×10⁶ cells/well) and cultured in medium containing propionate (1-10 mM) to induce 2-methylcitric acid accumulation. Cell lysates and culture medium are collected, and 2-methylcitric acid levels are measured by LC-MS/MS using the d3-labeled internal standard. For mitochondrial respiration studies, cells are treated with 2-methylcitric acid (0.1-5 mM) for 24-48 hours, and oxygen consumption rate (OCR) is measured using a Seahorse XF analyzer. Cell viability is assessed by MTT assay.
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| Animal Protocol |
For in vivo animal experiments, rodent models of propionic acidemia (e.g., PCCA or PCCB knockout mice) or methylmalonic acidemia (e.g., Mut-/- mice) are used. These models exhibit elevated urinary 2-methylcitric acid levels. For diagnostic method development, urine and plasma are collected from patients or animal models. 2-Methylcitric acid is extracted with acetonitrile or methanol, and 2-Methylcitric acid-d3 is added as internal standard. Analysis is performed by LC-MS/MS as described in non-cellular assays. No administration of the labeled compound is required for diagnostic applications. For toxicokinetic studies, animals can be administered propionate (100-500 mg/kg, IP) to induce acute metabolic disturbances, and 2-methylcitric acid levels in blood and urine are measured over time using the deuterated internal standard.
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| ADME/Pharmacokinetics |
2-Methylcitric acid-d3 has a molecular weight of 209.17, with three deuterium atoms providing a mass shift of +3 Da relative to unlabeled 2-methylcitric acid (MW 206.15). The compound is soluble in water and methanol. It should be stored as a powder at -20degC for up to 3 years, and in solution at -80degC for up to 6 months or at -20degC for up to 1 month. The deuterium label is stable and non-radioactive. The compound is the deuterated form of 2-methylcitric acid, a tricarboxylic acid that is a key metabolite in the propionyl-CoA catabolism pathway. It has three carboxylic acid groups (pKa values approximately 3.1, 4.4, and 5.6).
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| Toxicity/Toxicokinetics |
2-Methylcitric acid-d3 is a stable isotope-labeled compound with minimal toxicity at analytical concentrations (ng-ug per sample). The non-deuterated parent compound, 2-methylcitric acid, at concentrations that accumulate in patients with propionic and methylmalonic acidemias (1-10 mM) inhibits mitochondrial respiration, contributing to the pathophysiology of these diseases. However, when used as an internal standard, the amount is negligible and poses no toxicity risk. Standard laboratory safety precautions for handling organic acids should be followed. The compound is non-radioactive and safe for use in research laboratories.
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| References |
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| Additional Infomation |
2-Methylcitric acid-d3 is an analytical standard and research tool, not an approved drug. No clinical trials have been conducted with the deuterated version for therapeutic purposes. The non-deuterated parent compound, 2-methylcitric acid, is a diagnostic and monitoring biomarker for propionic acidemia and methylmalonic acidemia, two rare inherited metabolic disorders. Early detection and monitoring of these disorders via measurement of 2-methylcitric acid in urine or dried blood spots by LC-MS/MS allow for timely intervention (dietary management, cofactor supplementation, liver transplantation) to prevent severe metabolic decompensation and neurological damage. 2-Methylcitric acid-d3 is used exclusively as an internal standard for quantitative LC-MS/MS analysis of 2-methylcitric acid in clinical and research settings. Available for research use only.
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| Molecular Formula |
C7H7D3O7
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| Molecular Weight |
209.17
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| Exact Mass |
209.061
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| CAS # |
146764-58-1
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| Related CAS # |
2-Methylcitric acid;6061-96-7
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| PubChem CID |
90470055
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| Appearance |
White to off-white solid powder
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| Density |
1.6±0.1 g/cm3
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| Boiling Point |
313.1±42.0 °C at 760 mmHg
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| Flash Point |
157.4±24.4 °C
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| Vapour Pressure |
0.0±1.5 mmHg at 25°C
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| Index of Refraction |
1.559
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| LogP |
-1.37
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
14
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| Complexity |
271
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| Defined Atom Stereocenter Count |
0
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| SMILES |
[2H]C([2H])([2H])C(C(=O)O)C(CC(=O)O)(C(=O)O)O
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| InChi Key |
YNOXCRMFGMSKIJ-FIBGUPNXSA-N
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| InChi Code |
InChI=1S/C7H10O7/c1-3(5(10)11)7(14,6(12)13)2-4(8)9/h3,14H,2H2,1H3,(H,8,9)(H,10,11)(H,12,13)/i1D3
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| Chemical Name |
4,4,4-trideuterio-2-hydroxybutane-1,2,3-tricarboxylic acid
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.7808 mL | 23.9040 mL | 47.8080 mL | |
| 5 mM | 0.9562 mL | 4.7808 mL | 9.5616 mL | |
| 10 mM | 0.4781 mL | 2.3904 mL | 4.7808 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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