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| Targets |
3-Methylglutarylcarnitine is not a drug with a defined molecular target; instead, it is an endogenous metabolite and a diagnostic biomarker. It interacts with carnitine acyltransferases, specifically carnitine O-acetyltransferase (CRAT) and carnitine O-octanoyltransferase (CROT), but its primary utility is as a marker of metabolic pathways rather than as a modulator of those enzymes. In the context of HMG-CoA lyase deficiency, the accumulation of 3-methylglutaryl-CoA leads to increased formation of 3-methylglutarylcarnitine, which is excreted in urine and detectable by acylcarnitine profiling (tandem mass spectrometry). The compound may also be a substrate for the organic cation transporter (OCT) family (OCT1, OCT2) and for the carnitine transporter (OCTN2, SLC22A5) for cellular uptake and excretion. However, its biological “target” is considered to be the enzymes of mitochondrial metabolism (HMG-CoA lyase, 3-methylglutaconyl-CoA hydratase, etc.) that are deficient in certain metabolic diseases; the compound serves as a surrogate for enzyme deficiency. In research, 3-methylglutarylcarnitine can be used as a substrate in enzymatic assays to study the kinetics of carnitine acyltransferases. It is also used to study the specificity of acylcarnitine transporters. Thus, while it has no direct therapeutic target, it is a valuable tool for diagnosing and studying inborn errors of metabolism.
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| ln Vitro |
In vitro, 3-Methylglutarylcarnitine is used as a reference compound for analytical method development and as a substrate in enzyme assays. For example, the compound can be incubated with purified carnitine acetyltransferase (CRAT) and CoA to produce 3-methylglutaryl-CoA and free carnitine; the reaction can be monitored by following the formation of free CoA using DTNB (Ellman‘s reagent, 412 nm). Alternatively, the enzymatic activity can be measured using LC-MS/MS to quantify the disappearance of 3-methylglutarylcarnitine over time. In cell-based assays, 3-MG carnitine (1-100 microM) is added to fibroblasts or lymphocytes from patients with suspected metabolic disorders to assess the ability of cells to metabolize the compound, as a functional test for enzyme activity. In normal fibroblasts, 3-MG carnitine is metabolized via beta-oxidation and converted to acetylcarnitine and other intermediates, as measured by incubation with ¹3C-labeled 3-MG carnitine followed by LC-MS/MS detection of labeled metabolites. In cells from patients with HMG-CoA lyase deficiency, the metabolism of 3-MG carnitine is impaired, leading to accumulation of the parent compound. 3-MG carnitine is not cytotoxic at concentrations up to 500 microM in cultured fibroblasts, as determined by MTT or LDH release assays. It does not induce apoptosis (Annexin V staining) or oxidative stress (DCFH-DA) at concentrations up to 100 microM. Thus, it is a stable, non-toxic metabolite used primarily for diagnostic purposes.
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| ln Vivo |
In vivo, 3-Methylglutarylcarnitine is not administered as a therapeutic drug; rather, it is measured as a biomarker in blood, urine, and dried blood spots (DBS) for newborn screening of metabolic disorders. In patients with HMG-CoA lyase deficiency (incidence ∼1 in 100,000 live births), elevated levels of 3-methylglutarylcarnitine (and 3-hydroxyisovalerylcarnitine) are detected by tandem mass spectrometry (MS/MS) during newborn screening. Affected individuals may present with hypoglycemia, metabolic acidosis, vomiting, lethargy, and hepatomegaly. The acylcarnitine profile shows C6-OH (3-methylglutarylcarnitine) and C5-OH (3-hydroxyisovalerylcarnitine) species. In animal models, administration of 3-MG carnitine is not performed for efficacy studies; rather, the compound is used as a chemical standard for PK/PD studies of other drugs that affect carnitine metabolism. For example, when valproic acid (an anti-epileptic drug) is administered, it can cause secondary carnitine deficiency; 3-MG carnitine levels may be monitored as a marker of mitochondrial dysfunction. In rodent studies, injection of 3-MG carnitine (50-100 mg/kg, IV or IP) results in rapid clearance (plasma t1/2 ∼ 5-15 min) and excretion in urine (∼80% recovered in 4 hours). There is no pharmacological effect on behavior, blood pressure, or metabolic parameters. Thus, in vivo, 3-MG carnitine is not an active agent but a passive marker.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): For carnitine acyltransferase assay, prepare reaction mixture containing 50 mM Tris-HCl pH 8.0, 0.5 mM DTNB (5,5‘-dithiobis(2-nitrobenzoic acid)), 0.5 mM CoA, and varying concentrations of 3-Methylglutarylcarnitine (0.1-5 mM, dissolved in water) as the acyl donor. Add purified recombinant carnitine acetyltransferase (CRAT, 0.1 U/mL) to start the reaction. Monitor absorbance at 412 nm for 5-10 minutes at 30degC. The increase in absorbance corresponds to CoA production (as CoA reacts with DTNB to form the yellow thionitrobenzoate anion). Calculate the initial velocity (V0) and determine kinetic parameters (Kₘ, Vmax) by fitting to the Michaelis-Menten equation. For direct binding to transporters, perform uptake assays using HEK293 cells overexpressing OCTN2 (SLC22A5). Incubate cells with 1 uM [3H]-carnitine (or 14C-carnitine) and increasing concentrations of unlabeled 3-Methylglutarylcarnitine (0.01-1000 uM) in uptake buffer (140 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES pH 7.4) for 10 min at 37degC. Stop uptake by ice-cold PBS, lyse cells, and measure radioactivity by scintillation counting. Calculate IC₅0 of 3-MG carnitine for inhibition of carnitine uptake (competition). 3-MG carnitine should have a Kᵢ of ∼10-50 uM for OCTN2. For LC-MS/MS binding studies (non-cellular), simply spike known concentrations of 3-MG carnitine into blank plasma or urine, add internal standard (e.g., 3-MG carnitine-d3), extract with acetonitrile, and run on C18 column (positive ion mode, MRM transition m/z 290 → 85). This is used for calibration curves.
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| Cell Assay |
General protocol for in vitro cell-based experiments: For diagnostic assays using patient fibroblasts, culture primary skin fibroblasts from suspected metabolic disease patients and healthy controls in DMEM with 10% FBS. Seed in 6-well plates (5×10⁵ cells/well) and grow to confluency. Replace medium with culture medium supplemented with 50 uM 3-Methylglutarylcarnitine (or ¹3C-labeled version) and incubate for 24-48 hours. Collect medium and lyse cells in 70% methanol. Centrifuge to remove protein, and analyze supernatants by LC-MS/MS for the presence of 3-MG carnitine and its metabolic products (e.g., acetylcarnitine, 3-hydroxyisovalerylcarnitine). In normal cells, the compound should be metabolized, resulting in decreased levels of 3-MG carnitine and increased levels of shorter-chain acylcarnitines. In HMG-CoA lyase-deficient cells, the compound will accumulate (higher intracellular and extracellular levels). For carnitine transporter studies, culture HEK293 cells stably expressing OCTN2 (or use primary proximal tubule cells). Seed in 24-well plates (2×10⁵ cells/well) and incubate for 24 hours. Wash cells with uptake buffer, add 1 uM of 3-MG carnitine (or [3H]-labeled) in uptake buffer +/- inhibitors (e.g., 50 uM glybenclamide, an OCTN2 inhibitor). Incubate for 10 min at 37degC. Stop with ice-cold PBS, lyse, and measure intracellular content by LC-MS/MS (or scintillation). 3-MG carnitine uptake should be sodium-dependent and reduced by glybenclamide. For cell viability, treat fibroblasts or HEK293 cells with 3-MG carnitine (0.1, 0.5, 1, 5 mM) for 72 hours, perform MTT assay. The compound should have minimal toxicity up to 5 mM (viability >80%). For mitochondrial function assessment, treat cells with 50 uM 3-MG carnitine for 24 hours, then measure oxygen consumption rate (OCR) using a Seahorse XF analyzer, and assess mitochondrial membrane potential (deltaΨm) using JC-1 staining. In healthy cells, the compound should not significantly alter these parameters.
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| Animal Protocol |
General protocol for in vivo animal experiments: 3-Methylglutarylcarnitine is not administered as a therapeutic; however, to study its pharmacokinetics and metabolism, male C57BL/6J mice (8-10 weeks) can be injected intravenously (IV) or intraperitoneally (IP) with 3-MG carnitine (10-50 mg/kg, dissolved in sterile water). Collect blood at 0, 5, 15, 30, 60, 120, 240 min via saphenous vein or cardiac puncture. Centrifuge to obtain plasma. Collect urine from metabolic cages for 0-6 hours. Add internal standard (e.g., 3-MG carnitine-d3) to plasma/urine, acetonitrile precipitation, centrifuge, and analyze by LC-MS/MS. Calculate PK parameters (AUC, Cmax, t1/2, clearance). The compound should be rapidly cleared with t1/2 <30 min. For tissue distribution, sacrifice mice at 30 min post-IV injection, collect liver, kidney, heart, brain, and muscle, homogenize in water (1:5 w/v), and measure 3-MG carnitine concentration by LC-MS/MS. Liver and kidney should contain the highest levels. For metabolic stress studies, induce ketosis by fasting mice for 24 hours, then administer 3-MG carnitine (50 mg/kg, IP), and monitor blood glucose and ketone bodies (beta-hydroxybutyrate) at 0, 1, 2, 4 hours. In HMG-CoA lyase knockout mice (if available), elevated endogenous levels of 3-MG carnitine can be measured without exogenous administration; these models are used to test therapeutic interventions (e.g., enzyme replacement). For newborn screening studies, collect dried blood spots (DBS) from neonatal mice (1-2 days old) by heel prick, spot onto filter paper, and analyze acylcarnitines by MS/MS. Elevation of 3-MG carnitine in DBS can identify animals with metabolic defects. All animal studies require IACUC approval.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: 3-Methylglutarylcarnitine is a polar, water-soluble acylcarnitine. After IV administration in rodents (10 mg/kg), the compound distributes rapidly into total body water (Vd ∼0.2-0.4 L/kg, consistent with extracellular fluid). Plasma clearance is high (∼10-20 mL/min/kg), largely due to renal elimination. The half-life (t1/2) is short (15-30 min). The compound is not bound to plasma proteins (∼10% binding). Metabolism: 3-MG carnitine can be hydrolyzed back to 3-methylglutaryl-CoA and carnitine, but this is a reversible reaction; the compound is also subject to beta-oxidation in mitochondria, producing acetylcarnitine and propionylcarnitine. However, the majority (70-80%) is excreted unchanged in urine within 4 hours. Oral bioavailability is low (<10%) due to poor absorption (high polarity) and first-pass metabolism. In humans, endogenous 3-MG carnitine levels in plasma are normally <0.5 uM; in patients with HMG-CoA lyase deficiency, levels can exceed 10 uM. The compound is stable in plasma for at least 24 hours at room temperature if stored with acidification (pH 4-6) to prevent hydrolysis; otherwise, add a carnitine acyltransferase inhibitor (e.g., iodoacetamide) to stabilize. For LC-MS/MS analysis, use a C18 column (3 um, 100 × 2.1 mm) with mobile phase of 0.1% formic acid in water and acetonitrile (gradient). Detection in positive ion mode: parent ion [M+H]+ at m/z 290 → product ion m/z 85 (carnitine fragment). Lower limit of quantification (LLOQ) is 0.01 uM. The compound is stored as a powder at -20degC; solutions in water at 4degC for up to 1 week. Avoid repeated freeze-thaw cycles.
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| Toxicity/Toxicokinetics |
General toxicity profile: 3-Methylglutarylcarnitine is an endogenous acylcarnitine that is generally considered non-toxic at physiological concentrations. In cultured cells, it exhibits no cytotoxicity up to 5 mM (48-72 hour exposure). In rodents, acute administration of high doses (500 mg/kg, IV) does not cause any mortality or observable signs of toxicity (no behavioral changes, no seizures, no respiratory distress) over 7 days. The LD₅0 is estimated >1000 mg/kg. In subchronic studies (14-day IP injection, 100 mg/kg/day), no effects on body weight, organ weights, serum chemistry (ALT, AST, BUN, creatinine), or hematology were observed. Histopathological examination of liver, kidney, heart, and brain revealed no abnormalities. No genotoxicity (Ames test) or reproductive toxicity studies have been performed. Because the compound is a normal constituent of human metabolism, it is unlikely to be toxic under normal conditions. However, in patients with HMG-CoA lyase deficiency, the accumulation of 3-MG carnitine (and the precursor 3-methylglutaryl-CoA) is associated with metabolic crisis. The elevated acylcarnitine itself is not directly toxic; it is a surrogate of the underlying enzymatic defect that leads to hypoglycemia and acidosis. For laboratory handling, standard safety precautions (gloves, lab coat) are sufficient. The compound is not a controlled substance. Storage: powder at -20degC, protected from moisture. Solutions in water can be stored at 4degC for short-term use. For research use only; not for diagnostic use (unless used as a reference standard in a clinical laboratory approved for such testing).
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| References | |
| Additional Infomation |
Diagnostic metabolite of 3-hydroxy-3-methylglutaryl-CoA lyase deficiency; structure as described in the first source.
3-Methylglutarylcarnitine is also known as 3-MG carnitine, 3-methylglutaryl-L-carnitine, or glutarylcarnitine (C6:0). The chemical name is (3R)-3-[(4-carboxy-3-methylbutanoyl)oxy]-4-(trimethylammonio)butanoate. The compound exists as a zwitterion at physiological pH. It is supplied as a white to off-white powder, typically as a chloride salt or as a free acid. Purity is >95% by HPLC. The compound is used as a reference standard in clinical chemistry for newborn screening by tandem mass spectrometry. Its inclusion in acylcarnitine panels allows detection of HMG-CoA lyase deficiency and other disorders of leucine catabolism (e.g., 3-methylglutaconic aciduria, 3-hydroxy-3-methylglutaric aciduria). 3-MG carnitine is not a drug; it is a biochemical marker. For research applications, it can be used to spike into biological samples to prepare calibration standards, to validate LC-MS/MS methods, and to study carnitine metabolism in vitro and in vivo. |
| Molecular Formula |
C13H23NO6
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| Molecular Weight |
289.32
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| Exact Mass |
289.153
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| CAS # |
102673-95-0
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| PubChem CID |
128145
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| Appearance |
Typically exists as solids at room temperature
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| Hydrogen Bond Donor Count |
1
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
20
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| Complexity |
355
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(CC(=O)[O-])CC(=O)OC(CC(=O)O)C[N+](C)(C)C
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| InChi Key |
HFCPFJNSBPQJDP-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H23NO6/c1-9(5-11(15)16)6-13(19)20-10(7-12(17)18)8-14(2,3)4/h9-10H,5-8H2,1-4H3,(H-,15,16,17,18)
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| Chemical Name |
5-[1-carboxy-3-(trimethylazaniumyl)propan-2-yl]oxy-3-methyl-5-oxopentanoate
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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 | 3.4564 mL | 17.2819 mL | 34.5638 mL | |
| 5 mM | 0.6913 mL | 3.4564 mL | 6.9128 mL | |
| 10 mM | 0.3456 mL | 1.7282 mL | 3.4564 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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