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| Targets |
4,6-Dioxoheptanoic acid (succinylacetone) is a potent inhibitor of porphobilinogen synthase (PBGS, also known as ALAD, delta-aminolevulinic acid dehydratase), the second enzyme in the heme biosynthetic pathway. By inhibiting PBGS, succinylacetone causes accumulation of delta-aminolevulinic acid (ALA) and leads to secondary porphyrinuria. The target is central to heme biosynthesis in erythroid cells and hepatocytes. The ¹3C-labeled version (13C5) shares the same target but is used for tracing rather than activity studies.
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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, 4,6-Dioxoheptanoic acid (succinylacetone) is a potent inhibitor of heme biosynthesis, with IC₅0 values in the low uM range for PBGS inhibition. In erythroid cell cultures, treatment with succinylacetone (0.1-1 mM) leads to accumulation of ALA, depletion of heme, and impaired hemoglobin synthesis. The compound is used to study heme-dependent processes such as erythropoiesis and mitochondrial function. The ¹3C-labeled version (4,6-Dioxoheptanoic acid-13C5) is not used for activity studies but serves as a tracer for metabolic flux analysis. |
| ln Vivo |
In vivo, 4,6-Dioxoheptanoic acid (succinylacetone) accumulates in patients with hepatorenal tyrosinemia type I, leading to liver and kidney dysfunction, neurological crises, and increased risk of hepatocellular carcinoma. In animal models, administration of succinylacetone induces biochemical abnormalities consistent with tyrosinemia. The ¹3C-labeled version (4,6-Dioxoheptanoic acid-13C5) is used as an internal standard for the quantitation of succinylacetone in dried blood spots and urine for the diagnosis of hepatorenal tyrosinemia and for follow-up of patients under treatment.
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| Enzyme Assay |
For non-cellular assays (analytical quantification), 4,6-Dioxoheptanoic acid-13C5 is prepared as a stock solution in methanol (1 mg/mL). For LC-MS/MS analysis, a calibration curve for succinylacetone is prepared in human dried blood spot extracts (0.1-100 ng/mL) with a fixed concentration of SA-13C5 (e.g., 10 ng/mL). Sample preparation: 3 mm dried blood spot punch is extracted with 100 uL methanol containing internal standard, sonicated for 30 minutes, and centrifuged. The supernatant is injected onto a C18 column with a mobile phase of 0.1% formic acid in water and methanol (gradient elution). MRM transitions: SA 159→97, SA-13C5 164→102.
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| Cell Assay |
For cell-based assays, human hepatoma cells (e.g., HepG2) or erythroleukemia cells (e.g., K562) are used. Cells are seeded in 6-well plates (1×10⁶ cells/well) in DMEM with 10% FBS. 4,6-Dioxoheptanoic acid (0.1-1 mM) is added for 24-72 hours. Heme content is measured by fluorescence (excitation 400 nm, emission 620 nm) after extraction with oxalic acid. ALA dehydratase (PBGS) activity is measured in cell lysates using ALA as substrate and quantifying porphobilinogen by Ehrlich‘s reagent. For metabolic tracing, 4,6-Dioxoheptanoic acid-13C5 (10-100 uM) is added, and ¹3C-labeled intermediates are analyzed by LC-MS.
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| Animal Protocol |
For in vivo animal experiments, hepatorenal tyrosinemia model mice (FAH knockout mice) are used. For diagnostic method development, dried blood spots are collected from patients or animal models on filter paper cards. Succinylacetone is extracted with methanol containing 4,6-Dioxoheptanoic acid-13C5 as internal standard. Analysis is performed by LC-MS/MS as described in non-cellular assays. This method is applied retrospectively and prospectively for the diagnosis of hepatorenal tyrosinemia and for follow-up of patients under treatment (e.g., with nitisinone). No administration of the labeled compound is required for diagnostic applications.
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| ADME/Pharmacokinetics |
4,6-Dioxoheptanoic acid-13C5 has a molecular weight of 163.12, with five carbon-13 atoms providing a mass shift of +5 Da relative to non-labeled succinylacetone (MW 158). The compound is soluble in methanol, DMSO, and water. It should be stored as a powder at -20degC, protected from light. The ¹3C label is stable (non-radioactive) and does not exchange under physiological conditions. Stability is maintained for at least 12 months under proper storage.
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| Toxicity/Toxicokinetics |
4,6-Dioxoheptanoic acid-13C5 is a stable isotope-labeled compound with minimal toxicity at analytical concentrations (ng-ug per sample). The non-deuterated parent compound, succinylacetone, at high concentrations can inhibit heme biosynthesis and cause cytotoxicity. However, when used as an internal standard or tracer, the amount is negligible and poses no significant toxicity risk. Standard laboratory safety precautions for handling organic compounds should be followed. The compound is non-radioactive and safe for use in research laboratories.
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| References |
[1]. Russak EM, et al. Impact of Deuterium Substitution on the Pharmacokinetics of Pharmaceuticals. Ann Pharmacother. 2019 Feb;53(2):211-216.
[2]. Ebert PS, et al. Succinylacetone, a potent inhibitor of heme biosynthesis: effect on cell growth, heme contentand delta-aminolevulinic acid dehydratase activity of malignant murine erythroleukemia cells. Biochem Biophys Res Commun. 1979 Jun 27;88(4):1382-90. |
| Additional Infomation |
4,6-Dioxoheptanoic acid-13C5 is an analytical standard and research tool, not an approved drug. It has not undergone clinical trials for therapeutic use. Its primary application is as an internal standard for the quantitative analysis of succinylacetone in dried blood spots and urine by LC-MS/MS, which is a critical diagnostic test for hepatorenal tyrosinemia type I (HT-1). Early diagnosis allows treatment with nitisinone and dietary restriction to prevent severe liver and kidney damage. The compound is also used in metabolic tracer studies to investigate heme biosynthesis pathways.
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| Molecular Formula |
C213C5H10O4
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| Molecular Weight |
163.12
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| Exact Mass |
163.075
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| CAS # |
881835-86-5
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| Related CAS # |
4,6-Dioxoheptanoic acid;51568-18-4
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| PubChem CID |
46782950
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| Appearance |
White to off-white solid powder
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| LogP |
0.399
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
11
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| Complexity |
183
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(=O)CC(=O)CCC(=O)O
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| InChi Key |
WYEPBHZLDUPIOD-JBSMRZEESA-N
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| InChi Code |
InChI=1S/C7H10O4/c1-5(8)4-6(9)2-3-7(10)11/h2-4H2,1H3,(H,10,11)/i1+1,2+1,4+1,5+1,6+1
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| Chemical Name |
4,6-dioxo(3,4,5,6,7-13C5)heptanoic 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 | 6.1305 mL | 30.6523 mL | 61.3046 mL | |
| 5 mM | 1.2261 mL | 6.1305 mL | 12.2609 mL | |
| 10 mM | 0.6130 mL | 3.0652 mL | 6.1305 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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