Mitochondrial complex V (ATP synthase). 3-UPA specifically inhibits complex V activity without affecting complexes I-IV or mitochondrial β-oxidation of fatty acids. The study did not report specific IC50, Ki, or EC50 values for this inhibition. [1]
As an endogenous metabolite, ureidopropionic acid itself does not exert pharmacological activity through specific therapeutic targets. However, computational prediction analyses (SEA algorithm) have identified potential interactions of this compound with multiple protein targets, including the GABA-B receptor (GABBR2), GABA transporter 3 (SLC6A11), and various lysine-specific demethylases (KDM2A, KDM3A, KDM4D, KDM5C, KDM6B, KDM7A). These predicted targets are associated with neurological conditions such as depression, epilepsy, and narcolepsy. It is important to note that these remain computational predictions requiring experimental validation, and the primary biological significance of ureidopropionic acid is as a catabolic intermediate rather than a drug-like ligand.

- Neuronal Damage: Exposure of cultured chicken neurons to 3-UPA (0.001-10 mmol/L, 1-24 hours) induced concentration- and time-dependent neurodegeneration as determined by trypan blue exclusion. Cell viability decreased significantly at all tested concentrations compared to control (P < 0.001). [1]
- Excitotoxicity Enhancement: Co-incubation with L-glutamate (0.01-1 mmol/L) and 1 mmol/L 3-UPA revealed an enhanced susceptibility of neurons to L-glutamate, with overadditive effects observed at 0.1 mmol/L (15.3% overadditive, P < 0.05) and 0.25 mmol/L (18.0% overadditive, P < 0.05) glutamate. [1]
- Reactive Oxygen Species (ROS) Production: 3-UPA (0.01-1 mmol/L) elicited a concentration- and time-dependent increase in mitochondrial ROS production (detected by dihydrorhodamine-123 fluorescence), which preceded the rise in intracellular calcium and was not prevented by the NMDA receptor antagonist MK-801. [1]
- Intracellular Calcium Concentration: 3-UPA (1 mmol/L) induced a delayed increase in intracellular calcium concentration ([Ca²⁺]i) after 4-24 hours of exposure, as measured by fura-2 fluorescence. This increase was partially reduced by MK-801. No early or transient increase was observed within 15 minutes of exposure. [1]

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Ureidopropionic acid is not typically evaluated for direct in vitro pharmacological activity as a therapeutic agent. Its primary in vitro applications are as an analytical standard for metabolic studies. The compound serves as a biomarker for the diagnosis of β-ureidopropionase deficiency, where elevated levels of ureidopropionic acid accumulate in urine and other biofluids. In the context of cancer chemotherapy, this compound is relevant as a metabolite whose accumulation can indicate DPD or β-ureidopropionase deficiency, which predisposes patients to severe toxicity from 5-fluorouracil treatment.

Ureidopropionic acid is not used as a therapeutic agent, so traditional in vivo pharmacodynamic data is not applicable. However, the compound serves as a critical diagnostic biomarker in vivo. In patients with β-ureidopropionase deficiency, ureidopropionic acid and β-ureidoisobutyric acid are persistently elevated in urine. Clinical symptoms associated with this deficiency include hypotonia and dystonic movements in affected infants, though asymptomatic cases have also been identified. The detection of elevated urinary ureidopropionic acid levels enables differential diagnosis from other pyrimidine degradation enzyme deficiencies (DHPDase deficiency and DHPase deficiency).

- Single Respiratory Chain Complex Activities: Submitochondrial particles from bovine heart were used to measure steady-state activities of complexes I-V spectrophotometrically. 3-UPA was tested at concentrations up to 10 mmol/L. Complex I activity was measured according to Okun et al. (1999a,b, 2000), complex II per Ziegler and Rieske (1967), complex III per Brandt and Okun (1997), complex IV per Sinjorgo et al. (1987), and complex V per Percy et al. (1985). Basal activities (U/mg protein) were: Complex I 1.24, Complex II 0.97, Complex III 24.0, Complex IV 15.0, Complex V 0.508. Standard inhibitors confirmed assay validity (e.g., oligomycin inhibited complex V by 93% at 80 μM). [1]
- Mitochondrial β-Oxidation of Fatty Acids: Human skin fibroblasts from healthy volunteers were incubated with 3-UPA (0, 1, and 10 mmol/L) for 24 hours, followed by 96-hour incubation with palmitic acid (200 μmol/L) and L-carnitine (400 μmol/L). Acylcarnitine profiles (C2-C16) were analyzed by electrospray ionization tandem mass spectrometry. No significant inhibition of β-oxidation was observed, with minor decreases in C4 and C12 at 1 mmol/L (P < 0.01) not considered biologically meaningful. [1]

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Ureidopropionic acid is used as a substrate or product in enzyme assays for pyrimidine catabolism enzymes. A representative protocol for β-ureidopropionase activity measurement: Prepare reaction buffer containing ureidopropionic acid as substrate (typically 0.1-5 mM) in an appropriate buffer system (e.g., 50 mM Tris-HCl, pH 7.5-8.0). Add enzyme source (purified β-ureidopropionase or tissue homogenate). Incubate at 37°C for 30-60 minutes. Terminate the reaction by heat inactivation (95°C for 5 minutes) or acidification. Quantify the product (β-alanine) or remaining substrate using HPLC with UV detection or LC-MS/MS. For diagnostic applications, urease pretreatment of urine samples without fractionation enables high-recovery GC/MS detection of ureidopropionic acid and related ureide compounds.

- Primary Neuronal Culture: Primary neuronal cultures were prepared from chick embryo telencephalons. Neurons were maintained in DMEM supplemented with 20% fetal bovine serum at 37°C and 5% CO2, and used for experiments after 5 days in vitro (DIV). This culture system was previously shown to express NMDA receptors and be susceptible to various neurotoxins. [1]
- Cell Viability Assay (Trypan Blue Exclusion): Cell viability was determined by trypan blue (0.4% in PBS) exclusion. Stained (non-viable) and unstained (viable) cells were counted under a microscope (600-800 neurons in eight randomly chosen subfields). Cell viability was expressed as the percent ratio of unstained to total cells. Control levels were normalized to 100%. [1]
- Measurement of Intracellular Calcium ([Ca²⁺]i): Neurons were bath-loaded with fura-2 acetoxymethyl ester (5 μmol/L) in the presence of 0.1% Pluronic F-127 for 30 minutes, followed by washing and an additional 30-minute incubation. [Ca²⁺]i was measured in single neurons (n=100) using an inverted microscope with fluorescence objective, CCD camera, and image processor. Fluorescence at 340 nm and 380 nm (emission 510 nm) was recorded after 0.5-24 hours of exposure to 1 mmol/L 3-UPA. Ratios were converted to [Ca²⁺]i per Grynkiewicz et al. (1985). [1]
- Measurement of Reactive Oxygen Species (ROS): ROS production was monitored in single neurons (n=100) after incubation with 3-UPA (0.01-1 mmol/L) for 0.5-24 hours using the oxidant-sensitive dye dihydrorhodamine-123 (5 μmol/L). Fluorescence was detected at excitation 490 nm and emission 510 nm. [1]
- Mitochondrial β-Oxidation in Fibroblasts: Human skin fibroblast cultures from healthy volunteers were grown to confluency in DMEM with 2 mmol/L L-glutamine, 10% fetal calf serum, and antibiotics. After reaching confluency, cells were incubated with 3-UPA (0, 1, 10 mmol/L) for 24 hours in F-12 HAM medium. Acylcarnitine profiles were then analyzed after 96-hour incubation in serum- and glutamine-free medium containing palmitic acid (200 μmol/L) and L-carnitine (400 μmol/L). [1]

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Ureidopropionic acid is not typically used directly in live cell assays. It can be quantified in cell culture supernatants or lysates as a metabolic endpoint. A representative protocol: Culture cells (e.g., hepatocytes or cancer cell lines) in appropriate medium. Collect culture medium or cell lysates. Deproteinize samples by adding acetonitrile or perchloric acid, followed by centrifugation. Analyze the supernatant by LC-MS/MS or HPLC-UV for ureidopropionic acid quantification. This approach is used to study pyrimidine metabolism in cellular models, particularly for assessing the impact of genetic deficiencies or drug treatments (e.g., 5-FU) on metabolic flux.

For animal studies, ureidopropionic acid is typically administered for pharmacokinetic or metabolic tracing experiments. A representative metabolic study protocol: Administer a pyrimidine precursor (e.g., uracil or dihydrouracil) to rodents via oral gavage or intraperitoneal injection. Collect blood samples at multiple time points (e.g., 0, 15, 30, 60, 120, 240 minutes) and urine over 24 hours. Quantify ureidopropionic acid and its metabolites in plasma and urine using LC-MS/MS or GC/MS. Compare levels between control and treatment groups or between wild-type and enzyme-deficient animals. Elevated ureidopropionic acid levels indicate impaired β-ureidopropionase or DPD activity.

Specific pharmacokinetic data for ureidopropionic acid as an administered compound is limited. However, its pharmacokinetic behavior as an endogenous metabolite is understood in the context of pyrimidine catabolism. Following the administration of pyrimidine precursors (e.g., uracil), ureidopropionic acid appears in plasma and urine as a downstream metabolite. The uracil/ureidopropionic acid or uracil/dihydrouracil ratio is used clinically to assess dihydropyrimidine dehydrogenase (DPD) activity. The compound is polar (predicted logP -0.98 to -1.4) and water-soluble (20.9 mg/mL), characteristics that limit passive diffusion across biological membranes and result primarily in renal excretion as a metabolic end product. Computational predictions suggest potential interactions with solute carriers SLC22A6, which may mediate renal handling.

- Neuronal Toxicity (in vitro): 3-UPA induced concentration- and time-dependent neuronal damage at concentrations ranging from 0.001 to 10 mmol/L. Significant cell death was observed after 24-hour exposure even at the lowest tested concentration (0.001 mmol/L, P < 0.001 vs. control). [1]
- Mechanism of Toxicity: The neurotoxicity of 3-UPA involves inhibition of mitochondrial complex V (ATP synthase) activity, leading to increased ROS production, delayed rise in intracellular calcium, and secondary excitotoxicity mediated by NMDA receptor activation. Neuroprotection was afforded by the antioxidant α-tocopherol (10 μmol/L) and the NMDA receptor antagonist MK-801 (10 μmol/L), but not by non-NMDA antagonist CNQX (50 μmol/L), metabotropic glutamate receptor antagonist L-AP3 (50 μmol/L), or succinate (0.1-100 mmol/L). [1]
- Mitochondrial Toxicity: 3-UPA specifically inhibited complex V activity in a concentration-dependent manner (shown in Fig. 5D). It did not affect the activity of complexes I, II, III, or IV, nor did it impair mitochondrial β-oxidation of fatty acids. Ureido compounds such as formamide and urea (up to 100 mmol/L) did not inhibit complex V, while L-citrulline (100 mmol/L) showed only slight inhibition (14%). [1]

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According to the Safety Data Sheet (SDS) for research-grade ureidopropionic acid, the compound is classified as "not a hazardous substance or mixture" under normal handling conditions. It has not been listed as a carcinogen by NTP, IARC, OSHA, or ACGIH. First aid measures include eye and skin rinsing with water, and medical attention if ingested. As an endogenous metabolite present in normal human physiology, ureidopropionic acid is not considered toxic at physiological concentrations. However, its pathological accumulation due to β-ureidopropionase deficiency can indicate underlying metabolic disease, and it serves as a marker for increased risk of severe toxicity from 5-fluorouracil chemotherapy. The compound is strictly intended for research use only, not for human therapeutic or diagnostic applications.

[1]. 3-Ureidopropionate contributes to the neuropathology of 3-ureidopropionase deficiency and severe propionic aciduria: a hypothesis. J Neurosci Res. 2001;66(4):666-673.

- Background: 3-UPA (synonym: N-carbamyl-β-alanine) is a physiological intermediate in pyrimidine degradation, catalyzed to β-alanine by 3-ureidopropionase (β-alanine synthase). Inherited deficiency of this enzyme leads to accumulation of 3-UPA in body fluids and causes severe neurological symptoms including dystonic dyskinetic movement disorder, global brain and cerebellar atrophy, delayed myelination, optic nerve atrophy, microcephaly, and psychomotor delay. Secondary accumulation also occurs in severe propionic aciduria due to inhibition of 3-ureidopropionase by propionic acid. [1]
- Proposed Pathophysiology: The study proposes that 3-UPA neurotoxicity results from inhibition of mitochondrial ATP synthase, leading to energy impairment, secondary excitotoxicity (via NMDA receptor activation), and oxidative stress. Limited availability of β-alanine (the end product of pyrimidine degradation) and its derivative carnosine (an antioxidant dipeptide) may further enhance susceptibility to 3-UPA toxicity by reducing endogenous defense mechanisms. [1]
- Comparison with 3-Nitropropionate (3-NPA): 3-UPA toxicity partially resembles that of 3-NPA, a known complex II inhibitor used as an animal model for Huntington disease. However, unlike 3-NPA, 3-UPA does not inhibit complex II but instead targets complex V. [1]

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N-Carbamoyl-β-alanine is a β-alanine derivative of propionic acid with a urea group at the 3-position. It is a metabolite, and also a mouse metabolite. Functionally, it is related to propionic acid. It is the conjugate acid of N-carbamoyl-β-alanine. Uretopropionic acid is a metabolite found or produced in Escherichia coli (K12 strain, MG1655 strain). 3-Uretopropionic acid has been reported in Daphnia frenulum, Drosophila melanogaster, and other organisms with relevant data.

C4H8N2O3

132.1179

132.053

462-88-4

FITC-Ureidopropionic acid

111

White to off-white solid powder

1.337g/cm3

324.8ºC at 760mmHg

170-175ºC (dec.)

150.2ºC

4.87E-05mmHg at 25°C

1.504

0.22

3

3

3

9

123

0

C(CNC(=O)N)C(=O)O

JSJWCHRYRHKBBW-UHFFFAOYSA-N

InChI=1S/C4H8N2O3/c5-4(9)6-2-1-3(7)8/h1-2H2,(H,7,8)(H3,5,6,9)

3-(carbamoylamino)propanoic acid

3-Ureidopropionic acid; 462-88-4; N-Carbamoyl-beta-alanine; 3-ureidopropionate; beta-Ureidopropionic acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~946.11 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (15.74 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (15.74 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (15.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)