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Orotic acid is an intermediate in the de novo synthesis of pyrimidine nucleotides, catalyzed by the enzyme orotate phosphoribosyltransferase (OPRT), which converts orotic acid to orotidine-5‘-monophosphate (OMP). OMP is then decarboxylated by orotidine-5‘-monophosphate decarboxylase (OMPDC) to form UMP. The combined activity of OPRT and OMPDC is referred to as UMP synthase. Orotic acid also affects carbohydrate and lipid metabolism. In mammals, orotic acid is involved in the regulation of uric acid excretion and has been used to treat kidney stones (it competes with uric acid for transport). As a pyrimidine precursor, it supports nucleotide synthesis in rapidly dividing cells. The compound is also used as a marker for metabolic disorders such as orotic aciduria (which occurs in defects of the urea cycle).
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| ln Vitro |
In vitro, orotic acid is used to study the de novo pyrimidine synthesis pathway. In hepatocytes or other cell types, supplementation with orotic acid (0.1-10 mM) can increase the flux through the pyrimidine synthesis pathway, leading to increased production of UMP, UDP, and UTP, which are essential for RNA synthesis and cellular energy metabolism. Orotic acid is also used as a substrate in enzyme assays for orotate phosphoribosyltransferase (OPRT) and for orotidine-5‘-monophosphate decarboxylase (OMPDC). In cell culture, orotic acid can cause fatty liver (hepatic steatosis) in certain conditions due to its effects on lipid metabolism (see in vivo). The compound is also used to study the regulation of pyrimidine synthesis and its interaction with the urea cycle. In addition, orotic acid has been reported to have antioxidant properties.
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| ln Vivo |
In vivo, orotic acid has been studied for its role in pyrimidine metabolism and its therapeutic potential in kidney stones and gout. In rodents, high doses of orotic acid (e.g., 1% in the diet) induce fatty liver (hepatic steatosis) by increasing the synthesis of pyrimidine nucleotides, which then promotes triglyceride accumulation. This model is used to study the mechanisms of fatty liver disease. Orotic acid is also used to treat hyperuricemia and gout, as it competes with uric acid for renal tubular reabsorption, thereby increasing uric acid excretion. In humans, orotic acid is an endogenous metabolite; elevated levels are observed in orotic aciduria, a rare genetic disorder of pyrimidine metabolism (UMP synthase deficiency) that presents with orotic acid crystalluria and megaloblastic anemia. The compound is also found in milk (hence the name “orotic acid,” from Greek oros for milk whey) and is used as a food supplement (vitamin B13).
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| Enzyme Assay |
For non-cellular assays (enzyme activity), orotate phosphoribosyltransferase (OPRT) activity is measured by incubating purified OPRT with orotic acid (0.1-1 mM) and PRPP (phosphoribosyl pyrophosphate, 1 mM) in 50 mM Tris-HCl buffer (pH 7.5) containing 5 mM MgCl2 for 30 minutes at 37degC. The reaction is stopped by heating. The product, orotidine-5‘-monophosphate (OMP), is quantified by HPLC with UV detection at 260 nm or by LC-MS. For OMP decarboxylase (OMPDC) activity, OMP (0.1-1 mM) is incubated with purified OMPDC in the same buffer, and UMP production is measured at 260 nm. For the coupled OPRT/OMPDC assay (UMP synthase), both enzymes are combined, and UMP production from orotic acid and PRPP is measured. For analytical quantification, orotic acid hydrate is dissolved in water or 0.1% formic acid (1 mg/mL). For LC-MS/MS, a calibration curve is prepared in human urine or plasma (0.1-1000 ng/mL). MRM transitions: orotic acid 155→111 (loss of CO2) and 155→67; orotic acid monohydrate (C5H6N2O5) 174→111 and 174→67.
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| Cell Assay |
For cell-based assays, hepatocytes (e.g., HepG2 cells) or fibroblasts are seeded in 6-well plates (1×10⁶ cells/well) in DMEM with 10% FBS. Cells are treated with orotic acid (0.5-5 mM) for 24-72 hours. Pyrimidine nucleotide levels (UMP, UDP, UTP) are measured by LC-MS/MS. For fatty liver studies, cells are treated with orotic acid (1-5 mM) for 48 hours, and intracellular lipid accumulation (triglycerides, cholesterol) is measured by Oil Red O staining or by biochemical lipid extraction and quantification. For toxicity studies, cell viability is assessed by MTT assay. For enzyme activity studies, cell lysates are prepared, and OPRT and OMPDC activities are measured as described above. For orotic aciduria studies, cells from patients with UMP synthase deficiency can be cultured and supplemented with uridine, and orotic acid accumulation in culture medium is measured by LC-MS.
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| Animal Protocol |
For in vivo animal experiments, rodents are used to study fatty liver induced by orotic acid. Male Sprague-Dawley rats are fed a diet containing 1% orotic acid for 7-14 days. At the end of the study, liver tissues are collected for histological analysis (H&E staining, Oil Red O staining for lipid droplets), triglyceride and cholesterol content (biochemical extraction), and gene expression analysis (qPCR for lipogenic genes such as FASN, SCD1, SREBP1c). Blood samples are collected for measurement of liver enzymes (ALT, AST) and serum lipids. For hyperuricemia studies, rats are administered orotic acid (100-500 mg/kg, oral) for 7-14 days, and blood samples are collected for measurement of uric acid levels (colorimetric or enzymatic assay). For pharmacokinetic studies, orotic acid is administered orally (10-100 mg/kg) to rats, and blood and urine are collected for LC-MS analysis. For orotic aciduria diagnosis, urine samples from patients or animal models are analyzed by LC-MS using a calibration curve and internal standards.
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| ADME/Pharmacokinetics |
Orotic acid monohydrate has a molecular weight of 174.11 (anhydrous: 156.10). The compound is a white to off-white crystalline powder with a melting point of 345degC (dec.). It is slightly soluble in water (1-3 mg/mL), soluble in hot water, and soluble in dilute alkali and ammonia solutions. The compound 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. Aqueous solutions may decompose over time; it is recommended to prepare fresh solutions. Orotic acid has pKa values of approximately 2.5 (carboxyl), 4.1 (pyrimidine NH), and 9.5 (pyrimidine NH). It is the monohydrate form that is most commonly used in research. The compound is an endogenous metabolite present in human urine and blood at low levels (micromolar range).
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| Toxicity/Toxicokinetics |
Orotic acid has low acute toxicity. The oral LD₅0 in rats is >5,000 mg/kg. In humans, orotic acid is generally recognized as safe (GRAS) as a food ingredient and dietary supplement at typical consumption levels (1-10 mg/day). At high doses (≥500 mg/day), it may cause mild gastrointestinal disturbances (diarrhea, abdominal discomfort). In animal studies, high doses (1% in diet) induce fatty liver without significant cytotoxicity or necrosis; this effect is reversible upon withdrawal of orotic acid. Orotic acid is not mutagenic or carcinogenic. Standard laboratory safety precautions for handling organic acids should be followed. The compound is non-radioactive and safe for research use.
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| Additional Infomation |
See also: Orytic acid (note moved to).
2,6-Dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylic acid hydrate (Orotic acid monohydrate) is a research compound and endogenous metabolite, not an approved drug. It has not undergone clinical trials as a drug. However, orotic acid (vitamin B13) has been used in the food industry as a dietary supplement (often in sports nutrition for its purported role in nucleic acid synthesis) and in the pharmaceutical industry as an ingredient in products for kidney stones and gout (in combination with other agents). Its primary research applications are in studying pyrimidine nucleotide biosynthesis (de novo pathway), hepatosteatosis (fatty liver disease) induced by orotic acid, and hyperuricemia (uric acid excretion). The compound is also used as a biochemical tool to study the urea cycle (orotic acid accumulates in orotic aciduria, a secondary consequence of urea cycle defects) and as a reference standard in clinical chemistry for the diagnosis of orotic aciduria (e.g., in patients with late-onset ornithine transcarbamylase deficiency). Available for research use only. |
| Molecular Formula |
C5H6N2O5
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| Molecular Weight |
174.11
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| Exact Mass |
174.027
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| CAS # |
50887-69-9
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| Related CAS # |
2,6-Dioxo-1,2,3,6-tetrahydropyrimidine-4-carboxylic acid hydrate-15N2
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| PubChem CID |
16218504
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| Appearance |
White to off-white solid powder
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| Density |
1.642 g/cm3
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| Boiling Point |
656.9ºCat 760 mmHg
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| Melting Point |
>300 °C(lit.)
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| Flash Point |
351.1ºC
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
12
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| Complexity |
268
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=C(NC(=O)NC1=O)C(=O)O.O
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| InChi Key |
YXUZGLGRBBHYFZ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C5H4N2O4.H2O/c8-3-1-2(4(9)10)6-5(11)7-3;/h1H,(H,9,10)(H2,6,7,8,11);1H2
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| Chemical Name |
2,4-dioxo-1H-pyrimidine-6-carboxylic acid;hydrate
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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) |
DMSO: 100 mg/mL (574.35 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (14.36 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 5.7435 mL | 28.7175 mL | 57.4350 mL | |
| 5 mM | 1.1487 mL | 5.7435 mL | 11.4870 mL | |
| 10 mM | 0.5743 mL | 2.8717 mL | 5.7435 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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