Endogenous Metabolite; folic acid receptor; Modulation of monoaminergic systems [2]

In order to stop chromosomal breakage and DNA hypomethylation, sodium folate is essential [1].

Folic acid (50 mg/kg i.p. for 7 days) significantly reduced immobility time in mouse forced swim test (p < 0.01) and tail suspension test (p < 0.05), indicating antidepressant-like effects [2]
Folic acid supplementation (5 mg/kg diet) prevented hepatic gene expression alterations induced by low-protein diet in pregnant rat offspring [3]
In a mouse model of this behavior, sodium folate (10, 50, or 100 mg/kg; orally) exhibits antidepressant-like effects [2]. Mice acclimated to their new surroundings do not exhibit any psychostimulant effect from sodium folate (1, 10 nmol/site) [2]. In rat pups, oral sodium folate (1, 5 mg/kg) inhibits epigenetic changes in hepatic gene expression [3].

Renal cell toxicity: HK-2 cells treated with folic acid (0-100 μM) for 24h. Viability assessed by MTT assay. ROS measured with DCFH-DA probe. Mitochondrial membrane potential evaluated via JC-1 staining. Protein expression analyzed by western blot [Free Radic Biol Med. 2020 Jul;154:18-32.]

Animal/Disease Models: 30-40 g Swiss mice [2]
Doses: 10, 50, 100 mg/kg
Route of Administration: Oral
Experimental Results: diminished immobility time in forced swim test (FST) (F324=11.21), produced significant Immobility time in tail suspension test (TST) (F3,20=5.71).

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Animal/Disease Models: 30-40 g Swiss mice [2]
Doses: 1-10 nmol/site
Route of Administration: Intracerebroventricular injection
Experimental Results: diminished mouse FST (F3,22=12.31) and TST (F3,22=5.50) immobile time).

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Animal/Disease Models: Virgin female Wistar rats [3]
Doses: 1, 5 mg/kg (180 g/kg protein plus 1 mg/kg folic acid or 90 g/kg casein plus 1, 5 mg/kg folic acid)
Route of Administration: Oral administration
Experimental Results: Prevention of epigenetic modifications in liver gene expression in offspring.

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Antidepressant study: Mice received daily intraperitoneal injections of folic acid (50 mg/kg dissolved in saline) for 7 days. Behavioral tests conducted 30 min post-last dose [2]
Epigenetics study: Pregnant rats fed low-protein diet (8% casein) ± folic acid-supplemented diet (5 mg/kg diet) throughout gestation. Offspring livers analyzed at 34 days [3]

Absorption, Distribution and Excretion
Folic acid is rapidly absorbed primarily in the proximal small intestine. Naturally occurring conjugated folic acid is enzymatically reduced to folic acid in the gastrointestinal tract before absorption. After oral administration, folic acid is detectable in plasma within approximately 15 to 30 minutes; peak concentrations are typically reached within 1 hour. In a small number of healthy adults, only trace amounts of folic acid are detected in urine after a single oral dose of 100 micrograms. In one study, oral administration of 5 mg of folic acid and in another, 40 micrograms per kilogram of body weight, resulted in approximately 50% of the dose appearing in the urine. After a single oral dose of 15 mg of folic acid, up to 90% of the dose is recovered in the urine. Most metabolites appear in the urine after 6 hours; they are usually completely excreted within 24 hours. Small amounts of orally administered folic acid may also be excreted in feces. Folic acid is also present in the breast milk of lactating mothers. Tetrahydrofolate derivatives are distributed throughout the body, but are primarily stored in the liver.
After oral administration, folic acid is rapidly absorbed from the gastrointestinal tract; the vitamin is primarily absorbed in the proximal small intestine.
Folic acid in its monoglutamate form, including folic acid, is transported across the proximal small intestine via a saturated, pH-dependent process. Higher doses of pteroylmonoglutamate (including folic acid) are absorbed via an unsaturated passive diffusion process. Pteroylmonoglutamate is absorbed more efficiently than pteroylpolyglutamate.
After oral administration, peak folic acid activity in the blood occurs within 30 to 60 minutes. When synthetic folic acid is taken on an empty stomach, its bioavailability is almost 100%. The bioavailability of natural folic acid from food is approximately 50%, while the bioavailability of synthetic folic acid taken after a meal is between 85% and 100%. Approximately two-thirds of folic acid in plasma is bound to proteins. …When pharmacological doses of folic acid are taken, a significant amount of unmetabolized folic acid is detected in the plasma. The liver stores more than 50% of the body's folic acid, approximately 6 to 14 mg. The total amount of folic acid in the human body is approximately 12 to 28 mg. For more complete data on the absorption, distribution, and excretion of folic acid (11 items in total), please visit the HSDB record page.

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Metabolism/Metabolites
Folic acid is metabolized in the liver by dihydrofolate reductase (DHFR) to cofactors dihydrofolate (DHF) and tetrahydrofolate (THF). Folic acid is converted to its metabolically active form (tetrahydrofolate) in the liver and plasma (in the presence of ascorbic acid) by dihydrofolate reductase. After absorption of 1 mg or less of folic acid, most of it is reduced and methylated in the liver to N-methyltetrahydrofolate… Folic acid is absorbed by the liver and metabolized to polyglutamate derivatives (primarily pteroylpentaglutamate), which are generated by the action of folic acid polyglutamate synthase. …Folic acid polyglutamate is released from the liver into the systemic circulation and bile. When released from the liver into circulation, the polyglutamate form is hydrolyzed by γ-glutamyl hydrolase and reverted to the monoglutamate form.

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Antidepressive study: Mice were intraperitoneally injected with folic acid (50 mg/kg, dissolved in saline) daily for 7 consecutive days. Behavioral tests were performed 30 minutes after the last administration [2]
Epigenetic study: Pregnant rats were fed a low-protein diet (8% casein) ± a folic acid-supplemented diet (5 mg/kg) throughout pregnancy. Offspring livers were analyzed at 34 days [3]

Toxicity Summary
Identification: Folic acid is an anti-anemia vitamin. Sources: Folic acid is isolated from leafy green vegetables, liver, yeast, and fruits. Synthetic folic acid is also available commercially. Folic acid is a yellow to orange-brown crystalline powder, odorless. It is readily soluble in alkalis, hydroxides, and carbonates. It is insoluble in ethanol, acetone, chloroform, and ether. The solution can be inactivated by ultraviolet light. Alkaline solutions are easily oxidized, and acidic solutions are easily heated. Indications: Used for the prevention and treatment of vitamin B deficiency. Used to treat megaloblastic and macrocytic anemia caused by folic acid deficiency. Low birth weight infants, infants breastfed by folic acid-deficient mothers, or infants with chronic diarrhea and infections may require folic acid supplementation. Other factors that may increase folic acid requirements include alcoholism, liver disease, hemolytic anemia, breastfeeding, use of oral contraceptives, and pregnancy. Folic acid has been used to reduce the risk of birth defects in fetuses born to pregnant women. Human Exposure: Major Risks and Target Organs: Folic acid toxicity is relatively low. However, adverse reactions have been reported after injectable administration. Allergic reactions to folic acid are rare. Clinical manifestations overview: Severe allergic reactions are characterized by hypotension, shock, bronchospasm, nausea, vomiting, rash, and erythema. Itching may also occur. Gastrointestinal and central nervous system adverse reactions have been reported. Apart from rare reports of allergic reactions, folic acid treatment is generally well tolerated. Bioavailability: After oral administration, folic acid is rapidly absorbed from the gastrointestinal tract. Peak serum folate levels are reached 30 to 60 minutes after oral administration. Contraindications: Use with caution in patients with impaired renal function. It is also contraindicated in patients with folic acid hypersensitivity. Folic acid should be used with caution in patients with possible folic acid-dependent tumors. Never use folic acid alone or in combination with insufficient doses of vitamin B12 to treat undiagnosed megaloblastic anemia. Although folic acid may induce a hematopoietic response in patients with megaloblastic anemia due to vitamin B12 deficiency, it does not prevent the occurrence of subacute combined spinal cord degeneration. Absorption route: Oral: After oral administration, folic acid is rapidly absorbed from the proximal gastrointestinal tract, primarily in the proximal small intestine. Naturally occurring folic acid polyglutamic acid is enzymatically hydrolyzed into monoglutamic acid in the gastrointestinal tract before absorption. After oral administration, peak folic acid activity in the blood is reached within 30 to 60 minutes. Enterohepatic circulation of folic acid has been confirmed. Distribution by exposure route: Tetrahydrofolate and its derivatives are distributed throughout all tissues of the body. The liver contains half of the body's folic acid and is the main storage site. Metabolism: After absorption, folic acid is converted to metabolically active tetrahydrofolate by hepatic dihydrofolate reductase. After absorption, folic acid is mainly reduced and methylated in the liver to N-5-methyltetrahydrofolate, which is the main transport and storage form of folic acid in the body. Larger doses of folic acid may not be metabolized by the liver and exist primarily in the blood as folic acid. Elimination by exposure route: Oral administration: In healthy adults, only trace amounts of folic acid are detected in urine after a single oral dose. After taking large doses, renal tubular reabsorption reaches its maximum, and excess folic acid is excreted unchanged in the urine. Small amounts of orally administered folic acid can be recovered in feces. Pharmacodynamics: Folic acid can be converted into various coenzymes, which mainly participate in various intracellular metabolic reactions, including the conversion of homocysteine to methionine, serine to glycine, thymidine synthesis, histidine metabolism, purine synthesis, and the utilization or generation of formic acid. In the human body, exogenous folic acid is required for nucleoprotein synthesis and the maintenance of normal erythropoiesis. Folic acid is a precursor to tetrahydrofolate, which is active and can act as a cofactor in the single-carbon transfer reaction in the biosynthesis of nucleic acids, purines, and thymidines. Adults: Currently, there is limited data on the toxicity of folic acid in humans. There have been reports of two patients experiencing exacerbated psychotic behavior during folic acid treatment. Researchers studied the cellular morphological effects of folic acid using in vitro established human oral epithelial cells. The results showed that folic acid at twice the clinical dose did not cause significant cytotoxic reactions in cultured cells. The most significant changes were the appearance of degenerative cells in the culture, characterized by edema, increased cytoplasmic transparency, cell flattening, and atypical filaments. Interactions: Folic acid treatment may increase phenytoin metabolism in folic acid-deficient patients, leading to decreased serum phenytoin concentrations. There are also reports that concomitant use of folic acid and chloramphenicol in folic acid-deficient patients may antagonize the hematopoietic response to folic acid. Concomitant use of ethoxytocin or mephenytoin with folic acid may reduce the effects of hydantoin-like drugs by increasing hydantoin metabolism. Trimethoprim acts as a folic acid antagonist by inhibiting dihydrofolate reductase; therefore, patients taking this drug must take folinic acid calcium instead of folic acid. Folic acid may also interfere with the effects of pyrimethamine. Amoxicillin (4-aminofolate) and methotrexate (4-amino-10-methylfolate) antagonize the reduction of folic acid to tetrahydrofolate. Methotrexate is still used as an antitumor drug; its activity may depend on blocking certain purine synthesis pathways (which require folic acid), thus depriving tumor cells of the compounds needed for proliferation. Calcium folinic acid is used therapeutically as a potent antidote for the toxicity of folic acid antagonists (used as antitumor drugs). Methotrexate, pyrimethamine, or triamterene can also exert folic acid antagonistic effects by inhibiting dihydrofolate reductase. Analgesics, anticonvulsants, antimalarial drugs, and corticosteroids may cause folic acid deficiency. Major adverse reactions: Rare reports of folic acid allergic reactions, including erythema, rash, pruritus, malaise, and bronchospasm. Gastrointestinal and central nervous system adverse reactions have been reported in patients taking 15 mg of folic acid daily for one month. Animal/plant studies: Mechanism of action: Folic acid toxicity is relatively low. Mouse toxicity studies have shown that folic acid can cause seizures, ataxia, and asthenia. Histopathological studies in certain strains of mice have shown that toxic doses may also cause acute tubular necrosis. Studies have shown a possible association between folic acid neurotoxicity and cholinergic receptors in the piriform cortex and amygdala.

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Interactions
Concomitant use of high doses of folic acid and pyrimethamine for the prevention of myelosuppression may antagonize the antiparasitic effects of pyrimethamine.
High-dose nonsteroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, indomethacin, naproxen, mefenamic acid, piroxicam, and sulindac, may have antifolate activity.
Folic acid supplementation in mice enhanced the therapeutic activity of the antifolate chemotherapy drug lometroxe and reduced its adverse effects.
Daily folic acid administration enhanced the antidepressant effect of fluoxetine.
For more information on interactions, please refer to the complete data on folic acid (17 items in total) on the HSDB record page.

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Acute toxicity: Rat LD₅₀ > 6000 mg/kg [1]
Nephropathy: A single high dose (250 mg/kg, intraperitoneal injection) induced renal tubular necrosis and chronic fibrosis in mice [6]
Neurological effects: A dose > 15 mg/day masked vitamin B₁₂ deficiency neuropathy [1]
Drug interactions: Decreased plasma concentrations of phenytoin and phenobarbital [1]

[1]. Folic acid safety and toxicity: a brief review. Am J Clin Nutr. 1989 Aug;50(2):353-8.

[2]. Folic acid administration produces an antidepressant-like effect in mice: evidence for the involvement of the serotonergic and noradrenergic systems. Neuropharmacology. 2008 Feb;54(2):464-73.

[3]. Dietary protein restriction of pregnant rats induces and folic acid supplementation prevents epigenetic modification of hepatic gene expression in the offspring. J Nutr. 2005 Jun;135(6):1382-6.

[4]. Folic acid and L-5-methyltetrahydrofolate: comparison of clinical pharmacokinetics and pharmacodynamics. Clin Pharmacokinet. 2010 Aug;49(8):535-48.

Therapeutic Uses
Folic acid is indicated for the prevention and treatment of folic acid deficiency, including megaloblastic anemia and nutritional anemia, as well as anemia during pregnancy, infancy, or childhood. Increased folic acid intake and/or possible folic acid supplementation are recommended in the following populations or conditions (based on a confirmed diagnosis of folic acid deficiency): alcoholism, hemolytic anemia, chronic fever, gastrectomy, chronic hemodialysis, infants (low birth weight, breastfed, or infants fed unfortified formula such as condensed milk or goat milk), intestinal diseases (celiac disease, tropical stomatitis, persistent diarrhea), malabsorption syndromes associated with hepatobiliary diseases (liver impairment, alcoholism with cirrhosis), and/or chronic stress. Pharmaceuticals (Veterinary): ...for the prevention of megaloblastic anemia, embryonic death, cervical paralysis, and periodontitis. Chicks. For more complete data on the therapeutic uses of folic acid (7 types), please visit the HSDB record page.

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Drug Warnings
Rare reports of allergic reactions to folic acid preparations, including erythema, rash, itching, malaise, and bronchospasm-induced dyspnea.
Rare reports of gastrointestinal adverse reactions, such as anorexia, nausea, bloating, flatulence, and bitter/unpleasant taste, in patients taking 15 mg of folic acid daily for one month; and central nervous system adverse reactions, such as altered sleep patterns, poor concentration, irritability, hyperactivity, excitement, depression, confusion, and impaired judgment.
Long-term use of folic acid may result in decreased serum vitamin B12 levels.
Folic acid should be used with extreme caution in patients with undiagnosed anemia, as it may mask the diagnosis. By alleviating the hematological manifestations of pernicious anemia while allowing neurological complications to progress, it can lead to serious neurological damage, potentially causing severe consequences even before diagnosis.
For more complete data on drug warnings for folic acid (7 in total), please visit the HSDB records page.

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Pharmacodynamics
Folic acid is a water-soluble B vitamin found in foods such as liver, kidneys, yeast, and leafy green vegetables. Also known as folate or vitamin B9, folic acid is an essential cofactor for enzymes involved in DNA and RNA synthesis. More specifically, the body needs folic acid to synthesize purines, pyrimidines, and methionines before they can be incorporated into DNA or proteins. Folic acid is a precursor to tetrahydrofolate, which acts as a cofactor in the formylation reactions in the biosynthesis of purines and thymidine in nucleic acids. Folic acid deficiency is thought to cause impaired thymidine synthesis, leading to defects in deoxyribonucleic acid (DNA) synthesis, which in turn leads to megaloblastic formation and megaloblastic anemia and megaloblastic anemia. Folic acid is particularly important during periods of rapid cell division, such as infancy, pregnancy, and erythropoiesis, and plays a protective role in the development and progression of cancer. Because the human body cannot synthesize folic acid endogenously, folic acid deficiency must be prevented through diet and supplements. For folic acid to function properly in the body, it must first be reduced to the cofactors dihydrofolate (DHF) and tetrahydrofolate (THF) by dihydrofolate reductase (DHFR). This crucial metabolic pathway is essential for the de novo synthesis of nucleic acids and amino acids, but antimetabolite therapies (e.g., [DB00563]) disrupt this pathway because these therapies, as dihydrofolate reductase (DHFR) inhibitors, prevent DNA synthesis in rapidly dividing cells, thereby inhibiting the formation of dihydrofolate (DHF) and tetrahydrofolate (THF). Typically, serum folic acid levels below 5 ng/mL indicate folic acid deficiency, and levels below 2 ng/mL often lead to megaloblastic anemia.

C19H17N7O6-2.2[NA+]

485.36118

463.122

6484-89-5

Folic acid;59-30-3

135398658

Yellowish-orange crystals; extremely thin platelets (elongated @ 2 ends) from hot water

482 °F (decomposes) (NTP, 1992)
250 °C

-1.1

6

10

9

32

767

1

C1=CC(=CC=C1C(=O)NC(CCC(=O)[O-])C(=O)[O-])NCC2=CN=C3C(=N2)C(=O)NC(=N3)N.[Na+].[Na+]

OVBPIULPVIDEAO-LBPRGKRZSA-N

InChI=1S/C19H19N7O6/c20-19-25-15-14(17(30)26-19)23-11(8-22-15)7-21-10-3-1-9(2-4-10)16(29)24-12(18(31)32)5-6-13(27)28/h1-4,8,12,21H,5-7H2,(H,24,29)(H,27,28)(H,31,32)(H3,20,22,25,26,30)/t12-/m0/s1

(2S)-2-[[4-[(2-amino-4-oxo-3H-pteridin-6-yl)methylamino]benzoyl]amino]pentanedioic 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)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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