Tetrahydrofolic acid (L-5,6,7,8-Tetrahydrofolic acid) trihydrochloride is a biologically active vitamin B9 folic acid derivative that is a carbon atom donor for amino acids, nucleic acids, and lipids. Tetrahydrofolic acid trihydrochloride acts as an acceptor for free formaldehyde to produce 5,10-methylenetetrahydrofolate-tetrahydrofolate.

Physicochemical Properties

Molecular Formula	C19H26CL3N7O6
Molecular Weight	554.81
CAS #	150731-85-4
Appearance	Typically exists as solids at room temperature
LogP	0
SMILES	O=C1N=C(N)NC2NCC(CNC3C=CC(C(=O)N[C@H](C(=O)O)CCC(=O)O)=CC=3)NC1=2.Cl
Synonyms	L-5,6,7,8-Tetrahydrofolic acid trihydrochloride; L-Tetrahydrofolic acid trihydrochloride
HS Tariff Code	2934.99.9001
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)

Biological Activity

Targets	Human Endogenous Metabolite
ln Vitro	Tetrahydrofolic acid (0-200 μM; 3 days; Adh5-/- DT40 cells) trihydrochloride exposure is cytotoxic to Adh5- and Fanconi anemia (FA)-deficient cells, producing extensive DNA damage and accumulation of chromosome breaks[1]. Tetrahydrofolic acid (0-100 μM; 16 hours; Adh5-/- DT40 cells) trihydrochloride treatment strongly promotes FANCD2 and ser139-H2AX foci formation in Adh5-/- cells in a dose-dependent manner[1]. Tetrahydrofolic acid trihydrochloride exposure activates the action of DNA damage response (DDR) thymidylate synthases due to uncontrolled activity, leading to depletion of essential nucleotides and promoting repair via homologous recombination mechanisms[1].
ln Vivo	Tetrahydrofolic acid trihydrochloride treatment (62.5 mg/kg; intraperitoneal injection; daily; Adh5-/- mice) disrupted hematopoietic function, increased ser139-H2AX phosphorylation, and decreased the survival rate of progenitor cells (HSPCs), indicating that excessive tetrahydrofolic acid trihydrochloride may be mutagenic and genotoxic to bone marrow cells [1].
Cell Assay	Cell Viability Assay[1] Cell Types: Adh5-/- DT40 cells Tested Tested Concentrations: 0-200 µM Incubation Duration: 3 days Experimental Results: Viability of Adh5-/-DT40 cells rapidly dropped. Western Blot Analysis[1] Cell Types: Adh5-/- DT40 cells Tested Tested Concentrations: 0-200 µM Incubation Duration: 16 hours Experimental Results: Strongly promoted FANCD2 and ser139-H2AX focus formation in Adh5-/- cells in a dose-dependent manner.
Animal Protocol	Animal/Disease Models: Adh5-/- mice[1] Doses: 62.5 mg/kg Route of Administration: Intraperitoneal injection; daily Experimental Results: Perturbed hematopoiesis, increased ser139-H2AX phosphorylation, and decreased the survival of progenitor cells (HSPCs).
References	[1]. Synthesis of Tetrahydropteridine C6-Stereoisomers, Including N6-Formyl-(68)-tetrahydrofolic Acid. The Journal of Organic Chemistry, 57(16), 4470-4477.

Solubility Data

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

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 (e.g. IP/IV/IM/SC) 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). 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) 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). 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 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.8024 mL	9.0121 mL	18.0242 mL
	5 mM	0.3605 mL	1.8024 mL	3.6048 mL
	10 mM	0.1802 mL	0.9012 mL	1.8024 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.