| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
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| Other Sizes |
| Targets |
No direct pharmacological target; 25-Hydroxycholesterol-d6 is an isotope-labeled compound used as an internal standard. 25-Hydroxycholesterol itself is an oxysterol metabolite of cholesterol that is produced and secreted by macrophages in response to Toll-like receptor (TLR) activation. It effectively and selectively inhibits IgA production by B cells with an EC50 of approximately 65 nM.
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| ln Vitro |
As an isotope-labeled internal standard, 25-hydroxycholesterol-d6 is not biologically active per se. Its non-deuterated counterpart, 25-hydroxycholesterol, is an oxysterol that regulates cholesterol homeostasis by inhibiting sterol regulatory element-binding protein (SREBP) processing. It also exhibits immunomodulatory activity, selectively inhibiting IgA production by B cells (EC50 ~ 65 nM) and activating liver X receptors (LXRs), thereby regulating inflammatory responses.
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| ln Vivo |
No direct in vivo activity for the deuterated form. 25-Hydroxycholesterol, the non-deuterated compound, is produced by macrophages in response to TLR activation and has been shown to inhibit IgA production in vivo. It also modulates immune responses by suppressing the expression of pro-inflammatory cytokines and promoting anti-viral responses. 25-Hydroxycholesterol-d6 is not intended for pharmacological use but as an analytical standard to quantify endogenous 25-hydroxycholesterol levels.
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| Enzyme Assay |
(1) Prepare calibration standards by spiking known concentrations of 25-hydroxycholesterol (analyte) with a fixed concentration of 25-Hydroxycholesterol-d6 (internal standard) into blank biological matrix (e.g., serum, plasma, or cell lysates). (2) Extract lipids by liquid-liquid extraction (ethyl acetate or hexane) or solid-phase extraction. (3) Derivatize if necessary (e.g., with MSTFA for GC-MS analysis). (4) Separate on a GC column (e.g., DB-5MS) or LC column (C18 reverse-phase). (5) Detect by MS in selected ion monitoring (SIM) or multiple reaction monitoring (MRM) mode, using the m/z ratio of the unlabeled compound and the m/z shift due to the 6 deuterium atoms. (6) Calculate the ratio of analyte peak area to internal standard peak area to quantify 25-hydroxycholesterol concentration.
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| Cell Assay |
(1) Seed B cells or macrophages in culture medium (e.g., RPMI-1640 with 10% FBS). (2) For 25-Hydroxycholesterol-d6: add as an internal standard to cell lysates or culture supernatants post-harvest, not as a treatment. (3) For 25-hydroxycholesterol treatment: stimulate B cells with LPS (1 ug/mL) and IL-4 (10 ng/mL) to induce IgA production, then treat with 0.01-1 uM 25-Hydroxycholesterol for 3-7 days. (4) Measure IgA levels in supernatant by ELISA. (5) For isotope tracing: add 25-Hydroxycholesterol-d6 to culture medium at 0.1-10 uM for 0-24 h, extract cellular lipids, and analyze the metabolic fate by LC-MS/MS.
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| Animal Protocol |
(1) Use 6-8 week old C57BL/6 mice (20-25 g). (2) For pharmacokinetic studies of unlabeled 25-hydroxycholesterol: administer IV or IP at 5-20 mg/kg, formulated in 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline. (3) Collect blood at predetermined time points (0, 0.5, 1, 2, 4, 8, 12, 24 h). (4) Add a fixed amount of 25-Hydroxycholesterol-d6 (internal standard) to each plasma sample before extraction. (5) Extract lipids, analyze by LC-MS/MS, and calculate 25-hydroxycholesterol concentration using the internal standard method. (6) Calculate PK parameters: t1/2, Cmax, AUC, CL.
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| ADME/Pharmacokinetics |
Standard formulation for 25-Hydroxycholesterol-d6 as an internal standard: prepare a stock solution in DMSO (1-10 mg/mL), store at -80degC. For LC-MS analysis, dilute the stock solution in methanol or acetonitrile to a working concentration of 100-1000 ng/mL. For biological sample preparation: add 10-50 uL of working internal standard solution to 100 uL of plasma or cell lysate. The deuterated compound has nearly identical physicochemical properties to the unlabeled compound, making it an ideal internal standard for accurate mass spectrometry-based quantification.
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| Toxicity/Toxicokinetics |
25-Hydroxycholesterol-d6 is considered non-toxic as it is a stable isotope-labeled endogenous metabolite. The unlabeled compound 25-hydroxycholesterol is non-toxic at physiological concentrations (low nM to uM). For in vitro toxicity of 25-hydroxycholesterol: CCK-8 assay on HEK293 cells shows IC50 > 50 uM. For in vivo toxicity: in mice, IP injection of 25-hydroxycholesterol up to 50 mg/kg is well tolerated with no significant weight loss or organ toxicity. The isotope-labeled version is for research use only, not for human therapeutic use.
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| References | |
| Additional Infomation |
25-Hydroxycholesterol-d6 is a high-purity stable isotope-labeled internal standard (≥98% purity, 99 atom % D) used for the quantification of 25-hydroxycholesterol in biological samples by GC-MS or LC-MS. It belongs to the class of oxysterols, which are oxygenated derivatives of cholesterol involved in cholesterol homeostasis, immune modulation, and inflammation. This compound is not a drug and is not approved by the FDA or EMA; it is strictly for research use in analytical method development, pharmacokinetic studies, and biomarker discovery.
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| Molecular Formula |
C27H40D6O2
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|---|---|
| Molecular Weight |
408.69
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| Exact Mass |
408.387
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| CAS # |
88247-69-2
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| Related CAS # |
25-Hydroxycholesterol;2140-46-7
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| PubChem CID |
42608393
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| Appearance |
White to off-white solid powder
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| Density |
1.0±0.1 g/cm3
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| Boiling Point |
513.1±23.0 °C at 760 mmHg
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| Flash Point |
213.5±17.2 °C
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| Vapour Pressure |
0.0±3.0 mmHg at 25°C
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| Index of Refraction |
1.538
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| LogP |
7.66
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
29
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| Complexity |
636
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| Defined Atom Stereocenter Count |
8
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| SMILES |
[2H]C([2H])([2H])C(CCC[C@@H](C)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC=C4[C@@]3(CC[C@@H](C4)O)C)C)(C([2H])([2H])[2H])O
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| InChi Key |
INBGSXNNRGWLJU-KHKDDIDZSA-N
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| InChi Code |
InChI=1S/C27H46O2/c1-18(7-6-14-25(2,3)29)22-10-11-23-21-9-8-19-17-20(28)12-15-26(19,4)24(21)13-16-27(22,23)5/h8,18,20-24,28-29H,6-7,9-17H2,1-5H3/t18-,20+,21+,22-,23+,24+,26+,27-/m1/s1/i2D3,3D3
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| Chemical Name |
(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-7,7,7-trideuterio-6-hydroxy-6-(trideuteriomethyl)heptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol
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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 | 2.4468 mL | 12.2342 mL | 24.4684 mL | |
| 5 mM | 0.4894 mL | 2.4468 mL | 4.8937 mL | |
| 10 mM | 0.2447 mL | 1.2234 mL | 2.4468 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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