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| Targets |
4-Hydroxyestrone-d4 is a stable isotope-labeled internal standard. Its unlabeled parent compound, 4-Hydroxyestrone (4-OHE1), is an endogenous estrogen metabolite. The primary target of 4-OHE1 is the estrogen receptor (ER), both ERalpha and ERbeta, although it has a lower binding affinity compared to the parent hormone, estradiol. Unlike estradiol, 4-OHE1 is metabolically activated to form reactive quinones (4-hydroxyestrone-3,4-quinone) that can react with DNA, forming depurinating adducts. This genotoxic mechanism distinguishes it from receptor-mediated actions and is implicated in the initiation of breast, prostate, and other cancers. 4-OHE1 is also a substrate for catechol-O-methyltransferase (COMT), which methylates and inactivates it. 4-Hydroxyestrone-d4 is used as a tracer to study these complex pathways without altering the biochemical targets.
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| ln Vitro |
Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as quantitative tracers while the drugs were being developed. Because deuteration may have an effect on a drug's pharmacokinetics and metabolic properties, it is a cause for concern [1].
The in vitro biological activity of 4-Hydroxyestrone-d4 is not directly studied, but its unlabeled parent, 4-Hydroxyestrone (4-OHE1), has significant activity. In MCF-7 human breast cancer cells, 4-OHE1 (1 nM-1 uM) has been shown to act as a weak estrogen agonist, inducing cell proliferation and the expression of estrogen-responsive genes (e.g., pS2) at concentrations above 10 nM. However, at high concentrations (10-50 uM), it induces cytotoxicity and apoptosis. Its most notable in vitro activity is its genotoxic potential. In the presence of cytochrome P450 (e.g., CYP1B1) or metal ions (Cu2+), 4-OHE1 is oxidized to a reactive quinone that forms DNA adducts, leading to apurinic sites and DNA strand breaks, as measured by the Comet assay. Furthermore, it has been shown to increase the frequency of mutations in the HPRT gene in human breast epithelial cells. The labeled version (d4) is used as an internal standard to quantify 4-OHE1 in these cell culture experiments by LC-MS. |
| ln Vivo |
4-Hydroxyestrone-d4 is a stable isotope-labeled internal standard. The in vivo activity of its unlabeled parent, 4-Hydroxyestrone (4-OHE1), is an area of intensive research. In female rats, injection of 4-OHE1 (0.5-5 mg/kg) has been shown to induce cell proliferation in the mammary gland and uterus, suggesting weak estrogenic activity. However, it is also a potent carcinogen. In the Syrian golden hamster kidney tumor model, chronic administration of 4-OHE1 (5 mg/kg, 3x/week for 6 months) leads to a 100% incidence of renal cell carcinomas. This is in contrast to estradiol, which does not cause kidney tumors in this model. The carcinogenic effect is attributed to the formation of DNA adducts and oxidative DNA damage in the kidney tissue rather than ER-mediated effects. 4-Hydroxyestrone-d4 is used as an internal standard in LC-MS to quantify the levels of 4-OHE1 and its DNA adducts in these animal tissues.
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| Enzyme Assay |
A generic non-cell-based assay for 4-Hydroxyestrone-d4 involves its use as an internal standard in an LC-MS/MS method for quantifying estrogen metabolites in serum. Prepare a standard stock solution of the unlabeled 4-Hydroxyestrone in methanol (1 mg/mL). Prepare a separate stock solution of the internal standard (4-Hydroxyestrone-d4) at the same concentration. Prepare calibration standards by spiking the unlabeled analyte into a blank matrix (e.g., charcoal-stripped human serum) to achieve concentrations ranging from 5 pg/mL to 1000 pg/mL. Add a fixed concentration of the internal standard (e.g., 50 pg/mL) to each calibration standard. Also prepare blank and double-blank samples. For sample preparation, perform liquid-liquid extraction with methyl tert-butyl ether (MTBE). Centrifuge and collect the organic layer. Evaporate the solvent under nitrogen. Derivatize the residue with dansyl chloride to improve ionization efficiency. Analyze the samples by LC-MS/MS in positive ion mode. Monitor the mass transitions: m/z 506 → 171 for 4-Hydroxyestrone (dansyl derivative), and m/z 510 → 175 for the d4 internal standard. Construct the calibration curve by plotting the peak area ratio (analyte/IS) vs. the nominal concentration.
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| Cell Assay |
A standard in vitro cell-based protocol for the unlabeled 4-Hydroxyestrone involves the assessment of DNA damage. Culture MCF-10A human breast epithelial cells in DMEM/F12 medium supplemented with 5% horse serum, EGF, hydrocortisone, insulin, and cholera toxin. Seed the cells in 6-well plates at 2×10⁵ cells per well. Treat the cells with various concentrations (0.1, 0.5, 1, 5, 10 uM) of unlabeled 4-Hydroxyestrone or vehicle (ethanol) for 24 hours. After treatment, harvest the cells and perform the alkaline single-cell gel electrophoresis (Comet) assay to measure DNA strand breaks. Use the CometScore software to analyze the percentage of DNA in the tail. For LC-MS analysis, collect a separate set of cell culture medium samples, add 4-Hydroxyestrone-d4 as the internal standard, and extract the samples. Analyze the extracts by LC-MS/MS to quantify the exact concentration of 4-OHE1 present in the medium, ensuring the correct dose is correlated with the degree of DNA damage. Also, measure the activity of the detoxification enzyme COMT in the cell lysate.
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| Animal Protocol |
A standard in vivo protocol for 4-Hydroxyestrone-d4 is a carcinogenicity study in the Syrian golden hamster. Use 6-8 week old female hamsters (n=15 per group). Administer unlabeled 4-Hydroxyestrone (5 mg/kg) or vehicle (sesame oil) via subcutaneous injection three times per week for 6 months. Monitor body weight and general health weekly. At the end of the study, euthanize the animals and harvest the kidneys. Fix one kidney in formalin for histopathological examination (H&E staining) to assess tumor formation. Freeze the other kidney in liquid nitrogen. To analyze DNA adducts, isolate DNA from the frozen kidney tissue. Hydrolyze the DNA to nucleotides and analyze by LC-MS/MS in positive ion mode. Use 4-Hydroxyestrone-d4 as an internal standard to quantify the levels of the 4-OHE1-DNA adducts. Also, measure the urinary and plasma levels of 4-OHE1 using LC-MS with the d4 internal standard to assess systemic exposure. This protocol is used to confirm the mechanism of estrogen-induced carcinogenesis.
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| ADME/Pharmacokinetics |
4-Hydroxyestrone-d4 is an internal standard for the quantification of 4-Hydroxyestrone (4-OHE1), an endogenous catechol estrogen metabolite. The pharmacokinetics (PK) of 4-OHE1 in animals have been studied. Following intravenous administration in rats, 4-OHE1 has a short half-life of approximately 20-40 minutes and a moderate volume of distribution (Vd ~ 1-2 L/kg). It is primarily metabolized by catechol-O-methyltransferase (COMT) to its methylated form, 4-methoxyestrone. It is also further hydroxylated by CYP450 enzymes and conjugated (glucuronidation, sulfation) for elimination. The majority of the compound and its metabolites are excreted in the urine and feces. The endogenous levels of 4-OHE1 in human serum are very low (picomolar range), and accurately measuring these requires a highly sensitive method. The labeled 4-Hydroxyestrone-d4 is critical for the accurate quantitation of these trace concentrations via isotope dilution mass spectrometry.
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| Toxicity/Toxicokinetics |
4-Hydroxyestrone-d4 is a research-grade stable isotope-labeled compound and is not a pharmaceutical drug. Its toxicity profile is inferred from its unlabeled parent, 4-Hydroxyestrone (4-OHE1). While 4-OHE1 is an endogenous compound, it is also a potent carcinogen. In animal models, it is a complete carcinogen capable of initiating and promoting tumors, particularly in the kidney of Syrian hamsters. The mechanism of toxicity is its metabolic conversion to a reactive quinone that forms DNA adducts, leading to mutations and genomic instability. It is not considered directly cytotoxic at low concentrations, but it causes cell death at high concentrations. 4-OHE1 is weakly estrogenic, meaning it could theoretically promote the growth of hormone-sensitive cancers. Therefore, while the labeled deuterated version is chemically stable and safe to handle using PPE, its biological effects mimic those of 4-OHE1. Standard precautions for handling potential carcinogens should be followed in the laboratory.
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| References | |
| Additional Infomation |
4-Hydroxyestrone-d4 (4-OHE1-d4) is the stable isotope-labeled version of 4-Hydroxyestrone, an endogenous catechol estrogen metabolite of estrone and estradiol. It is intended for research use only as an internal standard for the accurate quantification of 4-Hydroxyestrone in biological samples (serum, urine, tissue) by LC-MS/MS. Unlike estradiol, which primarily acts through estrogen receptor-mediated transcriptional activation, 4-Hydroxyestrone has been identified as a genotoxic metabolite. It can be oxidized to reactive quinones that form depurinating DNA adducts, which are implicated in the initiation of breast, prostate, and cervical cancers. Measuring its levels is thus critical for understanding estrogen-induced carcinogenesis and for assessing the risk of hormone-related cancers. The d4 internal standard ensures precise and reliable quantitation in complex biological matrices.
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| Molecular Formula |
C18H18D4O3
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| Molecular Weight |
290.39
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| Exact Mass |
290.182
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| CAS # |
81586-98-3
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| Related CAS # |
4-Hydroxyestrone;3131-23-5
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| PubChem CID |
71749017
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| Appearance |
Off-white to gray solid powder
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| LogP |
3.523
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
21
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| Complexity |
448
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| Defined Atom Stereocenter Count |
4
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| SMILES |
C[C@@]12C(=O)C([2H])([2H])C[C@H]1[C@@H]1CCC3C(=C(C([2H])=C(C=3[C@H]1CC2)[2H])O)O
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| InChi Key |
XQZVQQZZOVBNLU-RFZGAVBWSA-N
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| InChi Code |
InChI=1S/C18H22O3/c1-18-9-8-11-10-4-6-15(19)17(21)13(10)3-2-12(11)14(18)5-7-16(18)20/h4,6,11-12,14,19,21H,2-3,5,7-9H2,1H3/t11-,12-,14+,18+/m1/s1/i4D,6D,7D2
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| Chemical Name |
(8R,9S,13S,14S)-1,2,16,16-tetradeuterio-3,4-dihydroxy-13-methyl-6,7,8,9,11,12,14,15-octahydrocyclopenta[a]phenanthren-17-one
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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 | 3.4436 mL | 17.2182 mL | 34.4364 mL | |
| 5 mM | 0.6887 mL | 3.4436 mL | 6.8873 mL | |
| 10 mM | 0.3444 mL | 1.7218 mL | 3.4436 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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