| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
The primary target of creatine, and by extension creatine-d3 as a tracer, is the creatine kinase (CK) enzyme system, which comprises several isoforms: CK-MM (expressed predominantly in skeletal muscle), CK-MB (expressed in cardiac muscle), and CK-BB (expressed in brain and smooth muscle). Creatine acts as a substrate for creatine kinase, accepting a phosphate group from ATP to form phosphocreatine, a high-energy phosphate reservoir that can rapidly donate its phosphate back to ADP to regenerate ATP during periods of high energy demand. The creatine/phosphocreatine system is essential for maintaining cellular ATP homeostasis, particularly in tissues with high and fluctuating energy requirements. Creatine also directly or indirectly interacts with various cellular processes, including modulation of mitochondrial function, regulation of cellular pH, and protection against oxidative stress. As a deuterated tracer, creatine-d3 targets the same metabolic pathways but does not interfere with endogenous creatine pools due to the isotopic labeling, which allows for precise quantitation via mass spectrometry.
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| ln Vitro |
Stable heavy isotopes of hydrogen, carbon, and other elements have been incorporated into drug molecules, largely as tracers for quantitation during the drug development process. Studies involving the human use of drugs labeled with deuterium suggest that these compounds may offer some advantages when compared with their nondeuterated counterparts. Deuteration has gained attention because of its potential to affect the pharmacokinetic and metabolic profiles of drugs. Deutetrabenazine is the first deuterated drug to receive Food and Drug Administration approval. This deuterated form of the drug tetrabenazine is indicated for the treatment of chorea associated with Huntington's disease as well as tardive dyskinesia. Ongoing clinical trials suggest that a number of other deuterated compounds are being evaluated for the treatment of human diseases and not merely as research tools.[1]
In vitro activity studies using creatine-d3 primarily involve its use as an internal standard for the quantification of creatine and its metabolites in cell culture systems. Typically, standard curves are constructed by spiking known amounts of unlabeled creatine into blank biological matrices (e.g., cell lysates, culture media), followed by the addition of a fixed amount of creatine-d3 as an internal standard (e.g., 10-50 ng/mL). The ratio of the peak area of unlabeled creatine to that of creatine-d3 is then used to calculate the absolute concentration of creatine in unknown samples by LC-MS/MS. This approach allows researchers to monitor dynamic changes in creatine metabolism under various experimental conditions, such as following treatment with metabolic modulators, during cellular differentiation (e.g., myogenesis or neurogenesis), or in response to oxidative stress. The in vitro activity of creatine itself includes promoting cell viability, enhancing ATP recovery, and protecting against excitotoxicity in neuronal cell cultures. However, as a stable isotope tracer, creatine-d3 is not evaluated for biological activity but rather for its utility as a quantitative internal standard. |
| ln Vivo |
Deuterated compounds may, in some cases, offer advantages over nondeuterated forms, often through alterations in clearance. Deuteration may also redirect metabolic pathways in directions that reduce toxicities. The approval of additional deuterated compounds may soon follow. Clinicians will need to be familiar with the dosing, efficacy, potential side effects, and unique metabolic profiles of these new entities.
In vivo activity of creatine-d3 is centered around its application in the D3-creatine dilution method for non-invasive assessment of total body creatine pool size and skeletal muscle mass. This method involves the oral administration of a single dose of creatine-d3 (typically 30-100 mg) to human subjects or laboratory animals. The labeled creatine is absorbed from the gastrointestinal tract, distributed throughout the body, and enters the creatine pool in skeletal muscle, where it is non-enzymatically converted to creatinine at a rate proportional to the total creatine pool size. The enrichment of D3-creatinine (the deuterated form of creatinine) in urine is measured by LC-MS/MS, and the dilution of the administered D3-creatine label by the endogenous unlabeled creatine pool is used to calculate total body creatine pool size and subsequently total skeletal muscle mass. This method has been validated in both humans and rodents and is considered a direct, non-invasive, and accurate alternative to imaging-based techniques such as MRI or CT for measuring muscle mass. The D3-creatine dilution method does not require ionizing radiation, is unaffected by hydration status, and can be repeated longitudinally to monitor changes in muscle mass over time. |
| Enzyme Assay |
To assess creatine kinase (CK) activity, an enzyme assay can be performed in cell-free systems using creatine-d3 as a tracer. In this assay, creatine kinase is isolated from tissue homogenates (e.g., rat heart or skeletal muscle) or obtained as a purified recombinant enzyme. The reaction mixture contains 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 5 mM ATP, 5 mM phosphocreatine, and 0.5-1 mM creatine-d3 or unlabeled creatine as a substrate. The reaction is initiated by the addition of the CK enzyme (0.1-1 U/mL) and incubated at 30-37degC for 5-30 minutes. The reaction is terminated by heating at 95degC for 5 minutes or by adding 0.5 M perchloric acid. Following centrifugation to remove precipitated proteins, the supernatant is analyzed by LC-MS/MS to detect the formation of D3-phosphocreatine (the phosphorylated product) or the disappearance of creatine-d3. The conversion rate is calculated based on the change in concentration of the substrate or product over time, and kinetic parameters such as Km and Vmax are determined by performing the assay at various substrate concentrations. This method enables the specific tracking of CK activity without interference from endogenous unlabeled creatine, as the isotopic label provides distinct mass shifts detectable by mass spectrometry.
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| Cell Assay |
For cell-based studies using creatine-d3, cells of interest (e.g., C2C12 myoblasts, L6 rat skeletal muscle cells, or primary hippocampal neurons) are cultured in appropriate growth medium (e.g., DMEM supplemented with 10% FBS and 1% penicillin-streptomycin) at 37degC in a 5% CO2 incubator. Once cells reach 70-90% confluence, the medium is replaced with fresh medium containing creatine-d3 at concentrations ranging from 0.1 to 10 mM for 24-72 hours. Following the incubation period, cells are washed twice with ice-cold PBS to remove extracellular creatine-d3, then harvested by scraping into lysis buffer (e.g., 50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail). The cell lysate is centrifuged at 12,000 × g for 10 minutes at 4degC, and the supernatant is collected. Proteins are precipitated by adding 3-5 volumes of ice-cold acetonitrile or methanol, and after centrifugation, the resulting supernatant is analyzed by LC-MS/MS. The concentration of creatine-d3 and its phosphorylated product (phosphocreatine-d3) in the cell lysate is measured relative to an external standard curve. This approach allows researchers to quantify the cellular uptake, phosphorylation, and metabolism of creatine-d3, as well as to assess the effects of various compounds or genetic manipulations on creatine metabolism.
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| Animal Protocol |
Cellular uptake of creatine-d3 can be studied in experimental animals, typically rodents (mice or rats). The standard protocol for D3-creatine dilution involves the oral administration of a single dose of creatine-d3 (dissolved in water or saline) via gavage at a dose of approximately 30-100 mg per kg body weight. After administration, animals are placed in metabolic cages for 24-72 hours to allow for separate collection of urine and feces. Alternatively, for time-course studies, blood samples are collected from the tail vein or via cardiac puncture at various time points (0, 1, 2, 4, 8, 12, 24, 48, and 72 hours post-dosing). At the end of the experiment, animals are euthanized, and tissues of interest (gastrocnemius muscle, soleus muscle, heart, brain, liver, and kidney) are harvested, snap-frozen in liquid nitrogen, and stored at -80degC for subsequent analysis. Tissues are homogenized in ice-cold water or acetonitrile, and after protein precipitation, the supernatant is analyzed by LC-MS/MS to measure the concentration of creatine-d3 and D3-creatinine. The enrichment of D3-creatinine in urine (typically expressed as the ratio of D3-creatinine to total creatinine) is the primary readout for calculating total body creatine pool size and skeletal muscle mass. In longitudinal studies, the D3-creatine dilution method can be applied repeatedly to monitor changes in muscle mass in response to interventions such as exercise training, aging, denervation, or drug treatment.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of creatine-d3 are assumed to be nearly identical to those of unlabeled creatine due to the minimal isotope effect of deuterium substitution on a methyl group. Following oral administration in humans, creatine is absorbed from the small intestine via the creatine transporter SLC6A8, with peak plasma concentrations (Cmax) achieved approximately 1-2 hours post-dose. The oral bioavailability of creatine is relatively low (approximately 30-40%) due to incomplete absorption and extensive first-pass metabolism in the liver. Creatine-d3 is distributed throughout the body, with the highest concentrations found in skeletal muscle (~95% of total body creatine pool), followed by the heart, brain, and other tissues with high energy demands. The volume of distribution is large (approximately 40-60 L in humans), reflecting extensive tissue binding. Plasma protein binding is minimal (<10%). Metabolism of creatine is minimal; the primary fate of creatine is non-enzymatic, irreversible cyclization to creatinine at a constant rate of approximately 1.5-2% of the total creatine pool per day. Creatinine is excreted unchanged in the urine via glomerular filtration, with no significant tubular reabsorption. The elimination half-life of creatine in humans is approximately 3 hours following intravenous administration, but the pharmacodynamic half-life of the creatine pool in muscle is much longer (~30-45 days), reflecting the slow turnover of muscle creatine stores. In the D3-creatine dilution method, the enrichment of D3-creatinine in urine reaches steady-state by approximately 48-72 hours post-dose, and the decline in urine D3-creatinine enrichment follows the turnover rate of the creatine pool.
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| Toxicity/Toxicokinetics |
The toxicity profile of creatine-d3 is expected to be similar to that of unlabeled creatine, which has an excellent safety profile and a long history of use as a dietary supplement at doses of 3-20 g per day. In clinical studies and animal experiments, creatine has been shown to be well-tolerated with minimal adverse effects. The most common side effects reported with high-dose creatine supplementation are gastrointestinal in nature, including bloating, nausea, diarrhea, and abdominal discomfort, likely due to osmotic effects in the intestinal lumen. At very high doses (≥20 g/day in humans or ≥5 g/kg in rodents), mild elevations in serum creatinine and blood urea nitrogen (BUN) may occur, but these are generally not indicative of true renal impairment. In fact, creatine has been shown to be safe in individuals with normal kidney function, and a Cochrane review concluded that creatine supplementation does not adversely affect renal function in healthy individuals. However, individuals with pre-existing kidney disease are advised to consult a healthcare professional before taking creatine supplements. Creatine-d3, as an isotopically labeled compound, is used at trace levels (microgram to milligram quantities) in research settings, making its toxicity negligible. No genotoxicity, carcinogenicity, or reproductive toxicity has been reported for creatine. As with all research chemicals, standard laboratory safety practices (wearing gloves, lab coat, and eye protection) should be followed when handling creatine-d3.
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| References | |
| Additional Infomation |
Creatine D3 is a deuterated compound.
The non-labeled parent compound, creatine, is an endogenous amino acid derivative that plays a crucial role in cellular energy homeostasis, particularly in tissues with high energy demands such as skeletal muscle and brain. Creatine is widely used as a dietary supplement to improve athletic performance, increase lean body mass, and enhance high-intensity exercise capacity. Creatine-d3 is the deuterium-labeled version of creatine, where the three hydrogens on the methyl group are replaced with deuterium atoms. The primary research application of creatine-d3 is as an internal standard for the quantitative analysis of creatine and its metabolites (primarily creatinine) in biological matrices such as plasma, serum, urine, tissue homogenates, and cell lysates using LC-MS/MS or GC-MS. The use of a stable isotope-labeled internal standard corrects for matrix effects, ionization suppression or enhancement, and variations in sample preparation, thereby improving the accuracy, precision, and reproducibility of quantitative bioanalytical methods. Additionally, creatine-d3 is the key reagent in the D3-creatine dilution method, a non-invasive technique for the measurement of total body creatine pool size and skeletal muscle mass in humans and laboratory animals. This method has gained significant attention as a direct, safe, and repeatable approach for assessing muscle mass in clinical research, including studies on sarcopenia (age-related muscle loss), cachexia (disease-related muscle wasting), obesity, and metabolic disorders. The D3-creatine dilution method has been validated against reference methods such as MRI and DEXA and is considered a promising tool for monitoring the efficacy of interventions aimed at preserving or increasing muscle mass. Creatine-d3 is intended for research use only and is not approved for diagnostic or therapeutic applications in humans. |
| Molecular Formula |
C4H6D3N3O2
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|---|---|
| Molecular Weight |
134.15
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| Exact Mass |
134.088
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| CAS # |
143827-19-4
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| Related CAS # |
Creatine; 57-00-1
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| PubChem CID |
12205340
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| Appearance |
Solid powder
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| LogP |
-1.2
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
9
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| Complexity |
134
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| Defined Atom Stereocenter Count |
0
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| SMILES |
[2H]C([2H])([2H])N(CC(=O)O)C(=N)N
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| InChi Key |
CVSVTCORWBXHQV-FIBGUPNXSA-N
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| InChi Code |
InChI=1S/C4H9N3O2/c1-7(4(5)6)2-3(8)9/h2H2,1H3,(H3,5,6)(H,8,9)/i1D3
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| Chemical Name |
2-[carbamimidoyl(trideuteriomethyl)amino]acetic acid
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| Synonyms |
N-(Aminoiminomethyl)-N-(methyl-d3)glycine
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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) |
H2O : 20.83 mg/mL (155.27 mM; with sonication (<60°C))
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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 | 7.4543 mL | 37.2717 mL | 74.5434 mL | |
| 5 mM | 1.4909 mL | 7.4543 mL | 14.9087 mL | |
| 10 mM | 0.7454 mL | 3.7272 mL | 7.4543 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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