Asymmetric-dimethylarginine-d6 has no independent pharmacological target as a stable isotope internal standard. The unlabeled ADMA is an endogenous inhibitor of nitric oxide synthase (NOS) and a metabolite in the arginine-citrulline pathway. ADMA is formed from arginine by protein arginine methyltransferases (PRMTs) and degraded by dimethylarginine dimethylaminohydrolases (DDAHs) and alanine-glyoxylate aminotransferase 2. Elevated ADMA levels are associated with endothelial dysfunction and cardiovascular disease risk.

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].

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As a stable isotope internal standard, Asymmetric-dimethylarginine-d6 dihydrochloride is not tested for in vitro pharmacological activity. It is added to biological samples to enable accurate quantification of ADMA by LC-MS/MS. The deuterium labeling provides a mass shift that allows the internal standard to be distinguished from the endogenous analyte, correcting for matrix effects, extraction recovery, and instrument variability without interfering with the biological activity of ADMA.

Asymmetric-dimethylarginine-d6 dihydrochloride has no in vivo pharmacological activity as a therapeutic agent. It is used as an internal standard for quantifying ADMA in biological samples obtained from animal and human studies, including plasma, urine, and tissue homogenates. ADMA is an important biomarker for cardiovascular disease, endothelial dysfunction, and chronic kidney disease, and the deuterated standard enables accurate measurement of this biomarker in clinical and preclinical research.

For in vitro LC-MS/MS quantification, Asymmetric-dimethylarginine-d6 dihydrochloride is dissolved in water, methanol, or 0.1% formic acid to prepare a stock solution (e.g., 1 mg/mL). The internal standard is added to biological samples (cell lysates, plasma, urine) at a fixed concentration (e.g., 10-500 ng/mL). For plasma samples, protein precipitation is performed by adding 3-5 volumes of methanol or acetonitrile containing the internal standard, followed by vortexing and centrifugation (10,000-15,000 rpm, 10 minutes). The supernatant is transferred and evaporated to dryness under nitrogen. The residue is reconstituted in mobile phase (e.g., 0.1% formic acid in water:methanol) and analyzed by LC-MS/MS. Derivatization with dansyl chloride or similar agents may be used to enhance ionization efficiency. The ADMA-d6 peak is monitored, and the ADMA/ADMA-d6 peak area ratio is used for quantification.

For cell-based studies, cells (e.g., endothelial cells, macrophages, or hepatocytes) are cultured in standard medium (DMEM or RPMI-1640 with 10% FBS). After experimental treatments affecting ADMA metabolism (e.g., with PRMT inhibitors or DDAH modulators), cell lysates or culture supernatants are collected. Asymmetric-dimethylarginine-d6 dihydrochloride is added as an internal standard at a fixed concentration (e.g., 10-100 ng/mL). Following protein precipitation with methanol or acetonitrile containing 0.1% formic acid and centrifugation, the supernatant is analyzed by LC-MS/MS to quantify endogenous ADMA levels. ADMA concentrations are normalized to protein content or cell number. For time-course studies, the internal standard is added to all samples at a consistent concentration to correct for sample handling and instrument variability.

For in vivo pharmacokinetic or biomarker studies, Asymmetric-dimethylarginine-d6 dihydrochloride is not administered to animals independently. It is used as an internal standard for quantifying ADMA in biological samples obtained from animals treated with experimental compounds affecting NOS activity or the arginine metabolic pathway. After collection of plasma (via tail vein or cardiac puncture), urine, or tissue homogenates, the internal standard is added at a fixed concentration (e.g., 10-500 ng/mL). For tissue samples, homogenization is performed in PBS or water, followed by protein precipitation with methanol or acetonitrile. After centrifugation, the supernatant is analyzed by LC-MS/MS to determine absolute ADMA concentrations. For clinical studies, ADMA-d6 is similarly added to patient plasma or urine samples.

Metabolism / Metabolites
Uremic toxins often accumulate in the blood due to overeating or poor kidney filtration. Most uremic toxins are metabolic waste products that are normally excreted through urine or feces.

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Asymmetric-dimethylarginine-d6 dihydrochloride is an internal standard and does not have independent pharmacokinetic parameters. ADMA is an endogenous metabolite with a plasma half-life of approximately 30-60 minutes in humans, primarily cleared by renal excretion and enzymatic degradation by DDAH. Normal plasma ADMA concentrations in healthy humans are 0.3-0.8 uM. Elevated ADMA levels are associated with cardiovascular disease, endothelial dysfunction, and chronic kidney disease. The deuterated version is used to calibrate analytical methods and does not alter the ADME properties of the endogenous analyte.

Toxicity Summary
Uremic toxins, such as asymmetric dimethylarginine, can be actively transported to the kidneys via organic ion transporters, particularly OAT3. Elevated uremic toxin levels can stimulate the production of reactive oxygen species (ROS). This appears to be mediated by the direct binding of uremic toxins to or inhibition of NADPH oxidases, particularly NOX4, which is abundant in the kidneys and heart (A7868). ROS can induce a variety of different DNA methyltransferases (DNMTs) involved in the silencing of the KLOTHO protein. KLOTHO has been shown to play an important role in anti-aging, mineral metabolism, and vitamin D metabolism. Multiple studies have shown that in acute or chronic kidney disease, KLOTHO mRNA and protein levels are decreased due to elevated local ROS levels (A7869).

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Asymmetric-dimethylarginine-d6 dihydrochloride is a stable isotope-labeled internal standard with low toxicity as it is used at trace concentrations (ug-mg quantities). ADMA is an endogenous metabolite present in all mammalian cells at low micromolar concentrations. At pathological elevated levels, ADMA contributes to endothelial dysfunction and cardiovascular disease risk by inhibiting nitric oxide synthase. The deuterated version is chemically identical except for isotopic substitution. Standard laboratory safety precautions for handling amino acid derivatives (gloves, safety glasses, fume hood) apply. Not intended for human consumption.

[1]. Impact of Deuterium Substitution on the Pharmacokinetics of Pharmaceuticals. Ann Pharmacother. 2019;53(2):211-216.

N(ω),N(ω)-dimethyl-L-arginine is a derivative of L-arginine, with two methyl groups attached to the primary amino group of its guanidine group. It is an EC 1.14.13.39 (nitric oxide synthase) inhibitor. It is a non-protein L-α-amino acid, belonging to the guanidine group, and is a derivative of L-arginine, as well as a dimethylarginine. It is the conjugate base of N(ω),N(ω)-dimethyl-L-arginine (1+). Asymmetric dimethylarginine (ADMA) is a naturally occurring chemical substance found in blood plasma. It is a metabolic byproduct of the ongoing protein modification process in the cytoplasm of all human cells and is closely related to the conditionally essential amino acid L-arginine. ADMA interferes with the production of nitric oxide from L-arginine, a key chemical for maintaining endothelial cell and cardiovascular health. N,N-dimethylarginine has been reported to be present in fruit flies, fission yeast, and other organisms with relevant data.
Asymmetric dimethylarginine (ADMA) is a dimethylated derivative of L-arginine, in which two methyl groups are asymmetrically linked to arginine residues. ADMA is a competitive inhibitor of NOS (nitric oxide), formed when S-adenosylmethionine protein N-methyltransferase transfers two methyl groups from S-adenosylmethionine to one of the two guanidino nitrogen atoms of the arginine residue in a protein. ADMA is released during protein degradation and is a substrate of dimethylarginine dimethylaminohydrolase (DDAH). Free ADMA in plasma competes with L-arginine for binding to heme in NOS, thereby inhibiting nitric oxide (NO) synthesis. Reduced NO synthesis inhibits vasodilation, leading to endothelial dysfunction. Elevated plasma ADMA levels can be caused by certain types of cancer, cardiovascular disease, hypertension, hyperlipidemia, type 2 diabetes, and increased oxidative stress.
Asymmetric dimethylarginine is a uremic toxin. Based on their chemical and physical properties, uremic toxins can be divided into three main categories: 1) small molecule, water-soluble, non-protein-bound compounds, such as urea; 2) small molecule, lipid-soluble and/or protein-bound compounds, such as phenols; and 3) larger so-called medium-molecule compounds, such as β2-microglobulin. Long-term exposure to uremic toxins can lead to a variety of diseases, including kidney damage, chronic kidney disease, and cardiovascular disease. Asymmetric dimethylarginine (ADMA) is a naturally occurring chemical substance found in blood plasma. It is a metabolic byproduct of a continuous protein modification process in the cytoplasm of all human cells. It is closely related to the conditionally essential amino acid L-arginine. ADMA interferes with the formation of nitric oxide from L-arginine, a key chemical substance for maintaining endothelial cell and cardiovascular health. Asymmetric dimethylarginine is produced during protein methylation, a common post-translational modification mechanism of proteins. This reaction is catalyzed by a group of enzymes called S-adenosylmethionine protein N-methyltransferases (protein methyltransferases I and II). The methyl group used to generate ADMA is derived from the methyl donor S-adenosylmethionine, an intermediate in homocysteine metabolism. (Homocysteine is an important blood chemical as it is also a marker of cardiovascular disease). After synthesis, ADMA migrates into the extracellular space and then enters the plasma. Asymmetric dimethylarginine was determined using high-performance liquid chromatography. Dimethyl-L-arginine is a metabolite found or produced in Saccharomyces cerevisiae. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor formed by the methylation of arginine residues in proteins and released upon proteolysis. In this reaction, S-adenosylmethionine is the methyl donor, and S-adenosylhomocysteine is the demethylation product. Therefore, ADMA and homocysteine are biochemically related. Both plasma homocysteine and ADMA concentrations are elevated in patients with renal insufficiency, likely due to impaired metabolic clearance (rather than urinary clearance). Hyperhomocysteinemia is associated with an increased risk of cardiovascular disease in patients with end-stage renal disease, particularly in those without malnutrition and inflammation. Furthermore, plasma ADMA levels are associated with cardiovascular disease in patients with renal failure. Both homocysteine and ADMA are believed to exert their adverse vascular effects by impairing endothelial cell-dependent nitric oxide function, leading to decreased vasodilation, increased smooth muscle cell proliferation, platelet dysfunction, and increased monocyte adhesion.

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Asymmetric-dimethylarginine-d6 dihydrochloride is not a drug but a deuterium-labeled stable isotope internal standard. It has no approved therapeutic status, no clinical trial history as a therapeutic agent, and is not intended for human consumption. This compound is used for research applications including as an internal standard for GC-MS or LC-MS quantification of ADMA in biological samples, clinical biomarker studies for endothelial dysfunction and cardiovascular disease, studies of the arginine-NO pathway and NOS regulation, and metabolic flux analysis of arginine methylation and ADMA metabolism. Available with ≥99% deuterated purity (d1-d6, ≤1% d0) and ≥98% chemical purity.

C8H14D6CL2N4O2

281.21

280.134

1313730-20-9

Asymmetric dimethylarginine;30315-93-6;Asymmetric dimethylarginine dihydrochloride;220805-22-1

123831

White to off-white solid powder

195 - 197 °C

-3.6

3

4

6

14

215

1

CN(C)C(=NCCC[C@@H](C(=O)O)N)N

YDGMGEXADBMOMJ-LURJTMIESA-N

InChI=1S/C8H18N4O2/c1-12(2)8(10)11-5-3-4-6(9)7(13)14/h6H,3-5,9H2,1-2H3,(H2,10,11)(H,13,14)/t6-/m0/s1

(2S)-2-amino-5-[[amino(dimethylamino)methylidene]amino]pentanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

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.)