The primary molecular targets of CML are receptors for AGEs (RAGE) and scavenger receptors such as SR-A and CD36. CML-modified proteins (CML adducts) are ligands for RAGE. Binding of CML-adducts to RAGE activates the transcription factor NF-kappaB, leading to the production of pro-inflammatory cytokines (TNF-alpha, IL-6) and reactive oxygen species (ROS). This contributes to inflammation, vascular damage, and insulin resistance. CML itself (free form) is not a ligand; it must be attached to proteins to bind RAGE. RAGE is the primary receptor for AGEs. It is a marker of protein damage.

In vitro, CML is used as a standard to quantify AGE levels in biological samples. It is not an active drug. There is no IC50 value for CML against a specific enzyme. It is a chemical marker of glycation. In vitro, using ELISA or HPLC, researchers measure CML levels in serum or tissue homogenates. An elevated CML level is an indicator of chronic inflammation, metabolic syndrome, or aging. It is also used as an antigen in ELISA to measure anti-CML antibodies. The compound is a white solid. It is soluble in water. It is stable. It is a biochemical assay reagent. It is a major component of advanced glycation end products (AGEs).

CML has no direct in vivo therapeutic activity. It is a harmful molecule that accumulates with age and in disease states. In animal models, infusion of CML-modified proteins induces insulin resistance and vascular dysfunction. However, the free amino acid CML is largely inert. It is excreted in the urine. Its concentration in plasma increases in diabetic patients and is correlated with diabetic complications (retinopathy, nephropathy). It is used as a biomarker to evaluate the efficacy of anti-glycation drugs (e.g., aminoguanidine, pyridoxamine). It is a biomarker of oxidative stress and aging. It is an advanced glycation end product (AGE).

Non-cellular assays for CML are analytical rather than biological. The standard protocol for quantifying CML in a protein sample is by LC-MS/MS. The protein sample (e.g., plasma) is hydrolyzed in 6N HCl at 110degC for 24 hours to release free CML. A known amount of an internal standard (e.g., CML-d4 or CML-¹3C) is added. The hydrolysate is dried, reconstituted in 0.1% formic acid, and injected into an LC-MS/MS system. A C18 column is used. The eluent is detected by positive electrospray ionization (ESI+) with multiple reaction monitoring (MRM) for m/z 205 → 84 (CML) and 209 → 88 (internal standard). The calibration curve is prepared using CML standard. The concentration in the sample is calculated. This is the gold standard method.

Cell-based assays for CML are typically used to study the toxicity of CML-modified proteins. A standard protocol uses RAW 264.7 macrophages. Cells are seeded in 96-well plates. They are treated with bovine serum albumin (BSA) that has been pre-incubated with glucose to generate CML-BSA adducts (e.g., 50-500 ug/mL) for 24-48 hours. The free CML amino acid is not used; it is too polar to enter cells and is not recognized by RAGE. After incubation, the medium is collected to measure TNF-alpha and IL-6 by ELISA. Cell lysates are used to measure ROS production by DCFH-DA staining. CML-BSA induces a significant increase in cytokine secretion and ROS, mimicking the pro-inflammatory effect of AGEs. The free CML standard is used to spike into the medium to verify the specificity of the ELISA detection.

In vivo animal experiments using CML are typically conducted to test the effect of a drug on AGE formation. A standard protocol: Male Sprague-Dawley rats are made diabetic by injection of streptozotocin (STZ, 60 mg/kg). The diabetic rats are then treated with an anti-glycation agent (e.g., pyridoxamine, 100 mg/kg/day, oral) for 8 weeks. Control diabetic rats receive the vehicle. At the end of the study, rats are euthanized. Serum and kidney cortex samples are collected. The samples are hydrolyzed. CML levels are measured by LC-MS/MS as described above. A significant decrease in CML levels in the kidney tissue of the treated group indicates that the test compound prevented glycation. CML is a biomarker for drug efficacy.

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 usually excreted through urine or feces.

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The pharmacokinetics of CML is as follows: CML is a small polar molecule (MW 204.22, LogP -1.16). It is a zwitterion. It is not highly protein-bound. It is freely filtered by the glomerulus. Its plasma concentration in healthy individuals is very low (< 100 nM). In patients with renal insufficiency, CML accumulates due to reduced clearance. The half-life is prolonged in chronic kidney disease. It is not metabolized. It is excreted unchanged in urine. It is an endogenous compound, but dietary intake (e.g., from cooked meat, dairy) contributes to plasma levels. It is a uremic toxin. It is stable in serum. The pKa is around 2.9 and 9.7. It is water-soluble (35 mg/mL). It should be stored at -20degC.

Toxicity Summary
Uremic toxins, such as carboxymethyl lysine, 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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The toxicology of CML is not applicable as a pure compound. The modified proteins (AGEs) are pro-inflammatory and cytotoxic. They contribute to diabetic complications (nephropathy, neuropathy) and atherosclerosis. However, the free amino acid is considered safe to handle. It is not classified as a hazardous chemical. It may be an irritant. Standard safety precautions (gloves, lab coat) are recommended. It is not a drug. It is a biochemical reagent. It is a white powder. It is a natural product (formed in the body). It is an advanced glycation end product (AGE) receptor (RAGE) ligand (when conjugated to proteins). It is a marker for aging. It is a translation product of lysine glycation.

[1]. The advanced glycation end product, Nepsilon-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions. J Biol Chem. 1996 Apr 26;271(17):9982-6https://pubmed.ncbi.nlm.nih.gov/8626637/.

Nepsilon-(Carboxymethyl)lysine is an L-lysine derivative in which the N6 position is replaced by a carboxymethyl group. It has antigenic activity. It is an L-lysine derivative and also a non-protein L-α-amino acid.
N(6)-carboxymethyl lysine has been reported in both Sagittaria pygmaea and Saccharomyces cerevisiae, and relevant data are available.
N(6)-carboxymethyl lysine is a lysine derivative found in proteins in which the ε-nitrogen atom of the amino acid residue is modified by a carboxymethyl group. This advanced glycation end product (AGE) is elevated in certain diseases and is associated with aging.
Carboxymethyl lysine is a uremic toxin. Based on their chemical and physical properties, uremic toxins can be divided into three main categories: 1) small, water-soluble, non-protein-bound compounds, such as urea; 2) small molecule, lipid-soluble and/or protein-bound compounds, such as phenols; 3) larger so-called medium molecules, such as β2-microglobulins. Long-term exposure to uremic toxins can lead to various diseases, including kidney damage, chronic kidney disease, and cardiovascular disease. N(6)-Carboxymethyl lysine (CML), also known as N(ε)-(carboxymethyl)lysine, is an advanced glycation end product (AGE). CML has been the most commonly used AGE marker in food analysis. The reference (RN) given here refers to the (L)-isomer; the structure is described in the first reference.

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The significance of Nε-(Carboxymethyl)-L-lysine (CML) is that it is the most widely studied and validated AGE. It is a measure of the "glycation stress" on a patient's body. Unlike other AGEs (like pentosidine, which is fluorescent), CML is non-fluorescent, but it is chemically stable. It is the standard used in food science to monitor the Maillard reaction (browning of food), which produces CML. It is also used in nutritional research to assess the intake of processed foods. In the pharmaceutical industry, it is used as a positive control in screening assays for RAGE antagonists. It is not a drug. It is a reference standard for analytical chemistry. It is an immunogen. It is a protein post-translational modification (PTM). It is a lysine derivative.

C8H16N2O4

204.22

204.111

5746-04-3

CML-d3;2699607-49-1;CML-d4;936233-18-0

123800

White to off-white solid powder

1.3±0.1 g/cm3

428.9±45.0 °C at 760 mmHg

280ºC (dec.)

213.2±28.7 °C

0.0±2.2 mmHg at 25°C

1.518

-1.16

4

6

8

14

196

1

C(CCNCC(=O)O)C[C@@H](C(=O)O)N

NUXSIDPKKIEIMI-LURJTMIESA-N

InChI=1S/C8H16N2O4/c9-6(8(13)14)3-1-2-4-10-5-7(11)12/h6,10H,1-5,9H2,(H,11,12)(H,13,14)/t6-/m0/s1

(2S)-2-amino-6-(carboxymethylamino)hexanoic acid

CML; N6-(Carboxymethyl)-L-lysine; Nε-(1-Carboxymethyl)-L-lysine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

H2O : ~35 mg/mL (~171.38 mM; with ultrasonication)

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.)