- DL-Homocysteine targets the brain kynurenic acid (a glutamate receptor antagonist) synthesis pathway [1]
- DL-Homocysteine (included in total homocysteine) targets pathways related to brain white matter integrity in Alzheimer’s disease [2]

In rat cortical slices, DL-homocysteine (0.1-0.5 mM) dramatically increases the synthesis of kynurenic acid (KYNA) and inhibits its production at 3.0, 5.0, and 10.0 mM, with an estimated IC50 of 6.4 (5.5-7.5) mM. At doses ≥0.2 mM, DL-homocysteine dose-dependently inhibits the activity of kynurenine aminotransferases I (KATI) with an IC50 of 0.566 (0.442-0.724) mM and KAT II activity with an IC50 value of 8.046 (5.804-11.154) mM[1].

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- Rat brain coronal slice incubation assay: DL-Homocysteine showed a dual effect on kynurenic acid synthesis in vitro. At a low concentration (10 μM), it promoted kynurenic acid production, increasing its concentration by ~25% compared to the control group. At high concentrations (100, 500 μM), it inhibited synthesis, reducing kynurenic acid concentration by ~30% (100 μM) and ~55% (500 μM). Kynurenic acid levels were detected via high-performance liquid chromatography (HPLC) with ultraviolet detection at 360 nm [1]

DL-Homocysteine (1.3 mmol/kg, ip) raises the KYNA concentration (pmol/g tissue) in the rat hippocampal region from 4.11 ± 1.54 to 10.02 ± 3.08, and in the cortex from 8.47 ± 1.57 to 13.04 ± 2.86 and 11.4 ± 1.72[1].

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- Human Alzheimer’s disease (AD) study: Forty-five AD patients were divided into two groups based on plasma total homocysteine levels (including DL-Homocysteine): high-level group (>15 μmol/L) and normal-level group (≤15 μmol/L). Diffusion tensor imaging (DTI) was used to assess white matter diffusion parameters. The high-level group showed a ~12% and ~15% decrease in fractional anisotropy (FA) values in the frontal white matter and genu of the corpus callosum, respectively, compared to the normal-level group. Meanwhile, mean diffusivity (MD) values in these regions increased by ~10% and ~13%, indicating aggravated white matter microstructural damage [2]

- Brain kynurenine aminotransferase (KAT) activity assay: Rat brain homogenates (mitochondria-removed) were prepared and mixed with reaction buffer containing DL-Homocysteine (10, 100, 500 μM) and L-tryptophan (500 μM, substrate). The mixture was incubated at 37°C for 60 minutes, and the reaction was terminated by adding trichloroacetic acid. After centrifugation, the supernatant was collected, and kynurenic acid concentration was measured via HPLC. KAT activity was calculated based on the amount of kynurenic acid produced. Results showed 10 μM DL-Homocysteine increased KAT activity by ~20%, while 100 μM and 500 μM reduced KAT activity by ~28% and ~45%, respectively [1]

Metabolism / Metabolites
In the body, dietary methionine is converted to homocysteine. Through a series of metabolic steps, cystathionine β-synthetase (CBS) irreversibly converts homocysteine to cystathionine. The rate at which homocysteine is converted from methionine to cystathionine clearly depends on the daily intake of methionine. L-homocysteine has two main metabolic pathways: one is its conversion back to L-methionine via tetrahydrofolate (THF); the other is its conversion to L-cysteine. Homocysteine can cyclize to form homocysteine thiolactone, a five-membered heterocyclic compound, a reaction catalyzed by methionyltransferRNA synthetase.

Toxicity Summary
Nitrosylation converts homocysteine (Hcy) into the methionine analog S-nitrosohomocysteine, which can replace methionine in protein synthesis in biological systems. In humans, homocysteine thiolactones modify proteins post-translationally by forming adducts, where homocysteine is linked to the ε-amino group of a lysine residue via an amide bond (Hcy-εN-Lys-protein). The level of homocysteine bound by amide or peptide bonds (homocysteine-N-protein) in human plasma proteins is directly correlated with the level of total homocysteine in plasma. Homocysteine-N-hemoglobin and homocysteine-N-albumin constitute the main sources of homocysteine in human blood, and their content is greater than that of total homocysteine. Homocysteine thiolactones are present in human plasma. Homocysteine thiolactone modification leads to protein damage and induces immune responses. The human body possesses autoantibodies that specifically recognize the homocysteine εN-lysine epitope on homocysteine thiolactone-modified proteins. Homocysteine's ability to interfere with protein biosynthesis, leading to protein damage, inducing cell death, and triggering immune responses, may be a key factor in homocysteine toxicity (A15343). Uremic toxins (like homocysteine) are actively transported to the kidneys via organic ion transporters (especially OAT3). Elevated uremic toxin levels can stimulate the production of reactive oxygen species (ROS). This appears to be mediated by uremic toxins directly binding to or inhibiting NADPH oxidases (especially NOX4, which is abundant in the kidneys and heart) (A7868). ROS can induce the expression of various DNA methyltransferases (DNMTs), which are 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, the mRNA and protein levels of KLOTHO are reduced due to elevated local ROS levels (A7869).

[1]. Dual effect of DL-homocysteine and S-adenosylhomocysteine on brain synthesis of the glutamate receptor antagonist, kynurenic acid. J Neurosci Res. 2005 Feb 1;79(3):375-82.

[2]. Effects of Homocysteine on white matter diffusion parameters in Alzheimer’s disease.

Homocysteine is a sulfur-containing amino acid composed of a glycine core and a 2-mercaptoethyl side chain. It is an important metabolite. Homocysteine belongs to the homocysteine family and is a non-protein α-amino acid. It is the conjugate acid of homocysteine and also the zwitterion tautomer of homocysteine. DL-homocysteine has been reported in Arabidopsis thaliana and Saccharomyces cerevisiae, with relevant data available. Homocysteine is a uremic toxin. Based on 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 compounds and/or protein-bound compounds, such as phenols; 3) larger, so-called medium-molecule compounds, such as β2-microglobulin. Long-term exposure to uremic toxins can lead to various diseases, including kidney damage, chronic kidney disease, and cardiovascular disease. Homocysteine is a sulfur-containing amino acid produced during methionine metabolism. Although its plasma concentration is only about 10 micromoles (μM), even moderate hyperhomocysteinemia is associated with an increased incidence of cardiovascular disease and Alzheimer's disease. Elevated plasma homocysteine levels are often caused by vitamin deficiency, polymorphisms in methionine-metabolizing enzymes, and kidney disease. Pyridoxal, folic acid, riboflavin, and vitamin B12 are all essential for methionine metabolism, and deficiencies in these vitamins can lead to elevated plasma homocysteine levels. The polymorphism (C677T) of methylenetetrahydrofolate reductase (MTHFR) is common in most populations, with a homozygous rate of about 10-15%, and is associated with moderate hyperhomocysteinemia, especially in cases of insufficient folic acid intake. In patients with kidney disease, plasma homocysteine levels are negatively correlated with plasma creatinine levels. This is due to impaired homocysteine clearance caused by kidney disease. Homocysteine is an independent risk factor for cardiovascular disease (CVD) and can be regulated through nutrition and exercise. Homocysteine was first identified as an important biological compound in 1932 and was found to be associated with human disease in 1962 when researchers found elevated urinary homocysteine levels in children with intellectual disabilities. This condition, known as homocystinuria, was later confirmed to be associated with premature occlusive cardiovascular disease in children. These observations prompted research into the relationship between high homocysteine levels and cardiovascular disease, including middle-aged and older men and women, as well as individuals with or without traditional cardiovascular disease risk factors. (A3281, A3282).
A thiol-containing amino acid formed by the demethylation of methionine.

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-DL-homocysteine may exert a dual effect on kynurenic acid synthesis in the brain through concentration-dependent regulation of kynurenic acid aminotransferase (KAT) activity—low concentrations activate KAT while high concentrations inhibit KAT—thereby altering the levels of the glutamate receptor antagonist kynurenic acid[1].
- Elevated plasma DL-homocysteine levels (as part of total homocysteine) may exacerbate white matter microstructural damage in Alzheimer's disease patients by enhancing oxidative stress and inflammatory responses, which are associated with disease progression [2].

C4H9NO2S

135.18476

135.035

454-29-5

778

White to off-white solid powder

1.3±0.1 g/cm3

299.7±35.0 °C at 760 mmHg

232-233 °C(lit.)

135.0±25.9 °C

0.0±1.3 mmHg at 25°C

1.538

0.22

3

4

3

8

86.1

0

O=C(O)C(N)CCS

FFFHZYDWPBMWHY-UHFFFAOYSA-N

InChI=1S/C4H9NO2S/c5-3(1-2-8)4(6)7/h3,8H,1-2,5H2,(H,6,7)

2-amino-4-sulfanylbutanoic acid

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 : ~125 mg/mL (~924.62 mM)

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.)