System xc- (cystine/glutamate antiporter, SLC7A11/SLC3A2). [1][6]
Keap1 (Kelch-like ECH-associated protein 1). L-cystine treatment increases Nrf2 protein half-life from 19.4 min to 30.9 min. [6]
FliY-YecSC (ABC transporter, high affinity, Km = 110 nM). [5]
YdjN (low affinity symporter, Km = 1.1 µM). [5]

In hepatocellular carcinoma Huh6 and Huh7 cells, growth inhibition by system xc- inhibitors (sulfasalazine, erastin) was greater in medium with a physiological level of 83 µM L-cystine than in commercial medium with 200 µM. RSL3 (GPX4 inhibitor) showed no difference. GI50 for sulfasalazine in Huh7 cells was 209 µM in 83 µM L-cystine vs. 371 µM in 200 µM L-cystine. [1]
In HeLa cells, L-cystine (0.1-1.6 mM) induced a dose-dependent increase in Nrf2 protein, with 0.8 mM being most effective. Time-course studies showed Nrf2 induction by 30 min, peaking at 4 h. [6]
L-cystine (0.8 mM, 16 h) upregulated multiple Nrf2 downstream genes (NQO1, HMOX1, GCLC, GCLM, SRXN1, TXNRD1, AKR1C1, OSGIN1) as shown by RNA-seq and RT-PCR. OSGIN1 showed the highest induction. [6]
L-cystine activated ARE-luciferase reporters (both mGST-ARE and hNQO1-ARE) in a dose-dependent manner. [6]
L-cystine treatment (0.8 mM, 4 h) increased Nrf2 protein half-life from 19.4 min to 30.9 min and decreased Nrf2 ubiquitination. [6]
In E. coli, two L-cystine importers were identified: YdjN (low affinity, Km = 1.1 µM, Vmax = 14.7 pmol [10^9 cells]^-1 min^-1) and FliY-YecSC (high affinity, Km = 110 nM, Vmax = 6.41 pmol [10^9 cells]^-1 min^-1). A ΔydjNΔyecS mutant could not grow with L-cystine as sole sulfur source. [5]
L-cystine (0.8 mM, 16 h) reduced H2O2-induced ROS generation and protected against H2O2- or doxorubicin-induced apoptosis in HeLa cells, dependent on Nrf2 as shown by siRNA knockdown. [6]
In E. coli, deletion of fliY or ydeD increased lipid peroxidation (MDA levels 2.1-2.3 fold higher than wild-type) under H2O2 stress. [5]
L-cystine treatment did not change Nrf2 or Keap1 mRNA levels. [6]
L-cysteine was incapable of inducing Nrf2. [6]

In rats, after intravenous injection of L-[^35S]cystine, plasma radioactivity disappearance followed a biphasic curve with a fast t½ of 7 ± 1 min and a slow t½ of 104 ± 5 min. Peak cellular accumulation in kidney cortex (Distribution Ratio 14.2) was reached at 30 min. [4]
Intracellular products of L-[^35S]cystine metabolism in rat kidney cortex included cystine, cysteine, reduced glutathione, and an unidentified compound (possibly taurine, cysteinesulphinate, or cysteic acid). Only cystine was found in plasma. [4]
Ureter ligation did not significantly affect cellular accumulation of L-cystine metabolic products in rat kidney. Co-administration of L-lysine (450 µmol) with L-cystine enhanced cellular accumulation of ^35S-products and increased cystine clearance (fractional clearance 24.0 ± 0.7% at 15-30 min vs. 4.02 ± 0.52% without lysine). [4]
In a patient with cystinosis, kidney biopsy analysis by FTIR, OPTIR, and AFM-IR revealed the presence of both L-cystine (hexagonal morphology) and L-cysteine (rectangular morphology) crystals at the micrometer and nanometer scales. [3]
In E. coli, Hpx- ΔydeDΔfliY mutant cells (lacking the Cys/CySS shuttle system) showed accumulation of H2O2 in the periplasm (~11% of cells) when stained with a membrane-impermeable H2O2 probe. [5]

L-cystine uptake assay in E. coli: Cells grown to mid-exponential phase were harvested, washed with KPM solution (10 mM MgSO4, 0.1 M K2HPO4, pH 6.5), and resuspended to OD660 = 0.4. Cell suspension was energized with D-glucose for 10 min at 37°C. Uptake was initiated by adding indicated concentrations of L-[^14C]-cystine. After 10 min incubation at room temperature, cells were collected by filtration through GF/C filters, washed three times, and radioactivity was determined by liquid scintillation. The initial rate was proportional for at least 20 min. Kinetic parameters (Km, Vmax) were calculated using Hanes-Woolf plots. [5]
Periplasmic H2O2 detection: Cells were stained with 1 mM pentafluorobenzenesulfonyl 2',7'-difluorourorescin (BES-H2O2, a cell-impermeant fluorescent probe) for 30 min at room temperature. After washing, stained cells were viewed under fluorescence microscope (excitation 485 nm, emission 530 nm). Staining patterns were categorized as no staining, cytoplasmic staining, or periplasmic staining. [5]
Lipid peroxidation (TBARS) assay: Cells were harvested, and TBARS solution (0.01% butylated hydroxytoluene, 20% trichloroacetic acid, 0.65% thiobarbituric acid) was added. Samples were heated at 100°C for 25 min, cooled, centrifuged, and absorbance at 532 nm was measured. MDA equivalent concentration was calculated from a standard curve. [5]

Cell viability (CellTiter-Glo): Cells were seeded in 96-well plates (2,000 cells/well). After 24 h, medium was replaced with medium containing 200 µM or 83 µM L-cystine with indicated drugs. Cells were treated with sulfasalazine (72 h for Huh6, 24 h for Huh7), erastin (72 h), or RSL3 (72 h). Viability was measured using CellTiter-Glo assay. GI50 was defined as dose reducing viability by 50%. [1]
PI staining for membrane permeabilization: After treatment, cells were washed with PBS and incubated with 1 µg/mL propidium iodide for 30 min in the dark. Bright field and fluorescence images (ex/em 535/617 nm) were acquired. Percentage of PI-positive cells was calculated from three random images per group. [1]
Lipid peroxidation (C11-BODIPY): After treatment, 1 µM C11-BODIPY probe was added for 30 min at 37°C. Cells were collected by trypsinization, and fluorescence (ex/em 488/525 nm) was measured from 5,000 cells by flow cytometry. [1]
Nrf2 protein detection by Western blot: Cells were harvested in Laemmli sample buffer containing 5% 2-mercaptoethanol. Samples were boiled, sonicated, separated by SDS-PAGE, transferred to PVDF membranes, and incubated with anti-Nrf2, anti-Keap1, anti-HMOX1, anti-SRXN1, anti-TXNRD1, anti-AKR1C, anti-GAPDH, or anti-Ubiquitin antibodies, followed by HRP-conjugated secondary antibodies. [6]
ARE reporter assay: Cells were transfected with pGL4.37-mGST-ARE luc or pGL4.37-hNQO1-ARE luc vector together with pCMV Renilla luciferase. At 24 h post-transfection, cells were treated with L-cystine or SFN for 16 h. Firefly and Renilla luciferase activities were measured using Dual-Glo Luciferase Assay System. [6]
Ubiquitination assay: Cells treated with 0.8 mM L-cystine or 5 µM SFN along with 10 µM MG132 for 4 h. Cells were lysed in TBS buffer with 1% SDS. After boiling, lysates were diluted and pre-cleared with protein A/G beads. Nrf2 was immunoprecipitated with anti-Nrf2 antibody overnight, then captured with protein A/G beads. Immunoprecipitated proteins were eluted and subjected to Western blot for ubiquitin detection. [6]
ROS detection (DCFDA): Cells were pre-treated with 0.8 mM L-cystine for 16 h, then incubated with 10 µM DCFDA for 30 min in the dark. H2O2 was added, and DCF fluorescence (ex/em 485/535 nm) was measured by plate reader. [6]
Caspase-3/7 activity assay: Cells were lysed in buffer (0.5% Nonidet P-40, 0.5 mM EDTA, 150 mM NaCl, 50 mM Tris, pH 7.5). Lysates were incubated with 80 µM Ac-DEVD-AMC substrate for 1 h at 37°C. Fluorescence intensity (ex/em 365/450 nm) was measured. [6]
MTT assay for cell viability: Cells were treated with L-cystine for 8 h then with doxorubicin for 24 h. MTT (2 mg/mL) was added for 2 h. Formazan was dissolved in acidified isopropanol, and absorbance at 570 nm was measured. [6]
siRNA transfection: Cells were transfected with control siRNA or Nrf2 siRNA using Lipofectamine 3000. After 6 h, DMEM with 20% FBS was added for 18 h, then replaced with fresh growth medium for 48 h before L-cystine treatment. [6]
Keap1 knockout by CRISPR-Cas9: HeLa cells were transfected with Keap1 CRISPR/Cas9 KO plasmid. GFP-positive cells were sorted by flow cytometry and seeded at single-cell density for colony formation. Keap1 knockout was validated by Western blot. Five clones (KO1-KO5) were tested for L-cystine response. [6]
Real-time RT-PCR: Total RNA (1 µg) was converted to cDNA. qPCR was performed with SYBR Green Master Mix using specific primers for Nrf2, Keap1, NQO1, HMOX1, GCLM, TXNRD1, SRXN1, AKR1C1, OSGIN1, GAPDH, or 18S rRNA. mRNA abundance was calculated using the ∆CT method. [6]

Rat model for L-cystine accumulation: Adult male Sprague-Dawley rats (150-250 g) were injected intravenously with L-[^35S]cystine (1.6 mM, 1.14 µCi per rat). At specified times (0-90 min), rats were stunned and decapitated. Blood was collected, and kidneys were removed. Kidneys were cut in half and sectioned serially with a Stadie-Riggs microtome. Four slices from outer pole to outer medulla were obtained. Intracellular and extracellular radioactivity was assessed. Distribution ratio (counts/min per mL intracellular fluid / counts/min per mL plasma) was calculated. [4]
Ureter ligation: Under anesthesia, left ureters were tied below the renal pelvis. After 1 h (or 10 min), rats were injected with L-[^35S]cystine. Tissue was taken 45 min (or 10 min) later. [4]
Clearance studies: Rats were anesthetized with Inactin. Tracheotomy, bladder, and jugular vein catheterization were performed. 10% mannitol in 0.9% NaCl was infused. [methoxy-^3H]inulin was administered as primer and sustaining infusion. After control urine samples, rats received IV injection of L-[^35S]cystine (1.6 mM, 8.0 µCi) with or without L-lysine (450 µmol). Three 15-min urine collections were obtained with midpoint blood samples. Fractional clearance (Cystine/Cinulin) was calculated. [4]

After IV injection in rats, plasma L-[^35S]cystine radioactivity decreased with a fast half-life of 7 ± 1 min and a slow half-life of 104 ± 5 min. [4]
The only ^35S-labeled compound found in rat plasma after L-[^35S]cystine injection was cystine. [4]

In HeLa cells, L-cystine at concentrations up to 1.6 mM induced Nrf2 without apparent toxicity. [6]
In E. coli, deletion of L-cystine transporter genes (ydjN, yecS, fliY) did not affect growth under normal conditions but increased sensitivity to H2O2 stress (prolonged lag-phase). [5]
L-cystine is considered a non-toxic amino acid derivative suitable for cytoprotection without safety concerns. [6]

[1]. Extracellular Concentration of L-Cystine Determines the Sensitivity to System xc- Inhibitors. Biomol Ther (Seoul). 2022 Mar 1;30(2):184-190.

[2]. Enhancement of antigen-specific immunoglobulin G production in mice by co-administration of L-cystine and L-theanine. J Vet Med Sci. 2007 Dec;69(12):1263-70.

[3]. Cystinuria and cystinosis are usually related to L-cystine: is this really the case for cystinosis? A physicochemical investigation at micrometre and nanometre scale. Comptes Rendus. Chimie, Volume 25 (2022) no. S1, pp. 489-502.

[4]. Cellular accumulation of L-cystine in rat kidney cortex in vivo. J Clin Invest. 1973 Feb;52(2):454-62.

[5]. Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli. PLoS One. 2015 Apr 2;10(3):e0120619.

[6]. Fresh Medium or L-Cystine as an Effective Nrf2 Inducer for Cytoprotection in Cell Culture. Cells. 2023 Jan 12;12(2):291.

In cystinuria (autosomal recessive disorder), L-cystine crystallites exhibit hexagonal morphology consistent with its crystallographic structure (hexagonal, P6₁22 space group). [3]
In cystinosis (lysosomal storage disorder), abnormal deposits were previously thought to be pure L-cystine but show rectangular crystal morphology. Physicochemical investigation at nanometre scale (OPTIR, AFM-IR) revealed both L-cystine and L-cysteine are present, suggesting cystinosis is related to rectangular crystals of L-cysteine which undergo phase transition to L-cystine. [3]
In E. coli, the Cys/CySS shuttle system (YdeD exporter and FliY-YecSC importer) provides reducing equivalents to the periplasm to scavenge H2O2, protecting membrane lipids from peroxidation. Both importers are induced by H2O2: ydeD (3.5-fold), fliY (2.5-fold), yecS and yecC (2-fold). [5]
L-cystine enters cells via the cystine/glutamate antiporter (SLC7A11). This uptake is blocked by erastin and sulfasalazine. Once inside, it is reduced to L-cysteine, which is used for GSH synthesis or protein production. [5][6]
Fresh DMEM (containing 0.2 mM L-cystine) induces Nrf2 protein in HeLa, HEK293, AC16, and MCF7 cells. This effect is reproduced by L-cystine alone but not by L-cysteine, glucose, or glutamine. [6]

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L-cysteine is the L-enantiomer of the sulfur-containing amino acid cysteine. It is used as a flour treatment agent, a human metabolite, a Saccharomyces cerevisiae metabolite, a mouse metabolite, and an inhibitor of EC 1.2.1.11 (aspartate semialdehyde dehydrogenase). It is a cysteine derivative of L-cysteine and a non-protein L-α-amino acid. It is the conjugate acid of the L-cysteine anion. It is the enantiomer of D-cysteine. It is the tautomer of the L-cysteine zwitterion. It is a covalently linked dimer, a non-essential amino acid formed by the oxidation of cysteine. Two cysteine molecules are linked by a disulfide bond to form cysteine. L-cysteine is present in or produced by Escherichia coli (K12 strain, MG1655 strain). L-cysteine has been reported in Spanish sage, soybean, and other organisms with relevant data. Cystine is not one of the 20 essential amino acids. Cystine is a sulfur-containing derivative formed by the oxidation of the thiol side chain of the cysteine amino acid. It has antioxidant properties, protecting tissues from radiation and pollution damage and slowing the aging process. It also contributes to protein synthesis. Cystine is abundant in many proteins in bone tissue and skin, and is also found in insulin and digestive enzymes such as chromaffinogen A, papain, and trypsinogen. (NCI04)
A covalently linked dimer, non-essential amino acid formed by the oxidation of cysteine. Two cysteine molecules are linked by a disulfide bond to form cystine.

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Drug Indications
L-cysteine is said to have anti-inflammatory properties, can combat various toxins, and may help treat osteoarthritis and rheumatoid arthritis. More research is needed before L-cysteine is approved for the treatment of any of these diseases. Research to date has primarily focused on animal models.

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Mechanism of Action
In certain situations, such as acetaminophen overdose, glutathione in the liver is depleted, subjecting tissues to oxidative stress and leading to loss of cellular integrity. L-Cysteine is a major precursor in the synthesis of glutathione.

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Pharmacodynamics
L-Cysteine is a covalently linked dimer, non-essential amino acid formed by the oxidation of cysteine. Two cysteine molecules are linked by a disulfide bond to form cystine. Cystine is a naturally occurring chemical in urine; when deposited in the kidneys, it can form stones (hard mineral deposits). Cystine is a compound formed by two cysteine molecules linked by a disulfide bond. Cystine is essential for the proper utilization of vitamin B6, and it also helps in the healing of burns and wounds, and can break down mucus deposits in diseases such as bronchitis and cystic fibrosis. Cystine also helps in the supply of insulin to the pancreas, which is essential for the absorption of carbohydrates and starches. It can increase glutathione levels in the lungs, liver, kidneys, and bone marrow, which may have an anti-aging effect on the body by reducing age spots, among other things.

C6H12N2O4S2

240.29

240.023

C, 29.99; H, 5.03; N, 11.66; O, 26.63; S, 26.68

56-89-3

L-Cystine-d4;1192736-38-1;L-Cystine-34S2;113512-08-6;(S)-L-Cystine-15N2;L-Cystine-3,3'-13C2;2483736-13-4

67678

White to off-white solid powder

1.6±0.1 g/cm3

468.2±45.0 °C at 760 mmHg

260-261ºC

237.0±28.7 °C

0.0±2.5 mmHg at 25°C

1.653

1.23

4

8

7

14

192

2

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

LEVWYRKDKASIDU-IMJSIDKUSA-N

InChI=1S/C6H12N2O4S2/c7-3(5(9)10)1-13-14-2-4(8)6(11)12/h3-4H,1-2,7-8H2,(H,9,10)(H,11,12)/t3-,4-/m0/s1

(2R)-2-amino-3-[[(2R)-2-amino-2-carboxyethyl]disulfanyl]propanoic acid

NSC-13203; L-cystine; 56-89-3; L-Dicysteine; beta,beta'-Dithiodialanine; NSC 13203; Cystine

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)

0.1 M NaOH : ~3.33 mg/mL (~13.86 mM)
0.1 M HCL : 2.5 mg/mL (~10.40 mM)
DMSO :< 1 mg/mL
H2O : < 0.1 mg/mL

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.)