Endogenous Metabolite

Background: New strategies are needed to combat multidrug-resistant bacteria. The restriction of iron uptake by bacteria is a promising way to inhibit their growth. We aimed to suppress the growth of Vibrio bacterial species by inhibiting their ferric ion-binding protein (FbpA) using food components. Methods: Twenty spices were selected for the screening of FbpA inhibitors. The candidate was applied to antibacterial tests, and the mechanism was further studied. Results: An active compound, rosmarinic acid (RA), was screened out. RA binds competitively and more tightly than Fe3+ to VmFbpA, the FbpA from V. metschnikovii, with apparent KD values of 8 μM vs. 17 μM. Moreover, RA can inhibit the growth of V. metschnikovii to one-third of the control at 1000 μM. Interestingly, Sodium citrate (SC) enhances the growth inhibition effect of RA, although SC only does not inhibit the growth. The combination of RA/SC completely inhibits the growth of not only V. metschnikovii at 100/100 μM but also the vibriosis-causative pathogens V. vulnificus and V. parahaemolyticus, at 100/100 and 1000/100 μM, respectively. However, RA/SC does not affect the growth of Escherichia coli. Conclusions: RA/SC is a potential bacteriostatic agent against Vibrio species while causing little damage to indigenous gastrointestinal bacteria. [1]

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Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and Sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. [2]

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The inhibitory effect of BP and Sodium citrate/SCT on proliferation of MGC-803 cells [2]
To evaluate the effect of BP and SCT on cell proliferation, we investigated the viability of MGC-803 cells after treatment of a range of concentrations of BP and SCT for 24 h and 48 h respectively by MTT assay. As showed in Fig. 1, the viability of MGC-803 cells was markedly decreased after exposure to BP and SCT in a dose- and time-dependent manner. The IC50 are 5.73 ± 0.75 μg/ml and 10.08 ± 0.87 mM after 48 h of exposure to BP and SCT respectively. In addition, direct observation of cells morphology with an inverted microscope revealed that numerous morphological changes occurred in cells after treatment with BP and SCT. In particular, cell shrinkage, condensation of cytoplasm and chromosomal condensation in a dose-dependent manner after the treatment of BP and SCT for 48 h.

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Promotion of apoptosis by BP and Sodium citrate/SCT in MGC-803 cells [2]
The effect of BP and SCT on cell viability in MGC-803 cells were analyzed by the method of Annexin V-PE and 7-ADD double staining at the same time. The results in Fig. 2A and C showed that both BP and SCT induced apoptosis in MGC-803 cells in a dose- and time-dependent manner. However, 5-FU also promoted MGC-803 cells to apoptosis remarkably.

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BP and SCT/Sodium citrate caused MGC-803 cells cycle arrest at G2/M phase [2]
The cell cycle plays key roles in cancer cell proliferation. The cell cycle repartition was analyzed with PI staining. As showed in Fig. 2B and D, more cells treated with BP and SCT were arrested at G2/M phase compared with control group, while cell numbers at G1 phase were decreased. The results presented concentration dependent. Meanwhile, 5-FU had the same effect on the cell cycle as BP and SCT.

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BP and SCT/Sodium citrate reduced intracellular ATP level of MGC-803 cells [2]
Intracellular ATP level was considered the key role in growth and proliferation of malignant tumor cells. To detect intracellular ATP content, MGC-803 cells were incubated with different concentrations of BP and SCT for 4 h and 8 h. As presented in Fig. 3A, both BP and SCT could prominently suppress the intracellular ATP generation in a dose- and time-dependent manner. In addition, 5-FU could also reduce ATP production in MGC-803 cells.

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BP and SCT/Sodium citrate suppressed lactate production of MGC-803 cells [2]
In order to study whether BP and SCT regulate the glycolytic metabolism in MGC-803 cells, we measured the generation of lactate in MGC-803 cells after treatment with BP and SCT for 24 h and 48 h respectively. As presented in Fig. 3B, the intracellular lactate production was significantly decreased in BP- and SCT-treated cells comparatively with untreated cells, and the results displayed dose- and time-dependent. However, no differences were perceived on the intracellular concentration of lactate between 5-FU group and control group.

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The effect of BP and SCT/Sodium citrate on the activity of glycolytic key enzymes [2]
To investigate the effect of BP and SCT on glycolytic metabolism, the activity of HK and PFK was taken into account. As showed in Fig. 3C and D, after incubated with BP for 24 h and 48 h, the activity of HK was down regulated in a dose- and time-dependent manner. However, no differences were observed on the HK activity in SCT- and 5-FU-treated groups compared with control group. In addition, the PFK activity was concentration- and time-dependently suppressed by SCT. Interestingly, BP and 5-FU had no distinct effect on the PFK activity in MGC-803 cells.

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The effect of BP and SCT/Sodium citrate on the mRNA and protein expressions of apoptosis related genes [2]
To study the effect of BP and SCT on apoptosis regulatory genes, the relative expression revel of anti-apoptotic Bcl-2 and Survivin, and pro-apoptotic Bax, caspase-3 and Cyt-c were taken into analysis. MGC-803 cells were exposed to BP and SCT for 24 h, then the expression level of mRNA and proteins of apoptosis related genes were detected with qPCR and western blot respectively. As showed in Fig. 4, both BP and SCT statistically decreased the mRNA and protein expressions of anti-apoptotic Bcl-2 and Survivin, whereas, increased the mRNA and protein expression level of pro-apoptotic Bax, caspase-3 and Cyt-c. The regulating effect of BP and SCT on the apoptosis related genes was in a dose-dependent manner. Meanwhile, 5-FU had also the same effect on these apoptosis related genes as BP and SCT.

The increased concentrations of blood urea nitrogen and serum creatinine in the model group were significantly decreased by Sodium citrate treatment. Hematoxylin-eosin and Masson staining showed that sodium citrate treatment apparently improved renal pathological changes in CRF rats. Western blot analysis showed that sodium citrate treatment decreased the protein levels of transforming growth factor-beta 1 and collagen type IV, which were increased in model rats. Moreover, immunohistochemical staining demonstrated that sodium citrate could effectively reduce the protein expression of glucose-regulated protein 78 kDa and CCAAT/enhancer-binding protein homologous protein in the model rats, which was consistent with western blot results. Additionally, the high dose of sodium citrate had a stronger protective effect in CRF rats than the low dose of sodium citrate.[3]

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Effects of Sodium citrate on Blood Biochemical Indexes [3]
At the 6, 8, 12, and 16 weeks of the experiment, blood was collected and the concentrations of BUN, Scr calcium, potassium, and phosphorus were examined in the different groups. The BUN levels in the model group were significantly increased as compared to the levels in the control group during experiment (p < 0.05). In addition, there was no significant difference between control group and sodium citrate control group (p > 0.05). Moreover, BUN levels were significantly decreased in the low- and high-dose sodium citrate-treated groups compared with the levels in the model group (p < 0.05). Additionally, a significant difference was observed between the low- and high-dose groups at 12 and 16 weeks of the experiment. Except in the normal control group, BUN levels peaked at 8 or 12 weeks during the experiment and then decreased to the lowest level at 16 weeks (fig. 1A). Regarding the Scr levels, the changes were consistent with BUN levels. They were increased in the model group compared with the normal group, and decreased in the sodium citrate-treated groups compared to the levels in the model group. Scr levels peaked at 8 weeks during the experiment except in the control group and then dropped to the lowest level at 16 weeks (fig. 1B). For serum potassium (online suppl. table S1; for all online suppl. material, see www.karger.com/doi/10.1159/000437235), calcium (online suppl. table S2), and phosphorus (online suppl. table S3) concentrations, there were no significant differences among the 5 groups (p > 0.05).

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Effect of Sodium citrate on Renal Pathological Changes [3]
According to the blood biochemical indexes results, there was no significant difference observed between the control group and sodium citrate control group. Thus, we eliminate sodium citrate group in further research. At 8 weeks of the experiment, renal damage was the most serious. Therefore, we chose the results at 8 and 16 weeks to observe the effect of citrate on adenine-induced CRF in rats. The rat kidneys at 8 weeks were inspected visually. In the control group, the kidneys were reddish-brown with luster, the capsule was easy to exfoliate, and had a smooth surface without swelling. As for the model group, the kidneys were pale and had dark spots on the surface and were of a larger size compared to those extracted from the control group. The kidneys had an unclear boundary between the cortex and medulla and were difficult to exfoliate (fig. 2A).

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Effect of Sodium citrate on the Protein Levels of TGF-β1 and CIV [3]
Renal injury was most serious after 8 weeks of the experiment; so we used tissue samples collected at the 8 week timepoint to assess the protein expression of TGF-β1 and CIV. The expression of renal fibrosis-related proteins TGF-β1 (fig. 3A) and CIV (fig. 3B) was significantly increased in the model group as compared with that of the control group, which was decreased by sodium citrate treatment. Moreover, the high-dose treated group showed a greater inhibitory effect on the expression of TGF-β1 and CIV than the low-dose treated group.

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Sodium citrate Regulates GRP78 and CHOP in the Adenine-Induced CRF Rat Model [3]
The GRP78 immunohistochemistry results showed that, compared with the control group, GRP78 protein expression was significantly increased in the model group. In the sodium citrate-treated groups, GRP78 protein expression was increased compared to that in the model group. After 8 weeks of the experiment, GRP78 protein expression was the highest in groups besides the control group when renal dysfunction was at its worst (fig. 4A and C). The CHOP immunohistochemistry results showed that, compared with the control group, CHOP protein expression was significantly increased in the model group, whereas it decreased in the citrate-treated groups compared with the model group. From 6 to 16 weeks of the experiment, CHOP expression levels increased except for the control group (fig. 4B and D). Furthermore, we detected the expression level of GRP78, PERK, ATF6, and CHOP at the 8-week using western blot method (fig. 4E and F). The results showed that the expression level of GRP78 was significantly increased in model group compared with that of the control group, and elevated with sodium citrate treatment. The expression of PERK, ATF6, and CHOP was apparently increased in model group compared with that of the control group, while it significantly decreased with sodium citrate treatment.

MTT assay [2]
Cell viability was assessed using MTT assay. Approximately 3 × 103 cells were seeded into each well of a 96-well culture plate and incubated for 24 h. Complete medium was used to dilute the drug to the desired concentrations for (1, 2, 3, 4, 5, 6, 8, 10 and 12 μg/ml) BP and (1.25, 2.5, 5, 10, 15, 20, 30, 40 and 60 mM) Sodium citrate/SCT, and the corresponding drug-containing medium was added to each well. Incubation in a CO2 incubator was allowed for another 24 h and 48 h respectively. 30 μl of MTT solution was added and followed by incubation for 4 h. Then supernatant aspiration and DMSO addition (150 μl/well) were done, and the OD value was measured at 490 nm with microplate reader.

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Flow cytometer analysis [2]
MGC-803 cells in the logarithmic growth phase were trypsinized and seeded into culture flasks. The corresponding drug-containing medium was added (4, 6, 8 μg/ml of BP; 5, 10, 20 mM of Sodium citrate/SCT and 0.5 mM of 5-FU) after cells were attached to the flasks for 24 h respectively, and negative control group was included at the same time. For cell cycle analysis, after incubation for 24 h, the cells were harvested and washed twice with PBS, and fixed in cold 70% ethanol overnight at 4 °C. After RNase A (0.2 mg/ml) digestion for 30 min at 37 °C, cells were stained with PI (50 μg/ml) for 30 min at 4 °C in dark. For apoptosis analysis, cells were collected after treatment for 24 h and 48 h, and washed twice with cold PBS. Then the cells were resuspended in 100 μl of binding buffer, PE and 7-ADD was added respectively and mixed well. The reaction was performed at room temperature for 30 min in dark. Another 400 μl 0f binding buffer was added after staining. Finally, a flow cytometer was used to detect the cell cycle repartition and apoptosis.

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Determination of the content of lactate and ATP and the activity of glycolytic enzymes [2]
MGC-803 cells were incubated with corresponding drugs (4, 6, 8 μg/ml of BP; 5, 10, 20 mM of Sodium citrate/SCT and 0.5 mM of 5-FU) respectively. For the detection of lactate and the activity of HK and PFK, the incubation was for 24 h and 48 h. And for the detection of ATP content, the cells were incubated for 4 h and 8 h. After incubation, cells were collected and washed twice with cold PBS. The content of lactate and ATP and the activity of HK and PFK were measured with Assay Kits respectively. And the detection was performed according to the manufacturer’s instructions.

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Reverse transcriptase-polymerase chain reaction assay [2]
MGC-803 cells were harvested after treatment with corresponding drugs (4, 6, 8 μg/ml of BP; 5, 10, 20 mM of Sodium citrate/SCT and 0.5 mM of 5-FU), and negative control group was included at the same time. Total RNA was prepared with an RNeasy kit and reverse-transcribed using PrimeScript® RT reagent Kit. The primers of Bcl-2, Bax, Survivin, Cyt-C, Casepase-3 and GAPDH were synthesized by xxx (see Table 1). The reactions were conformed by SYBR Green PCR Master Mix. Real-time PCR assays were performed using Applied Biosystems® 7500 Real-Time PCR Systems according to the manufacturer’s instructions. Real-time PCR data were quantified using the 2−△△Ct method.

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Western blot analysis [2]
MGC-803 cells were incubated with BP, Sodium citrate/SCT and 5-FU for 24 h respectively and washed twice with cold PBS after collection, then lysed in cell lysis reagent. The reaction was performed on ice for 20 min, then the lysate was centrifugated at 4 °C 12,000 rpm for 30 min. The supernatant was saved, and the protein concentration of samples was measured with BCA method. The proteins were resolved on a 15% SDS-PAGE gel and transferred to a nitrocellulose membrane. Then the membrane was blocked overnight with 5% non-flat milk, and the primary antibody (1:200) was added to incubated overnight at 4 °C. After incubation of primary antibody, the membrane was washed three times for 10 min with PBST, and then incubated with the secondary antibody (1:4000) for 1 h. Finally, the gray scale values of the protein bands were determined.

Establishment of the Animal Model [3]
A total of 48 healthy male Sprague-Dawley rats, weighting 180-250 g, were reared in a temperature-controlled room (22-24°C) with a 12-12 h light-dark cycle. Before the experiment, all rats were housed with food and water freely available for 3 days to adapt the environment. Animals were randomly divided into 5 groups: the control group, normal rats; the Sodium citrate control group, normal rats with high dose treatment of sodium citrate; the model group, rats with renal failure; the low-dose group, model rats with low dose treatment of sodium citrate; and the high-dose group, model rats with high dose treatment of sodium citrate. Rats in the control group and sodium citrate control group received distilled water at 10 ml/kg/day by gavage during the experiment. In the other three groups, a 0.75% adenine suspension was given at a dose of 5.0 ml/kg/day by gavage every morning to induce CRF. For sodium citrate administration, rats were treated with either a low dose (216 mg/kg) or a high dose (746 mg/kg) of Sodium citrate by gavage. The rats were euthanized after 6, 8, 12, and 16 weeks, and blood and renal tissues were collected for analysis.

Exposure Routes
The substance can be absorbed into the body by inhalation of its aerosol and by ingestion.

[1]. Rosmarinic Acid and Sodium Citrate Have a Synergistic Bacteriostatic Effect against Vibrio Species by Inhibiting Iron Uptake. Int J Mol Sci. 2021 Dec 1;22(23):13010.

[2]. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway. Biochem Biophys Res Commun. 2016 Jun 17;475(1):37-43.

[3]. Sodium Citrate Inhibits Endoplasmic Reticulum Stress in Rats with Adenine-Induced Chronic Renal Failure. Am J Nephrol. 2015;42(1):14-21.

Sodium citrate dihydrate is the dihydrate of trisodium citrate. It has a role as an anticoagulant. It contains a sodium citrate.
Sodium salts of citric acid that are used as buffers and food preservatives. They are used medically as anticoagulants in stored blood, and for urine alkalization in the prevention of KIDNEY STONES.
See also: ... View More ...

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Interestingly, E. coli responds to iron restriction by producing citrate and has an Fec(CD)E/A transporter system for ferric citrate uptake, which is absent in V. metschnikovii (Table 1). Furthermore, FecB, the periplasmic subunit of the Fec(CD)E/B system, binds a variety of Fe-citrate complexes (citrate, Fe3+-Cit, [Fe2(Cit2)]2-, and [Fe(Cit)2]5-) as well as other citrate complexes (Ga3+, Al3+, Sc3+, In3+, and Mg2+) with a Kd value in the micromolar range. When FbpAs in E. coli and V. metschnikovii were inhibited by 1000 μM RA, Fe3+ was reduced to Fe2+. Subsequently, adding citrate chelates free Fe2+ into Fe2+-citrate complexes. These Fe2+-citrate complexes can be taken up by E. coli via the Fec(CD)E/B transporter system, whereas the Fe2+-citrate complexes are not bioavailable for V. metschnikovii. This could be why E. coli is more resistant to RA and the combination of RA and SC than V. metschnikovii. Therefore, RA combined with SC could inhibit V. metschnikovii without deleterious effects on indigenous gastrointestinal bacteria. [1]

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In summary, the results of the present study reveal that both BP and Sodium citrate/SCT could inhibit growth and proliferation of MGC-803 cells and block glycolytic pathway and regulated the Bcl-2 family genes to induce mitochondria-regulated apoptosis. These observations will add new light in the field of developing therapeutic strategies for gastric cancer in the near future on BP and SCT. [2]

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Background/aims: Endoplasmic reticulum stress (ERS) is an important self-protective cellular response to harmful stimuli that contribute to various diseases, including chronic renal failure (CRF). Sodium citrate plays an important role in antioxidant and cellular immunity, but whether it improves ERS in CRF is unclear.
Methods: The rats were randomly divided into five groups: the control group, the sodium citrate control group, the model group, model rats with low dose sodium citrate (216 mg/kg), and model rats with a high dose of sodium citrate (746 mg/kg). The rats were euthanized at 6, 8, 12, and 16 weeks with their blood and renal tissue in detection.
In conclusion, this study demonstrates that sodium citrate has a protective effect on CRF rats by inhibiting ERS. In addition, this protective effect was stronger with higher doses of the sodium citrate in CRF rats. Our findings may provide a new therapeutic strategy for treatment of CRF. [3]

C6H9NA3O9

294.0996

293.993

6132-04-3

Lithium citrate tetrahydrate;6080-58-6;Citric acid;77-92-9;Hydroxycitric acid tripotassium hydrate;6100-05-6

71474

White to off-white solid powder

1.76

309.6ºC at 760 mmHg

>300 °C(lit.)

173.9 °C

1.58

3

9

2

18

211

0

C(C(=O)[O-])C(CC(=O)[O-])(C(=O)[O-])O.O.O.[Na+].[Na+].[Na+]

NLJMYIDDQXHKNR-UHFFFAOYSA-K

InChI=1S/C6H8O7.3Na.2H2O/c7-3(8)1-6(13,5(11)12)2-4(9)10;;;;;/h13H,1-2H2,(H,7,8)(H,9,10)(H,11,12);;;;2*1H2/q;3*+1;;/p-3

trisodium;2-hydroxypropane-1,2,3-tricarboxylate;dihydrate

Trisodium citrate dihydrate; B22547B95K; Nauzene; DTXSID1049437; N-1560; Natrii citras, dehydrate; TRISODIUM CITRATE DIHYDRATE (II); ...; 6132-04-3;

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)

H2O : ~125 mg/mL (~425.03 mM)

Solubility in Formulation 1: 100 mg/mL (340.02 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)