Ferric Ammonium Citrate Upregulates PD-L1 Expression through Generation of Reactive Oxygen Species

Iron plays an important role in macrophage polarization by altering metabolic and redox status. However, the impact of iron on the immune status of macrophages is still controversial. In this study, we report that ferric ammonium citrate (FAC) upregulates PD-L1 expression in macrophages.
FAC not only altered the phenotype of macrophages but also led to enriching immune-modulatory T cell subsets. Since iron is known to be a constituent of coenzymes facilitating metabolic processes in mitochondria, we examined the metabolic status of FAC-overloaded macrophages by measuring the oxygen consumption rate (OCR) and the represented coenzyme, aconitase.
In addition to enhancement of metabolic processes, FAC accelerated the Fenton reaction in macrophages, which also contributed to the facilitation of oxygen consumption. We reasoned that the enhancement of the OCR leads to the production of reactive oxygen species (ROS), which are directly linked to PD-L1 induction.
Using ferrostatin, rotenone, and N-acetyl-L-cysteine, we confirmed that metabolic and redox regulation is responsible for FAC-mediated PD-L1 expression.
Furthermore, we suggested that FAC-induced ROS production may explain FAC-mediated pro- and anti-inflammatory responses in macrophages. These findings may extend our understanding of regulating iron concentration during immune checkpoint therapy in cancer patients.

Ferric ammoniumcitrate (FAC)-induced inhibition of osteoblast proliferation/differentiation and its reversal by soybean-derived peptides (SDP)

Ferric citrate has been used to treat hyperphosphatemia, a prevalent symptom in patients with chronic kidney disease while ferric ammonium citrate (FAC), a more dissolvable format, is widely used as food additive. However, excess iron is associated with osteoporosis.
Dietary soybean products have been shown to prevent the progression of osteoporosis. In this study, a group of peptides, referred as P3, was identified from the enzymolysis of soybean protein isolates, and its biological functions were investigated. The results showed that MC3T3-E1 cell cycle progression from G0/G1 to S phase was accelerated by P3 treatment. MC3T3-E1 cell proliferation was enhanced by P3 via ERK1/2 activation.
Importantly, P3 treatment abolished the antiproliferative effect of FAC on MC3T3-E1 cell. In addition, P3 treatment increased the expression of ALP, COL-1, OCN, consequently promoting the differentiation and mineralization of MC3T3-E1 cells via activation of p38 MAPK pathway.
Consequently, P3 treatment was able to reverse the inhibitory effect of FAC on osteoblasts differentiation and mineralization. Our findings suggest P3, as a dietary supplement, has a potential therapeutic function to attenuate the adverse effects of FAC on bone metabolism and to prevent osteoporosis progression.

Ammonium Ferric Citrate induced Ferroptosis in Non-Small-Cell Lung Carcinoma through the inhibition of GPX4-GSS/GSR-GGT axis activity

The morbidity and mortality rates associated with non-small-cell lung carcinoma (NSCLC) are increasing every year, placing new demands on existing therapies and drugs. Ammonium ferric citrate (AFC) is often used as a food additive for iron supplementation; however, to our knowledge, no studies have investigated whether AFC can induce ferroptosis in NSCLC.
In this study, we demonstrated that specific concentrations of AFC effectively inhibit the proliferation and invasion of lung cancer cell lines in vitro using a cell proliferation inhibition test, a transwell assay, and flow cytometry analysis of cell cycle and apoptosis. In addition, AFC significantly induced oxidative stress injury in lung cancer cell lines.
A quantitative polymerase chain reaction assay showed that AFC markedly reduced the expression levels of cell growth factors, negative regulators of ferroptosis, and autophagy regulators. Lastly, a protein-protein interaction analysis revealed that glutathione peroxidase 4 (GPX4) exerted its biological role through the regulation of the GSS/GSR complex and downstream GGT family proteins.
When the expression of GPX4 changes, its biological activities, such as the glutathione metabolic process, cellular biosynthetic process, cellular response to chemical stimulus, and antioxidant activity, change accordingly, thereby affecting the survival quality and physiological and biochemical activities of cells.
Overall, this study verifies that AFC has the biological activity of activating oxidative stress injury in NSCLC cell lines, leading to a decrease in their autophagy and inducing ferroptosis. We also confirmed that the GPX4-GSS/GSR-GGT axis is a crucial target of AFC-induced ferroptosis.

Increasing Intracellular Levels of Iron with Ferric Ammonium Citrate Leads to Reduced P-glycoprotein Expression in Human Immortalised Brain Microvascular Endothelial Cells

Purpose: P-glycoprotein (P-gp) at the blood-brain barrier (BBB) precludes the brain penetration of many xenobiotics and mediates brain-to-blood clearance of β-amyloid, which accumulates in the Alzheimer’s disease (AD) brain. Zinc and copper are reported to modulate BBB expression and function of P-gp; however, the impact of exogenous iron, which accumulates in AD, on P-gp dynamics remains unknown.
Methods: P-gp protein and MDR1 transcript levels were assessed in immortalised human cerebral microvascular endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC; 250 μM, 72 h), by Western blotting and RT-qPCR, respectively. P-gp function was assessed using rhodamine-123 and [3H]-digoxin accumulation. Intracellular reactive oxygen species (ROS) levels were determined using 2′,7′-dichlorofluorescin diacetate and intracellular iron levels quantified using a ferrozine assay.
Results: FAC treatment significantly reduced P-gp protein (36%) and MDR1 mRNA (16%) levels, with no significant change in rhodamine-123 or [3H]-digoxin accumulation. While P-gp/MDR1 downregulation was associated with elevated ROS and intracellular iron, MDR1 downregulation was not attenuated with the antioxidant N-acetylcysteine nor the iron chelators desferrioxamine and deferiprone, suggesting the involvement of a ROS-independent mechanism or incomplete iron chelation.
Conclusions: These studies demonstrate that iron negatively regulates P-gp expression at the BBB, potentially impacting CNS drug delivery and brain β-amyloid clearance.
Keywords: P-glycoprotein; blood-brain barrier; efflux transporter; iron; reactive oxygen species.

Structures of disodium hydrogen citrate mono-hydrate, Na 2 HC 6 H 5 O 7(H 2 O), and di-ammonium sodium citrate, (NH 42 NaC 6 H 5 O 7, from powder diffraction data

The crystal structures of disodium hydrogen citrate monohydrate, Na2HC6H5O7(H2O), and di-ammonium sodium citrate, (NH4)2NaC6H5O7, have been solved and refined using laboratory X-ray powder diffraction data, and optimized using density functional techniques. In NaHC6H5O7(H2O), the NaO6 coordination polyhedra share edges, forming zigzag layers lying parallel to the bc plane.
The hydro-phobic methyl-ene groups occupy the inter-layer spaces. The carb-oxy-lic acid group makes a strong charge-assisted hydrogen bond to the central carboxyl-ate group. The hydroxyl group makes an intra-molecular hydrogen bond to an ionized terminal carboxyl-ate oxygen atom. Each hydrogen atom of the water mol-ecule acts as a donor, to a terminal carboxyl-ate and the hydroxyl group. Both the Na substructure and the hydrogen bonding differ from those of the known phase Na2HC6H5O7(H2O)1.5.

Ammonium citrate, dibasic

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Ammonium citrate, tribasic

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AB0060 | 250g: 60.44 EUR

Ammonium bismuth citrate

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GX7991-100G | 100 g: 90.00 EUR

Ammonium bismuth citrate

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GX7991-250G | 250 g: 150.00 EUR

Ammonium citrate tribasic

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Ammonium citrate tribasic

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GL7832-1KG | 1 kg: 178.00 EUR

Ammonium citrate tribasic

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Ammonium citrate tribasic

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Ammonium citrate tribasic

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GL7832-500G | 500 g: 118.00 EUR

di-Ammonium hydrogen citrate

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di-Ammonium hydrogen citrate

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di-Ammonium hydrogen citrate

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di-Ammonium hydrogen citrate

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Ammonium iron(III) citrate (Red)

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GK1122-100G | 100 g: 41.00 EUR
In (NH4)2NaC6H5O7, the NaO6 coordination octa-hedra share corners, making double zigzag chains propagating along the b-axis direction. Each hydrogen atom of the ammonium ions acts as a donor in a discrete N-H⋯O hydrogen bond. The hydroxyl group forms an intra-molecular O-H⋯O hydrogen bond to a terminal carboxyl-ate oxygen atom.