{"id":"e6df8b69-a118-56c6-ba7c-2ac7fb246756","stable_key":"cb568d28-484a-5c2e-9fcc-2d780358e514:c-reg-extracellular-protein-oxidation","predicate":"increases-ascorbate-associated","statement":"Adding iron to pharmacological ascorbate increased measured extracellular protein oxidation about threefold in the RPMI/FCS experiment while preserving intracellular thiols. The authors interpreted this as extracellular interception of Fenton-derived oxidants; direct hydroxyl-radical spin trapping was unsuccessful because ascorbate reduced the spin adducts.","claim_class":"mechanistic","status":"source_derived_draft","evidence_grade":"ungraded","direction":"positive","is_public":true,"mechanism_event_id":"2f817934-cefd-56de-bc99-f32630c86e2a","mechanism_event_label":"Extra iron shifted more oxidation onto proteins in the culture liquid while protecting the cells. The proposed short-lived radical intermediate could not be directly measured.","subject":{"id":"80537c07-6aa7-5a3b-9148-f7033ce3c0fd","slug":"ferric-ammonium-citrate","display_name":"Ferric ammonium citrate","entity_type_key":"chemical_species"},"object":{"id":"eb50742e-0b01-5d3f-b3d6-3b3813fab5cd","slug":"extracellular-protein-oxidation","display_name":"Extracellular protein oxidation","entity_type_key":"cellular_process"},"evidence_count":1,"mechanism_event":{"id":"2f817934-cefd-56de-bc99-f32630c86e2a","stable_key":"cb568d28-484a-5c2e-9fcc-2d780358e514:c-reg-extracellular-protein-oxidation-event","event_type":"biochemical_relationship","label":"Extra iron shifted more oxidation onto proteins in the culture liquid while protecting the cells. The proposed short-lived radical intermediate could not be directly measured.","description":"Adding iron to pharmacological ascorbate increased measured extracellular protein oxidation about threefold in the RPMI/FCS experiment while preserving intracellular thiols. The authors interpreted this as extracellular interception of Fenton-derived oxidants; direct hydroxyl-radical spin trapping was unsuccessful because ascorbate reduced the spin adducts.","status":"provisional","compartment":null,"participants":[{"entity":{"id":"da1a7137-08f2-5a36-8bb6-377fbd32e327","slug":"vitamin-c","display_name":"Vitamin C","entity_type_key":"chemical_species"},"role":"nutrient context","stoichiometry":null,"state_label":"","sequence_order":0,"notes":""},{"entity":{"id":"38d0c4d9-53d1-5239-bfe3-c1b5e9b79085","slug":"ascorbate","display_name":"L-Ascorbate","entity_type_key":"small_molecule"},"role":"reducing agent","stoichiometry":null,"state_label":"","sequence_order":1,"notes":""},{"entity":{"id":"da9d64bc-69d4-5d97-90a4-8f0ed03e0ac8","slug":"hydrogen-peroxide","display_name":"Hydrogen peroxide","entity_type_key":"small_molecule"},"role":"Fenton substrate","stoichiometry":null,"state_label":"","sequence_order":2,"notes":""},{"entity":{"id":"80537c07-6aa7-5a3b-9148-f7033ce3c0fd","slug":"ferric-ammonium-citrate","display_name":"Ferric ammonium citrate","entity_type_key":"chemical_species"},"role":"subject","stoichiometry":null,"state_label":"","sequence_order":3,"notes":""},{"entity":{"id":"eb50742e-0b01-5d3f-b3d6-3b3813fab5cd","slug":"extracellular-protein-oxidation","display_name":"Extracellular protein oxidation","entity_type_key":"cellular_process"},"role":"target","stoichiometry":null,"state_label":"","sequence_order":4,"notes":""}]},"contexts":[{"dimension":"cross_nutrient","value_text":"true","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"evidence_location","value_text":"Figure 3a–c; Results discussing failed EPR spin trapping","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"experimental_model","value_text":"Biochemical extracellular protein-oxidation assay and intracellular thiol-sensitive RSSR EPR assay","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"exposure","value_text":"5 mM ascorbate ±5 µM FAC iron for 2 h in RPMI-1640 +10% FCS; additional 30 µM iron experiment gave similar results. Thiols assayed using 100 µM RSSR probe.","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"limitations","value_text":"Observed protein oxidation supports but does not directly prove the proposed hydroxyl-radical buffering mechanism. FAC speciation and medium composition limit tissue extrapolation.","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"nutrient_topic","value_text":"Vitamin C research collection; topical membership is not evidence of a direct dietary effect.","comparator":null,"unit":null,"notes":"","entity":{"slug":"vitamin-c","display_name":"Vitamin C","entity_type_key":"chemical_species"}},{"dimension":"organism","value_text":"Homo sapiens LNCaP/PC-3 cells; bovine-serum-containing extracellular medium","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"plain_language","value_text":"Extra iron shifted more oxidation onto proteins in the culture liquid while protecting the cells. The proposed short-lived radical intermediate could not be directly measured.","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"primary_references","value_text":"[c-reg-mojic] Extracellular iron diminishes anticancer effects of vitamin C: an in vitro study. (2014). https://pubmed.ncbi.nlm.nih.gov/25092529/ DOI: 10.1038/srep05955","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"tissue_or_cell_type","value_text":"Prostate cancer-cell cultures and their extracellular RPMI/FCS medium","comparator":null,"unit":null,"notes":"","entity":null}],"evidence":[{"id":"842e930f-4be2-54ce-a386-cd0930c9f212","evidence_kind":"source_excerpt","locator":"Lines 1313-1325","start_line":1313,"end_line":1325,"excerpt":"### c-reg-extracellular-protein-oxidation\nAdding iron to pharmacological ascorbate increased measured extracellular protein oxidation about threefold in the RPMI/FCS experiment while preserving intracellular thiols. The authors interpreted this as extracellular interception of Fenton-derived oxidants; direct hydroxyl-radical spin trapping was unsuccessful because ascorbate reduced the spin adducts.\nCondition category: normal\nnutrient_topic: Vitamin C research collection; topical membership is not evidence of a direct dietary effect.\nplain_language: Extra iron shifted more oxidation onto proteins in the culture liquid while protecting the cells. The proposed short-lived radical intermediate could not be directly measured.\norganism: Homo sapiens LNCaP/PC-3 cells; bovine-serum-containing extracellular medium\ntissue_or_cell_type: Prostate cancer-cell cultures and their extracellular RPMI/FCS medium\nexperimental_model: Biochemical extracellular protein-oxidation assay and intracellular thiol-sensitive RSSR EPR assay\nlimitations: Observed protein oxidation supports but does not directly prove the proposed hydroxyl-radical buffering mechanism. FAC speciation and medium composition limit tissue extrapolation.\nexposure: 5 mM ascorbate ±5 µM FAC iron for 2 h in RPMI-1640 +10% FCS; additional 30 µM iron experiment gave similar results. Thiols assayed using 100 µM RSSR probe.\ncross_nutrient: true\nevidence_location: Figure 3a–c; Results discussing failed EPR spin trapping\n[c-reg-mojic] Extracellular iron diminishes anticancer effects of vitamin C: an in vitro study. (2014). https://pubmed.ncbi.nlm.nih.gov/25092529/ DOI: 10.1038/srep05955","model_system":"Biochemical extracellular protein-oxidation assay and intracellular thiol-sensitive RSSR EPR assay","directness":"author_interpretation","verification_status":"source_derived_draft","notes":"Exact curation-document quotation, not publisher quotation. Study references: [c-reg-mojic] Extracellular iron diminishes anticancer effects of vitamin C: an in vitro study. 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