Gulcin 2025 — Antioxidants: a comprehensive review

Gulcin, at the Department of Chemistry, Atatürk University (Erzurum, Türkiye), surveys the chemistry and in vitro methodology of antioxidant capacity measurement in a 105-page narrative review. The paper is organised around (i) reactive oxygen and nitrogen species (ROS, RNS) and the biochemistry of oxidative stress, (ii) enzymatic antioxidants (superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase) and their metalloenzyme cofactors, (iii) non-enzymatic antioxidants (vitamins, polyphenols, carotenoids, sulfur-containing peptides), (iv) the principal in vitro antioxidant capacity assays — DPPH·, ABTS·⁺, DMPD·⁺, FRAP, CUPRAC, FCR, ORAC, O₂·⁻ scavenging, NO· scavenging, ONOO⁻ scavenging, H₂O₂ scavenging, ¹O₂ quenching, metal chelating, and TBARS — with chemical principles, advantages, disadvantages, and limitations of each, and (v) a closing discussion of method selection. There are no original experimental measurements, no food or biological sampling, and no contamination occurrence data; the heavy-metals relevance is methodological (the metal-chelating antioxidant assay and the Fenton/Haber-Weiss chemistry of iron- and copper-mediated ROS generation) and conceptual (the role of heavy metals as a source of oxidative stress).

Why this matters

• It is the current (2025) Springer Nature comprehensive reference for in vitro antioxidant assay methodology. For wiki readers asking why metal chelation is treated as an antioxidant activity — and why peptide chelators like glutathione and metallothionein are framed as endogenous antioxidants in the parallel peptide literature in this folder — Gulcin’s review provides the chemical rationale.
• It frames the Fenton (Fe²⁺ + H₂O₂ → Fe³⁺ + OH⁻ + OH·) and Haber-Weiss (O₂·⁻ + H₂O₂ + Fe³⁺ → O₂ + Fe²⁺ + OH⁻ + OH·) reactions as the dominant pathways by which transition metals (Fe, Cu, Cr³⁺, Co²⁺, Ti³⁺) generate the most damaging ROS species (hydroxyl radical, OH·). This is the chemical mechanism by which dietary and environmental heavy-metal exposure translates into oxidative cellular damage, which the metal pages and the chelation/remediation pages reference as background.
• It catalogues the ferrozine, 2,2′-bipyridine, and EDTA-based metal chelating assays as the standard methods for quantifying the iron-chelating capacity of food constituents and plant extracts — the same chemistry that underlies how dietary phenolics, polyphenols, and sulfur-containing peptides reduce iron-mediated lipid peroxidation in food matrices and in vivo.
• It notes the FDA-approved clinical use of EDTA for lead and mercury poisoning treatment and the use of calcium-sodium-EDTA for treating mercury and lead poisoning, anchoring the regulatory-pharmacology vocabulary that more clinically-focused reviews in this folder (Sears 2013, Luo 2024) build on.
• It defines heavy metals operationally as “elements with a density greater than 5 g/cm³” (after Duffus 2002), examples being Pb, Cd, Hg, and As, while flagging that “some heavy metals are essential in trace amounts for biological processes (e.g., zinc, iron, and copper), others are toxic even at low concentrations (e.g., Cd, Hg, and Pb).” This taxonomy is the vocabulary HMI’s metal pages already operate in.

Heavy-metals-relevant content in the review

Gulcin’s review treats heavy metals as a background topic, not a primary subject. The directly metal-relevant material is concentrated in three locations:

• Fenton and Haber-Weiss chemistry (Introduction, ROS section): The review traces hydroxyl radical (OH·) generation to the iron-catalysed decomposition of hydrogen peroxide. The Fenton reaction is given as Fe²⁺ + H₂O₂ → Fe³⁺ + OH⁻ + OH·. The Haber-Weiss reaction is given as O₂·⁻ + H₂O₂ + Fe³⁺ → O₂ + Fe²⁺ + OH⁻ + OH·. The review notes that “numerous metal ions in lower oxidation states, such as Cu⁺, Ti³⁺, Cr³⁺, and Co²⁺, also react with H₂O₂ in a manner similar to Fe²⁺. These combinations with H₂O₂ are termed ‘Fenton-like’ reagents.” Hydroxyl radical half-life is given as ≈10⁻⁹ s; the radical is described as the most reactive ROS species.
• Metal chelating assay (Methods section, pp. 1971-1976): The review describes three principal chelating-agent classes — ferrozine, 2,2′-bipyridine (Bpy), and EDTA — and the chemistry by which antioxidants are evaluated by their capacity to disrupt the Fe²⁺-chelator complex. Ferrozine forms a 3:1 ferrozine:Fe²⁺ red complex with molar absorptivity 27,900 M⁻¹·cm⁻¹ at 562 nm, stable across pH 4-9. Chelating capacity is measured as percent reduction in absorbance after antioxidant pretreatment. The review notes that “EDTA is a hexadentate chelating agent, commercially available as its disodium salt” and “the FDA has approved EDTA for treating lead and mercury poisoning.” Calcium-sodium EDTA “has been employed for treating mercury and lead poisoning.” Renal toxicity is named as the principal EDTA adverse effect.
• Definition and toxicity of heavy metals (Metal chelating assay introduction): “Heavy metals are generally defined as elements with a density greater than 5 g/cm³. These elements are found in the periodic table and often have high atomic weights (Duffus 2002). Examples include lead (Pb), cadmium (Cd), mercury (Hg), and arsenic (As), among others. While some heavy metals are essential in trace amounts for biological processes (e.g., zinc, iron, and copper), others are toxic even at low concentrations (e.g., Cd, Hg, and Pb). Their toxicity often stems from their ability to bioaccumulate and disrupt cellular functions by binding to proteins and enzymes.” Sources of metal pollution named: mining activities, industrial wastewater, urban waste, acid rain, fossil fuel residues, fertilizers, and pesticides.

Antioxidant capacity assays catalogued

The review’s main reference contribution is an organised vocabulary of the in vitro antioxidant capacity assays in current use, with chemical principles, advantages, disadvantages, and limitations. The methods listed below are those with direct heavy-metals or metal-chelation methodology relevance; the review covers an additional ~15 methods focused on radical scavenging (DPPH·, ABTS·⁺, DMPD·⁺, O₂·⁻, NO·, ONOO⁻, ¹O₂, H₂O₂) that are not summarised here because they do not bear on metal-chelation chemistry.

The review notes that compounds containing -OH, -SH, -COOH, -NH₂, -SR, -PO₃H₂, C=O, -NR₂, -S- and -O- functional groups in favourable structure-function configuration can exhibit metal chelation activity (citing Yuan et al. 2005, Gülçin 2006a). Specific food constituents identified as Fe²⁺-chelating: curcumin (chelates Fe²⁺ via -OH and -OCH₃ groups), resveratrol (two molecules chelate one Fe²⁺ before ferrozine), L-adrenaline (binds Fe²⁺ through amine and hydroxyl groups), L-carnitine (chelates Fe²⁺ through carbonyl and hydroxyl functional groups), quercetin (chelates Cu²⁺ and Fe²⁺ through carbonyl groups, per Kazazica et al. 2006; Fiorucci et al. 2007), and usnic acid (chelates Fe²⁺ through hydroxyl and carboxyl groups attached to its phenolic ring, per Çetin Çakmak & Gülçin 2019).

Enzymatic antioxidant biochemistry (with metal cofactors)

The review describes four enzymatic antioxidants, three of which are metalloenzymes:

• Superoxide dismutase (SOD; E.C. 1.15.1.1) — metalloenzyme catalysing O₂·⁻ → H₂O₂. Isoforms classified by metal cofactor: Cu/ZnSOD (cytosolic, eukaryotes including humans), MnSOD (peroxisomes and mitochondria), Fe/Ni SOD, and extracellular SOD. The dismutation reaction for Cu/ZnSOD is shown as O₂·⁻ + Cu²⁺-SOD → O₂ + Cu⁺-SOD followed by O₂·⁻ + 2H⁺ + Cu⁺-SOD → H₂O₂ + Cu²⁺-SOD. A general form is given for M(n+1) and M(+) where n=1 for Cu and n=2 for Mn, Fe, Ni.
• Catalase (CAT; E.C. 1.11.1.6) — iron-dependent heme-containing homotetramer. Decomposes H₂O₂ → 2H₂O + O₂. The reaction proceeds via Fe³⁺-E (the iron centre of the heme group) → Fe⁴⁺-E(·⁺) resonance form (heme as radical cation). High catalytic efficiency; high concentrations in erythrocytes, kidneys, and liver.
• Glutathione peroxidase (GPx; E.C. 1.11.1.9) — selenocysteine-containing peroxidase. Reduces H₂O₂ via glutathione (GSH) → GSSG + 2H₂O. Five isoforms identified: GPx1 (cytosolic, predominantly in erythrocytes, kidneys, and liver), GPx2 (cytosolic, colon and liver), GPx3 (extracellular, plasma and proximal kidney tubule), GPx4 (phospholipid hydroperoxide-specific), and GPx5 (epididymis). All contain a single selenocysteine residue essential for activity.
• Glutathione reductase (GR; E.C. 1.8.1.7) — FAD-dependent NADPH-utilising flavoprotein oxidoreductase. Reduces GSSG → 2GSH. Not a metalloenzyme but tied to the antioxidant metalloenzyme network through its role in regenerating reduced glutathione for GPx.

Non-enzymatic antioxidants relevant to metal chelation

The review enumerates classes of non-enzymatic antioxidants with metal-chelating properties:

• Glutathione (GSH) — tripeptide γ-Glu-Cys-Gly. Neutralises O₂·⁻, NO·, OH·, and ONOO·. Composed of glutamic acid, cysteine, and glycine. The cysteine thiol is the metal-binding site.
• Polyphenols — “some polyphenols exhibit metal-chelating properties, effectively inhibiting Fenton-type oxidation reactions by binding transition metal ions in their free states” (citing Rice-Evans et al. 1996, Karaman et al. 2009, Gulcin 2012). Specific phenolic compounds named as metal chelators: gallic acid, catechins, chlorogenic acid, caffeic acid, ferulic acid, tannic acid.
• Flavonoids — including catechins, proanthocyanidins, anthocyanins, flavonols (quercetin); metal-chelation activity attributed to carbonyl and ortho-dihydroxy groups in their aromatic ring structures.
• Sulfur-containing antioxidants — taurine, methionine, N-acetylcysteine (NAC), α-lipoic acid; named in the context of being thiol-containing antioxidants that the CUPRAC assay (but not FRAP) can detect.
• Tocopherols and tocotrienols (vitamin E forms), carotenoids (β-carotene, lycopene, lutein, zeaxanthin, astaxanthin, β-cryptoxanthin), and vitamin C (ascorbic acid) are described as primarily radical-scavenging rather than metal-chelating antioxidants.

Methods (brief)

Narrative review with 122 figures and 9 tables. The author is Faculty of Sciences, Department of Chemistry, Atatürk University, Erzurum, Türkiye. No declared systematic search strategy, no PRISMA-style inclusion/exclusion criteria, no risk-of-bias assessment, no quantitative synthesis. The review synthesises the author’s prior published methodological work (~30 self-citations to Gülçin and co-author publications spanning 2002-2025) along with the broader antioxidant-chemistry literature. The reference list exceeds 700 entries.

Funding: Open access funding provided by the Scientific and Technological Research Council of Türkiye (TÜBİTAK). No conflict of interest declared.

Implications

• Certification: The review reports no occurrence data and contributes nothing directly to HMTc threshold-setting work. Its indirect value is methodological: it documents the chemistry by which polyphenol-, peptide-, and chelator-rich dietary components can reduce iron- and copper-mediated lipid peroxidation in food matrices, which is the chemical rationale behind shelf-life extension claims for antioxidant-enriched products. The source is excluded from pool admission for any percentile-selection arithmetic. No HMTc rationale tag is appropriate.
• App: No routing to ingredient or product pages. The source contributes background reading for iron, copper, lead, cadmium, mercury-total, and arsenic-total on the topic of oxidative stress chemistry and metal-chelation as an antioxidant mechanism, not on contamination occurrence.
• Courses: Useful as a primary teaching reference for an educator-audience module on (i) the Fenton/Haber-Weiss chemistry by which dietary and environmental heavy metals generate ROS in vivo, (ii) the design and limitations of the in vitro antioxidant capacity assays (DPPH, ABTS, FRAP, CUPRAC, ORAC, metal chelating) that food-industry QA labs use to characterise antioxidant ingredients, and (iii) the role of metal-coordinating functional groups (-OH, -SH, -COOH, carbonyl) in food-grade polyphenols and peptide chelators. Quote-sized claims should be traced to the named primary references (Apak, Benzie & Strain, Stookey, Halliwell & Gutteridge) rather than to this review.
• Microbiome: Marginal. The review does not engage gut-microbiome-metal axis topics.
• Mitigation evidence: Most relevant to remediation-evidence as a methodology reference for the in vitro assays used to characterise metal-binding capacity of food constituents and plant extracts, complementing the more clinically-focused chelation review by sears2013-chelation-heavy-metal-detoxification-review.

Limitations

• Narrative review without declared search strategy, inclusion criteria, or risk-of-bias assessment. The reference selection skews heavily toward the author’s own prior publications (~30 self-citations to Gülçin et al. across 2002-2025), which is acceptable for a single-author comprehensive review by an established methodologist in the field but means the review is best treated as a state-of-the-art summary at 2025 from one prominent perspective, not as an evidence map.
• No primary occurrence measurements. The review contains no food or biological sampling and no contamination concentrations; it cannot be admitted to any HMTc per-analyte percentile pool.
• The heavy-metals discussion is background-grade. The operational definition of heavy metals (>5 g/cm³, after Duffus 2002), the brief sources-of-pollution list, and the FDA-EDTA-for-Pb/Hg statement are all standard introductory framing. Downstream uses requiring quantitative heavy-metal information should source primary studies (cited in Tchounwou et al. 2012, ref in this review) or agency monographs, not Gulcin’s review.
• Some method advantages/disadvantages are reported as expert opinion without primary-study attribution. The methodological recommendations (FRAP at pH 3.6 is non-physiological, CUPRAC at pH 7.0 is closer to physiological) are well-grounded but presented in a summary style.
• The 122-figure scheme density (mostly chemical reaction mechanisms) substantially exceeds what is reproducible in a wiki source page; readers needing the full mechanistic detail should consult the original PDF.

Wiki pages this source may touch

• iron
• copper
• lead
• cadmium
• mercury-total
• arsenic-total
• remediation-evidence

Verification notes

Existing-page check. DOI grep (10.1007/s00204-025-03997-2), raw_handle grep (MFK_35-antioxidants), and cite-key glob (gulcin2025-*) over wiki/sources/ on 2026-06-08 returned no hits. The only Cd-toxicity/antioxidant-vitamin page (sitek2022) is a different paper by different authors. This is a NEW source page with no prior version to merge-enhance.

Evidence tier. B (peer-reviewed narrative review). The paper reports no primary measurements, declares no systematic search strategy, and offers no risk-of-bias assessment. A-tier is reserved for primary peer-reviewed studies and authoritative agency monographs; this is neither. The author is an established methodologist at Atatürk University with a substantial publication record in antioxidant chemistry methodology, and Archives of Toxicology is a Springer Nature toxicology journal indexed in PubMed/MEDLINE, which supports B over C.

Metals frontmatter. Recording [Pb, Cd, tAs, tHg] — the four HMTc-priority metals the review explicitly names as toxic heavy metals (in the metal-chelating-assay introduction: “Examples include lead (Pb), cadmium (Cd), mercury (Hg), and arsenic (As)”) and that EDTA is described as treating (“FDA has approved EDTA for treating lead and mercury poisoning”). The paper uses “Hg” and “As” without speciation; recording as tHg and tAs (the unspeciated/total tags) per the speciation discipline in conventions. iAs and MeHg are not separately discussed and are omitted. Cr is mentioned only in the Fenton-like reagent context (Cr³⁺ + H₂O₂) without contamination relevance and is omitted; Cr-VI is not discussed. Ni, Al, Sn are not in scope of the review. Out-of-HMTc-vocabulary metals heavily discussed (Fe, Cu, Zn, Mn) drive the review’s chemistry content but do not appear in HMTc analyte vocabulary frontmatter.

Ingredients, products, matrices frontmatter. All empty. The review measures nothing in any food, beverage, personal-care, or environmental matrix; it surveys antioxidant assay chemistry without primary sampling. Food constituents discussed (fruits, vegetables, tea, olive oil, polyphenol-rich extracts, curcumin, resveratrol, quercetin, usnic acid) are referenced in the context of antioxidant assay performance, not heavy-metal occurrence. Routing these to ingredient pages would misrepresent the source as occurrence-side evidence.

Jurisdictions. [TR]. The author is at Atatürk University in Erzurum, Türkiye; open-access funding from TÜBİTAK (Scientific and Technological Research Council of Türkiye). The review cites US regulatory framing (FDA approval of EDTA) but the review itself is Turkish-sited methodological work, not a US, EU, or international monograph. Recording TR only.

Sample size. Null. This is a methodology review with no sampling frame of its own.

Audience. [regulator, educator]. Regulator audience because the chemistry of FDA-approved EDTA chelation and the operational definition of heavy metals are referenced. Educator audience because the review’s primary contribution is method documentation suitable for course material. Consumer audience excluded because the review is methodological and produces no consumer-translated quantitative claims. App audience excluded because no contamination_profile values are derivable from the review.

Brand firewall (Part 12). No commercial brand names appear in the review body. The review names reagent/instrument vendors implicitly (DPPH, ABTS, ferrozine, TPTZ, neocuproine, EDTA-disodium) but these are scientific-method-name terms (Exception 2 of the brand firewall, locked 2026-05-17): they identify the chemicals doing the measuring, not contamination values of sampled consumer products. No firewall action required.

HMTc firewall (Part 2). The review contains no HMTc-threshold language and no consumer-audience risk advisories. The closest is the statement that EDTA “is also effective in eliminating excess iron from the body” and “the FDA has approved EDTA for treating lead and mercury poisoning” — clinical-pharmacology context, not a consumer or threshold claim. The review is upstream of any HMTc rationale tagging.

Speciation discipline. The review uses unspeciated “Hg,” “mercury,” “As,” and “arsenic” throughout the heavy-metals background section. The chelation-target list (“lead, cadmium, mercury, and arsenic”) is unspeciated. Following conventions, tHg and tAs (total/unspeciated tags) are recorded in frontmatter; iAs and MeHg are not added since the paper does not separately distinguish the species.

Date arithmetic. Received 31 January 2025, accepted 18 February 2025, published online 15 April 2025 — consistent with year frontmatter (2025). Article DOI 10.1007/s00204-025-03997-2 resolves to Archives of Toxicology Volume 99, pp 1893-1997.

Open-access license. The paper is published by Springer Nature under the Creative Commons Attribution 4.0 International License (“This article is licensed under a Creative Commons Attribution 4.0 International License, which permits any use, sharing, adaptation, distribution and reproduction in any medium or format…”). Recorded as CC-BY-4.0.

Reviewer note on scope fit. This paper is in scope per the 2026-06-02 commit 3f47f95 — scope: mitigation/remediation is in-scope, not a skip. The folder context (heavy_metals_peptides under Kimi_Agent_Black Market Peptide Metal Survey) is peptide-mediated metal binding and detoxification; this review’s coverage of glutathione (γ-Glu-Cys-Gly tripeptide chelator) and selenocysteine-containing glutathione peroxidase, together with the metal-chelating assay methodology that underpins how peptide chelation activity is quantified in vitro, is the direct fit. The review is methodology-side, not occurrence-side, which is recorded accurately in the Implications section.

Page history

The five most recent substantive edits to this page. The full version history lives in git; when DOI minting comes online (see schema docs), each entry below will also link to a version-pinned DataCite DOI.