Oxidation (HSA Cys34 — HMA/HNA1/HNA2)
Definition
Oxidation of HSA refers to the chemical modification of cysteine-34 (Cys34) — the only free thiol group on the albumin molecule — by reactive oxygen species (ROS) and reactive nitrogen species (RNS). It exists along a reversibility continuum from mild (reversible) to severe (irreversible), captured by the HMA/HNA1/HNA2 nomenclature.
The HMA/HNA1/HNA2 Nomenclature
| Form | Full name | Cys34 state | Δmass | Reversibility | Fraction (healthy) |
|---|---|---|---|---|---|
| HMA | Human Mercaptalbumin | Free reduced thiol (–SH) | 0 Da | — (reference) | 70–80% |
| HNA1 | Human Nonmercaptalbumin-1 | Disulfide bond to small thiol (Cys, homocysteine, glutathione) | +119 Da (cysteinylation most common) | Reversible — disulfide bond can be reduced | 20–30% |
| HNA2 | Human Nonmercaptalbumin-2 | Irreversibly oxidized to sulfenic (–SOH, +16 Da), sulfinic (–SO₂H, +32 Da), or sulfonic acid (–SO₃H, +48 Da) | +16 / +32 / +48 Da | Irreversible — permanent loss of antioxidant function | ~5% |
Note: In the top-down LC-MS nomenclature used in ALBOM (el-balkhi-2025), HNA1 corresponds primarily to the HSA+CYS isoform (+119 Da); HNA2 corresponds to the sulfonylated form (HSA+SULF, +48 Da). The IEC-based HMA/HNA1/HNA2 fractionation (oettl-2013 method) captures this at lower isoform resolution.
Mechanism
ROS-driven oxidation at Cys34
HSA-Cys34-SH (HMA, antioxidant active)
↓ + ROS (H₂O₂, HOCl, ONOO⁻)
HSA-Cys34-S-S-Cys (HNA1 / cysteinylation) — if small thiol mixes
or
HSA-Cys34-SOH (sulfenic acid, +16 Da) — transient, unstable
↓ + further ROS
HSA-Cys34-SO₂H (sulfinic acid, +32 Da = HNA2 partial)
↓ + further ROS
HSA-Cys34-SO₃H (sulfonic acid, +48 Da = HNA2 full, irreversible)
Homodimerization pathway (alternative fate of HMA)
HSA-Cys34-SH + HSA-Cys34-SH → (HD-HSA) disulfide homodimer
— see baldassarre-2016-dimers; both Cys34 residues consumed, both monomers lose antioxidant function
Mass spectrometry signature
| Modification | Δmass (Da) | Residue | Detection |
|---|---|---|---|
| Cysteinylation (HNA1) | +119 Da | Cys34 | TD: HSA+CYS; BU: Cys34 +119 peptide |
| Sulfenic acid (intermediate) | +16 Da | Cys34 | Unstable; rarely detected intact |
| Sulfinic acid (HNA2 partial) | +32 Da | Cys34 | TD: HSA+SO₂H; noted in domenicali-2014 |
| Sulfonic acid (HNA2 full) | +48 Da | Cys34 | TD: HSA+SULF (ALBOM nomenclature); most stable irreversible form |
| Methionine oxidation | +16 Da | Met residues | BU approach; detectable by rahali-2022 |
Biological significance
HMA (reduced):
- Free thiol at Cys34 = primary extracellular antioxidant — scavenges H₂O₂, peroxynitrite, hypochlorous acid
- Metal chelation at N-terminus (Cu, Fe) — prevents Fenton reaction
- Drug binding preserved at Sudlow sites
HNA1 (reversibly oxidized):
- Antioxidant function lost at Cys34 — thiol consumed
- HNA1 actively triggers cytokine production in leukocytes via p38 MAPK → alcaraz-quiles-2018
- Reversible: in favorable redox environment, can theoretically be reduced back to HMA
HNA2 (irreversibly oxidized):
- Permanent loss of Cys34 antioxidant function
- Elevated HNA2 > 12% = poor 30/90-day prognosis in cirrhosis → oettl-2013
- Correlates with MELD, bilirubin, INR, CRP
Clinical relevance
| Disease | Oxidation finding | Clinical significance |
|---|---|---|
| Liver fibrosis / cirrhosis | HNA1↑↑, HNA2↑, HMA↓ | Progressive increase with stage; HNA2 >12% predicts 90-day mortality |
| ACLF | HNA1 and HNA2 highest | Worst outcome group; multiple mechanisms of Cys34 loss |
| Alcoholic hepatitis (AAH) | HNA1 elevated | Acute oxidative burst; see montomoli-2026-aah, das-2017-sah |
| DILI | Early HNA1 elevation | Precedes enzyme elevation (D3 vs D7); el-balkhi-2024-seb |
| Diabetes mellitus | HNA1 moderately elevated | Oxidative stress from metabolic disease |
| Sepsis | HNA2 elevated | Systemic oxidative stress, not liver-specific |
Affected proteins in our research
- HSA — Cys34 is the primary site; 99% of relevant oxidative changes
- Other proteins oxidized in systemic oxidative stress (Met oxidation etc.) — detectable by BU but not our TD panel
Analytical approaches
- IEC-HPLC (UV): separates HMA / HNA1 / HNA2 fractions; oettl-2013 method; clinical-grade; limited to 3 fractions
- RP-LC-HR-MS top-down: full isoform landscape; +16/+32/+48 Da resolved at protein level; el-balkhi-2025 method; 10 isoforms
- RP-LC-MS bottom-up: site-level confirmation at Cys34; oxidation states confirmed by peptide MS; rahali-2022
- SEB test: functional assay detecting binding site disruption by oxidation at Cys34 → el-balkhi-2024-seb
Key references
- oettl-2013 — HNA2 >12% cut-off, survival prediction
- domenicali-2014 — HSA+SO₂H in cirrhosis; isoform profiling; native HSA predicts survival
- naldi-2017-review — comprehensive analytical review
- alcaraz-quiles-2018 — HNA1 drives cytokine storm via p38 MAPK
- das-2017-sah — HNA2 hyperoxidation modulates neutrophils in SAH
- el-balkhi-2025 — ALBOM: HSA sulfonylation (HNA2) pattern in CLD stages