Albumin as a dynamic extracellular redox regulator: from Cys34 oxidation biomarkers to polysulfide-mediated homeostatic mechanisms and clinical applications

Abstract

Human serum albumin (HSA) is the most abundant circulating protein and a central mediator of extracellular redox homeostasis. Its antioxidant function has been attributed primarily to the free thiol at cysteine residue 34 (Cys34), whose progressive oxidation—yielding nonmercaptoalbumin isoforms HNA1 and HNA2—serves as a sensitive biomarker of systemic oxidative stress. Advances in electrospray ionisation time-of-flight mass spectrometry have enabled precise quantification of Cys34 modifications, establishing cysteinylated albumin as a clinically applicable marker in chronic kidney disease, chronic liver disease, diabetes, and ageing-related functional decline. Beyond its role as a biomarker, oxidized albumin is now recognized as an active pathogenic mediator in preclinical models, with emerging evidence implicating ferroptosis as a potential mechanism of renal tubular cell injury, and contributing to cardiovascular risk and sarcopenia. A previously unrecognized dimension of albumin redox biology has also emerged: the presence of endogenous oxidized polysulfide bonds (Ox-PSS; RSS_n-R', n > 1) across multiple intramolecular cysteine bridges. Quantified using the newly developed Elimination Method for Sulfide from Polysulfide, albumin polysulfides are dynamically regulated by oxidative stress and are significantly depleted in chronic kidney disease and early hepatitis, even when conventional Cys34-based markers remain unchanged. Polysulfide depletion impairs both Site II drug-binding capacity and reactive oxygen species scavenging activity, with direct implications for pharmacokinetics and therapeutic efficacy. These findings collectively reframe HSA as a dynamic polysulfide reservoir and extracellular redox regulator, highlighting albumin polysulfide modulation as a compelling emerging therapeutic strategy.

1. Introduction

Human serum albumin (HSA) is the most abundant protein in serum, accounting for approximately 60%–65% of total serum proteins [1]. It plays pivotal roles in diverse physiological functions, including maintenance of colloid osmotic pressure, transport of long-chain fatty acids and drugs, and antioxidant activity as a radical scavenger [1]. Approximately 10–15 g of HSA is synthesized daily in the liver, maintaining a plasma half-life of approximately 20 days as it circulates between the intravascular space and the interstitial fluid [1]. During this prolonged circulation, HSA sensitively reflects the internal milieu and undergoes various post-translational modifications (PTMs), including oxidation, glycation, carbamylation, truncation, and dimerization [2–4].

In recent years, the concept of ‘redox albuminomics’ has been proposed to evaluate not only the quantitative changes in HSA but also the qualitative changes induced by these PTMs [5]. In particular, the cysteine residue at position 34 (Cys34) accounts for approximately 80% of free thiol groups in serum and serves as a major antioxidant site; its redox state has consequently attracted considerable attention as a sensitive biomarker of systemic oxidative stress [2,3]. The present review comprehensively discusses Cys34 oxidation, covering advances in measurement technologies, clinical significance in renal disease, diabetes and liver disease, the pathogenic properties of oxidized albumin, nutritional and physical function assessment, and applications in pharmaceutical quality control. Building on this foundation, we then introduce the emerging biology of albumin polysulfides—a previously unrecognized redox dimension with important biomarker and therapeutic implications.

2. Albumin structure and redox states

2.1. Cys34 of human serum albumin

HSA is a single-chain protein consisting of 585 amino acids. It contains 17 intramolecular disulfide bonds; uniquely, Cys34 exists as a free thiol (–SH) [1]. Based on the redox state of Cys34, circulating HSA is classified into three isoforms [1,6–9] (Figure 1):

Figure 1.

Characterization of HSA redox states based on Cys34 modification. HSA exists in three primary forms: reduced mercaptoalbumin [10], in which Cys34 is in the free thiol state, and two oxidized variants. HNA1 represents a reversible oxidized state in which Cys34 forms a mixed disulfide with low-molecular-weight thiols (cysteine, homocysteine, or glutathione). Further oxidation yields HNA2, characterized by irreversible formation of sulfinic (─SO₂H) or sulfonic (─SO₃H) acids at Cys34. The progressive shift from HMA toward HNA2 reflects increasing systemic oxidative stress and is associated with impaired antioxidant and drug-binding capacity.

2.1.1. Human mercaptoalbumin [10]

HSA in which Cys34 is in the reduced state (–SH). It accounts for approximately 70%–80% of total HSA in healthy individuals and is considered ‘functional albumin’, retaining potent antioxidant capacity and ligand-binding affinity.

2.1.2. Human nonmercaptoalbumin 1 (HNA1)

HSA in which Cys34 forms a mixed disulfide with low-molecular-weight thiols, such as cysteine, homocysteine, or glutathione. It accounts for approximately 20%–30% in healthy individuals.

2.1.3. Human nonmercaptoalbumin 2 (HNA2)

HSA in which Cys34 is irreversibly oxidized to sulfinic acid (─SO₂H) or sulfonic acid (─SO₃H). It represents approximately 5% in healthy individuals and increases under conditions of elevated oxidative stress.

2.2. Discovery of HSA polysulfides as endogenous supersulfides

Extensive studies have established that Cys34 undergoes diverse oxidative modifications—including nitrosylation, sulfenylation, sulfonylation, and cysteinylation—in response to systemic oxidative stress, and the oxidized fraction of Cys34 has been proposed as a sensitive biomarker for ageing and oxidative stress–related diseases. However, several observations challenge the traditional view that albumin's redox activity is regulated by Cys34 alone. Recombinant albumin mutants lacking Cys34 retain substantial antioxidative activity, indicating the involvement of additional redox-active elements beyond Cys34 [11]. Furthermore, specific intramolecular cysteine–cysteine bridges, including Cys392–Cys438 and Cys558–Cys567, exhibit redox or chemical reactivity that cannot be readily explained by classical disulfide chemistry [12,13].

In parallel with these findings, increasing attention has been directed toward sulfur-catenated molecules known as supersulfides. Supersulfides are characterized by polysulfide bonds and are endogenously generated during protein translation through cysteinyl-tRNA synthetase–mediated sulfur insertion [14]. These molecules possess unique chemical properties, exhibiting both nucleophilic and electrophilic reactivity, and participate in diverse biological processes, including antioxidative defense, redox signaling, and mitochondrial bioenergetics [10,15,16]. Although polysulfidation has been reported in various intracellular proteins, it has rarely been documented in extracellular proteins.

For clarity throughout this review, we distinguish between the oxidized form of polysulfides (Ox-PSS; R-SS_n-R', n > 1) and the reduced form of polysulfides (Red-PSS; R-SS_n-H). Both Ox-PSS and Red-PSS are subspecies within the broader supersulfide family. Unlike conventional thiols or disulfides, both forms exhibit enhanced chemical reactivity due to the presence of sulfane sulfur atoms (S⁰), enabling participation in a wide range of redox reactions [17].

Taking these observations together, we hypothesized that albumin may be polysulfidated. Albumin is distinguished by its extraordinary abundance, evolutionary conservation of cysteine residues, and prolonged circulation time. Evolutionary comparisons reveal that cysteine positions are highly conserved across species, whereas methionine residues show considerable variability (Figure 2). This conservation is consistent with the hypothesis that polysulfide formation at cysteine residues is a biologically regulated and evolutionarily conserved feature of albumin, though direct experimental validation of this hypothesis in species beyond human and rodent models is lacking. We propose that albumin may serve not merely as a passive carrier protein but as a candidate systemic regulator of extracellular redox homeostasis through polysulfide chemistry.

Figure 2.

Amino acid sequences of various albumins and location of sulfur-containing amino acids. Closed and open circles indicate the positions of Cys and Met residues, respectively, across albumins from multiple species. The high degree of conservation of Cys positions relative to the variable distribution of Met residues supports the view that polysulfide formation at Cys residues is a biologically regulated, evolutionarily conserved feature of albumin.

Reduced-form polysulfides (Red-PSS), which are prevalent in intracellular proteins, have been extensively studied owing to the availability of alkylation-based assays and sulfane sulfur probes. In contrast, Ox-PSS remained poorly characterized for many years due to the lack of reliable quantitative methods, and its biological significance in extracellular proteins, such as albumin, was consequently overlooked.

3. Analytical advances

3.1. Advances in measurement technology: from HPLC to ESI-TOFMS

High-performance liquid chromatography (HPLC) was previously the primary method for evaluating the redox state of Cys34 [9]. Although HPLC has contributed substantially to pathophysiological analysis, it is limited by long measurement times and difficulty in identifying specific oxidative modification structures.

Methods developed using electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) have enabled high-sensitivity, high-throughput analysis of HSA PTMs [18–20]. This approach has revealed that the major molecular species of oxidized albumin is cysteinylated albumin (Cys-Cys34-HSA), in which a single cysteine molecule is attached to Cys34 (Figure 3). The proportion of cysteinylated albumin measured by ESI-TOFMS shows a strong positive correlation with results from conventional HPLC methods, establishing it as a clinically applicable oxidative stress marker [18].

Figure 3.

Mass spectrometric profiling of HSA microheterogeneity. Deconvoluted ESI-TOFMS spectra illustrating the diversity of HSA species in chronic kidney disease. Identified peaks correspond to reduced HSA and oxidized HSA bearing a Cys34 mixed disulfide with cysteine (cysteinylated albumin), the predominant oxidized molecular species.

3.2. Quantification of oxidized polysulfides in albumin

A major obstacle to elucidating the physiological role of Ox-PSS had been the absence of appropriate analytical tools. Conventional assays for hydrogen sulfide and sulfide ions—including the methylene blue method [21], electrode-based techniques, and derivatisation assays [22]—are not directly applicable to Ox-PSS embedded within protein structures. To overcome this limitation, the Elimination Method for Sulfide from Polysulfide (EMSP) was developed [23]. This approach exploits the susceptibility of Ox-PSS to alkaline hydrolysis, resulting in the release of sulfide ions that are quantitatively detected under stabilized conditions; ascorbic acid is employed to prevent sulfide oxidation during the assay.

Application of EMSP to purified HSA revealed that a single albumin molecule endogenously contains approximately 15 mol/mol of Ox-PSS–derived sulfide [23]. Comparative analyses across diverse proteins demonstrated a strong correlation between Ox-PSS content and cysteine residue number, whereas no such correlation was observed with methionine content. This finding provides the first direct evidence that polysulfide bridges are abundant structural features of albumin rather than rare post-translational modifications. Complementary mass spectrometric analyses further suggest that multiple intramolecular cysteine–cysteine bridges participate in polysulfide bond formation [24].

Although EMSP provides the first robust quantitative approach to Ox-PSS measurement in proteins, several important limitations and unresolved questions must be acknowledged. First, EMSP measures total Ox-PSS content and cannot currently resolve which specific cysteine bridges carry polysulfide bonds within albumin's structure; complementary mass spectrometric data [17,24] implicate multiple sites but do not provide definitive residue-level assignments. Second, the physiological stability of Ox-PSS bonds under the redox conditions prevailing in circulating plasma—including variation in pH, temperature, and the presence of competing thiol species—has not been systematically characterized. Third, the quantitative data on albumin polysulfide content reported to date derive predominantly from the authors' own research groups, and independent replication by other laboratories will be essential to validate both the reported absolute values and the disease-associated changes. Fourth, the selectivity of alkaline hydrolysis for Ox-PSS over other labile sulfur-containing species present in albumin has not been exhaustively validated, and orthogonal analytical approaches will be important for confirming specificity. Addressing these gaps will be critical to establishing albumin polysulfides as a robust and reproducible dimension of protein redox biology with translational relevance.

4. Albumin polysulfides: functional significance and redox mechanism

4.1. Effect of polysulfides on albumin structure and function

To clarify the functional significance of albumin polysulfides, selectively and partially Ox-PSS–depleted albumin preparations were generated using Sulfane Sulfur Probe 4. Partial depletion was employed to avoid albumin aggregation. Even partial removal of Ox-PSS induced detectable alterations in both secondary and tertiary structures as assessed by circular dichroism spectroscopy, accompanied by increased exposure of hydrophobic surface regions, indicating destabilization of the native conformation. Functionally, partial Ox-PSS depletion markedly impaired albumin's drug-binding capacity at Site II—the binding site responsible for a wide range of acidic and neutral drugs at subdomain IIIA of HSA [25]. Reductions in Site II binding capacity have also been reported in oxidative stress–related diseases, such as acute kidney injury and chronic kidney or liver disease [26,27], suggesting that reduced albumin polysulfides may contribute to disease-related changes in pharmacokinetics and an increased incidence of drug side effects.

In addition to drug binding, removal of even a small fraction of Ox-PSS significantly reduced albumin's antioxidative activity, including hydrogen peroxide scavenging and free radical quenching, indicating that specific polysulfide sites may play dominant roles in redox buffering.

4.2. Dynamic polysulfide redox cycling

A recent report demonstrated that Ox-PSS reacts directly with reactive oxygen species (ROS) to be converted to Red-PSS, which transiently enhances antioxidant activity [24]. Under sustained pathological oxidative stress, irreversible oxidation of albumin occurs through consumption of Red-PSS, leading to a progressive decline in both antioxidant capacity and drug-binding capacity (Figure 4). These findings demonstrate the existence of a novel in vivo redox regulation mechanism mediated by albumin polysulfides and establish HSA as a dynamic Ox-PSS reservoir capable of responding to fluctuating redox conditions.

Figure 4.

Biosynthesis of albumin polysulfides and their relationship to antioxidant and drug-binding activities. Under physiological conditions, albumin contains oxidized form of polysulfides (Ox-PSS) at multiple intramolecular cysteine bridges. Hydrolysis reaction converts Ox-PSS to the reduced form of polysulfides (Red-PSS), transiently enhancing antioxidant activity. Sustained pathological oxidative stress causes irreversible consumption of Red-PSS, resulting in a progressive decline in both antioxidant capacity and Site II drug-binding capacity.

From a chemical perspective, the reactivity of Ox-PSS toward ROS is rooted in the unique electronic properties of polysulfide bonds. These bonds exhibit both nucleophilic and electrophilic character, enabling versatile interactions with a range of oxidants, including hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl), and superoxide (O₂⁻•). Central to this reactivity is the sulfane sulfur atom within the polysulfide chain, which possesses pronounced electrophilicity [24]. This makes it particularly susceptible to nucleophilic attack by ROS-derived species, leading to cleavage or rearrangement of the polysulfide bond. Such reactions generate transient Red-PSS intermediates that can further participate in redox cycling. Insights from persulfide chemistry suggest that these transformations are not merely degradative but functionally adaptive, allowing polysulfides to act as both sensors and scavengers of oxidative stress. Experimental evidence specifically demonstrates that albumin-bound Ox-PSS undergoes rapid ROS-dependent conversion, reinforcing the concept of albumin as an active participant in extracellular redox chemistry rather than a passive carrier protein.

The role of albumin polysulfides must also be considered within the broader thiol–disulfide and hydrogen sulfide (H₂S)-related redox network. Circulating low-molecular-weight supersulfide species, such as cysteine persulfide and glutathione polysulfides, contribute to systemic sulfane sulfur pools. Given its high plasma concentration, albumin likely serves as a major extracellular reservoir of sulfane sulfur, capable of buffering and redistributing these reactive sulfur species. The H₂S- and/or polysulfide-producing enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) may indirectly support the replenishment of albumin-bound polysulfides by generating circulating sulfane sulfur species and related sulfur metabolites. Additionally, potential exchange reactions between albumin polysulfides and small-molecule thiols or persulfides could facilitate dynamic sulfur trafficking. However, it is important to note that direct evidence for such exchange mechanisms in the extracellular compartment remains limited, and further investigation is needed to clarify these pathways.

Importantly, the dynamics of albumin polysulfides are mechanistically distinct from, yet closely linked to, the well-characterized redox states of the Cys34 residue. Under conditions of mild oxidative stress, Ox-PSS serves as a first-line defense system: its conversion to Red-PSS enhances ROS-scavenging capacity without immediately altering the redox state of Cys34. This positions polysulfide metabolism upstream in the redox response hierarchy. In contrast, during prolonged or severe oxidative stress, the depletion of Red-PSS diminishes this buffering capacity, leading to subsequent oxidation of Cys34. This progression results in the formation of human nonmercaptoalbumin species (HNA1 and HNA2), which reflect increasingly irreversible oxidative modifications. Thus, a two-tiered model emerges: an early, reversible polysulfide-mediated response followed by a later, thiol-centered oxidative shift. Supporting this framework, experimental observations indicate that depletion of polysulfides can precede detectable changes in the mercaptoalbumin ratio, highlighting their role as sensitive and early responders to redox imbalance (Figure 5).

Figure 5.

Mercaptoalbumin ratio and serum supersulfides in healthy individuals and patients with early hepatitis. Serum samples were obtained from healthy volunteers (n = 10) and patients with early hepatitis in the context of chronic liver disease (n = 10). The mercaptoalbumin ratio and serum supersulfides were analyzed by LC–MS/MS and EMSP, respectively. Serum polysulfides were significantly reduced in the early hepatitis group despite no significant change in the mercaptoalbumin ratio, demonstrating that polysulfide assessment detects redox impairment at an earlier stage than conventional Cys34-based markers.

Taken together, albumin-bound polysulfides emerge as a critical and previously underappreciated component of extracellular redox regulation. Their capacity to directly quench ROS, interface with systemic sulfur metabolism, and influence downstream thiol oxidation supports a multilayered defense network that sustains redox homeostasis. When this system is disrupted under pathological oxidative stress, the consequences extend beyond a loss of antioxidant buffering to include impairment of albumin's ligand-binding and transport functions, thereby potentially contributing to disease progression and altering therapeutic outcomes.

Despite these advances, key mechanistic gaps remain. In particular, little is known about whether HSAs differing in ‘redox quality’ (e.g. Ox-PSS versus Red-PSS) exhibit distinct cellular uptake profiles (e.g. Megalin/cubilin on renal tubular cells or neonatal Fc receptor on endothelial cells, etc.), or whether albumin-bound polysulfides can directly mediate intracellular sulfur transfer. At present, these concepts remain hypothetical, as illustrated in Figure 6. Addressing these uncertainties will require rigorous experimental validation, highlighting an urgent need for studies that provide direct and quantitative evidence of these processes.

Figure 6.

Proposed model of extracellular albumin polysulfide dynamics and cellular interactions. In the blood compartment (upper panel), albumin bearing oxidized polysulfides (Ox-PSS; SSnS) undergoes ROS-driven conversion to the reduced polysulfide form (Red-PSS; SSnH), transiently enhancing antioxidant activity while decreasing Ox-PSS content. Low-molecular-weight supersulfide donors, including cysteine persulfide (CysSSH) and glutathione persulfide (GSSH), may replenish albumin polysulfide content via sulfane sulfur exchange in the extracellular space. In the cellular compartment (lower panel), endothelial cells and renal proximal tubular cells interact with circulating albumin through FcRn-mediated recycling and reabsorption pathways, respectively. Enzymatic sources of sulfane sulfur, including cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST), may further supplement albumin polysulfide levels. Whether albumin-bound polysulfides can directly transfer sulfane sulfur to intracellular targets following cellular uptake remains a hypothesis requiring experimental validation.

5. Pathophysiological significance

In the field of nephrology in particular, oxidized albumin could be emerging not only as a biomarker but also as a therapeutic target and active pathogenic substance. The evidence reviewed in this section encompasses a diverse range of study designs, spanning from longitudinal human cohorts (e.g. the PREVEND study [28]), and cross-sectional observational data to in vitro and animal models. Consequently, the strength of causal inference derived from these findings varies accordingly. It should be explicitly noted that oxidized albumin may function as a biomarker, a mediator, or an epiphenomenon depending on the clinical context. This distinction remains incompletely resolved for most of the conditions discussed herein.

5.1. Chronic kidney disease and cardiovascular risk

A 10-year follow-up study of 4745 healthy individuals (the PREVEND study) demonstrated that lower serum free thiol levels are significantly associated with a higher risk of developing CKD [28]. Since approximately 80% of these thiol groups originate from Cys34, a decrease in thiol content reflects a shift from reduced to oxidized albumin, suggesting that systemic oxidative stress and accumulation of modified albumin are associated with renal decline, though whether oxidative modification of albumin is a driver, accelerant, or consequence of disease progression has not been established in interventional studies. It is well established that HMA decreases and oxidized albumin (HNA1, HNA2) increases as renal function declines in patients with non-dialysis CKD and those on dialysis [9,29,30]. Low HMA levels in dialysis patients significantly correlate with the risk of cardiovascular disease (CVD) onset and CVD-related mortality, consistent with a contribution of oxidative stress to atherosclerosis progression, though causality remains to be demonstrated. Additionally, in peritoneal dialysis patients, higher serum HMA levels have been reported to correlate with higher peritoneal transport status [31].

5.2. Highly oxidized albumin in focal segmental glomerulosclerosis

In patients with active focal segmental glomerulosclerosis (FSGS), highly oxidized albumin—in which Cys34 is irreversibly oxidized to sulfonic acid (─SO₃H)—has been specifically detected in plasma [32]. This oxidative modification correlates with disease activity and may be useful for predicting recurrence after transplantation.

5.3. Prognostic prediction in diabetic kidney disease and diabetic retinopathy

Cysteinylated albumin has been shown to predict early progression of diabetic kidney disease (DKD)—specifically the transition from stage G2 to G3a—with higher accuracy than urinary albumin excretion [33]. Regarding diabetic retinopathy [34], a 2025 exploratory retrospective cohort pilot study demonstrated that baseline HNA% in patients with simple diabetic retinopathy was associated with improvement in retinopathy after 5 years [35]. The baseline HNA% was significantly lower in the improvement group, and this association was independent of HbA1c levels during follow-up, suggesting that HNA% may reflect cumulative oxidative stress, including past glycaemic fluctuations (the legacy effect)—a prognostic dimension distinct from HbA1c. These preliminary findings require confirmation in larger prospective cohorts.

5.4. Alport syndrome and Nrf2 activation therapy

A 2025 study by Kaseda et al. demonstrated that UD-051, an Nrf2 activator (Keap1–Nrf2 interaction inhibitor), dose-dependently suppressed decline in renal function, glomerular sclerosis, inflammation, and fibrosis in a mouse model of Alport syndrome, thereby extending survival [36]. Plasma oxidized albumin ratio showed a strong inverse correlation with expression levels of Nrf2 target genes (Nqo1, Gclc) in renal tissue, indicating that plasma oxidized albumin is a sensitive biomarker reflecting antioxidant capacity (Nrf2 activity) within renal tissue.

5.5. Induction of ferroptosis: a novel pathogenic mechanism

Although it has long been suggested that oxidized albumin possesses intrinsic toxicity capable of exacerbating renal damage, a 2025 study identified ferroptosis as the specific underlying mechanism [37]. Exposure of renal tubular epithelial cells (NRK-52E) to oxidized albumin induced cell death characterized by intracellular albumin accumulation, increased lipid peroxidation, intracellular iron accumulation, and decreased expression of antioxidant factors GPX4 and xCT, together with increased expression of the lipid metabolism enzyme ACSL4; these changes were suppressed by ferroptosis inhibitors. Administration of oxidized albumin to a doxorubicin-induced nephropathy mouse model exacerbated renal function, proteinuria, and tubular injury in vivo. These findings raise the possibility that oxidized albumin may function not merely as a consequence of oxidative stress but as an active contributor to renal tubular injury. However, these conclusions are based on a single preclinical study using one cell line and one mouse model; independent replication across a range of nephropathy models and, ultimately, clinical validation will be required before the ferroptosis mechanism can be established as a general pathogenic pathway.

5.6. Impact of AOPPs on renal health and sarcopenia

Advanced oxidation protein products (AOPPs) are formed when hypochlorous acid (HOCl), produced by neutrophils during inflammation, oxidatively modifies serum proteins—primarily albumin. AOPPs trigger tubular disorders by inducing abnormal fatty acid metabolism in renal cells [38] and contribute to muscle wasting and weakness (sarcopenia) by causing mitochondrial dysfunction in skeletal muscle [39,40]. Serum AOPPs and oxidized albumin levels are significantly elevated in dialysis patients with sarcopenia compared with those without [39], suggesting utility as diagnostic tools for sarcopenia in the CKD population. Since the link between AOPPs and sarcopenia is supported by mechanistic data from animal models [39,40] and human cross-sectional associations [39], further longitudinal or interventional studies in humans are warranted.

5.7. Decline of functional albumin in liver disease

In chronic liver disease (CLD), cysteinylated albumin increases progressively with advancing Child–Pugh classification stage [18,41]. Oxidatively modified HSA exhibits reduced binding affinity for bilirubin and drugs, including warfarin and diazepam, impairing its function as ‘functional albumin’ [18,27,34,41,42]. Administration of branched-chain amino acid (BCAA) preparations has been confirmed not only to promote albumin production but also to restore the proportion of HMA and improve ligand-binding capacity [18,41], demonstrating that therapeutic intervention can produce measurable qualitative improvement in circulating albumin.

A critical caveat applies across the pathophysiological evidence reviewed in this section. With few exceptions, associations between albumin oxidation and disease outcomes derive from observational human studies, in vitro experiments, or limited animal models. These data are insufficient to establish definitively whether oxidized albumin functions as a causative driver, a consequential biomarker, or an innocent bystander in disease pathogenesis. Human interventional studies demonstrating that restoration of reduced albumin or prevention of Cys34 oxidation translates into improved clinical outcomes are largely absent and represent an important research priority. Until such evidence is available, claims about the pathogenic role of oxidized albumin should be interpreted with appropriate caution.

6. Clinical and pharmaceutical applications

6.1. Reduced albumin as a sensitive biomarker for nutrition and physical function

Two recent studies highlight the proportion of reduced albumin as a more sensitive indicator of health status in older adults than total serum albumin concentration. Motokawa et al. reported that while conventional serum albumin levels in community-dwelling older adults do not correlate with protein intake, the ratio of reduced albumin is significantly associated with insufficient dietary protein intake, suggesting its utility as an indicator of mild malnutrition [43]. Shibasaki et al. demonstrated that reduced albumin is linked to physical function during resistance training interventions in middle-aged and older adults; a positive correlation was observed between improvement in maximum walking speed and the increase in reduced albumin following training [44]. Together, these findings suggest that reduced albumin reflects both protein nutritional status and motor function or training efficacy in older adults.

6.2. Clinical implications of albumin polysulfides

Oxidative stress is a hallmark of numerous chronic diseases, including CKD, CLD, cardiovascular disease, and chronic inflammatory disorders [45]. Clinical studies have demonstrated that polysulfide levels in circulating albumin are significantly reduced in patients with CKD compared with healthy controls. Notably, polysulfide levels increase rapidly following hemodialysis, reflecting the removal of oxidative stress–inducing substances, such as ROS and uremic toxins, highlighting the dynamic responsiveness of albumin polysulfides to systemic redox conditions [46].

Beyond CKD, alterations in albumin redox state have been reported in CLD, sepsis, and acute inflammatory conditions [26,27]. Importantly, serum polysulfides are significantly reduced in patients with early hepatitis in the context of CLD, even when the mercaptoalbumin ratio—a conventional oxidative stress marker—remains unchanged (Figure 5). This observation suggests that albumin polysulfide assessment may provide additional qualitative information that enhances risk stratification and disease monitoring at earlier stages of oxidative stress than conventional Cys34-based markers allow. Low serum albumin levels have also been associated with poor outcomes in severe infections, such as COVID-19 [47,48]; in this context, assessment of albumin polysulfide content may offer further qualitative insight beyond total albumin concentration.

It should be noted that current clinical data do not permit definitive discrimination between causative and consequential roles of polysulfide alterations in disease progression. The strong association between polysulfide depletion and oxidative stress, however, supports albumin polysulfide as a sensitive biomarker reflecting functional impairment beyond what protein concentration alone can indicate.

6.3. Quality evaluation of albumin preparations and recombinant products

The degree of PTMs in clinical albumin preparations may vary depending on manufacturing processes and storage conditions. Comparative analyses of commercial preparations have demonstrated that preparations with a higher proportion of cysteinylated albumin exhibit lower ligand-binding and antioxidant capacities [49], indicating that the degree of Cys34 oxidation may be a useful quality indicator for evaluating the functional quality of such preparations.

6.4. Pharmacological and biopharmaceutical perspectives

From a pharmaceutical standpoint, albumin polysulfides have important implications for drug development and therapy. Albumin is widely exploited as a drug carrier through both non-covalent binding and covalent conjugation strategies. Changes in albumin structure and binding properties resulting from altered polysulfide content may therefore influence drug distribution, efficacy, and toxicity. Furthermore, modulation of albumin polysulfides represents a potential therapeutic strategy: enhancement of polysulfide content may improve albumin's antioxidative capacity and stabilize drug-binding functions under pathological conditions. Proof-of-concept has already been demonstrated—albumin with increased polysulfide content has been engineered to alleviate kidney injury in a rhabdomyolysis model [50].

Albumin polysulfides represent a previously unrecognized qualitative dimension of albumin redox biology that extends our understanding beyond classical antioxidant mechanisms. Further evidence will clarify how albumin functions as a dynamic polysulfide reservoir contributing to systemic redox homeostasis and influencing both drug-binding and antioxidant properties.

7. Conclusion

The framework of ‘redox albuminomics’ provides a crucial approach for evaluating qualitative changes in HSA—particularly the oxidation state of Cys34—as sensitive biomarkers of systemic oxidative stress. Advanced analytical techniques, such as ESI-TOFMS, have enhanced our ability to identify specific oxidative modifications, establishing oxidized albumin as a clinically relevant marker across a range of diseases. Beyond its role as a biomarker, oxidized albumin exhibits intrinsic pathogenicity in CKD, contributing to cardiovascular risk, sarcopenia, and—as recently demonstrated—ferroptosis-mediated renal tubular cell death. Its utility further extends to nutritional and physical function assessment in older adults and to pharmaceutical quality control.

Complementing this established biology, the assessment and regulation of albumin polysulfides opens new opportunities for biomarker development and therapeutic intervention in oxidative stress–related diseases. Albumin polysulfides are dynamically regulated, respond more sensitively than Cys34-based markers at early disease stages, and directly influence drug-binding and antioxidant function. Future research targeting oxidized albumin and albumin polysulfides as therapeutic objectives holds significant potential for improving clinical outcomes. Advances in protein polysulfide measurement techniques and the development of agents that promote albumin polysulfide formation are anticipated to become increasingly important research priorities.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Funding

No funding was received for this work

Data availability statement

No primary research results, software or code have been included, and no new data were generated or analyzed as part of this review.