Binding and transport functions of human serum albumin and its clinical implications in liver disease: a narrative review.

Introduction
Liver cirrhosis (LC) represents the advanced stage of several liver diseases, including hepatitis B and C infections, metabolic dysfunction-associated steatotic liver disease, chronic alcohol consumption, autoimmune disorders, and other related conditions. Cirrhosis and other chronic liver diseases account for over 1.4 million deaths globally each year. At the national level, China reports the highest number of new cases annually, with over 400,000 individuals diagnosed with cirrhosis and other chronic liver conditions (1). In 2020, the Disease Surveillance Point System of the Chinese Center for Disease Control and Prevention reported a total of 15,375 deaths attributed to cirrhosis (4.57/100,000 people) (2). In Southern China, common LC-related complications typically involve ascites, hepatocellular carcinoma (HCC), upper gastrointestinal bleeding, hepatopulmonary syndrome, hepatic encephalopathy, and hepatorenal syndrome (HRS) among others. The occurrence of complications may lead to an increased risk of death in patients with LC (3). Currently, human serum albumin (HSA) is recommended for the management of complications associated with LC, including HRS, large-volume paracentesis, and spontaneous bacterial peritonitis (SBP) (4).
HSA, the most abundant protein in human plasma, contributes 75% of total plasma oncotic pressure and is therefore used to increase circulating blood volume (5). However, the importance of HSA in liver disease is not limited to its colloid functions. Recently, increasing research has focused on its non-colloid functions. HSA also possesses many other biological functions, including binding, transporting, and detoxifying endogenous and exogenous compounds, antioxidant activity, and regulating inflammation and immune responses.
Currently, LC has a high incidence and mortality rate in China. To effectively reduce the burden of LC, we need to focus on strategies for its development and progress. This article aims to review the clinical application of HSA’s binding and transport functions in the diagnosis and treatment of liver diseases, providing insights into the comprehensive management of these conditions. We present this article in accordance with the Narrative Review reporting checklist (available at https://tgh.amegroups.com/article/view/10.21037/tgh-25-72/rc).

Methods
We performed a computerized search in PubMed and Embase, restricting the results to articles published in English and Chinese from January 2000 to March 2025. The search keywords use Medical Subject Headings and related entry terms, including terms related to “serum albumin, human”, “recombinant human albumin”, “antineoplastic agents”, “analgesics”, “anti-bacterial agents”, “diuretics”, “antiviral agents”, “fatty acids”, “ferritins”, “bilirubin”, “protein conformation”, and “Amino Acid Sequence” (Table 1). We evaluated in-text references of relevant publications and conducted inverse searches based on the identified papers in these databases to uncover further relevant studies that were not captured by the automated search process.

Structural basis and potential mechanisms of binding and transport functions of HSA

The structural features of HSA
HSA is primarily synthesized in the liver by hepatocytes. The process begins with the production of preproalbumin, which contains a 24-amino-acid N-terminal extension, including an 18-amino-acid signal peptide and a 6-amino-acid pro-peptide (6,7). The signal peptide is cleaved in the endoplasmic reticulum to form proalbumin (6). Proalbumin is then transported to the Golgi apparatus, where it undergoes additional post-translational modifications (PTMs) to become mature HSA before being secreted into the bloodstream (7). Mature HSA consists of 585 amino acids with a molecular weight of 66.5 kDa and features a long half-life of about 18 days (8).
HSA is an asymmetric, heart-shaped molecule, approximately 7 nm in diameter, composed of three structurally similar homologous α-helical domains: Domain I (residues 1–195), Domain II (residues 196–383), and Domain III (residues 384–585). All the domains consist of ten α-helices and are further divided into two subdomains, which are typically designated as subdomains IA, IB, IIA, IIB, IIIA, and IIIB. Each subdomain A is composed of six α-helices, while each subdomain B comprises four α-helices (9). Furthermore, HSA contains 35 cysteine residues that participate in the formation of 17 disulfide bridges, thereby stabilizing the conformation of this globular protein. This structural organization facilitates the allosteric properties of monomeric HSA, enabling it to bind multiple ligands effectively (8).

Binding sites for HSA
Pioneering research conducted by Sudlow and colleagues in 1975 and 1976, employing a fluorescent probe displacement technique, demonstrated the presence of two distinct potential drug-binding sites on HSA (10,11). These sites were identified as drug-binding site I located in subdomain IIA, and binding site II situated in subdomain IIIA (9).
Binding site I is a pre-formed hydrophobic cavity, encompassing all six helices of the subdomain IIA and a loop-helix feature contributed by IB (residues 148–154). The interior of the cavity is predominantly nonpolar, featuring two clusters of polar residues: an inner cluster at the base (Tyr150, His242, and Arg257) and an outer cluster at the entrance (Lys195, Lys199, Arg218, and Arg222). In addition to hydrophobic contacts, drug-binding site I compounds engage in several specific interactions with residues from both the inner and outer polar clusters. The abundance of basic residues defines the ligand-binding specificity of this site (12). Ligands that exhibit strong affinity for drug-binding site I are typically dicarboxylic acids and/or bulky heterocyclic molecules characterized by a centrally localized negative charge (13).
Binding site II also comprises a largely pre-formed hydrophobic cavity with distinct polar features, but there are significant differences between the two drug-binding pockets. This drug-binding pocket is primarily hydrophobic and features distinctive electrostatic characteristics. Polar residues are located at one side of the entrance to the binding pocket. Key residues within this site, such as Arg410, Ser489, and Lys414, play crucial roles in interacting with associated ligands (12). Drug-binding site II predominantly accommodates aromatic carboxylates, which tend to adopt extended conformation (13).
A new drug-binding site, designated as drug-binding site III within subdomain IB, has been identified as the principal binding location for various compounds, including a photoisomer of hemin, steroid antibiotics like fusidic acid, and sulphonamide derivatives (14,15). This site has a higher affinity for endogenous ligands and heterocyclic compounds, with Tyr138, Tyr161, Arg141, and Lys190 being key residues at the ligand binding site (16).

Clinical value of the binding and transporting ability of HSA to endogenous substances

The physiological function of HSA
Normally, HSA levels fluctuate between 35 and 55 g/L. This range is typically maintained through the regulation of albumin synthesis, distribution, and degradation. HSA synthesis is self-regulated and occurs within vesicles in hepatocytes, with approximately 10–15 g of HSA synthesized daily under physiological conditions. However, in cases of advanced liver disease, the loss of hepatocytes and ongoing inflammation can impair albumin synthesis (17).
The primary physiological and biological functions of HSA in both health and disease states include: (I) anticoagulant and antithrombotic effects—HSA promotes the binding of nitric oxide (NO) to the domain of cysteine, enhancing the antiaggregatory property of platelets by preventing rapid inactivation of NO, while also resisting inflammation and scavenging plasma free radicals (18,19); (II) binding and transportation of drugs, ions, fatty acids (FA), bile acids, bilirubin, gases, ligands, hormones, and calcium ions (20).
According to the current research status, more research focuses on the binding and transport of HSA with FA, bilirubin and iron ions. Next, we will elaborate on the function of albumin in binding and transporting molecules.

PTMs of HSA
The PTMs of HSA generate multiple isoforms with distinct structures, consequently altering its functional properties. To date, 123 distinct PTMs of HSA have been identified, including O-phosphorylation, acetylation, glycosylation, methylation, carbonylation, oxidation, and homocysteinylation (21).
The PTMs sites of HSA (e.g., phosphorylation, glycosylation, acetylation, and carboxylation sites) exhibit a pan-domain distribution pattern. Phosphorylation sites are strategically positioned to potentially interfere with lipid binding and structural stability. The introduction of negatively charged phosphate groups into the C-terminal domain may significantly disrupt binding affinity and conformational integrity, while increased hydrophilic surface exposure could impair the functionality of two critical lipid-binding sites. Glycosylation sites are implicated in modulating metal binding, lipid interaction sites, and protein stability. Lysine glycosylation reduces basic charge by blocking amino groups and introducing bulky glycan moieties. Domain-specific glycosylation may elevate the isoelectric point, redistribute hydrophobic/hydrophilic surface regions, and ultimately compromise HSA functionality (21). Acetylation similarly affects lipid-binding capacity. Carbonylation, an irreversible non-enzymatic PTMs, introduces aldehyde/ketone groups into HSA under oxidative stress conditions, leading to functional impairment (22). The high reactivity of cysteine thiol (-SH) groups in HSA’s amino acid sequence enables diverse modifications, including oxidation, nitrosylation, homocysteinylation, and Cys-Cys dimerization. The PTM’s status of Cys34 may reflect competitive modification dynamics under varying physiological and pathological conditions (21). Furthermore, circulating HSA undergoes minor truncation at the N-terminal and/or C-terminal regions. Naldi et al. identified aspartate-alanine residue deletion as the predominant N-terminal truncation form in cirrhotic patients, which significantly impairs antioxidant capacity (23). The most frequent C-terminal truncation involves leucine residue loss, typically reducing protein stability and shortening its half-life (24,25). PTMs serve as the core pathophysiological mechanism underlying the reduction in effective albumin concentration (eAlb) (26). Concurrently, the resultant structural alterations significantly impair its binding capacity for bilirubin, FAs, chelated metal ions, and drugs (e.g., warfarin).

HSA binding with FA
Binding site I is specific for heterocyclic anions with dispersed charges, while site II is specific for aromatic carboxylates. These sites are also referred to as “warfarin sites” and “ibuprofen sites”, reflecting their preference for two significant drug molecules. He and Carter utilized X-ray crystallography to locate these binding sites within subdomain IIA (Sudlow site I) and subdomain IIIA (Sudlow site II) of the HSA structure (27). Additionally, Bhattacharya et al. speculated, based on X-ray crystallography, that there may be as many as seven different FA binding sites in HSA (28). Using a 7-nitrobenzo-2-oxa-1,3-oxadiazol-4-yl (NBD)-C12 FA, a single molecule was identified by Wenskowsky et al. within the crucial transport HSA (29). This study focused on high-affinity FA binding sites and their characterization. The binding ratio of this ligand to the HSA complex is 1:1, indicating a highly site-specific interaction. Titration experiments and radioactive equilibrium dialysis assays confirmed that the newly identified binding site is distinct from binding sites I and II. Furthermore, the X-ray crystal structure analysis facilitated the localization of the binding site within HSA subdomain IIA. Under normal physiological conditions, approximately three FA molecules are bound to each HSA (30).
The pathogenesis of metabolic dysfunction-associated steatohepatitis (MASH) is characterized by lipotoxicity, inflammation, and fibrosis (31). Reduced levels of HSA diminish FA binding capacity, thereby elevating the concentration of free FAs (FFAs) in the bloodstream (32). Elevated FFAs can lead to inflammation, oxidative stress, mitochondrial dysfunction, and uncoupled oxidative phosphorylation, triggering a fibrogenic response in hepatic stellate cells that may progress to MASH and cirrhosis (33,34). FFAs have the capacity to bind to and activate Toll-like receptor 4, which is expressed by both parenchymal and non-parenchymal cell types in the liver (35). This activation initiates pro-inflammatory cytokine cascades that play a significant role in the pathophysiology and clinical outcomes associated with severe liver injuries (35). HSAs facilitate the predominant release of FFAs into systemic circulation, delaying the progression of MASH (36).

HSA binding with bilirubin
Bilirubin is one of the end products of human hemoglobin catabolism. It acts as a high-affinity endogenous ligand for HSA (37). Like many weakly polar and poorly soluble compounds, bilirubin is transported in the blood predominantly bound to HSA, with less than 0.01% of total bilirubin existing in its unbound form (i.e., free bilirubin) (38,39). In physiological conditions, HSA-bilirubin complex consists of binding of one molecule of HSA with one molecule of bilirubin, but in liver diseases with high bilirubin concentrations, HSA-bilirubin complex is loaded with three molecules of bilirubin (20). The binding of bilirubin to subdomain IB of HSA can inhibit lipid peroxidation (40). Once HSA binds with bilirubin, the complex circulates through the bloodstream to the liver, where bilirubin is absorbed and subsequently secreted into bile for excretion (41).
All liver lesions induce a decrease in the hepatocyte cell count, which may cause hyperbilirubinemia (42). The binding of unconjugated bilirubin to HSA prevents isomerization and aids its transport to the liver (42). The interaction between HSA and bilirubin is essential for liver detoxification, particularly in acute and chronic liver diseases, as it helps maintain bilirubin balance and minimize toxic damage. When the concentration of HSA decreases, a larger proportion of bilirubin remains unbound, facilitating its crossing of the blood-brain barrier and potentially leading to neurological damage (43). Additionally, the HSA-bilirubin binding capacity dynamically indicates liver function reserve. Bilirubin is primarily transported to the liver for metabolism via HSA, making the albumin-bilirubin (ALBI) score a valuable index for assessing liver function (44,45). This score aids in prognostic assessment for chronic liver diseases, including cirrhosis and HCC, and helps predict risks such as post-hepatectomy liver failure (46-48). While the ALBI score’s specificity for liver function lacks definitive mechanistic validation, its clinical utility parallels other indices like the Model for End-Stage Liver Disease and Child-Pugh scores, remaining pragmatically valuable despite theoretical limitations. In liver diseases (e.g., cirrhosis or acute injury), hepatocyte mass is reduced, leading to hyperbilirubinemia (49). Reduced HSA levels impair bile acid binding capacity, exacerbating cholestasis and intrahepatic inflammation, thereby perpetuating liver disease progression (50,51). HSA binding prevents bile acid-induced cytotoxicity to intestinal mucosa and hepatocytes (52).

HSA binding with Fe2+
HSA is essential for hemoglobin-iron clearance, providing protection against oxidative damage caused by free hemoglobin-iron, limiting pathogen exposure to hemoglobin-iron, and promoting iron homeostasis through the recycling of hemoglobin-iron atoms (46,53). Notably, within the first few seconds after hemoglobin-iron enters the plasma, over 80% binds to high-density lipoprotein (HDL) and low-density lipoprotein (LDL), leaving only 20% to bind with hemopexin (HPX) and HSA. Subsequently, HPX and HSA slowly transport hemoglobin-iron from HDL and LDL (54). The HSA-hemoglobin-iron complex is then internalized via endocytosis mediated by cluster of differentiation (CD) 71 and CD91 receptors in liver macrophages, allowing its release into liver parenchymal cells (53).
Iron overload occurs in 10–30% of chronic liver disease patients, with up to one-third of MASH patients showing increased liver iron (55). Excess or free iron in the liver contributes to the formation of reactive oxygen species (ROS) via the Fenton reaction, increasing oxidative stress, which is a known cause of liver damage (56-58). HSA demonstrated binding affinities ranging from 60% and 20% of the available iron when the iron concentration was varied across the typical clinical range of free iron concentrations (1–10 µM), indicating HSA as a significant ligand for free iron (59,60). The sequestration of free iron by proteins can modulate its reactivity and restrict its availability for participation in the Fenton reaction (60). HSA demonstrates a strong affinity for binding free heme, thereby enhancing its scavenging and playing a crucial role in maintaining cellular homeostasis (53).

Clinical value of the binding and transporting ability of HSA to exogenous substances
HSA is employed in combination therapy for various liver diseases, including circulatory dysfunction associated with common complications of decompensated LC, by leveraging its dual permeability and non-permeability properties. To date, research on the mechanisms underlying HSA combined with other therapeutic modalities remains limited. Therefore, our focus will be on commonly used drugs for treating LC and its complications—including antiviral agents, antibiotics, diuretics, and HCC therapeutic drugs—with particular attention to their binding affinity and transport mechanisms when interacting with HSA.

HSA and antiviral drugs
The liver serves as the primary site of replication for various viruses, including hepatitis viruses (e.g., hepatitis A virus, hepatitis B virus), cytomegalovirus, Epstein-Barr virus, and herpes simplex virus. Infection with these viruses can lead to a range of pathological conditions, including both acute and chronic hepatitis, LC, hepatic failure, and HCC (61). Without antiviral treatment, the risk of LC in hepatitis B patients can reach as high as 40%, whereas in hepatitis B patients, the risk of cirrhosis ranges from 10% to 20% (62). Currently, several antiviral agents, including lamivudine, entecavir, and ribavirin, are recommended for the treatment of chronic hepatitis B and chronic hepatitis C (63,64). Recently, the interactions between these drugs and HSA have been increasingly investigated and revealed. Li et al. [2014] investigated the interactions between ribavirin, lamivudine, and HSA using fluorescence spectroscopy and X-ray crystallography. They reported that both ribavirin and lamivudine bind to the IIA subdomain of HSA, primarily through hydrogen bonding and hydrophobic interactions. Furthermore, both drugs share binding sites within the IIA subdomain of HSA, exhibiting competitive inhibition (65). Abubakar et al. [2024] elucidated the molecular association between entecavir and HSA, revealing that entecavir forms a stable complex with HSA through hydrogen bonds, hydrophobic interactions, and van der Waals forces, with docking studies indicating that entecavir binds to subdomain IIA (site I) (66). These findings provide detailed structural and biochemical insights into the interactions between HSA and antiviral drugs, offering a scientific basis for the design and delivery of subsequent drug developments. An observational cohort study conducted by Perumalswami and colleagues found that among hepatitis C patients who were stable over the past year and treated with regimens containing sofosbuvir, lower baseline levels of HSA and higher levels of total bilirubin were identified as risk factors for decompensation and severe adverse events during treatment with sofosbuvir, with an adjusted odds ratio of 0.12 per g/dL decrease (P=0.01) (67). Normal baseline albumin is a surrogate marker for better overall synthetic liver function and a more robust patient health status, which are the reasons for a better therapeutic response (68). A study evaluated the effectiveness and safety of peg-interferon and ribavirin for hepatitis C virus infection. Results showed that patients with high baseline HSA levels (≥3.5 g/dL) had significantly better antiviral responses, with 84.5% achieving sustained virologic response (SVR) compared to those with lower HSA levels (P=0.03). Multivariate analysis confirmed HSA as an independent predictor of SVR (69).

HSA and antibiotic drugs
Based on clinical practice, patients with liver disease complicated by infection may require the use of antibiotics. Some of these antibiotics exhibit high protein binding rates, such as ceftriaxone sodium, levofloxacin, and imipenem. When determining the dosing regimen, it is crucial to consider albumin concentration and drug binding affinity, as this directly affects the free active concentration of the drug in the blood, thereby influencing the dosage, dosing frequency, and efficacy of the drug. Therefore, we focus on the binding sites of commonly used antibiotics with HSA to provide a theoretical basis for subsequent clinical studies.

Beta-lactam antibiotics
Kawai et al. investigated the binding of ceftriaxone and cefazolin to HSA. Their findings revealed that both antibiotics specifically bind to subdomain IB of HSA (70). Sabour et al. examined the interaction between aztreonam, a monobactam antibiotic, and HSA using various biophysical and computational techniques. Their results indicated that aztreonam binds to HSA with moderate affinity at the subdomain IB, causing conformational changes that reduce HSA’s α-helical content. The interaction is mainly stabilized by non-covalent interactions, such as hydrogen bonds, van der Waals forces, and π-anion interactions, as confirmed by molecular docking and dynamic simulations (71). Based on Fontana’s findings, a pharmacokinetic (PK) study compared the binding of two cephalosporins—ceftriaxone (high binding affinity for HSA) and cefotaxime (low binding affinity for HSA)—to penicillin-binding proteins (PBPs; β-lactam antibiotics inhibit bacterial growth by inactivating PBPs) in the presence of HSA at a concentration comparable to plasma levels. The results showed that 24 hours after administration, the serum concentration of ceftriaxone remained significantly higher than the minimum inhibitory concentrations and minimum bactericidal concentrations of most Enterobacteriaceae isolates. (72).

Quinolone antibiotics
Levofloxacin is a representative compound from the fluoroquinolone class of antibiotics (73). It is a synthetic chemotherapeutic agent that demonstrates a broad spectrum of antibacterial activity. Bhat et al. investigated the binding interactions between HSA and synthesized choline-based levofloxacin conjugates. Their results revealed that the binding was both electrostatic and hydrophobic, leading to a static quenching effect on HSA fluorescence. The study also demonstrated a conformational change in HSA and a decrease in its native protein functionality upon ligand binding (74). The binding of ciprofloxacin (CFX) to HSA was investigated using fluorescence displacement and induced circular dichroism in the study conducted by Varshney et al. Fluorescence displacement measurements, in the presence of various marker ligands, identified site I (subdomain IIA) as the primary binding site for CFX, near the chloroform binding site (75). Fluoroquinolones are advised for infection prevention in cirrhosis or liver failure patients (76). Extended norfloxacin effectively prevents primary and secondary SBP and Gram-negative infections, potentially reducing systemic inflammation (77). Notably, fluoroquinolone therapy has been associated with an elevated risk of acute liver injury, particularly within the initial two months of administration (78).

Carbapenem antibiotics
Imipenem was found to bind at two sites on HSA: a high-affinity site in subdomain IIIA (site I) and a low-affinity site in subdomains IIA–IIB, with electrostatic, hydrogen bonding, and hydrophobic interactions stabilizing the complex at the high-affinity site. The binding process was spontaneous, with binding constants between 104 and 105 M⁻¹, and led to conformational changes in HSA, affecting its esterase-like activity and folding pathway (79).

HSA and diuretics
Ascites is the most prevalent complication of LC, associated with a 5-year mortality rate of up to 30%. Its development is attributed to portal hypertension, systemic inflammation, and splanchnic arterial vasodilation. Treatment aimed at addressing the underlying cause, along with the use of non-selective beta-blockers, can prevent the onset of ascites in patients with compensated cirrhosis. Current management strategies for ascites focus on controlling fluid overload through options such as diuretics, sodium restriction, and/or paracentesis. Previous studies have shown that loop diuretics (e.g., furosemide, bumetanide, piretanide), hydrochlorothiazide, and indapamide are capable of binding to subdomain IA or IIA of HSA, and spironolactone binds specifically to site I (80-83). The long-term administration of albumin, norfloxacin prophylaxis, and transjugular intrahepatic portosystemic shunting may mitigate the risk of further decompensation and enhance survival rates (84,85). Romanelli conducted a study in which 100 patients admitted for the first time with ascites were randomly assigned to two groups. The first group received a diuretic plus HSA 25 g/week for the first year and 25 g HSA every two weeks thereafter. The others received diuretics alone. They found patients treated with albumin combined with diuretics had a significantly higher cumulative survival rate (P=0.008) and a lower probability of recurrence of ascites (51% vs. 94%, P<0.001). Long-term albumin infusion extends survival by an average of 16 months (86). Nakamura et al. demonstrated that the combination of HSA and diuretics in treating ascites among patients with advanced cirrhosis could further alleviate the condition (87). The potential mechanisms by which HSA combined with diuretics ameliorates ascites in cirrhotic patients include: firstly, HSA elevates plasma colloid osmotic pressure, enhances binding capacity with sodium and potassium ions, and facilitates renal excretion of ascites; secondly, it replenishes effective circulating blood volume, improves renal function, and increases urine output; thirdly, it activates diuretic sensitivity to potentiate diuretic efficacy (88).

HSA and anticancer drugs in HCC
The latest advancements and research directions in the field of liver cancer treatment aim to improve the survival rates and quality of life of liver cancer patients through multidisciplinary collaboration and precision medicine. Building on this foundation, exploring more possibilities to achieve these goals is extremely welcome. Accordingly, we summarize the clinical practice of HSA in liver cancer. Research on targeted therapy, chemotherapy, immunotherapy, radiotherapy and liver transplantation reveals HSA’s crucial role in influencing treatment effectiveness for HCC. Further research is needed to refine HSA formulations and understand their interactions with cancer cells and the immune system.

Targeted therapy
The interaction between anticancer drugs and HSA can result in alterations in the concentration of the free, biologically active components of these drugs. HSA, whether administered exogenously or synthesized endogenously, prolongs the circulatory half-life of anticancer agents and facilitates tumor targeting via the enhanced permeability and retention effect. It is important to note that only the unbound fraction of the drug within tissues exhibits pharmacological activity. While stronger binding to HSA diminishes the concentration of the pharmacologically active free drug, it concurrently extends the drug’s half-life, as the unbound form is more susceptible to hepatic metabolism (89,90). HCC is closely associated with hypoalbuminemia, a condition that can enhance the proportion of unbound drugs in the plasma (91,92). Patients with LC or HCC exhibit diminished expression of drug-metabolizing enzymes and transporters, thereby compromising the liver’s capacity to process drugs. Consequently, both conditions may lead to increased levels of free drugs, heightening the risk of toxicity and potentially requiring modifications in drug dosage (93). Recently, for advanced HCC, atezolizumab combined with bevacizumab has become the new first-line treatment standard, and the overall survival rate, progression-free survival rate, and progression-free survival rate have all improved (94,95). The results revealed that 10% of HSA could preserve the activity of bevacizumab (96). Therefore, how to prolong the efficacy of bevacizumab has become a key issue. Luis de Redín et al. developed glutaraldehyde-crosslinked HSA-bevacizumab nanoparticles to enhance the drug-loading capacity of bevacizumab (97). It can significantly extend the release time of bevacizumab, enhancing its anti-angiogenic effect. Therefore, constructing HSA-bevacizumab recombinant protein can further enhance the anti-tumor effect of bevacizumab. In cases where contraindications exist for the use of atezolizumab plus bevacizumab, sorafenib or lenvatinib may be considered as first-line treatment options for patients with advanced HCC who are classified as Child-Pugh class A and have an Eastern Cooperative Oncology Group Performance Status of 0–1 (98,99). The interaction site for sorafenib on HSA has been identified within subdomain IIA (site I) (87). Numerous studies indicate that, among patients undergoing sorafenib treatment for HCC, a lower ALBI score correlates with a greater therapeutic benefit from the drug (100-102). However, antioxidant HSA has been observed to negate sorafenib-induced viability in hepatic stellate cells by diminishing sorafenib-induced vacuolation and cell death (103). Lenvatinib exhibits a high binding rate to HSA, with 96.6% to 97.1% of the drug being bound. This substantial binding facilitates the primary transport of lenvatinib in the bloodstream via HSA, consequently extending its half-life and potentially affecting its distribution across various tissues within the body (104). In a study involving 82 HCC patients treated with lenvatinib, Ueshima et al. reported a high objective response rate among patients who presented with an ALBI grade 1 at baseline (105).

Chemotherapy
HCC is known for its resistance to chemotherapy, which is associated with increased mortality rates in liver cancer patients (106). Consequently, enhancing sensitivity to chemotherapy drugs is a critical factor in the treatment of HCC. Arsenic trioxide (ATO, As2O3) has been approved by the U.S. Food and Drug Administration as an effective treatment for acute promyelocytic leukemia. However, its application in treating advanced solid tumors, including HCC, is limited due to its modest efficacy and intolerable side effects. Zhang et al. synthesized a series of ATO-based nanoparticles with uniform single albumin size using HSA as a template (107). A multifunctional drug delivery system based on MnAs/HSA, named MnAs/indocyanine green (ICG)/HSA-arginylglycylaspartic acid (RGD), was developed, and its efficacy was evaluated both in vitro and in vivo. The results indicate that the photothermal effect of MnAs/ICG/HSA-RGD not only induces irreversible damage to HCC but also accelerates the release of As and Mn2+ ions, facilitating secondary tumor destruction. Furthermore, treatment inhibited the expression of heat shock protein 90, vimentin, and matrix metalloproteinase (MMP)-9 in tumor cells, thereby reducing metastasis and recurrence (107). L-HSA-doxorubicin (DOXO) is another drug delivery system based on HSA, designed to enhance the uptake of docetaxel by HCC cells. This system demonstrated comparable activity to sorafenib, resulting in a clinical overall survival benefit of approximately three months (108).

Immunotherapy
HSA can work as a carrier for nanomaterials. By constructing recombinant proteins of HSA and materials or cytokines, the tumor microenvironment can be reshaped (109,110). For example, Zheng et al. constructed a peptide nano-vaccine based on HSA and biodegradable MnO2 (111). The vaccine could improve the stability and immunogenicity of the antigenic peptide and promote its uptake by dendritic cells (DCs). The efficient uptake of the nanovaccine by DCs is anticipated to significantly enhance DCs’ maturation and antigen presentation, thereby potentially stimulating T cell immune responses. Meanwhile, Mn2+ degraded by nano-vaccines can activate the STING pathway and further promote DC maturation, and effectively mediate the immune response of T cells, thereby improving the effect of immunotherapy. Warmuth et al. used scMATCH3 technology and constructed a recombinant fusion protein of programmed cell death ligand 1 (PD-L1), 4-1BB and HSA, and designed the candidate drug NM21-1480 (112). NM21-1480 inhibits PD-L1/programmed cell death protein 1 (PD-1) signaling and effectively stimulates 4-1BB signaling, thereby stimulating CD8+ T cells and DCs, only in the presence of PD-L1.

Radiotherapy
HSA can also be used as a carrier for photosensitizers to enhance the efficacy of radiation therapy. Strózik et al. designed and constructed 5,10,15,20-tetrakis (4-sulfonatophenyl)-porphyrin (TSPP), a potential photosensitizer for photodynamic therapy and radiotherapy, in combination with HSA recombinant protein (113). Jiao et al. designed chimeric peptide OGS [PD-1/PD-L1 peptide OPBP-1 combined linker GS with vascular endothelial growth factor receptor (VEGFR) antibody] targeting PD-L1 and VEGFR2 (114). OGS can bind PD-L1 with high affinity, block PD-1/PD-L1 interaction, and inhibit human umbilical vein endothelial cells migration and tube formation in wound healing and tube formation experiments (114). To further extend the half-life of OGS, we modified it by coupling it with peptide DSP that both have high binding affinity to HSA to form peptide DSPOGS. DSPOGS can elicit an effective anti-tumor immune response and inhibit tumor angiogenesis, enhance tumor-infiltrating CD8+ T cells and IFN-γ-secreting CD8+ T cells in the spleen and tumor-draining lymph nodes, and inhibit tumor growth. In addition, the combination of radiotherapy and DSPOGS can significantly improve the treatment effect (114). Lin et al. designed a radiotherapy-mediated redox homeostasis-controllable nanomedicine to enhance the sensitivity of ferroptosis in tumor treatment (115). The nanomedicine is co-assembled with the ferroptosis inducer heme and the thioredoxin 1 (Trx-1) inhibitor 1-methylpropyl 2-imidazolyl disulfide (PX-12) with HSA. For our nanomedicine, heme converts H2O2 into ROS through the Fenton reaction, thereby inducing ferroptosis, while PX-12 effectively inhibits the activity of the antioxidant Trx-1 and inhibits ROS consumption, thereby leading to ferroptosis amplification. Combining radiotherapy with HSA-carrier nanomedicines further enhances the ferroptosis effect.

Liver transplantation
Liver transplantation can cure both HCC and the underlying liver disease (116). Immunosuppressive therapy affects the incidence of allograft rejection and graft dysfunction in liver transplant recipients, which is essential for improving transplant success rates (117). However, long-term outcomes in liver transplant recipients may be related to immunosuppressant use (118), making optimal immunosuppression crucial (119). Common post-transplant immunosuppressants include calcineurin inhibitors, mycophenolate mofetil (MMF), and mechanistic target of rapamycin mammalian target of rapamycin inhibitors. Recent studies have reported the binding and transport functions between these drugs and HSA. Only approximately 30% of cyclosporine is bound to HSA (120). HSA levels affect the PK of tacrolimus. Multiple PK studies have shown that lower HSA levels increase the clearance of tacrolimus, suggesting that the dosage of tacrolimus should be adjusted when HSA levels rise to avoid adverse events (121,122). MMF and its active metabolite, mycophenolic acid (MPA), primarily bind to site I (subdomain IIA) of HSA, with docking studies revealing significant molecular binding differences in the binding of MMF and MPA (123). For MPA, the aromatic moiety would be in close contact with Trp214; for MMF, the carboxylate group of the chain would be fixed near Trp214 through electrostatic interactions with residues Arg218 and Arg222. PK studies have demonstrated the significant impact of HSA levels on MPA disposition. In pediatric liver transplant recipients, the immediate post-transplantation period showed significantly lower median albumin levels compared to the stable post-transplantation period (26.8 vs. 33.0 g/L, P=0.04), which was associated with a higher unbound MPA fraction (2.8% vs. 1.0%, P=0.0496) and increased apparent clearance (16.0 vs. 11.2 L/h, P=0.03) (124). These findings were further supported by a broader population PK analysis in pediatric patients receiving MPA therapy, which identified HSA as a significant factor affecting both clearance and distribution (125). Khodaei et al. employed a specific probe displacement method to demonstrate that sirolimus primarily binds to site I of HSA (126).

Conclusions
HSA, abundant in plasma, binds, transports, and detoxifies compounds, has antioxidant properties, and modulates immune responses. HSA synthesis in liver cells is complex, with various drug binding sites that facilitate the synthesis and transport of ions, FA, bile acids, bilirubin, calcium ions, hormones, gases, ligands, and drugs. In the context of LC and its associated complications, the ability of HSA to sequester toxic metabolites, modulate oxidative stress, and regulate drug PK highlights its therapeutic significance. Moreover, the administration of HSA has been shown to improve clinical outcomes in conditions such as ascites, HRS, and HCC by enhancing drug delivery, extending circulation time, and reducing systemic inflammation. Despite its demonstrated efficacy, challenges such as hypoalbuminemia, financial constraints, and variability in the functionality of recombinant HSA (rHSA) necessitate further investigation. Future research should aim to optimize HSA-based therapies through advanced drug delivery systems and elucidate the molecular mechanisms underlying ligand interactions. Incorporating the non-oncotic functions of HSA into precision medicine frameworks has the potential to transform the management of liver diseases, particularly in resource-constrained environments.

Definition and clinical application of effective albumin
The clinical significance of HSA extends beyond its absolute serum levels to its functional efficacy in vivo. This insight led to the concept of “eAlb”, which highlights that albumin’s physiological roles (e.g., ligand binding and transport functions beyond colloid effects) are as critical as its absolute serum levels. The higher the proportion of modified albumin relative to total albumin concentration (tAlb), the lower the fraction of intact, structurally preserved albumin—termed “effective albumin”—which is significantly lower than the serum albumin concentration routinely measured in clinical settings (26). In pathological conditions such as cirrhosis, eAlb is markedly lower than tAlb, reflecting both quantitative depletion and qualitative dysfunction of albumin (127). The ANSWER trial demonstrated that prolonged HSA administration not only elevates total albumin levels (3.1 vs. 4.1 g/dL, P<0.001) but also enhances eAlb (0.77 vs. 0.93 g/dL, P=0.02), restoring functional capacity (128). A prospective observational study, evaluating 319 patients with cirrhosis hospitalized because of acute decompensation (AD), demonstrated that patients with cirrhosis complicated by bacterial infections, renal failure, and ascites exhibited significantly low eAlb levels (129). As compared to tAlb, eAlb is more closely associated with disease severity and albumin dysfunction and distinguishing patients with AD from those with acute-on-chronic liver. These results suggest future research assessing eAlb as a biomarker for predicting prognosis and treatment response. Currently, well-established functional detection and evaluation methods for albumin include: the hydroxyl radical antioxidant capacity assay, which evaluates the overall antioxidant capacity of albumin; the albumin binding capacity assay, which assesses the binding capacity of albumin’s critical drug-binding sites; the ischemia-modified albumin assay, which evaluates the binding ability of albumin’s N-terminal region to chelate metal ions; and the serum enhanced binding assay, which evaluates the level of PTMs (130-134). There are some emerging technologies for eAlb, including detection liquid chromatography-tandem mass spectrometry, and electron paramagnetic resonance, showing promising clinical application prospects with research in patients with cirrhosis still in the preliminary stage (129,135). Nevertheless, clinical implementation of eAlb faces challenges: standardization of its measurement and pathological thresholds remains unresolved, while its utility in guiding albumin therapy indications and real-time efficacy monitoring requires further validation through large-scale studies (26,136).

Differences in substance binding and transport functions between HSA and rHSA
Recombinant HSA, referred to as rHSA, is HSA produced through genetic engineering technology. Currently, rHSA under research in clinical trials includes ScrHSA expressed in Saccharomyces cerevisiae, OsrHSA expressed in rice endosperm cells, and RecrHSA expressed in Escherichia coli. Since the complementary DNA (cDNA) of rHSA was first reported in the world in 1981, the development and exploration of rHSA prepared through genetic engineering technology has gone through 40 years (137). Domenicali et al. found that the physiological function of HSA is not only related to its protein concentration, but also to the maintenance of its structural integrity (138). A comparative analysis of HSA and rHSA revealed nuanced structural variations within subdomains IA and IIA. These differences between the recombinant and plasma-derived forms of HSA (pHSA) were identified in the peptide backbone, disulfide bridges, and specific amino acid residues (139). Furthermore, another study investigating PTMs in pHSA and rHSA identified six PTMs: acetylation, succinylation, crotonylation, phosphorylation, beta-hydroxybutyrylation, and lactylation. While both pHSA and rHSA exhibited these PTMs, they differed in site characteristics and modification levels, potentially affecting their function, efficacy, and safety. Further research is needed to understand these differences (140) (Table 2).

Supplementary
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