Background: Although total 25-hydroxyvitamin D (25(OH)D) measurements may not accurately reflect functional vitamin D status, vitamin D deficiency is common in hepatocellular carcinoma (HCC). The contribution of altered vitamin D-binding protein (VDBP) and albumin to impaired bioavailability is poorly characterized in liver cancer. Methods: We measured total, free, and bioavailable 25(OH)D, VDBP, and albumin in 46 HCC patients and 87 healthy controls during winter and summer. Correlations with Child-Pugh score, Barcelona Clinic Liver Cancer (BCLC) stage, and disease aetiology were evaluated. Results: HCC patients exhibited significantly lower VDBP (177.3± 237.0 vs. 239.9 ± 141.9 mg/L, p < 0.001) and albumin (35.9 ± 5.4 vs. 48.0 ± 3.9 g/L, p < 0.001) compared to winter controls. Total 25(OH)D was lower in HCC (39.3 ± 22.1 nmol/L) versus summer controls (75.0 ± 22.8 nmol/L, p < 0.001) but comparable to winter controls (p = 0.061). HCC patients lacked seasonal variation in vitamin D fractions, unlike the controls. VDBP negatively correlated with free (ρ = -0.606, p < 0.001) and bioavailable 25(OH)D (ρ = -0.541, p < 0.001). Child-Pugh score correlated positively with BCLC stage (ρ = 0.378, p = 0.012) and inversely with albumin (ρ = -0.565, p< 0.001). Conclusions: Free and bioavailable vitamin D are profoundly compromised in HCC, reflecting impaired hepatic synthetic function and systemic inflammation. These fractions may serve as novel metabolic biomarkers superior to total 25(OH)D for assessing vitamin D deficiency and guiding individualized supplementation strategies in patients with liver cancer.

Vitamin D is a pleiotropic hormone with established roles in mineral homeostasis and increasingly recognized functions in immune modulation, cellular proliferation control, and cancer biology [1–3]. Hepatocellular carcinoma (HCC), the most common primary liver malignancy, predominantly arises in cirrhotic livers subject to chronic inflammation and hepatic dysfunction— conditions that profoundly perturb vitamin D metabolism and transport [4–6]. The liver is essential for two critical steps in vitamin D homeostasis: first, the 25-hydroxylation of vitamin D to generate the major circulating metabolite, 25-hydroxyvitamin D (25(OH)D); second, the synthesis of vitamin D-binding protein (VDBP) and albumin, which carry vitamin D in serum [7– 9]. In HCC and advanced cirrhosis, both processes are compromised by reduced hepatic synthetic capacity and systemic inflammatory cytokines such as IL-6 and TNF-α, which suppress VDBP production [10–12]. Despite evidence suggesting that low total 25(OH)D portends a worse HCC prognosis, few studies have systematically profiled the spectrum of vitamin D metabolites—including free and bioavailable fractions—in relation to clinical stage, hepatic function, and inflammation in HCC [13, 14]. This gap has direct therapeutic implications, as vitamin D supplementation regimens tailored to total 25(OH)D may be insufficient or potentially unsafe in the context of significant protein-binding alterations [15]. Vitamin D metabolism and bioavailability are profoundly influenced by liver function, particularly in hepatocellular carcinoma (HCC). The liver not only converts vitamin D into its circulating form, 25(OH)D, but also synthesizes the principal VDBP and albumin, which regulate the free and bioavailable fractions accessible to target tissues. In HCC, hepatic dysfunction and systemic inflammation disrupt these pathways, leading to a decrease in the production of VDBP and albumin, thereby diminishing vitamin D bioavailability despite serum levels that may not fully reveal the deficiency [13,16,17]. This complex interplay is summarized schematically in Figure 1.

Figure 1: Schematic representation of altered vitamin D metabolism and transport in hepatocellular carcinoma. This schematic illustrates the normal and disrupted pathways of vitamin D metabolism in hepatocellular carcinoma (HCC). Under physiological conditions, the liver hydroxylates vitamin D into 25-hydroxyvitamin D (25(OH)D) and synthesizes vitamin D-binding protein (VDBP) and albumin, which bind 25(OH)D in serum. These bound forms regulate the free and bioavailable fractions that can enter cells to activate vitamin D receptor  (VDR)-mediated signalling critical for immune modulation and antitumor activity. In HCC, impaired liver function reduces hydroxylation capacity and leads to decreas in the synthesis of VDBP and albumin. Additionally, inflammation downregulates VDBP production. These alterations lower circulating total, free, and bioavailable 25(OH)D, impairing vitamin D bioactivity in tumour and immune cells. Arrows represent metabolic and transport pathways; dashed arrows indicate disrupted or diminished processes in HCC. Conventionally, vitamin D status is assessed by measuring total serum 25(OH)D. However, the ʺfree hormone hypothesisʺ posits that only the unbound or bioavailable fractions of hormones can cross cell membranes and activate receptors [18,19]. In populations with low albumin and VDBP— such as patients with HCC total 25(OH)D may substantially underestimate true vitamin D deficiency [20–23]. Recent epidemiological and translational studies support the role of vitamin D in HCC prevention and possibly prognosis, yet few investigations have comprehensively characterized all vitamin D fractions or their carrier proteins in relation to clinical liver function markers [5,24,25].The aim of this study was to systematically profile total, free, and bioavailable 25(OH)D alongside VDBP and albumin in HCC patients compared to healthy controls across seasons, and to evaluate their correlations with measures of hepatic reserve (Child-Pugh score), tumour burden (BCLC stage), and disease aetiology. We hypothesized that free and bioavailable 25(OH)D, rather than total measurements alone, would better reflect functional vitamin D deficiency and correlate with clinical disease severity in HCC.

2.1.Patient Characteristics:

The HCC cohort (n = 46) consisted predominantly of male patients (39 males and 7 females) with a mean age of 71.4 ± 7.5 years, significantly older than the healthy control group (14 males and 73 females, age 35.9 ± 12.5 years, p < 0>

2.2.Vitamin D Status and Binding Proteins:

In healthy controls, serum vitamin D levels showed expected seasonal variation (Table 1). Total 25(OH)D was significantly higher in summer (75.0 ± 22.8 nmol/L) compared to winter (44.1 ± 17.8 nmol/L, p < 0 xss=removed xss=removed>

Table 1:Vitamin D Metabolites, Binding Proteins, and Clinical Characteristics in HCC Patients and Healthy Controls.

* Kruskal-Wallis test used for comparisons due to non-normal distribution. Data presented as mean±SD. 25(OH)D – 25-hydroxyvitamin D; F – female; HCC – hepatocellular carcinoma; M – male; VDBP – vitamin D- binding protein. In contrast, HCC patients exhibited persistently low vitamin D fractions year-round without significant seasonal increase. Total 25(OH)D in HCC (39.3 ± 22.1 nmol/L) was comparable to winter controls (p = 0.061) but markedly lower than summer controls (p < 0 xss=removed>

2.3.Disease Aetiology and Clinical Staging:

HCC aetiology distribution included alcoholic liver disease (28 patients; 25/3 male/female), HBV (3 patients; 1/2 male/female), HCV (5 patients; 4/1 male/female), hemochromatosis (1 patient; 1/0 male/female), metabolic liver disease (6 patients; 6/0 male/female), cryptogenic cirrhosis (2 patients; 2/0 male/female), and primary biliary cholangitis (1 patient; 0/1 male/female). Gender distribution across aetiology groups differed significantly (p = 0.031). Barcelona Clinic Liver Cancer staging revealed stage 0 in 2 patients, stage 1 in 9 patients, stage 2 in 27 patients, stage 3 in 4 patients, and stage 4 in 2 patients, with significant differences in stage distribution across aetiologies (p = 0.012). Distribution across aetiologies and cancer stages is detailed in Table 2.

Table 2: HCC Aetiology and Barcelona Clinic Liver Cancer (BCLC) Staging Distribution.

BCLC – Barcelona Clinic Liver Cancer; F – female; HBV – hepatitis B virus ; HCV – hepatitis C virus; M – male; PBC – primary biliary cholangitis.

2.4.CORRELATION ANALYSES:

Strong positive correlations were identified between VDBP and albumin (ρ = 0.395, p = 0.007) and between VDBP and total 25(OH)D (ρ = 0.347, p = 0.018). Notably, VDBP showed robust negative correlations with free 25(OH)D (ρ = -0.606, p < 0 xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed>

Table 3: Spearmanʹs Rank Correlation Coefficients in HCC Cohort.

25(OH)D – 25-hydroxyvitamin D; BCLC – Barcelona Clinic Liver Cancer; HCC – hepatocellular carcinoma; VDBP – vitamin D-binding protein.

3.1.Principal Findings and Interpretation:

This study demonstrates profound alterations in vitamin D metabolism in HCC, characterized by significantly reduced total, free, and bioavailable 25(OH)D fractions despite markedly elevated calculated free 25(OH)D values in the context of severe albumin and VDBP depletion [9,40]. The paradoxically high free 25(OH)D reflects the mathematical consequence of extreme protein binding partner depletion rather than true vitamin D sufficiency. In healthy individuals, approximately 85– 90% of circulating 25(OH)D is bound to VDBP, 10–15% is albumin-bound, and less than 1% exists as truly free hormone [9,40,41]. VDBP acts as a reservoir and prolongs the half-life of circulating 25(OH)D, and this critical transport function is severely compromised in HCC. Our HCC patients exhibited dramatically reduced VDBP (177.3 ± 237.0 mg/L vs. 239.9 ± 141.9 mg/L in winter controls, p< 0 xss=removed>

3.2.Mechanisms Underlying Vitamin D Perturbation in Hcc:

Multiple interconnected mechanisms account for the observed vitamin D deficiency in HCC: Hepatic Synthetic Dysfunction: The liver is the primary site of 25-hydroxylation, converting cholecalciferol to 25(OH)D. In HCC and advanced cirrhosis, a reduced hepatic mass, fibrosis, and impaired function (as reflected in Child-Pugh scores and albumin levels) decrease this critical enzymatic step. Our finding that albumin strongly correlates with Child-Pugh score (ρ = -0.565, p < 0 xss=removed>

3.3.Comparison With Prior Studies:

Our findings extend and confirm prior investigations into vitamin D metabolism in HCC. Chiang et al. and others documented reduced total 25(OH)D in HCC versus cirrhotic controls [33,34]. Critically, Fang et al. demonstrated in a large prospective Chinese cohort that bioavailable 25(OH)D was a superior prognostic marker of HCC survival compared to total vitamin D, even after adjustment for albumin and VDBP [35]. Their finding that bioavailable fractions predicted outcomes better than total 25(OH)D aligns precisely with our data: bioavailable 25(OH)D (8.5 ± 6.3 nmol/L in HCC vs. 13.1 ± 8.3 nmol/L in controls, p < 0 xss=removed xss=removed xss=removed xss=removed>

3.4.Clinical Implications and Limitations of Current Vitamin D Assessment:

Limitations of total 25(OH)D Measurement: Current clinical guidelines typically target a total 25(OH)D level of ≥75 nmol/L or ≥100 nmol/L for optimal bone health and general well-being. However, our data demonstrate that a total 25(OH)D alone is profoundly misleading in HCC. An HCC patient with a total 25(OH)D of 39.3 ± 22.1 nmol/L and severe hypoalbuminemia (35.9 ± 5.4 g/L) and VDBP depletion (177.3 ± 237.0 mg/L) faces compounded vitamin D deficiency at both circulating and bioavailable levels. Yet, calculated bioavailability equations using depleted carrier proteins yield paradoxically elevated free fractions, creating diagnostic confusion. Direct measurement of free 25(OH)D by equilibrium dialysis would provide clarity but is rarely performed clinically. Superior Utility of Free and Bioavailable Fractions: The strong correlations between VDBP/albumin and bioavailable 25(OH)D (ρ = -0.541 and -0.327, respectively, p < 0>

3.5.Study Strengths:

This study possesses several important strengths: (1) Comprehensive simultaneous measurement of all physiologically relevant vitamin D fractions (total, free, bioavailable) alongside their principal carrier proteins (VDBP, albumin) in a well-characterized HCC cohort (n=46) compared to healthy controls (n=87). (2) Dual-season design enabling assessment of environmental (sun exposure) versus disease-related determinants of vitamin D status. The striking absence of seasonal variation in HCC, despite robust seasonal increases in control isolates suggests disease-driven mechanisms. (3) Detailed clinical staging and correlations with established metrics of hepatic reserve (Child-Pugh score, ρ = -0.565 with albumin) and tumour burden (BCLC stage, ρ = 0.378 with Child- Pugh), integrating vitamin D findings with clinical disease severity. (4) Application of validated equations for calculating free/bioavailable 25(OH)D based on measured VDBP, albumin, and total 25(OH)D, with binding affinities derived from published literature and previously validated in large epidemiological cohorts [29,41]. (5) Integration of mechanistic literature linking vitamin D pathway dysregulation (VDR suppression, CYP24A1 upregulation) to HCC biology, supporting interpretation of observed biochemical patterns.

3.6.Study Limitations:

Key limitations merit acknowledgement: (1) Cross-sectional design precludes causal inference and limits longitudinal outcome assessment. We cannot determine whether vitamin D depletion contributes to HCC progression, serves as a prognostic marker, or is merely an epiphenomenon of hepatic dysfunction. (2) Calculated rather than directly measured free/bioavailable 25(OH)D. Although validated equations employing measured VDBP and albumin were used, direct measurement by equilibrium dialysis or high-performance liquid chromatography (HPLC) coupled with mass spectrometry would provide greater accuracy, particularly in extreme hypoalbuminemia.[3]Moderate sample size with single-center recruitment, potentially limiting generalizability to other geographic, ethnic, or healthcare settings. The HCC aetiology distribution (predominantly alcoholic liver disease, with fewer HBV and HCV cases) reflects local referral patterns and may not represent global HCC epidemiology. (4) Incomplete assessment of potential confounders such as dietary vitamin D intake, occupational and leisure sunlight exposure, use of vitamin D supplements or skincare products, and genetic VDBP polymorphisms (Gc1f, Gc1s, Gc2). While we stratified by season, individual-level variability in sun exposure was not quantified. (5) No direct clinical outcome assessment. In our study we measured associations between vitamin D fractions and clinical disease severity markers (Child-Pugh, BCLC) but did not assess survival, time-to-progression, treatment response, or immune parameters (IL-6, TNF-α, regulatory T cells). Longitudinal follow-up with outcome data is essential to validate vitamin D fractions as independent prognostic biomarkers. (6) Potential survivor bias if sicker patients were less likely to participate in the study, though this is mitigated by enrolment of consecutive patients across all BCLC stages (0–4) with representation of both early and advanced disease.

4.1.Study Population and Design:

This was a cross-sectional observational study conducted at University Medical Centre Ljubljana between 2022 and 2024. The study enrolled 46 patients with biopsy-proven or imaging-confirmed HCC according to EASL/EORTC criteria and 87 age-stratified healthy controls. Blood samples were collected during winter (December–February) and summer (June–August) seasons to assess seasonal variations in vitamin D metabolism.

4.2.Inclusion And Exclusion Criteria:

HCC patients were eligible if they had a confirmed diagnosis, were at least 18 years old, and had complete clinical and laboratory data available. Exclusion criteria included severe concurrent renal dysfunction (eGFR <30>

4.3.Ethical Approval and Informed Consent:

The study was conducted in accordance with the Declaration of Helsinki and approved by the institutional ethics committee of University Medical Centre Ljubljana. Written informed consent was obtained from all participants prior to enrolment.

4.4.Clinical Data Collection:

For HCC patients, the following clinical data were recorded: age, gender, aetiology of liver disease (alcoholic, hepatitis B virus [HBV], hepatitis C virus [HCV], hemochromatosis, metabolic, cryptogenic, primary biliary cholangitis [PBC]), Child-Pugh score, Barcelona Clinic Liver Cancer (BCLC) stage, and time since HCC diagnosis.

4.5.Biochemical Measurements:

Serum samples were collected by venepuncture in the morning after an overnight fast. All samples were processed and stored at -80°C until analysis.Measurements were performed at the Clinical Institute of Clinical Chemistry and Biochemistry (University Medical Centre, Ljubljana). 25(OH)D3, S-albumin and S-DBP in serum, were measured in all participants using the following methods: The concentration of 25(OH)D3 vitamin was measured using competitive luminescent immunoassay with intra-laboratory CV < 6>

4.6.Statistical Analysis:

Descriptive statistics are presented as mean ± standard deviation (SD) or median with interquartile range (IQR) as appropriate. Given the non-normal distribution of key variables (as assessed by the Shapiro-Wilk test), the Kruskal-Wallis test was used for comparisons between groups and seasons. Spearmanʹs rank correlation coefficient (ρ) was employed to assess associations between continuous variables. Two-sided p-values <0>

Vitamin D metabolism is profoundly disrupted in HCC, with severe reductions in total, free, and bioavailable 25(OH)D fractions occurring concurrently with depleted VDBP and albumin levels, which reflect hepatic synthetic dysfunction. The absence of seasonal vitamin D variation in HCC patients, in stark contrast to healthy controls, demonstrates that this disruption is a disease-driven phenomenon reflecting impaired hepatic 25-hydroxylase activity, inflammatory suppression of carrier protein synthesis, and dysregulation of vitamin D-metabolizing enzymes—not merely reduced sun exposure. Measurement of free and bioavailable 25(OH)D alongside VDBP and albumin may provide a superior assessment of functional vitamin D status and hepatic reserve compared to total 25(OH)D measurement alone. Integration of these biomarkers with clinical staging (Child-Pugh, BCLC) could enhance risk stratification in HCC. These fractions warrant prospective validation as independent risk factors for HCC progression, mechanistic investigation of their relationships to VDR-mediated antitumor immunity, and evaluation as targets for intervention to improve outcomes in liver cancer. Future research should include: (1) randomized controlled trials of vitamin D supplementation strategies titrated to free/bioavailable 25(OH)D targets with survival and quality-of-life endpoints; [2] investigation of VDBP genetic polymorphisms and their influence on vitamin D bioavailability and HCC outcomes; [3] mechanistic studies of VDR signalling and antitumor immunity as a function of free 25(OH)D levels; and [4] integration of vitamin D fractions with inflammatory cytokine panels (IL-6, TNF-α) and immune cell analysis to construct multidimensional metabolic-immune risk scores for HCC prognostication and personalized therapy. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/doi/s1, Figure S1: title; Table S1: title; Video S1: title.

Conceptualization, D.S. and J.O.; Methodology, D.S., M.R., M.Z., S.Š., B.Š., A.J., and J.O.; Software, D.S.; Validation, J.O.; Formal Analysis, D.S., and J.O.; Investigation, D.S., M.R., M.Z., S.Š., B.Š.; Data Curation, D.S., M.R., M.Z., S.Š., B.Š., A.J., and J.O.; Writing—Original Draft Preparation, D.S.; M.R., and J.O.; Writing—Review and Editing, B.Š., and J.O.; Visualization, J.O.; Supervision, B.Š. and J.O.; Project Administration, D.S. and J.O. All authors have read and agreed to the published version of the manuscript.

This research received no external funding.

Institutional Review Board Statement: The Ethics Committee approved the study protocol for Human Research of the Medical Ethics Commission of the Republic of Slovenia (Protocol ID: 0120-60/2021/5, 22 March 2021).Informed Consent Statement: All patients gave written informed consent for this study before inclusion.

The raw data supporting the conclusions of this article will be made available by the corresponding author on request.

N/A

: The authors declare no conflicts of interest.

 The following abbreviations are used in this manuscript: