Role of Serum Ferritin, Vitamin D Status, and C-Reactive Protein in Nonalcoholic Fatty Liver Disease: A Case-control Study

1. Associate Professor, Department of Biochemistry, Sree Balaji Medical College and Hospital, Chennai, Tamil Nadu, India. 2. Professor, Department of Biochemistry, Sree Balaji Medical College and Hospital, Chennai, Tamil Nadu, India. 3. Tutor, Department of Biochemistry, Sree Balaji Medical College and Hospital, Chennai, Tamil Nadu, India. 4. Professor, Department of Biochemistry, Sree Balaji Medical College and Hospital, Chennai, Tamil Nadu, India.

Abstract

Introduction: Nonalcoholic Fatty Liver Disease (NAFLD) is an increasingly prevalent metabolic liver disorder associated with chronic low-grade inflammation. Altered iron metabolism, Vitamin D deficiency, and systemic inflammatory markers have been implicated in its pathogenesis; however, their relationship with disease severity remains incompletely understood.

Aim: To evaluate the association of serum ferritin, Vitamin D status, and C-Reactive Protein (CRP) with the presence and severity of NAFLD.

Materials and Methods: This hospital-based case-control study was conducted at the Department of Biochemistry, Sree Balaji Medical College and Hospital, Chennai, Tamil Nadu, India, from January 2023 to December 2023. A total of 200 participants were enrolled, including 100 NAFLD patients diagnosed by clinical, biochemical, and ultrasonographic criteria and 100 age- and gender-matched healthy controls. Serum ferritin, 25-hydroxyvitamin D, and CRP levels were estimated using standard laboratory methods. Data were analysed using Statistical Package for Social Sciences (SPSS) version 26.0. Normality was assessed using the Shapiro-Wilk test. Mann-Whitney U test and Chi-square/Fisher’s-exact tests were used for group comparisons, while Spearman’s rank correlation was used to assess associations with NAFLD severity.

Results: The mean age of NAFLD patients was 32.27±3.99 years, and that of controls was 31.46±4.19 years, with equal gender distribution in both groups (50 males and 50 females). NAFLD patients had significantly higher median serum ferritin [183.0 (148.0-254.75) vs 145.0 (97.75-236.25) ng/mL; p=0.0001] and CRP levels [6.5 (5.0-9.5) vs 1.5 (0.8-2.0) mg/L; p<0.0001], and significantly lower Vitamin D levels [13.0 (8.57-17.0) vs 24.0 (22.0-30.5) ng/mL; p<0.0001] than controls. Vitamin D deficiency was present in 79% of NAFLD patients. Serum ferritin (r=-0.024, p=0.813) and Vitamin D (r=0.149, p=0.139) showed no significant correlation with NAFLD severity, whereas CRP demonstrated a significant positive correlation (r=0.604, p<0.001).

Conclusion: NAFLD is associated with elevated serum ferritin and CRP levels and reduced Vitamin D concentrations. While ferritin and Vitamin D were associated with the presence of NAFLD, CRP correlated significantly with disease severity, suggesting its potential utility as a marker of disease progression and inflammatory burden.

Hepatic steatosis, Hypovitaminosis D, Iron metabolism, Metabolic syndrome, Systemic inflammation

Nonalcoholic Fatty Liver Disease (NAFLD) is the most prevalent chronic liver disorder worldwide and represents the hepatic manifestation of metabolic syndrome, encompassing obesity, insulin resistance, dyslipidaemia, and type 2 diabetes mellitus (1). The disease spectrum ranges from simple steatosis to Nonalcoholic Steatohepatitis (NASH), progressive fibrosis, cirrhosis, and hepatocellular carcinoma (2). Increasing evidence suggests that chronic low-grade inflammation and oxidative stress play central roles in the initiation and progression of NAFLD, making inflammatory biomarkers crucial for understanding disease pathophysiology and risk stratification (3).

Serum ferritin, traditionally regarded as a marker of iron storage, has emerged as an important acute phase reactant and indicator of systemic inflammation. Elevated ferritin levels are frequently observed in patients with NAFLD and have been associated with hepatic iron deposition, insulin resistance, and increased disease severity (4). Excess iron promotes oxidative stress through the generation of reactive oxygen species, leading to hepatocellular injury, lipid peroxidation, and activation of inflammatory pathways (5). Thus, hyperferritinaemia may reflect both iron overload and inflammatory burden in NAFLD.

Vitamin D deficiency is highly prevalent in patients with NAFLD and is increasingly recognised as a contributor to metabolic and inflammatory dysregulation. Beyond its classical role in calcium homeostasis, Vitamin D exerts immunomodulatory, anti-inflammatory, and antifibrotic effects through regulation of cytokine production and inhibition of hepatic stellate cell activation (6). Low serum 25-hydroxyvitamin D levels have been linked to increased hepatic steatosis, insulin resistance, and progression to NASH, suggesting a potential role in disease severity and prognosis (7).

C-Reactive Protein (CRP), a sensitive marker of systemic inflammation, has been widely studied in metabolic disorders and cardiovascular disease. Elevated CRP levels are commonly observed in NAFLD and correlate with obesity, insulin resistance, and hepatic inflammation (8). The combined assessment of CRP with ferritin and Vitamin D status may therefore provide a more comprehensive understanding of the inflammatory and metabolic milieu underlying NAFLD.

Despite accumulating evidence linking iron metabolism, Vitamin D deficiency, and systemic inflammation with NAFLD, several studies have evaluated these biomarkers largely in isolation (9),(10),(11). Previous investigations have demonstrated an association between elevated serum ferritin and disease severity, as well as between low Vitamin D levels and hepatic steatosis and insulin resistance (12),(13),(14). Similarly, increased CRP levels have been reported to reflect systemic inflammation and cardiometabolic risk in NAFLD (15),(16). However, most of these studies have focused on individual biomarkers rather than exploring their combined role within a single patient cohort (17),(18).

The present study differs from previous reports by simultaneously evaluating serum ferritin, Vitamin D status, and CRP in the same NAFLD cohort, providing a more integrated assessment of metabolic and inflammatory interactions. By combining continuous, categorical, and correlation analyses, it offers deeper insight into their interrelationship. This multi-biomarker approach may improve risk stratification and help identify potential therapeutic targets in NAFLD. Therefore, the present study was conducted to evaluate the association of serum ferritin, Vitamin D status, and CRP as biomarkers of inflammatory burden in patients with NAFLD. The objectives were to estimate serum ferritin, 25-hydroxyvitamin D, and CRP levels and assess the prevalence of Vitamin D deficiency in patients with NAFLD and to evaluate the association with NAFLD.

Material and Methods

This was a hospital-based case-control study conducted at the Department of Biochemistry, Sree Balaji Medical College and Hospital, Chrompet, Chennai Tamil Nadu, India, from January 2023 to December 2023. The study protocol was approved by the Institutional Human Ethics Committee and Research Committee of Sree Balaji Medical College and Hospital (Approval Reference No: 002/SBMC/IHEC/2021/1662). The study was conducted in accordance with ethical principles.

Inclusion criteria:

• For Cases: It included NAFLD individuals aged 20-40 years based on a combination of clinical evaluation, biochemical investigations, and imaging findings. Ultrasonography of the abdomen was used as the primary imaging modality for the diagnosis of NAFLD (19),(20). Liver biopsy was not performed due to its invasive nature and higher cost (21).

• For Controls: It consisted of age- and gender-matched healthy individuals without evidence of NAFLD on ultrasonography and without clinical or biochemical features suggestive of liver disease.

Exclusion criteria: Participants were excluded, if they had a history of alcohol consumption, chronic liver disease or liver failure, renal disease, diabetes mellitus, thyroid disorders, or were receiving steroids or hepatotoxic medications.

Sample size calculation: The sample size for this case-control study was calculated to detect significant differences in biomarker prevalence between NAFLD cases and controls using the formula for comparing two proportions:

n=(Zα/2+Zβ)²×[p1(1-p1)+p2(1-p2)] / (p1-p2)²

n =sample size required per group, Zα/2=1.96 (95% confidence level), Zβ=0.84 (80% power), p₁=prevalence among cases, p₂=prevalence among controls

Using a 95% confidence level and 80% power, sample sizes were estimated based on reported prevalence of Vitamin D deficiency (45% vs. 25%), elevated serum ferritin (40% vs. 15%), and elevated CRP (50% vs. 30%) among cases and controls, respectively (22).

The largest calculated sample size was 91 participants per group based on CRP. Participants were recruited using consecutive sampling, wherein eligible NAFLD patients attending the outpatient services of the Department of Medicine and apparently healthy age- and gender-matched controls attending routine health examinations were consecutively enrolled until the required sample size of 200 was achieved.

Study Procedure

All participants were informed about the nature and purpose of the study, and written informed consent was obtained before enrolment. A patient information leaflet detailing the objectives and procedures of the study was provided to willing participants. Demographic and clinical details, including age, gender, height, weight, Body Mass Index (BMI), general medical history, family history, medication history, and blood pressure, were recorded. A routine clinical examination was performed for all participants before blood sample collection. Ultrasonographic evaluation was performed to confirm the presence or absence of NAFLD in all study participants (23). NAFLD was diagnosed based on the presence of hepatic steatosis on ultrasonography, characterised by increased hepatic echogenicity compared with the renal cortex, blurring of intrahepatic vessel margins, and deep attenuation of the ultrasound signal, in the absence of significant alcohol intake and other secondary causes of fatty liver disease (24).

Based on the conventional ultrasonographic grading criteria, NAFLD was classified into Grade 0 (normal), Grade 1 (mild), Grade 2 (moderate), and Grade 3 (severe) hepatic steatosis (24).

Grade 0 – Normal

Grade 1 (mild) - Represented by slight diffuse increase in fine echoes in the hepatic parenchyma with normal visualisation of the diaphragm and intrahepatic vessel borders.

Grade 2 (moderate) - Represented by a moderate diffuse increase in fine echoes with slightly impaired visualisation of the intrahepatic vessels and diaphragm.

Grade 3 (severe) - Represented by a marked increase in fine echoes with poor or no visualisation of the intrahepatic vessel borders, diaphragm, and posterior portion of the right lobe of the liver.

Anthropometric assessment: Anthropometric measurements including height and weight were obtained using standard procedures, and BMI was calculated as weight in kilograms divided by height in meters squared (kg/m²).

Sample collection: After an overnight fast of 12 hours, approximately 5 mL of venous blood was collected from all participants by aseptic venipuncture using sterile vacutainers. The samples were allowed to clot and were centrifuged at 3000 rpm for 10 minutes to separate serum. The obtained serum was used for biochemical analysis. Serum ferritin levels were measured using a Chemiluminescent Immunoassay (CLIA) method. The normal reference range for ferritin was considered as 30-300 ng/mL for males and 15-150 ng/mL for females, with values >200 ng/mL taken as elevated for analysis. Serum 25-hydroxyvitamin D3 levels were estimated by chemiluminescent immunoassay (CLIA) Vitamin D status was classified as deficient (<20 ng/mL), insufficient (20-29 ng/mL), and sufficient (≥30 ng/mL) (25).C-reactive protein (CRP) levels were determined using a high-sensitivity immunoturbidimetric assay. CRP levels were interpreted as normal (<1 mg/L), low-grade inflammation (1-3 mg/L), and elevated (>3 mg/L) (26),(27). Serum ferritin and 25-hydroxyvitamin D3 levels were estimated by chemiluminescent immunoassay (CLIA) using commercially available assay kits on a Mindray immunoanalyser (Mindray CL-960i, Shenzhen, China). High-sensitivity C-reactive protein (hs-CRP) was measured using an immunoturbidimetric assay (in Mispa i3) with reagents from Agappe Diagnostics Ltd. All assays were performed strictly according to the respective manufacturer’s instructions, and appropriate internal quality-control procedures were followed to ensure the accuracy and reliability of the results.

STATISTICAL ANALYSIS:

Data were analysed using SPSS version 26.0. Continuous variables were tested for normality using the Shapiro-Wilk test. Normally distributed variables were expressed as mean±Standard Deviation (SD), whereas non normally distributed variables were presented as median with interquartile range (IQR). Comparisons between NAFLD cases and healthy controls were performed using the independent t-test for normally distributed variables and the Mann-Whitney U test for non-normally distributed variables. Categorical variables, including the prevalence of Vitamin D deficiency, elevated ferritin, and raised CRP, were compared using the Chi-square test or Fisher’s exact test, as appropriate. Correlations between biochemical parameters and NAFLD severity were assessed using Spearman’s rank correlation coefficient. A two-tailed p-value <0.05 was considered statistically significant.

Results

The case-control study included a total of 200 participants, comprising 100 patients diagnosed with NAFLD attending the outpatient services of the Department of Medicine and 100 apparently healthy age-matched controls. The significant Shapiro-Wilk test results (p<0.001 for all three biomarkers) indicate deviation from a normal distribution. Hence, non parametric statistical methods were considered appropriate for analysing serum ferritin, Vitamin D, and CRP levels.

The mean age and gender distribution were comparable between NAFLD cases (32.27±3.99 years; 50 males and 50 females) and controls (31.46±4.19 years; 50 males and 50 females) (p>0.05). NAFLD patients had significantly higher BMI compared to controls (p<0.001). Postprandial blood glucose levels were significantly elevated in NAFLD cases, while fasting blood glucose did not show a statistically significant difference. Lipid profile analysis revealed significantly higher total cholesterol, triglycerides, and LDL levels, along with significantly lower HDL levels in NAFLD patients compared to controls (p<0.001), indicating an adverse metabolic profile (Table/Fig 1).

The majority of participants were classified as Grade 1 (mild steatosis), accounting for 68 (68%) cases, while 22 (22%) had Grade 2 (moderate steatosis) and 10 (10%) had Grade 3 (severe steatosis). These findings indicate that most patients were in the early stage of hepatic steatosis, with a smaller proportion demonstrating advanced disease severity (Table/Fig 2).

Among NAFLD patients, 79 (79%) had Vitamin D deficiency, and 21 (21%) had insufficient levels, while none had sufficient Vitamin D status (Table/Fig 3). Serum ferritin levels were ≥200 ng/mL in 40 (40%) patients, indicating a substantial proportion with elevated iron stores or inflammatory burden. CRP was elevated (>3 mg/L) in 95 (95%) patients, while 5 (5%) had low-grade inflammation and none had normal CRP levels, reflecting marked systemic inflammation in the study group.

A significantly higher proportion of NAFLD patients exhibited Vitamin D deficiency (79%) compared to controls (13%), and this difference was highly significant (χ²=85.04, p<0.001), indicating a strong association between hypovitaminosis D and NAFLD. Although elevated serum ferritin levels were more frequent among NAFLD cases (40%) than controls (31%), this difference did not reach statistical significance (χ²=1.76, p=0.185). In contrast, elevated CRP levels were observed in 95% of NAFLD patients, whereas none of the controls had raised CRP levels (0%), and this association was highly significant on Fisher’s exact testing (p<0.001), reflecting a pronounced inflammatory state in NAFLD (Table/Fig 4).

The Mann-Whitney U test demonstrated significant differences in all three biomarkers between NAFLD cases and controls. NAFLD patients had significantly higher median serum ferritin [183.0 (148.0-254.75) ng/mL] and CRP levels [6.5 (5.0-9.5) mg/L] compared to controls [145.0 (97.75-236.25) ng/mL and 1.5 (0.8-2.0) mg/L, respectively]. Conversely, median Vitamin D levels were significantly lower in NAFLD patients [13.0 (8.57-17.0) ng/mL] than in controls [24.0 (22.0-30.5) ng/mL], indicating increased inflammatory burden and Vitamin D deficiency in NAFLD (Table/Fig 5).

Although mean serum ferritin values showed minor variations across grades (205.4±96.8 ng/mL in Grade 1, 210.7±101.3 ng/mL in Grade 2, and 214.9 ±108.6 ng/mL in Grade 3), the differences were small and did not indicate a consistent relationship with disease severity. Similarly, Vitamin D levels demonstrated only modest variation across grades (13.1±5.7, 14.0±6.1, and 14.6±6.4 ng/mL for grades 1, 2, and 3, respectively), suggesting no clear trend with increasing severity. In contrast, CRP levels increased from 6.2±2.4 mg/L in Grade 1 to 9.1±2.8 mg/L in Grade 2 and 12.8±3.1 mg/L in Grade 3, indicating a possible association between systemic inflammation and worsening NAFLD (Table/Fig 6).

Spearman’s rank correlation analysis demonstrated no significant correlation between serum ferritin levels and NAFLD severity (r=-0.024, p=0.813), indicating that ferritin levels did not vary systematically with increasing ultrasonographic grade of NAFLD. No significant correlation was observed between Vitamin D levels and NAFLD severity (r=0.149, p=0.139). In contrast, CRP exhibited a moderately strong positive correlation with NAFLD grade (r=0.604, p<0.001), suggesting that systemic inflammation increases significantly as the severity of hepatic steatosis worsens. These findings indicate that among the biomarkers studied, CRP was the only marker significantly associated with NAFLD severity, whereas serum ferritin and Vitamin D appeared to be associated with the presence of NAFLD rather than the extent of disease severity (Table/Fig 7).

Discussion

The present case-control study evaluated serum ferritin, Vitamin D status, and CRP as biomarkers of inflammatory burden in patients with NAFLD. The findings demonstrated that NAFLD patients had significantly higher serum ferritin and CRP levels and significantly lower Vitamin D concentrations than healthy controls, highlighting the co-existence of metabolic dysregulation and systemic inflammation in NAFLD.

Serum ferritin levels were significantly higher in NAFLD patients than in controls, supporting previous evidence linking iron metabolism to NAFLD pathogenesis (28),(29),(30). Elevated iron stores may promote oxidative stress, lipid peroxidation, and insulin resistance, thereby contributing to hepatic injury and disease progression. However, although ferritin levels differed significantly between cases and controls, the proportion of subjects with elevated ferritin (>200 ng/mL) was not significantly different between groups. Furthermore, serum ferritin showed no significant correlation with ultrasonographic NAFLD severity. These findings suggest that ferritin may be associated with the presence of NAFLD and underlying metabolic disturbances but may have limited utility as an indicator of disease severity.

Vitamin D deficiency was highly prevalent among NAFLD patients, with 79% of cases exhibiting deficient levels. This observation is consistent with previous studies reporting an association between low Vitamin D status and hepatic steatosis, insulin resistance, and metabolic dysfunction (31),(32),(33). Vitamin D exerts anti-inflammatory and immunomodulatory effects and has been implicated in the regulation of glucose and lipid metabolism. The significantly lower Vitamin D levels observed in NAFLD patients support its potential role in disease susceptibility. However, no significant correlation was observed between Vitamin D levels and NAFLD severity, suggesting that Vitamin D deficiency may be more closely related to disease occurrence than progression.

Among the biomarkers evaluated, CRP emerged as the strongest indicator of disease activity. CRP levels were markedly elevated in NAFLD patients, and 95% of cases exhibited CRP concentrations above the defined cut-off value. In addition, CRP demonstrated a significant positive correlation with NAFLD severity (r=0.604, p<0.001), indicating that systemic inflammation increases with worsening hepatic steatosis. These findings are consistent with previous reports identifying chronic low-grade inflammation as a key driver of NAFLD progression and cardiometabolic complications (34),(35). The absence of significant correlations between NAFLD severity and serum ferritin or Vitamin D levels suggests that these biomarkers may reflect metabolic and inflammatory alterations associated with disease presence rather than disease progression. In contrast, CRP appears to better represent ongoing inflammatory activity and severity of hepatic involvement. Therefore, the combined assessment of serum ferritin, Vitamin D, and CRP may provide complementary information, with ferritin and Vitamin D serving as indicators of disease burden and CRP acting as a marker of disease severity.

Overall, the findings underscore the multifactorial nature of NAFLD and support the integration of inflammatory and metabolic biomarkers in its clinical evaluation. Given their affordability and widespread availability, these biomarkers may aid in identifying at-risk individuals and monitoring disease activity. Further longitudinal studies are required to clarify causal relationships and determine whether interventions targeting Vitamin D deficiency, iron dysregulation, and systemic inflammation can improve clinical outcomes in NAFLD.

Limitation(s)

The present study has certain limitations. Its case-control design does not allow the establishment of causal relationships between serum ferritin, Vitamin D, CRP, and NAFLD. The study was conducted at a single tertiary care centre with a relatively modest sample size, which may limit the generalisability of the findings. NAFLD diagnosis and grading were based on ultrasonography rather than liver biopsy, the gold standard for disease assessment. Additionally, the absence of longitudinal follow-up precluded evaluation of temporal changes in biomarker levels and their relationship with disease progression. Fibrosis scores such as FIB-4, NAFLD Fibrosis Score, and ELF were not calculated due to lack of required biochemical parameters.

Conclusion

The present study demonstrated that NAFLD exhibits significantly higher serum ferritin and CRP levels and significantly lower Vitamin D concentrations compared with healthy controls. Vitamin D deficiency was highly prevalent among NAFLD patients, while elevated CRP levels were observed in the majority of cases, indicating a substantial inflammatory burden. Serum ferritin and Vitamin D levels were not significantly correlated with disease severity. In contrast, CRP demonstrated a strong positive correlation with NAFLD severity, suggesting that it may serve as a useful marker of disease progression and inflammatory activity. The combined assessment of serum ferritin, Vitamin D, and CRP may provide a simple, cost-effective approach for identifying patients with increased metabolic and inflammatory burden. Further prospective studies are required to validate these findings and to determine the clinical utility of these biomarkers in monitoring disease progression and guiding therapeutic interventions in NAFLD.

Authors’ contribution: The present study was done by Dr. Mary Chandrika Anton under the guidance of Dr. B. Shanthi. Mr. Vasudevan Elumalai contributed with sample collection and Dr. K. Sumathi contributed in discussion.

DOI and Others

DOI: 10.7860/JCDR/2026/87939.24394

Date of Submission: Feb 03, 2026
Date of Peer Review: Mar 17, 2026
Date of Acceptance: Jul 08, 2026
Date of Publishing: Sep 01, 2026

Author declaration:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA

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