Type 2 diabetes mellitus (T2DM) is a complex and heterogeneous metabolic disorder characterized by insulin resistance, progressive pancreatic β-cell dysfunction, and persistent hyperglycemia. Its pathogenesis extends beyond abnormalities in insulin secretion and action and involves chronic low-grade inflammation, oxidative stress, altered lipid metabolism, and disrupted communication among metabolically active organs [1,2]. The increasing global prevalence of T2DM has intensified interest in identifying circulating molecules that may improve understanding of its pathophysiology and reflect the metabolic disturbances accompanying the disease [1]. Among these molecules, hepatokines have emerged as important mediators linking hepatic metabolism with systemic insulin sensitivity and glucose homeostasis [3]. The liver functions not only as a central organ for glucose and lipid metabolism but also as an endocrine organ capable of secreting proteins that influence distant tissues [3,4]. These liver-derived proteins, collectively termed hepatokines, can modulate insulin signaling, inflammation, energy metabolism, and cardiometabolic risk. Dysregulated hepatokine production has therefore been proposed as an important mechanism connecting hepatic metabolic dysfunction with insulin resistance, obesity, metabolic dysfunction-associated steatotic liver disease, and T2DM [3–5]. Fetuin-A, also known as alpha-2-HS-glycoprotein (AHSG), is a glycoprotein synthesized predominantly by hepatocytes and released into the systemic circulation [6]. Fetuin-A has attracted considerable attention because of its diverse metabolic and physiological functions. Experimental and clinical evidence indicates that fetuin-A may inhibit insulin-receptor tyrosine kinase activity and thereby interfere with downstream insulin signaling, providing a potential mechanism through which elevated circulating concentrations could contribute to insulin resistance [6–8]. Fetuin-A may also interact with free fatty acids and inflammatory pathways, potentially promoting metabolic inflammation and impaired insulin action [7,8]. Clinical studies have consequently investigated fetuin-A as a possible biomarker of metabolic dysfunction. Higher circulating concentrations have been associated with insulin resistance, metabolic syndrome, obesity, dyslipidemia, and abnormalities of glucose metabolism [6,9]. A systematic review and meta-analysis demonstrated an association between circulating fetuin-A and metabolic syndrome, supporting its potential role in the clustering of cardiometabolic abnormalities [9]. Studies in patients with newly diagnosed T2DM have additionally linked fetuin-A with hepatic steatosis and metabolic abnormalities, further emphasizing the close relationship between hepatic dysfunction and systemic glucose regulation [10]. Nevertheless, the biological significance of fetuin-A is complex and should not be interpreted exclusively in terms of adverse metabolic effects. Fetuin-A is an important systemic inhibitor of pathological mineral precipitation and ectopic calcification and participates in the formation and stabilization of calciprotein particles [6]. Thus, its physiological and pathological effects may vary according to metabolic state, inflammation, renal function, mineral metabolism, and underlying cardiovascular risk. These multiple functions may partly explain differences in fetuin-A concentrations and associations reported across clinical populations. Despite growing evidence linking fetuin-A with insulin resistance and T2DM, its relationship with the duration and degree of glycemic disturbance remains incompletely defined, particularly in local populations. Evaluating fetuin-A alongside measures such as HbA1c and blood glucose may provide additional insight into its relationship with chronic metabolic dysregulation. Therefore, the present study aimed to compare serum fetuin-A concentrations between adults with T2DM and apparently healthy controls and to evaluate its associations with diabetes duration, age, HbA1c, and blood glucose.

A hospital-based case-control study was conducted at Kirkuk Teaching Hospital, Kirkuk, Iraq, from 1 December 2025 to 30 April 2026. The study included 150 participants, comprising 100 adults with established type 2 diabetes mellitus (T2DM) as the case group and 50 apparently healthy individuals as the control group. Eligible participants were aged 40–69 years. Among patients with T2DM, 60 (60.00%) were men and 40 (40.00%) were women. Patients with T2DM were recruited from individuals attending Kirkuk Teaching Hospital during the study period. The control group consisted of apparently healthy adults without a known diagnosis of diabetes mellitus. Demographic and clinical information was recorded for each participant, including age and sex. Among patients with T2DM, the duration of diabetes was also documented. Glycemic status was assessed using blood glucose and glycated hemoglobin (HbA1c). Blood Collection and Sample Preparation Approximately 5 mL of venous blood was collected from each participant under standard aseptic conditions. Approximately 1 mL was transferred into an ethylenediaminetetraacetic acid (EDTA) tube for HbA1c measurement, while the remaining 4 mL was transferred into a gel-containing serum-separation tube. After clot formation, serum was separated and used for biochemical testing and measurement of serum fetuin-A. Measurement of Serum Fetuin-A Serum fetuin-A concentrations were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit supplied by Bioassay Technology Laboratory, China. The assay was performed according to the manufacturer's sandwich ELISA principle. Serum samples were added to antibody-coated wells, allowing fetuin-A in the specimens to bind to the corresponding specific antibody. Following the prescribed incubation and washing procedures, the detection reagents and substrate were added, producing a color reaction proportional to the amount of fetuin-A present in each sample. Optical density was measured spectrophotometrically at 450 nm, and fetuin-A concentrations were determined from the corresponding standard calibration curve. The source laboratory protocol described a 40 nmol/mL working standard, with serial standards of 20, 10, 5, and 2.5 nmol/mL, whereas the final clinical results were recorded in mg/mL. No retrospective conversion between these units was performed because an accurate conversion requires confirmation of the original assay specifications, dilution procedures, and calculation method. Therefore, the reported study units were retained, but they should be verified against the original ELISA kit insert, standard curve, and laboratory worksheets before final manuscript submission. Measurement of Glycemic Parameters HbA1c was measured in EDTA-anticoagulated whole blood using the i-CHROMA II fluorescence immunoassay system and was expressed as a percentage. Blood glucose concentration was determined using a commercial Biolabo enzymatic method, according to the manufacturer's instructions. These parameters were used to characterize glycemic status and to investigate their relationships with circulating serum fetuin-A concentrations. Study Variables and Outcomes The principal outcome of the study was serum fetuin-A concentration. The primary analysis compared mean serum fetuin-A concentrations between adults with T2DM and apparently healthy controls. Secondary analyses evaluated the relationships of serum fetuin-A with duration of diabetes, age, HbA1c, and blood glucose among patients with T2DM. Serum fetuin-A concentrations were also examined according to sex within the diabetic group. Statistical Analysis Statistical analyses were performed using IBM SPSS Statistics version 23.1. Continuous variables were summarized as mean ± standard deviation (SD), whereas categorical variables were expressed as frequencies and percentages. The independent-samples Student's t-test was used for comparisons of continuous variables between two independent groups. Analysis of variance (ANOVA) was used for comparisons involving more than two groups when appropriate, while categorical variables were evaluated using the chi-square test. Correlation analysis was performed to determine the direction and strength of the relationships between serum fetuin-A and continuous clinical and biochemical variables, including duration of diabetes, age, HbA1c, and blood glucose. Correlation coefficients (r) and corresponding P values were reported. Statistical tests were two-sided, and P≤0.05 was considered statistically significant. To provide an additional measure of the magnitude of the difference in fetuin-A between the T2DM and control groups, the mean difference with its 95% confidence interval (CI) and Hedges' g standardized effect size were calculated from the reported sample sizes, means, and SDs. These values represent secondary calculations based on published summary statistics rather than participant-level reanalysis.

The study included 150 participants, comprising 100 adults with T2DM and 50 apparently healthy controls. The age distribution was generally comparable between the two groups, and no statistically significant difference in the reported age categories was observed (P>0.05). The largest proportion of patients with T2DM was aged 50–55 years (35.00%), whereas the largest proportion of controls was aged 45–49 years (34.00%). Table 1. Age distribution of patients with T2DM and healthy controls Age (years) T2DM, n (%) Controls, n (%) 45–49 22 (22.00%) 17 (34.00%) 50–55 35 (35.00%) 13 (26.00%) 56–60 23 (23.00%) 16 (32.00%) 61–66 20 (20.00%) 4 (8.00%) Total 100 (100.00%) 50 (100.00%) The T2DM group included 60 men (60.00%) and 40 women (40.00%). The available data do not provide the corresponding sex distribution of the control group; therefore, a formal comparison of sex distribution between cases and controls cannot be presented. Table 2. Sex distribution among patients with T2DM Sex Number Percentage Male 60 60.00% Female 40 40.00% Total 100 100.00% Serum fetuin-A concentrations were substantially higher among patients with T2DM than among healthy controls. The mean concentration was 20.10 ± 6.74 mg/mL in the T2DM group compared with 9.08 ± 1.52 mg/mL in controls. The difference was highly statistically significant (P<0.001), indicating a clear elevation of circulating fetuin-A in the diabetic group. Table 3. Serum fetuin-A concentration in patients with T2DM and healthy controls Measure T2DM (n=100), mean ± SD Controls (n=50), mean ± SD P value Fetuin-A (reported mg/mL) 20.10 ± 6.74 9.08 ± 1.52 <0.001 To quantify the magnitude of the observed case-control difference, additional effect estimates were calculated from the reported summary statistics. The mean difference in serum fetuin-A between the T2DM and control groups was 11.02 mg/mL, with a 95% CI of 9.63–12.41 mg/mL. The corresponding Hedges' g was 1.96, demonstrating a large standardized difference between the two groups. Table 4. Magnitude of the difference in serum fetuin-A between T2DM patients and controls Parameter Estimate Mean difference 11.02 mg/mL 95% CI 9.63–12.41 mg/mL Hedges' g 1.96 Effect-size interpretation Large Reported P value <0.001 The mean difference, 95% CI, and Hedges' g were reconstructed from the reported sample sizes, means, and SDs. Correlation analysis demonstrated significant positive relationships between serum fetuin-A and all four evaluated clinical and glycemic variables among patients with T2DM. The strongest correlation was observed with duration of diabetes (r=0.762, P=0.001), followed by age (r=0.634, P=0.001) and HbA1c (r=0.549, P=0.001). Blood glucose showed a weaker but statistically significant positive correlation with fetuin-A (r=0.249, P=0.012). Table 5. Correlations of serum fetuin-A with clinical and glycemic variables among patients with T2DM Variable r P value Strength Duration of diabetes 0.762 0.001 Strong positive Age 0.634 0.001 Strong positive HbA1c 0.549 0.001 Moderate-to-strong positive Blood glucose 0.249 0.012 Weak positive Ranking the correlations according to their absolute magnitude demonstrated that diabetes duration had the strongest relationship with circulating fetuin-A. Age was the second most strongly associated variable, followed by HbA1c, whereas blood glucose demonstrated the weakest association. These findings suggest that fetuin-A may be more closely related to chronic clinical and metabolic characteristics than to a single measurement of circulating glucose. Table 6. Ranking of clinical and glycemic factors associated with serum fetuin-A Rank Variable r P value Relative association 1 Duration of diabetes 0.762 0.001 Strongest 2 Age 0.634 0.001 Second strongest 3 HbA1c 0.549 0.001 Third strongest 4 Blood glucose 0.249 0.012 Weakest Sex-stratified analysis showed that women with T2DM had a numerically higher mean serum fetuin-A concentration than men (23.19 ± 9.20 vs 21.37 ± 7.92 mg/mL). However, the difference was not statistically significant (P>0.05), indicating that the available data did not demonstrate a significant sex-related difference in circulating fetuin-A among patients with T2DM. Table 7. Sex-stratified serum fetuin-A concentrations among patients with T2DM Sex n Fetuin-A, mean ± SD (mg/mL) P value Female 40 23.19 ± 9.20 Male 60 21.37 ± 7.92 >0.05 The overall case-control comparison showed that mean fetuin-A was approximately 2.21-fold higher in patients with T2DM than in healthy controls. This marked elevation was accompanied by a large standardized effect size and significant positive correlations with diabetes duration, age, HbA1c, and blood glucose. Table 8. Summary of the principal serum fetuin-A findings Analysis Main finding Statistical result T2DM vs controls Higher fetuin-A in T2DM P<0.001 T2DM/control mean ratio 2.21-fold higher — Mean difference 11.02 mg/mL 95% CI: 9.63–12.41 Standardized difference Large Hedges' g=1.96 Fetuin-A vs diabetes duration Strong positive correlation r=0.762; P=0.001 Fetuin-A vs age Strong positive correlation r=0.634; P=0.001 Fetuin-A vs HbA1c Moderate-to-strong positive correlation r=0.549; P=0.001 Fetuin-A vs blood glucose Weak positive correlation r=0.249; P=0.012 Sex difference Not statistically significant P>0.05 conversion should be made without these records.

1. Galicia-Garcia U, Benito-Vicente A, Jebari S, et al. Pathophysiology of type 2 diabetes mellitus. Int J Mol Sci. 2020;21(17):6275. doi:10.3390/ijms21176275. 2. Lu X, Xie Q, Pan X, et al. Type 2 diabetes mellitus in adults: pathogenesis, prevention and therapy. Signal Transduct Target Ther. 2024;9:262. 3. Meex RCR, Watt MJ. Hepatokines: linking nonalcoholic fatty liver disease and insulin resistance. Nat Rev Endocrinol. 2017;13(9):509-520. 4. Stefan N, Häring HU. The role of hepatokines in metabolism. Nat Rev Endocrinol. 2013;9(3):144-152. 5. Icer MA, Yıldıran H. Effects of fetuin-A with diverse functions and multiple mechanisms on human health. Clin Biochem. 2021;88:1-10. doi:10.1016/j.clinbiochem.2020.11.004. 6. Trepanowski JF, Mey J, Varady KA. Fetuin-A: a novel link between obesity and related complications. Int J Obes (Lond). 2015;39(5):734-741. 7. Pal D, Dasgupta S, Kundu R, et al. Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance. Nat Med. 2012;18(8):1279-1285. doi:10.1038/nm.2851. Nature 8. Srinivas PR, Wagner AS, Reddy LV, et al. Serum alpha 2-HS-glycoprotein is an inhibitor of the human insulin receptor at the tyrosine kinase level. Mol Endocrinol. 1993;7(11):1445-1455. 9. Mohammed SK, Taha EM, Muhi SA. A case-control study to determination FBXW7 and Fetuin-A levels in patients with type 2 diabetes in Iraq. J Diabetes Metab Disord. 2021;20(1):237-243. doi:10.1007/s40200-021-00738-x. PubMed 10. Roshanzamir F, Miraghajani M, Rouhani MH, Mansourian M, Ghiasvand R, Safavi SM. The association between circulating fetuin-A levels and type 2 diabetes mellitus risk: systematic review and meta-analysis of observational studies. J Endocrinol Invest. 2018;41(1):33-47. PubMed 11. Pan X, Wen SW, Bestman PL, Kaminga AC, Acheampong K, Liu A. Fetuin-A in metabolic syndrome: a systematic review and meta-analysis. PLoS One. 2020;15(3):e0229776. doi:10.1371/journal.pone.0229776. PLOS 12. Mathews ST, Singh GP, Ranalletta M, et al. Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene. Diabetes. 2002;51(8):2450-2458. 13. Dasgupta S, Bhattacharya S, Biswas A, et al. NF-κB mediates lipid-induced fetuin-A expression in hepatocytes that impairs adipocyte function effecting insulin resistance. Biochem J. 2010;429(3):451-462. 14. Stefan N, Fritsche A, Weikert C, et al. Plasma fetuin-A levels and the risk of type 2 diabetes. Diabetes. 2008;57(10):2762-2767. 15. Lanthier N, Lebrun V, Molendi-Coste O, van Rooijen N, Leclercq IA. Liver fetuin-A at initiation of insulin resistance. Metabolites. 2022;12(11):1023. doi:10.3390/metabo12111023. PubMed 16. Lee-Ødegård S, Ueland T, Thorsby PM, Aukrust P, Michelsen AE, Halvorsen B, Drevon CA, Birkeland KI. Fetuin-A mediates the difference in adipose tissue insulin resistance between young adult Pakistani and Norwegian patients with type 2 diabetes. BMC Endocr Disord. 2022;22(1):208. doi:10.1186/s12902-022-01127-y. PubMed 17. Yamasandhi PG, Dharmalingam M, Balekuduru A. Fetuin-A in newly detected type 2 diabetes mellitus as a marker of non-alcoholic fatty liver disease. Indian J Gastroenterol. 2021;40(6):556-562. doi:10.1007/s12664-021-01176-6. PubMed 18. Sun Q, Cornelis MC, Manson JE, Hu FB. Plasma levels of fetuin-A and hepatic enzymes and risk of type 2 diabetes in women in the U.S. Diabetes. 2013;62(1):49-55. 19. Jahnen-Dechent W, Heiss A, Schäfer C, Ketteler M. Fetuin-A regulation of calcified matrix metabolism. Circ Res. 2011;108(12):1494-1509. 20. Ketteler M, Bongartz P, Westenfeld R, et al. Association of low fetuin-A levels in serum with cardiovascular mortality in patients on dialysis: a cross-sectional study. Lancet. 2003;361(9360):827-833. 21. Ix JH, Shlipak MG, Brandenburg VM, Ali S, Ketteler M, Whooley MA. Association between human fetuin-A and the metabolic syndrome: data from the Heart and Soul Study. Circulation. 2006;113(14):1760-1767. 22. Jensen MK, Bartz TM, Djoussé L, et al. Genetically elevated fetuin-A levels, fasting glucose levels, and risk of type 2 diabetes: the Cardiovascular Health Study. Diabetes Care. 2013;36(10):3121-3127. 23. Mori K, Emoto M, Yokoyama H, et al. Association of serum fetuin-A with insulin resistance in type 2 diabetic and nondiabetic subjects. Diabetes Care. 2006;29(2):468. 24. Ismail NA, Ragab S, El Dayem SMA, et al. Fetuin-A levels in obesity: differences in relation to metabolic syndrome and correlation with clinical and laboratory variables. Arch Med Sci. 2012;8(5):826-833. 25. Roshanzamir F, Miraghajani M, Rouhani MH, Mansourian M, Ghiasvand R, Safavi SM. Fetuin-A levels and risk of type 2 diabetes mellitus: a systematic review and meta-analysis. Evidence from prospective and observational studies supports an association between higher circulating fetuin-A and increased T2DM risk