INTRODUCTION
β-Thalassemia is among the most common genetic hemoglobin production disorders that require regular blood transfusions to sustain patient survival [1, 2]. However, ongoing blood transfusions lead to iron overload in vital tissues, particularly the liver, causing oxidative stress and cellular injury, as evidenced by persistently elevated serum aminotransferase levels, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) [3]. Thus, these enzymes are the most important clinical biomarkers for assessing the extent of oxidative stress and hepatic damage in these patients [1]. Zinc oxide nanoparticles (ZnO NPs) have unique optical and physicochemical properties, and their interactions with biological systems have led to a significant increase in their medical and biological applications [4, 5]. The high surface activity of these particles and their ability to release zinc ions upon entering or coming into contact with biological fluids such as blood and serum make them highly prone to binding to biomolecules [6, 7]. The direct in vitro interaction between nanoparticles and enzymes is governed by distinct biochemical mechanisms compared with the direct in vivo cytotoxic effect, which usually results in enzyme release and increased serum concentrations [8, 9]. When ZnO nanoparticles come into contact with serum, their surface is rapidly covered by a dynamic layer of adsorbed proteins, known as the protein corona [10]. This physicochemical interaction may alter the three-dimensional conformation of the enzymes or occlude their active sites, thereby directly inhibiting their enzymatic activity [11, 12]. Furthermore, the presence of nanoparticles in biological specimens can interfere with spectroscopic reagents used in routine laboratory assays, leading to false-negative results that prevent an accurate diagnostic evaluation [13–15].Despite the critical diagnostic importance of ALT and AST enzymes in thalassemia patients, who possess a serum matrix characterized by specific biochemical properties such as elevated levels of ferritin and free iron, the impact of direct exposure to ZnO NPs on the activity of these enzymes in the patients’ serum has not been adequately investigated. Accordingly, this study aims to systematically evaluate, in vitro, the concentration and time-dependent effects of ZnO NPs on the enzymatic activities of ALT and AST in the serum of thalassemia patients, while elucidating the kinetic variations in the enzymes’ sensitivity to nanoparticle-induced inhibition and the underlying biochemical mechanisms.

MATERIALS AND METHODS
Synthesis of ZnO Nanoparticles
Zinc oxide nanoparticles (ZnO NPs) were prepared via chemical precipitation. Briefly, Zn(NO₃)₂·6H₂O (4.0 g, 0.54 M) and NaOH (1.0 g, 1.0 M) were dissolved separately in 25 mL of deionized water. The precursor solution was heated to 60 °C, and the NaOH solution was added dropwise over 30 min under continuous stirring until reaching pH 7. The mixture was ultrasonicated for 15 min, magnetically stirred at 90–100 °C for 1 h, and filtered under vacuum (Whatman No. 42). The precipitate was washed with absolute ethanol (2×) and deionized water (3×), dried at 120 °C for 6 h, and calcined at 600 °C for 4 h to yield ZnO NPs.

Study Design and Experimental Workflow
Serum samples were collected from 30 transfusion-dependent β-thalassemia major patients (n = 30) and 30 age- and sex-matched healthy controls (n = 30) recruited from Diyala Governorate, Iraq. Equal amounts of each donor were treated with ZnO NPs at concentrations of 0 (control), 25, 50, and 100 µg/mL and incubated for 6, 12, 18, or 24 h. Samples were centrifuged after incubation to sediment nanoparticles and to reduce analytical interferences. The clarified supernatants were immediately analyzed for ALT and AST activity using a Mindray BS-120 auto-analyzer. All experimental assays were performed in triplicate (3×).

Ethical Approval and Patient Selection
Diyala Health Directorate, Iraq, and conducted in accordance with the Declaration of Helsinki. This study was approved by the Institutional Review Board (IRB). All participants or their legal guardians provided written informed consent. The cohort included 30 patients with iron overload and transfusion-dependent beta-thalassemia major, as well as 30 healthy controls. Exclusion criteria were active viral hepatitis (HBV/HCV), HIV, liver cirrhosis, or concurrent use of hepatotoxic drugs. Venous blood was drawn after an overnight fast, allowed to clot at room temperature, and centrifuged (3000 × g, 10 min). Clarified serum aliquots were either analyzed immediately or stored at −80 °C [16].

Biochemical Measurement of Serum ALT and AST Activities
Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured at 37 °C using an automatic clinical chemistry analyzer (Mindray BS-120, Shenzhen Mindray Bio-Medical Electronics Co., Ltd., China) and the corresponding commercial reagent kits, according to the IFCC standardized UV kinetic methods without pyridoxal phosphate activation [17–19]. Enzyme activity was determined in both assays by measuring the rate of decrease in NADH absorbance at 340 nm [17–19]. The measuring ranges were linear from 4 to 1000 U/L for ALT and 4 to 800 U/L for AST [17–19]. Samples exceeding upper linearity limits were diluted in 9 g/L NaCl saline, re-assayed, and multiplied by the corresponding dilution factor [17–19]. System calibration and analytical accuracy were regularly verified using Mindray Multi Sera Calibrator and two levels of Mindray ClinChem Multi Control materials according to the manufacturer’s specified acceptable ranges [17–19].

In Vitro Hemocompatibility Evaluation
To evaluate cell membrane integrity and establish the biosafety profile of ZnO nanoparticles prior to downstream serum-exposure assays, an in vitro hemolysis test was conducted across serial concentrations (32, 64, 128, and 256 µg/mL) [20]. Phosphate-buffered saline (PBS) and 1% Triton X-100 served as the negative control (baseline 0% lysis) and positive control (complete 100% cellular disruption), respectively [20, 21]. After incubating the RBC suspensions at 37 °C for 1 h, samples were centrifuged at 1500 × g for 10 min, and the released hemoglobin in the supernatant was quantified spectrophotometrically at 540 nm using a UV-Vis spectrophotometer. The quantitative percentage of hemolysis was calculated according to the following standard equation [20, 21]:

Hemolysis (%) = (Asample - Anegative control)/(Apositive control-Anegative control) × 100

Statistical Analysis
Data were analyzed using GraphPad Prism (v9.0) and SPSS (v26.0). Continuous metrics are expressed as mean ± SD or median (IQR), and percentage reductions are reported with 95% CIs. Enzymatic activities and treatment-to-control ratios were log-transformed to meet parametric assumptions and back-transformed to report geometric mean ratios (GMR). The main and interaction effects of ZnO NP concentration (0–100 µg/mL) and incubation duration (6–24 h) on ALT and AST activity were evaluated by two-way RM-ANOVA with Greenhouse-Geisser correction and partial η 2 effect sizes. Pairwise comparisons between treatment and control were conducted using planned contrasts with Holm’s adjustment of p-values and Cohen’s d effect sizes. Statistical significance was defined as two-tailed p < 0.05.

RESULTS AND DISCUSSION
FT-IR Spectroscopic Analysis of ZnO Nanoparticles
FT-IR spectroscopy (Fig. 1) of synthesized ZnO nanoparticles was used to validate the functional groups and phase formation. The characteristic absorption bands at 501.49 and 416.62 cm-1 are attributed to the Zn-O stretching vibrations, confirming the hexagonal wurtzite structure [22]. Symmetric C–O/C=O stretching or residual precursor nitrates (NO₃⁻) [23] are assigned to a sharp band at 1404.18 cm⁻¹. Moreover, the broad high-frequency bands at 3201.83 cm⁻¹ and 3140.11 cm⁻¹ are attributed to O–H stretching of surface-adsorbed hydroxyl groups and moisture, respectively, thereby indicating the structural purity of the nanoparticles [24].

Structural Analysis of ZnO Nanoparticles by XRD
The crystalline phase purity and structural parameters of synthesized ZnO nanoparticles were determined by X-ray diffraction (XRD) analysis (Fig. 2). The XRD pattern reveals sharp and highly intense diffraction peaks, which are in full agreement with the standard crystallographic reference (COD Entry No. 96-230-0113), indicating a single-phase hexagonal wurtzite structure with 100.0% phase purity and space group P63mc [25]. The refined lattice parameters were a = 3.2494 Å and c = 5.2054 Å with a calculated theoretical density of 5.677 g/cm 3 and a stoichiometric elemental distribution of 80.3 wt% Zn and 19.7 wt% O. No secondary phases or crystalline impurities from unreacted precursor materials were observed, indicating high phase purity. The average crystallite size (D) was calculated as 24.61 nm using the Debye–Scherrer equation across the characteristic Bragg reflection angles (2θ = 27.53°–68.18°, Cu-Kα radiation, λ = 1.54178 Å, K = 0.94), which confirmed the successful fabrication of ultra-small nanoscale crystallites [26].

EDX Elemental Analysis of ZnO Nanoparticles
Fig. 3 shows the Energy-Dispersive X-ray (EDX) spectroscopy analysis of the elemental composition and chemical purity of synthesized ZnO nanoparticles. The EDX spectrum clearly shows the characteristic optical absorption peaks for Zn (Zn Lα at ~1.01 keV, Zn Kα at ~8.63 keV, and Zn Kβ at ~9.57 keV) and O (O Kα at ~0.52 keV), confirming the synthesis of zinc oxide nanostructures unequivocally [27]. Quantitative elemental analysis demonstrated a weight composition of 86.82 wt % Zn and 13.18 wt % O, corresponding to atomic percentages of 61.71 at % Zn and 38.29 at % O, respectively. The nonexistence of additional impurity peaks in the spectrum confirms the high chemical purity and phase stoichiometry of the prepared nanoparticles, therefore strongly supporting the XRD and FT-IR observations [28].

Morphological Analysis by SEM of the synthesized ZnO nanoparticles
Scanning Electron Microscopy (SEM) was used to characterize the surface morphology, grain shape and particle size distribution of the synthesized ZnO nanoparticles (Fig. 4). The SEM micrograph shows quasi-spherical and faceted polyhedral nanoparticles with sharp boundaries. There is also significant agglomeration of the nanoparticles owing to high surface energy and cohesive electrostatic forces during the synthesis process [31]. The particle size distribution is shown in the inset histogram ranging from 35-75 nm and the calculated mean particle size is 58.23 nm [29]. The discrepancy between the SEM average particle size (58.23 nm) and the XRD crystallite size (24.61 nm) is explained by the aggregation of several single sub-nanometer crystallites to form larger polycrystalline grains observed by electron microscopy [30].

Descriptive Statistics of Serum ALT and AST Activities
Table 1 shows the serum ALT and AST activities after in vitro exposure to ZnO NPs at different concentrations (0, 25, 50 and 100 µg/mL) and incubation times (6, 12, 18 and 24 h). The unexposed baseline control (0 µg/mL) showed increased enzyme activities (ALT: 415.02-422.98 U/L; AST: 410.14-416.90 U/L), which suggest baseline hepatic involvement and chronic iron overload in patients with β-thalassemia major [1, 3]. Incubation with ZnO NPs resulted in a progressive reduction of the measured transaminase activities, in a concentration and time dependent manner. At 6 h, exposure to 25 µg/mL ZnO NPs decreased mean ALT activity from 421.46 ± 712.91 U/L to 205.72 ± 358.37 U/L, reaching a minimum of 24.88 ± 46.48 U/L after 24 h at 100 µg/mL. Similarly, mean AST activity fell from 410.14 ± 636.06 U/L in baseline controls to 268.52 ± 401.42 U/L under maximum exposure conditions (24 h, 100 µg/mL). Unlike in vivo toxicological pathways, where enzyme leakage is caused by cell lysis, this direct in vitro suppression of activity is caused by physical interactions on the surface such as protein corona formation, conformational changes, or catalytic site blockage [8, 10, 11].

Percentage Reduction in Serum Transaminase Activities
Table 2 shows the percent reductions in serum ALT and AST activities compared with the unexposed controls and indicates a significant difference in kinetic sensitivity to nanoparticles. The ALT had high susceptibility to ZnO NPs with mean reductions from 51.6 ± 9.1% (6 h, 25 µg/mL) to 93.8 ± 5.2% (24 h, 100 µg/mL). In contrast, AST was more stable, with decreases of 12.9 ± 3.7% to 30.3 ± 8.5% under the same conditions. This differential inhibition indicates differences in structural binding affinities, protein conformations or surface charge interactions with the nanoparticle protein corona [9, 12]. Furthermore, colloidal optical interference at 340 nm during UV kinetic measurements may result in lower detectable enzyme activity [13–15].

Repeated-Measures Analysis of Variance (RM-ANOVA)
As presented in Table 3, two-way repeated-measures ANOVA with Greenhouse-Geisser correction revealed significant main and interactive effects of ZnO NP concentration and incubation duration on ALT and AST activities. For ALT, highly significant main effects were observed for concentration (F = 74.077, p < 0.001, partial η² = 0.949) and incubation time (F = 53.106, p < 0.001, partial η² = 0.930), as well as their interaction (F = 22.432, p < 0.001, partial η² = 0.849). Similarly, AST showed significant main effects for concentration (F = 68.625, p = 0.001, partial η² = 0.945) and time (F = 15.738, p = 0.003, partial η² = 0.797), along with a significant interaction effect (F = 12.457, p = 0.003, partial η² = 0.757). High effect sizes across all factors (partial η² > 0.75) confirm that nanoparticle dose and exposure duration independently and synergistically drive transaminase inactivation in thalassemic serum [7, 11].

Pairwise Treatment-to-Control Comparisons for ALT
As shown in Table 4, pairwise contrast analyses of log-transformed treatment-to-control geometric mean ratios (GMRs) with Holm’s correction confirmed significant reductions in ALT activity across all evaluated conditions (adjusted p ≤ 0.007). Increasing ZnO NP concentration and exposure time caused a progressive decrease in GMR from 0.477 (52.3% reduction; 6 h, 25 µg/mL) to a minimum of 0.050 (95.0% reduction; 24 h, 100 µg/mL). Very large standardized effect sizes (absolute Cohen’s d from 3.00 to 4.57) are indicative of significant nanoparticle interference that may result in dramatic false-negative underestimation of serum ALT levels in routine automated clinical assays [13–15].

Pairwise Treatment-to-Control Comparisons for AST
Table 5 shows that pairwise contrast analyzes of log-transformed treatment-to-control GMRs with Holm’s correction confirmed statistically significant reductions in AST activity for all conditions evaluated (adjusted p ≤ 0.009). The GMR gradually decreased from 0.870 (13.0% reduction; 6 h, 25 µg/mL) to a minimum of 0.693 (30.7% reduction; 24 h, 100 µg/mL) with increasing ZnO NP concentration and exposure time. Standardized effect sizes were large at all timepoints with a range of absolute Cohen’s d from 2.92 to 6.05. The percentage of total reduction of AST was observed to be less than that of ALT, which further confirms the higher enzymatic stability of AST and its different structural binding dynamics with ZnO nanoparticles [9, 12, 13-15].

In Vitro Hemocompatibility Evaluation of ZnO Nanoparticles
An in vitro hemolysis evaluation was performed using serial ZnO NP concentrations (32, 64, 128 and 256 µg/mL) to evaluate the biosafety profile and red blood cell (RBC) membrane integrity prior to downstream serum-exposure assays [20, 21]. As illustrated in Fig. 5, 1% Triton X-100 (positive control) treated erythrocytes were completely disrupted with marked release of hemoglobin in the supernatant (100% hemolysis). Conversely, PBS incubation (negative control) resulted in a clear supernatant with intact pelleted RBCs. The supernatants of all tested concentrations of ZnO NP (32–256 µg/mL) were clear with well-defined RBC pellets with visual profiles comparable to that of the negative control (Fig. 5). Quantitative spectrophotometric analysis at 540 nm confirmed that the hemolysis was minimal (< 2.0% for all tested concentrations), which is much lower than the 5.0% hemolysis threshold for non-hemolytic biomaterials according to ASTM F756 [20, 21]. These results agree with the good hemocompatibility and structural integrity of RBC membranes in the presence of ZnO NPs and show that the decrease in activities of ALT and AST is due to direct physical interactions of enzymes with nanoparticles and not due to artifacts like membrane-disruption [8, 20].

CONCLUSION
The present study clearly demonstrates that the direct in-vitro exposure of synthesized hexagonal wurtzite ZnO nanoparticles significantly suppresses the serum ALT and AST activities in a dose and time-dependent manner in transfusion-dependent β-thalassemia major patients. The red blood cell hemolysis assays confirmed good hemocompatibility (< 2.0% lysis) and preservation of the membrane. However, a significant difference in the enzyme vulnerability kinetics was revealed. ALT was highly susceptible to nanoparticle-induced inactivation (up to 93.8% reduction), while AST was significantly more structurally stable (up to 30.3% reduction). Two-way RM-ANOVA revealed that nanoparticle concentration and incubation time are independent and synergetic factors for transaminase suppression (partial η2 > 0.75). Mechanistically, loss of activity is caused by surface physical interactions including protein corona formation, masking of the catalytic active site, and colloidal light scattering that interferes with standard automated UV kinetic measurements at 340 nm. Therefore, clinical biochemists and hematologists should be careful in evaluating the liver function of thalassemic patients exposed to zinc-based nanomaterials, as direct nanoparticle-assay interactions could lead to profound false-negative diagnostic misinterpretations.

ACKNOWLEDGEMENTS
The authors sincerely thank the Department of Chemistry, College of Education for Pure Sciences, University of Diyala for providing the necessary laboratory facilities and support to perform this study. We would also like to thank the staff at the Diyala Health Directorate, the patients and healthy volunteers who participated in this research.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.