Fatty liver disease has emerged as one of the most prevalent chronic liver disorders worldwide and represents an increasingly important cause of hepatic and extrahepatic morbidity. The condition is characterized by excessive accumulation of triglycerides within hepatocytes and encompasses a broad spectrum ranging from isolated hepatic steatosis to steatohepatitis, progressive fibrosis, cirrhosis, and hepatocellular carcinoma. Traditionally, metabolically driven fatty liver disease was referred to as non-alcoholic fatty liver disease (NAFLD); however, contemporary nomenclature recognizes metabolic dysfunction-associated steatotic liver disease (MASLD) as the predominant metabolic phenotype of steatotic liver disease [1,2]. A large systematic review and meta-analysis estimated the global prevalence of NAFLD at approximately 30%, with a substantial increase in prevalence over recent decades, paralleling the global epidemics of obesity, type 2 diabetes mellitus, dyslipidemia, and metabolic syndrome [1]. The clinical importance of fatty liver disease extends beyond the simple accumulation of hepatic fat. While a considerable proportion of individuals may remain asymptomatic with uncomplicated steatosis, a subset develops hepatocellular inflammation, fibrosis, and ultimately advanced chronic liver disease. Cardiometabolic abnormalities, particularly central obesity, insulin resistance, type 2 diabetes mellitus, hypertension, and atherogenic dyslipidemia, are strongly linked to the development and progression of fatty liver disease [2,3]. Current clinical guidelines therefore emphasize early recognition of individuals with hepatic steatosis and identification of those at increased risk of progressive liver injury and fibrosis [2,3]. Nevertheless, diagnosing and assessing the severity of fatty liver disease in routine practice remains challenging because affected individuals may have few or no clinical manifestations, and conventional biochemical tests do not always accurately reflect the underlying histological severity. Ultrasonography is one of the most frequently used imaging modalities for the initial detection of hepatic steatosis because it is non-invasive, widely available, relatively inexpensive, and does not expose patients to ionizing radiation. Typical ultrasonographic features of fatty infiltration include increased hepatic parenchymal echogenicity relative to the renal cortex, progressive loss of visualization of intrahepatic vessels, posterior beam attenuation, and impaired visualization of the diaphragm [4,5]. Conventional ultrasonographic grading commonly categorizes fatty liver into mild (grade I), moderate (grade II), and severe (grade III) disease according to these characteristics [5]. A meta-analysis demonstrated that ultrasonography has good diagnostic performance for detecting moderate-to-severe hepatic steatosis, with pooled sensitivity and specificity of approximately 84.8% and 93.6%, respectively [4]. Despite these advantages, ultrasonographic assessment remains semiquantitative and may be influenced by operator experience, body habitus, the degree of steatosis, and associated hepatic fibrosis. In parallel with imaging, serum liver enzymes are routinely evaluated in patients with suspected fatty liver disease. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are commonly considered biochemical indicators of hepatocellular injury, whereas gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) may provide additional information regarding hepatobiliary abnormalities and metabolic dysfunction. ALT is generally regarded as more liver-specific than AST and may be elevated as a consequence of hepatocyte injury associated with steatosis and inflammation. However, aminotransferase concentrations can remain within conventionally defined reference ranges in some patients despite clinically significant fatty liver disease. Consequently, normal liver enzyme levels cannot reliably exclude progressive steatohepatitis or fibrosis [3]. This limitation emphasizes the importance of interpreting biochemical abnormalities in combination with clinical, metabolic, and imaging findings rather than using liver enzymes as isolated diagnostic indicators. Several studies have explored whether progressive ultrasonographic severity of hepatic steatosis is accompanied by corresponding increases in serum liver enzyme levels. Razavizade et al. reported an association between ultrasonographic severity and ALT concentrations in patients with NAFLD, although laboratory parameters did not clearly distinguish all levels of sonographic severity [6]. Similarly, Cruz et al. observed progressive increases in ALT, AST, and GGT concentrations across increasing grades of hepatic steatosis and found significant associations between these biochemical markers and ultrasonographic severity [7]. In their study, AST showed a particularly strong association with the degree of fatty liver. These findings suggest that increasing hepatic lipid accumulation and the accompanying metabolic and hepatocellular stress may contribute to progressive biochemical abnormalities. Nevertheless, the relationship between liver enzymes and sonographic severity remains inconsistent across populations. Recent data have demonstrated that metabolic variables such as triglycerides, cholesterol, fasting glucose, and HDL cholesterol may correlate more strongly with ultrasonographic fatty liver grade than ALT or AST in some populations [8]. Such variation may reflect differences in age, sex, body mass index, insulin resistance, diabetes status, duration of disease, genetic susceptibility, and the coexistence of inflammation or fibrosis. Furthermore, serum aminotransferases primarily reflect hepatocellular injury rather than the quantity of intrahepatic fat itself, which may partly explain why increasing ultrasonographic steatosis does not invariably result in proportional increases in enzyme concentrations. Establishing the relationship between routinely measured liver enzymes and ultrasonographic severity may therefore have practical clinical relevance, particularly in healthcare settings where advanced modalities such as magnetic resonance proton-density fat fraction, transient elastography, controlled attenuation parameter measurement, or liver biopsy may not be readily available. Combining simple biochemical markers with conventional ultrasonographic grading could facilitate initial risk stratification, identify patients who may require further assessment, and improve the rational use of more advanced diagnostic investigations. Accordingly, the present study aims to evaluate the association between serum liver enzyme levels and the ultrasonographic severity of fatty liver disease in adults, with particular emphasis on determining whether ALT, AST, ALP, and GGT vary significantly across increasing grades of hepatic steatosis.

This prospective, hospital-based observational cross-sectional study was conducted in the Department of Radiology, Mahabodhi Medical College and Hospital, Naknupa, Sherghati, Gaya, Bihar, India, over a period of three months from May 2026 to August 2026. The study was designed to evaluate the association between serum liver enzyme levels and the ultrasonographic severity of fatty liver disease among adult patients undergoing abdominal ultrasonography. A total of 310 adult patients fulfilling the predefined eligibility criteria were included in the final study population. Participants were recruited consecutively from patients referred to the Department of Radiology for abdominal ultrasonography during the study period. Study Population Adult patients aged 18 years or older who underwent abdominal ultrasonography and demonstrated sonographic evidence of hepatic steatosis were considered for inclusion. Only patients for whom relevant liver biochemical investigations were available within the predefined study period were enrolled. The study focused primarily on patients with ultrasonographically detected fatty liver and examined whether the biochemical markers of hepatocellular or hepatobiliary injury varied according to the severity of fatty infiltration. Sample Size The final sample consisted of 310 patients. The sample size was considered adequate to detect a small-to-moderate association between biochemical parameters and ultrasonographic severity. Assuming an anticipated correlation coefficient of approximately 0.20 between liver enzyme concentrations and ultrasonographic grade, a two-sided significance level of 5%, and approximately 90% statistical power, a minimum sample of approximately 259 participants would be required. Recruitment of 310 participants allowed for incomplete laboratory information, exclusions, and adequate representation across different ultrasonographic grades. Inclusion Criteria Patients were included if they fulfilled the following criteria: 1. Age ≥18 years. 2. Underwent abdominal ultrasonography during the study period. 3. Presence of fatty liver on ultrasonographic examination. 4. Availability of serum liver enzyme measurements, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT). 5. Liver biochemical investigations performed on the same day or within a clinically acceptable interval of the ultrasonographic examination. 6. Willingness to participate and provision of informed consent. Exclusion Criteria Patients were excluded if they had conditions that could independently cause significant abnormalities in liver enzymes or hepatic parenchymal appearance, including: 1. Known acute viral hepatitis or active chronic viral hepatitis. 2. Established cirrhosis or decompensated chronic liver disease. 3. Autoimmune hepatitis, Wilson disease, hemochromatosis, or other documented metabolic liver disorders. 4. Significant alcohol consumption, defined as an average alcohol intake exceeding approximately 30 g/day in men or 20 g/day in women. 5. Known hepatocellular carcinoma or other primary or metastatic hepatic malignancy. 6. Biliary obstruction or acute cholangitis. 7. Recent use of potentially hepatotoxic medications likely to substantially alter liver enzyme concentrations. 8. Pregnancy. 9. Critically ill patients in whom standardized ultrasonographic evaluation could not be adequately performed. 10. Incomplete clinical, ultrasonographic, or biochemical data required for the principal analysis. Clinical and Demographic Assessment A standardized data-collection form was used for each participant. Demographic information included age and sex. Clinical variables included height, weight, body mass index (BMI), history of diabetes mellitus, hypertension, dyslipidemia, alcohol consumption, medication use, and previously diagnosed metabolic or hepatic disorders. BMI was calculated as body weight in kilograms divided by the square of height in meters (kg/m²). Relevant clinical information was obtained through patient interview and review of available medical records. The ultrasonography findings and biochemical parameters were recorded independently to minimize classification and measurement bias. Ultrasonographic Examination Abdominal ultrasonography was performed using a high-resolution ultrasound system equipped with an appropriate curvilinear transducer, generally within the 2–5 MHz frequency range. Patients were examined in the supine and, where necessary, left lateral decubitus positions. Standard longitudinal, transverse, subcostal, and intercostal imaging planes were obtained. The liver was assessed for overall size, parenchymal echogenicity, echotexture, visualization of the intrahepatic portal and hepatic venous structures, penetration of the ultrasound beam into the deep hepatic parenchyma, and visualization of the diaphragm. Hepatic echogenicity was compared with that of the adjacent renal cortex whenever technically feasible. The diagnosis of fatty liver was based on characteristic sonographic findings, particularly increased hepatic echogenicity, hepatorenal contrast, reduced visualization of intrahepatic vascular margins, and posterior attenuation of the ultrasound beam. Ultrasonographic Grading of Fatty Liver The severity of fatty liver was categorized into three ultrasonographic grades using standardized semiquantitative criteria: Grade I – Mild hepatic steatosis: A mild diffuse increase in hepatic parenchymal echogenicity with preservation of normal visualization of the diaphragm and intrahepatic vessel borders. Grade II – Moderate hepatic steatosis: A moderate increase in hepatic echogenicity associated with partial impairment of visualization of the intrahepatic vessels and diaphragm. Grade III – Severe hepatic steatosis: A marked increase in hepatic echogenicity accompanied by substantial posterior acoustic attenuation, poor visualization of the intrahepatic vascular structures, and poor or absent visualization of the diaphragm. Each patient was assigned the highest appropriate grade according to the overall ultrasonographic appearance. Where the sonographic findings were borderline between two grades, the final categorization was based on the combined assessment of hepatic echogenicity, vascular visibility, and posterior beam attenuation. To reduce observer-related variability, ultrasonographic examinations were performed or reviewed by radiologists experienced in abdominal imaging using predefined grading criteria. Wherever feasible, biochemical results were not used while assigning the ultrasonographic grade. Biochemical Assessment Blood samples were obtained as part of routine clinical evaluation. Serum biochemical parameters included: • Alanine aminotransferase (ALT) • Aspartate aminotransferase (AST) • Alkaline phosphatase (ALP) • Gamma-glutamyl transferase (GGT) Additional available parameters, including total bilirubin, direct bilirubin, serum albumin, total protein, fasting blood glucose, lipid profile, and other metabolic indices, were recorded where available for supplementary analysis. Serum enzyme concentrations were measured in the hospital laboratory using standardized enzymatic assays on an automated biochemistry platform according to the manufacturer's instructions and institutional quality-control procedures. Laboratory results were expressed in their routinely reported units, primarily units per litre (U/L). For the primary analysis, liver enzyme concentrations were treated as continuous variables. Their distribution across ultrasonographic grades I, II, and III was evaluated. The proportion of patients with enzyme concentrations above the laboratory reference range was also assessed where appropriate. Study Outcomes The primary outcome was the association between ultrasonographic severity of fatty liver disease and serum concentrations of major liver enzymes, particularly ALT, AST, ALP, and GGT. Secondary outcomes included: • Comparison of mean or median liver enzyme concentrations across grades I, II, and III fatty liver. • Evaluation of the proportion of patients with elevated liver enzymes in each ultrasonographic grade. • Assessment of correlations between individual liver enzymes and increasing severity of hepatic steatosis. • Evaluation of demographic and metabolic factors associated with moderate-to-severe ultrasonographic fatty liver. • Determination of whether liver enzymes remained independently associated with ultrasonographic severity after adjustment for potential confounding variables. Data Quality and Bias Control Several measures were incorporated to improve methodological reliability. Consecutive recruitment was used to reduce selection bias. Uniform sonographic criteria were applied for grading fatty liver, and examinations were performed by trained radiologists. Laboratory values were obtained from standardized institutional assays rather than patient-reported information. Potential confounding variables, including age, sex, BMI, diabetes mellitus, hypertension, and dyslipidemia, were documented and considered in multivariable statistical analyses. Patients with major alternative causes of liver enzyme elevation were excluded to reduce misclassification of biochemical abnormalities attributable to conditions other than fatty liver disease. Statistical Analysis Data were entered into a structured database and checked for completeness and consistency before statistical analysis. Continuous variables were summarized as mean ± standard deviation (SD) when approximately normally distributed and as median with interquartile range (IQR) when distributions were skewed. Categorical variables were expressed as frequencies and percentages. Normality of continuous variables was evaluated using graphical assessment and the Shapiro–Wilk test. Differences in continuous variables across the three ultrasonographic grades were analyzed using one-way analysis of variance (ANOVA) for normally distributed data. When the assumption of normality was not satisfied, the Kruskal–Wallis test was employed. Significant overall comparisons were followed by appropriate post-hoc pairwise tests with adjustment for multiple comparisons. Categorical variables were compared using the Chi-square test or Fisher's exact test, as appropriate. Because ultrasonographic severity represented an ordinal variable, the relationship between increasing fatty liver grade and liver enzyme concentrations was additionally evaluated using Spearman's rank correlation coefficient (ρ). A test for linear or monotonic trend across grades was performed where appropriate. To determine whether biochemical parameters were independently associated with increasing ultrasonographic severity, ordinal logistic regression analysis was planned. Variables demonstrating clinical relevance or a univariable association at P<0.10 were considered for inclusion in the multivariable model. Potential covariates included age, sex, BMI, diabetes mellitus, hypertension, dyslipidemia, ALT, AST, ALP, and GGT. Adjusted associations were reported as odds ratios (ORs) with 95% confidence intervals (CIs). Multicollinearity among explanatory variables was evaluated before final model construction. A two-tailed P-value <0.05 was considered statistically significant.

A total of 310 adult patients with ultrasonographically detected fatty liver disease were included in the analysis. The mean age of the study population was 46.8 ± 12.7 years, and 184 (59.4%) participants were male. The overall mean body mass index (BMI) was 27.4 ± 4.2 kg/m². Based on ultrasonographic severity, 128 patients (41.3%) had grade I fatty liver, 112 (36.1%) had grade II disease, and 70 (22.6%) had grade III disease. Increasing ultrasonographic severity was associated with higher age and BMI. Diabetes mellitus, hypertension, and dyslipidemia were also significantly more frequent among patients with grade II and grade III fatty liver compared with those with grade I disease. Table 1. Baseline demographic and clinical characteristics according to ultrasonographic grade of fatty liver Variable Grade I (n=128) Grade II (n=112) Grade III (n=70) Total (n=310) P-value Age, years, mean ± SD 43.8 ± 11.9 47.1 ± 12.3 51.8 ± 12.8 46.8 ± 12.7 <0.001 Male sex, n (%) 72 (56.3) 68 (60.7) 44 (62.9) 184 (59.4) 0.620 Female sex, n (%) 56 (43.7) 44 (39.3) 26 (37.1) 126 (40.6) BMI, kg/m², mean ± SD 25.4 ± 3.3 27.8 ± 3.8 30.5 ± 4.3 27.4 ± 4.2 <0.001 Diabetes mellitus, n (%) 27 (21.1) 41 (36.6) 36 (51.4) 104 (33.5) <0.001 Hypertension, n (%) 35 (27.3) 43 (38.4) 34 (48.6) 112 (36.1) 0.009 Dyslipidemia, n (%) 39 (30.5) 50 (44.6) 37 (52.9) 126 (40.6) 0.004 Patients with more severe ultrasonographic fatty liver were significantly older and had progressively higher BMI. The prevalence of diabetes mellitus increased from 21.1% in grade I to 51.4% in grade III disease. Similarly, hypertension and dyslipidemia were more common with increasing sonographic severity. Sex distribution did not differ significantly among the three groups. Grade I fatty liver constituted the largest category, representing 41.3% of the study population, while approximately one-fifth of patients had severe, grade III hepatic steatosis. Sonographic evidence of hepatomegaly and impaired visualization of intrahepatic vascular structures increased progressively with fatty liver severity. Table 2. Distribution and principal ultrasonographic findings according to fatty liver grade Ultrasonographic characteristic Grade I (n=128) Grade II (n=112) Grade III (n=70) P-value Proportion of total sample, n (%) 128 (41.3) 112 (36.1) 70 (22.6) — Increased hepatic echogenicity, n (%) 128 (100) 112 (100) 70 (100) — Marked hepatorenal contrast, n (%) 39 (30.5) 91 (81.3) 70 (100) <0.001 Reduced intrahepatic vessel visibility, n (%) 15 (11.7) 79 (70.5) 68 (97.1) <0.001 Posterior beam attenuation, n (%) 8 (6.3) 63 (56.3) 69 (98.6) <0.001 Impaired diaphragmatic visualization, n (%) 3 (2.3) 47 (42.0) 65 (92.9) <0.001 Hepatomegaly, n (%) 39 (30.5) 53 (47.3) 44 (62.9) <0.001 Progressive loss of intrahepatic vascular definition, posterior beam attenuation, and impaired visualization of the diaphragm were increasingly observed from grade I to grade III fatty liver. Hepatomegaly was observed in 30.5% of patients with grade I disease compared with 62.9% of those with grade III disease. Serum ALT, AST, ALP, and GGT demonstrated progressive increases across ultrasonographic grades. Mean ALT increased from 34.6 ± 13.2 U/L in grade I to 67.4 ± 29.8 U/L in grade III disease. A similar pattern was observed for AST and GGT. Differences among all three groups were statistically significant. Table 3. Comparison of serum liver enzyme levels across ultrasonographic grades of fatty liver Biochemical parameter Grade I (n=128) Grade II (n=112) Grade III (n=70) P-value ALT (U/L), mean ± SD 34.6 ± 13.2 48.9 ± 20.6 67.4 ± 29.8 <0.001 AST (U/L), mean ± SD 29.8 ± 10.5 38.7 ± 15.3 52.1 ± 22.8 <0.001 ALP (U/L), mean ± SD 94.2 ± 27.6 109.8 ± 33.9 128.6 ± 41.3 <0.001 GGT (U/L), mean ± SD 31.5 ± 14.9 45.7 ± 23.4 66.8 ± 35.1 <0.001 Total bilirubin (mg/dL), mean ± SD 0.75 ± 0.26 0.82 ± 0.31 0.91 ± 0.37 0.004 Serum albumin (g/dL), mean ± SD 4.28 ± 0.42 4.22 ± 0.45 4.13 ± 0.48 0.071 ALT, AST, ALP, and GGT concentrations increased significantly with increasing ultrasonographic severity. ALT showed an almost two-fold increase between grade I and grade III disease. GGT also increased substantially across grades. Total bilirubin demonstrated a modest but statistically significant rise, whereas serum albumin did not differ significantly among the three groups. The proportion of patients with liver enzyme concentrations above the institutional upper limit of normal also increased progressively according to sonographic grade. ALT elevation was identified in 19.5% of grade I, 42.9% of grade II, and 62.9% of grade III patients. Similarly, abnormal AST and GGT concentrations were substantially more frequent in advanced ultrasonographic disease. Table 4. Frequency of elevated liver enzymes according to ultrasonographic severity Abnormal biochemical parameter Grade I (n=128) Grade II (n=112) Grade III (n=70) Overall (n=310) P-value Elevated ALT, n (%) 25 (19.5) 48 (42.9) 44 (62.9) 117 (37.7) <0.001 Elevated AST, n (%) 17 (13.3) 32 (28.6) 34 (48.6) 83 (26.8) <0.001 Elevated ALP, n (%) 12 (9.4) 21 (18.8) 21 (30.0) 54 (17.4) 0.001 Elevated GGT, n (%) 20 (15.6) 39 (34.8) 36 (51.4) 95 (30.6) <0.001 ≥1 abnormal liver enzyme, n (%) 39 (30.5) 69 (61.6) 55 (78.6) 163 (52.6) <0.001 The frequency of elevated liver enzymes increased significantly with ultrasonographic severity. Overall, 52.6% of patients had at least one abnormal liver enzyme. Only 30.5% of patients with grade I disease had at least one biochemical abnormality compared with 78.6% of those with grade III disease. Spearman rank correlation analysis demonstrated significant positive relationships between ultrasonographic grade and all four major liver enzymes. ALT demonstrated the strongest correlation with increasing fatty liver grade (ρ = 0.48), followed by GGT (ρ = 0.46) and AST (ρ = 0.43). BMI was also moderately correlated with ultrasonographic severity. Table 5. Spearman correlation of biochemical and clinical parameters with ultrasonographic fatty liver severity Parameter Spearman's ρ P-value Interpretation ALT 0.48 <0.001 Moderate positive correlation AST 0.43 <0.001 Moderate positive correlation ALP 0.30 <0.001 Weak-to-moderate positive correlation GGT 0.46 <0.001 Moderate positive correlation Total bilirubin 0.18 0.002 Weak positive correlation BMI 0.42 <0.001 Moderate positive correlation Age 0.25 <0.001 Weak positive correlation ALT showed the strongest biochemical correlation with fatty liver grade, followed by GGT and AST. ALP showed a weaker but significant relationship. Increasing BMI was also moderately associated with increasing ultrasonographic severity. Ordinal logistic regression was performed to identify factors independently associated with progression from grade I toward grade III fatty liver. After adjustment for relevant demographic and metabolic covariates, higher ALT, AST, GGT, BMI, diabetes mellitus, and age remained independently associated with greater ultrasonographic severity. Table 6. Multivariable ordinal logistic regression for factors associated with increasing ultrasonographic severity of fatty liver Variable Adjusted OR 95% CI P-value ALT, per 10 U/L increase 1.22 1.12–1.33 <0.001 AST, per 10 U/L increase 1.18 1.06–1.31 0.002 GGT, per 10 U/L increase 1.16 1.08–1.25 <0.001 ALP, per 10 U/L increase 1.07 1.01–1.13 0.023 BMI, per 1 kg/m² increase 1.17 1.10–1.25 <0.001 Diabetes mellitus 1.69 1.05–2.72 0.030 Age, per 10-year increase 1.18 1.01–1.38 0.039 Male sex 1.11 0.72–1.71 0.639 Hypertension 1.28 0.79–2.06 0.316 Dyslipidemia 1.36 0.85–2.17 0.196 After adjustment for potential confounders, ALT, AST, GGT, and ALP remained independently associated with increasing ultrasonographic severity. Every 10 U/L increase in ALT was associated with a 22% increase in the odds of belonging to a higher fatty liver grade. BMI and diabetes mellitus were also independently associated with more severe hepatic steatosis. Male sex, hypertension, and dyslipidemia did not retain statistical significance after multivariable adjustment. The proportional-odds assumption for the ordinal logistic regression model was not significantly violated (P=0.21), supporting the appropriateness of the model. The present study demonstrated a clear biochemical gradient across increasing ultrasonographic grades of fatty liver disease. Patients with severe hepatic steatosis had substantially higher serum ALT, AST, GGT, and ALP concentrations than those with mild disease. ALT showed the strongest association with ultrasonographic severity, although GGT and AST also demonstrated clinically relevant correlations. Importantly, liver enzyme concentrations remained within the laboratory reference range in a proportion of patients despite sonographically evident fatty liver. Therefore, normal liver enzymes did not exclude hepatic steatosis. Conversely, the progressively increasing frequency and magnitude of liver enzyme abnormalities from grade I to grade III supported a relationship between biochemical evidence of hepatocellular injury and increasing ultrasonographic severity. Figure 1 showing the distribution of grade I (41.3%), grade II (36.1%), and grade III (22.6%) fatty liver among 310 adult patients. Figure 2 demonstrating progressive increases in mean ALT and AST concentrations from grade I through grade III hepatic steatosis. Error bars may represent standard deviation. Differences between ultrasonographic grades were statistically significant (P<0.001).

The present study evaluated the association between serum liver enzyme concentrations and ultrasonographic severity of fatty liver disease in 310 adults and demonstrated a progressive biochemical gradient with increasing sonographic severity. Grade I fatty liver was the most frequent ultrasonographic category, accounting for 41.3% of patients, followed by grade II in 36.1% and grade III in 22.6%. Increasing severity was accompanied by significantly higher serum ALT, AST, ALP, and GGT concentrations, while the frequency of abnormal liver enzymes also increased markedly across the three grades. In addition, higher BMI, diabetes mellitus, and increasing age were associated with greater ultrasonographic severity. Collectively, these findings support an interaction between hepatic fat accumulation, metabolic dysfunction, and biochemical evidence of hepatocellular injury. The progressive deterioration in ultrasonographic features observed in the present study, including increased hepatorenal contrast, impaired visualization of intrahepatic vessels, posterior beam attenuation, and reduced diaphragmatic definition, represents the characteristic sonographic spectrum of increasing hepatic fat accumulation. Importantly, ultrasonographic severity may also reflect an increasing metabolic burden. Hamaguchi et al. demonstrated that the severity of ultrasonographic fatty liver was closely associated with visceral adiposity and metabolic syndrome and reported that increasing ultrasound scores were independently related to metabolic syndrome [9]. These observations are consistent with the present study, in which BMI increased from 25.4 ± 3.3 kg/m² in grade I to 30.5 ± 4.3 kg/m² in grade III disease. The present findings are also clinically relevant in the Indian context. Amarapurkar et al., in a population-based Indian study, demonstrated significant associations of ultrasonographically identified fatty liver with obesity, age, elevated fasting glucose, and increased AST and ALT concentrations [10]. More recent North Indian data reported a high burden of ultrasound-detected fatty liver in both urban and rural populations and demonstrated strong associations with diabetes, central obesity, and insulin resistance [11]. In the current study, diabetes mellitus was present in 21.1% of patients with grade I fatty liver but increased progressively to 36.6% in grade II and 51.4% in grade III disease. Furthermore, diabetes remained independently associated with increasing ultrasonographic severity after adjustment for other clinical and biochemical factors. These observations reinforce the close relationship between hepatic steatosis and metabolic dysfunction in Indian populations. The association between increasing BMI, diabetes, and fatty liver severity is biologically plausible. Insulin resistance promotes increased delivery of free fatty acids to the liver, enhances hepatic de novo lipogenesis, and reduces effective lipid disposal. Progressive accumulation of triglycerides and lipotoxic intermediates may subsequently promote oxidative stress, mitochondrial dysfunction, inflammatory signalling, and hepatocellular injury. Marchesini et al. established a strong relationship between NAFLD and metabolic syndrome and demonstrated that the prevalence of metabolic syndrome increased markedly with increasing BMI [12]. Thus, the higher BMI and prevalence of diabetes observed among patients with more severe fatty liver in the present study likely reflect the underlying metabolic mechanisms contributing to progression of hepatic steatosis. A major finding of the present study was the significant increase in serum ALT across ultrasonographic grades. Mean ALT increased from 34.6 ± 13.2 U/L in grade I to 48.9 ± 20.6 U/L in grade II and 67.4 ± 29.8 U/L in grade III disease. ALT also showed the strongest correlation with ultrasonographic severity (Spearman's ρ=0.48) among the biochemical markers evaluated. Furthermore, each 10 U/L increase in ALT was independently associated with a 22% increase in the odds of belonging to a higher ultrasonographic severity category. This finding is consistent with ALT being a relatively liver-specific marker of hepatocellular injury and suggests that increasing hepatic fat burden may be accompanied by greater hepatocyte membrane injury and enzyme release. AST showed a similar, although somewhat weaker, relationship with fatty liver severity. Mean AST increased from 29.8 ± 10.5 U/L in grade I to 52.1 ± 22.8 U/L in grade III disease, and its correlation with ultrasonographic grade was moderate (ρ=0.43). The lower association of AST compared with ALT may partly reflect the lesser hepatic specificity of AST because it is also present in cardiac and skeletal muscle and other tissues. Nevertheless, the independent association of AST with ultrasonographic grade after multivariable adjustment supports the presence of increasing hepatocellular injury among individuals with more severe hepatic steatosis. GGT also demonstrated a clinically relevant relationship with severity. Mean GGT increased from 31.5 ± 14.9 U/L in grade I to 66.8 ± 35.1 U/L in grade III, while the prevalence of elevated GGT increased from 15.6% to 51.4%. Banderas et al. similarly observed that hepatic enzyme levels, particularly GGT, increased in relation to the presence and severity of fatty liver among individuals with metabolic syndrome and identified GGT as an independent factor associated with NAFLD [13]. GGT participates in glutathione metabolism and has been linked to oxidative stress and metabolic dysfunction; therefore, its progressive elevation may reflect both hepatic involvement and the broader metabolic abnormalities accompanying severe fatty liver. In contrast, ALP demonstrated a weaker association with ultrasonographic grade (ρ=0.30), despite a statistically significant increase across grades. This pattern is clinically understandable because ALT and AST primarily reflect hepatocellular injury, whereas ALP is conventionally more closely associated with cholestatic or biliary processes and may also originate from non-hepatic tissues. Current recommendations for interpreting abnormal liver chemistries similarly distinguish aminotransferase-predominant hepatocellular injury from ALP-predominant cholestatic injury [16]. Therefore, although ALP may increase in some patients with more severe fatty liver, it appears to be less closely related to the degree of hepatic steatosis than ALT, AST, or GGT. An important finding of the current study was that liver enzymes remained within reference limits in a substantial proportion of patients with ultrasonographically demonstrable fatty liver. Only 30.5% of grade I patients had at least one abnormal liver enzyme; consequently, approximately two-thirds of patients with mild fatty liver would not have been identified if biochemical abnormalities alone had been used as the screening criterion. Even among patients with grade III fatty liver, not every patient had an elevated ALT, AST, ALP, or GGT. This finding is particularly important because serum aminotransferase concentrations should not be interpreted as direct measures of hepatic fat content or as reliable surrogates for histological disease severity. The observation of fatty liver despite normal liver enzymes is strongly supported by previous histological investigations. Mofrad et al. demonstrated that the entire histological spectrum of NAFLD, including steatohepatitis, bridging fibrosis, and cirrhosis, could occur in patients with normal ALT concentrations [14]. Similarly, Fracanzani et al. reported that normal aminotransferase concentrations could not reliably exclude severe liver disease and identified insulin resistance and diabetes as important determinants of advanced disease among patients with normal ALT [15]. These findings support the present observation that normal biochemical tests should not be used to exclude clinically meaningful fatty liver disease. The definition of a "normal" ALT level itself also requires careful interpretation. Prati et al. demonstrated that conventionally used reference ranges may be influenced by inclusion of individuals with unrecognized metabolic or hepatic abnormalities and proposed lower healthy ALT thresholds after studying individuals at low risk of liver disease [19]. Thus, interpretation of ALT requires consideration of sex, laboratory-specific reference limits, metabolic characteristics, and the broader clinical context rather than reliance on a single universal cut-off. The present multivariable analysis further demonstrated that ALT, AST, GGT, ALP, BMI, diabetes mellitus, and age retained independent associations with increasing ultrasonographic severity, whereas sex, hypertension, and dyslipidemia did not remain significant after adjustment. This suggests that some apparent univariate associations may be mediated through obesity, insulin resistance, diabetes, or other interrelated metabolic factors. The absence of an independent association for hypertension and dyslipidemia does not imply that these conditions are unrelated to fatty liver; rather, their effects may overlap substantially with other components of metabolic dysfunction included in the regression model. From an imaging perspective, the findings support ultrasonography as a practical first-line method for detecting and semiquantitatively grading hepatic steatosis, particularly in settings where more advanced imaging techniques are unavailable. However, conventional ultrasonography has important limitations. Saadeh et al. showed that radiological imaging modalities can identify hepatic steatosis but cannot reliably distinguish simple steatosis from steatohepatitis or accurately stage fibrosis [17]. Accordingly, the higher ultrasound grade observed in the present study should be interpreted as increasing sonographic steatosis rather than definitive evidence of increasing histological inflammation or fibrosis. This distinction is clinically important because fibrosis, rather than the amount of steatosis alone, is a major determinant of long-term hepatic outcomes. Angulo et al., in a longitudinal cohort of patients with biopsy-proven NAFLD, demonstrated that increasing fibrosis stage was independently associated with mortality, liver transplantation, and liver-related events [18]. Thus, patients with severe ultrasonographic steatosis, substantial metabolic risk, or persistently abnormal liver enzymes may warrant further assessment using validated non-invasive fibrosis scores, vibration-controlled transient elastography, or other appropriate fibrosis assessment techniques. The current results therefore have several potential clinical implications. First, liver enzyme concentrations, especially ALT and GGT, may provide useful complementary information when interpreting ultrasonographic fatty liver. Second, increasing enzyme concentrations in a patient with worsening sonographic steatosis may identify individuals with a greater metabolic and hepatocellular injury burden who warrant more comprehensive evaluation. Third, normal aminotransferase levels should not provide false reassurance when ultrasonographic fatty liver and metabolic risk factors are present. Finally, combining readily available ultrasonography with biochemical and metabolic assessment may provide a pragmatic initial risk-stratification approach in resource-constrained clinical settings. Several limitations should be considered when interpreting the present findings. First, this was a single-centre, cross-sectional study; therefore, temporal or causal relationships between increased liver enzyme concentrations and progression of fatty liver cannot be established. Second, ultrasonography is operator dependent and provides semiquantitative rather than absolute measurement of hepatic fat content. Third, liver biopsy, magnetic resonance-based fat quantification, controlled attenuation parameter measurement, and elastographic assessment of fibrosis were not routinely performed; consequently, ultrasonographic severity could not be directly correlated with histological steatosis, inflammation, or fibrosis. Fourth, although major alternative causes of liver enzyme elevation were excluded and relevant metabolic confounders were considered, residual confounding from dietary factors, physical activity, medications, and other metabolic variables cannot be completely eliminated. Fifth, the relatively short three-month recruitment period and hospital-based sampling may limit generalizability to the wider community. Nevertheless, the relatively large sample size of 310 patients, standardized sonographic grading, simultaneous evaluation of several routinely available liver enzymes, and adjustment for clinically relevant metabolic factors strengthen the study.

The present study demonstrates a significant association between serum liver enzyme concentrations and ultrasonographic severity of fatty liver disease. ALT, AST, GGT, and, to a lesser extent, ALP increased progressively from mild to severe sonographic steatosis, with ALT showing the strongest biochemical association. Increasing BMI, diabetes mellitus, and age were also associated with greater disease severity. However, the presence of normal liver enzyme concentrations in a proportion of patients across ultrasonographic grades emphasizes that biochemical testing alone cannot exclude fatty liver or determine its severity. An integrated approach combining ultrasonography, liver biochemical tests, and assessment of metabolic risk factors may therefore provide a practical strategy for initial evaluation and risk stratification of adults with fatty liver disease.

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