Correlation between Cumulative Methotrexate Dose, Metabolic Syndrome and Hepatic Fibrosis Detected by FibroScan in Rheumatoid Arthritis Patients

Background and Objectives: Methotrexate (MTX) is routinely prescribed for rheumatoid arthritis (RA) patients, but high cumulative doses may lead to hepatic fibrosis. Additionally, a high proportion of RA patients suffer from metabolic syndrome, which also increases the risk of hepatic fibrosis. This cross-sectional study aimed to explore the association between a cumulative MTX dose, metabolic syndrome, and hepatic fibrosis in patients diagnosed with RA. Materials and Methods: RA patients undergoing treatment with MTX were examined using transient elastography (TE). All patients, regardless of having hepatic fibrosis, were compared to identify the risk factors. Results: Two hundred and ninety-five rheumatoid arthritis patients were examined using FibroScan. One hundred and seven patients (36.27%) were found to have hepatic fibrosis (TE > 7 kPa). After multivariate analysis, only BMI (OR = 14.73; 95% CI 2.90–74.79; p = 0.001), insulin resistance (OR = 312.07; 95% CI 6.19–15732.13; p = 0.04), and cumulative MTX dosage (OR 1.03; 95% CI 1.01–1.10; p = 0.002) were associated with hepatic fibrosis. Conclusions: While the cumulative MTX dose and metabolic syndrome are both the risk factors of hepatic fibrosis, metabolic syndrome, including a high BMI and insulin resistance, poses a greater risk. Therefore, MTX-prescribed RA patients with metabolic syndrome factors should be attentively monitored for signs of liver fibrosis.


Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory arthritis that attacks and destroys synovial joints. RA is one of the most common types of arthritis. Early intervention to reduce inflammation can minimize joint damage. Disease-modifying antirheumatic drugs (DMARDs) are the principal treatment to reduce the progression and symptoms of RA.
Methotrexate (MTX) is considered the first-line treatment for RA and is recommended by both the European League Against Rheumatism (EULAR) and the American College of Rheumatology (ACR) [1,2]. It is prescribed as a backbone of DMARDs for RA [3]. MTX reduces inflammation by increasing adenosine. It also suppresses dihydrofolate reductase (DHFR) and prevents purine and pyrimidine synthesis, generates reactive oxygen species (ROS), decreases adhesion molecules, alters cytokine profiles, and inhibits polyamine [4,5].
Metabolic syndromes and obesity are known risk factors for hepatic fibrosis [12]. The proportion of metabolic syndrome is higher within RA patients than it is among the overall population. RA patients with metabolic syndromes, including a high BMI and fatty liver, have a higher risk of fibrosis from MTX than RA patients who do not have metabolic syndrome [8]. In this study, we measured hepatic fibrosis in RA patients who received MTX with non-invasive transient elastography (TE) and attempted to explore the association between metabolic syndrome parameters, cumulative MTX dose, and hepatic fibrosis.

Study Population
This was a cross-sectional study of RA patients who had received at least 1000 mg cumulative dose of MTX. There were 1288 rheumatoid arthritis patients during the study period. Nine hundred and ninety-three patients who did not meet the inclusion criteria, had the exclusion criteria, or rejected the participation were excluded from the study. The 295 patients were enrolled at Thammasat University Hospital, Thailand between November 2020 and November 2021. The diagnosis was based on ACR criteria, 2010 [13], and the treatment adhered strictly to the current ACR and EULAR RA guidelines [1,2].
The exclusion criteria were age less than 18 years; severe comorbidities, such as chronic liver, heart, or kidney disease; aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels two times greater than ULN; current hepatotoxic drug use besides MTX and other DMARDs; alcohol consumption over 7 units per week; and contraindications such as pregnancy ( Figure 1).

Demographics and Clinical Parameters
The patient information collected included demographics, comorbidities, RA and MTX factors, concurrent medications, and laboratory parameters. Metabolic syndrome parameters included the waist, body mass index (BMI), impaired fasting glucose (IFG), triglyceride, and high-density lipoprotein HDL. The blood samples were tested (i.e., aspartate aminotransferase (AST), alanine aminotransferase (ALT), fasting plasma glucose, etc.). The ultrasound was conducted to rule out chronic liver disease and for more information on liver imaging morphology (i.e., fatty liver) ( Table 1).

Demographics and Clinical Parameters
The patient information collected included demographics, comorbidities, RA and MTX factors, concurrent medications, and laboratory parameters. Metabolic syndrome parameters included the waist, body mass index (BMI), impaired fasting glucose (IFG), triglyceride, and high-density lipoprotein HDL. The blood samples were tested (i.e., aspartate aminotransferase (AST), alanine aminotransferase (ALT), fasting plasma glucose, etc.). The ultrasound was conducted to rule out chronic liver disease and for more information on liver imaging morphology (i.e., fatty liver) ( Table 1).

Transient Elastography (TE)
A 3.5 MHz ultrasound transducer probe (FibroScan, EchoSens, Paris, France) was used to measure liver stiffness. Patients were fasting (nil per os; NPO) for at least 3 h prior to the study. The shear wave velocity was measured ten times in each 6 cm thick portion of the right liver lobe, 1 cm in diameter and 2-4 cm long. The average, with an interquartile range of less than 20% of the median value, was used to calculate the stiffness. Any value greater than or equal to 7 kilopascals indicated fibrosis [14]. The controlled attenuation parameter (CAP)-a measure of fat storage in the liver-was also measured by FibroScan and a value of 248 or more was considered excessive.

Metabolic Syndrome
The patients were diagnosed with metabolic syndrome according to the modified National Cholesterol Education Program Adult Treatment Panel III (NCEPT ATP III criteria) [15], a variation of the modified NCEP ATP III criteria. According to the modified NCEP ATP III criteria [16], the presence of at least three of the following five factors is needed for diagnosing metabolic syndrome: abdominal obesity (waist circumference > 102 cm (40 inches) in men or >88 cm (35 inches) in women); hypertriglyceridemia (triglycerides > 1.7 mmol/L); low HDL cholesterol (HDL < 1.03 mmol/L for men and <1.29 mmol/L for women); elevated blood pressure (>85 mmHg) or current use of antihypertensive medications; and impaired fasting glucose (fasting plasma glucose > 5.6 mmol/L). Specific waist circumference cut-off points were used appropriately for Asians: 90 cm for men and 80 cm for women [15].

Statistical Analysis
Descriptive statistics were used to describe the baseline characteristics of the cohort. Comparison between people with and without hepatic fibrosis was performed using Student's t-test or X 2 test, depending on the type of data.
A univariate analysis was performed using Stata Package (Stat Statistical Software, College Station, TX, USA), including all collected parameters as independent variables and hepatic fibrosis as the dependent variable (primary outcome). All variables with p-value < 0.10 were selected for inclusion in the subsequent multivariate logistic regression, using the stepwise approach. We reported the odds ratios (OR), their 95% confidence interval (CI), and the p-values for all the variables. The variables with p-value < 0.05 in the multivariate logistic regression analysis would be considered independently associated with hepatic fibrosis.

Ethics Approval
The study protocol was reviewed and approved by the human research ethics committee of Thammasat University (Medicine), in compliance with the declaration of Helsinki (protocol code MTU-EC-IM-6-141/60, date of approval 27 December 2017).

Baseline Characteristics
Of the 295 RA patients enrolled in this study, 107 (36.27%) had hepatic fibrosis ( Figure 1). The group of patients with fibrosis was older (p = 0.02) and had higher values for albumin (p = 0.007), AST (p = 0.002), ALT (p = 0.001), creatinine (p < 0.001), INR (p = 0.02), and uric acid (p < 0.001) compared to the group with no fibrosis. There was no difference in sex, alcohol consumption, hemoglobin, white blood cell count, or platelet count. The values are given in Table 1.

Rheumatoid Arthritis
The group of patients with hepatic fibrosis had RA longer (p = 0.009). Leflunomide (p = 0.03), a conventional synthetic DMARD, and steroids (p = 0.04) were prescribed more in the fibrosis group.

Factors Associated with Hepatic Fibrosis
The factors significantly associated with hepatic fibrosis according to univariate analysis are listed in Table 2. These variables were subsequently included in the multivariate analysis. Multivariate regression analysis found that BMI (OR = 14.73; 95% CI 2.90-74.79; p = 0.001); insulin resistance (OR = 312.07; 95% CI 6.19-15732.13; p = 0.04); and cumulative MTX dose (OR 1.03; 95% CI 1.01-1.10; p = 0.002) were significantly associated with hepatic fibrosis. (Table 3) Table 3. The multivariate logistic regression analysis of the factors associated with significant hepatic fibrosis.

Discussion
In our study, we compared RA patients receiving MTX who had hepatic fibrosis to those who did not. The patients with fibrosis were significantly older and their levels of albumin, AST, ALT, creatinine, and INR were significantly higher. The fibrosis patients had a longer duration of RA, longer duration of MTX, higher cumulative MTX, higher average MTX dose per weight, more prescriptions of leflunomide or prednisolone, and higher ESR levels. In our study, multivariate regression analysis indicated that a high BMI, insulin resistance, and cumulative MTX dose were associated with hepatic fibrosis.
Meta-analysis suggests that MTX could cause transient elevations of ALT, and at least one study found somewhat elevated ALT in up to 50% of MTX users, but significant elevations are rare [6,26,27]. Folic acid has been reported to reduce ALT and lower the risk of hepatotoxicity [9,27]. However, liver enzymes and synthetic function may be normal even in severe hepatic fibrosis [5,9]. Furthermore, some studies indicate that fibrosis could result from oxidative stress [28].
Higher cumulative doses of MTX are increasingly toxic to the liver [11]. Lower doses can cause similar damage if patients have preexisting liver disease (e.g., hepatitis B and hepatitis C), or risk factors such as metabolic syndrome [5,6,12,17,26,27,29].
The most prescribed treatment for many autoimmune diseases such as inflammatory myositis, psoriasis, psoriatic arthritis, and RA is MTX. MTX can synergistically augment preexisting hepatic diseases [8]. A high cumulative MTX dose, obesity, old age, and alcohol can further increase liver damage. Smaller cumulative MTX doses in combination with metabolic syndrome or nonalcoholic steatohepatitis (NASH) can also increase the risk of fibrosis [19]. A high BMI, high cholesterol, and long duration of inflammatory arthritis can increase the risk of MTX-induced liver fibrosis [8].
We previously demonstrated that RA patients with metabolic syndrome features (high BMI, IFG, and fatty liver) carry a higher risk of developing hepatic fibrosis [8]. The current study found some association between hepatic fibrosis and body weight, high BMI, IFG, fatty liver, and statin use from univariate analysis. Only BMI and insulin resistance, however, were retained in significant association with hepatic fibrosis after multivariate analysis.
RA patients have more metabolic syndrome, obesity, dyslipidemia, diabetes, and cardiovascular disorders than the general population [19]. RA patients with NASH or metabolic syndrome should be closely monitored during MTX use [8]. Although AST and ALT were not statistically associated with liver fibrosis, they may remain clinically valuable due to their low price and wide availability. Advice on lifestyle modification and weight reduction should be provided for all RA patients with metabolic syndrome taking MTX. The risk of liver fibrosis in patients with RA has been found to be more closely associated with metabolic syndrome than with a cumulative MTX dose [30,31]. Our current study also found that hepatic fibrosis was more significantly associated with metabolic syndrome than with cumulative MTX dose.
This study has some limitations. Our study results may have limited applicability due to the cross-sectional, single-center design involving a homogeneous group of participants (ethnic Thai). We did not categorize liver fibrosis by severity due to the lack of a standardized threshold for liver elasticity in Asian populations taking MTX. Another limitation is that we did not include a histopathological examination and a Child-Pugh score assessment for our study cohort.
In conclusion, a cumulative MTX dose is a contributing risk factor for hepatic fibrosis, especially when the patients have metabolic syndrome. In particular, the metabolic syndrome components, including BMI and insulin resistance, significantly correlated with hepatic fibrosis in MTX-prescribed RA patients. These patients are at high propensity and should be attentively monitored by a physician. A hepatologist consultation should be considered in complicated cases.