Differential risk factor patterns for a positive treadmill test among subjects with and without ultrasound-based fatty liver

Introduction
Non-alcoholic fatty liver disease (NAFLD) is considered the hepatic consequence of metabolic syndrome (MS) [1]. Secondary to the high prevalence of obesity and MS, NAFLD is today one of the most common causes of gastroenterology consultations in the clinical setting [2]. In this context, gastroenterologists are exposed to large numbers of subjects with increased risk factors for the development of cardiovascular disease (CVD).
Cardiovascular disease is the most common cause of death and will continue to be so for at least the next 30 years [3]. Because MS is an important risk factor for CVD, it is logical to expect an increased risk of CVD among subjects with NAFLD. Several lines of evidence indicate that subjects with NAFLD have increased values for (a) indirect markers of CVD, such as mean intima-media thickness, even without abnormal liver function tests [4] and ultra-sensitive C-reactive protein (US-CRP) [5]; and (b) direct markers of CVD (coronary angiography studies demonstrating that NAFLD is independently associated with CVD) [6].
Recently, the Firenze Bagno a Ripoli study showed that g-glutamyl transpeptidase or aspartate aminotransferase (AST) is an independent predictor of CVD [7], and this finding was confirmed in other European [8] and American populations [9].
However, it is not clear whether the risk factors for CVD in subjects with NAFLD are the same for subjects without NAFLD. The aim of this study was to analyze the risk factor patterns for a positive treadmill test in asymptomatic subjects with and without ultrasound-based fatty liver.
Material and methods
This was a cross-sectional study performed in ambulatory voluntary subjects from January 2005 to January 2007. Adult asymptomatic subjects with alcohol consumption below 20 γ per day were included. Patients with liver disease from any other cause were excluded. Anthropometric, biochemical, ultrasound imaging, and exercise treadmill testing data were collected for all subjects. The study was approved by the Human Subjects Committee as conforming to the ethical guidelines of the 1975 Declaration of Helsinki, and written informed consent was obtained from all participants before entry.
Physical examination
Body weight was measured in light clothing and without shoes, to the nearest 0.10 kg. Height was measured to the nearest 0.5 cm. Body mass index (BMI) was calculated as weight in kilograms divided by height in metres squared. “Overweight” was defined as a BMI of 25-29.9 kg/m² and “obesity” as a BMI of ł 30 kg/m². Three blood pressure readings were taken at 1 min intervals, and the average of the first and third readings was used in the analysis.
Analytical procedures
Blood samples were collected from all patients after a fast of 8 h, for the determination of serum concentrations of glucose, total cholesterol, cholesterol associated with high-density lipoprotein (HDL cholesterol), cholesterol associated with low-density lipoprotein (LDL cholesterol), triglycerides, AST, alanine aminotransferase (ALT), albumin, and total bilirubin. Plasma glucose was measured in duplicate in the fasting state using an automated analyzer. The coefficient of variation for a single determination was 1.5%. Cholesterol, HDL cholesterol, and triglycerides were measured by enzymatic colorimetric methods, using CHOL, HDL–C plus (second generation), and triglyceride assays, respectively (Roche Diagnostics Co., Indianapolis, IN, USA). LDL cholesterol concentrations were calculated using the Friedewald formula [11] or measured by an enzymatic colorimetric method when triglyceride levels were higher than 150 mg/dl.
Non-alcoholic fatty liver disease definition
The diagnosis of NAFLD was based on ultrasonographic findings compatible with hepatic steatosis. Real-time ultrasonographic studies were performed while the subjects were fasting. A 3.5 MHz transducer (Elegra, Siemens Medical Systems, Mountain Grove, CA, USA) was used to obtain the following images: sagittal view of the right lobe of the liver and right kidney; transverse view of the left lateral segment of the liver and spleen; transverse view of the liver and pancreas, and any focal areas of altered echotexture. A bright liver echo pattern that signified a discrepancy higher than expected in the echo amplitude between the liver and kidney parenchyma was considered to indicate steatosis. In the second evaluation, all studies for each subject were viewed side-by-side in a masked fashion (k = 0.92).
Metabolic syndrome
Metabolic syndrome was defined according to the criteria of the Executive Summary of the Third Report of the National Cholesterol Education Program as the presence of three or more of the following criteria [12].
Abdominal obesity: waist circumference ł 102 cm in men and > 88 cm in women, but because the waist circumference measure was not available for all patients, we used a validated ideal BMI (> 22 kg/m² in men and > 23 kg/m² in women), which detects diabetes mellitus and high blood pressure with similar efficacy to waist circumference [13]. Hypertriglyceridaemia: triglycerides ł 150 mg/dl. HDL cholesterol: HDL < 40 mg/dl in men and < 50 mg/dl in women. High blood pressure: ł 130/85 mm Hg. High fasting glucose: ł 100 mg/dl.
Ultra-sensitive C-reactive protein
Ultra-sensitive C-reactive protein was measured in 1 ml of blood following an overnight fast. The serum was frozen at –73°C and processed within 30 days using a chemiluminescent immunoassay system (Immulite 2000, Diagnostic Products Corporation, Los Angeles, CA, USA) with a dynamic range of 0.02-250 mg/l and a coefficient of variation of less than 15% [14].
Cardiovascular disease
For the purpose of this paper, CVD was investigated in all subjects by exercise treadmill testing, using the Q-Stress system (Quinton Instrumentation Technologies, Mexico, Mexico), following the Bruce protocol [15]. We recorded: blood pressure and heart rate at each exercise stage and at peak exercise, time to onset of angina, and 1 mm ST-segment depression, ST-segment depression at peak exercise, maximal ST-segment depression, presence of cardiac arrhythmias; metabolic equivalents and double product (heart rate in bpm × systolic blood pressure in mm Hg), and total exercise duration. Myocardial ischaemia was defined as the presence of 0.1 mV horizontal or downsloping ST-segment depression 80 ms after the J-point during exercise or recovery. Cardiac arrhythmias were defined as ventricular premature beats of Lown grade II or higher. All studies were performed and interpreted by the same cardiologist on two separate occasions. We decided to use the exercise treadmill test as a marker of CVD because it is a non-invasive test with good performance in assessing coronary pathology [16].
Statistical analysis
Continuous variables are presented as means ± standard deviations (SD). Quantitative data were analyzed using Student’s t test and one-way analysis of variance (ANOVA) for two or more independent groups, respectively. Differences in the proportions of categorical data were found with Fisher’s exact test when the number of patients was Ł 5, and with the c2 test for 2 × 2 tables when the number of subjects in each cell was > 5. Univariate analyses identified the clinical and biochemical variables that predicted a positive exercise treadmill test. All variables with a p value < 0.2 in univariate analyses were included in a multivariate backward stepwise logistic regression analysis. A p value of 0.05 or less was considered significant. All data were analyzed using SPSS, PC version 12.0 (Chicago, IL, USA).
Results
A total of 1421 subjects were included during the study period, with mean age 46 ±11 years and a predominance of men (n = 920, 64.7%). The mean BMI was 26.7 ±4.1 kg/m², and 259 subjects (18.2%) were obese (BMI ł 30 kg/m²). The prevalence of MS was 37.3%; that of NAFLD was 30.2%. Patients with NAFLD had a higher prevalence of obesity (37.4 vs. 9.8% for patients without NAFLD, p < 0.001) and MS (61.4 vs. 26.8%, p < 0.001) and higher values for US-CRP (49 vs. 28%, p < 0.001). Interestingly, in the total sample, no statistical differences were observed between these two groups in the treadmill test results (1.3% for the non-NAFLD group vs. 2.3% for the NAFLD group, p = 0.176) (Table I).
The subjects were classified according to treadmill test result and NAFLD status. In patients with a positive treadmill test, differences were observed in weight (p = 0.002), BMI (p = 0.008), and the prevalence of obesity (p = 0.019) (Table II). In subjects with a negative treadmill test, similar differences were observed as in Table I.
In subjects with NAFLD, univariate analysis was used to identify those patients with a high risk of CVD. The differences between patients with negative and positive treadmill test were systolic blood pressure ł 130 mm Hg (p = 0.029), diastolic blood pressure ł 85 mm Hg (p = 0.092), combination of both (p = 0.036), and obesity (p = 0.183) (Table III). On multivariate analysis, only systolic blood pressure ł 130 mm Hg remained a significant factor (OR = 4.705, 95% CI 1.285-17.231, p = 0.019) even with the inclusion of smoking status in the model (OR = 3.859, 95% CI 1.070-13.922, p = 0.039) (Table IV).
Using these findings to identify patients with NAFLD and a high risk of CVD, we observed that a high systolic blood pressure value was associated with a higher prevalence of positive treadmill tests (7.1%, p = 0.011).
To corroborate the differential risk patterns for CVD among subjects with and without NAFLD, we performed univariate and multivariate analyses to detect the risk factors for CVD among subjects without NAFLD and with normal levels on liver function tests (Table V). In this subgroup, waist circumference (OR = 8.750, 95% CI 1.830-41.843, p = 0.007) and MS (OR = 3.802, 95% CI 1.121-12.987, p = 0.032) were independently associated with a positive treadmill test (Table IV).
Discussion
In this study we analyzed a sample of asym-ptomatic subjects using non-invasive tools, and identified a high-risk group for CVD within the group of patients with NAFLD. According to our results, subjects with NAFLD and high systolic blood pressure have an increased risk of CVD.
Although there was no significant difference (only a trend) in the prevalence of positive treadmill tests among subjects with and without NAFLD, we identified different risk factors for CVD in the two groups. The risk factors for patients without NAFLD were the same as those for the general population [18, 19], which emphasizes the differences in risk patterns according NAFLD status. This distinction could be useful in the clinical setting to identify high-risk subjects.
According to our results, high blood pressure emerges as a new discriminatory factor among subjects with NAFLD with which to identify those patients at risk of CVD. To the best of our knowledge, this is the first study to demonstrate that blood pressure levels are associated with CVD in NAFLD patients. This finding is in agreement with previous results showing that high blood pressure has an effect on peripheral vascular structural and functional parameters, independently of MS [20], and that the utility of MS in predicting death from CVD is limited [21], contrary to the high utility of blood glucose or blood pressure [22]. In fact, in patients with diet-controlled type 2 diabetes mellitus, NAFLD increases the risk of CVD (assessed by carotid artery intima-media thickness), which is mainly attributed to elevated blood pressure [23].
This cross-sectional study has the intrinsic limitations of this kind of study, and only epidemiological associations could be derived from this design. Prospective studies must be performed to confirm these initial findings. Additionally, the low number of events could limit the power of the statistical analysis. In this work, we found different risk factor patterns for CVD considering the NAFLD status. This is particularly important considering that, at this time, there are no other recommendations for preventative measures or diagnostic approaches to CVD in subjects with NAFLD, apart from those described for MS [28-30]. Consequently, identifying groups at high risk of CVD could have important implications, and a more aggressive approach may be required.
In conclusion, although some evidence demonstrates that subjects with NAFLD had increased risk for CVD, these risk factors are different from those observed in the general population. Future prospective studies are necessary to confirm this initial epidemiological evidence.
Acknowledgments
Part of this study was presented in the Digestive Disease Week, San Diego, CA, on May 21, 2008.
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