Study population and data collection
Patient enrollment in this study was shown in Figure 1. This prospective study enrolled 529 Japanese outpatients with T2DM from August 2011 to December 2016. Patients had no history of cardiovascular disease but were referred to our hospital with suspected coronary artery disease. We excluded patients who consumed >20 g of alcohol per day, patients with known liver disease, patients currently using oral corticosteroids or amiodarone, and patients with a coexisting active tumor. All of the patients underwent blood tests, measurement of CACS, and abdominal CT on the same day.
Assessment of coronary calcification
CT imaging was performed using a Somatom Definition Flash scanner (Siemens Medical Solutions, Erlangen, Germany). CACS was measured using the following parameters: 120kV, 150mA, and 3-mm thickness. CACS was calculated using an automated computerized system (Virtual Place, Raijin; AZE Inc., Tokyo, Japan) and the Agatston method, which involved multiplying the area of each calcified plaque by the density factor determined by a peak pixel intensity within the plaque. The plaque-specific scores for all the slices were added together. The density factor was 1, 2, 3, or 4 for plaques with peak intensities of 130 to 199 Hounsfield Units (HU), 200 to 299 HU, 300 to 399 HU, or ≥400 HU, respectively [13]. In addition, patients were divided into three groups by CACS: CACS 0, CACS (1–99) and CACS (≥100).
Assessment of visceral adipose tissue and NAFLD
Abdominal non-contrast CT scans were carried out alongside cardiac CT, at the level that contained images of the liver, spleen, and umbilicus. The visceral adipose tissue area was assessed using the semi-automatic segmentation technique at the umbilical level [14]. Hepatic and splenic Hounsfield attenuations were measured using circular regions of interest in the liver and spleen [15]. In the liver, we located regions of interests at two segments (right anterior and right posterior). The ratio of hepatic:spleen attenuation was calculated by using the mean HU measurement of the two right liver lobe regions of interest. In this study, we defined hepatic steatosis as a hepatic:spleen attenuation ratio <1.0 [16]. NAFLD was finally diagnosed after other causes of hepatic steatosis were ruled out.
Assessment of other risk factors
Hypertension was defined as having a seated blood pressure over 140/90 mmHg or undergoing current treatment with antihypertensive medication. Dyslipidemia was defined as one or more of the following: ≥150 mg/dL serum triglyceride, <40 mg/dL high-density lipoprotein cholesterol, ≥140 mg/dL low-density lipoprotein cholesterol, or current treatment with a lipid-lowering drug. Smoking status was defined as currently smoking or not smoking. Obesity was defined as a body mass index ≥30 kg/m2. FRS was calculated according to the Wilson et al. algorithm to estimate the 10-year risk of a coronary heart disease event [17]. In addition, patients were classified into three groups according to the European Society of Cardiology (ESC) recommendation: very-high risk, high risk, and moderate risk [18].
Outcomes and follow up
The patients were followed up prospectively from the date of CT. Follow-up clinical information was obtained from review of medical records or telephone interviews by attending physicians. The study endpoint was cardiovascular events defined as cardiovascular death, nonfatal myocardial infarction, late coronary revascularization, nonfatal stroke, or hospitalization due to heart failure. The diagnosis of myocardial infarction was made using the criteria of typical acute chest pain and persistent ST-segment elevation or positive cardiac enzymes. Late coronary revascularization was defined as percutaneous coronary intervention or coronary artery bypass grafting as indicated by the treating physician due to stable angina with a newly positive functional test. Patients with scheduled revascularization within 90 days of the CACS measurement were not counted as events. These patients were censored at the time of the first revascularization. Hospitalization for heart failure was defined as any unplanned stay overnight or longer in a hospital environment, for which the principal reason for admission was heart failure. Nonfatal stroke was defined as a sudden onset nonconvulsive and focal neurological deficit persisting for more than 24 hours.
Statistical analysis
Continuous variables are expressed as mean ± standard deviation or median with interquartile range. Dichotomous variables are expressed as number and percentage. Differences in continuous variables between the two groups were analyzed by Student’s t-test or Mann–Whitney U-test as appropriate. Categorical data were compared by chi-squared analysis. In subsequent analysis, triglyceride data were log-transformed because they did not show a normal distribution. Similarly, because the distribution of the Agatston score data was also highly skewed, CACS was log-transformed after adding 1 to all calcium scores to manage values of 0 (log[CACS+1]). Kaplan–Meier curves were used to estimate cumulative event rates of cardiovascular events. Differences between time-to-event curves were compared by log-rank test. Annual event rates were calculated by dividing the 4-year Kaplan–Meier event rates by 4 and comparing them. The effect of variables on cardiovascular events was assessed using Cox proportional hazard analysis, and the results were reported as hazard ratios (HR) with 95% confidence intervals (CI). The incremental value of NAFLD was assessed by calculating the global chi-squared test and the receiver operating characteristic (ROC) curve analysis. C-statistics were calculated from the ROC curves and compared using Delong test. The category-free net reclassification improvement was also calculated. All reported p values were two-sided and p<0.05 was considered statistically significant. Statistical analyses were performed using SPSS statistical software (version 24; IBM Corp., Armonk, NY, USA) and the R statistical package (version 3.5.2; R Foundation for Statistical Computing, Vienna, Austria).