Association of plasma free fatty acids levels with the presence and severity of coronary and carotid atherosclerotic plaque in patients with type 2 diabetes mellitus

From December 2016 to November 2018, 1026 consecutive patients with T2DM were screened out from Dyslipidemia and Cardiovascular Disease center in FuWai Hospital. The inclusion criteria for patients with T2DM were as follows: (1) men and women over 18 years old; (2) underwent coronary angiography and carotid ultrasonography. Exclusion criteria included: (1) patients whose data is not entire; (2) severe liver or/and renal insufficiency; (3) thyroid dysfunction; (4) malignant disease; (5) familial hypercholesterolemia (FH). As a result, a total of 302 eligible patients were recruited in the present study.

The study protocol was in compliance with the Declaration of Helsinki, and was approved by the hospital’s ethics review committee (Fu Wai Hospital and the National Center for Cardiovascular Diseases, Beijing, China, approval number: 2013–442). Informed written consents were obtained from all patients enrolled in this study.

The clinical characteristics after admission were collected from all enrolled patients including age, gender, body mass index (BMI), the medical history of hypertension (HTN), stroke, smoking status and the usage of aspirin, statins, angiotensin converting enzyme inhibitors (ACEI), angiotensin receptor blocker (ARB) and Calcium Blocker. DM was defined by fasting plasma glucose (FPG) ≥7.0 mmol/L or the 2-h plasma glucose of the oral glucose tolerance test ≥11.1 mmol/L or currently using hypoglycemic drugs or insulin. BMI was calculated as BMI (kg/m²) = body weight (kg)/body height (m²). HTN was diagnosed as a self-reported hypertension, systolic blood pressure (SBP) ≥140 mmHg and/or diastolic blood pressure (DBP) ≥90 mmHg for three or more consecutive times or currently taking antihypertensive drugs. Smoking was ascertained as participants who had smoked regularly within the previous 12 months. CAD was defined as coronary stenosis more than 50% of at least one coronary artery.

Blood samples of all the enrolled patients were collected from cubital vein after a 12 h overnight fasting and collected into EDTA-containing tubes. Plasma concentrations of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein A (apoA), apolipoprotein B (apoB), FPG and FFAs were measured using an automatic biochemistry analyzer (Hitachi 7150, Tokyo, Japan) as our previous published papers [12, 13]. Plasma hemoglobin A1C (HbA1c) levels were measured using the Tosoh G7 Automate HPLC Analyzer (TOSOH Bioscience, Japan).

All enrolled patients in the present study underwent coronary angiography using standard Judkins techniques according to our previous studies [12, 14]. CAD was mainly manifested as coronary artery diameter stenosis of more than 50%. The severity of coronary atherosclerosis was evaluated by the Gensini score, which assigned the severity score based on the degree and location of stenosis based on our previous publications [15]. In detail, the narrowing was scored as 32 for 100% occluded artery, 16 for 91–99%, 8 for 76–90%, 4 for 51–75%, 2 for 26–50%, and 1 for 1–25%. The score was then multiplied by a coefficient to indicate the functional importance of each segment. The coefficient was as follows: the left main coronary artery was 5, the proximal left anterior descending artery and the proximal circumflex artery were 2.5, the middle left anterior descending artery was 1.5, the right coronary artery, the distal left anterior descending artery, the posterolateral artery and obtuse artery were 1.0, and the residual major segments were 0.5 [16]. Finally, the Gensini score was calculated by the sum of the integral of each coronary artery segment [16].

Each patient’s bilateral carotid arteries were examined by two experienced ultrasound physicians who were blind to the clinical characteristics of the patients using 128 System (Acuson, Mountain View, CA, USA) with a high-resolution 7.5–10.0 MHz transducer accrdoing to our previous study [12, 17]. The intima-media thickness (IMT) of proximal and distal common carotid artery, bifurcation of left and right common carotid artery and carotid sinus were measured respectively. Carotid plaque was defined as the IMT exceeding 1.5 mm, or local IMT enlargement exceeding 50% of surrounding IMT, and recorded as presence or absence. Crouse score for carotid plaques was defined as the sum of maximum thicknesses of all plaques as previously reported [18].

We divided 302 patients into CAD group and without CAD group and CAP group and without CAP group. The baseline characteristics and laboratory data of all patients were shown in Table 1. As is shown in the Table 1, the average age of the studied patients was 57.52 ± 10.56 years, of which 64.9% were males. The mean plasma FFA level was 0.49 ± 0.17 mmol/l.

As is shown in Table 2, the 302 patients were divided into three groups according to the tertiles of plasma level of FFAs: tertile 1 (< 0.41 mmol/L, n = 107), tertile 2 (0.41–0.56 mmol/L, n = 96), tertile 3 (> 0.56 mmol/L, n = 99). Patients with the higher levels of FFAs appeared to have increased concentrations of FPG and HbA1C when compared with the first tertile (all p value for trend < 0.05).

As is shown in Table 1, CAD was present in 232 patients (76.8%) and there were more often men (70.3%) with a higher history of HTN (76.3%) and smoking (54.3%). Importantly, plasma FFA levels were significantly elevated in patients with CAD compared with that without (0.51 ± 0.17 mmol/l vs 0.41 ± 0.15 mmol/L, p < 0.001). There were lower left ventricular ejection fraction (LVEF) and higher FPG, HbA1c and rates of aspirin and statins in patients with CAD (all p for trend < 0.05). Besides, comparing the patients with low, medium and high FFA levels, we found significant differences in the prevalence of CAD and Gensini scores (p < 0.001 in all, Table 2, Fig. 1a and b).

In addition, the results showed that FFAs were positively associated with CAD presence (r = 0.257, p < 0.001) and Gensini scores (r = 0.268, p < 0.001) in the Spearman correlation analysis (Table 3). Moreover, statin therapy, FPG and HbA1c were positively associated with the presence of CAD and Gensini scores. In the multivariable model (Table 4), FFA per SD increase was associated with increased risk of the presence of CAD (OR = 1.83, 95%CI: 1.27–2.65, p = 0.001) after adjusting traditional risk factors including age, gender, BMI, statin therapy, HTN, smoking, FPG, HbA1C, TC, HDL-C and LDL-C. Besides, gender, HTN, statin therapy and HbA1c were also predictors for the presence of CAD.

The data from Table 1 indicated that patients with CAP were older (59.10 ± 11.01 vs 55.61 ± 9.70, p = 0.004), more often male (68.5%) and had higher rate of HTN (77.0%). Plasma FFA levels were significantly elevated in patients with CAP compared with that without (0.54 ± 0.17 mmol/l vs 0.44 ± 0.15 mmol/L, p < 0.001). We also compared the patients with low, medium and high FFA levels and found significant differences in the prevalence of CAP and Crouse scores (all p value < 0.001; Table 2, Fig. 1c and d).

Moreover, spearman correlation analysis (Table 3) showed that FFAs were positively correlated with the presence of CAP (r = 0.254, p < 0.001) and Crouse scores (r = 0.359, p < 0.001). Additionally, data suggested that age and FPG were positively correlated with Crouse scores. After adjusting traditional risk factors including age, gender, BMI, statin therapy, HTN, smoking, FPG, HbA1C, TC, HDL-C and LDL-C, FFA per SD increase was associated with increased risk of the presence of CAP in multivariable regression analysis (OR = 1.62, 95%CI: 1.22–2.14, p = 0.001, Table 4). Besides, age and gender were also predictors for the presence of CAP.

As is shown in Fig. 2 a and b, the area under the curve (AUC) indicated that the FFAs had an appropriate discriminate ability in diagnosing the presence of CAD (AUC = 0.68, 95% CI: 0.61–0.75, p < 0.001) and CAP (AUC = 0.65, 95% CI: 0.59–0.71, p < 0.001) in patients with T2DM.