This study was carried out at the Zhongnan Hospital of Wuhan University and Renmin Hospital of Wuhan University. The research protocol was approved by the Medical Ethics Committee of the Zhongnan Hospital of Wuhan University. All individuals provided informed consent with verbally or in writing. The normal group population was selected from those who underwent a comprehensive health examination at Zhongnan Hospital of Wuhan University, Renmin Hospital of Wuhan University. The study subjects included 103 T2DM and 59 AMI patients who had been hospitalized at two departments: Department of endocrinology and cardiovascular medicine in Zhongnan Hospital of Wuhan University. The 21 volunteers were graduate students from the Center for Gene Diagnosis of Zhongnan Hospital of Wuhan University. The health examination individuals were selected for participation based on the following criteria: liver, kidney function and blood test were in the normal range for the most parameters, and without other systemic disease history.

Type 2 diabetes was defined according to the American Diabetes Association (ADA) [12]. Any one of the following criteria meets the diagnosis for T2DM (In the absence of unequivocal hyperglycemia, criteria 1–3 should be confirmed by repeat testing): (1) HbA_1c ≥6.5%. (2) FPG ≥7.0 mmol/l. (3) Two-hour plasma glucose ≥11.1 mmol/l during an OGTT. (4) In a patient with classic symptoms of hyperglycemia or hyperglycemic crisis, a random plasma glucose ≥11.1 mmol/l. Patients were excluded for the following reasons: (1) Patients who were diagnosed with type 1 diabetes mellitus (2) Patients who were complicated with cardiovascular disease, viral hepatitis, tumor, autoimmune disease, serious liver or kidney dysfunction (3) patients who use lipid-lowering drugs (e.g. Lipitor and Zocor, etc, which are in a class of drugs known as statins).

AMI was defined according ESC/ACCF/AHA/WHF [13, 14]. Detection of a rise of cardiac troponin (cTn) with at least one above the 99th percentile upper reference limit (cTnT ≥0.01 ng/L) and with at least one of the following: (1) Symptoms of ischaemia: chest pain lasting 30min or longer. (2) New or presumed new significant ST-segment-T wave (ST-T) changes or new left bundle branch block (LBBB). (3) Development of pathological Q waves in the ECG. (4) Imaging evidence of new loss of viable myocardium or new regional wall motion abnormality. (5) Identification of an intracoronary thrombus by angiography or autopsy. Patients were excluded for the following reasons: (1) Previous myocardial infarction. (2) Patients who were complicated with diabetes mellitus, viral hepatitis, tumor, autoimmune disease, serious liver or kidney dysfunction. (3) Patients who used lipid-lowering drugs (e.g. Lipitor and Zocor, etc. which are in a class of drugs known as statins).

Height (cm) and weight (kg) were measured in the standing position when health examination or admission. Venous blood samples were collected after overnight fasting for T2DM patients, healthy examination individuals and volunteers. Venous blood samples were collected for AMI patients within 4 hours of admission. Serum samples were prepared by centrifugation at 2000 rpm for 10 min at 4°C. Blood routine parameters were measured using the automatic hematology analyzer (Beckman-CoulterLH750, USA). The inter-assay coefficients of variation (CV%) amounts to 3.6% and 2.9% in the higher normal and lower normal range respectively. Fasting serum glucose (GLU), total cholesterol (TC), triglyceride (TG) and high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), Serum aminotransferase (ALT) and aspartate aminotransferase (AST), serum urea nitrogen(BUN), uric acid concentrations were measured using the automated chemistry analyzer (Olympus AU5400 biochemistry analyzer, Japan). The inter-assay coefficients of variation (CV%) amounts to 3.9% and 2.7% in the higher normal and lower normal range respectively. Serum samples for FFAs assays were stored at -80°C until analysis. Serum FFA levels were measured with high pressure liquid chromatography (HPLC) within one hour after rehydration.

SPSS 13.0 software was used for statistical analysis. Categorical variables were expressed as proportions and compared between groups using the X² test. Continuous data are expressed as mean ± SD for normally distributed variables or median (inter quartile range) for others. The paired t-test was used to compare continuous variables. Unavailable relationships were calculated with Pearson correlation coefficients. Comparison of the groups by ANOVA was followed by SNK-q test to determine differences between individual groups. Multivariable stepwise regression analyses were performed to determine the independent correlation factors to FFA. Because of skewed distribution, the values of FFA were transformed in logarithmically for analysis. The level of significance was set at P < .05.

A total of 21 volunteer subjects were enrolled in this study. Mean age was 26.7 ± 2.3 years, and 33% were men. The serum sample was stored at room temperature (1 h, 4 h, 8 h, 24 h) and at 4°C 4 h, and plasma sample at room temperature 1 h and at 4°C 4 h. There was no significant difference in serum FFA levels between the room temperature 1 h and 4°C 4 h (407.3 ± 148.4 mmol/L vs. 406.2 ± 148.1 mmol/L, P = 0.19). The FFA levels began to increase in those groups under room temperature (4 h: 416.9 ± 149.2 mmol/L; 8 h: 419.5 ± 149.2 mmol/L; 24 h: 513.8 ± 151.2 mmol/L; P < .001) compared to room temperature 1 h (407.3 ± 148.4 mmol/L) (Figure 1A). Plasma FFA levels were significant lower compared to the serum levels (room temperature 1 h: 351.3 ± 137.9 mmol/L vs. 407.3 ± 148.4 mmol/L; P < .001).The scatter plot also showed an elevated of FFA levels with the storing time lasted (R² = 0.957, P < .001, Figure 1B).

We divided 540 healthy individuals into six sub-groups (Figure 2). There was a significant difference among the six sub-groups after adjusted for sex and BMI, the FFA levels had a trend of increasing with the age, but not enough to achieved statistically significant among the age among 20 ~ 50 years and 60 ~ years. We found no difference between sexes (Table 1).

Baseline characteristics were shown in Table 2. A total of 242 eligible consecutive individuals were included which age was over 60 years between June 2013 to October 2013. There were 143, 52 and 47 subjects in the control group, T2DM and AMI, respectively. All data were in a normal distribution exclude ALT, AST and FFAs.

In this part, 242 subjects were chosen according to age greater than or equal to 60 (129/242 were men; 71.0 ± 6.8 years). There were 143, 52 and 47 subjects in the control group, T2DM and AMI, respectively. We compared the three groups with one-way ANOVA (Figure 3). Serum levels of the triglyceride (TG) and glucose (GLU) were significantly higher in individuals with T2DM and AMI compared to control, as TG (2.02 ± 1.29 mmol/L&1.63 ± 0.88 mmol/L vs. 1.12 ± 0.33 mmol/L, P < .001&P < .001) (Figure 3A) and glucose (7.75 ± 2.93 mmol/L&5.78 ± 1.32 mmol/L vs. 4.90 ± 0.49 mmol/L, P < .001&P < .01) (Figure 3D). The HDL levels were lower in T2DM (0.98 ± 0.18 mmol/L, P < .001) and AMI (1.01 ± 0.22 mmol/L, P < .001) compared to control group (1.30 ± 0.22 mmol/L) (Figure 3B). And serum FFAs and AST obtained significant difference in AMI compared to T2DM or control (P < .001) (Figure 3E and F).

The participants were divided the participants into four groups based on the age as shown in Table 3. Stepwise multivariable regression models were used to find the association of FFA with each of the other factors with adjusted sex and BMI. Those factors ALT, AST, TC, TG, HDL, LDL, BUN, TP and fasting glucose were incorporated into the regression models.

For stepwise multivariable regression analysis, we selected the variables that were allowed to enter the model in advance. HDL and TG were associated with The FFAs in the control group (20 ~ 50 years) (R = 0.28, P < .001) and were allowed to enter the multivariable model, so was HDL was associated with FFAs in the control group (≥60 years) (R = 0.26, P < .01) and T2DM (≥60 years) (R = 0.36, P < .05), AST was associated with The FFAs in the AMI (≥60 years) (R = 0.487, P < .01).