Change in glucose intolerance status and risk of incident cardiovascular disease: Tehran Lipid and Glucose Study

The TLGS is a large longitudinal prospective population-based study of a representative urban sample of Tehran population. Details of study design, sampling and rationale is published elsewhere [15].

In brief, TLGS includes 15,005 participants at first visit (1999–2002), with additional 3550 recruitments in the second visit (2002–2005) of study. Follow up visits happened at approximately 3 year intervals. In the current study 9558 individuals, who were 30 years or older were included [7927 from baseline (1999–2002) and 1631 from second phase (2002–2005)]. After excluding those with diabetes (n = 1354), prevalent CVD (n = 406) or incident CVD before the second examination (n = 128), 7670 individuals remained. Other exclusion criteria included those who did not participate in the second examination i.e., 2002–2005 for those entered in the first phase and 2005–2008 for participants recruited in the second phase (n = 2810), those with missing data on covariates, i.e., age, sex, smoking, education, physical activity, creatinine, fasting plasma glucose (FPG), 2-h post challenge plasma glucose (2 h-PCPG), total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C), body mass index (BMI), waist circumference (WC), systolic blood pressure (SBP) and diastolic blood pressure (DBP), at baseline and second examination (n = 766), leading to 4094 participants with complete data (respondents) who were followed until March 2016.

Demographic information, medical history, smoking habits and history of CVD were obtained from participants during interviews, using a pretested questionnaire at baseline and each follow-up. Details of anthropometric measurements including weight, height and WC have been previously described elsewhere [15]. BMI was calculated as weight in kilograms divided by square of height (m²). Blood pressure was measured using a standardized mercury sphygmomanometer (calibrated by the Iranian Institute of Standards and Industrial Researches), twice on the right arm in a seated position after at least 15 min rest and the mean of these two measurements, was considered as the participant’s blood pressure.

At the first phase of the TLGS, assessment of physical activity level was performed using the Lipid Research Clinics questionnaire; however, due to inexactness of this questionnaire, it was replaced by the Modifiable Activity Questionnaire in the second phase, which measures all 3 forms of activities including job, leisure time and household activities in the last year [15]. After 12 to 14 h of overnight fasting, a blood sample was taken for the biochemical analysis on the same day. FPG and 2 h-PCPG (using 75 g glucose, for those without history of taking glucose-lowering medications) were measured by the glucose oxidation enzymatic colorimetric method. Further details regarding laboratory measurements including FPG, 2-h PCPG, TC and HDL-C have been described before [15].

As reported in our first article regarding CVD outcomes in the TLGS cohort [16], each participant is followed-up for any medical event leading to hospitalization during the past year by telephone call and he/she is asked for any medical conditions by a trained nurse. If a related event has occurred, a trained physician collects complementary data regarding that event during a home visit and by gathering data from participant’s medical files. In the case of mortality, data is collected from the hospital or death certificate by an authenticated local physician. Collected data is then evaluated by an outcome committee consisting of an internist, endocrinologist, cardiologist, epidemiologist, and other experts, when needed, to assign a specific outcome for every event. Importantly, the outcome committee is blinded to the status of baseline risk factors.

In the current study, CHD events included cases of (1) definite myocardial infarction (MI) diagnosed by diagnostic electrocardiogram (ECG) and biomarkers (including CK, CK-MB, CK-MBm, troponin (cTn) and myoglobin), (2) probable MI distinguished by positive ECG findings plus cardiac symptoms or signs and biomarkers showing negative or equivocal results, (3) unstable angina pectoris, who admitted the hospital and developed new cardiac symptoms or showed changing symptom patterns and positive ECG findings with normal biomarkers [17], (4) angiography proven CHD defined as ≥ 50% stenosis in at least one major coronary vessel [18], and (5) CHD death (any death due to CHD according to the above-mentioned criteria or sudden cardiac death caused by cardiac disease occurring ≤ 1 h after onset of symptoms according to verbal autopsy documents outside of hospital). Details of stroke definition in TLGS cohort has been addressed elsewhere [19]. Accordingly, definite stroke was defined using the World Health Organization’s definition as “rapidly developing clinical signs of focal or global disturbance of cerebral function, lasting > 24 h or leading to death with no apparent cause other than that of vascular origin” [20]. Moreover, another criterion of definite stroke was imaging suggestive of stroke in cases of acute clinically relevant brain injuries accompanied by rapidly vanishing symptoms. Possible stroke was defined as any acute neurologic deficit with no imaging that is indicative of stroke or with data that were not fully consistent with the World Health Organization’s definition for definite stroke. When symptoms resolved within 24 h, cases were labeled as transient ischemic attack. In the current study, all cases of definite or possible stroke or transient ischemic attack were defined as stroke. Furthermore, CVD was defined as a composite measure of any CHD events, stroke or cerebrovascular death.

We used the 2003 American Diabetes Association (ADA) criteria as our reference for categorization of our study. Therefore, we defined normal fasting glucose (NFG) as FPG < 5.6, normal glucose tolerance (NGT) as 2 h-PCPG < 7.8 mmol/L, IFG: 5.6 ≤ FPG < 7 mmol/L and IGT: 7.8 ≤ 2 h-PCPG < 11 mmol/L. NFG/NGT was defined as those with both NFG and NGT states. IFG/IGT was defined as those with IFG and/or IGT. Diabetes was defined as FPG ≥ 7 mmol/L, 2 h-PCPG ≥ 11.1 mmol/L or using anti-hyperglycemic agents.

Estimated glomerular filtration rate (eGFR) presented as mL/min/1.73 m², was estimated using the CKD Epidemiology Collaboration (CKD-EPI) equation [21].

Smoking was defined as occasional or daily use of any kind of tobacco and smoking status was classified as current versus past or never smoker. Low physical activity was classified as subjects participating in physical activity < 3 day/week for participants recruited in first phase or < 600 metabolic equivalent task–minutes (MET)/week for those who entered in the second phase [22]. Education was classified into three groups: < 6 years, 6–12 years and > 12 years. Marital status was categorized as single, married and widowed/divorced.

The baseline characteristics were presented as mean (standard deviation, SD) for continuous variables and frequencies (%) for categorical variables. Comparison of baseline characteristics between NFG/NGT versus IFG/IGT was done by Student’s T-test for continuous variables and the Chi-square test for categorical variables. The required assumptions to conduct the T-test are normal distribution of data and homogeneity of the variance. Normality was tested using Kolmogorov–Smirnov (K–S) test. Moreover, we also used histograms with fitted normal curves to check the normality of data. Homogeneity of variances was tested using the Levene test.

Mean difference [95% Confidence interval (CI)] of continuous variables and the mean differences in the prevalence [95% CI] of each category of categorical variables were estimated to compare respondents with non-respondents [those who did not participate in the first follow-up visit and those with missing data of FPG, 2 h-PCPG and other covariates (n = 3576)].

Cox proportional hazard regression was used to assess the hazard ratios (HRs) of changes in glucose tolerance status for CVD/CHD. Time to event was defined by time of censoring or the event occurring, whichever came first. We censored participants in the case of other causes of death, leaving the district or being in the study until 20 March 2016, without any event.

Univariable Cox analysis was performed for each potential risk factor including age, sex, education, using lipid lowering or anti-hypertensive drug, being in the intervention group, smoking, SBP, DBP, marital status, TC, HDL-C, BMI, WC, eGFR, physical activity as well as changes in BMI, WC, SBP, DBP, eGFR, TC and HDL-C; then, covariates with a P-value < 0.2 in the initial univariable analysis were selected to enter the multivariable model.

The following categories both at baseline and 3 years later were defined: NFG, NGT, NFG/NGT, IFG, IGT and IFG/IGT as well as incident type 2 diabetes (only in the first follow-up). We checked for changes for each different category over the next examination.

For those with NFG, NGT and NFG/NGT at baseline, changes include remaining in normoglycemia or progression to IFG, IGT or IFG/IGT. For those with IFG, IGT and IFG/IGT, regression to normoglycemia, remaining in previous status and progression to diabetes, were assessed (Fig. 1). Considering the limited number of those with NFG, NGT and NFG/NGT at baseline who directly converted to diabetes [NFG to diabetes (n = 60), NGT to diabetes (n = 52), NFG/NGT to diabetes (n = 24)], we excluded these groups in the data analysis.

Three models were defined: model 1 was adjusted for age, sex and model 2 was further adjusted for SBP, DBP, TC, HDL-C, WC, eGFR, physical activity, smoking, education and use of anti-hypertensive drug. In model 3, covariates in model 2 plus change of WC, TC and HDL-C were adjusted.

The proportional hazards assumption in the Cox model was assessed using Schoenfild residual test and all proportionality assumptions were appropriate. Statistical analysis was performed using SPSS for windows version 20 and STATA version 14. P-values ≤ 0.05 were considered statistically significant.