The Isfahan Cohort Study: Rationale, Methods and Main Findings

Authors: Nizal Sarrafzadegan, Mohammad Talaei, Masoumeh Sadeghi, Roya Kelishadi,

Shahram Oveis Gharan, Noushin Mohammadifard, Amir Reza Sajjadieh, Payam Kabiri, Tom

Marshall, G. Neil Thomas, Aliakbar Tavasoli

Isfahan Cardiovascular Research Center (WHO- Collaborating Center for Research and

Training in Cardiovascular Diseases Control, Prevention, and Rehabilitation for Cardiac

Patients in the Eastern Mediterranean region), Isfahan University of Medical Sciences,

Isfahan, Iran and Public Health, Epidemiology and Biostatistics, Health and Population

Sciences, The University of Birmingham, Birmingham, UK

Correspondence to:

Nizal Sarrafzadegan, MD

Professor in Medicine and Director

Isfahan Cardiovascular Research Center

Isfahan University of Medical Sciences

Address: P.O.Box: 81465-1148, Isfahan, Iran

Phone: +98 311 3377888-9

Fax: +98 311 3373435

Website: www.crc.mui.ac.ir , www.ihhp.ir

1 Abstract

A 10-year longitudinal population-based study, entitled the Isfahan Cohort Study (ICS) is being conducted. The ICS commenced in 2001, recruiting individuals aged 35+ living in urban and rural areas of three counties in central Iran, to determine the individual and combined impact of various risk factors on the incidence of cardiovascular events. After

24379 person-years of follow-up with a median follow-up of 4.8 years, we documented 219 incident cases of ischemic heart disease (IHD) (125 in men and 94 in women) and 57 incident cases of stroke (28 in men and 29 in women). The absolute risk of IHD was 8.9 (7.8-10.2) per

1000 person-years for all participants, 10.6 (8.8-12.5) per 1000 person-years for men and 7.4

(6.0-9.0) per 1000 person-years for women. The respective risk of ischemic stroke was 2.3

(1.7-3.0), 2.3 (1.6-3.3) and 2.3 (1.5-3.2) per 1000 person-years. The risk of IHD was approximately 3.5-fold higher in the presence of hypertension, followed by diabetes mellitus and hypercholesterolemia with near 2.5- and 2-fold higher risk, respectively.

This cohort provides confirmatory evidence of the ethnic differences in the magnitude of the impact of various risk factors on cardiovascular events. The differences may be due to varying absolute risk levels among populations and the existing ethnic disparities for using

Western risk equations to local requirements.

2 Introduction

Chronic non-communicable diseases (CNCDs), particularly cardiovascular disease (CVD) are no longer limited to the industrialized world. According to the World Health Organization

(WHO) estimates; in 2003 low- and middle-income countries accounted for 86% of the global

CVD disease burden, and by 2010, CVD will be the leading cause of death in developing countries (1). CVD has no geographic, gender or socio-demographic margins, and the major risk factors are well-documented. There is a large body of evidence supporting strong ethnic differences in CVD risk factor levels and their synergetic effects on CVD, even from an early age (2).

It has been suggested that environmental-gene interactions along with socioeconomic and behavioral factors underlie ethnic disparities in CVD risk factor profiles (3). Most studies have described cross-sectional associations. The INTERHEART study, the largest study to date, was conducted in 52 countries and revealed that the relative risks associated with different CVD risk factors are consistent in both sexes and at all ages and in the different regions. The main difference was due to the underlying distribution of the risk factors (4). However the impact of risk factors in various ethnicities can be best verified by longitudinal studies as the distribution of risk factors and in turn their impact on CVD events may vary between populations.

The scarcity of such longitudinal evidence in low- and middle-income countries and the abovementioned ethnic differences underscore the necessity of conducting cohort studies to determine not only the risk factor prevalence, but also the impact of each or different combinations of risk factors on the incidence of coronary and cerebrovascular events in diverse populations.

3 Middle Eastern countries are of special concern in this context, as in the next two decades they face the world’s greatest increment in the absolute burden of diabetes and in turn other

CNCDs, especially CVDs (5). As one of these countries, Iran has experienced both a rapid epidemiologic transition and change in the health and disease profile from predominantly infectious diseases to CNCDs (6). Several cross-sectional studies have documented alarming prevalence rates of CVDs and their risk factors among the Iranian population (7-12), but there is very limited data from longitudinal studies and those are limited to a metropolitan area of the capital city (13).

The Isfahan Cohort Study (ICS) is the first study of its kind, not only in Iran, but also in the Eastern Mediterranean region. It is a 10-year longitudinal study, conducted from 2001 in urban and rural areas of three counties in central Iran. It aims to determine the individual and combined impact of various risk factors on the incidence of CVD events including fatal and non-fatal myocardial infarction (MI), fatal and non-fatal stroke and sudden cardiac death.

Here, we report the methodology and some of the main results of this ongoing population- based cohort study.

4 Materials / Subjects and Methods

The ICS is a population-based, longitudinal ongoing study of 6504 adults aged equal or greater than 35 years at baseline, living in urban and rural areas from three counties in central

Iran (Isfahan, Arak and Najafabad, Figure 1) who had participated in the baseline survey of a community trial for CVD prevention and control, entitled Isfahan Healthy Heart Program

(IHHP) (14-15). They were recruited from January 2 to September 28, 2001 and will be followed up for at least 10 years. Figure 2 summarizes the methodology of the ICS including the variables measured, sample sizes and follow up.

Baseline Survey of the IHHP:

The baseline survey of the IHHP was conducted in a representative population of adults aged

≥19 years who were living in urban and rural areas of Isfahan, Arak and Najafabad. These areas are shown on the map of Iran (Figure 1). Participants were selected by multistage random sampling. The study population was first stratified by their living area (urban vs. rural) according to the regional population distribution derived from a national population census conducted in 1999. Census blocks were then randomly selected from each county with the probability of selection proportional to the expected number of households and divided into clusters of approximately 1000 households. Within each cluster approximately 5-10 percent of households were randomly selected for enumeration. From each household one eligible individual aged ≥19 years was randomly selected, provided they were of Iranian nationality, mentally competent and not pregnant. The response rate in home interviews was

98%; however 95% attended the examination clinic. Given that individual’s cardiovascular health was not considered in the exclusion criteria of the IHHP sampling framework, the baseline participants included 108 (2.8%) cases with a history of MI, stroke or heart failure who were excluded from the ICS baseline survey.

5 The sample for the IHHP was recruited into different age and sex groups to reflect the age, sex distribution of the community. It was estimated that the prevalence of cardiovascular risk factors would be 0.2 in the control area and sample size for the IHHP was determined to have a 90% power to detect a relative risk of 0.75 at a significance of 0.05. Taking account of clustering, the overall sample size was 4828 in each area, allowing for losses to follow up it was intended to recruit 6300 in each area (12600 subjects). A total of 12514 individuals were included in the baseline survey.

Selection of subjects for the ICS:

Ethical approval was obtained from the Ethics Committee of Isfahan Cardiovascular

Research Center (ICRC), a WHO-collaborating center. Of the 12514 individuals from the baseline survey, there were 6640 adults aged 35 years and over in the baseline survey who were enrolled into the ICS. We intend to follow up the volunteers for at least ten years.

Data collection:

After obtaining informed written consent, a 30-minute full structured interview was conducted by trained health professionals using a validated questionnaire including questions on demographic characteristics, socioeconomic status, behaviors, attitudes, skills and knowledge about CNCDs, as well as related lifestyle behaviors (including smoking, physical activity, and nutritional habits). Thereafter participants were invited to the nearest health center, where a 15 -minute medical interview and a 20-minute physical examination was conducted by trained physicians and nurses. Measurement of blood pressure and anthropometric parameters were carried out following standard protocols (16, 17) and using calibrated instruments. Body mass index (BMI) was calculated as weight (kg) divided by height squared (m2). A twelve-lead electrocardiogram was recorded at the primary health care centers for all participants.

6 Fasting (12 hours) blood samples (10mL) were obtained from all participants and were examined at the ICRC central laboratory with adherence to external national and international quality controls. Serum total cholesterol (TC), triglycerides (TG) and fasting blood glucose

(FBG) were measured enzymatically (18), using an autoanalyzer (Eppendorf, Germany) and serum high-density lipoprotein-cholesterol (HDL-C) was determined after precipitation of low-density and very low-density lipoproteins with dextran sulfate-magnesium (19). C- reactive protein level was determined by the same autoanalyzer. Serum low-density lipoprotein-cholesterol (LDL-C) was calculated using the Friedwald equation in subjects with

TG less than 400 mg/dl, otherwise were measured using standard kits (20). A 75 gram oral glucose tolerance test was performed on non-diabetic individuals to determine their 2-hour post-load plasma glucose level (2hpp). Blood samples were centrifuged immediately in each county, samples obtained in Isfahan and Najafabad were transported to the central laboratory within about 1 hour; in the reference area, FBG and 2hhp were measured immediately, and serum frozen at -20°C, then by a 3-hour ground transport with cold chain (-20°C) they were transported to the central laboratory and kept frozen until assayed within 72 hours.

Dyslipidemia was defined as if LDL-C ≥130 mg/dl, TC ≥200mg/dl, TG ≥150 mg/dl, or

HDL-C <40 mg/dl in men or <50 mg/dl in women (21). Diabetes mellitus was defined as if

FBG ≥126 mg/dl or the patient was receiving anti-diabetic agents; impaired glucose tolerance (IGT) was identified if the 2-hour postprandial (2hPP) ≥140 but less than 200 mg/dl

(22). The BMI value ≥25 kg/m2 but less than 30 kg/m2 was classified as overweight and those who had BMI ≥30 kg/m2 were classified as obese (23). According to International Diabetes

Federation (IDF) cut points, WC ≥94 cm in men or ≥80 cm in women were defined as abdominal obesity (24). We also considered the cut points suggested for abdominal obesity in the Iranian population of WC ≥91.5 cm and WC ≥85.5 cm in men and women, respectively

(25). Waist to hip ratio (WHR) ≥0.95 in men and ≥0.8 in women was considered as a high

7 WHR (23). Blood pressure ≥140/90 mmHg or if the patients were receiving antihypertensive drugs was defined as hypertension (26).

Follow up surveys

After the baseline survey in 2001, follow up of the volunteers has been carried out every two years. Telephone interviews were carried out in 2003 and in 2005-2006. In 2007, full structured interviews; physical and biochemical measurements were repeated in the same way as for the baseline survey. Figure 2 shows the study algorithm. A third telephone interview follow up has been finished recently and will be repeated in 2011. For the telephone follow up interviews, at least 5 attempts are made to contact all living participants or their first degree relatives if they are deceased. If telephone interviews are unsuccessful, the participants are visited at their home address for follow-up. After confirmation of participants’ identity, structured primary interviews were performed based on a questionnaire with 3 main questions; “is he/she alive?”, “has he/she been hospitalized for any reason? (With specific focus on cardiovascular and cerebrovascular events), and “has the participant experienced any of the following 5 neurological symptoms (hemiparesis, dysarthria, facial asymmetry, imbalance and transient monoocular blindness)?”

If death, hospitalization or neurological symptoms have occurred, the date of the events, physician diagnosis and the hospital’s name are obtained during the interview. If any event has occurred, the related questionnaire was checked alongside the relevant health records. In the case of out of hospital deaths, death certificates were obtained from the provincial mortality database and verbal autopsies were performed by a trained expert nurse in a secondary interview with surviving family members. The verbal autopsy used a pre defined questionnaire including medical history, signs and symptoms prior to death. Expert nurses

8 conducted additional secondary interviews for hospitalized cases where information was incomplete or inconsistent.

Confirmation of End Points:

The reported events were checked with the MI and stroke registry database of the

Surveillance Department, ICRC. The registry is collected monthly in the three aforementioned counties. In the case of any inconsistency in dates or diagnoses, or unobtainable records, original medical records were investigated. If hospitalization data were not found in the registry database, trained nurses investigated relevant hospital medical records. Two separate panels of specialists consisting of four cardiologists and neurologists, reviewed all relevant documents of every patient (primary questionnaires, registry records, medical records, secondary interviews, verbal autopsies or death certificates) and made the final decision on all of the five main events (fatal and non-fatal MI, fatal and non-fatal stroke and sudden cardiac death) and unstable angina (UA).

The diagnosis of acute MI (AMI) was based on the presence of at least two of the following criteria: 1) typical chest pain lasting more than 30 min, 2) ST elevation >0.1 mV in at least 2 adjacent electrocardiograph leads and 3) an increase in the serum level of cardiac biomarkers (27). The definition of UA required typical chest discomfort lasting more than 20 minutes within the 24 hours preceding hospitalization and representing a change in the usual pattern of angina or pain: occurring with a crescendo pattern, being severe and described as a frank pain (28). The diagnosis of UA might be new or be based on dynamic ST-segment or T- wave changes in at least two adjacent ECG leads. Sudden cardiac death was defined as death within 1 hour of onset, a witnessed cardiac arrest, or abrupt collapse not preceded by >1 hour of symptoms. IHD included definite or probable MI, UA, and sudden cardiac death.

Moreover, the WHO stroke definition was used, i.e. stroke was defined as a rapid-onset focal neurological disorder persisting at least 24 hours and had probable vascular origin. The

9 diagnosis of incident stroke was conducted based on the clinical criteria. CVDs were defined as combination of IHD and stroke. Although the in-hospital diagnoses of clinicians were taken into account, the final decisions of the panel were made independently.

Statistical Analysis:

Data entry was carried out using EPI info™. All data were analyzed using the SPSS for

Windows software (SPSS Inc., Chicago, IL, USA; version 15.0). Student’s t-test was used for the comparison of means of independent groups and the 2 test for the comparison of proportions and categorical variables. The residency areas were included in the model as a variable in 6 levels for the urban and rural areas of the three counties being studied.

Cox proportional hazards modeling was used with time to outcome as the dependent variable and the presence of defined risk factors as independent dichotomous variables for the calculation of hazard ratios (HR) and 95% confidence intervals (CI). Individuals were censored at the first cardiovascular event. Kaplan–Meier analysis was used to evaluate time to outcome as a function of dichotomous independent variables with log-rank statistic for determining statistically significant differences. For all analyses, statistical significance was assessed at a level of 0.05 (2-tailed) and a p-value less than 0.1 was considered of borderline significance (marginal significance).

Results

The response rate for house interviews was 98%; however 95% attended the examination clinic. Given that individual’s cardiovascular health was not considered in the exclusion criteria of the IHHP sampling framework, the baseline participants included 108 (2.8%) cases with a history of MI, stroke or heart failure who were excluded from the ICS baseline survey.

10 From the 6640 individuals aged 35 years and older studied in the baseline cohort, 996

(15%) of telephone interviews were not successful, and address follow-ups were carried out.

Of the whole sample, 978 (15%) and 464 (7.1%) participants were missed after first and second stage of follow-up, respectively. The baseline characteristics and the prevalence of

CVD risk factors were not significantly different among those participants lost to follow up compared those remained in the follow up surveys.

The questionnaires of the 1014 (20%) reports for events were investigated by a general practitioner to identify 620 (12.2%) relevant reports including 132 (2.6%) deaths, 396 (7.8%) hospitalizations and 92 (1.8%) reports of only neurological symptoms and referred them for a further search of more authentic documents including medical records, death certificates and verbal autopsies. Of the total deaths, 77 (36%) occurred out of hospital. Expert nurses conducted additional secondary interviews for 196 (3.9%) of 5063 available hospitalized cases with incompatible or incomplete information.

After 24379 person-years of follow-up (with median follow-up of 4.8 years, 4.6 and 5 years for the 25th and the 75th quartiles, respectively), we documented 219 incident cases of

IHD (125 in men and 94 in women) and 57 incident cases of stroke (28 in men and 29 in women). IHD comprised 13 (8 in men and 5 in women) fatal and 45 (30 in men and 15 in women) non-fatal MI, 113 (54 in men and 59 in women) UA, and 48 (33 in men and 15 in women) sudden cardiac deaths.

Ischemic stroke comprised 13 (7 in men and 6 in women) fatal and 44 cases (21 in men and 23 in women) of non-fatal stroke. Absolute risk of IHD was 8.9 (95%CI: 7.8-10.2) per

1000 person-years for total participants, 10.6(8.8-12.5) per 1000 person-years for men and 7.4

(6.0-9.0) per 1000 person-years for women. The respective risk of ischemic stroke was 2.3

(1.7-3.0), 2.3 (1.6-3.3) and 2.3 (1.5-3.2) per 1000 person-years.

11 Table 1 shows the baseline characteristics of participants and CVD risk factors. In general, the participants who suffered from defined CVD events were urban men from older age groups with higher rates of smoking, over 3 cm greater WC, higher WHR and a two-fold higher prevalence of diabetes or hypertension than those without CVD event. Except HDL-C, all other lipid levels were significantly higher in those with CVD events than those without an event. The mean BMI value was higher in the participants with CVD events, whereas the corresponding figure was not significantly different for the prevalence of obesity.

The risk of CVD was approximately 3.5-fold higher in the presence of hypertension, followed by diabetes mellitus and hypercholesterolemia with near 2.5- and 2-fold higher risk, respectively (Table 2). Multivariable adjustment attenuated the extent to which these risk factors increased the risk of CVD but the ranking remained constant. Abdominal obesity according to IDF and Iranian definitions yielded marginally significant associations with similar hazard ratios. Nevertheless, in the multivariable analysis, none of the definitions resulted in significant association with an increased risk of CVD events. Moreover, a unit increase in the proportion of TC to HDL-C ratio, considered as a quantitative variable, was associated with a significant increase the risk in CVD even in multivariable adjusted model.

Figure 3 shows the age- and sex-adjusted effect of multiple risk factors on the level of risk for CVD events. While adding high TC to the analysis did not change the HR of hypertension and diabetes combination, high LDL-C with the previous combination increased the sex- and age-adjusted HR to nearly 3. A combination of the first two variables with high TG and low

HDL-C level resulted in a HR of more than 4. The combination of hypertension, diabetes, high LDL-C and smoking were documented in 9 participants, and resulted in what was considered an unstable HR of 7.41 with wide CIs of 2.36-23.27, and was thus not shown in the figure.

12 Discussion

This large population-based longitudinal study provides confirmatory evidence about the ethnic differences in the magnitude of the impact of various risk factors on CVD events.

Globally, in most population-based studies, the prediction of the absolute risk of CVD events has been based on risk prediction equations originating from the Framingham Heart Study, as the oldest longitudinal study for CVD risk (29). However, the limitations for the applicability of the Framingham risk function in diverse populations are well documented. The

Framingham Heart study has developed mathematical functions for predicting the risk of

CHD events. This cohort consisted of 2439 men and 2812 women free of CVD. The 5- and

10-year CHD event rates were 3.7% and 8.0% for men and 1.4% and 2.8% for women, respectively (28). This and most other cohorts in the Western countries consist of white middle class individuals; therefore there are concerns about the generalizability of their findings to other populations and societies particularly in low- and middle-income countries.

To our knowledge, ICS is the first community-based study of CVD risk from any Eastern

Mediterranean country that is being conducted in a geographic area with a large, stable, well- defined population living in urban and rural areas with high participation rates and using valid case ascertainment. The number lost to follow up, especially in the third phase, was low, and it necessitated much technical effort and cost to achieve this. A major reason for loss to follow up in the earlier phases was changes in telephone numbers introduced by the

Government. This was a part of network capacity development and did not have special distribution, so it was likely to be random and thus not bias the follow up of the volunteers.The baseline characteristics and the prevalence of CVD risk factors were not significantly different among those participants lost to follow up compared those remained in the follow up surveys, and this strengthen the ICS findings.

13 Although after correction for length of follow up, the observed number of CVD events in ICS was not significantly different from the expected number of events derived from Framingham study, but CVD events documented in ICS, i.e. 5.4% among men and

4.7% among women, showed a considerably higher rate of events among women when compared to the Framingham Heart Study. However, this difference might be because of higher admission rates for UA that are not included among CVD events in some other studies.

As described before, the ICS was designed to evaluate the relative risk of CVD risk factors in Iranians with its possible extrapolation to the greater Eastern Mediterranean population. Available evidence suggests that absolute risk varies among different populations independent of their major risk factors. For instance, the absolute risk among South Asians

(Indians and Pakistanis) living in Western countries appears to be about two times higher than that of whites, even when the two populations are matched for major CVD risk factors (30).

Moreover, it is possible that some populations would have lower baseline levels of the risk factors than those observed in the Framingham Heart Study population. Results from the

Honolulu Heart Study and the Seven Countries Study showed that the population of Eastern

Asian ancestry and Japanese exhibit a lower risk for CHD for a given set of risk factors in comparison to other populations (31). These marked differences between various populations provide sufficient evidence for adjustments of absolute risk in different racial and ethnic groups; however, the relative risk estimates can probably be reliable across various groups

High blood pressure was seen in 29% of the population studied in ICS in comparison to

31.3% in Americans in 1999 to 2000 (33). Hypertension in both univariate and multivariate analysis had the most powerful effect on CVD events with HR of 3.4 and 2.4, respectively.

The relative risk ranged from 1.1 to 2.5 in men and 0.5 to 1.8 in women studied in

Framingham and that higher levels of BP are typically associated with abnormalities in other

14 risk factors and increased prevalence of CVD events (34). The Atherosclerosis Risk in

Communities (ARIC) study revealed that among black women, the RR of hypertension was significantly higher (8.9) than that documented in the populations studied in the Framingham

Heart Study and other cohorts, however in other ethnic groups, as Hispanic, Japanese-

Americans and native Americans, the RR of CVD events was not significantly different in hypertensive individuals (34). Contrary to most other cohort studies that underscored the role of hypertension on stroke, in our study it significantly increased the risk of MI, and this shows the importance of conducting longitudinal studies in different ethnic groups.

Among lipids disorders, high TC and LDL-C had significant relative risk to CVD events even after adjustment for other factors. Low HDL-C was not associated with CVD events even in the crude analyses. The prevalence of low HDL-C in the population is high which may be explained by a number of measures including genetic predisposition, sedentary lifestyle and adverse quality of fat intake in Iranians.

In our study, when hypertension was added to other risk factors, there was an exponential increase in the RR for CVD events which reached levels as high as 7.4 when considering a combination of high blood pressure combined with diabetes mellitus, high LDL-C and smoking. Overall, 78% of all CVD events happened in those individuals who suffered from hypertension, diabetes and high LDL-C. Our findings are consistent with the results of the

INTERHEART study in regards of the association of risk factors with MI, which showed the effect of multiple risk factors increase the risk of MI. It showed that major CVD risk factors as dyslipidemia, smoking, hypertension, diabetes mellitus and abdominal obesity, as well unhealthy lifestyle behaviors account for most of the risk of myocardial infarction worldwide in both sexes and at all ages in all regions. Furthermore it found that smoking, hypertension and diabetes mellitus increase the odds ratio for MI up to near 12 fold compared to those without these risk factors (4). Although this global study found similar results in various

15 regions the associations were cross-sectional. In contrast, variations in the hazard ratios and distribution of risk factors between populations in the current cohort study, more importantly verified the longitudinal impact of the risk factors. The Prospective

Collaborative Study Group, which pooled 61 observational studies in more than one million volunteers with a collective experience of more than 12 million person-year showed a powerful predictive value of systolic and diastolic blood pressure for vascular death down to levels of 115 mmHg and 75 mmHg, respectively (35). A recent analysis of the same study evaluated the joint importance of cholesterol and hypertension on vascular mortality, and revealed that TC was positively associated with IHD mortality in both middle and old age and at all blood pressure levels (36).

Our findings describing the combined effects of risk factors are in line with other large cohorts with long-term follow up. For instance with the 38 year follow-up of 19,000 men in the Whitehall study that documented a three-fold higher rate of dying from vascular disease, a two-fold higher rate of dying from non-vascular causes, and a nearly 10-year shorter life expectancy at age 50 for those individuals with three CVD risk factors compared with those without any risk factor (37). Such findings confirm that an approach for risk assessment of

CVD events by considering the combined effect of risk factors instead of focusing on single risk factors would account for the multifactorial origin of CVD, and would also be practical for the comprehensive management of patients at high risk. In our cohort, a combination of hypertension and diabetes with dyslipidemia in terms of high LDL-C levels increased the HR of CVD events, whereas the corresponding figure was not significant for high TC levels alone. It is also worth mentioning that the combination of the abovementioned risk factors, i.e. hypertension, diabetes and high LDL-C in the presence of smoking resulted to the highest risk.

16 Conclusion: The ICS provides an optimal base for assessing determinants of cardiovascular risk in Iranians which would likely be pertinent to other countries in the region. This study used internationally-recognized standard methodology and quality control protocols both in the baseline survey and in the periodic follow-up studies. The study was conducted in a large population of urban and rural residents, and also used a valid case ascertainment. Similar to other cohort studies in various populations, the ICS confirmed the impact of traditional risk factors, notably hypertension, and the different combinations of risk factors on CVD events. Future analyses by gender and living area (urban vs. rural) might result in different HR and impact on CVD events; nonetheless as a general assumption, this cohort confirms the need for risk estimation in different populations, particularly in this region where data are currently minimal.

Acknowledgements

This cohort study was conducted by ICRC affiliated with the Isfahan University of Medical

Sciences. We are thankful to the team of the ICRC, Isfahan Provincial Health Center, Najaf-

Abad Health Office and Arak University of Medical Sciences. We would like also to extend our sincere thanks to the ICS team, especially to Mrs. Mansoureh Boshtam, Mr. Hossein

Balouchi, Dr. Hossein Heidari and Dr. Ahmad Bahonar for their technical assistance.

Conflict of interest: None to disclose.

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22 Table 1- Characteristics of the study participants according to the development of cardiovascular event: ICS, 2001-2006.

Characteristics CVD Event No CVD Events P value (N= 276) (N=5099) Age (years) 58.8±11.2 50.2±11.5 <0.001 Male (%) 153 (55.4) 2635 (51.7) 0.021 Fasting Blood Glucose (mg/dl) 100±47 88±32 <0.001 2-hour Post Prandial Glucose (mg/dl) 129±73 110±56 <0.001 Systolic Blood Pressure (mmHg) 138±25 121±20 <0.001 Diastolic Blood Pressure (mmHg) 85±14 78±11 <0.001 Total Cholesterol (mg/dl) 227±52 211±49 <0.001 Triglycerides (mg/dl) 218±118 189±102 <0.001 LDL-C (mg/dl) 140±46 127±42 <0.001 HDL-C (mg/dl) 46.5±10.3 46.9±10.4 0.534 Total /HDL-C 5.04±1.44 4.66±1.31 <0.001 C-reactive protein (mg/dl) 3.32±1.30 3.31±1.49 0.965 Body Mass Index (kg/m2) 27.3±4.9 26.7±4.4 0.024 Waist Circumference (cm) 97.6±12.6 94.5±12.2 <0.001 Waist to Hip Ratio 0.95±0.07 0.92±0.07 <0.001 Urban residence area 217 (78.6) 3680 (72.2) 0.019 Diabetes Mellitus* 59 (21.8) 459 (9.1) <0.001 Impaired Glucose Test† 31 (12.6) 436 (8.9) 0.055 Hypertension‡ 165 (59.8) 1395 (27.4) <0.001 Current Smoking 71 (25.7) 1050 (20.6) 0.041 Dyslipidemia § 252 (92.3) 4355 (86.9) 0.009 Overweight║ 112 (41.5) 2031 (40.8) Obesity ¶ 65 (24.1) 1078 (21.6) Abdominal Obesity (IDF cut points)** 206 (75.5) 3542 (69.7) 0.044 Abdominal Obesity (Iranian cut-points) †† 203 (75.2) 3438 (68.9) 0.030 High Waist to Hip Ratio‡‡ 194 (71.3) 3322 (65.4) 0.046 Mean±SD or proportion (%). CVD: Cardiovascular disease * Diabetes Mellitus: Fasting Blood Glucose ≥126 mg/dl or receiving anti-diabetic agents † Impaired Glucose Test: 2-hour post prandial glucose (2hPP) ≥140 but less than 200 mg/dl ‡ Hypertension: Systolic blood pressure 140 mmHg, Diastolic blood pressure 90 mmHg, or current treatment for hypertension § Dyslipidemia: LDL-C ≥130 mg/dl, Total Cholesterol ≥200mg/dl, Triglyceride ≥150 mg/dl, HDL-C <40 mg/dl in men and <50 mg/dl in women ║ Overweight: 25 kg/m2 ≤Body Mass Index <30 kg/m2 ¶ Obesity: Body Mass Index ≥30 kg/m2 ** Abdominal Obesity (IDF cut-points): Waist Circumference ≥94 cm in men and ≥80 cm †† Abdominal Obesity (Iranian cut-points): Waist Circumference ≥91.5 cm in men and ≥85.5 cm ‡‡ High Waist to Hip Ratio: Waist to hip ratio ≥0.95 in men and ≥ 0.8 in women

23 Table 2- Crude and adjusted hazard ratios of risk factors for cardiovascular events: ICS, 2001-2006

Crude HR Adjusted HR Characteristic P-Value P-Value (95% CI) (95% CI)* Sex (Male) 1.40 (1.10-1.79) 0.005 1.41 (1.05-1.90) 0.021 Diabetes mellitus† 2.66 (1.97-3.57) <0.001 1.75 (1.27-2.39) <0.001 Impaired glucose tolerance‡ 1.57 (1.07-2.31) 0.020 1.16 (0.78-1.73) 0.452 Hypertension§ 3.42 (2.68-4.37) <0.001 2.10 (1.60-2.76) <0.001 Current smoking 1.37 (1.04-1.80) 0.022 1.41 (1.03-1.93) 0.032 Overweight║ 1.05 (0.79-1.39) 0.718 0.97 (0.72-1.30) 0.867 Obesity¶ 1.13 (0.81-1.56) 0.462 1.04 (0.73-1.49) 0.789 Abdominal obesity (IDF cut- 1.31 (0.99-1.73) 0.059 1.18 (0.85-1.63) 0.304 points)** Abdominal obesity (Iranian cut- 1.36 (1.02-1.80) 0.031 1.10 (0.80-1.50) 0.545 points)†† High waist to hip ratio‡‡ 1.27 (0.97-1.66) 0.78 1.18 (0.86-1.63) 0.297 Hypercholesterolemia§§ 1.91 (1.46-2.49) <0.001 1.46 (1.09-1.96) 0.010 High LDL-cholesterol ║║ 1.81 (1.39-2.35) <0.001 1.57 (1.20-2.07) 0.001 Low HDL-cholesterol ¶¶ 1.03 (0.81-1.32) 0.769 1.26 (0.97-1.65) 0.076 Hypertriglyceridemia*** 1.72 (1.32-2.25) <0.001 1.23 (0.92-1.65) 0.161 High total cholesterol/HDL-C††† 1.27 (1.17-1.39) <0.001 1.19 (1.09-1.31) <0.001 C-Reactive Protein 0.96 (0.85-1.08) 0.582 0.94 (0.84-1.06) 0.362 * Adjusted Model including: Residency, age, sex, smoking status, BMI categories, hypertension, diabetes, high cholesterol, high triglyceride, low HDL-cholesterol † Fasting blood glucose ≥126 mg/dl or being on anti-diabetic agents ‡ 2-hour postprandial (2hPP) ≥140 mg/dl, but less than 200 mg/dl § Systolic blood pressure 140 mmHg, Diastolic blood pressure 90 mmHg, or current treatment for hypertension ║ 25 kg/m2 ≤Body mass index <30 kg/m2 ¶ Body mass index ≥30 kg/m2 ** IDF cut-points: Waist circumference ≥94 cm in men and ≥80 cm †† Iranian cut-points: Waist circumference ≥91.5 cm in men and ≥85.5 cm ‡‡ Waist to hip ratio ≥0.95 in men and ≥ 0.80 in women §§ Total cholesterol≥240mg/dl ║║ LDL-cholesterol ≥130mg/dl ¶¶ HDL-cholesterol <40 mg/dl in men and <50 mg/dl in women *** Hypertriglyceridemia: Triglyceride ≥150 mg/dl ††† High total cholesterol/HDL-cholesterol ≥5 mg/dl

24 Table 3: Summary

What is known about this topic

 The impact of cardiovascular disease (CVD) risk factors varies in different populations.

What this study adds

 “Isfahan Cohort Study” is the first long term cohort study that has been conducted in

urban and rural areas of not only Iran, but also in the Eastern Mediterranean region,

aimed at assessing the relative risk of each or combination of CVD risk factors among

men and women aged ≥35 years.

25 Figure 1- Map of Iran and locations of 3 areas of study (Isfahan, Najafabad and Arak) and Tehran (Iran’s capital)

26 Figure 2- Algorithm for the Isfahan Cohort Study

27 Figure 3- Age and sex adjusted hazard ratios for risk factors combinations*

HTN: Hypertension DM: Diabetes Mellitus HCh: Hypercholesterolemia H. TG: Hypertriglyceridemia