Dietary fat intake and risk of coronary heart disease: the Strong Heart Study

¹ From the Center for American Indian Health Research, University of Oklahoma Health Sciences Center, Oklahoma City, OK (JX and ETL); Medstar Research Institute, Hyattsville, MD (SE-A, BVH, and CM); the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD (CL and RRF); the Indian Health Service, Aberdeen Area Office, Aberdeen, South Dakota (EMZ); and the Department of Epidemiology and Preventive Medicine, Sackler Medical Faculty, Tel Aviv University, Tel Aviv, Israel (UG)

² The opinions expressed here are those of the authors and do not necessarily reflect the views of the Indian Health Service.

³ Supported by cooperative agreement grants (no. U01HL-41642, U01HL-41652, and U01HL-41654) from the National Heart, Lung, and Blood Institute.

⁴ Address reprint requests to J Xu, Center for American Indian Health Research, University of Oklahoma Health Sciences Center, College of Public Health, 801 NE 13th Street, Room 112, Oklahoma City, OK 73190. E-mail: susan-xu{at}ouhsc.edu.

ABSTRACT  
Background: The results of previous studies on the association between dietary fat intake and coronary heart disease (CHD) incidence are inconsistent.

Objective: The aim of this study was to examine the association between dietary fat intake and CHD incidence in American Indians in the Strong Heart Study.

Design: A total of 2938 participants aged 47–79 y and free of CHD at the second examination (1993–1995) were examined and followed for CHD, nonfatal CHD, and fatal CHD events to 31 December 2002. Dietary intake was assessed by using a 24-h diet recall and was calculated as percentages of energy.

Results: Participants were followed for a mean (±SD) of 7.2 ± 2.3 y. During follow-up, 436 incident CHD cases (298 nonfatal CHD and 138 fatal CHD events) were ascertained. Participants aged 47–59 y in the highest quartile of intake of total fat, saturated fatty acids, or monounsaturated fatty acids had higher CHD mortality than did those in the lowest quartile [hazard ratio (95% CI): 3.57 (1.21, 10.49), 5.17 (1.64, 16.36), and 3.43 (1.17, 10.04), respectively] after confounders were controlled for. These associations were not observed for those aged 60–79 y.

Conclusions: Total fat, saturated fatty acid, and monounsaturated fatty acid intake were strong predictors of CHD mortality in American Indians aged 47–59 y, independent of other established CHD risk factors. It may be prudent for American Indians to reduce their fat intake early in life to reduce the risk of dying from CHD.

Key Words: Dietary fat intake • fatty acids • cholesterol • coronary heart disease • mortality • American Indians

INTRODUCTION  
The classic diet-heart hypothesis posits that diets high in saturated fatty acids (SFAs) and cholesterol and low in polyunsaturated fatty acids (PUFAs) raise serum total and LDL cholesterol, which in turn increase the risk of coronary heart disease (CHD) (1). Despite decades of intensive interest and research in the diet-heart hypothesis, however, investigations of dietary fat and CHD incidence from prospective epidemiologic studies have resulted in inconsistent findings. Some studies found a significantly positive association between total fat intake and CHD incidence (2-4) and CHD death (5, 6); other studies could not demonstrate this association (7-10). SFA intake was found to be positively associated with CHD incidence (4, 11) and CHD death (5, 6, 12, 13), whereas other studies found no association between SFAs and CHD death (7, 9, 10, 14). Monounsaturated fatty acid (MUFA) intake was inversely associated with CHD incidence in 2 studies (11, 15), whereas other studies reported a positive association between MUFA intake and CHD incidence (3, 4) and CHD death (5). An inverse association between PUFA intake and CHD incidence (10, 15) and CHD death (13, 14) was found in some studies, but other studies found no association (2-5, 7-9, 16). A positive association between trans fatty acids (TFAs) and CHD incidence was seen in some studies (9, 10, 15, 17). Age-related differences in the association between dietary fat and CHD incidence was shown in some studies (3-5, 10), which reported that CHD risk differed among age groups. The inconsistency of the findings from prospective epidemiologic studies may be partly explained by small sample sizes; use of different diet assessment methods; incomplete adjustment for potential confounders, such as energy intake and other types of fat (13, 15); and effect dilution due to high intraindividual variability (18). To the best of our knowledge, no previous analysis has been conducted of the association between dietary fat intake and CHD incidence or CHD death among American Indians.

The Strong Heart Study (SHS), a longitudinal study of cardiovascular disease and its risk factors in American Indian men and women (19), has shown that CHD incidence is now higher in this population than in other US populations (20), which suggests that this population's lifestyle, including diet, has undergone profound changes. The objective of the current analysis was to examine the association between dietary fat intake and CHD risk in this population.

SUBJECTS AND METHODS  
Subjects
The SHS is a population-based study of American Indians who reside in central Arizona, Oklahoma, and North and South Dakota. The SHS cohort consisted of 4549 American Indians aged 45–74 y at the baseline examination (1989 –1991). We used data from 3638 participants, who returned to the second SHS examination in 1993–1995 as the basis for our analysis. Dietary data were collected from all participants at the second examination at the Strong Heart clinics by means of a single 24-h dietary recall; 3450 participants completed the second examination and had complete nutrition data. After excluding adults who had definite myocardial infarction (MI) or CHD (definite CHD, definite MI, or definite electrocardiogram-evident MI) before or at the second examination (n = 233), whose total reported energy intake was 600 kcal/d or >8000 kcal/d for men or 6000 kcal/d for women (n = 124), who were under dialysis treatment, or had a kidney transplant or liver cirrhosis (n = 154), and who were older than 79 y (n = 1), the final subsample consisted of 2938 participants aged 47–79 y.

To compare the nutrient intakes of the SHS participants with those of the general US population as obtained in the third National Health and Nutrition Examination Survey (NHANES III) 1988–1994 (21), which occurred at about the same time as the SHS, a subsample of 6687 persons aged 47–79 y was obtained from the NHANES III data set (22). As in the SHS, total energy intake was estimated in NHANES III by use of a single 24-h dietary recall. We excluded those who reported having angina or a history of MI (n = 1099), as described previously (23), and those whose total reported energy intake was missing or 600 kcal/d or >8000 kcal/d for men or 6000 kcal/d for women (n = 494). Because the SHS participants' blood samples were drawn in the morning, we excluded those in NHANES III who were assigned to an afternoon or evening session, and plasma glucose values were obtained after an overnight fast of <9 h (n = 2750). The final subsample included 2344 participants from NHANES III.

Data collection
The second examination in the SHS included demographic data, personal medical history, health habits, family history of cardiovascular disease and diabetes, blood pressure, body mass index (BMI), and the collection of fasting blood samples for measurements of lipids and lipoproteins. Details about the data collection and laboratory procedures are published elsewhere (19, 20). Diabetes was defined according to the American Diabetes Association criteria (24), ie, as taking insulin or oral antidiabetic medication or having a fasting glucose concentration 126 mg/dL. Cigarette smoking and alcohol consumption were determined by questionnaire.

The second examination also included a 24-h dietary recall for all participants. The interviews were conducted by local field staff who were trained and supervised by Indian Health Service dietitians and the principal investigator according to standardized methods (25). Detailed information about staff training, project supervision, and quality assurance was previously reported (26). Dietary intake data were collected and analyzed by using the Minnesota Nutrition Data System (NDS version 2.1) developed by the Nutrition Coordinating Center (NCC), University of Minnesota (Minneapolis, MN; Food Database version 4A; Nutrient Database version 18; 27, 28). TFA were not available in NDS version 2.1; therefore, to include them in the nutrient data, final calculations were completed by using NCC Nutrient Database Version 36 (NDS-R 2005). The NDS-R time-related database updates analytic data while retaining nutrient profiles true to the version used for data collection (29).

The Indian Health Service, participating institutional review boards, and the participating tribes approved the SHS. Informed consent was obtained from all participants.

Ascertainment of endpoints
The primary endpoints for this study were CHD, nonfatal CHD, and fatal CHD events. CHD events comprised the first nonfatal CHD or fatal CHD event occurring after the second examination but before 31 December 2002. Nonfatal CHD events included definite MI, definite CHD, and electrocardiogram-evident definite MI events. Fatal CHD for the purpose of this study was defined as death from definite fatal MI, definite sudden death due to CHD, definite fatal CHD, and possible fatal CHD event. Detailed definitions of the nonfatal and fatal events were described previously (30, 31). CHD events that occurred during the follow-up period were ascertained from the annual mortality and morbidity surveillance or at the third examination (1998–1999). Medical records were abstracted, and fatal CHD events were ascertained and confirmed by mortality and morbidity review committees using specific criteria (32). Records of those who did not participate in the third examination (n = 525) were also reviewed. Follow-up for mortality was 99.8% complete.

Statistical analysis
Dietary intake data for all SHS analyses were based on a data set obtained from NCC Nutrient Database Version 36. To compare dietary intake with that reported in NHANES III, the dietary intake data for the SHS were obtained from NCC Nutrient Database version 18, which did not include TFA information. Data are presented as means ± SDs for the SHS by CHD category and ages: 47–59 y and 60–79 y at the second examination. Previous studies showed an age-related difference in the association between fat intake and CHD incidence (3-5, 10). Means and SEMs are presented for the NHANES III data and were obtained by using the Proc DESCRIPT procedure in SAS-Callable SUDAAN (version 9.0; Research Triangle Institute, Research Triangle Park, NC), which is specifically designed for the analysis of complex survey data. Means or proportions of CHD risk factors and mean intakes of nutrients were compared by age or CHD category by using t tests or Wilcoxon's rank-sum tests or chi-square tests whenever appropriate for the SHS, and mean intakes of nutrients were compared by age by using the DIFFVAR statement in the Proc DESCRIPT procedure for NHANES III, in which the sampling weights were provided by the National Center for Health Statistics. Mean nutrient intakes in the SHS and NHANES III for the same age group were compared by t tests.

Time to CHD event or death or censoring for each participant was calculated from the second examination until the first nonfatal CHD, fatal CHD, or 31 December 2002. Dietary fat intakes were calculated as a percent of energy and divided into quartiles. The Cox proportional hazards model (33) was used to study associations between quartiles of dietary fat intake and CHD, either nonfatal or fatal CHD event over time separately. The effect of isocaloric substitution of each dietary fat for carbohydrate was evaluated by multivariate nutrient-density models that simultaneously included energy intake and percent of energy from protein (15, 34) and other confounders, including age, sex, study center (Arizona, Oklahoma, North and South Dakota), diabetes status (diabetes versus nondiabetes), BMI, HDL cholesterol, LDL cholesterol, total triacylglycerol, smoking status (current versus past and never), alcohol consumption (current versus past and never), and hypertension. Triacylglycerol was log-transformed to stabilize the variance. Interactions were examined in the multivariate-adjusted models for quartiles of dietary fat intake with age group, sex, or diabetes, as well as interaction for age group and all other confounders separately. Tests for trend were conducted by modeling the median of each quartile-defined category as a continuous variable in Cox proportional hazards models. The proportionality assumption of the Cox model was assessed by generating the time-dependent covariate by creating interaction of each covariate and a function of survival time and including it in the model. There was no evidence for the violation of this assumption for any covariates. We also repeated the analyses by modeling dietary fat intakes as continuous variables. All analyses were performed with SAS version 9.00 (SAS Institute Inc, Cary, NC). All P values were 2-tailed, and statistical significance was defined as P < 0.05 for all tests.

RESULTS  
Participants were followed for an average (±SD) of 7.2 ± 2.3 y. During follow-up (21 101 person-years), 436 CHD events were ascertained (298 first nonfatal CHD events and 138 fatal CHD events). CHD risk factors at baseline are presented by follow-up CHD category (yes or no) and baseline age group (47–59 and 60–79 y) in Table 1. Within each age group, those who developed CHD were more likely to be male, diabetic, and hypertensive and to have lower HDL cholesterol and higher triacylglycerol concentrations than those who did not develop CHD. Participants aged 47–59 y who developed CHD were also older and more frequently smokers than were those who did not develop CHD. Participants aged 60–79 y who developed CHD also had higher LDL cholesterol than did those who did not develop CHD. Comparing those who developed CHD by age groups, participants aged 47–59 y had higher BMI and triacylglycerol and lower HDL and were more likely to be current smokers and drinkers, but were less likely to be hypertensive, than were those aged 60–79 y. When comparing those who did not develop CHD by age group, participants aged 47–59 y were more likely to be male and current smokers and drinkers, were less likely to be hypertensive, and had higher BMIs than did those aged 60–79 y.

View this table:
TABLE 1. Coronary heart disease (CHD) risk factors at baseline of 2938 American Indian men and women included in the Strong Heart Study¹

 
The mean percentages of energy from dietary fat, SFAs, MUFAs, PUFAs, and TFAs were 35.1%, 11.9%, 13.4%, 6.5%, and 2.4%, respectively. Specific types of fat intake were positively correlated with one another, with the highest correlation between SFAs and MUFAs (r = 0.77, P < 0.0001). Associations between TFAs and MUFAs (r = 0.46, P < 0.0001) were also high, but those between TFAs and SFAs (r = 0.25, P < 0.0001) and between TFAs and PUFAs (r = 0.13, P < 0.0001) were lower. Also summarized in Table 1 are dietary intake differences by CHD category and age group. There was no significant univariate difference in dietary intake between those who developed CHD and those who did not during follow-up in either age group. As expected, participants aged 47–59 y consumed significantly more calories and a higher percentage of calories from fat (total, SFA, MUFA, and PUFA) than did participants aged 60–79 y regardless of whether they developed CHD during follow-up. Among those who developed CHD, participants aged 47–59 y consumed significantly more cholesterol than did those aged 60–79 y. There was no significant difference in TFA intake as a percent of energy between the 2 age groups among either those who developed CHD or those who did not.

Mean dietary intakes by age group from the SHS and NHANES III are presented in Table 2. In both the SHS and NHANES III, dietary intakes followed a similar pattern in each age group. When comparing mean dietary intake between participants from the SHS and NHANES III, mean energy intake was lower, but cholesterol intake was higher, in the SHS participants than in the NHANES III participants in both age groups. Mean total fat intake, SFA intake, and MUFA intake (all % of energy) were also higher in the SHS participants, but PUFA intake was higher in the NHANES III participants, in both age groups. All differences were significant (P < 0.01).

View this table:
TABLE 2. Macronutrient and cholesterol intakes of the Strong Heart Study (SHS) sample and the third National Health and Nutrition Examination Survey (NHANES III) sample by age¹

 
In multivariate analyses, there were no statistically significant interactions for CHD incidence between quartiles of dietary fat intake and sex, diabetes, or age group (P values for interactions varied from 0.11 to 0.98). Intake of total fat and its components were not associated with CHD incidence after the analysis was controlled for other confounders (Table 3).

View this table:
TABLE 3. Hazard ratio (HR) and 95% CI of each quartile of dietary fat intake associated with coronary heart disease (CHD) and nonfatal CHD events for all participants in the Strong Heart Study¹

 
Statistically significant interactions for CHD death were detected between quartiles of dietary fat intake and age group (Table 4). A higher intake of total fat, SFAs, and MUFAs was associated with higher CHD mortality among participants aged 47–59 y but not among those aged 60–79 y. Participants aged 47–59 y in the highest quartile of total fat, SFAs, and MUFAs had adjusted hazard ratios (HRs) of 3.57 (95% CI: 1.21, 10.49), 5.17 (95% CI: 1.64, 16.36), and 3.43 (95% CI: 1.17, 10.04), respectively. Omitting HDL cholesterol and LDL cholesterol from the model did not change the results. When evaluating the association between SFAs and MUFAs and CHD death separately, both SFAs and MUFAs remained significant predictors of CHD death among persons aged 47–59 y [HR for increasing quartiles of SFAs: 1.00, 3.31 (95% CI: 1.04, 10.55), 1.69 (95% CI: 0.43, 6.60), and 5.65 (95% CI: 1.71, 18.68), P for trend < 0.01; for MUFAs: 1.00, 1.32 (95% CI: 0.38, 4.58), 3.56 (95% CI: 1.07, 11.81), and 5.16 (95% CI: 1.45, 18.32), P for trend < 0.01] after adjustment for the above risk factors as well as PUFAs and TFAs as a percent of energy. In the similar model including all fat components simultaneously and adjusted for the above risk factors, SFAs and MUFAs did not predict CHD death independently of each other in this younger age group [HRs for increasing quartiles of SFAs: 1.00, 2.52 (95% CI: 0.66, 9.65), 0.98 (95% CI: 0.20, 4.87), and 2.98 (95% CI: 0.66, 13.58), P for trend = 0.19; for MUFAs: 1.00, 0.94 (95% CI: 0.24, 3.67), 2.52 (95% CI: 0.60, 10.64), and 2.88 (95% CI: 0.59, 14.21), P for trend = 0.13]. PUFAs, TFAs, and cholesterol intake were not associated with CHD death (Table 4). There were no statistically significant interactions for CHD death between quartiles of dietary fat intake and sex or diabetes. In addition, there were no statistically significant interactions for risk of CHD, either nonfatal or fatal, between age group and all other confounders.

View this table:
TABLE 4. Hazard ratio (HR) and 95% CI of each quartile of dietary fat intake associated with coronary heart disease (CHD) death by age group in the Strong Heart Study¹

 
Shown in Table 5 are the associations of CHD mortality and dietary fat intake modeled as a continuous variable (% of energy), adjusted for the risk factors listed above. As with quartiles of dietary intake, total fat, SFAs, and MUFAs were significantly associated with CHD death in participants aged 47–59 y. Among these same persons, SFAs and MUFAs, in separate analyses, remained significant predictors of CHD death independent of PUFAs and TFAs and the other CHD risk factors listed above [HR for a 5% increase in energy from SFAs: 0.66 (95% CI: 1.15, 2.42); from MUFAs: 1.68 (95% CI: 1.11, 2.53)]. In analyses modeling all fat components simultaneously, the associations between SFAs and MUFAs and CHD death were attenuated, and SFAs and MUFAs were not independently predictive of CHD death among those aged 47–59 y [HR for a 5% increase in energy from SFAs: 1.45 (95% CI: 0.84, 2.51); from MUFAs: 1.25 (95% CI: 0.68, 2.29)]. PUFAs and TFAs were not associated with CHD death in this younger age group. No association was found between dietary fat and its components and CHD death among participants aged 60–79 y. To test the robustness of our models, we repeated our analyses after excluding individuals (n = 27) whose first event occurred within the first 6 mo of follow-up, and the results did not change.

View this table:
TABLE 5. Hazard ratio (95% CI) associated with coronary heart disease death per macronutrient intake (5% increment in percentage of energy as a continuous variable) in the Strong Heart Study¹

 

DISCUSSION  
In this large longitudinal study, higher intakes of total fat, SFAs, and MUFAs at baseline were associated with higher CHD mortality among American Indians aged 47–59 y but not among those aged 60–79 y. There was no association between total fat intake or its components and nonfatal CHD events in this population. Additionally, we found no association between PUFAs, TFAs, and cholesterol intake and total CHD incidence (fatal or nonfatal event). Our findings were consistent regardless of how we categorized macronutrient intake, either as quartiles or as continuous intake. American Indians aged 47–59 y who were in the highest quartile of intake from total fat (>42.6% of energy), SFAs (>14.6% of energy), or MUFAs (>16.6% of energy) had a higher risk of dying from CHD than did those in the lowest quartile. Substituting 5% of energy from either MUFAs or SFAs with the same amount of energy from carbohydrate was associated with 62–68% higher CHD mortality. However, the relations of CHD death to SFAs and MUFAs were not independent of each other.

The inconsistencies in epidemiologic studies of the association between dietary fat and CHD incidence are summarized in Table 6. Our findings of positive associations between total fat, SFAs, and MUFAs and CHD death are consistent with some studies (5, 6, 12). Although some animal studies suggest that MUFAs may induce atherosclerosis (35), it is also very likely that our finding for MUFAs is confounded with SFA intake. In our study population, the main sources of MUFAs were meat, poultry, and fish (46%) and these same food groups provided almost the same contributions of SFAs (45%). These mutual food sources for both fatty acids may explain the high correlation between SFAs and MUFAs and the lack of independent associations of them with CHD death. For most previous studies that found MUFAs to be associated with lower CHD death, the main MUFA source was olive oil (1), whereas in our study, olive oil and olives contributed only 0.3% of MUFA intake. This suggests that in other studies, the beneficial effects may have been related to other components in the olive oil rather than MUFAs, and that the source of MUFAs may be important in determining CHD risk. The higher intake of total fat, SFAs, and MUFAs and lower intake of PUFAs in the SHS than in the US population suggests a less healthy eating pattern among American Indians that contributed to higher CHD death.

View this table:
TABLE 6. Major prospective studies of dietary fat intake and risk of coronary heart disease (CHD)¹

 
Although several studies have reported a relation between TFA intake and CHD incidence (9, 10, 15, 17), there was no association between TFA intake and incident CHD, either nonfatal or fatal, in our study. The mean TFA intake in our study was similar to that calculated by using a 24-h recall and a 2-d food record in the Continuing Survey of Food Intakes by Individuals (36). Another study in middle-aged men and women reported lower percentages of TFA intake: in UK men and women, 1.6% and 1.3% of energy, respectively, and in US men and women, 2.0% and 1.9% of energy, respectively (37). The lack of association in our study merits further investigation, including the examination of the contributions of naturally occurring versus hydrogenated vegetable oil sources on CHD incidence, because TFAs from the 2 sources might be differentially associated with risk factors or progression of heart disease (38, 39).

Epidemiologic studies on age-related differences in the association between dietary fat intake and CHD incidence are sparse. As far as we are aware, only 4 prospective studies examined differences by age (3-5, 10). Our findings are consistent with their findings that CHD death increases with higher intakes of total fat, SFAs, and MUFAs among participants aged 47–59 y but not among participants aged 60–79 y. This may arise from differences in baseline risk, because the magnitude of baseline risk determines the magnitude of the relative effect (40). Another possibility is that participants aged 60–79 y comprise a selected group given that many older cohort members were excluded because of existing CHD. In addition, older cohort members who did not have preexisting CHD may have been more likely than younger cohort members to have one or more CHD risk factors, such as diabetes, and may have changed their diets as a consequence.

Our positive findings for CHD death and lack of findings for CHD incidence are supported by data from the -Tocopherol, ß-Carotene Cancer Prevention Study (13), which showed that TFA intake was associated with CHD death but not with CHD incidence. This may be due to the difficulty in collecting complete event data, because participants may not report it and hospital records could not be located, which may lead to a possible misclassification and lower risk estimates. In contrast, death data are usually more complete.

Our results support the hypothesis that a high intake of dietary fat is associated with CHD death among middle-aged American Indian adults. The strengths of the present study include a large sample size and adjustment for energy intake and protein intake in addition to other established CHD risk factors. To the best of our knowledge, this is the first study to quantify the association between dietary fat intake and CHD incidence in American Indians.

The current study had several limitations: a single-day's diet is a poor descriptor of an individual's usual intake because of intraindividual variability. However, we found similar results when using intake as a continuous measure or categorized into quartiles, which assumes that bias was of a relatively constant magnitude. Multiple 24-h recalls or some other assessment method that measures usual intake would reduce random error and further increase the strength of the associations reported here (5, 41). In addition, we only included diet at the second SHS examination in our models, and diet may have changed during the follow-up period. Because this is an epidemiologic analysis, we cannot determine a causal association between fat intake and CHD incidence. It is possible that individuals at high CHD risk, for whatever reason, tend to consume more fat in their diet. Consumption of a diet lower in fat and SFAs might reflect high compliance to medications or lifestyle modification, which would improve general health in these persons. Thus, dietary intake alone may not be the sole factor underlying lower CHD incidence.

In conclusion, the results of this epidemiologic study suggest that CHD death, in middle-aged but not elderly American Indians, is related to both the quantity and the quality of dietary fats. A higher fat intake as a percent of energy from SFAs and MUFAs is associated with increased CHD mortality in this population subgroup. It may be prudent for American Indians to reduce their fat intake early in life to reduce the risk of dying from CHD.

ACKNOWLEDGMENTS  
We acknowledge the assistance and cooperation of the Ak-Chin Tohono O'Odham (Papago)/Pima, Gila River, and Salt River Pima/Maricopa communities in Arizona; the Apache, Caddo, Comanche, Deleware, Fort Sill Apache, Kiowa, and Wichita communities in Oklahoma; and the Oglala Sioux, Cheyenne River Sioux, and Spirit Lake communities in North and South Dakota, without whose support this study would not have been possible. We also thank the Indian Health Service hospitals and clinics at each center; the directors of the Strong Heart Study clinics, Betty Jarvis, Tauqeer Ali, Alan Crawford, and Marcia O'Leary, and their staffs; and the physicians who performed the mortality and morbidity reviews.

All authors were responsible for the critical revision of the manuscript for important intellectual content. Additionally, JX and SE-A were responsible for the hypothesis, the analysis concept, the data analysis and interpretation, and drafting of the manuscript. CL and UG assisted with the analysis and interpretation of data. BVH, RRF, EMZ, and ETL were responsible for the study design and data collection. None of the authors had a conflict of interest.

REFERENCES