Sample

For more than 50 years, the National Health and Nutrition Examination Survey has been conducted by the Centers for Disease Control and Prevention to provide estimates of the health and nutritional status of non-institutionalized civilians living in the United States. For data collection, NHANES employs a complex, multistage, probability sampling design [18]. In stage 1, primary sampling units (PSUs) are selected. These are mostly single counties. For stage 2, PSUs are divided into segments, generally city blocks or their equivalent. In stage 3, households within each block are chosen randomly. Finally, in stage 4, participants are selected from a list of all individuals residing in selected households. Individuals are drawn at random within age-sex-race screening subdomains [18].

NHANES data containing telomere length measures are available for a 4-year period only, 1999–2002. These data became available to the public in November, 2014. NHANES 1999–2002 oversampled individuals 12–19 years, persons age 60 and older, African Americans, Mexican Americans, and low-income individuals to provide more accurate estimates of these groups. All of the data are cross-sectional [18]. The NHANES data sets used by the present study are available to the public online at no cost [19].

DNA samples were requested from all respondents of NHANES 1999–2000 and NHANES 2001–2002, ages 20 years and older. Of the 10,291 participants who were eligible to give DNA samples, 3567 adults from NHANES 1999–2000 and 4260 from NHANES 2001–2002 consented to have their DNA used for future research and provided a useable DNA sample (n = 7827, 76%). Because survival bias is a potential issue among the extremely old, and because individuals 85 years and older were all given the age of 85 by NHANES, participants 85 years old and older were excluded from the data set. Only participants with complete data, including values for telomere length, caffeine and coffee consumption, and the potential mediating variables, were included in the analyses, a total of 5826 participants, 2741 men and 3085 women. The National Center for Health Statistics Ethics Review Board at the Centers for Disease Control and Prevention approved collection of the NHANES data and posting of the NHANES public-use files, which were required for this investigation. All NHANES participants provided written informed consent.

Measures

Data were collected on 13 variables in the present investigation: leukocyte telomere length, caffeine intake, age, gender, race, education, marital status, housing, body mass index (BMI), smoking, physical activity, alcohol use, and coffee intake.

Telomere length

The procedure used to measure telomere length has been described in detail by NHANES [20] and elsewhere [21, 22]. Blood samples were collected as part of the NHANES survey and used for DNA analysis. According to NHANES [20], DNA was extracted from the samples and stored at -80°C at the Division of Health Sciences Laboratory, CDC. Specimens were then shipped to the laboratory of Dr. E. Blackburn (University of California, San Francisco) for analysis. Using the quantitative polymerase chain reaction method, leucocyte telomere length was measured and compared to standard reference DNA (T/S ratio). Five 96-well quality control plates, representing 5% of the complete set, were provided. The investigators were blinded regarding the duplicate samples. Each specimen was assayed 3 times on 3 different days. Each specimen was assayed using duplicate wells resulting in 6 values. The six measurements were used to determine the mean and standard deviation of the T/S ratio. Plates containing the samples were assayed in groups of 3, with no 2 plates grouped together more than once. Control DNA measures were employed to normalize between-run variability. If more than 5% of the duplicate samples on the quality control plates were discordant with their pair in the complete set, the variant failed the quality control. For each sample, potential outliers were flagged and excluded from calculations (<2% of samples). The inter-assay coefficient of variation was 6.5% [20]. Mean T/S ratio values were converted to base pairs using the formula: 3274 + 2413 × (T/S). It is important to note that base pair estimates are only comparable for T/S ratio data produced using the same reference standard and the same lab procedures [20].

Caffeine and coffee consumption

According to NHANES [23], dietary intake data were collected via a 24-h dietary recall using a computer-assisted dietary interview system, which was administered by an NHANES interviewer. Many studies have used the NHANES 24-h dietary recall system [24–26]. Interviewers were bilingual, trained, and each had a college degree in Food and Nutrition or Home Economics, with at least 10 credits in food and nutrition. Interviews were conducted in a private setting in an NHANES Mobile Examination Center [23].

The dietary assessment was used to collect detailed information about all foods and beverages consumed, including a complete description of each food and the amount consumed. Nutrients and non-nutrient food components, including caffeine intake and coffee consumption, were calculated from foods and beverages that were consumed during the 24-h period prior to the interview (midnight to midnight). Coffee consumption included all forms of coffee. The interview used a multi-pass format. Food probes that were used in previous NHANES and USDA surveys were part of the built-in features of the system. The computer-assisted system provided a standardized interview format. Interviewers followed scripts provided in the system to explain the dietary interview component to the participant [27].

Covariates

NHANES used five categories to differentiate among races and ethnicities: Non-hispanic White, Non-hispanic Black, Mexican American, Other race or Multi-racial (Other), and Other Hispanic. Education level was defined using three categories: Less than high school, high school diploma (including GED), and more than high school. Marital status was defined by seven categories: married, widowed, divorced, separated, never married, living with partner, or other. Housing arrangements, a measure of social economic status, was defined using three categories: home owned or being bought, home being rented, and other.

Several lifestyle factors were measured in the present study to serve as potential mediating variables. Participation in leisure time physical activity was quantified using met-minutes of activity per week during the past 30 days. Specifically, participants were asked to report from a list, which of 62 physical activities they participated in regularly, whether the activity was moderate or vigorous, the number of times in the past 30 days they engaged in the activity, and the average duration of the activity. Durations of less than 10-min were not counted. A MET score was produced for each activity and total MET-minutes per week were calculated by NHANES for each participant using the compendium of physical activities [28]. Although a majority of the sample was sedentary, non-sedentary participants were divided into sex-specific tertiles, resulting in four categories of physical activity: sedentary, low, moderate, and high.

Smoking was indexed using a measure of pack-years, representing cumulative exposure to tobacco smoke. Pack years were calculated as the mean number of cigarettes smoked per day times the number of years smoked, divided by 20.

Body mass index (BMI) represents participants’ weight in kilograms divided by the square of their height in meters. BMI (kg/m2) allows the comparison of body weights independent of height. BMI categories were used to differentiate among participants who were underweight (<18.5), normal weight (≥18.5 and < 25.0), overweight (≥25.0 and < 30.0), obese (≥30.0) or missing.

Three categories were used to define alcohol use: abstainers, moderate drinkers, and heavy drinkers. Abstainers were those reporting no alcohol use in the past 12 months. Moderate drinkers were defined as women who reported drinking more than 0 but less than 2 drinks per day over the past 12 months, or men who reported drinking more than 0 and less than 3 alcoholic beverages per day during the past 12 months. Heavy drinkers were women who reported drinking 2 or more alcoholic beverages per day over the past 12 months or men who reported drinking 3 or more alcoholic drinks per day over the past 12 months.

Statistical analysis

NHANES assigns an individual-level sample weight to each participant [29]. It is a measure of the number of people in the population represented by that sample-person in NHANES, reflecting the unequal probability of selection, nonresponse adjustment, and adjustment to independent population controls. When unequal selection probability is applied, the sample weights are used to produce an unbiased national estimate. For the present study, sample weights were based on 4 years of diet data, which included the caffeine and coffee consumption variables.

In the present study, frequencies for categorical variables, and means (±SE) for continuous variables, were reported to describe the data. Each descriptive value included adjustments based on the complex sampling design of NHANES by incorporating strata, primary sampling unit (PSU) indicators, and sample weights [29]. The SAS SurveyMeans procedure was employed to generate weighted means that represent values for the U.S. population, and SAS SurveyFreq was used to calculate weighted frequencies, which are also generalizable to the U.S. adult population.

For the current study, total caffeine consumption (mg per day) was the primary exposure variable. Coffee consumption (g per day) was also evaluated as a secondary exposure variable. Coffee intake was measured in grams by NHANES. To convert to cups, grams of coffee can be divided by 225.

The extent of the linear associations between caffeine intake and telomere length and coffee consumption and telomere length were measured using regression analysis and the SAS SurveyReg procedure. Regression estimates for each model were based on the complex, multistage, probability sampling process of NHANES. To test the extent to which the relationships between the exposure variables and telomere length were mediated by differences in age, race, marital status, education, housing, smoking, alcohol use, body mass index, physical activity, and coffee consumption (or caffeine intake), these factors were controlled statistically using partial correlation and the SAS SurveyReg procedure. Throughout the paper, statements that adjustments were made for “the covariates” indicates that all these covariates were controlled simultaneously using partial correlation.

Separate models were used to determine the magnitude of the linear relationship between caffeine intake and telomere length among all participants, coffee drinkers only, and those reporting no coffee intake. When treated as a categorical variable, coffee intake was divided into four groups. Quartiles could not be used to divide coffee intake because 48% of the subjects reported no coffee consumption. Beyond those reporting no coffee intake, coffee drinkers were divided into tertiles, resulting in four categories: 1) no coffee intake, 2) 1–355 g, 3) 356–639 g, 4) 640 or more grams.

To study the relationships between telomere length and the covariates, including coffee intake, mean differences in telomere length were compared across each level of the covariates. Additionally, to afford an additional perspective of the caffeine intake and telomere length relationship, mean differences in telomere base pairs were tested across four categories of caffeine consumption based on 150 mg increments: 0 mg, 1–149 mg, 150–299 mg, 300–449 mg, and 450 mg or more per day. Mean telomere differences across the caffeine categories were studied within three separate samples: all participants, coffee drinkers only, and those reporting no coffee intake.

All P-values were two-sided and statistical significance was accepted when alpha was < 0.05. Statistical analyses were performed using SAS Version 9.4 (SAS Institute, Inc., Cary, NC).