British Medical Journal 2014 (Dec 2); 349: g6674 ~ FULL TEXT
Marta Crous-Bou, Teresa T Fung, Jennifer Prescott, Bettina Julin, Mengmeng Du, Qi Sun,
Kathryn M Rexrode, Frank B Hu, Immaculata De Vivo
Channing Division of Network Medicine,
Department of Medicine, Brigham and Women's Hospital
and Harvard Medical School, Boston, MA 02115, USA
Department of Epidemiology, Program in Genetic Epidemiology
and Statistical Genetics,
Harvard School of Public Health,
Boston, MA 02115, USA.
OBJECTIVE: To examine whether adherence to the Mediterranean diet was associated with longer telomere length, a biomarker of aging.
DESIGN: Population based cohort study.
SETTING: Nurses' Health Study, an ongoing prospective cohort study of 121,700 nurses enrolled in 1976; in 1989-90 a subset of 32,825 women provided blood samples.
PARTICIPANTS: 4676 disease-free women from nested case-control studies within the Nurses' Health Study with telomere length measured who also completed food frequency questionnaires.
MAIN OUTCOME MEASURE: Association between relative telomere lengths in peripheral blood leukocytes measured by quantitative real time polymerase chain reaction and Alternate Mediterranean Diet score calculated from self reported dietary data.
RESULTS: Greater adherence to the Mediterranean diet was associated with longer telomeres after adjustment for potential confounders. Least squares mean telomere length z scores were -0.038 (SE 0.035) for the lowest Mediterranean diet score groups and 0.072 (0.030) for the highest group (P for trend = 0.004).
CONCLUSION: In this large study, greater adherence to the Mediterranean diet was associated with longer telomeres. These results further support the benefits of adherence to the Mediterranean diet for promoting health and longevity.
From the FULL TEXT Article:
The traditional Mediterranean diet is characterized by a high intake of vegetables, fruits, nuts, legumes, and grains (mainly unrefined); a high intake of olive oil but a low intake of saturated lipids; a moderately high intake of fish; a low intake of dairy products, meat, and poultry; and a regular but moderate intake of alcohol (specifically wine with meals).  Observational studies and intervention trials have consistently shown the health benefits of a high degree of adherence to the Mediterranean diet, including reduction of overall mortality [2-4]; reduced incidence of chronic diseases, especially major cardiovascular diseases ; and increased likelihood of healthy aging. 
Telomeres are repetitive DNA sequences at the ends of eukaryotic chromosomes that undergo attrition each time a somatic cell divides.  Telomeres prevent the loss of genomic DNA at the ends of linear chromosomes and in turn protect their physical integrity. [8, 9] Telomere attrition has been shown to be accelerated by oxidative stress and inflammation. [10, 11] Telomere length is considered to be a biomarker of aging; shorter telomeres are associated with a decreased life expectancy and increased rates of developing age related chronic diseases. [12-14] Telomere length decreases with age and varies considerably among individuals.  Studies suggest that telomere attrition is modifiable, as substantial variability exists in the rate of telomere shortening that is independent of chronological age. [16-18] Therefore, variability of telomere length may be partially explained by lifestyle practices, including dietary patterns.  As accelerated telomere attrition may underlie many chronic diseases, identifying modifiable factors that affect telomere dynamics is important.
Given that fruits, vegetables, and nuts, key components of the Mediterranean diet, have well known antioxidant and anti-inflammatory effects, and that telomere length is affected by both of these processes, we hypothesized that greater adherence to the Mediterranean diet would be associated with longer telomere length. Therefore, the main objective of this study was to examine the association between greater adherence to the Mediterranean diet and leukocyte telomere length in US women within the Nurses’ Health Study cohort. For comparison, we also evaluated the association between other existing dietary patters (prudent pattern, Western pattern, and Alternative Healthy Eating Index) and leukocyte telomere length.
The Nurses’ Health Study is a prospective cohort study of 121,700 female registered nurses in 11 US states, aged 30-55 years at enrollment. The study was established in 1976; since then, participants have completed biennial questionnaires to update information on demographic characteristics, lifestyle factors, and newly diagnosed diseases. [20, 21] Between 1989 and 1990 32,825 cohort participants provided blood samples. Details of the blood collection and archival methods have been described previously. 
Multiple nested case-control studies have been conducted within the Nurses’ Health Study blood sub-cohort to investigate the association between leukocyte telomere length and cancer, cardiovascular disease, and cognitive function, among others. [23-28] The analysis reported here included data on 4,676 women selected as healthy controls (free of major chronic diseases, including cancer and cardiovascular disease) from nested case-control studies, with previously measured leukocyte telomere length and completed food frequency questionnaires at the time of blood draw.
In 1980 participants completed a 61 item semi-quantitative food frequency questionnaire to assess their diet in the previous year. In 1984, 1986, and every four years thereafter, an expanded food frequency questionnaires containing 116-130 food items was sent to the nurses to update their dietary information. Participants were asked about the frequency of consumption of each food item of a pre-specified standard portion size in the previous year. The validity of these questionnaires has been previously described.  We calculated all dietary patterns and diet quality scores from self reported dietary data on the 1990 food frequency questionnaire, the closest to the blood draw (1989-90).
Trichopoulou adapted the Alternate Mediterranean Diet score from the Mediterranean Diet Score to assess adherence to the traditional Mediterranean diet.  The Alternate Mediterranean Diet score includes the following nine components: vegetables (excluding potatoes), fruits, nuts, whole grains, legumes, fish, monounsaturated:saturated fatty acid ratio, red and processed meats, and moderate alcohol intake. The possible score range is 0-9, with a higher score representing a closer resemblance to the Mediterranean diet. We dichotomized each of the nine dietary components at the median. We gave participants with intake above the median 1 point each for vegetables, fruits, nuts, whole grains, legumes, fish, and monounsaturated:saturated fatty acid ratio; we scored intake equal to or below the median as 0 points for each component. We scored red and processed meat consumption below the median as 1 point, with 0 points for intake equal to or above the median. Participants received 1 point for alcohol intake between 5 and 15 g/day; otherwise, they got 0 points. Details of the Alternate Mediterranean Diet scoring system are described elsewhere. 
We used principal component analysis of the food frequency questionnaires to identify prudent and Western dietary patterns. [30, 31] The prudent dietary pattern is characterized by high intakes of fruits, vegetables, legumes, fish and other seafood, poultry, and whole grains; the Western dietary pattern includes high intakes of red and processed meats, butter, high fat dairy products, eggs, sweets and desserts, French fries, and refined grains. Details of the reproducibility and validity of dietary patterns assessed with a food frequency questionnaire have been previously described. [30, 31]
The Alternative Healthy Eating Index measures adherence to a dietary pattern based on foods and nutrients most predictive of risk for chronic disease in the literature.  The Alternative Healthy Eating Index ranges from 0 to 110 points. Each of the 11 components of the score (vegetables, fruits, whole grains, sugar sweetened drinks, nuts and legumes, red and processed meat, trans fat, long chain (n-3) fats, polyunsaturated fatty acids, sodium, and alcohol) has a minimum score of 0 (worst) and a maximum score of 10 (best), according to component specific criteria reflecting either the current dietary guidelines or associations reported in the literature. Details of component selection and Alternative Healthy Eating Index calculation have been previously described. 
In addition to age at blood draw, we collected information on factors potentially associated with Mediterranean diet and telomere length. Using the biennial questionnaires completed proximal to blood collection and a supplemental questionnaire administered at blood collection, we assessed various anthropometric, reproductive, and lifestyle factors, as well as factors that have been related to telomere length in the Nurses’ Health Study elsewhere: body mass index (kg/m2), cigarette smoking (pack years), [33-35] physical activity (total metabolic equivalent hours of activity per week).  We calculated dietary factors, including energy intake (total calories), from the 1990 food frequency questionnaire.
Telomere length measurement
Genomic DNA was extracted from peripheral blood leukocytes using the QIAmp 96-spin blood protocol (Qiagen, Chatsworth, CA, USA). Pico-Green quantification of genomic DNA was performed using a Molecular Devices 96 well spectrophotometer (Sunnyvale, CA, USA).
Relative telomere length was determined using a modified, high throughput version of the quantitative real time polymerase chain reaction based telomere assay. [36, 37] The quantitative real time polymerase chain reaction telomere assay was run on the Applied Biosystems 7900HT Sequence Detection System (Foster City, CA, USA). Laboratory personnel were blinded to participants’ characteristics, and all assays were processed in triplicate by the same technician and under identical conditions.
The average relative leukocyte telomere length was calculated as the ratio of telomere repeat copy number to a single gene (36B4) copy number. Leukocyte telomere length is reported as the exponentiated ratio of telomere repeat copy number to a single gene copy number corrected for a reference sample. In all nested case-control studies, the telomere and single gene assay coefficients of variation for triplicates were less than 4%. Coefficients of variation for the exponential ratio of telomere repeat copy number to a single gene copy number were under 18%. In the Nurses’ Health Study, the three year and 10 year interclass correlations for reliability were determined to be 0.80 and 0.60. Although this assay provides a relative measurement of telomere length, ratios of telomere repeat copy number to a single gene copy number highly correlate with absolute telomere lengths determined by Southern blot (r=0.677; P<0.001). 
Telomere length was assayed in various batches corresponding to each study. To minimize the impact of potential batch effect on leukocyte telomere length measurements across different studies, we calculated z scores of log transformed leukocyte telomere length by standardizing the leukocyte telomere length in comparison with the mean within each individual study. [18, 19, 38]
We used Spearman’s partial rank correlation coefficients to examine age adjusted associations between characteristics of the study population, quarters of leukocyte telomere length, and Alternate Mediterranean Diet score groups.
We used multivariable linear regression models to determine the cross sectional association between leukocyte telomere length and adherence to the Mediterranean diet. We calculated multivariable adjusted least squares mean leukocyte telomere length z scores (and their corresponding 95% confidence intervals) across all dietary patterns score groups by using generalized linear models. Firstly, only age at blood draw was included in the models. Models were then additionally adjusted for other potential confounders (body mass index, smoking, physical activity, energy intake, and batch). Finally, other variables were included as potential covariates (for example, postmenopausal hormone therapy, history of hypertension, and socioeconomic status), but these were removed from the final models as estimates remained the same. All P values are two sided, and an α level of 0.05 was used. We used SAS version 9.2 for all statistical analyses. Reported STROBE guidelines have been the basis for reporting our results. 
Table 1 shows the main age standardized characteristics of the study population by quarters of telomere length. The mean age of the participants was 59 (SD 6.6) years, and the exact age range of the participants included in our study was 42-70 years. As expected, a statistically significant inverse correlation existed between age at blood draw and telomere length: younger women had longer telomeres (P<0.001). Moreover, women with shorter telomere length smoked slightly more (P=0.02). Distributions of main lifestyle factors between the study participants (n=4,676), in comparison with the rest of the Nurses’ Health Study blood sub-cohort (n=32,825) after age standardization, were largely similar (results not shown).
Age standardized characteristics of study population at blood draw, by quarters of leukocyte telomere length z score (n=4676). Values are mean (SD)
|Baseline characteristics||Quarters of leukocyte telomere length z score||P for trend|
|Age at blood draw*, years||59.6 (6.3)||59.2 (6.6)||58.6 (6.6)||57.7 (6.8)||<0.001|
|Body mass index at blood draw, kg/m2||25.5 (4.7)||25.2 (4.4)||25.0 (4.5)||25.3 (4.3)||0.13|
|Pack years†||23.9 (20.8)||22.8 (18.8)||23.5 (20.4)||22.9 (18.9)||0.024|
|Physical activity, metabolic equivalent hours of activity/week||16.4 (24.4)||16.7 (21.5)||16.4 (19.5)||16.6 (18.8)||0.87|
|Total energy, kcal||1790 (508)||1757 (485)||1743 (487)||1768 (494)||0.72|
|Vegetables, servings/day||3.1 (1.8)||3.1 (1.6)||3.1 (1.7)||3.2 (1.7)||0.51|
|Fruits, servings/day||2.4 (1.3)||2.4 (1.3)||2.4 (1.5)||2.5 (1.4)||0.81|
|Whole grains, servings/day||1.5 (1.3)||1.5 (1.2)||1.5 (1.2)||1.5 (1.3)||0.77|
|Fish, servings/day||0.4 (0.4)||0.3 (0.3)||0.3 (0.3)||0.4 (0.3)||0.68|
|Meats, servings/day||0.8 (0.5)||0.7 (0.5)||0.7 (0.5)||0.8 (0.5)||0.54|
|Legumes, servings/day||0.4 (0.3)||0.4 (0.3)||0.4 (0.3)||0.4 (0.4)||0.72|
|Nuts, servings/day||0.4 (0.6)||0.4 (0.5)||0.3 (0.5)||0.4 (0.6)||0.91|
|Alcohol, g/day||5.5 (9.8)||5.7 (9.9)||5.3 (10.1)||5.5 (9.2)||0.90|
|Total fat, g/day||62.5 (22.5)||61.4 (23.2)||60.3 (21.4)||61.4 (22.7)||0.76|
|Total saturated fat, g/day||21.0 (8.0)||20.7 (8.6)||20.3 (8.0)||20.5 (8.1)||0.95|
|Total monounsaturated fat, g/day||24.0 (9.2)||23.6 (9.4)||23.2 (8.7)||23.6 (9.3)||0.78|