Essential Nutrients, Added Sugar Intake, and Epigenetic Age in Midlife Black and White Women

This cross-sectional study uses data from the NHLBI Growth and Health Study and its follow-up to examine the association of dietary patterns, such as intakes of essential nutrients and added sugar, and scores of nutrient indices with epigenetic age among Black and White women at midlife in the US.


Introduction
Epigenetic clocks powerfully predict biological age independent of chronological age.These clocks reflect altered gene and protein expression patterns, particularly those resulting from differential DNA methylation (DNAm) at CpG (5′-C-phosphate-G-3′) sites.2][3] These alterations often result in pathogenic processes (eg, genomic instability, systemic inflammation, and oxidative stress) characteristic of aging and chronic disease. 1,4,5As such, myriad clocks reflecting epigenetic age have been developed for a range of age-or disease-related targets. 4,6The GrimAge series contains second-generation markers of epigenetic aging that account for clinical and functional biomarkers, and is most notable for its robust associations with human mortality and morbidity risk, including time to death and comorbidity counts. 6,7The recently developed version 2 of the GrimAge clock (hereafter, GrimAge2) improved on the first's predictive abilities and confirmed its applicability for people at midlife and of different racial and ethnic backgrounds. 1,6igenetic changes are modifiable and efforts to counter epigenetic alteration in humans have centered on lifestyle factors including diet, inspiring concepts of an "epigenetic diet" and "nutriepigenetics." 8,9So far, 2 epidemiological studies have found inverse associations between higher diet quality and slower epigenetic aging using clock measures related to mortality, including the first version of GrimAge. 7,10In those studies, diet measures were reflective of healthy dietary patterns (eg, the Dietary Approaches to Stop Hypertension [DASH] diet, the Alternate Mediterranean Diet [aMED] score) emphasizing consumption of fruits, vegetables, whole grains, nuts and seeds, and legumes. 8,11For example, the Mediterranean-style diet is largely plant-based with emphasis on extra virgin olive oil and seafood.This makes it replete with bioactive nutrients and phytotherapeutic compounds and low in highly processed, high fat, and nutrient-poor foods, a mixture hypothesized to be protective against low-grade chronic inflammation ("inflammaging"), oxidative stress, intracellular and extracellular waste accumulation, and disrupted intracellular signaling and protein-protein interactions.[14] Dietary Reference Intakes (DRIs) are an established set of nutrient-specific reference values determined by experts that guide population intakes for adequacy and toxic effects. 15Recent thinking, however, suggests that diets may not always adequately supply nutrients and other bioactives, particularly relative to the amounts necessary to fully condition gene expression or counteract epigenetic alterations to ensure optimal physiological metabolism. 8Macronutrients and micronutrients play crucial roles in DNA replication, damage prevention, and repair, whereas nutrient deficiencies (and excesses) can cause genomic damage to the same degree as physical or chemical exposures. 167][18] Diet quality inventories, such as those for Mediterranean-style diets, have not generally incorporated DRIs, although such considerations could clarify how food-based indices compare against requirements for related nutrients (eg, those with epigenetic properties) and refine epidemiological and intervention efforts.
0][21] However, in diet quality indices often studied in the epigenetic context (eg, the aMED), sugar is noticeably unaccounted for, and it

JAMA Network Open | Genetics and Genomics
has also yet to be examined alone.Given the high consumption of sugar globally and the demographic variations within, [22][23][24]  For inclusion in current analyses, the participants needed valid diet records and epigenetic data at midlife along with age and race and ethnicity information (participant self-reported); after excluding 5 women with epigenetic data quality issues, 342 individuals were included in the analytic sample.Complete case analyses were done.Among the 624 women who were followed up, the women composing the analytic sample were younger (39.2 years vs 39.9 years; P < .001)and had greater body mass index (BMI, calculated as weight in kilograms divided by height in meters squared) compared with women without complete diet and epigenetic data (32.5 vs 30.7;P = .02)(Table 1).
No differences were otherwise observed.

Epigenetic Clock: GrimAge2
Participants provided saliva samples used for DNAm analyses performed by the University of California, Los Angeles Neuroscience Genomics Core (UNGC) of the Semel Institute for Neuroscience and Human Behavior using the Infinium HumanMethylation450 BeadChip platform (Illumina, Inc).
DNAm profiles were generated by Horvath's online calculator, 27  C-reactive protein (logCRP) and hemoglobin A 1c (log A 1c )-beyond the original 7. Linear transformation of results from these models allows GrimAge2 to be taken as an epigenetic age

JAMA Network Open | Genetics and Genomics
Essential Nutrients, Added Sugar Intake, and Epigenetic Age in Black and White Women estimate (in years).Further information can be accessed from studies on DNA treatment and isolation and advanced analysis options for generating output files 28 or GrimAge2. 1

Dietary and Nutritional Assessment
The participants were instructed by the NGHS study staff to self-complete a 3-day food record at follow-up for 3 nonconsecutive days. 29 followed published scoring methodology 30 reflecting the degree of adherence to 9 components of an anti-inflammatory, antioxidant-rich diet.The AHEI-2010 was assessed following published scoring

JAMA Network Open | Genetics and Genomics
Essential Nutrients, Added Sugar Intake, and Epigenetic Age in Black and White Women instructions 31 and reflects the degree of adherence to 11 dietary components associated with decreased risk for chronic disease.

Epigenetic Nutrient Index
This study developed a novel nutrient index (ENI) after the Mediterranean-style diet, but via a nutrient-based approach rather than a food-based one.Nutrient selection was done a priori based on antioxidant and/or anti-inflammatory capacities as well as roles in DNA maintenance and repair documented in the literature. 16,32,33Scores can range from 0 to 24, with higher scores reflecting higher DRI adherence (Table 2). 34The internal consistency of the ENI was acceptable (Cronbach α = 0.79).The ENI also demonstrated convergent validity with r = 0.51 ENI-aMED correlation as well as higher ENI scores in women from childhood households with higher annual incomes (13.9 vs 11.7, for Ն$40 000/y vs <$10 000/y, respectively) and parental educational attainment (14.7 vs 12.3, for Նcollege graduate vs < high school graduate, respectively), corresponding to the literature. 36arson correlations between the ENI and diet scores and added sugar intake were also calculated.
The ENI score was moderately correlated with the AHEI-2010 score (r = 0.44) but not correlated with added sugar intake.The aMED and AHEI-2010 scores were highly correlated at r = 0.73.Added sugar intake had moderate correlation with the AHEI-2010 score (r = −0.44)and low correlation with the aMED score (r = −0.28).

Added Sugar Intake
Added sugar intake was calculated as the mean across valid food records using NDSR output.The NDSR defines added sugar intake as the total sugar added to foods (eg, as syrups and sugars) during food preparation and commercial food processing.Monosaccharides and disaccharides naturally occurring in foods are not included. 35

Covariates
To maximize internal validity and minimize confounding, several covariates were included.Age and sample batch were controlled for as well as naive CD8 and CD8pCD28nCD45Ran memory and effector T-cell counts, thus accounting for normal cell count variation.To control for baseline factors (currently yes for any of the following conditions: diabetes, hypertension, hypercholesterolemia, or thyroid), BMI (measured), having ever smoked (yes or no), and mean daily total energy intake (as higher diet quality scores might result from higher energy intake) 37 were also included.

Statistical Analysis
Descriptive analyses provided summary statistics.Linear regression models estimated unadjusted and adjusted cross-sectional associations between each of the 4 dietary exposures with GrimAge2.

Study Participant Characteristics
The  1).The participants were well distributed across socioeconomic status categories at baseline (9-10 years old).The participants presented with low to moderate levels of diet quality; the mean (SD) scores were 3.9 (1.9) (possible range, 0-9) on the anti-inflammatory, antioxidant Mediterranean-style pattern (aMED); 55.4 (14.7) (possible range, 0-110) on the AHEI-2010 for chronic disease risk; and 13.5 (5.0) (possible range, 0-24) on the ENI for intakes of epigenetic-relevant nutrients relative to DRIs.The participants also reported mean (SD) daily added sugar intake of 61.5 (44.6) g, although the score range was large (2.7-316.5 g).

Associations Between Diet and Epigenetic Age
Table 3 provides the overall unadjusted and adjusted associations between each dietary exposure of interest and GrimAge2 resulting from multivariable linear regression models.In both unadjusted and adjusted models, all dietary exposures were statistically and significantly associated with GrimAge2 in the hypothesized, anticipated direction.In adjusted models, the associations observed for each dietary exposure were slightly attenuated.Each unit increase in the scores was associated with year changes in GrimAge2, as follows: aMED (β, −0.41; 95% CI, −0.69 to −0.13), AHEI-2010 (β, −0.05; 95% CI, −0.08 to −0.01), and ENI (β, −0.17; 95% CI, −0.29 to −0.06), indicating that healthier diets were associated with decelerated epigenetic aging.Each gram increase in added sugar intake was associated with a 0.02 (95% CI, 0.01 to 0.04) increase in GrimAge2, reflecting accelerated epigenetic aging.measure and added sugar intake with GrimAge2 in the context of each other.In all instances, healthier diet measures and added sugar intake appeared to maintain their independent associations with GrimAge2 in the expected directions.Associations were statistically significant for added sugar intake in all models as well as for aMED scores; 95% CIs were more imprecise for AHEI-2010 and ENI scores.

Discussion
The findings of this cross-sectional study are among the first, to our knowledge, to demonstrate the association of added sugar intake with an epigenetic clock.Further, to our knowledge, it is the first study to examine the associations of diet with GrimAge2 and extend the applicability of such results to a cohort of Black and White women at midlife.As hypothesized, measures of healthy dietary patterns (aMED, AHEI-2010 scores), and high intakes of nutrients theoretically related to epigenetics (ENI) were associated with younger epigenetic age, while a higher intake of added sugar was  b Adjusted for current age, sample batch, naive CD8 and CD8pCD28nCD45Ran memory and effector T-cell counts, race (Black or White), ever diagnosis of a chronic condition, current medication use, baseline (age 9-10 years) household income, baseline (age 9-10 years) highest parental educational attainment, number of parents in household at baseline (age 9-10 years), number of siblings at baseline (age 9-10 years), body mass index at midlife, mean total kilocalories, and ever smoking status assessed at midlife (yes or no).As a complete cases analysis approach was taken, adjusted vs unadjusted analyses differed by n = 17 women with missing baseline household income data.
c Statistically significant result (P < .05).Although the magnitudes of associations were diminished and some 95% CIs became wider, their statistical significance generally persisted, supporting the existence of independent epigenetic associations of both healthy and less healthy diet measures.This approach is informative, as dietary components are often examined singularly or in indices, which can lead to erroneous conclusions if key contextual dietary components are not accounted for or are obscured.From these findings, even in healthy dietary contexts, added sugar still has detrimental associations with epigenetic age.
Similarly, despite higher added sugar intake, healthier dietary intakes appear to remain generally associated with younger epigenetic age.
The number of published nutriepigenetic studies, particularly on examining second-generation epigenetic clock markers, is still relatively small.However, the results of the present study are consistent with the literature.Two other studies 7,10 have examined GrimAge1-associated outcomes and found higher diet quality scores, including the DASH and aMED, were associated with slower epigenetic aging.However, those studies were limited to older (>50 years) and White populations, limiting their demographic generalizability.Analyses of epigenetic aging and added sugar intake are new, but findings are consistent with the larger body of epidemiological work that has drawn connections between added sugar intake and cardiometabolic disease, 19,20 perhaps suggesting a potential mechanism underlying such observations.Granted, point and 95% CI estimates for the added sugar-GrimAge2 associations were close to zero, suggesting a smaller role for added sugar compared with healthy dietary measures; however, more studies are needed.Nevertheless, their statistical significance was persistent.
Nutrient-based inventories can provide epidemiological contributions for genomic health studies.The idea of epigenetically critical nutrients is important for 2 reasons.7][18] In the novel ENI constructed for the present study, points were awarded based on comparisons of average daily intakes with: (1) estimated average requirements, or the requirement considered adequate for half of the healthy individuals in a population, and (2) recommended dietary allowances or adequate intakes, or where 97% to 98% or essentially all of a population's healthy individuals' requirements for a nutrient are met. 15Future iterations could test varying ENI scoring parameters relative to DRIs for epigenetic benefit.Second, taking a nutrient approach suggests that any dietary pattern rich in vitamins, minerals, and other bioactives could be useful for preserving epigenetic health.This is helpful because dietary patterns are socioculturally influenced, but a nutrient focus rather than a focus on foods could help bridge cultures, class, and geography. 9The Okinawan diet, for example, is nutritionally similar to the Mediterranean-style diet but more aligned to Asian tastes. 38In general, the sociodemographic determinants of diet should not be discounted.
Across the US population, for instance, it is known that overall diet quality is mediocre and relatively low while added sugar intake is considerably high, as also observed in the sample of the present study.However, specific nutrient intakes will vary based on the particulars of dietary patterns. 22,36As dietetics and medicine progresses into the era of personalized nutrition and personalized medicine, the role of social factors including diet will be important to consider in epigenetic studies and could figure prominently in work on health disparities.

Strengths and Limitations
Strengths of this study are its inclusion of a diverse group of women as well as use of robust measures of diet and DNAm.It was also possible to control for several potential sociodemographic confounders.
This study also has limitations.As a cross-sectional study, it is not possible to infer causality without temporality, and therefore longitudinal studies are needed.Additionally, diet was selfreported via 3-day food records, which may lead to underestimates and overestimates of intakes

JAMA Network Open | Genetics and Genomics
Essential Nutrients, Added Sugar Intake, and Epigenetic Age in Black and White Women depending on the nutrient.Therefore, augmenting dietary assessment with food frequency questionnaires and/or biomarkers could be helpful. 39Also, other nutrients with pro-epigenetic properties were not included in the current ENI.Still, the Cronbach α for this first ENI version was acceptable at 0.79 and it demonstrated good convergent validity with customary socioeconomic and demographic characteristics.The tolerable upper intake levels of the DRIs were not considered in constructing the ENI.Future work should assess the prevalence of intakes beyond upper limits to assess whether toxicity could be a concern.

Conclusions
To our knowledge, the findings of this cross-sectional study are among the first to find associations between indicators of healthy diet as well as added sugar intake and second-generation epigenetic aging markers and one of the first to include a cohort of Black women.Higher diet quality and higher consumption of antioxidants or anti-inflammatory nutrients were associated with younger epigenetic age, whereas higher consumption of added sugar was associated with older epigenetic age.Promotion of healthy diets aligned with chronic disease prevention and decreased added sugar consumption may support slower cellular aging relative to chronological age, although longitudinal analyses are needed.
which provided (1) estimates of epigenetic age based on GrimAge2 estimation methods; and (2) assessments of data quality (again, 5 observations did not pass quality checks).GrimAge2 uses Cox proportional hazards regression models that regress time to death (due to all-cause mortality) on DNAm-based surrogates of plasma proteins, a DNAm-based estimator of smoking pack-years, age, and female sex.It was updated from GrimAge, version 1 6 by including 2 new DNAm-based estimators of plasma proteins-high-sensitivity Data were entered into and analyzed by the Nutrition Data System for Research (NDSR) software, version 2018 (University of Minnesota Nutrition Coordinating Center).Diet Quality Indices: aMED and AHEI-2010 Mean nutrient and food intakes were calculated across valid food records for each woman based on the NDSR 2018 output.These values were used to calculate the scores of 2 overall diet quality nutrient indices (aMED and the Alternate Healthy Eating Index [AHEI]-2010) and a novel index (Epigenetic Nutrient Index [ENI]) score as described below.The aMED (Mediterranean-style diet) = 342) a Adjusted (n = 325) b Alternate Mediterranean diet score −0.62 (−0.91 to −0.34) c −0.41 (−0.69 to −0.13) c Alternate Healthy Eating Index -2010 score −0.10 (−0.13 to −0.06) c −0.05 (−0.08 to −0.01) c Epigenetic Nutrient Index score −0.19 (−0.29 to −0.08) c −0.17 (−0.29 to −0.06) c Added sugar, g 0.02 (0.01 to 0.04) c 0.02 (0.01 to 0.04) c a Adjusted for factors identified through epigenetic clock best practices and expert recommendations: current age, sample batch, and naive CD8 and CD8pCD28nCD45Ran memory and effector T-cell counts.
Essential Nutrients, Added Sugar Intake, and Epigenetic Age in Black and White Women To date, nutriepigenetic work has mostly involved older White populations and focused on healthy dietary aspects.It is therefore important to examine the associations between nutrition and epigenetic aging in more diverse samples and to better understand what specific dietary aspects could be underlying the observed associations.Nutrients with established epigenetic action should be examined, especially considering intakes relative to amounts set forth in the DRIs and nutritional recommendations.Similarly, sugar is an established pro-inflammatory and oxidative agent that has JAMA Network Open.2024;7(7):e2422749.doi:10.1001/jamanetworkopen.2024.22749(Reprinted) July 29, 2024 2/12 Downloaded from jamanetwork.comby guest on 08/01/2024 created and its associations with epigenetic aging were tested alongside established diet quality indices.

Table 1 .
Characteristics of 624 Participants Within the Overall Follow-Up Sample, Analytic vs Nonanalytic Sample a Unless otherwise noted, data are given as number (percentage).bStatisticallysignificant result (P < .05).c Epigenetic clock measure.

Table 2 .
Scoring Rubric for the ENI as Based on the Dietary Reference Intakes a influence on diet and epigenetic age over time, the following parameters assessed at age 9 or 10 years (mostly parent or caregiver reported) were further adjusted for annual household income, highest parental educational attainment, number of parents in household, and number of siblings.Additionally, self-reported race (Black or White) as well as the current health and lifestyle factors of self-reported chronic conditions (yes to any of the following ever: cancer, diabetes [including gestational, prediabetes], hypertension, or hypercholesterolemia) or medication use 35breviations: AI, adequate intake; EAR, estimated average requirement; ENI, Epigenetic Nutrient Index; median as , median of the analytic sample; MUFA, monounsaturated fatty acids; NA, not applicable; RDA, recommended dietary allowance; SFA, saturated fatty acids.aValuescorresponding to the EAR, RDA, and AI for each nutrient component are noted (in parentheses) as based on age-and sex-based intake estimates put forth by the Food and Nutrition Board of the US Institute of Medicine and the National Health Service of the United Kingdom (here, values correspond to those for women aged 36-43 years). 34 accounting for differences in vitamin A activity of carotenoids.35cNotaccountingfordifferences in folate absorption between natural and synthetic forms.35JAMANetworkOpen | Genetics and GenomicsEssential Nutrients, Added Sugar Intake, and Epigenetic Age in Black and White Women JAMA Network Open.2024;7(7):e2422749. do0.1001/jamanetworkopen.2024.22749 (Rinted) July 29, 2024 5/12 Downloaded from jamanetwork.comby guest on 08/01/2024 and their potential

Table 3 .
Overall Unadjusted and Adjusted Associations Between Dietary Exposure and GrimAge2

Table 4 .
Overall Adjusted Associations for Diet Quality Scores and Added Sugar With GrimAge2 Where Dietary Exposures Are Combined Among 325 Participants Adjusted for current age, sample batch, naive CD8 and CD8pCD28nCD45Ran memory and effector T-cell counts, race (Black or White), ever diagnosis of a chronic condition, current medication use, baseline (age 9-10 years) household income, baseline (age 9-10 years) highest parental educational attainment, number of parents in household at baseline (age 9-10 years), number of siblings at baseline (age 9-10 years), body mass index at midlife, mean total kilocalories, and ever smoking status assessed at midlife (yes or no).
b c Statistically significant result (P < .05).JAMA Network

Open | Genetics and Genomics Essential
Nutrients, Added Sugar Intake, and Epigenetic Age in Black and White Women Downloaded from jamanetwork.comby guest on 08/01/2024 associated with older epigenetic age.Additionally, this study examined indicators of healthy and less healthy diets in the same model, allowing simultaneous evaluation of each in the presence of the other.