TABLE 1.

Characteristics of included studies assessing the effect of nutritional biomarkers on biological aging in older adults with findings, conclusions, and assessment of the validity of conclusions¹

First author (ref), year, country	Study design and primary outcome	Participants and baseline characteristics	Assessments (for NRSI), intervention and control (for RCT)	Findings	Conclusions	Validity of conclusions
Alonso-Pedrero (28), 2020, Spain	Retrospective cross-sectional analysis; telomere attrition	n = 886 Spanish; mean age: 67.7 ± 6.1 y; mean BMI (in kg/m2): 25.85; 72.8% men; Spanish; 5.3 y at university (4 in 5 undergraduate degree); 3 in 4 married; 1 in 2 former smokers	UPF consumption assessed via a self-administered 136-item semi-quantitative FFQ, validated in Spain	Highest UPF consumption group [4.1 (±1.1) servings/d] had shorter telomeres vs. lowest consumption group [1.1 (±0.5) servings/d] (adjusted OR: 1.82; 95% CI: 1.05, 3.22; P-trend = 0.03). Participants in the highest UPF consumption group were more likely to have family history of CVD, diabetes, and dyslipidemia prevalence, and to snack more in between meals, and to consume more fats, SFAs, PUFAs, sodium, dietary cholesterol, fast food, and processed meat, and less carbohydrates, proteins, fiber, potassium, magnesium, calcium, phosphorus, olive oil, fruits, and vegetables, and overall adhere less to the Mediterranean diet, compared with participants in the lowest UPF consumption group.	Higher UPF consumption (≥3 servings/d) is associated telomere attrition in elderly Spanish	Conclusions are robust since a validated FFQ (that mitigates bias due to self-reporting) and a standard method to measure telomere length was used. But underreporting cannot be excluded due to the FFQ not including certain foods, e.g., energy bars
Atzmon (29), 2002, USA	Retrospective correlational analysis; cognitive function (MMSE)	n = 139 Ashkenazi Jews; mean age: F, 97.7 ± 0.2 y; M, 97.6 ± 0.4 y; 26.4% men	Plasma HDL cholesterol, TG, apo A-I	Plasma HDL-cholesterol concentrations correlated significantly with MMSE (r = 0.32; P < 0.0001).Each decrease in plasma HDL-cholesterol tertile (74.9 ± 2.1, 50.6 ± 0.5, and 36.8 ± 1.0 mg/dL) was associated with a significant decrease in MMSE (23.4 ± 1.5, 17.7 ± 1.8, and 12.4 ± 1.8; P < 0.04 for each plasma HDL cholesterol tertile). Increased plasma apo A-I and decreased plasma TG concentrations were also correlated with a significantly superior cognitive function. Biological markers of hydration and nutritional status did not differ between the groups with the higher or lower plasma HDL cholesterol or MMSE.	Cognitive dysfunction in centenarians is associated with a progressive decline in plasma HDL cholesterol	The study is robust, but the population studied is very homogeneous, limiting the applicability of findings to diverse populations
Bowman (30), 2012, USA	Retrospective cross-sectional analysis; cognitive function (MMSE, MRI, clinical dementia rating)	n = 104; mean age: 87±10 y; mean BMI: 25 ± 4; 38% men; 15±3 y of education; 2% smokers; 39% drinkers; 44% hypertension; 5% diabetes; 21% depression	30 plasma dietary biomarkers that were grouped into 8 distinct NBPs via multivariate analysis (PCA)	Two NBPs associated with more favorable cognitive and MRI measures: one high in plasma B vitamins (thiamin, riboflavin, vitamin B-6, folate, and vitamin B-12) and vitamins C, D, and E, and another high in plasma marine ω-3 fatty acids. A third pattern characterized by high trans fat was associated with less favorable cognitive function and less total cerebral brain volume. Depression attenuated the relation between the marine ω-3 pattern and white matter hyperintensity volume.	Distinct plasma NBPs are associated with cognitive function and brain volume	Limitations are 1) the PCA that may require decisions with the data from the investigator; 2) selecting a priori nutrient biomarkers based on knowledge of their association with neurodegeneration; 3) the restriction of the sample population to a relatively healthy and well-educated cohort of white, non-Hispanic elders with minimal genetic risk for AD. Therefore, the results cannot be generalized
Chang (31), 2020, Taiwan	Case control post hoc analysis; leukocyte telomere attrition and TERRA expression	n = 72; sarcopenia (S), non-sarcopenia (NS); mean age: S, 75.7 ± 5.5 y; NS, 74.9 ± 6.7 y; mean BMI: S, 25.6 ± 2.9; NS, 21.5 ± 2.3; 22% men	Lower body strength exercise 2x per week + BCAAs (0.36 g valine, 0.54 g leucine, 0.43 g isoleucine), 0.65 g glutamine, 0.61 g arginine; 1.01 g of other amino acids + 600 mg calcium and 800 IU vitamin D-3	No significant difference in telomere length between S and NS. TERRA expression was lower in S compared with NS (5.18 ± 2.98 vs. 2.51 ± 1.89; P < 0.001). Intervention significantly increased TERRA expression in the S group, but not telomere length. The GEE analysis demonstrated that TERRA expression was negatively associated with sarcopenia (β coefficient= –2.705, P < 0.001) but positively associated with intervention (β coefficient = 1.599, P = 0.023).	Sarcopenia is associated with decrease in TERRA expression in leukocytes. Rebound TERRA expression (to the level similar to the NS controls) was observed in the S group after exercise and nutrition intervention	Physical activity habits were not considered at baseline and their heterogeneity could be a confounder. Another limitation is the use of leukocyte telomere length as a surrogate marker for sarcopenia. Although muscle and leukocyte telomere lengths correlate, these are affected in different ways by aging. These limitations restrict the generalizability of the findings and the validity of the conclusion
Freitas-Simoes (32), 2019, Spain	Retrospective cross-sectional analysis; leukocyte telomere attrition	n = 344 Spanish; mean age: 68.8 ± 3.3 y; mean BMI: 27.1 ± 3.8; 31.4% men; 55% hypertension; 54% dyslipidemia, 35% on statins; 11% diabetes, 6% on metformin; 72% never smoked; PA (METs/wk): 2510 (1606–3888); 10 (8–15) y of education	RBC proportions of PUFAs	In multivariate models adjusted for age and gender, the RBC proportions of dietary PUFAs were unrelated to telomere length. In contrast, the RBC proportion of arachidonic acid was inversely related to telomere length (r = 0.10, 95% CI: 0.19, 0.01, P = 0.023).	Increased levels of C20:4 ω-6 in RBCs are associated with shorter telomeres	Strengths of the study include the methods of assessment of biomarkers and of the outcome. The main limitation is the relatively small number of participants and the homogeneity of the population. Further studies need to test the findings in more ethnically diverse and larger populations, as well as in more people without metabolic syndrome
García-Calzón (33), 2015, Spain	Retrospective cross-sectional analysis; leukocyte telomere attrition	n = 520; mean age: 67 ± 6 y; mean BMI: 29.1; 45% men; 83% hypertension; 66% dyslipidemia; 37% diabetes; 10% current smokers; PA (METs-min/d): T1 = 307.5 ± 205.9, T2 = 293.5 ± 206.8, T3 = 230.1 ± 165.0	DII calculated from self-reported data using a 137-item FFQ that has been previously validated in Spain	Cross-sectional associations: Participants following a more anti-inflammatory diet (lowest DII score) had longer telomeres at baseline (P -trend = 0.012). Longitudinal associations: Greater anti-inflammatory potential of the diet (i.e., a decrease in the DII) significantly slowed down the rate of telomere shortening.Greater DII (greatest proinflammatory values) after 5 y of follow-up was associated with an almost 2-fold higher risk of accelerated telomere attrition compared with the highest decrease in DII (greatest anti-inflammatory values) during this period (P-trend = 0.025).	The cross-sectional and longitudinal associations between DII and telomere length in people with high CVD risk suggest that diet may modulate telomere length via pro- or anti-inflammatory mechanisms	The conclusions are strengthened by the standardized and validated assessment methods in this population but need to be tested in more diverse populations and also assessed for confounding, e.g., subjects with highest proinflammatory index had higher BMI and lower levels of PA (P < 0.001), and these may have confounded the effect of diet
Gensous (34), 2020, Italy and Poland	Retrospective cross-sectional analysis; epigenetic age (Horvath's clock)	n = 120; mean age: Italy: 72.2 ± 3.8 y, Poland: 71.1 ± 4 y; mean BMI: Italy, 26.99 ± 3.60; Poland, 28.07 ± 3.37; 73.9% men	Mediterranean diet assessed using 7-d food records	Epigenetic rejuvenation was seen after nutritional intervention. The effect was statistically significant in the group of Polish females (P = 0.0013) and in subjects who were epigenetically older at baseline. A genomewide association study of epigenetic age changes after the intervention did not return significant loci, but small-effect alleles were identified, mapping in genes enriched in pathways related to energy metabolism, regulation of cell cycle, and of immune functions. After 1 y of nutritional intervention, adherence to a Mediterranean diet significantly increased in both Italian and Polish participants, and a significant decrease in BMI was observed in Italian males (paired Student's t test, P = 0.008).	Mediterranean diet can promote epigenetic rejuvenation but with country-, sex-, and individual-specific effects, thus highlighting the need for a personalized approach to nutritional interventions	The small sample size limits the generalizability of the conclusions. The lack of analyses by demographic characteristics of the sample, other than sex and country (e.g., BMI, physical activity status, conditions), does not allow for exclusion of confounders
Handing (35), 2019, USA	Retrospective cross-sectional analysis; cognition (immediate and delayed story recall and word memory, digit subtraction, orientation questions; adapted from MMSE)	n = 1308; mean age: 70.8 y; 47% men; 68% overweight; 47% hypertension; 34% dyslipidemia; 13% diabetes; 70% self-rated their health good or excellent; 64% White; 74% less than college education; 33% drinkers; 14% smokers; 41% did PA	% of calories from fat, protein, and carbohydrate (via 24-h dietary recall), serum concentrations of vitamins B-12, C, D, and E, folate, iron, homocysteine, β-carotene, and inflammatory biomarkers (serum CRP, plasma fibrinogen, and serum ferritin)	Serum folate was positively significantly associated with cognitive score. Specifically, the interaction between age-cognition and folate indicated the associations of higher age and lower global cognition and lower immediate story recall were weaker in those with higher folate values (P values <0.05). A significant interaction between age and plasma fibrinogen indicated that the association between age and worse digit subtraction was stronger with values >3.1 g/L.	Folate and fibrinogen were significant moderators between age and cognition	The large sample size and the diversity of cognitive performance instruments strengthen the conclusion. Limitations are the cross-sectional design that does not allow for assessing causation, the inaccurate recall bias of the 24-h dietary recall, the non-inclusion of other inflammatory biomarkers linked to cognition, e.g., IL-1, IL-6, and TNF-α and ω-6 PUFAs and trans fats
Nettleton (36), 2008, USA	Retrospective cross-sectional analysis; leukocyte telomere attrition	n = 840; mean age: 65.3 ± 0.7 y; mean BMI: 29.0 ± 0.4; 48% men; 54% Hispanic adults; 27% African American; 19% White; 76.3 ≥high school; 37.6 to <$25K income; 11% smoking	Dietary intake patterns (whole grains, fruit and vegetables, low-fat dairy, nuts or seeds, non-fried fish, coffee, refined grains, fried foods, red and processed meat, sugar-sweetened soda) via 120-item FFQ	After adjustment for age, other demographics, lifestyle factors, and intakes of other foods or beverages, only processed meat intake was associated with telomere length. Categorical analysis showed that participants consuming 1 serving of processed meat each week had 0.017 smaller T/S ratios vs. nonconsumers.Age was strongly associated with telomere length. For every 1-y increment in age, the T:S ratio was 0.005 lower (β ± SE: –0.007 ± 0.001, P < 0.001).After adjustment for age, longer telomere length was associated with female sex, shorter duration of smoking, and less time spent in inactive leisure activities (P < 0.001–0.04).	Processed meat intake was inversely associated with telomere length, but other diet features did not display any associations	Limitations of the study include the lack of validation of the FFQ in the study population and the lack of analysis by SES, since people in lower SES may be likely to consume processed meat due to cost. Lower SES may be associated with greater life stress, and shorter telomeres. These limitations restrict the generalizability of the findings
O'Callaghan (37), 2014, Australia	Double-blinded, parallel-group RCT; 6 mo duration and 75% retention; telomere attrition	n = 44; mean age: C, 73 ± 4 y; DHA, 74.2 ± 7 y; EPA, 74.9 ± 5.1 y; mean BMI: C, 28.1 ± 5.3; DHA, 26.8 ± 2.6; EPA, 28.1 ± 4.1; C, 47% men; DHA, 72% men; EPA, 82% men; 14% diabetes; prehypertensive; mild cognitive impairment; C group slightly higher education (≥year 12) than I (years 11–12)	I group received 4 capsules/d of an ω-3 PUFA supplement; EPA group received 1.67 g EPA + 0.16 g DHA and DHA group received 1.55 g DHA + 0.40 g EPA; C group received ω-6 PUFA linoleic acid (LA) at 2.2 g/d for 6 mo	The intervention did not show an increase in telomere length with treatment and there was a trend toward telomere shortening during the intervention period. Telomere shortening was greater in the LA group (d = 0.21) than in the DHA (d = 0.12) and EPA groups (d = 0.06). Increased erythrocyte DHA concentrations were associated with reduced telomere shortening (r = –0.67; P = 0.02) in the DHA group.	Telomeric shortening may be attenuated by ω-3 PUFA supplement-ation, requiring further investigation in larger samples	Strength of the study is the blood sample collection method (in the morning, after an overnight fast, and before breakfast). Limitations include the use of whole blood to measure telomere length, as certain immune cell subpopulations have different proliferative rates and telomere lengths. The study was statistically underpowered to allow for meaningful conclusions, and this may be a reason for the lack of statistically significant differences
Praveen (38), 2020, India	Retrospective cross-sectional analysis; telomere attrition and mtCN	n = 428 Indian; median age (IQR): 65.0 (62.0–70.0) y; median BMI (IQR): 24.7 (22.1–27.2); 51.2% men; 83% abdominal obesity; 45% overweight; 22% obesity; 26% hypertension; 18% anemia; urban residents	Plasma folate and vitamin B-12	Significant positive correlation was found between the telomere length and mtCN (r = 0.162, P < 0.001), and both of them were positively associated with plasma folate (r = 0.221, P < 0.001) and vitamin B-12 concentrations (r = 0.187, P < 0.001).	Folate and vitamin B-12 may delay aging by preventing the reduction in telomere length and mtCN	The homogeneous small sample restricts the generalizability of the conclusions. The lack of accounting for confounders, such as physical activity, smoking, and others, limits the validity of the conclusions
Seesen (39), 2020, Thailand	Nonrandomized intervention/control study; aging biomarkers (CRP, IL-6, IGF-I, and CD4+:CD8+ T-cell ratio) and cognitive status (MMSE)	n = 122; mean age: bran supplement (Br), 68.80 (±2.82) y; Exercise (Ex), 68.17 (±2.65) y; Br+Ex, 68.20 (±2.41) y; C, 69.06 (±2.74) y; 12% men; 49% with frailty; 10% smoking; 12.5% alcohol; 65% married	I group received 10 g black rice germ, Br providing 300 mg anthocyanins/d and Ex	During the 24-wk intervention, the combined Br+Ex group significantly (P < 0.05) decreased the inflammatory biomarkers (CRP and IL-6) and significantly increased IGF-I (P < 0.001). Additionally, the Br+Ex group significantly improved physical performance and muscle strength (P < 0.05).	A synergistic effect of the combined intervention produced sustainable improvement in physical performance, lower-body muscle strength, and positive inflammatory and endocrine changes	The study produced strong associations that warrant further investigation due to the small sample size and the lack of randomization. There was no reporting on habitual physical activity of participants and BMI and these are additional limitations
Zwilling (40), 2019, USA	Retrospective cross-sectional analysis; cognitive function (memory, executive function)	n = 116; mean age: 69.0 ± 3.2 y; mean BMI: 26.0 (±3.6); 31% men; Caucasian; 72% college degree; 54% to >$54K income	NBPs: plasma carotenoids, tocopherols, lipids, riboflavin, folate, vitamin B-12, and vitamin D	Five NBPs were associated with enhanced cognitive performance: 1) ω-3 and ω-6 PUFAs, 2) lycopene, 3) ω-3 PUFAs, 4) carotenoids, and 5) B vitamins (riboflavin, folate, vitamin B-12) and vitamin D. Three NBPs were associated with enhanced functional brain network efficiency: 1) ω-6 PUFAs, 2) ω-3 PUFAs, and 3) carotene.	NBPs account for a significant proportion of variance in measures of cognitive performance and functional brain network efficiency	Limitations are 1) the PCA that may require decisions with the data from the investigator; 2) selecting a priori NBP from the Mediterranean Diet based on knowledge of its association withneuroprotection;
				ω-3 PUFAs moderated the frontoparietal network and general intelligence, while ω-6 PUFAs and lycopene moderated the dorsal attention network and executive function.		3) the limitation of an observational study for causal relations; 4) the restriction of the sample to well-educated, Caucasian, neurologically healthy older adults. Therefore, the results cannot be generalized

¹AD, Alzheimer disease; BCAA, branched-chain amino acid; C, control; CD4⁺/8⁺, cluster of differentiation 4⁺/8⁺; CRP, C-reactive protein; CVD, cardiovascular disease; d, Cohen's d; DII, Dietary Inflammatory Index; GEE, generalized estimating equation; I, intervention; IGF-I, insulin-like growth factor-I; MET, metabolic equivalent of task; MMSE, Mini-Mental State Examination; mtCN, mitochondrial DNA copy number; NBP, nutrient biomarker pattern; NRSI, nonrandomized studies of interventions; PA, physical activity; PCA, principal component analysis; r, Pearson's correlation coefficient; RCT, randomized controlled trial; ref, reference; SES, socioeconomic status; TERRA, telomeric repeat-containing RNA; TG, triglyceride; T/S: ratio of telomeric DNA (T) to the amount of a single-copy control DNA (S); UPF, ultra-processed food.