Reliance on self-reports and estimated food composition data in nutrition research introduces significant bias that can only be addressed with biomarkers.

The chemical composition of foods is complex, variable, and dependent on many factors. This has a major impact on nutrition research as it foundationally affects our ability to adequately assess the actual intake of nutrients and other compounds. In spite of this, accurate data on nutrient intake are key for investigating the associations and causal relationships between intake, health, and disease risk at the service of developing evidence-based dietary guidance that enables improvements in population health. Here, we exemplify the importance of this challenge by investigating the impact of food content variability on nutrition research using three bioactives as model: flavan-3-ols, (-)-epicatechin, and nitrate. Our results show that common approaches aimed at addressing the high compositional variability of even the same foods impede the accurate assessment of nutrient intake generally. This suggests that the results of many nutrition studies using food composition data are potentially unreliable and carry greater limitations than commonly appreciated, consequently resulting in dietary recommendations with significant limitations and unreliable impact on public health. Thus, current challenges related to nutrient intake assessments need to be addressed and mitigated by the development of improved dietary assessment methods involving the use of nutritional biomarkers.

Studies about the health benefits of foods or nutrients are often inconsistent. One study may find a health benefit of a particular food and may recommend that people increase their consumption of this food to reduce their disease risk. Yet another study may find the opposite. Inconsistent study results fuel confusion and frustration, and reduce trust in research. Limitations in the studies' designs are likely to be blamed for the inconsistent findings. For example, many studies rely on participants to self-report their food intake and on databases of the nutritional content of food. But people may not accurately report their food intake. Foods vary in their nutritional content, even between two items of the same food such as two apples. And how individuals metabolize foods can further affect the nutrients they receive. Nutritional biomarkers are a potential alternative to measuring dietary intake of specific nutrients. Biomarkers are compounds the body produces when it metabolizes a specific nutrient. Measuring biomarkers therefore give scientists a more accurate and unbiased assessment of nutrient intake. Ottaviani et al. conducted a study to test the differences when estimating nutrient intake using nutritional biomarkers compared with more conventional tools. They analyzed data from a nutrition study that involved over 18,000 participants. The experiments used computer modelling to assess study results using self-reported food intake in combination with food composition database information, or measures of three biomarkers estimating the intake of flavan-3-ols, epicatechin, and nitrates. The models showed that self-reported intake and food database information often led to inaccurate results that did not align well with biomarker measurements. Measuring nutritional biomarkers provides a more accurate and unbiased assessment of nutritional intake. Using these measurements instead of traditional methods for measuring nutrient intake may help increase the reliability of nutrition research. Scientists must work to identify and confirm biomarkers of nutrients to facilitate this work. Using these more precise nutrient measurements in studies may result in more consistent results. It may also lead to more trustworthy recommendations for consumers.

Impact of polyphenol oxidase on the bioavailability of flavan-3-ols in fruit smoothies: a controlled, single blinded, cross-over study.

Flavan-3-ols are bioactive compounds found in a variety of fruits and vegetables (F&V) that have been linked to positive health benefits. Increasing habitual flavan-3-ol intake is challenged by the generally low consumption of F&V. While smoothies are a commonly endorsed, consumer-accepted means to increase the daily intake of these important foods, fruits used for smoothie preparation can have a high polyphenol oxidase (PPO) activity and thus potentially affect the content and bioavailability of flavan-3-ols. To assess whether or not consuming freshly prepared smoothies made with different PPO-containing fruit impacts the bioavailability of the flavan-3-ols, a controlled, single blinded and cross-over study was conducted in healthy men (n = 8) who consumed a flavan-3-ol-containing banana-based smoothie (high-PPO drink), a flavan-3-ol-containing mixed berry smoothie (low-PPO drink) and flavan-3-ols in a capsule format (control). The peak plasma concentration (Cmax) of flavan-3-ol metabolites after capsule intake was 680 ± 78 nmol L-1, which was similar to the levels detected after the intake of the low PPO drink. In contrast, the intake of the high PPO drink resulted in a Cmax of 96 ± 47 nmol L-1, 84% lower than that obtained after capsule intake. In a subsequent study (n = 11), flavan-3-ols were co-ingested with a high-PPO banana drink but contact prior to intake was prevented. In this context, plasma flavan-3-ol levels were still reduced, suggesting an effect possibly related to post-ingestion PPO activity degrading flavan-3-ols in the stomach. There was a substantial range in the PPO activity detected in 18 different fruits, vegetables and plant-derived dietary products. In conclusion, bioavailability of flavan-3-ols, and most likely other dietary polyphenol bioactives, can be reduced substantially by the co-ingestion of high PPO-containing products, the implications of which are of importance for dietary advice and food preparation both at home and in industrial settings.

5-(3',4'-Dihydroxyphenyl)-γ-Valerolactone Is a Substrate for Human Paraoxonase: A Novel Pathway in Flavan-3-ol Metabolism.

SCOPE: Dietary flavan-3-ols are known to mediate cardiovascular benefits. Currently, it is assumed that the levels of flavan-3-ol catabolites detected in humans, 5-(3',4'-dihydroxyphenyl)-γ-valerolactone (γVL) and 5-(3',4'-dihydroxyphenyl)-γ-valeric acid (γVA), and their corresponding phase II metabolites, are determined exclusively by the action of the gut microbiome. However, a family of human proteins, paraoxonase (PON), can theoretically hydrolyze γVL metabolites into the corresponding γVAs. This study aims to determine if PON is involved in γVL and γVA metabolism in humans. METHODS AND RESULTS: A rapid conversion of γVL into γVA is detected in serum ex vivo (half-life = 9.8 ± 0.3 min) that is catalyzed by PON1 and PON3 isoforms. Phase II metabolites of γVL are also reacted with PON in serum. Following an intake of flavan-3-ol in healthy males (n = 13), the profile of γVA metabolites detected is consistent with that predicted from the reactivity of γVL metabolites with PON in serum. Furthermore, common PON polymorphisms are evaluated to assess the use of γVL metabolites as biomarkers of flavan-3-ol intake. CONCLUSION: PONs are involved in flavan-3-ol metabolic pathway in humans. PON polymorphisms have a minor contribution to inter-individual differences in the levels of γVL metabolites, without affecting their use as a nutritional biomarker.

Dietary flavanols restore hippocampal-dependent memory in older adults with lower diet quality and lower habitual flavanol consumption.

Dietary flavanols are food constituents found in certain fruits and vegetables that have been linked to cognitive aging. Previous studies suggested that consumption of dietary flavanols might specifically be associated with the hippocampal-dependent memory component of cognitive aging and that memory benefits of a flavanol intervention might depend on habitual diet quality. Here, we tested these hypotheses in the context of a large-scale study of 3,562 older adults, who were randomly assigned to a 3-y intervention of cocoa extract (500 mg of cocoa flavanols per day) or a placebo [(COcoa Supplement and Multivitamin Outcomes Study) COSMOS-Web, NCT04582617]. Using the alternative Healthy Eating Index in all participants and a urine-based biomarker of flavanol intake in a subset of participants [n = 1,361], we show that habitual flavanol consumption and diet quality at baseline are positively and selectively correlated with hippocampal-dependent memory. While the prespecified primary end point testing for an intervention-related improvement in memory in all participants after 1 y was not statistically significant, the flavanol intervention restored memory among participants in lower tertiles of habitual diet quality or habitual flavanol consumption. Increases in the flavanol biomarker over the course of the trial were associated with improving memory. Collectively, our results allow dietary flavanols to be considered in the context of a depletion-repletion paradigm and suggest that low flavanol consumption can act as a driver of the hippocampal-dependent component of cognitive aging.

Measuring the intake of dietary bioactives: Pitfalls and how to avoid them.

Bioactives are food constituents that, while not essential to human life, can affect health. Thus, there is increased interest in developing dietary recommendations for bioactives. Such recommendations require detailed information about the long-term association between habitual intake and health at population scale, and these can only be provided by large-scale observational studies. Nutritional epidemiology relies on the accurate estimation of intake, but currently used methods, commonly based on a 2-step process involving self-reports and food composition tables, are fraught with significant challenges and are unable to estimate the systemic presence of bioactives. Intake assessments based on nutritional biomarkers can provide an advanced alternative, but there are a number of pitfalls that need to be addressed in order to obtain reliable data on intake. Using flavan-3-ols as a case study, we highlight here key challenges and how they may be avoided. Taken together, we believe that the approaches outlined in this review can be applied to a wide range of food constituents, and doing so will improve assessments of the dietary intake of bioactives.

Flavan-3-ols and Cardiometabolic Health: First Ever Dietary Bioactive Guideline.

Guideline recommendation for a plant bioactive such as flavan-3-ols is a departure from previous recommendations because it is not based on deficiencies but rather improvement in health outcomes. Nevertheless, there is a rapidly growing body of clinical data reflecting benefits of flavan-3-ol intake that outweigh potential harms. Thus, the objective of the Expert Panel was to develop an intake recommendation for flavan-3-ols and cardiometabolic outcomes to inform multiple stakeholders including clinicians, policymakers, public health entities, and consumers. Guideline development followed the process set forth by the Academy of Nutrition and Dietetics, which includes use of the Evidence to Decision Framework. Studies informing this guideline (157 randomized controlled trials and 15 cohort studies) were previously reviewed in a recently published systematic review and meta-analysis. Quality and strength-of-evidence along with risk-of-bias in reporting was reviewed. In drafting the guideline, data assessments and opinions by authoritative scientific bodies providing guidance on the safety of flavan-3-ols were considered. Moderate evidence supporting cardiometabolic protection resulting from flavan-3-ol intake in the range of 400-600 mg/d was supported in the literature. Further, increasing consumption of dietary flavan-3-ols can help improve blood pressure, cholesterol concentrations, and blood sugar. Strength of evidence was strongest for some biomarkers (i.e., systolic blood pressure, total cholesterol, HDL cholesterol, and insulin/glucose dynamics). It should be noted that this is a food-based guideline and not a recommendation for flavan-3-ol supplements. This guideline was based on beneficial effects observed across a range of disease biomarkers and endpoints. Although a comprehensive assessment of available data has been reviewed, evidence gaps identified herein can inform scientists in guiding future randomized clinical trials.

Impact of Lipid Genetic Risk Score and Saturated Fatty Acid Intake on Central Obesity in an Asian Indian Population.

Abnormalities in lipid metabolism have been linked to the development of obesity. We used a nutrigenetic approach to establish a link between lipids and obesity in Asian Indians, who are known to have a high prevalence of central obesity and dyslipidaemia. A sample of 497 Asian Indian individuals (260 with type 2 diabetes and 237 with normal glucose tolerance) (mean age: 44 ± 10 years) were randomly chosen from the Chennai Urban Rural Epidemiological Study (CURES). Dietary intake was assessed using a previously validated questionnaire. A genetic risk score (GRS) was constructed based on cholesteryl ester transfer protein (CETP) and lipoprotein lipase (LPL) genetic variants. There was a significant interaction between GRS and saturated fatty acid (SFA) intake on waist circumference (WC) (Pinteraction = 0.006). Individuals with a low SFA intake (≤23.2 g/day), despite carrying ≥2 risk alleles, had a smaller WC compared to individuals carrying <2 risk alleles (Beta = −0.01 cm; p = 0.03). For those individuals carrying ≥2 risk alleles, a high SFA intake (>23.2 g/day) was significantly associated with a larger WC than a low SFA intake (≤23.2 g/day) (Beta = 0.02 cm, p = 0.02). There were no significant interactions between GRS and other dietary factors on any of the measured outcomes. We conclude that a diet low in SFA might help reduce the genetic risk of central obesity confirmed by CETP and LPL genetic variants. Conversely, a high SFA diet increases the genetic risk of central obesity in Asian Indians.

A Nutrigenetic Update on CETP Gene-Diet Interactions on Lipid-Related Outcomes.

PURPOSE OF REVIEW: An abnormal lipid profile is considered a main risk factor for cardiovascular diseases and evidence suggests that single nucleotide polymorphisms (SNPs) in the cholesteryl ester transfer protein (CETP) gene contribute to variations in lipid levels in response to dietary intake. The objective of this review was to identify and discuss nutrigenetic studies assessing the interactions between CETP SNPs and dietary factors on blood lipids. RECENT FINDINGS: Relevant articles were obtained through a literature search of PubMed and Google Scholar through to July 2021. An article was included if it examined an interaction between CETP SNPs and dietary factors on blood lipids. From 49 eligible nutrigenetic studies, 27 studies reported significant interactions between 8 CETP SNPs and 17 dietary factors on blood lipids in 18 ethnicities. The discrepancies in the study findings could be attributed to genetic heterogeneity, and differences in sample size, study design, lifestyle and measurement of dietary intake. The most extensively studied ethnicities were those of Caucasian populations and majority of the studies reported an interaction with dietary fat intake. The rs708272 (TaqIB) was the most widely studied CETP SNP, where 'B1' allele was associated with higher CETP activity, resulting in lower high-density lipoprotein cholesterol and higher serum triglycerides under the influence of high dietary fat intake. Overall, the findings suggest that CETP SNPs might alter blood lipid profiles by modifying responses to diet, but further large studies in multiple ethnic groups are warranted to identify individuals at risk of adverse lipid response to diet.

Validation of a high-throughput method for the quantification of flavanol and procyanidin biomarkers and methylxanthines in plasma by UPLC-MS.

Nutritional biomarkers are critical tools to objectively assess intake of nutrients and other compounds from the diet. In this context, it is essential that suitable analytical methods are available for the accurate quantification of biomarkers in large scale studies. Recently, structurally-related (-)-epicatechin metabolites (SREMs) and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone metabolites (gVLMs) were identified as biomarkers of intake of flavanols and procyanidins, a group of polyphenol bioactives. This study aimed at validating a high throughput method for the quantification of SREMs and gVLMs in plasma along with methylxanthines (MXs), dietary compounds known to interact with flavanol and procyanidin effects. To accomplish this, a full set of authentic analytical standards were used to optimize a micro solid phase extraction method for sample preparation coupled to HPLC-MS detection. Isotopically-labelled standards for all analytes were included to correct potential matrix effects on quantification. Average accuracies of 101%, 93% and 103% were obtained, respectively, for SREMs, gVLMs and MXs. Intra- and inter-day repeatability values were <15%. The method showed linear responses for all analytes (>0.993). Most SREMs and gVLMs had limits of quantifications <5 nM while limits of quantification of MXs were 0.2 µM. All analytes were stable under different tested processing conditions. Finally, the method proved to be suitable to assess SREMs, gVLMs and MXs in plasma collected after single acute and daily intake of cocoa-derived test materials. Overall, this method proved to be a valid analytical tool for high throughput quantification of flavanol and procyanidin biomarkers and methylxanthines in plasma.

Gut microbiota composition in relation to intake of added sugar, sugar-sweetened beverages and artificially sweetened beverages in the Malmö Offspring Study.

PURPOSE: It has been suggested that a high intake of sugar or sweeteners may result in an unfavorable microbiota composition; however, evidence is lacking. Hence, in this exploratory epidemiological study, we aim to examine if intake of added sugar, sugar-sweetened beverages (SSBs) or artificially sweetened beverages (ASBs) associate with the gut microbiota composition. METHODS: Participants (18-70 years) in the Malmö Offspring Study have provided blood, urine, and fecal samples and completed both web-based 4 day food records and short food frequency questionnaires. The gut microbiota was assessed by 16S rRNA sequencing, processed in QIIME and matched to Greengenes (v.13.8), giving 64 included genera after filtering. Intake of added sugar (n = 1371) (also supported by the overnight urinary sugar biomarker in a subgroup n = 577), SSBs (n = 1086) and ASBs (n = 1085) were examined as exposures in negative binomial regressions. RESULTS: Various genera nominally associated with intake of added sugar, SSBs, and ASBs. Only the negative association between SSB intake and Lachnobacterium remained significant after multiple testing correction. A positive association between SSB intake and the Firmicutes:Bacteroidetes ratio was also observed. CONCLUSION: In this wide population, the cross-sectional associations between added sugar and sweet beverage intake and the gut microbiota are modest, but the results suggest that SSB intake is associated negatively with the genus Lachnobacterium and positively with the Firmicutes:Bacteroidetes ratio. Larger studies, preferably using metagenomic sequencing, are needed to further evaluate if a link exists between intake of sugars and sweeteners and the human gut microbiota.

Biomarker-estimated flavan-3-ol intake is associated with lower blood pressure in cross-sectional analysis in EPIC Norfolk.

Flavan-3-ols are a group of bioactive compounds that have been shown to improve vascular function in intervention studies. They are therefore of great interest for the development of dietary recommendation for the prevention of cardio-vascular diseases. However, there are currently no reliable data from observational studies, as the high variability in the flavan-3-ol content of food makes it difficult to estimate actual intake without nutritional biomarkers. In this study, we investigated cross-sectional associations between biomarker-estimated flavan-3-ol intake and blood pressure and other CVD risk markers, as well as longitudinal associations with CVD risk in 25,618 participants of the European Prospective Investigation into Cancer (EPIC) Norfolk cohort. High flavan-3-ol intake, achievable as part of an habitual diet, was associated with a significantly lower systolic blood pressure (- 1.9 (- 2.7; - 1.1) mmHg in men and - 2.5 (- 3.3; - 1.8) mmHg in women; lowest vs highest decile of biomarker), comparable to adherence to a Mediterranean Diet or moderate salt reduction. Subgroup analyses showed that hypertensive participants had stronger inverse association between flavan-3-ol biomarker and systolic blood pressure when compared to normotensive participants. Flavanol intake could therefore have a role in the maintenance of cardiovascular health on a population scale.

Comparing Self-Reported Sugar Intake With the Sucrose and Fructose Biomarker From Overnight Urine Samples in Relation to Cardiometabolic Risk Factors.

Studies on sugar intake and its link to cardiometabolic risk show inconsistent results, partly due to dietary misreporting. Cost-effective and easily measured nutritional biomarkers that can complement dietary data are warranted. Measurement of 24-h urinary sugars is a biomarker of sugar intake, but there are knowledge gaps regarding the use of overnight urine samples. We aim to compare (1) overnight urinary sucrose and fructose measured with liquid chromatography-tandem mass spectrometry, (2) self-reported sugar intake measured with web-based 4-day food records, (3) their composite measure, and (4) these different measures' (1-3) cross-sectional associations with cardiometabolic risk factors in 991 adults in the Malmö Offspring Study (18-69 years, 54% women). The correlations between the reported intakes of total sugar, added sugar and sucrose was higher for urinary sucrose than fructose, and the correlations for the sum or urinary sucrose and fructose (U-sugars) varied between r≈0.2-0.3 (P < 0.01) in men and women. Differences in the direction of associations were observed for some cardiometabolic risk factors between U-sugars and reported added sugar intake, as well as between the sexes. In women, U-sugars, but not reported added sugar intake, were positively associated with systolic and diastolic blood pressure and fasting glucose. Both U-sugars and added sugar were positively associated with BMI and waist circumference in women, whereas among men, U-sugars were negatively associated with BMI and waist circumference, and no association was observed for added sugar. The composite measure of added sugars and U-sugars was positively associated with BMI, waist circumference and systolic blood pressure and negatively associated with HDL cholesterol in women (P < 0.05). Conclusively, we demonstrate statistically significant, but not very high, correlations between reported sugar intakes and U-sugars. Results indicate that overnight urinary sugars may be used as a complement to self-reported dietary data when investigating associations between sugar exposure and cardiometabolic risk. However, future studies are highly needed to validate the overnight urinary sugars as a biomarker because its use, instead of 24-h urine, facilitates data collection.

Evaluation of (-)-epicatechin metabolites as recovery biomarker of dietary flavan-3-ol intake.

Data from dietary intervention studies suggest that intake of (-)-epicatechin mediates beneficial vascular effects in humans. However, population-based investigations are required to evaluate associations between habitual intake and health and these studies rely on accurate estimates of intake, which nutritional biomarkers can provide. Here, we evaluate a series of structurally related (-)-epicatechin metabolites (SREM), particularly (-)-epicatechin-3'-glucuronide, (-)-epicatechin-3'-sulfate and 3'-O-methyl-(-)-epicatechin-5-sulfate (SREMB), as flavan-3-ol and (-)-epicatechin intake. SREMB in urine proved to be a specific indicator of (-)-epicatechin intake, showing also a strong correlation with the amount of (-)-epicatechin ingested (R2: 0.86 (95% CI 0.8l; 0.92). The median recovery of (-)-epicatechin as SREMB in 24 h urine was 10% (IQR 7-13%) and we found SREMB in the majority of participants of EPIC Norfolk (83% of 24,341) with a mean concentration of 2.4 ± 3.2 µmol/L. Our results show that SREMB are suitable as biomarker of (-)-epicatechin intake. According to evaluation criteria from IARC and the Institute of Medicine, the results obtained support use of SREMB as a recovery biomarker to estimate actual intake of (-)-epicatechin.

Oat bran, but not its isolated bioactive ß-glucans or polyphenols, have a bifidogenic effect in an in vitro fermentation model of the gut microbiota.

Wholegrain oats are known to modulate the human gut microbiota and have prebiotic properties (increase the growth of some health-promoting bacterial genera within the colon). Research to date mainly attributes these effects to the fibre content; however, oat is also a rich dietary source of polyphenols, which may contribute to the positive modulation of gut microbiota. In vitro anaerobic batch-culture experiments were performed over 24 h to evaluate the impact of two different doses (1 and 3 % (w/v)) of oat bran, matched concentrations of ß-glucan extract or polyphenol mix, on the human faecal microbiota composition using 16S RNA gene sequencing and SCFA analysis. Supplementation with oats increased the abundance of Proteobacteria (P <0·01) at 10 h, Bacteroidetes (P <0·05) at 24 h and concentrations of acetic and propionic acid increased at 10 and 24 h compared with the NC. Fermentation of the 1 % (w/v) oat bran resulted in significant increase in SCFA production at 24 h (86 (sd 27) v. 28 (sd 5) mm; P <0·05) and a bifidogenic effect, increasing the relative abundance of Bifidobacterium unassigned at 10 h and Bifidobacterium adolescentis (P <0·05) at 10 and 24 h compared with NC. Considering the ß-glucan treatment induced an increase in the phylum Bacteroidetes at 24 h, it explains the Bacteriodetes effects of oats as a food matrix. The polyphenol mix induced an increase in Enterobacteriaceae family at 24 h. In conclusion, in this study, we found that oats increased bifidobacteria, acetic acid and propionic acid, and this is mediated by the synergy of all oat compounds within the complex food matrix, rather than its main bioactive ß-glucan or polyphenols. Thus, oats as a whole food led to the greatest impact on the microbiota.

Dietary Intervention Modifies DNA Methylation Age Assessed by the Epigenetic Clock.

SCOPE: Alterations in DNA methylation patterns are correlated with aging, environmental exposures, and disease pathophysiology; the possibility of reverting or preventing these processes through dietary intervention is gaining momentum. In particular, methyl donors that provide S-adenosyl-methionine for one-carbon metabolism and polyphenols such as flavanols that inhibit the activity of DNA methyltransferases (DNMTs) can be key modifiers of epigenetic patterns. METHODS AND RESULTS: DNA methylation patterns are assessed in publicly available Illumina Infinium 450K methylation datasets from intervention studies with either folic acid + vitamin B12 (GSE74548) or monomeric and oligomeric flavanols (MOF) (GSE54690) in 44 and 13 participants, respectively. Global DNA methylation levels are increased in unmethylated regions such as CpG islands and shores following folic acid + vitamin B12 supplementation and decreased in highly methylated regions, including shelves and open-seas, following intervention with MOF. After supplementation with folic acid + vitamin B12, epigenetic age, estimated by the Horvath "epigenetic clock" model, is reduced in women with the MTHFR 677CC genotype. CONCLUSIONS: The effects of supplementation with folic acid + vitamin B12 and MOF on DNA methylation age are dependent upon gender and MTHFR genotype. Additionally, the findings demonstrate the potential for these dietary factors to modulate global DNA methylation profiles.