BARITONE Trial Protocol Aims to Map Dietary Biomarkers

Researchers at the University of Gothenburg in Sweden have outlined a protocol for the BARITONE project, a study designed to identify and validate metabolomic biomarker patterns that reflect habitual dietary intake. The project addresses a persistent challenge in nutrition research: the difficulty of accurately assessing what people eat, as most epidemiological studies rely on self-reported data that are subject to significant measurement errors. By combining a randomized controlled crossover trial with observational data, the study aims to provide objective biological markers for different protein sources and the consumption of vegetables, fruits, and berries.

The BARITONE project consists of two distinct but integrated components. The first is a cross-sectional screening study involving a larger sample of participants who undergo comprehensive baseline phenotyping, including the completion of a 7-day weighed food diary. The second component is a randomized controlled three-period crossover dietary intervention trial involving a selected subgroup of approximately 70 participants from the screening sample. This trial is registered on ClinicalTrials.gov under the identifier NCT07128940 and is conducted at the Metabolic Kitchen and Laboratory at Sahlgrenska Academy.

In the intervention trial, participants consume three distinct dietary patterns that differ primarily in their protein source: meat-based, fish-based, and plant-based. Each dietary period lasts one week and is separated by a one-week washout period during which participants resume their habitual diets. The design of the trial ensures that the overall meal structure and nutritional composition, including energy intake and macronutrient distribution, remain comparable across all three conditions. The only major variable is the type of protein provided at lunch and dinner, while breakfast and snacks remain identical across all intervention weeks.

Participants are selected based on specific eligibility criteria, including age, body mass index, and habitual intake levels of vegetables, fruits, and berries. The study prioritizes an equal distribution by sex and varying intake levels of these food groups to better identify biomarker patterns related to changes in consumption. Habitual intake is categorized into high, medium, and low groups based on data from the Swedish national dietary intake survey, Riksmaten 2010–2011. High intake is defined as greater than 500 grams per day, while low intake is defined as less than 200 grams per day.

The primary objective of the trial is to identify differences in plasma metabolite patterns at the end of each intervention week. Metabolomics involves the comprehensive analysis of low-molecular-weight metabolites in biological samples such as blood and urine, which reflect both exogenous compounds from food and endogenous metabolic processes. By using metabolomic patterns rather than single biomarkers, the study aims to better capture the complexity of dietary exposures and distinguish between different dietary patterns with greater precision than traditional self-report methods.

Biological samples are collected at screening and before and after each intervention period. Fasting venous blood and spot urine samples are processed and stored at -80 degrees Celsius until analysis. Metabolomic and lipidomic profiling will be performed at the Swedish Metabolomics Center in Umeå, Sweden, using advanced techniques such as gas chromatography–time-of-flight mass spectrometry and ultra-high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry. These platforms generate thousands of metabolite features, which will be analyzed using both unsupervised and supervised statistical approaches.

The statistical analysis plan includes unsupervised Principal Component Analysis and supervised Orthogonal Partial Least Squares methods to characterize metabolomic patterns associated with dietary intake. The primary comparison will focus on plasma metabolomic profiles at the end of the three diet intervention periods. Predictive biomarker models will be developed for each dietary protein source using linear elastic net regression, with the assigned intake expressed in grams per day as the outcome. These models will be internally validated using repeated nested participant-level cross-validation to ensure robustness and reliability.

Following the development of predictive models using the intervention data, the final models will be applied to screening metabolomic profiles from the same participants to evaluate performance under habitual dietary conditions. Subsequently, the locked models will be evaluated in an additional group of approximately 30 eligible screening participants who did not enter the intervention trial. This step is crucial for assessing the generalizability of the biomarkers in a real-world setting where participants are not under strict dietary control.

The study acknowledges several limitations, including the fact that participants are a self-selected group with a special interest in diet and health, which may limit the representativeness of the findings. Additionally, the intervention targets broad food categories rather than single foods, which may reduce the ability to identify biomarkers common to an entire category. The researchers note that residual variation in food composition and preparation cannot be fully quantified, although standardized recipes and prespecified products are used to minimize these factors.

Despite these limitations, the BARITONE project provides a rigorous framework for identifying and validating metabolomic biomarkers of present dietary intake. By combining controlled feeding studies with observational data on habitual intake, the study aims to improve the accuracy of dietary assessment in nutritional epidemiology. The findings may contribute to a better understanding of diet–health relationships and support future research on the metabolic effects of different dietary patterns.

The project is part of a broader effort to develop objective tools for measuring dietary intake, which is essential for advancing nutrition science and informing public health strategies. The BARITONE study’s focus on fasting samples is particularly relevant for epidemiological research, where such samples are most commonly collected.

The results of the trial will be published on ClinicalTrials.gov and the project homepage, ensuring transparency and accessibility of the findings. By addressing the methodological challenges of biomarker validation, the BARITONE project aims to bridge the gap between controlled laboratory findings and real-world dietary practices.

The BARITONE project’s findings will provide valuable insights into the metabolic responses to different protein sources and the consumption of vegetables, fruits, and berries, ultimately contributing to a more precise understanding of how diet influences health.

Detailed participant criteria define the baseline population. Investigators invited 287 eligible individuals identified through pre-screening questionnaires to attend baseline testing. Eligible participants were adults aged 18 to 65 years with a body mass index between 18.5 and 30 kilograms per square meter who considered themselves healthy. Exclusion criteria eliminated volunteers with metabolic or gastrointestinal disorders such as diabetes, inflammatory bowel disease, celiac disease, or hypothyroidism, as well as those who were pregnant, lactating, or diagnosed with life-threatening conditions. People taking metabolism-altering medications like insulin or lipid-lowering drugs, and those who used illicit drugs, alcohol, or nicotine products including cigarettes, snus, or electronic cigarettes, were also excluded. In addition, although volunteers could continue their habitual dietary supplements, investigators prohibited creatine monohydrate because it directly influences metabolomic profiles linked to amino acid and protein metabolism.

The intervention menus reflect standardized energy targets derived from the 2023 Nordic Nutrition Recommendations for adults aged 25 to 50 years with moderate physical activity. Diets are calibrated to two distinct intake tiers: an energy level of approximately 2,151 kilocalories per day and a higher tier of 2,701 kilocalories per day, with each target permitting a five percent tolerance margin. Across all intervention diets, macronutrient distributions supply 45 to 60 percent of daily energy from carbohydrates, 10 to 20 percent from protein, and 25 to 40 percent from fat. To isolate the effects of the primary protein sources, total vegetable, fruit, and berry consumption is held constant at roughly 350 grams per day across every arm. To support protocol adherence, participants receive their first day of intervention meals during their laboratory visit, while the remaining groceries are shipped directly to their homes through an online grocery delivery service. Volunteers track adherence through continuous deviation reports, documenting any uneaten study items or non-study foods consumed beyond permitted unsweetened tea, coffee, and water.

Beyond circulating metabolites, the protocol incorporates gut microbiome profiling and secondary biofluid assessments to examine individual variation. Participants provide frozen stool samples for full-length 16S ribosomal RNA gene sequencing using Oxford Nanopore technology at Umeå University to evaluate baseline gut microbial taxonomy. In addition to primary plasma profiles, secondary trial outcomes evaluate fasting urine metabolomic patterns and plasma lipid profiles, which researchers consider complementary rather than interchangeable. The investigative team plans exploratory cluster analyses using principal component analysis and Ward’s hierarchical clustering to identify discrete metabotypes, or subgroup metabolic response patterns, and evaluate their links to gut microbiota and clinical baseline traits. To test analytical robustness, per-protocol sensitivity evaluations will restrict analysis to participants who consumed at least 90 percent of provided protein and produce while maintaining absolute body weight within two percent. Finally, researchers plan external validation of their locked protein-source prediction models in the independent VOV observational study of vegans, omnivores, and vegetarians, which collected serum, urine, and four-day weighed food records.

Subject of Research: Biomarker patterns reflecting habitual food intake: the BARITONE sample and randomized controlled crossover trial protocol

Article Title: Biomarker patterns reflecting habitual food intake: the BARITONE sample and randomized controlled crossover trial protocol

Article References: Oberrauter, L.-M., Lindqvist, H. M., & Winkvist, A. (2026). Biomarker patterns reflecting habitual food intake: the BARITONE sample and randomized controlled crossover trial protocol. Frontiers in Nutrition, 13, Article 1977025. https://doi.org/10.3389/fnut.2026.1977025

Image Credits: AI Generated

DOI: 10.3389/fnut.2026.1977025

Keywords: Biomarker, patterns, reflecting, habitual, food, intake, BARITONE, sample, randomized, controlled, crossover, trial