Main objective
To characterize and validate single and combined metabolomic and proteomic biomarkers of diet, physical and sedentary activity, and sleep, as well as their interaction, from early infancy to youth, using machine learning, and to translate findings for practical application into an easily usable online tool for broad dissemination.
Funding
This project has received funding from JPI HDHL STAMIFY call and the following national funders: Instituto de Salud Carlos III (ISCIII); Next Generation EU funds for actions of the Recovery, Transformation and Resilience Plan (PRTR); Bundesministerium für Bildung und Forschung (BMBF) - Federal Ministry of Education and Research; Österreichische Forschungsförderungsgesellschaft mbH (FFG) and the "MissionERA" program.
Methodology
Study cohorts
The original population for BiomarKid project includes 3330 generally healthy subejects from five European countries. This includes participants from two longitudinal deeply phenotyped cohort studies CHOP (n=1700 0-18 years old) and ToMI (n=1600 1-6 years old).
European Childhood Obesity Project (CHOP)( Koletzko, B et al. 2009, Weber, M et al. 2014) is a cohort initiated as a randomized controlled trial to investigate how varying dairy protein levels in infant formula influence body composition later in life. Between October 2002 and July 2004, 1,678 healthy newborns aged ≤8 weeks were recruited across five European countries (Germany, Italy, Belgium, Poland, and Spain). Participants were randomized to receive either a higher-protein formula, a lower-protein formula, or to serve as controls in the exclusively breastfed group. Following the one-year intervention, participants completed multiple follow-up assessments until the age of ~ 18 years old.
The Toddler Milk Intervention trial (ToMI) ( Grote, V et al. 2020) is a randomized trial developed following the CHOP study to examine the role of milk protein intake during the second year of life in child growth and later obesity risk. Children were recruited at approximately 12 months of age in Munich (Germany) and Tarragona/Reus (Spain), and were assigned to receive toddler milk with either higher or lower protein content for consumption throughout the second year of life. Participants were so far followed until 6 years of age.
Physical Activity and Sleep Assessment
Habitual physical activity in CHOP and ToMI cohort was assessed at multiple ages using the SenseWear Armband 2 (Body Media Inc., Pittsburgh, PA) and ActiGraph wGT3X-BT accelerometer (ActiGraph LLC, Pensacola FL, USA). Physical activity data from the SenseWear Armband 2 were collected in 1-minute epochs over three consecutive days (≥20 h/day) and processed using InnerView software to estimate activity intensity and sleep parameters (Trost, SG et al. 2011, Schwarzfischer, P et al. 2018). ActiGraph data were collected at 30 Hz over seven consecutive days, processed in R with non-wear periods excluded using Choi’s algorithm (Choi, L et al. 2011), and activity intensity classified using age-appropriate validated cut-points (Trost, SG et al. 2012; Evenson, KR et al. 2008; Ferry, JM, et al. 2025).
Nutrition assessment
Dietary intake was assessed at multiple ages and was based on repeated 3-day dietary records and food frequency questionnaires in both CHOP and ToMI. Based on dietary data, KIDMED scores (Triebswetter & Erhardt et al. 2026) and data-driven dietary pattern scores (Luque et al. 2018) were generated.
Clinical health assessment
Measured clinical variables include anthropometry, body composition, blood pressure, and several blood measurements including IGF-1 Axis, lipid profile, and insulin resistance. More on the methods will be added shortly.
Metabolomics
Metabolomics analyses for all CHOP (5.5, 8, 11, 18 years) and TOMI (1,2,6 years) visits were conducted at LMU Hospital Munich (Dr. von Hauner Children’s Hospital) over multiple years due to the longitudinal study design. Plasma samples were analysed by targeted mass spectrometry-based approaches including HPLC-ESI-MS/MS and flow-injection analysis (FIA-ESI-MS/MS). Detailed descriptions of metabolomics analyses were published before (Kirchberg et al. 2020;Hao et al. 2025).
Proteomics
The Team from the Medical University of Vienna performed proteomics analyses of the ToMI trial. This included main protein and peptide analysis and quantitation (labelled and non-labelled) based on proteomics approach using nano chromatography and mass spectrometry. Multidimensional HPLC method was developed to increase the number of identified peptides and proteins, alongside the application of methods for single-cell proteomics.