Identification of Age-Associated Circulating Proteins and Lipids in 3800 Comorbidity-Enriched Older Adults from Japan-Based Cohorts Using Olink Assays and MRM Mass Spectrometry

Aging entails complex physiological changes, yet large-scale evidence among older Japanese individuals, especially those with comorbidities, remains limited. We analyzed serum and plasma samples from approximately 3800 Japanese aged 40 years and older to identify age-associated proteins and lipids, focusing on reproducibility and robustness. Chemokines CXCL9 and CCL11 and phosphatidylcholines PC 31:0 and PC 32:0 were positively associated with age across five cohorts, whereas lysophosphatidylcholines LPC-LA and LPC-AA showed negative associations. These molecular relationships were consistently reproduced across serum and plasma matrices and replicated in independent cohorts. Cross-platform consistency was confirmed between Olink Target 96 (relative NPX) and Target 48 (absolute quantification), with direct validation in Cohort 2. To our knowledge, this is the largest study to demonstrate reproducibility of age-associated molecular biomarkers in a comorbidity-enriched Japanese population. The principal contribution is technical─defining a set of robust, cross-platform, cross-matrix biomarkers of aging in older adults. Unlike previous Western studies which focused on younger or healthier populations, this work establishes reproducibility and generalizability in real-world aging. These validated biomarkers provide a valuable reference for clinical and translational research, including risk stratification and biological age assessment in comorbidity-enriched settings.