Comparative Analysis of Metabolic Pathway Enzyme Activity in Aging Liver

Thesis Abstract

Liver aging is accompanied by coordinated shifts in metabolic enzyme activities that compromise energy homeostasis and xenobiotic handling, yet comparative cross-sectional analyses of these enzymatic changes across aging cohorts remain underexplored. The study aims to elucidate age-related differences in metabolic pathway enzyme activities in the liver and to identify mechanistic relationships with systemic metabolic markers. Specifically, the objectives are (1) to quantify activities of key hepatic enzymes involved in glycolysis (hexokinase, phosphofructokinase), gluconeogenesis (phosphoenolpyruvate carboxykinase, glucose-6-phosphatase), beta-oxidation (carnitine palmitoyltransferase I), and detoxification (cytochrome P450 3A4, glutathione-S-transferase) in young, middle-aged, and elderly adults; (2) to compare enzymatic profiles across age groups using standardized liver tissue samples obtained from 90 participants undergoing clinically indicated hepatic biopsies, evenly represented by age strata (30 per group) and balanced for sex; (3) to correlate enzyme activities with systemic markers including fasting glucose, insulin, lipid profile, inflammatory cytokines (IL-6, TNF-?), and oxidative stress indicators (malondialdehyde, 8-oxo-dG); and (4) to describe associations with body composition and pharmacokinetic parameters inferred from concomitant medication clearance data. The methodology employs a cross-sectional, observational design within a biobank-supported framework. Liver tissue homogenates will be assayed for enzyme activities using spectrophotometric and fluorometric methods validated for clinical samples, with quality control procedures including blinded duplicate analyses. Protein concentration will be determined by the BCA assay to express enzyme activities per milligram of total protein. Systemic variables will be collected from participants’ medical records and validated research-wide questionnaires. Data analysis will utilize multivariate analysis of covariance (MANCOVA) to compare enzyme activities across age groups while adjusting for sex, BMI, and comorbidities, complemented by multiple linear regression to explore relationships between hepatic enzymes and systemic markers. A partial least squares regression model will be used to identify latent patterns linking metabolic enzyme profiles with inflammatory and oxidative stress parameters. The theoretical framework integrates the “Rate-Limiting Step” concept from metabolic control analysis with the inflammaging paradigm to interpret age-associated enzyme shifts as drivers of altered hepatic metabolic flux. Anticipated findings include reduced activity of gluconeogenic enzymes and beta-oxidation pathways in older groups, with compensatory or elevated phase I/II detoxification enzyme activity in some individuals, and a pattern of increased inflammatory and oxidative stress correlates with diminished mitochondrial-linked enzymatic capacity. The study is expected to contribute to knowledge by providing a comprehensive cross-sectional map of hepatic metabolic enzyme reprogramming with age, clarifying which pathways are most susceptible to aging-related decline and how systemic metabolic health interfaces with hepatic function. Such insights have implications for precision dosing, management of metabolic disorders in the elderly, and targeted interventions to preserve hepatic metabolic resilience. The conclusions will emphasize the necessity of age-stratified considerations in pharmacometabolic assessments and propose recommendations for personalized therapeutic strategies, including lifestyle or pharmacological interventions aimed at sustaining mitochondrial and detoxification enzyme activities in aging populations.