Protein turnover half-life is a critical determinant of cardiac homeostasis and can reveal previously unknown disease mechanisms. We recently developed a theoretical method to quantify protein half-life in human using stable isotopes in deuterium oxide (D2O). Our goal in the present study is to demonstrate the immediate clinical translation potential of the method by evaluating its safety, feasibility, efficacy, and reproducibility in 10 healthy human subjects.
The enrolled human subjects (4 females/6 males; age 22 - 51 y/o; body weight 51 - 108 kg) were labeled with a tailored protocol (UCLA IRB#12-000899), wherein each subject orally consumed weight-adjusted doses of (~45 mL each) 70% D2O daily for 14 days to enrich body water and proteins with deuterium. Throughout labeling, the subjects maintained regular food and fluid intake, and normal daily activities. Vital signs and medical health questionnaires were taken daily till 14 days post-labeling. We followed long-term physical conditions and the physiological clearance of D2O from body water for up to 240 days post-labeling, finding no physiological effects or signs of discomfort.
To monitor label enrichment and post-labeling clearance in the subjects, we measured the D2O level of plasma and saliva samples with GC-MS. Both body fluids were reliable sources for monitoring label enrichment kinetics, giving consistent values of individual enrichment levels (0.9-2.2%) and rates (0.15-0.40 d−1) in all subjects. Post-labeling D2O level naturally subsided with a characteristic half-life of ~7 days (0.1 d−1). With LC-MS and the in-house informatics platform Proturn, we successfully characterized the turnover dynamics of >600 human plasma proteins, the largest such human dataset to-date. We detected diverse protein half-life in plasma, e.g., from albumin (18.3 d) to IGF2 (8 h). Importantly, the method can quantify protein half-life with only a single time point, suggesting it can be used to study the dynamics of one single cardiac biopsy (e.g., during heart transplant).
In summary, D2O labeling is a safe, accessible, and effective technique for widespread clinical investigations of protein turnover dynamics. We further discuss its implications in understanding cardiac disease mechanisms.