Stable Isotope Assays for Collagen Turnover and Fibrosis

In animal studies, deuterium oxide (5%) is administered in drinking water for one week to label hydroxyproline in collagen.

At the time of tissue collection, a small plasma sample (~25 µL) is taken to measure precursor enrichment in body water. Harvested tissues are hydrolyzed to determine deuterium enrichment and hydroxyproline concentration. From these measurements, fractional synthesis rates of new collagen are calculated. 

This approach enables evaluation of fibrosis progression and therapeutic effects, including antifibrotic drugs or toxicant exposures such as carbon tetrachloride.

Flooding dose infusions of 13C- or deuterium-labeled amino acids can be applied to biopsy materials to quantify collagen synthesis rates. For example, Babraj et al. administered 13C-proline to human subjects and measured collagen fractional synthesis in muscle using gas chromatography–combustion isotope ratio mass spectrometry (GC-C-IRMS). 

This technique separates derivatized amino acids by gas chromatography, combusts them to CO₂ gas at 1000 °C, and measures isotopic enrichment with high precision.  

With this method, enrichment levels as low as 0.001% 13C can be detected, enabling an accurate assessment of collagen synthesis in human and animal studies.