Application of Neutron-Irradiated ⁶Li (n, α) ³H Reaction to a Protein-Based Fibrous Non-Woven Fabric Biopolymer: Radiolabeling of Cross-Linked Natural Fibrous Chicken Eggshell Membranes and Their Whole-Body Tissue Distribution after Oral Ingestion in Mice

Chicken eggshell membrane (ESM) is composed of a non-woven fibrous biopolymer fabric crosslinked by collagen (mainly types X and V), lysyl oxidase, and other molecules, including glycoproteins and lipids, which are secreted by cells in the hen’s fallopian tube. ESM has been reported to improve osteoarthritic knee pain when taken as a supplement and to have anti-fibrotic effects in rodents. ESM has physiological effects in vivo after digestion, absorption, and metabolism, but the mechanism of ESM efficacy remains unclear. We aimed to determine whether ESM is digested and absorbed by the body, although its metabolic mechanism has not been described. Although it is possible to create compounds labeled with single molecules,ESMs are insoluble, fibrillar, natural composite materials, and the molecular form of digested and absorbed ESM is unknown, so it is not possible to prepare synthetically labeled compounds. We developed a method for direct tritium labeling of ESM and investigated the absorption of the labeled ESM. Organic compounds, such as proteins, were mixed with lithium carbonate and then irradiated to label with 3H produced by the 6Li (n, α) 3 H reaction. Mice were orally administered tritium-labeled ESM, and blood and tissue radioactivity were measured using a liquid scintillation counter. Tritium-labeled ESM was distributed in the blood and all tissues after oral administration. Our method enabled successful tritium labeling of fibrous ESM, which allowed for metabolic evaluation of the labeled ESM product as it was digested and absorbed by the organs of experimental mice. This advanced tritium recoiling labeling method can be used for in vivo tracking of complex acellular extracellular matrix bioscaffolds used in biomedical research.