Estimation of the ratio of exchangeable organically bound hydrogen to total organically bound hydrogen in flatfish flesh

The exchangeability of hydrogen atoms within organic molecules in vivo with ambient water hydrogen is uncertain. We assessed this by administrating the tracer deuterium to the fish. Flatfish were retained in deuterium-elevated seawater for 48 h, and the deuterium-to-hydrogen (D/H) ratios in the flesh samples were measured. Rapid in vivo equilibration between exchangeable organically bound hydrogen (Ex-OBH) and tissue-free water (TFW) hydrogen was indicated by the linear relationship between the D/H ratios of total organically bound hydrogen (t-OBH) and TFW. The estimated ratio of Ex-OBH to t-OBH (fe) in vivo was 7.2 ± 0.0%, while there was no in vivo production rate of non-exchangeable organically bound deuterium (Nx-OBD). In contrast to this estimation of no production of Nx-OBD in vivo, the D/H values of Nx-OBH measured by equilibration in vitro increased, consequently reducing the value of fe in vitro to some extent compared to that of fe in vivo. This indicated that a certain portion of Ex-OBH in vivo was not exchangeable during the equilibration in vitro.