Determination of intrinsic creatine transporter (SLC6A8) activity and creatine transport function of leukocytes in rats

Creatine transporter (CRT) deficiency (CRT-D) results in a significant reduction of brain creatine levels, which causes various neurological symptoms in early childhood, and diagnosis of the severity of CRT-D based on the residual CRT transport activity in liquid biopsy samples would be beneficial for early intervention. The apparent reduction in creatine transport activity in CRT-D is thought to be due to reduced intrinsic CRT-mediated creatine transport per CRT protein and/or reduced absolute CRT protein expression on the plasma membranes. The purpose of this study was thus to determine the normal level of intrinsic CRT-mediated creatine transport activity based on absolute CRT protein quantification using rat CRT-overexpressing HEK293 cells (CRT/HEK293 cells), and to clarify creatine transport in erythrocyte- and leukocyte-enriched fractions isolated from the circulating blood of rats. The intrinsic creatine transport rate was calculated to be 0.237µL/(min·fmol CRT) based on the initial uptake rate and the absolute CRT protein level in CRT/ HEK293 cells. Taking into account Avogadro’s constant, the creatine transport activity per CRT protein is estimated to be 1190 creatine/(min·CRT molecule) in the presence of [¹⁴C]creatine at an extracellular concentration of 5µM. Isolated leukocyte-enriched fraction exhibited mRNA expression of CRT and partially Na⁺dependent [¹⁴C]creatine transport, whereas erythrocytes showed neither. These characteristics suggest that the leukocytes contain the CRT-mediated creatine uptake system, and are available for evaluation of residual CRT transport activity in CRT-D patients.