Creatine transporter is currently the focus of renewed interest with emerging roles in brain neurotransmission and physiology, and the bioenergetics of
cancer metastases. We here report on amendments of a standard
creatine uptake assay which might help clinical chemistry laboratories to extend their current range of measurements of
creatine and metabolites in body fluids to functional
enzyme explorations. In this respect, short incubation times and the use of a stable-
isotope-labeled substrate (D3-creatine) preceded by a
creatine wash-out step from cultured fibroblast cells by removal of
fetal bovine serum (rich in
creatine) from the incubation medium are recommended. Together, these measures decreased, by a first order of magnitude,
creatine concentrations in the incubation medium at the start of
creatine-uptake studies and allowed to functionally discriminate between 4 hemizygous male and 4 heterozygous female patients with X-linked
SLC6A8 deficiency, and between this cohort of eight patients and controls. The functional assay corroborated genetic diagnosis of
SLC6A8 deficiency. Gene anomalies in our small cohort included splicing site (c.912G > A [p.Ile260_Gln304del], c.778-2A > G and c.1495 + 2 T > G), substitution (c.407C > T) [p.Ala136Val] and deletion (c.635_636delAG [p.Glu212Valfs*84] and c.1324delC [p.Gln442Lysfs*21]) variants with reduced
creatine transporter function validating their pathogenicity, including that of a previously unreported c.1324delC variant. The present assay adaptations provide an easy, reliable and discriminative manner for exploring
creatine transporter activity and disease variations. It might apply to drug testing or other evaluations in the genetic and metabolic horizons covered by the emerging functions of
creatine and its transporter, in a way, however, requiring and completed by additional studies on female patients and blood-brain barrier permeability properties of selected compounds. As a whole, the proposed assay of
creatine transporter positively adds to currently existing measurements of this transporter activity, and determining on a large scale the extent of its exact suitability to detect female patients should condition in the future its transfer in clinical practice.