Quantification of tissue creatine content using capillary electrophoresis

Abstract

AbstractCreatine plays a fundamental role in cellular energy homeostasis. The current protocol describes an alternative method for creatine quantification in biological tissue samples using capillary electrophoresis, with high separation efficiency that enables rapid analysis with low sample volumes. The protocol involves homogenization of snap-frozen tissue in phosphate buffer, followed by electrophoresis through a bare-fused capillary (75 µm internal diameter) and measurement at 200 nm on the Agilent 7100 CE system. Under the optimised conditions, there was excellent linearity in creatine standards between 6.3 – 100 µM. The overall intra-assay variability for concentrations between 6.3 – 100 µM was 1.5 %, and the inter-assay variability was 6.4 %, with a limit of detection at 6 nmol/mg protein. The protocol was further benchmarked against a commercially available enzyme assay kit using lung samples from lambs that received continuous creatine or saline supplementation. There was good agreement between the two methods (mean difference = 0.42 [-0.26-1.1] nmol/mg protein). Importantly, capillary electrophoresis enables reliable detection of creatine in biological samples from just ∼1.5 mg of wet-weight lung tissue. Capillary electrophoresis enables rapid (<10 minutes) and highly efficient analysis of tissue samples and avoids challenges faced with traditional enzymatic assays. The current protocol was developed and optimised with ovine lung tissue, but it can be easily adapted to analyse various tissue types. For tissues with higher baseline creatine content, such as the skeletal muscles or brain, <1 mg wet weight tissue would be sufficient to detect creatine using capillary electrophoresis.