Identification and analysis of calcium speciation in red blood cells by ultrasonic-dialysiscapillary electrophoresisinductively coupled plasma optical emission spectrometry

Abstract

Ultrasonic dialysis and capillary electrophoresis inductively coupled plasma optical emission spectrometry were developed for the identification and determination of calcium species in human red blood cells (HRBC). Separation was optimized at 20 kV with a 40 mmol L⁻¹ pH 7.40 Tris-HCl buffer. The free Ca²⁺ concentration in HRBC cytoplasm was found to be 112 nmol L⁻¹. The recovery for Ca²⁺ spiked cytoplasm samples ranged from 96.0–103%. Eight primary calcium-containing species (concentrations 1.21–21.8 mg L⁻¹) were found in HRBC. One species had a molecular weight <15 kDa, one between 15–50 kDa, three between 50–100 kDa, and three >100 kDa. The calcium species with the greatest mobility was not free Ca²⁺, but a calcium-erythrocyte membrane species of 50–100 kDa. This method is sensitive and more selective than other common methods for free Ca²⁺ determination in HRBC.