Validity of the Alcian Blue Binding Test as an Indicator of Red Blood Cell and Glomerular Membrane Negative Charges

A. Bernard, Unit of Industrial Toxicology and Occupational Medicine, School of Medicine, Catholic University of Louvain, Brussels (Belgium) Dear Sir, A controversy presently exists concerning the possibility of assessing the glomerular polyanion charge on the basis of the Alcian blue (AB) binding to red blood cell (RBC) membrane. Studies by Levin et al. [1] and Boulton-Jones et al. [2] have suggested that the binding of the cationic dye AB to RBC surface reflects the loss of glomerular polyanion in patients with nephrotic syndrome. The enhanced protein excretion in these patients would be due at least partly to a reduced electrostatic repulsion by the glomerular filter of anionic macromolecules like albumin. This hypothesis is supported by our observations showing that the microalbuminuria of diabetes or chronic cadmium poisoning is associated with a parallel decrease of AB binding to RBCs [3, 4]. Several authors, however, have questioned the validity of the AB binding test to RBCs. Feehally [5] could not confirm the reduction of AB binding to RBCs from patients with nephrotic syndrome. Sewell et al. [6] have put forward reservations on the analytical validity of the AB binding test because of the instability of the dye in solution and the possible interference of contaminating albumin. More recently, Ghiggeri et al. [7] studied the physi-cochemical interaction between AB and proteins and concluded that the binding of AB to proteins is hydrophobic and therefore cannot be used for monitoring variations in surface charge due to sialic acid residues. This conclusion, however, is refuted by the experimental evidence presented here that the AB binding to RBC and glomerular membrane correlates with the sialic acid content of these structures. These data were obtained in rats given 100 ppm cadmium in the drinking water for 10 months and in their controls. The AB binding to RBC or glomerular membrane was determined by the method of Levin et al. [1] with the following modifications. RBCs were washed 4 times with phosphate-buffered saline. The AB solution (Alcian Blue 8GX, 54% purity, Sigma, St. Louis, Mo.) was prepared just before use by dissolving the dye (4 mg/ml) in a solution containing 25 mM MgCl2 and 0.15 M NaCl. This solution was vigorously vortex-mixed for 10 min and the undissolved dye removed by centrifugation (2,000 g × 10 min) and filtration on Whatman No. 1 filter. The final AB concentration was determined from the extinction coefficient (Ei0/om 68.4 for the crude AB