Determination of erythrocyte glucose 1,6-bisphosphate--a comparison of two methods using a centrifugal analyzer

Glucose 1,6-bisphosphate is a key effector of human erythrocyte glycolysis, yet to date its assay has been problematical. Two methods for measuring glucose 1,6-bisphosphate were modified and adapted to a centrifugal analyser and the inaccuracy and imprecision of each method were compared. One assay, based on stimulation of phosphoglucomutase, was shown to underestimate the erythrocyte levels by approximately 5% due to inhibition of the mutase by endogenous 2,3-bisphosphoglycerate. An alternative chemical/enzymic method, consisting of acid hydrolysis of glucose 1,6-bisphosphate to glucose 6-phosphate and subsequent determination of the monophosphate was modified by omitting an initial alkaline hydrolysis step and by increasing the duration of acid hydrolysis. The modified method also enabled the determination of erythrocyte glucose 6-phosphate. The normal concentration of glucose 1,6-bisphosphate in whole blood and in washed human erythrocytes, determined using the more accurate chemical/enzymic method was 83 +/- 5 mumol/l cells and 86 +/- 4 mumol/l cells, respectively; the corresponding concentrations of glucose 6-phosphate were 26 +/- 2 mumol/l cells and 15 +/- 3 mumol/l cells.