Enzymatic assay of D-mannose in serum

Abstract

We describe a new and improved enzymatic assay for determining the concentration of D-mannose in sera. Serum D-glucose is selectively converted to glucose-6 phosphate with the highly specific thermostable glucokinase (EC 2.7.1.2) from Bacillus stearothermophilus. The anionic reaction products and excess substrates are removed by a rapid and simple anion-exchange chromatography step in microcentrifuge spin columns. D-Mannose in the glucose-depleted sample is then assayed spectrophotometrically by using coupled enzymatic reactions. The quantitative elimination of glucose from the serum samples allowed the accurate and reproducible assay of serum mannose in the 0-200 mumol/L range. Recovery of mannose added to serum (5-200 mumol/L) was 94% +/- 4.4%. The intraassay CV was 6.7% at 40 mumol/L mannose (n = 5; 39.6 +/- 1.6 mumol/L) and 4.4% at 80 mumol/L (n = 11; 75.0 +/- 1.8 mumol/L); the interassay CV at these concentrations was 12.2% (n = 7; 36.9 +/- 2.1 mumol/L) and 9.8% (n = 7; 74.2 +/- 2.7 mumol/L), respectively. Sera from 11 healthy human volunteers contained an average of 54.1 +/- 11.9 mumol/L mannose (range 36-81 mumol/L).