Microanalysis of the metabolic intermediates of lactose synthesis in human milk and plasma using bioluminescent methods

Sensitive bioluminescent methods were developed to measure the metabolites glucose, glucose 6-phosphate (G6P), glucose 1-phosphate (G1P), UDP-glucose, and UDP-galactose in human milk and lactose and galactose in human plasma. The bioluminescent methods measured NADH produced by coupled enzymatic assays derived from equivalent spectrophotometric methods. We found that the long chain fatty acids in human milk (C10-C16) inhibited the bioluminescent reactions. This inhibition was overcome by adding defatted bovine serum albumin to the reaction mixture containing the bioluminescent enzymes. It also was necessary to modify methods of deproteinizing milk and blood plasma to accommodate small sample volumes. In the development of these assays emphasis was given to simplicity of reagent preparation, minimizing cost, and ease of use. The detection limit for the bioluminescent method for NADH was 0.28 nM for a 20-microliters sample. For the assays of the metabolites, recoveries ranged from 91 to 107%. For sample sizes of 2 to 5 microliters of protein free sample, the detection limits for milk were G1P, 0.09 microM; G6P, 0.05 microM; UDPhexose, 0.07 microM; UDP-Glc, 0.03 microM; glucose, 9 microM; and for plasma, lactose, 0.76 microM, galactose, 0.31 microM. The bioluminescent methods gave equivalent results to spectrophotometric methods for the measurement of blood lactose and milk glucose.