Galactose-1-phosphate (Gal-1-P) levels in RBCs are measured enzymatically in a 2-part reaction. In the first reaction aliquot, galactose dehydrogenase converts the endogenous galactose in the specimen to a galactonolactone, with the simultaneous production of nicotinamide adenine dinucleotide (NADH; reduced form) from nicotinamide adenine dinucleotide (NAD). The absorbance of NADH at 340 nm is used to determine the amount of galactose present in the specimen. In the second reaction aliquot, both alkaline phosphatase and galactose dehydrogenase are added. The alkaline phosphatase converts Gal-1-P into galactose and the galactose dehydrogenase converts the galactose into the galactonolactone, while producing NADH. The absorbance of NADH at 340 nm represents the galactose and Gal-1-P present in the specimen. By subtracting the absorbance of the first reaction aliquot from that of the second reaction aliquot, the Gal-1-P level can be determined.
The Gal-1-P levels are reported per gram of hemoglobin. In the hemoglobin analysis, RBCs are combined with aqueous ammonia to form a compound that absorbs light at 540 nm. The Beer-Lambert Law is applied to calculate the hemoglobin concentration from the measured absorbance.(Gitzelmann R: Estimation of galactose-1-phosphate in erythrocytes; a rapid and simple enzymatic method. Clin Chim Acta 1969 November;26:313-316)