Development of a new HPLC diagnostic method for galactosemia using 8-amino-2-naphthalenesulfonic acid

In a metabolic pathway from galactose to glucose-1-phosphate, there are thr ee major enzymes, galactokinase, galactose-1-phosphate uridyl transferase ( GALT) and UDP-galactose-4-epimerase. The deficiency of one of these enzymes causes accumulation of galactose in blood, which provides a pathognomonic marker. A new reversed-phase HPLC method with fluorescence detector has bee n developed for the measurement of galactose in 50 muL of blood on Guthrie filter paper using 8-amino-2-naphthalenesulfonic acid (8,2-ANS) as derivati zation reagent for the diagnosis of Galactosemia. Galactose was extracted f rom blood spotted on filter paper and derivatized with 8,2-ANS to produce S chiff bases, and reduced with sodium cyanoborohydride. Linear range was fro m 2 mg dL(-1) to 20 mg dL(-1) (r = 0.9998). Detection limit (S/N = 3) of th is method was 90 ng dL(-1). The mean recovery of galactose was 102.7% (SD = 0.3%, n = 14). The normal range of blood galactose in Korean neonates (spe cimen collected within 7 days after birth) was below 6 mg dL(-1) (n = 5 for each gender) without any gender difference. When applied to 11 anonymous b lood spots of heterogeneous genotypes of GALT deficiency all of the patient s' blood samples showed abnormal elevation of galactose, The results indicate that the new method using 8,2-ANS yields consistent an d correct galactose determination that is simple and practical as a rapid f irst screening tool for patients with galactosemia.