Neonatal screening for galactosemia by quantitative analysis of hexose monophosphates using tandem mass spectrometry: a retrospective study.

BACKGROUND: Classic galactosemia (OMIM 230400) is an inherited disorder in the metabolism of galactose caused by deficiency of the enzyme galactose 1-phosphate uridyl transferase (EC 2.7.7.12). Galactosemia leads to accumulation of galactose and galactose 1-phosphate (gal-1-P) in blood and tissues and, if untreated, produces neonatal death or severe mental retardation, cirrhosis of the liver, and cataracts. Hence, the disorder is included in many neonatal screening programs. METHODS: We retrospectively analyzed filter-paper blood samples obtained 4-8 days postpartum for routine neonatal screening from 12 galactosemia patients and 2055 random controls. Total hexose monophosphates (HMPs) were used as a marker of gal-1-P and were assayed by negative-ion mode electrospray tandem mass spectrometry (tandem MS) with settings biased toward gal-1-P detection. The predominant precursor/product ion pair m/z 259/79 was used to quantify total HMPs by external standardization. RESULTS: Linear calibration curves were obtained in the range 0-8 mmol/L gal-1-P. The detection limit was 0.1 mmol/L HMP, and total CVs ranged from 13% at the detection limit to <8% at >1 mmol/L HMP. The method was in agreement with an alkaline phosphatase-galactose dehydrogenase method. All samples from galactosemia patients contained increased HMP concentrations (range for patients, 2.6-5.2 mmol/L; range for reference group, <0.10-0.94 mmol/L). The diagnostic sensitivity and specificity were 100% at a cutoff of 1.2 mmol/L HMP. A Duarte/classic galactosemia compound heterozygous sample could be discriminated clearly from both patient and reference samples. CONCLUSION: Quantitative analysis of HMPs by tandem MS can be used in laboratory investigations of galactosemia.

A new method of blood galactose estimation for mass screening of galactosemia.

A new method for quantitative determination of galactose in blood by fluorescence of NADH was described. The assay system consisted of beta-galactose dehydrogenase, NAD, buffer and a denatured blood disc (3 mm diameter), and the reaction was carried out for 1 hr at 37 degrees C. Denaturation of hemoglobin was accomplished by exposing the blood disc to a vapor of formic acid in an air-tight container; this procedure completely eliminated false positive cases of galactosemia. This method can be applied in a wide range of galactose concentration from low (0 mg%) to high levels (200-1,000 mg%) with the accuracy of 8.0 +/- 0.3 mg% from a coefficient of variation of 3.5%. Semi-quantitative assay was also possible by using a spot test like Beutler's method. The galactose content in one disc paper (3 mm diameter) of blood containing 5 mg% galactose is approximately 0.1 micrograms. The newly developed method is satisfactorily applicable in neonatal mass screening and clinical cases.