An interference-free two-step enzyme assay with UPLC-tandem mass spectrometric product measurement for the clinical diagnosis of uridine diphosphate galactose-4-epimerase deficiency.

We present a robust clinical assay for the measurement of red blood cell uridine diphosphate galactose-4-epimerase enzyme activity for the diagnostic confirmation of patients positive for a newborn screen for inherited galactosemia in whom galactose-1-phosphate uridyltransferase activity is normal. Previous assays required the use of ion-pairing reagents and frequent need for system maintenance that was not appropriate for heavy clinical use where patient results should be quickly available. We have designed a two-step enzyme assay which converts stable-isotope-labeled UDP-galactose to isotope-labeled-UDP-glucose which is converted in the second reaction to the final product of [(13)C6]-UDP-glucuronic acid. Measurement conditions t remove potential interference from endogenous UDP-glucose and UDP-galactose. We also report a significant ion suppression effect of the red cell preparation for which we have optimized assay sample volume to minimize this effect.

Liquid chromatography-tandem mass spectrometry enzyme assay for UDP-galactose 4'-epimerase: use of fragment intensity ratio in differentiation of structural isomers.

BACKGROUND: Distinction between asymptomatic and potentially clinically significant forms of galactosemia due to UDP-galactose 4'-epimerase (GALE) deficiency requires enzyme measurement in erythrocytes and other cells. We sought to develop a GALE assay using a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. METHODS: The reversible GALE assay was conducted with UDPGal as a substrate. The coeluting reaction product, uridine diphosphate glucose (UDPGlc), and its isomeric substrate, uridine diphosphate galactose (UDPGal), were detected by MS/MS at mass transitions 565 > 280, 565 > 241 and 565 > 403. The UDPGal was enriched in mass transition 565 > 403 compared with UDPGlc, whereas the UDPGlc was enriched in the mass transition 565 > 241 compared with UDPGal. The percentage of UDPGal in the reaction mixture was calculated by use of the ratio of ion intensities of the 2 daughter ions and a fourth-order polynomial calibrator curve. RESULTS: The method yielded a mean (SD) GALE activity of 9.8 (2.2) µmol · g(-1) hemoglobin · h(-1) in erythrocyte extracts from 27 controls. The apparent Km of the substrate, UDPGal, was 0.05 mmol/L. The GALE activity ranged from 433 to 993 µmol · g(-1) protein · h(-1) in control lymphoblast extracts. In a blinded test of 22 subjects suspected of GALE deficiency, we identified 6 individuals whose residual activities were below the range of controls, compatible with intermediate GALE deficiency. CONCLUSIONS: This assay can be used to distinguish the different forms of GALE deficiency. From an analytical standpoint, differentiating isomers on the basis of fragment intensity ratios should also prove useful for analogous enzymatic studies involving substrates and products that are structural isomers.

Results of newborn screening for galactose metabolic disorders.

A screening strategy has been used which uses the Paigen and Beutler methods for the determination of galactose and galactose-1-phosphate. A blood spot test for epimerase has also been developed. In the last 10 years, 265,019 samples from newborns have been tested by these methods. Among the 154 screening positives, we have detected seven cases of epimerase-deficient galactosaemia (Type III), seven cases of Duarte/galactosaemia heterozygotes, 48 cases of other various types of heterozygotes, four cases of persistent hypergalactosaemia, three cases of hepatitis and one case of congenital atresia of the bile duct. These results indicate that our screening system has effectively detected the infants with galactose metabolic disorders.

Galactosaemia: a new severe variant due to uridine diphosphate galactose-4-epimerase deficiency.

A baby presented on day 5 with symptoms of classical galactosaemia which are believed to be owing to a lack of uridine diphosphate-4-epimerase, rather than to the usual galactose-1-phosphate uridyl transferase defect. Apart from galactosaemia the condition was characterised biochemically by a red cell accumulation of galactose-1-phosphate and uridine diphosphate galactose. Galactose restriction modified the acute clinical and biochemical abnormality, but it appears essential to include some galactose in the diet in this condition to allow synthesis of galactosides, including the brain gangliosides.

A new mass screening method of detecting UDP-galactose-4-epimerase deficiency.

A new mass screening method for detecting UDP-galactose-4-epimerase deficiency in newborn infants was devised so as to make a useful addition to the Guthrie test. Like Beutler's method, this method enables one to perform convenient spot tests on the basis of fluorometry of NADH. The preferred procedure was determined to be as follows: To a mixture of 10 microliter of 5-10 mM UDP-galactose, 10 microliter of 26 mM NAD, 10 microliter of a 1 M tris-HCl buffer (pH 8.0) and 10 microliter of a 1/100 dilution of a 5 mg/ml UDP-glucose dehydrogenase stock solution, distilled water is added until the total volume reaches 100 microliter. Then a blood-bearing filter paper disc is added to the mixture and, the reaction mixture is incubated at 37 degrees C for 6-17 hr. The resulting test solution is spotted on a Whatman No. 1 filter paper with a capillary tube, air-dried and tested for fluorescence with an ultraviolet light detector. No cases of UDP-galactose-4-epimerase deficiency were found in the present study.

A simple assay for uridine diphosphate galactose 4-epimerase activity.

A simple fluorescent test for the activity of blood uridine diphosphate galactose 4-epimerase which converts uridine diphosphate galactose to uridine diphosphate glucose is described. The enzyme activity is visually estimated by to reduction of NAD+ (non fluorescent) to NADH (fluorescent) in a coupled reaction with uridine diphosphate glucose dehydrogenase. The appearance of fluorescence indicates that epimerase activity is present in the sample. The usefulness of this test in newborn screening programs for inborn errors of galactose metabolism is stressed.

Uridine diphosphate galactose 4'-epimerase deficiency. IV. Report of eight cases in three families.

Eight persons who had no activity of uridine diphosphate galactose 4'-epimerase in their circulating blood cells are known today. They were healthy members of three different families. Propositi were newborns discovered in a mass screening test for blood galactose which registered high levels of erythrocyte galactose-1-phosphate. Epimerase deficiency was restricted to circulating blood cells, but in liver biopsy specimens, in cultured skin fibroblasts and in activated lymphocytes epimerase was found to be normally active. Heterozygotes had intermediate red cell epimerase activity. There were no symptoms of galactose intolerance and no pathology related to the enzyme defect. All 8 epimerase deficient persons had a ccdee Rhesus genotype. Attempts at demonstrating genetic linkage between the epimerase and Rhesus loci were unsuccessful because of the difficulty encountered in ascertaining epimerase heterozygosity. Individuals with hereditary UDP-galactose 4'-epimerase deficiency appear to produce an unstable mutant enzyme requiring higher NAD concentration for maximum activity.

Reversal of UDP-galactose 4-epimerase deficiency of human leukocytes in culture.

Stimulation with phytohemagglutinin of the leukocytes from six of the seven known individuals with UDP-galactose 4-epimerase (= UDP-glucose 4-epimerase; EC 5.1.3.2) deficiency consistently resulted in the appearance of epimerase activity in the cultured cells. A long-term lymphoblast culture derived from one proband also contained an active epimerase enzyme. A comparison of the properties of this enzyme with those of epimerase produced by control lymphoblast lines revealed comparable Km values for UDP-galactose and NAD and identical behavior on polyacrylamide electrophoresis. However, a difference in the NAD requirement for heat stability at 40 degree provided some evidence for a structural defect in this enzyme. Possible explanations for the appearance of UDP-galactose 4-epimerase activity in stimulated lymphocytes include an increased rate of synthesis of a mutant enzyme and a derepression of an epimerase locus during lymphocyte transformation.