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Problems in the Diagnosis of Transferase and Galactokinase Deficient Galactosemia

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Problems in the Diagnosis of Transferase and Galactokinase Deficient Galactosemia ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 10, No. 1 Copyright © 1979, Institute for Clinical Science, Inc. Problems in the Diagnosis of Transferase and Galactokinase Deficient Galactosemia M IC H A EL A. PESC E, Ph .D.* and SELMA H. BODOURIAN, M.S. Special Chemistry Laboratory, Columbia-Presbyterian Medical Center, New York, NY 10032 ABSTRACT Galactose in serum and galactose-l-phosphate in erythrocytes were measured in six transferase deficient children to determine if these metabo­ lites could be used in detecting transferase deficient galactosemia. In all six children the galactose levels were normal and the galactose-l-phosphate elevated. The galactose level depends on diet and the rate of metabolism to galactose-l-phosphate and, therefore, should not be used to predict trans­ ferase deficient galactosemia. The galactose-l-phosphate level was ele­ vated in all the transferase deficient children because once formed it can­ not be metabolized. Measurement of galactose-l-phosphate is difficult and is usually requested to determine whether or not the child is following the galactose restricted diet. In transferase deficient galactosemia, the enzyme hexose-l-phosphate uridylyltransferase is absent. The diagnosis should be determined by measurement of the activity of the enzyme hexose-l- phosphate uridylyltransferase in erythrocytes. In galactokinase deficient galactosemia, the enzyme galactokinase is ab­ sent. Galactose levels are elevated but the amount present depends on diet and how soon the blood was collected after the ingestion of galactose con­ taining foods. The diagnosis of galactokinase deficient galactosemia is based on the measurement of the enzyme galactokinase in erythrocytes. Introduction tose is metabolized to uridine diphospho- glucose by the series of reactions shown in Transferase and galactokinase deficient table I. In transferase deficient galacto­ galactosemia are inborn errors of galac­ semia, the enzyme hexose-l-phosphate tose metabolism. The main source of uridylyltransferase* is absent. In this dis­ galactose is from the disaccharide lactose order, vomiting, diarrhea, jaundice and which is found in milk. Lactose is hy­ failure to thrive are the earliest and most drolyzed in the intestine by the enzyme lactase to galactose and glucose. Galac- * This enzyme is frequently referred to as galactose-l-phosphate uridylyltransferase. Hexose- * Address correspondence to Michael A. Pesce, l-phosphate uridylyltransferase (EC 2.7.7.12) is the Ph.D., Special Chemistry Laboratory, Columbia- name designated by the Commission On Bio­ Presbyterian Medical Center, 622 West 168th Street, chemical Nomenclature and will be used through­ Box 253, New York, NY 10032. out this article. 26 0091-7370/0100-0026 $01.20 © Institute for Clinical Science, Inc. GALACTOSEMIA 2 7 TABLE I Normal Metabolic Pathway for Galactose Galactokinase Galactose + ATP --------------------------------------------- ^ Galactose-l-phosphate + ADP Mg++ ' Hexose-l-phosphate uridylyltransferase Galactose-l-phosphate + ---------------------------------------------^ Glucose-l-phosphate + uridine diphosphoglucose ^ -------------------------------- 1---------- uridine diphosphogalactose Uridine diphosphogalactose-4-epimerase Uridine diphosphogalactose ---------------------------------- -----------^ Uridine diphosphoglucose NAD+ Uridine diphosphoglucose pyrophosphorylase Uridine diphosphoglucose + PP ---------------------------------------------- ^ Glucose-l-phosphate + UTP common symptoms and usually appear usually confirmed by measuring the activ­ after milk is given to the infant. If milk ity of the enzymes hexose-l-phosphate feedings are continued, hepatic failure re­ uridylyltransferase and galactokinase in sults, infection may occur, cataracts and erythrocytes. It has been suggested that mental retardation may develop and death elevated levels of galactose5,7 or can result. In galactokinase deficient galactose-l-phosphate3 could be used to galactosemia, the enzyme galactokinase* detect transferase deficient galactosemia is absent. In this disorder, cataracts are the because their rate of metabolism would be only symptom. None of the severe slow and, therefore, would remain in the symptoms associated with transferase de­ blood for a considerable length of time ficient galactosemia are observed. after the consumption of milk. To deter­ Treatment for both of these disorders is mine if this assumption was correct, a galactose restricted diet. If initiated in hexose-l-phosphate uridylyltransferase the newborn period, the clinical problems activity* galactose and galactose-l- can usually be prevented. Therefore, it is phosphate levels were measured in nor­ important that the diagnosis be deter­ mal and transferase deficient children. mined as soon as possible after birth. These results and some of the pitfalls that The diagnosis of transferase and can be encountered in the diagnosis of galactokinase deficient galactosemia is transferase and galactokinase deficient • * (EC 2.7.1.6). galactosemia will be presented. TABLE II Reaction Scheme for Measurement of Hexose-l-phosphate Uridylyltransferase Activity Hexose-l-phosphate uridylyltransferase Galactose-l-phosphate + ------------------------------------------^ Glucose-l-phosphate + uridine diphosphoglucose uridine diphosphogalactose Pho sphog1ucomutase Glucose-l-phosphate ------------------------------------------- -------- — ^ Glucose-6-phosphate Glucose-6-phosphate dehydrogenase Glucose-6-phosphate + NADP+ ----------------------------------------- ^ 6-Pho sphogluconate + NADPH Phosphogluconate dehydrogenase 6-Phosphogluconate + NADP+ ------------------------------------------> Ribulose-5-phosphate + NADPH + C02 28 PESCE AND BODOURIAN M ethods formed was measured fluorometrically Hexose-1-phosphate uridylyltrans- and related to the concentration of the ferase activity was measured in erythro­ galactose-l-phosphate in the sample. cytes by a kinetic enzymatic system9 Galactose-l-phosphate concentration is based on the reaction scheme shown in reported as /u, g of hemoglobin. table II. Blood was collected in am­ monium heparinized microhematocrit R esults tubes, the erythrocytes lysed with water and the hexose-l-phosphate uridylyl- Hexose-l-phosphate uridylyltrans­ transferase activity determined by mixing ferase activity, galactose and galactose- the hemolysate with a reagent consisting l-phosphate values were measured in ten of galactose-l-phosphate, uridine diphos- normal children who had been fasting phoglucose, NADP+, ethylene diamine- from 8 to 12 hours and in six transferase tetraacetate (EDTA) and the enzymes deficient children. The results for the phosphoglucomutase and glucose-6- normal group are shown in table III. The phosphate dehydrogenase. Phospho- galactose levels in serum ranged between gluconate dehydrogenase was not added 0.0 and 0.6 mg per dl, the galactose-l- to the reagent system because it is present phosphate levels in erythrocytes were in sufficient quantities in the hemolysate from 14 to 42 fig per g of hemoglobin and to convert all of the 6-phosphogluconate the hexose-l-phosphate uridylyltrans­ produced to ribulose-5-phosphate. EDTA ferase activity was between 18 and 28 activates the enzyme hexose-l-phosphate units per gram of hemoglobin. uridylyltransferase and must be added to The results for the transferase deficient the reagent system. This reaction mixture group are shown in table IV. In all six was incubated for 30 minutes at 37°C and children, the galactose-l-phosphate the activity of hexose-l-phosphate level was elevated, as compared to in­ uridylyltransferase determined by dividuals with normal hexose-l- measuring the rate of increase in the ab­ phosphate uridylyltransferase activity, sorbance of the NADPH for ten minutes. and ranged between 84 and 422 fig p er g of Enzyme activity is expressed as units per hemoglobin. All these children were sup­ gram of hemoglobin. One unit of activity posedly following a galactose restricted is defined as the number of micromoles of diet. Galactose-l-phosphate levels in substrate consumed per hour at a tempera­ transferase deficient patients who are ture of 37° C. adhering to their galactose restricted diet Galactose in serum was determined by should b e about 100 fig p er g of hem oglo­ measuring the increase in absorbance of bin. In all the transferase deficient chil­ the NADH formed from the reaction of dren, the galactose level in serum was galactose and NAD+ catalyzed by the en­ normal. The first four children were fol­ zyme galactose dehydrogenase.6 lowing their diet and, therefore, their Galactose-l-phosphate levels in galactose level in serum was expected to erythrocytes were measured by a new be normal. The last two children were fluorometric method.8 With this assay, consuming galactose containing foods, as blood was collected in ammonium indicated by their galactose-l-phosphate heparinized microhematocrit tubes, the level, but their galactose level in serum packed cells lysed with water and imme­ was also normal. diately deproteinized in tris buffer at The data in table IV indicate that 100°C. Galactose-l-phosphate in the whether or not the transferase deficient supernatant was measured by the series of child is on a galactose restricted diet, the reaction shown in table II. The NADPH galactose level in serum will be normal GALACTOSEMIA 29 and the galactose-l-phosphate level in TABLE I I I erythrocytes will be elevated. Galactose and Galactose-l-phosphate Levels in Ten Children with Normal Hexose-l-phosphate Uridylyltransferase
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