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 Activity D iscussion

Hexose-l- M easurements W h i c h P r o v i d e phosphate D i a g n o s is o f T r a n s f e r a s e D e f i c i e n t Uridylyl- Galactose-l- transferase G alactosemia Galactose phosphate Activity Sex mg/dl )ig/gHb U/gHb Hexose-l-phosphate uridylyltrans- ferase activity. Since the enzyme F 0.5 39 26 hexose-l-phosphate uridylyltransferase is F 0.4 26 22 M 0.4 42 28 absent in transferase deficient galac­ F 0.6 40 19 tosemia, the diagnosis should be based on M 0.5 32 26 F 0.0 41 21 the estimation of the activity of this en­ M 0.4 16 18 M 0.4 41 26 zyme in erythrocytes. However, meas­ F 0.3 14 18 urement of hexose-l-phosphate uridylyl­ F 0.6 40 19 Mean 0.4 33 22 transferase activity is not an easy task and is subject to various problems. For exam­ ple, the enzyme activity in normal indi­ viduals is low as compared to other natant is measured by reaction with erythrocyte enzymes. The activity of uridine diphosphoglucose dehydro­ hexose-l-phosphate uridylyltransferase genase and NAD+. The activity of in normal children is about 25 times hexose-l-phosphate uridylyltransferase is lower than the normal activity of related to the amount of uridine diphos­ glucose-6-phosphate dehydrogenase. phoglucose consumed. In patients with Therefore, a sensitive method must be transferase deficient galactosemia, no employed to measure transferase activity. uridine diphosphoglucose should be con­ With the kinetic system described in the sumed and the amount of uridine diphos­ Method Section, sufficient sensitivity for phoglucose found in the supernatant measuring hexose-l-phosphate uridylyl­ should be the same as that added into the transferase activity is achieved because hemolysate. The reason for detection of two moles of NADPH are formed for each transferase activity in this patient was the mole of glucose-6-phosphate oxidized. absence of NADase in the erythrocytes. It was recently shown that the analyt­ ical method employed for measuring TABLE IV hexose-l-phosphate uridylyltransferase Galactose and Galactose-l-phosphate Levels activity can influence the results because in Six Transferase Deficient Children some enzyme activity was detected in the Hexose-l- erythrocytes of a transferase deficient in­ phosphate fant when measured by the uridine di- Uridylyl- Galactose-l- transferase phosphoglucose (UDPG) consumption Age Galactose phosphate Activity assay.2 The UDPG consumption assay1 is Years Sex mg/dl \ig/gHb U/gHb based on the series of reactions shown in 11 M 0.6 84 1 table V. With this method, the hemolysate 4 M * 94 0 18 M 0.0 100 0 is incubated with galactose-l-phosphate 7 M 0.3 114 1 and uridine diphosphoglucose for a Newborn M 0.4 185 0 8 0.0 specified time and the reaction stopped ' M 422 1 by precipitation of the proteins. The *There was not enough serum to determine a galactose uridine diphosphoglucose in the super- level. 3 0 PESCE AND BODOURIAN

TABLE V

Reaction Scheme for the Uridine Diphosphoglucose Consumption Assay

Hexose-l-phosphate uridylyltransferase Galactose-l-phosphate + ------— Glucose-l-phosphate + uridine diphosphoglucose uridine diphosphogalactose

Uridine diphosphoglucose dehydrogenase Uridine diphosphoglucose ------^ Uridine diphosphoglucuronic acid + 2 NAD+ + 2 NADH

Uridine diphosphate galactose-4-epiiaerase Uridine diphosphoglucose ...— ■ — — ■ ...... - ■ — ^ Uridine diphosphogalactose

As shown in table V, uridine diphospho­ Galactose in Serum. Galactose is usu­ glucose can also be converted to uridine ally absent or present in small quantities diphosphogalactose by the erythrocyte in the serum of normal individuals. As enzyme uridine-diphosphate galactose- shown in table IV, the galactose level in 4-epimerase. This reaction is catalyzed by serum was normal in the transferase defi­ the NAD+ present in the hemolysate. cient individuals, including those chil­ NADase inactivates NAD+; therefore, no dren who had consumed galactose con­ uridine diphosphoglucose can be con­ taining foods, because the blood was sumed. Because of the absence of drawn several hours after the diet viola­ NADase in the erythrocytes of this pa­ tion. During this time, galactose was tient, uridine diphosphoglucose was con­ metabolized to galactose-l-phosphate by verted to uridine diphosphogalactose, er­ the enzyme galactokinase which is the roneously indicating that there was some first enzyme in the normal metabolic hexose-l-phosphate uridylyltransferase pathway of galactose (table I). Elevated activity. The absence of NADase in the galactose levels will be found in the erythrocytes will not interfere with the serum of transferase deficient individuals kinetic assay for measuring hexose-l- only if the blood is drawn soon after the phosphate uridylyltransferase activity be­ child has ingested galactose containing cause sufficient uridine diphospho­ foods. Galactose levels should not be used glucose has been added to assure that the to detect transferase deficient galacto­ small amount of uridine diphospho­ semia because the results depend on diet glucose converted to uridine diphospho­ and rate of metabolism. galactose will not alter zero order kinetics. Galactose-l-phosphate in Erythro­ For accurate enzyme results, hexose- cytes. Galactose-l-phosphate is present in l-phosphate uridylyltransferase activity small quantities in the erythrocytes of in­ should be measured soon after collection dividuals with normal transferase activity, of the blood. It was recently shown that 25 the upper limit of normal is about 42 ¡ig percent of the enzyme activity was lost if per g of hemoglobin. As shown in table IV, the blood or erythrocytes were stored at galactose-l-phosphate levels are elevated 4°C or atroom temperature for one day.9 A in all the transferase deficient children loss of about 10 p ercent in enzym e activity whether or not they have deviated from was observed if the erythrocytes were their diet because galactose-l-phosphate stored at — 20° C for one day.9 If the deter­ once formed by the phosphorylation of mination cannot be performed immedi­ galactose cannot be metabolized. ately, the packed cells can be stored for Galactose-l-phosphate levels of greater one day at — 20° C without producing a than 120 /xg per g o f hem oglobin are found significant change in enzyme activity. in transferase deficient individuals who GALACTOSEMIA 3 1 are consuming galactose containing foods. jaundice, liver damage or mental retarda­ Galactose-l-phosphate levels ofabout 100 tion that is associated with transferase de­ fig p er g of hem oglobin are observed even ficient galactosemia. In this disorder, in individuals who are following their galactose cannot be metabolized and is galactose restricted diet because they are usually elevated in serum. However, the probably ingesting, either by chance or galactose level depends on when the knowingly, small quantities of galactose blood was taken after the patient had con­ containing foods. sumed milk. It was reported that the galac» An elevated galactose-l-phosphate tose level in the serum of a galactokinase level does not necessarily indicate trans­ deficient individual who has fasted over­ ferase deficient galactosemia because in night was only 3 mg per dl.10 In galacto­ patients with erythrocyte uridinediphos- kinase deficient individuals, galactose- phate galactose-4-epimerase deficiency4 l-phosphate is absent because it cannot the galactose-l-phosphate level was b e formed. markedly elevated. Uridinediphosphate The diagnosis of galactokinase defi­ galactose-4-epimerase is the third enzyme cient galactosemia should be determined involved in the normal pathway of galac­ by measuring the activity of the enzyme tose metabolism and converts uridine di- galactokinase in erythrocytes. In order to phosphogalactose to uridine diphospho- measure galactokinase activity, a very glucose (table I). All these patients were sensitive method is required because the asymptomatic with normal hexose-1- activity of galactokinase in the erythro­ phosphate uridylyltransferase activity cytes of normal individuals is about 100 and with either normal or slightly ele­ times lower than the activity of glucose- vated galactose levels. The diagnosis was 6-phosphate dehydrogenase. An indirect confirmed by measuring the activity of the way to determine the possibility of enzyme uridinediphosphate galactose- galactokinase deficiency is to measure 4-epimerase in erythrocytes. galactose, galactose-l-phosphate levels Galactose-l-phosphate measurements and hexose-l-phosphate uridylyltrans­ are difficult to perform and are generally ferase activity. If the patient has cataracts, used to determine if the transferase defi­ elevated levels of galactose with the ab­ cient individual has deviated from the sence of galactose-l-phosphate and nor­ galactose restricted diet. mal hexose-l-phosphate uridylyltrans­ Conclusion. The possibility of trans^ ferase activity, the possibility of galacto­ ferase deficient galactosemia should be kinase deficient galactosemia should be considered based on the clinical considered. However, the only way to symptoms of the patient and the finding of confirm the diagnosis of this disorder is to elevated galactose and galactose-l- measure the activity of the enzyme phosphate levels. However, the diagnosis galactokinase in erythrocytes. must be confirmed by measurement of the activity of the enzyme hexose-1- R eferences phosphate uridylyltransferase in erythro­ 1. Anderson, E. P., K a lc k a r , H. M., K u r a k a sh i, cytes. K., and ISSELLACHER, K. J.: A specific enzyma­ tic assay for the diagnosis of congenital galacto­ semia. I. The consumption test. J. Lab. Clin. D i a g n o s is o f G alactokinase Med. 50:469-477, 1957. D e f i c i e n t G alactosemia 2. D e B r u y n , C. H. M. M., R a y m a k e r s , C., WENSING, A., and O e i , T. L.: Galactose-l- G alactokinase deficient galactosem ia is phosphate uridylyltransferase activities in a rare disorder and is defined as absence erythrocytes from a patient with galactosemia: of the enzyme galactokinase (table I). Discrepancy between two methods. Clin. Chim. Acta 78:145-150, 1977. Cataracts aré the only clinical symptom. 3. Gitzelmann, R.: Estimation of galactose-l- There is none of the vomiting, diarrhea, phosphate in erythrocytes: A rapid and simple 3 2 PESCE AND BODOURIAN

enzymatic method. Clin. Chim. Acta 26:313- 7. M a s o n , G. A., Su m m e r , G . K., D u t t o n , H. H., 316, 1969. and SCHWANER, R. C., J r .: Automated fluoro- 4. G it z e l m a n n , R., St e in m a n n , B., M it c h e l l , metric analysis of galactose in blood. Clin. B., and HaiGIS, E.: Uridine diphosphate Chem. 23:971-974, 1977. galactose-4-epimerase deficiency. IV. Report of 8. PESCE, M. A. and BODOURIAN, S. H.: A new eight cases in three families. Helv. Pediat. Acta method for measuring galactose-l-phosphate in 31:441-451, 1976. erythrocytes. Clin. Chem. 23:1166, 1977. 5. GRENIER, A. and LABERGE, C.: Rapid method 9. Pesce, M. A., Bodourian, S. H., H a r r is , R. H., for screening for galactosemia and galacto- and NICHOLSON, J. F.: Enzymatic micromethod kinase deficiency by measuring galactose in for measuring galactose-l-phosphate uridylyl- whole blood spotted on paper. Clin. Chem. transferase activity in human erythrocytes. 19:463-465, 1973. Clin. Chem. 23:1711-1717, 1977. 6. H je l m , M. and TENGSTROM, B.: Enzymatic de­ 10. T h a l h a m m e r , O., G i t z e l m a n n , R., and termination of blood galactose with galactose P antlitschko , M .: Hypergalactosemia and oxidase and galactose dehydrogenase. galactosemia due to galactokinase deficiency in Biochem. Med. 2:174—176, 1968. a newborn. Pediatrics 42:441-445, 1968.