Problems in the Diagnosis of Transferase and Galactokinase D eficient Galactosemia

Transcription

1 AN NALS O F CLINICA L A N D LABORATORY SC IENCE, Vol. 10, No. 1 Copyright 1979, Institute for Clinical Science, Inc. Problems in the Diagnosis of Transferase and Galactokinase D eficient Galactosemia M IC H A EL A. PE SC E, P h.d.* and SELM A H. BODOURIAN, M.S. Special Chemistry Laboratory, Columbia-Presbyterian Medical Center, New York, NY ABSTRACT G alactose in serum and g alactose-l-phosphate in erythrocytes w ere m easured in six transferase deficient children to determ ine if th ese m etabolites could be used in detecting transferase deficient galactosem ia. In all six children the galactose levels w ere norm al and the galactose-l-phosphate elevated. T he galactose level depends on d iet and the rate o f m etabolism to galactose-l-phosphate and, therefore, should not be u sed to predict transferase deficient galactosem ia. T he galactose-l-phosphate level was elevated in all the transferase deficient children because once form ed it cannot be m etabolized. M easurem ent o f galactose-l-phosphate is difficult and is usually requested to determ ine w hether or not the child is follow ing the galactose restricted diet. In transferase deficien t galactosem ia, the enzym e hexose-l-phosphate uridylyltransferase is absent. T he diagnosis should be d eterm in ed by m easu rem en t o f th e activity o f th e enzym e h ex o se-lpho sph ate uridylyltransferase in erythrocytes. In galactokinase d efic ien t galactosem ia, the enzym e galactokinase is absent. Galactose levels are elevated b u t the am ount p resen t depends on d iet and how soon the blood was collected after the ingestion of galactose containing foods. T he diagnosis of galactokinase deficien t galactosem ia is based on the m easurem ent o f the enzym e galactokinase in erythrocytes. Introduction T ransferase and galactokinase deficient galactosem ia are inborn errors o f galacto se m etab o lism. T h e m ain so u rce of galactose is from the disaccharide lactose w hich is found in m ilk. L actose is hydrolyzed in the intestine by the enzym e lactase to galactose and glucose. Galac- * Address correspondence to Michael A. Pesce, Ph.D., Special Chem istry Laboratory, Columbia- Presbyterian Medical Center, 622 W est 168th Street, Box 253, New York, NY tose is m etabolized to uridine diphosphoglucose by the series of reactions show n in tab le I. In transferase d eficien t galactosem ia, th e enzym e hexose-l-phosphate uridylyltransferase* is absent. In this disorder, vom iting, diarrhea, jaundice and failure to thrive are the earliest and m ost * T his enzym e is freq u en tly re fe rre d to as galactose-l-phosphate uridylyltransferase. Hexosel-phosphate uridylyltransferase (EC ) is the nam e designated by the C om m ission On Biochemical Nomenclature and will be used throughout this article / $01.20 Institute for Clinical Science, Inc.

2 GALACTOSEM IA 2 7 TABLE I Normal Metabolic Pathway for Galactose Galactokinase Galactose + ATP ^ Mg++ ' Galactose-l-phosphate + ADP Hexose-l-phosphate uridylyltransferase Galactose-l-phosphate ^ Glucose-l-phosphate + uridine diphosphoglucose ^ uridine diphosphogalactose Uridine diphosphogalactose-4-epimerase Uridine diphosphogalactose ^ Uridine diphosphoglucose Uridine diphosphoglucose pyrophosphorylase Uridine diphosphoglucose + PP ^ Glucose-l-phosphate + UTP NAD+ com m on sym ptoms an d usually appear after m ilk is given to the infant. If milk feedings are continued, hepatic failure re sults, infection may occur, cataracts and m ental retardation may develop and death can re su lt. In g alacto k in ase d e fic ie n t galactosem ia, the enzym e galactokinase* is absent. In this disorder, cataracts are the o n ly sy m p to m. N o n e o f th e se v e re sym ptom s associated w ith transferase d e ficien t galactosem ia are observed. T reatm en t for both of these disorders is a galactose restricted diet. If initiated in the new born period, the clinical problem s can usually be prevented. Therefore, it is im portant that th e diagnosis b e d eterm in ed as soon as possible after birth. T h e d ia g n o sis o f tra n s fe ra s e an d galactokinase d efic ien t g alactosem ia is * (EC ). usually confirm ed by m easuring the activity of the enzym es hexose-l-phosphate uridylyltransferase and galactokinase in erythrocytes. It has been suggested that e le v a te d le v e ls o f g a la c to s e 5,7 or galactose-l-phosphate3 could be used to d etect transferase deficient galactosem ia because their rate of m etabolism w ould be slow and, therefore, w ould rem ain in the blood for a considerable length of tim e after the consum ption of milk. To d eterm ine if th is assu m p tio n w as co rrect, h ex o se-l-p h o sp h ate uridylyltransferase activity* g ala c to se an d g a la c to s e -lpho sph ate levels w ere m easu red in normal and transferase deficient children. T hese results and som e of the pitfalls that can be encountered in the diagnosis of transferase and galactokinase deficient galactosem ia w ill be presented. T A BLE 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 6-Phosphogluconate + NADP+ Phosphogluconate dehydrogenase > Ribulose-5-phosphate + NADPH + C02

3 28 PESC E AND BODOURIAN M ethods H e x o se -1 -p h o sp h a te u rid y ly ltra n s - ferase activity was m easu red in eryth rocytes by a k in etic en zy m atic sy ste m 9 based on the reaction schem e show n in ta b le II. B lood w as c o lle c te d in am m onium h e p a rin iz e d m icro h em ato crit tubes, the erythrocytes lysed w ith w ater an d th e h e x o se -l-p h o sp h a te u rid y ly l- transferase activity d eterm ined by m ixing the hem olysate w ith a reagent consisting o f galactose-l-phosphate, uridine diphosphoglucose, N A D P+, ethylene diam inetetra aceta te (ED TA ) and th e enzym es p h o s p h o g lu c o m u ta s e an d g lu co se-6 - p h o s p h a te d e h y d ro g e n a se. P h o sp h o - gluconate dehydrogenase was n o t added to the reag en t system because it is p re sen t in sufficient quantities in the hem olysate to convert all of the 6-phosphogluconate produced to ribulose-5-phosphate. ED TA activates the enzym e hexose-l-phosphate uridylyltransferase and m ust be added to th e reagent system. This reaction m ixture was incubated for 30 m inutes at 37 C and th e a c tiv ity o f h e x o s e -l-p h o s p h a te u rid y ly ltra n s fe ra s e d e te rm in e d by m easuring the rate of increase in the absorbance of the N A D PH for ten m inutes. Enzym e activity is expressed as units per gram of hem oglobin. O ne unit of activity is defined as th e num ber of m icrom oles of substrate consum ed per hour at a tem perature of 37 C. Galactose in serum was determ ined by m easuring the increase in absorbance of the NADH form ed from the reaction of galactose and NAD+ catalyzed by the enzym e galactose dehydrogenase.6 G a la c to s e -l-p h o s p h a te le v e ls in erythrocytes w ere m easured by a new fluorom etric m eth o d.8 W ith this assay, b lo o d w as c o lle c te d in am m o n iu m heparinized m icrohem atocrit tubes, the packed cells lysed w ith w ater and im m e d iately d e p ro te in iz e d in tris b u ffer at 100 C. G a la c to s e -l-p h o s p h a te in th e supernatant was m easured by the series of reaction show n in table II. T he NADPH form ed was m easu red fluorom etrically and related to the concentration o f the g a la c to se -l-p h o sp h a te in th e sam p le. G alactose-l-phosphate concentration is reported as /u, g of hem oglobin. R esults H e x o s e -l-p h o s p h a te u rid y ly ltr a n s ferase activity, galactose and galactosel-phosphate values w ere m easured in ten norm al children w ho had b e e n fasting from 8 to 12 hours and in six transferase d efic ien t child ren. T h e resu lts for th e norm al group are show n in table III. T he galactose levels in serum ranged b etw een 0.0 and 0.6 mg p er dl, the galactose-lp h o sp h ate levels in erythrocytes w ere from 14 to 42 fig p er g of hem oglobin and th e h e x o s e -l-p h o sp h a te u rid y ly ltra n s ferase activity was b etw een 18 and 28 u nits p er gram of hem oglobin. T he results for the transferase deficient group are show n in table IV. In all six c h ild re n, th e g a la c to s e -l-p h o s p h a te level was elevated, as com pared to in d iv id u a ls w ith n o rm al h e x o s e -lp h o sp h ate u rid y ly ltran sferase activity, and ranged b etw een 84 and 422 fig p er g of hem oglobin. All these children w ere supposedly follow ing a galactose restricted d iet. G a la c to se -l-p h o sp h a te le v e ls in tran sferase d efic ien t p a tie n ts w ho are adhering to th eir galactose restricted d iet should b e about 100 fig p er g of hem oglobin. In all the transferase deficient children, the galactose level in serum was norm al. T he first four children w ere follow ing th e ir d iet and, th erefo re, th eir galactose level in serum was expected to be norm al. T he last two children w ere consum ing galactose containing foods, as indicated by th eir galactose-l-phosphate level, b u t their galactose level in serum was also norm al. T h e d ata in ta b le IV in d ic a te th a t w h eth er or not the transferase deficient child is on a galactose restricted diet, the galactose level in serum w ill be norm al

4 GALACTOSEM IA 29 and the galactose-l-phosphate level in erythrocytes w ill be elevated. D iscussion M e a s u r e m e n t s W h i c h P r o v i d e D i a g n o s i s o f T r a n s f e r a s e D e f i c i e n t G a l a c t o s e m i a H exose-l-phosphate uridylyltransferase activity. S in c e th e e n z y m e hexose-l-phosphate uridylyltransferase is a b s e n t in tran sfera se d e fic ie n t g alactosem ia, the diagnosis should be based on the estim ation o f the activity of this en zym e in erythrocytes. H ow ever, m easurem ent of hexose-l-phosphate uridylyltransferase activity is not an easy task and is subject to various problem s. F or exam ple, th e en zym e activity in norm al in d i v id u a ls is low as co m p a re d to o th e r ery th ro c y te en zy m es. T h e activ ity of h ex o se-l-p h o sp h ate uridylyltransferase in norm al c h ild re n is ab o u t 25 tim es lo w e r th a n th e n o rm al a c tiv ity o f g lu c o s e -6 -p h o s p h a te d e h y d ro g e n a se. T herefore, a sensitive m ethod m ust be em ployed to m easure transferase activity. W ith the kinetic system described in the M ethod Section, sufficient sensitivity for m easuring hexose-l-phosphate uridylyltransferase activity is achieved because two m oles of N ADPH are form ed for each m ole o f glucose-6-phosphate oxidized. Sex TABLE I I I Galactose and Galactose-l-phosphate Levels in Ten Children with Normal Hexose-l-phosphate Uridylyltransferase Activity Galactose mg/dl Hexose-lphosphate Uridylyltransferase Activity U/gHb Galactose-lphosphate )ig/ghb It was recently show n that the analytical m eth o d e m p lo y ed for m easu rin g h ex o se-l-p h o sp h ate u ridylyltransferase activity can influence the results because som e enzym e activity was d etected in the erythrocytes of a transferase d eficien t infant w hen m easured by the uridine diphosp h o g lu co se (U D PG ) co n sum ption assay.2 T he U D P G consum ption assay1 is based on the series of reactions show n in table V. W ith this m ethod, the hem olysate is incubated w ith galactose-l-phosphate an d u rid in e d ip h o s p h o g lu c o s e fo r a specified tim e and the reaction stopped by p re c ip ita tio n of th e p ro tein s. T h e u rid in e dipho spho glucose in th e super- F F M F M F M M F F Mean n a ta n t is m e a s u re d by re a c tio n w ith u rid in e d ip h o s p h o g lu c o s e d e h y d ro g e n a se a n d N A D +. T h e a c tiv ity o f hexose-l-phosphate uridylyltransferase is related to the am ount of uridine diphosphoglucose consum ed. In patients w ith tran sferase d e fic ie n t galactosem ia, no uridine diphosphoglucose should b e consum ed and the am ount of uridine diphosp h o g lu co se fo u n d in th e s u p e rn a ta n t should be the same as that added into the hem olysate. T he reason for detection of transferase activity in this p atien t was the absen ce o f N A D ase in the erythrocytes. Age Years TABLE IV Galactose and Galactose-l-phosphate Levels in Six Transferase Deficient Children Sex Galactose mg/dl Hexose-lphosphate Uridylyltransferase Activity U/gHb Galactose-lphosphate \ig/ghb 11 M M * M M Newborn M ' M *There was not enough serum to determine a galactose level.

5 3 0 PESC E 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 diphosphoglucose can also be converted to uridine diphosphogalactose by th e erythrocyte enzym e u rid in e-d ip h o sp h ate galactose- 4-epim erase. This reaction is catalyzed by th e N A D + p re s e n t in th e hem olysate. NADase inactivates N A D +; therefore, no u rid in e diphosphoglucose can b e consu m ed. B ecause o f th e a b s e n c e o f N A D ase in th e erythrocytes of this p a tient, u rid in e diphosphoglucose was converted to uridine diphosphogalactose, erroneously indicating that th ere was som e h ex o se-l-p h o sp h ate uridylyltransferase activity. T he absence of NADase in the erythrocytes will not interfere w ith the k in etic assay for m easu rin g h ex o se -lphosphate uridylyltransferase activity b e cause s u ffic ie n t u rid in e d ip h o s p h o glucose has b een added to assure that the sm all a m o u n t o f u rid in e d ip h o s p h o glucose converted to uridine diphosphogalactose w ill not alter zero o rd er kinetics. For accurate enzym e results, hexosel-p h o sp h ate uridylyltransferase activity should b e m easured soon after collection o f the blood. It was recently show n that 25 percent of the enzym e activity was lost if the blood or erythrocytes w ere stored at 4 C or atroom tem perature for one day.9 A loss of about 10 p ercen t in enzym e activity was observed if th e erythrocytes w ere stored at 20 C for one day.9 I f the d eterm ination cannot be perform ed im m ediately, the packed cells can be stored for one day at 20 C w ithout producing a significant change in enzym e activity. Galactose in Serum. G alactose is u su ally absent or p resen t in sm all quantities in the serum of norm al individuals. As show n in table IV, the galactose level in serum was norm al in the transferase deficien t individuals, in clu d in g those children w ho had co n su m ed galactose contain in g foods, b ecau se th e b lo o d was d raw n several hours after the d ie t violatio n. D u rin g th is tim e, g alacto se was m etabolized to galactose-l-phosphate by the enzym e galactokinase w hich is the first en zy m e in th e norm al m etab o lic pathw ay of galactose (table I). E levated galactose lev els w ill b e fo u n d in th e serum of transferase deficient individuals only if the blood is draw n soon after the child has ingested galactose containing foods. Galactose levels should not be used to d etect transferase d efic ien t galactosem ia because the results d ep end on diet and rate of m etabolism. G alactose-l-phosphate in E rythrocytes. G alactose-l-phosphate is p re sen t in sm all quantities in the erythrocytes of individuals w ith norm al transferase activity, the u pper lim it o f norm al is about 42 ig per g of hem oglobin. As show n in table IV, g alactose-l-phosphate levels are elevated in all the transferase deficient children w h eth er or not th ey have deviated from th eir d iet because galactose-l-phosphate once form ed by the phosphorylation of g alacto se c a n n o t b e m e ta b o liz e d. G alactose-l-phosphate levels of greater th an 120 /xg per g o f hem oglobin are found in transferase d eficien t in dividuals w ho

6 GALACTOSEM IA 31 are consum ing galactose containing foods. G alactose-l-phosphate levels o fabout 100 fig p er g of hem oglobin are observed even in in dividuals w ho are follow ing th eir galactose restricted d ie t because they are probably ingesting, eith er by chance or knowingly, sm all q uantities of galactose containing foods. A n e le v a te d g a la c to s e -l-p h o s p h a te level does not necessarily indicate transferase deficient galactosem ia because in patients w ith erythrocyte uridinediphosphate galactose-4-epim erase deficiency4 th e g a la c to s e -l-p h o s p h a te le v e l w as m arkedly elevated. U ridinediphosphate galactose-4-epim erase is the third enzym e involved in the norm al pathw ay of galactose m etabolism and converts uridine diphosphogalactose to uridine diphosphoglucose (table I). All th ese patients w ere asy m p to m atic w ith n o rm al hexose-1- p h o sp h ate u rid y ly ltra n sfe ra se activity and w ith e ith er norm al or slightly elevated galactose levels. T he diagnosis was confirm ed by m easuring the activity of the enzym e u rid in e d ip h o sp h ate galactose- 4-epim erase in erythrocytes. G alacto se-l-p h o sp h ate m easurem ents are difficult to perform and are generally used to determ ine if the transferase deficien t individual has d ev iated from the galactose re stric ted diet. Conclusion. T he possibility of trans^ ferase deficient galactosem ia should be c o n s id e re d b a s e d on th e c lin ic a l sym ptom s of the p atien t and the finding of e le v a te d g a la c to se an d g a la c to s e -lphosphate levels. H ow ever, the diagnosis m ust be confirm ed by m easurem ent of the a c tiv ity o f th e e n z y m e h ex o se-1 - phosphate uridylyltransferase in erythrocytes. D i a g n o s i s o f G a l a c t o k i n a s e D e f i c i e n t G a l a c t o s e m i a G alactokinase deficient galactosem ia is a rare disorder and is defined as absence o f the enzym e galactokinase (table I). Cataracts aré the only clinical symptom. T here is none o f the vom iting, diarrhea, jaundice, liver dam age or m ental retardation that is associated w ith transferase d e ficie n t galactosem ia. In th is d iso rd er, galactose cannot be m etabolized and is usually elevated in serum. H ow ever, the galactose lev el d ep e n d s on w h en th e blood was taken after the p atien t had consum ed milk. It was reported that th e galac» tose level in th e serum of a galactokinase deficient individual who has fasted overnight was only 3 m g per d l.10 In galactokinase d eficien t individuals, galactosel-phosphate is ab sent because it cannot b e formed. T h e diagnosis o f galactokinase d eficien t galactosem ia should be d eterm in ed by m easuring the activity of the enzym e galactokinase in erythrocytes. In order to m easu re galactokinase activity, a very sensitive m ethod is required because the activity o f galactokinase in th e ery th rocytes of norm al individuals is about 100 tim es low er than the activity of glucose- 6-phosphate dehydrogenase. An indirect w ay to d e te rm in e th e p o s s ib ility of galactokinase deficiency is to m easure galactose, g alactose-l-phosphate levels an d h ex o se -l-p h o sp h ate u rid y ly ltran s ferase activity. If the p atient has cataracts, e lev ated levels o f galactose w ith th e absence of galactose-l-phosphate and norm al h ex o se -l-p h o sp h ate u rid y ly ltran s ferase activity, th e possibility o f galactokinase deficient galactosem ia should be considered. H ow ever, th e only way to confirm the diagnosis of this disorder is to m e a s u re th e a c tiv ity o f th e en z y m e galactokinase in erythrocytes. R eferences 1. A n d e r s o n, E. P., K a lc k a r, H. M., K u r a k a sh i, K., and ISSELLACHER, K. J.: A specific enzymatic assay for the diagnosis of congenital galactosemia. I. The consumption test. J. Lab. Clin. Med. 50: , D e B r u y n, C. H. M. M., R a y m a k e r s, C., W ENSING, A., and O e i, T. L.: G alactose-lphosphate uridylyltransferase activities in erythrocytes from a patient with galactosemia: D iscrepancy betw een two m ethods. C lin. Chim. Acta 78: , G it z e lm a n n, R.: Estimation of galactose-lphosphate in erythrocytes: A rapid and simple

7 3 2 PESCE AND BODOURIAN enzymatic method. Clin. Chim. Acta 26: , 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, B., and HaiGIS, E.: U rid in e d ip h o sp h ate galactose-4-epimerase deficiency. IV. Report of eight cases in three families. Helv. Pediat. Acta 31: , GRENIER, A. and LABERGE, C.: Rapid method for screening for galactosem ia and galactokinase deficiency by measuring galactose in whole blood spotted on paper. Clin. Chem. 19: , H j e l m, M. and TENGSTROM, B.: Enzymatic determination of blood galactose w ith galactose oxidase and galactose d ehydro g en ase. Biochem. Med. 2: , M a s o n, G. A., Su m m e r, G. K., D u t t o n, H. H., and SCHWANER, R. C., J r.: Automated fluorom etric analysis of galactose in blood. Clin. Chem. 23: , PESCE, M. A. and BODOURIAN, S. H.: A new method for m easuring galactose-l-phosphate in erythrocytes. Clin. Chem. 23:1166, P e s c e, M. A., B o d o u r ia n, S. H., H a r r is, R. H., and NICHOLSON, J. F.: Enzymatic micromethod for m easuring galactose-l-phosphate uridylyltransferase activity in hum an erythrocytes. Clin. Chem. 23: , T h a l h a m m e r, O., G i t z e l m a n n, R., and P a n t l it s c h k o, M.: Hypergalactosemia and galactosemia due to galactokinase deficiency in a newborn. Pediatrics 42: , 1968.