Galactose-1-phosphate uridyl transferase: A new manometric assay specially suited to erythrocyte studies in galactosaemia

Clinica Chimica Acta, ~9 (1973) 435-443 ri>Elscvicr Scientific Publishing Company,

.\mstcrdam

- Printed

435

in The Netherlands

CCA 6127

GALACTOSE-I-PHOSPHATE A NEW

MANOMETRIC

STUDIES

Institute

URIDYL ASSAY

TRANSFERASE:

SPECIALLY

SUITED

TO

ERYTHROCYTE

IN GALACTOSAEMIA

ofJilrntal

Subnormality, Lea Castle;Hospital,

Wolverley, Near Kiddevminster,

(U.K.)

L). ?;. RAINE Department of Clinical Chemistry, B.16 8ET (li.Ii.) (Received

.+\uguSt 28,

The Children’s Hospital,

Ladywood

Middleway,

Birmingham,

1973)

SUMMARY

Spectrophotometric assay of galactose-r-phosphate uridyl transferase, when applied to erythrocytes for studies relating to galactosaemia, is complicated by the high background optical density of the haemolysate. For this reason a sensitive manometric method for the assay of this enzyme has been developed. The reaction rate is followed continuously, and the enzyme measured under optimal kinetic conditions. The method is precise (C.V. = 5.4%) and the results correlate well (Y = 0.91) with the uridine diphosphoglucose consumption test.

INTRODUCTIOS

Although galactose-r-phosphate uridyl transferase (GPUT, E.C. 2.7.7.10) in erythrocytes can be assayed by spectrophotometric, manometric and radiochemical methods, the last two have not been widely used in clinical chemistry laboratories, mainly because they have been time-consuming and require capital equipment not always available. However, the alternative spectrophotometric assay of this enzyme in erythrocytes is not ideal because haemolysates of the required strength provide a high background optical density. This precludes the use of the preferred methods based on measurement of reaction rate, for example that of Isselbacherl which measures the rate of NADPH production at 340 nm. Consequently, the methods most widely used have been various modifications of the uridine diphosphoglucose (UDPG) consumption test first described by Anderson et aZ.2. In these, interfering erythrocyte proteins are first precipitated and UDPG is measured spectrophotometrically, using the highly specific enzyme UDPG dehydrogenase (UDPGD, E.C. I.I.I.ZZ) before and after incubation with the haemolysate. Galactose-I-phosphate+UDPG

?!% --

Glucose-I-phosphate+UDP-Galactose

[I]

WESTWrOOL),k4INIi

436 VDPG+N

‘4D ‘zlz

rDP-Glucuronic

Acid-S.WH

2’

The need to precipitate protein means that the reaction rate cannot be mcasurctl continuously. Moreover these methods suffer from two further disadvantngcs : fiirstl~~, in order to maintain a useful sensitivitv, a sub-optimal concentration of 1TDPG must be used” ; and secondly the enzyme u&line dil~l~ospl~oglycos~~l-4-epimerase (ITDPGE, E.C. 5.1.3.2) present in erytlirocytes J, interferes with the transferasc assav. UDP-Galactosc

?!?”

lDP(;

The use of radioactively labelled substrates for the assay of galactose-r-plrosphate uridyl transferasc provides very sensitive methods 5,6 but the labelled substrate and product must be scparntcd and so again continuous measurement of tile reactic)n rate cannot be made. Onlv the manometric methods, therefore, allow continuous monitoring of the reaction rate. In the method to I)e dt~scribed transferasc activity is coupled with S,ZDPH production using the enzymes l~llospllogluco~~~utase (PGM, 1S.C. 2.7.5.1) and glucose6-phosphate deh~drogcnase (GOPD, E.C. I. 1.1,_1-q)and, via tllc artificial electron carrier methylcnc blue, with oxygen consuml~tion. ~~DPG~~~~nlactose-~-l~l~~~sl~liate ‘;“‘;T ---7 C’DP-~~alactose~l-(~lucos~-~-I~l~osl~liatt~

r

Glucose-I-l)llos;plrate

-I_I

2

CTlucosc-I,(,-dil~Iiosl,llate

TT

Crlucosc-i)-1,11osphatc

Glucose-6-l~I~ospl~atc~ -NADP (;(,‘u O-l’l~osl~l~ogluro~~ic acid

S:1DPH

51

The first method reagents

of this tyrpe was time-consuming and cxpensivc, both iii and blood, and the results were not conlparable with the UDPG consmnption

tests. Moreover, the only manometric method since devclopecl, which avoided tlic use of methylene blue9, does not correlate well \vitIr either the 1TDPG consumption test or the early method in which methylcnc blue was used. Indeed, in the manometric method without methy-lene blue, ox!-gen consumption using galactose-I-l~Iiosl~l~~~t~~ and UDPG as substrates \vas sometimes as high as that using glucose-I-plrosI,Ilatc as the substrate, suggesting that the transferase was not always the rate limiting enzyirir~ in this assay system. Thus, although manometric methods should offer advantages over the more commonly used VDPG consumption tests, the results reported so far are neither comparable nor entirely satisfactory. The ability to classify heterozygotes for galactosaemia, which have about half the enzyme activity of homozygous normal subjects I0 depends on both the accuraq and precision of the enzyme assay. We therefore felt it important to explore the extent to which this could be achieved, even if this required a somewhat more elaborate method of assay. MATERIALS ANI) METHOU Preearatiort of haemolysatc Blood was collected by venepuncture, anticoagulated with heparin, transfered to the laboratory at 4’, centrifuged as soon as possible at IOOOg for 20 min at the same temperature, the buffy layer removed, and the erythrocytes washed three times with 2-3 volumes of ice-cold isotonic saline. The packed erythrocytes were then lysed by freezing (methylcellosolvc-solid CO,) and thawin g three times. The haemolysate was

GALACTOSE-I-PHOSPHATE URIDYL TRANSFERASE

437

kept until analysed at -zoo, at which the enzyme is stable for at least two weeks. The haemolysate at this concentration was used for the manometric assay but was diluted with one volume of de-ionised water for the UDPG consumption test. The haemoglobin concentration of both haemolysates was determined, in duplicate, by the cyanmethaemoglobin method”.

&agents.

De-ionised

water and AR.

grade chemicals

(BDH

Chemicals

Ltd.,

Poole, Dorset, England) were used to prepare the following reagents: (I) Sodium chloride solution, 9 g/l. (2) Methylene blue solution, 2 g/l. (3) Potassium hydroxide solution, 200 g/l. (4) Isotonic phosphate buffer. Potassium clihydrogen orthophosphate, 0.16 g, magnesium sulphate 7H,O, 0.29 g, potassium chloride, 0.35 g, disodium hydrogen orthophosphate, anhydrous 5.92 g, and sodium chloride, 6.90 g, were dissolved in approximately 800 ml de-ionised water and the pH was adjusted to 7.4 with hydrochloric acid (I 31). The solution was made up to one litre and kept in a dark bottle at 4’. The following Hilton

House,

chemicals

Uxbridge

were obtained

Road,

Baling,

from the Boehringer

London,

Corporation

and were dissolved

Ltd,

in de-ion&cd

water:

(5) fi-Kicotinamide

adenine

dinucleotide

phosphate

(NADP),

disodium

salt,

12 mg/ml (14 mM). (6) Adenosine-s’-diphosphate (ADP), trisodium salt, 5 mg/ml (IO mnl). (7) Galactose-r-phosphate, dipotassium salt, 43 mg/ml (IOO m&I). (8) Glucose-r-phosphate, dipotassium salt, 37 mg/ml (IOO mill). (9) Uridine-5’-diphosphoglucose (UDPG) disodium salt, 20 mg/ml (30 mM). Pvoccdure. Oxygen consumption was measured using a Warburg apparatus, Braun Model V85 (Shandon Scientific Company Ltd, London) using flasks with one side arm and approximately 5 ml capacity. The water bath temperature was set to 37.0”. The flasks were filled with reagents according to Table I.

Flask l@)

Reaction well NADP solution 50 .-\DP solution 50 2jO hlethylene blue solution 200 Phosphate buffer o Galactose-I-phosphate UDPG solution 50 De-ionised water 200 200 Haemolysate Flask sidearm. 0 Glucose-I-phosphate solution Centre well. Potassium hydroxide solution (with a small piece of filter paper, approx. I cm2) 50

A (blank)

Flask (Pl)

50 50 2.50 200

50 50 IjO

200 SO

50

B (test)

438

WESTWOOD,

RAINE

One flask is left completely empty and used to correct for fluctuations in atmospheric pressure and bath temperature during the incubation. Each flask is fitted to its manometer with the taps open, and then fitted to the Warburg apparatus and allowed to equilibrate in the water bath for about 15 min. After this equilibration, to bring all the reagents to the bath temperature, the manometer taps are closed and readings taken every 5 min for the next 45-60 min. These readings give the oxygen uptake for the transferase assay-, but in order to ensure that the transferase is the rate limiting step in the system the contents of the flask sidearm are now tipped into the reaction well, and manometer readings taken at the same intervals for a further 20-30 min. The oxygen consumption rate is calculated from the manometer readingsI” and galactose-I-phosphate uridyl transferase activity expressed

as pmole oxygen

consumed

per hour per gram haemoglobin.

UDPG consumption test The test described by Beutler and Baluda3 was used without modification. The haemoglobin concentration in the haemolysate was determined by the cyanmethaemoglobin method and galactose-r-phosphate uridyl transferase was expressed as pmole UDPG consumed per hour per gram haemoglobin. RESULTS

The assay conditions used in the manometric method are based on those of Kirkman and Bynum’ and Schwarz et a1.9. Unlike the latter group, we found that, when methylene blue was omitted from the reaction mixture, oxygen consumption was verl low. In fact unless the concentration of methylene blue exceeded about 300 pg/ml oxygen consumption by haemolysates was limited using glucose-I-phosphate as substrate

(Fig. I).

Oi 0

100 Methylene

200 blue

400

300 Lug/ml

500

1

Fig. I. The addition of methylene blue to haemolysates respiring on glucose-l-phosphate as substrate. In the manometric method described in the present study a concentration of 500 [Lg/ml was used.

GALACTOSE-I-PHOSPHATE TABLE

URIDYL TRANSFERASE

439

II

ADDITIONOF SUPPLEMEXTARYENZYMESAND METABOLITES GALACTOSE-I-PHOSPHATE

URIDYL

TRANSFERASE

TO HAEMOLYSATES

Transferme pl OJh/200

Concentration added to reaction mixture

Glucose-6.phosphate

dehpdrogenase*

0 3.j

Phosphoglucomutase*

@-Xicotinamide adenine dinuclcotide phosphate Galactose-I-phosphate Uridine-5’.diphosphoglucose

2.0 U/ml o I4 clM o

2 j0 /l’1\1 750 @I 2.5 mhf 5.0 mhZ 0.8 mM

from the Boehringer

Corporation

activity pl erythrocytes

31.3 33.3

1.6 mM * Reagents obtained Ealing, London.

MANOMETRIC

45.5 42.5 45.5 43.0 45.5 44.5 II.3 36.0 36.6 38.0 38.0

U/ml

0

Glucose-1,6-diphosphate*

IN THE

ASSAY

Ltd.,

Bilton

House,

Uxbridge

Road,

The enzymes involved in the couple with oxygen consumption, phosphoglucomutase and glucose-6-phosphate dehydrogenase were not rate limiting in the concentrations normally found in haemolysates, and they were not routinely added to the reaction mixture. Similarly, the addition of glucose-r,6_diphosphate was unnecessary and the concentrations of NADP, UDPG and galactose-r-phosphate used were not rate limiting (Table II). In common with Hsia et ah8 we found the addition of glucose-6-phosphate dehydrogenase eliminates the lag period of about 10-15 min which otherwise occurs after the addition of the substrates before oxygen consumption commences. However, this lag period was not normally observed in the method as described since it is completed during the equilibration while the manometer taps are open and before readings are taken. Since the coupling enzymes mentioned were not added to the reaction mixture, after the readings for the transferase activity had been made glucose-r-phosphate was tipped into the reaction well from the flask sidearm, and readings were taken for a further 20-30 min. This resulted in a a-5-fold increase in oxygen consumption (Fig. 2) and served to ensure that the galactose-r-phosphate uridyl transferase was the rate limiting enzyme in the system. The rate of oxygen uptake is constant for at least 45-60 min using galactose-rphosphate as substrate, and for at least 20 min using glucose-r-phosphate as substrate (Fig. 2). Oxygen consumption is also directly proportional to the amount of normal haemolysate added to the reaction flask. This was tested by mixing a normal haemolysate with one from a patient with galactosaemia who had no transferase activity as measured by the UDPG consumption test. In this way, the only enzyme in the normal haemolysate which was appreciably diluted was the transferase (Fig. 3). The precision of the assay was estimated by replicate analysis of a normal haemolysate. This was assayed II times during three consecutive days. The mean transferase activity of the haemolysate was 37.75 ,umole 0,/h/g haemoglobin with a standard deviation of 2.04, which is 5.4% of the mean. The UDPG consumption test was always performed in duplicate, so the precision was estimated from seventeen duplicate determinations using the formula

WESTWOOD,

440

RAINIS

A

120 glu-1-P

5

90

I

F

gal-l-P

I 30

ji/

NJ

0

0 0

30

60

Time

// /@

,d,’

I

60

s E 0"

B

/

," .!

90

imlnutesi

120

k 0

/Ij 50

Normal

100

150

haemolysate

200

full

Fig. 2. Oxygen consumption by normal hacmolysatcs measured by the method described in the text. Blanks ( x ) and tests (0) are shown. Galactose-r-phosphate (gal-r-I’) was added to the tests and after a 15.min equilibration period, the oxygen consumption was measured at 5.min intervals for 45-60 min. Glucose-I-phosphate (@u-r-P) was then added and oxygen consumption was mcaswed for a further 15-20 min. Fig. 3. Linearity of the manometric galactose-r-phosphate uridyl transfcrasc assay. A haemolysate from a patient with galactosaemia (o ,umole liDPG/h/g haemoglobin by the ITDPG consumption test) was mixed with a normal haemolysate, so that the only enzyme appreciably diluted was the transferase. The oxygen uptake is plotted against the volume of normal haemolysate in the Warburg flask.

s2 =

2 (x,-x,)” N-I

.

The mean transferase activity was 22.79 pmole UDPG/h/g haemoglobin, with a standard deviation of 1.32, which is 5.8% of the mean. In both the manometric assay and the UDPG consumption test five patients withgalactosaemia had no detectable galactose-r-phosphate uridyl transferase activit! in their erythrocytes. Ten parents of these patients (obligate heterozygotes for galactosaemia) had a mean transferase activity which, in the case of the UDPG consumption test, was 450/6 of the normal mean, and in the manometric assay was 510/o of the normal mean (Table III). The correlation between the two methods was examined by analysing erythrocytes from all these galactosaemia homozygotes and heterozygotes and from zz normal individuals by both methods (Fig. 4). The calculated regression between the two methods is: manometric assay (pmole 0,/h/g haemoglobin) = 4.12 + I .17 UDPG consumption test (pmole UDPG/h/g haemoglobin), and the correlation coefficient is o.~I. Both the correlation and regression coefficients are significantly different from zero at the 10% level. DISCUSSION Spectrophotometric and radiochemical assays of galactose-r-phosphate uridyl transferasc have disadvantages which have already been outlined. The consumption

GALACTOSE-I-PHOSPHATE TABLE

441

URIDYLTRANSFERASE

III

GALACTOSE-I-PHOSPHATE URIDYI. TRAixsFERAsE ASSAYS IN GALACTosAEhlIA HOMOZYGOTES ASD HETERoZYGoTEs AND IN NORMALHOMOZYGOTES _~ GalactosaemiaGalactosam~za Normal MEthod homozy@?s hamzygotes hrtevozygotes

Ma110111etric assay

(,umole C&/h/ghaemoglobin) mea” standard deviation no. individuals CIDPG consumption test (,umole IJDPG/h/g haemoglobin) mean standard deviation no. individuals

0

18.0~

0 5

4.36 IO

0

II.03

0

2.90

5

IO

35.32 5.42 37

24.85 6.40 40

60

1

0, 0

20 UDPG iumol

Consumption UDPGlhlg

LO

60

Test haemoglobinl

of the results of the ITDPG consumption test and the manometric galactose-rphosphate uridyl transfcrase assay in galactosaemia homozygotcs, galactosaemia heterozygotes and apparently normal inclividuals. Fig.

1.

Comparison

tests too have disadvantages, particularly the non-linearity which is a result of the low substrate concentrations which must be used, the interference from the enzyme uridine-disphosphoglycosyl-4-epimerase and the inability to follow the reaction continuously. Beutler and Baluda3 improved the consumption test by including a preincubation step designed to reduce the interference from the epimerase and by calculating correction factors to compensate for the non-linearity. The manometric methods do not suffer from these same disadvantages and although they have been compared with the UDPG consumption test once8, this was shortly after the introduction of the consumption test and before the modifications of Beutler and Baluda which seem to have improved its precision3. For this reason the manometric method developed in the present study has been compared with this more recent version of the consumption test. There is no significant difference between the precision of the two assays. The

442

WIS’IWOOI),

RAINE

coefficient of variation for the consumption test is estimated to be 5,8$1,, compared with 5.4% for the manomctric method. Ellis and GoldbergI found a similar precisioll for the same consumption test (4.496) and tl1is was the most precise of the consuml,tion tests they examined. Kirkman and Bynum rcportetl tl1e first manomctric assay. and estimated the coefficient of variation to be about 576, wliicli is similar to tlr(L figure found for the manomctric metllod used in the present study. The correlation between the two assays is high (Y = the correlation between tl1e consumption test of Brctthauer assay of Kirkman and Bynum which was reported by Hsia likely that this 11igher correlation reflects improvements in and tl1e manometric assay since this early work.

o.c)I), 11igher in fact tlrall rt n1.l” and the nlanometricrt nl.” (Y = 0.79). It seems both the consumption tfsbt

Using tl1e manometric assa!- developed in this study the mean transferase activity for normal subjects is 43.5 ,LLI0,/h/200 ,LL~erytllrocytes. This is close to tllc figure (44.5 ~1 0,/h/200 ~1 erythrocytes) reported by Schwarz L$ al.!‘, ~.ho UN] a substantially different method, but is considerably higher than the activities rel)ortcd by Kirkman and BJmum7 (21.2 $ OJh/zoo ,LL~erythrocytes) and by Hsia of (I/.” (19.5 ~1 O,/h/aoo ~1 erythrocytes). Schwarz c,t aZ.9 considered that their incrctasetl activity was the result of higher substrate concentrations, but this seems unlikely bccause, in the present study, the substrate concentrations used, whic~h were appro.yimately the same as those used by Kirkman and Bynum and by Hsia f:t c~Z.8,ai-<’ approximately ten times the Michaelis constantIS for each substrate, and have been shown not to be rate limiting. Moreover, in all the manometric mcthotls so far ticscribed, oxygen consumption is linear with respect to time, which would not by the case if the concentration of substrate was limiting. However, it ma!; by that the n,(‘thylene blue concentration used by these workers limited ox\-gcn consumption, since: in the present study oxygen uptake was found to depend markedly on methylcne t)lu(, concentration up to an optimum of about 3~) ,uglml (Fig. I). The meth>+nc I)IU(concentration usctl by Kirkman and Bynum and by Hsia c,t crl. was 50 j~g/ml. The manomctric method described differs from those reported earlier in anot]lcr important respect. The previous methods have given a mean transferase acti\.itv for galactosaemia heterozygotes of 62-Q ;{I of that for homozygous normal subjclcts. activit\- for the galactosaemi;~ Using the present method, tlic mean transferase heterozygotes was 51 ?;, of normal, a value closer to that given by the c~onsunil~tion test (45-55 :10) and closer to the 50% which would he expected if tllese hetcrozygotcs had one gene producing functional enzyme compared with two such gcnr~ in normal homozygotes. The increased separation of the results in normal 11omoz>-gates ant1 heterozygotes reI)resented by this fall from 65O::, to 50 ‘I(, is of particular importance: in the recognition among the otherwise healthy population of hetcrozygotes for galactosaemia. The efficiency of this classification is sensitive to even slight improvements in the accuraclr and precision of the assaysI”. This aspect of the study will bc rtporte([ in full at a later date. Perhaps the main drawback of both tl1e manonletric and consumption tests is the lack of opportunity to automate either of them. Tlrc 111anometric~ method is neither more time consuming nor expensive than the consumption test, but the apparatus required is less versatile in clinical chemistry laboratories than that required for the UDPG consumption test. For this reason, the consumption test will probably remain that most widely used for diagnosis of galactosaemia but the present method is preferred for heterozygote recognition.

GALACTOSE-I-PHOSPHATE

URIDYL

TRANSFERASE

443

ACKNOWLEDGEMENTS We are indebted to the Birmingham Regional Hospital Board Research Fund and to the United Birmingham Hospital Endowment Fund for grants with which some of the apparatus was purchased.

K. J. ISSELBACHER, in H. U. BERGMEYER (Ed.), Methods of Enzymatic Analysis, Academic Press, Xew York, 1963, p. 863. E.P.ASDERSON,H.M.KALCKAR, K. KLJRAHASHIAXIK. J. ISSELBACHER,J. Lab. CZi?z.Med.,jo

(1957)469.

I<.BEUTLER AND M. C. BALUDA, Clin.Chiw Acta, 13 (1966) 369. I~.J.ISSELBACHER,~.P.ANDERSON,K.KURAHASHIANDH.~~.KALCKAR,SC~~~ZC~,

1z3(1956)

635. A. ROBINSON, J. Exp. ,Vf&.,118 (1963) 359. D. BERTOLI AND S. SEGAL, J. Biol. Chem., 241 (1966) 4023. H. X. KIRKMAN AND E. BYNUM, Ann. Hum. Genet., 23 (1959) 117. U. Y. T. HSIA, &I. TAXXENBAUM, J. A. SCHNEIDER, I. HUANG AND K. SIMPSON, J. Lab. Clin. Med.,

V.

56 (1960)

368.

R. WELLS, A. HOLZEL, G. M. KOMROWER AND I.M. N. SI~IPSO~-, Ann. Hum. (1961) 179. D. T. Y. HSIA, AVfed.Clin.N&h. Amer., 53 (1969) 857. International Committee for Standardisation in Haematology of The European Society of Haematology, J. CZin. Path., 18 (1965) 353. \v. 1%‘. UMBREIT, R. H. UURRIS APZU J. 1;.STAUFFER, Manowtvic Techniques, Burgess, Ninnesots, 1964. G. ELLIS ALYD D. fir. GOLDBERG, Ann. CZin. Biochem., 6 (1969) 70. I<. Ii. URETTHAUER, R. G. HANSEN, G. N. DOKNELL AYD XV'.R. BERGREN, Proc. Nat. Acud. SC;., dj (1959) 328. E. HEUTLER ASD M. C. BALUDA, J. Lab. CZin. 2Vfed., 67 (1966) 947. n. WESTWOOD AND D. N. RAINE, in J. W. T. SEAKINS, R. X. SAUNDERS AND C. TOOTHILL (Eds.), PIW~YPSS and Treatment in Inherited Metabolic Disease, Churchill Livingstone, London, 1973, I’. 63. SCHWARZ,

Genrt.,

2j

A.