CLIN. CHEM.23/9, 1711-1717(1977)

CLINICALCHEMISTRY,Vol. 23, No. 9, 1977 1711

Enzymatic Micromethod for Measuring Galactose-1-phosphate

UridylyltransferaseActivity in Human Erythrocytes

Michael A. Pesce, Selma H. Bodourian, Ruth C. Harris, and John F. Nicholson

A kinetic microspectrophotometric assay for galactose-1-phosphate uridylyitransferase (EC 2.7.7. 12) activity inerythrocytes is described. BlOOd is collected in ammoniumheparinized microhematocrit tubes, centrifuged and theerythrocytes are lysed with water. Galactose-1-phosphateuridylyltransferase activity is determined by mixing 25 ulof hemolysate with a reagent consisting of galactose-1-phosphate, uridine diphosphogiucose, NADP, ethyl-enediaminetetraacetate (disodium salt), phosphogluco-mutase, and glucose-6--phosphate dehydrogenase. Thereaction medium is maintained at 37 #{176}C.The increase inabsorption of the NADPH formed (340 nm) is recorded for9 mm. Under these conditions two moles of NADPH areproduced per mole of glucose-6-phosphate oxidized.Activity is referred to hemoglobin, measured as cyan-methemoglobin. The chelator is added to activate theenzyme. Stability studies show that the transferase isstable for several days in frozen erythrocytes. Goodcomparisons were obtained when this assay was com-pared to the uridine diphosphogiucose consumptionmethod. Because the method requires only a small amountof blood and is rapid, it can be used routinely to quantitateerythrocyte galactose-1 -phosphate uridylyltransf eraseactivity in newborn infants.

Addftlonal Keyphrases pediatric chem!sty . ga!actosemia- inheriteddlsorders screeningneonates sample stability

nor,nal values

In galactosemia, there is marked reduction in activityof the enzyme galactose-1-phosphate uridylyltrans-ferase (EC 2.7.7.12, UDPglucose:a-D-galactose- 1-phosphateuridylyltransferase),resulting in the inabilityto convert galactose to glucose.

The clinical disease produced by this deficiency hasvarious manifestations: hepatic failure, cataracts, renaltubular dysfunction, and failure to thrive. While specificconstellations of signs and systems are predominant atvarious ages in the growing child, the greatest threat to

central nervous system development, to the lens of theeye, and to life occurs when the disease is symptomaticin the neonatal period. For this reason there has beengreat interest in rapid and accurate laboratory methodsfor the diagnosis of galactosemia in the newborn in-fant.

The assay most commonly used for quantitation ofgalactose-1-phosphate uridylyltransferase activity(GAL-i-PUT) is the uridine diphosphoglucose (UDPG)consumption assay (1, 2). Manometric (3) and radio-chemical techniques (4, 5) have also been used to mea-sure GAL-i-PUT activity. All of these methods are te-dious and require large amounts of blood. Recently,fluorometric assays (6, 7) have been used to measureGAL-i-PUT activity. With these techniques, incuba-tions of 30 mm to 1 h are required.

A kinetic spectrophotometric assay for determiningGAL-i-PUT activity was described by Colombo et al.(8). They measured the NADPH produced in 40 mmfrom the first three reactions shown in Table 1. Withtheir procedure they assumed that 1 mol of NADPHwas formed per mole of glucose-6-phosphate oxidized.However, since another enzyme, phosphogluconatedehydrogenase (decarboxylating)(EC 1.1.1.44)ispresent in hemolysates, more than 1 mol of NADPH isproduced per mole of glucose-6-phosphate oxidized, asshown by reaction 4 in Table 1. Therefore with theirmethod, GAL-i-PUT activity was overestimated.

In this article, we present a kinetic micromethod formeasuring GAL-i-PUT activity in erythrocytes. Theassay is based on the reaction scheme shown in Tablei. Under the conditions of the assay, 2 mol of NADPHisproducedpermole of galactose-1-phosphate reacting.Each sample is preincubated for 20 mm and then as-sayed, recording continuously for 9 mm the rate of in-crease in absorbance of NADPH. Because this proce-

Microchemistry Laboratory, Babies Hospital, The Children’sMedical and Surgical Center of New York at Columbia-PresbyterianMedical Center, 3959 Broadway, Box 54, New York, N. Y. 10032.

Presented at the 9th International Congress on Clinical Chemistry,Toronto, Canada, July 13-18, 1975.

Received Oct. 11, 1976; accepted May 30, 1977.

‘Nonstandard abbreviations used: GAL-i-PUT, galactose-1-phosphate uridylyltransferase; UDPG, uridine diphosphoglucose;EDTA, (disodium) ethylenediaminetetraacetate; GAL-i-P. galac-thee-i-phosphate; and 6-PGD, 6-phosphogluconate dehydrogenase(EC 1.1.1.43).

Table 1. Reaction Scheme for Analysis of Galactose-1-phosphate Urldylylti Activity

1712 CLINICALCHEMISTRY,Vol. 23, No. 9, 1977

GAL-i-PUTGalactose-1-phosphate + uridine diphosphoglucose glucose-i-phosphate + uridine diphosphogalactose

phosploghicomutaseGlucose-i-phosphate glucose-6-phosphate

Glucose-6-phosphate + NADPglucose-8-phospl’iate dehyogenase

+ NADPH

6-phosphogluconate dehybogenase6-Phosphogluconate + NADP ‘ ribulose-5-phosphate + NADPH

dure is rapid and accurate, and can be performed withsmall samples, it is suitable for use in surveying new-borns for GAL-i-PUT activity.

Materials and Methods

An LKB Reaction Rate Analyzer (LKB InstrumentsInc., Hicksville, N. Y. ii8Ol) was used in these stud-ies.

Reagents

Buffer: Glycine (0.05 mol/liter, pH 8.6) with addeddisodium ethylenediaminetetraacetate (EDTA) immol/liter. Dissolve 3.76 g of glycine (Sigma ChemicalCo., St. Louis, Mo. 63i78) and 0.372 g of EDTA (Sigma)in 900 ml of distilled water. Adjust the pH to 8.6 anddilute to i liter.

Magnesium chloride, 0.1 mol/liter. Dissolve 20.3 gof magnesium chloride in i liter of distilled water.

NADP, disodium salt, 25 mmol/liter. Dissolve 20mg of NADP (Boehringer Mannheim Corp., India-napolis, md. 46250) in i ml of water.

Galactose-1-phosphate, dipotassium salt, 80mmol/liter. Dissolve 69 mg of galactose-1-phosphate(Boehringer Mannheim Corp.) in 2 ml of distilledwater.

Uridine-5’-diphosphoglacose, disodium salt(UDPG), 22 mmol/liter. Dissolve 40 mg of UDPG(Boehringer Mannheim) in 3 ml of distilled water.

Phosphoglucomutase (EC 2.7.5.1) from rabbitmuscle, 2000 U/mi (Boehringer Mannheim).

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49),Grade I, from yeast, 700 kU/liter (BoehringerMannheim).

The working reagent is prepared by mixing 5 ml ofbuffer, 200 sl of magnesium chloride, 100 il of NADP,200 il of galactose-1-phosphate, 20 il of PGM, and 20l of glucose-6-phosphate dehydrogenase.

Procedure

Preparation of hemolysate. Whole blood iscollectedin ammonium heparinized microhematocrit capillarytubes (i.d. i.i mm) and centrifuged. A 20-mm portionof the capillary tube (about 20 il of packed cells) is cutoff below the plasma and buffy layer and the contentsare lysed with 200 l of water.

Analysis. To determine GAL-i-PUT activity, 800 lof working reagent, 25 ,l of hemolysate, and 50 il ofUDPG are pipetted into disposable cuvets. The back-ground absorbance of the LKB Analyzer is set at 0.7 A,the range at 0.05, and recorder chart speed at 20 mm/mill. Each sample is incubated at 37 #{176}Cfor 20 mm, afterwhich the increase in absorbance at 340 nm of theNADPH formed is recorded for 9 mm. The rate between6 and 9 mm is divided by three, to determine #{163}4/mm.Pre-incubation of the samples is necessary because ofa lag phase at the beginning of the reaction. Under theconditions of the assay, the reaction rate is linear for atleast 25 mm after the incubation period.

Hemoglobin assay. Hemoglobin isdeterminedby thecyanmethemoglobin method (9). Hemolysate, 5 gil, ismixed with 1.250 ml of Drabkin’s reagent. After 5 mmat room temperature, the absorbances are measured at550 nm. The concentration of hemoglobin in the he-molysate is calculatedfrom cyanomethemoglobmnstandards. GAL-i-PUT activity is calculated as fol-lows:

U/g of hemoglobin =#{163}4X TV X 10 X 60

E X 2 x SV x [Hb]

where #{163}4is the change in absorbance per minute, e isthe molar absorptivity of NADPH at 340 nm (6.22 Xi0) in liters/mole, TV is the total volume of the assaymixture (0.875 ml), SV is the sample volume (0.025 ml),2 is the number of moles of NADPH produced, [HbJ isthe hemoglobin concentration in the hemolysate ing/iOO ml, U is the activity of galactose-1-phosphateuridylyltransferase (in micromoles/hour).

Results and Discussion

Because the activity of GAL-i-PUT in erythrocytesis relatively low and the absorbance of hemoglobin at340 nm is rather high, instrumental requirements foraccurate spectrophotometric kinetic assay of the en-zyme in hemolysates are quite stringent. Under theconditions of this assay the hemoglobin concentrationis between 25 and 30 g/liter, the initial absorbancesrange from i.3 to 1.7, and the observed changes in ab-sorbances are 0.0005 to 0.006 per minute. Several cur-rently available spectrophotometric systems have suf-

Table 2. Comparison of Mlchaeils ConstantsObtained by the Present Method and with

Reported AssaysKm GAL.1..P Km UDPO

Procidurmmol/lft#{149}r

0.06

0.05

V0.04

0.03

KMOI64MM0L/L 002

CUOPO] MMOL/L

Fig. 2. Lineweaver-Burk plot for uridine diphosphoglucose, forthree hemolysates

CLINICALCHEMISTRY,Vol. 23, No. 9, 1977 1713

010

5uO4tMMOL/L

AL-I-PO4 MMOL/L

Fig. 1. Llneweaver-Burk plot for galactose-1-phosphate, for threehemolysates

ficient blanking ability and sensitivity to be used forenzyme assays under these conditions.

We determined the optimal conditions for the assayon a pooled hemolysate at 37 #{176}Cby varying one con-stituent while keeping all other conditions constant.

Buffer. Maximum activity for the transferase wasobtained using glycine, 50 mmolfliter, as buffer. As themolarity of the buffer increases, transferase activitydecreases. When 1 mol of glycine was used per liter notransferase activity was recorded. When tris(hydroxy-methyl)methylamine buffer was used, transferase ac-tivity was less than that obtained with glycine buffer.

pH. When the buffer pH was 8.6, the pH of theworking reagent was maintained in the range 7.8 to 8.0and activity was maximum. If the pH of the workingreagent was not in this range, considerably less activitywas observed.

Activators. With EDTA, 1 mmol/liter, in the buffer,activity of the transferase was maximum. At EDTAconcentrations greater than 5 mmol/liter, less activitywas observed, probably because of chelation of mag-nesium. Sulfhydryl compounds were also tested as ac-tivators of the transferase. Thioglycol and mercapto-ethanol inhibited the enzyme system. Dithiothreitol at0.1 mol/liter or cysteine at 0.2 mol/liter activated theenzyme, but less so than did EDTA, in either freshlydrawn or stored blood samples. When 0.2 mol/liter di-thiothreitol was used, spurious increases in absorbancewere observed. A combination of EDTA and cysteinewas less effective than EDTA alone in activating theenzyme.

Magnesium chloride and NADP. With magnesiumchloride 3.58 mmol/liter in the working reagent, theactivity was highest. Higher concentrations of magne-sium chloride resulted in lower activities. With NADP,0.45 mmol/liter, in the working reagent, activity wasmaximum.

Enzymes. Phosphoglucomutase, 7.2 kU/liter, wasused in our working reagent to completely equilibrateglucose-i-phosphate with glucose-6-phosphate. Addi-tion of glucose-i,6-diphosphate did not affect the rate

Present assay 0.455 0.164Colombo et al. ref. 8 0.192 not reportedUDPG consumption, ref. 11 0.39 0.16UDPG consumptIon,ref. 10 0.56 0.111

of NADPH production. Glucose-6-phosphate dehy-drogenase (2.5 kU/liter in the working reagent) wasadded to ensure that the oxidation of glucose-6-phos-phate to 6-phosphogluconate would not be rate limit-ing.

Substrates. Michaelis’ constant for GAL-1-P, andUDPG were determined with use of three hemolysates.The Km for GAL-i-P was measured by using 2.64, 1.32,0.66, 0.33, and 0.i65 mmol/liter of GAL-i-P and 0.65mmolfliter of UDPG. By Lineweaver-Burk analysis(Figure 1) the Km for GAL-i-P was 0.455 mmol/liter,in agreement with Km’s obtained by the UDPG-con-sumption assays (1 0, 11 ) but in disagreement with Co-lombo et al. (Table 2). Maximum activity was obtainedwith 2.64 mmol/liter GAL-i-P (5.8 X Km). Higherconcentrations of GAL-i-P resulted in slower rates ofreaction. The Km value for UDPG was obtained byusing0.57,0.285,0.142,0.071,and 0.36 mmol of UDPGand 2.64 mmol of GAL-i-P per liter. By Lineweaver-Burk analysis (Figure 2) the Km for UDPG was 0.164mmol/liter, in agreement with values previously re-ported (10, 11) (Table 2). The UDPG concentrationused in our assay was i.26 mmol/liter (8 X Km). AtUDPG concentrations between 8 and 12 X Km, transferase activity was maximum. Higher UDPG concen-trations resulted in a loss of transferase activity. IfUDPG was omitted from the working reagent, there wasno reaction, indicating that the method specificallymeasures transferase activity. Table 3 shows the opti-mum conditions for measurement of GAL-i-PUT ac-tivity.

The sensitivity and accuracy of this assay depends on

Table 3. Final Concentration of

pHGlycine buffer

Constituents

?‘

0.041 mol/Ilter0.82 mmol/Ilter3.3 mmol/Ilter

Table 5. Analysis for Galactose-1-phosphateUrldylyltransferase Activity In the Presence of6-Phosphogiuconate Dehydrogenase Added to

Hemolysate6 POD addd

L-1-PU1

Disodlum ethylenedlamlnetetraacetateMgCI2

NADP+ 041 mmol/Iiter h.rnolys.t.5 U/g Hbb Chang.

Glucose-6-phosphate dehydrogenase 2.3 kU/liter Hemolysate 1Phosphoglucomutase 66 kU/liter - 21.1Galactose-1-phosphate 2.64 mmol/llter 320 20.0

Uridlne-5-dlphosphoglucose 1.26 mmol/llter 500 19.71000 17.6

Table 4. AnalytIcal Recovery of Galactose-1-phosphate Uridylyltransferase Added to

HemolysateGAL-i-PUT In Add.dh.molys.t. GAL-i-PUT Found EXpCtId

R.cov.ry,

-5

-6-17

224500 20.9 -71000 17.8 -211500 15.1 -33

a Micromoles of 6-phosphogluconateconsumed per minute.b Micromoles of galactose-1-phosphate consumed per hour per gram of

hemoglobin.10.0

U/lIter of

3.667.32

14.63

h.molys.t.a

13.5217.0123.90

13.6617.3224.63

969695

8.82 2.563.85

10.24

11.2012.3217.92

11.3812.6719.06

939689

9.8 3.787.569.45

13.3018.2020.30

13.8517.3619.25

93111

111

10.39 5.610.1220.24

15.8621.1628.87

15.4520.5130.63

10810698

Table 6. Effect of Low PhosphogluconateDehydrogenase Activity on the Present Assay

Galacto..-i-phosphat. urldylyttr.nsfsraseactIvity

In h.niolysat. Added Found5 ExpeCt.da

a Micromoles of galactose-1-phosphate consumed per minute.

U/Ifterb

Hemoiysate 10.69 14.21 25.04 24.90Diluted 3.81 14.21 17.59 18.02

hemolysate

a Results calculated assuming that 2 mol of NADPH Is formed.b Micromoles of galactose-i-phosphate consumed per minute.

1714 CLINICALCHEMISTRY,Vol. 23, No. 9, 1977

Hemolysate 11

the formation of 2 mol of NADPH. One mole ofNADPH is formed when glucose 6-phosphate is oxi-dized to 6-phosphogluconate by glucose-6-phosphatedehydrogenase. The second mole of NADPH is pro-duced from the reaction of 6-phosphogluconate withNADP, catalyzed by 6-phosphogluconate dehydroge-nase. We assume that the hemolysate contains sufficient6-PGD to oxidize 6-phosphogluconate to ribulose-5-phosphate. To test the validity of this assumption, wemeasured the recovery of GAL-i-PUT2 (from yeast,Sigma) added to hemolysates. When the activities werecalculated assuming that 2 mol of NADPH is formedper mole of GAL-i-P consumed, recoveries between 89and iii% were obtained (Table 4). Although these re-suits indicate that there is usually sufficient 6-PGD inthe hemolysate to account for complete conversion of6-phosphogluconic acid to ribulose-5-phosphate withconcurrent formation of NADPH, an inborn absence of

2 The activity of galactose-1-phosphate uridylyltransferase ob-

tamed was measured by mixing an aliquot of the aqueous transferasesolution with our working reagent. The transferase activity was cal-culated by assuming that 1 mol of NADPH is formed per mole ofglucose-6-phosphate oxidized.

6-PGD would result in GAL-i-PUT activities corn-patible with heterozygosity for galactosemia.

Addition of 6-PGD to the working reagent wouldassure that 2 mol of NADPH was formed per mole ofglucose-6-phosphate oxidized. Hemolysates were ana-lyzed for transferase activity by use of reagents with andwithout added 6-PGD (from yeast, BoehringerMannheim). The results (Table 5) indicate that addi-tion of 6-PGD inhibited transferase activity. This in-hibition was not reversed by dialyzing the 6-PGDovernight at 4 #{176}Cin glycylglycine buffer. 6-PGD alsoinhibited transferase from yeast (Sigma) or calf liver(Boehringer Mannheim). Because 6-PGD cannot beadded to the system, individuals with moderate 6-PGDdeficiency could exhibit falsely low transferase activityin our assay. However, in a survey of a large Americanpopulation (12) it was shown that less than i% havereductions of erythrocyte 6-PGD to the range of 42 to65% of normal; there was no instance of complete ab-sence of 6-PGD.

In the normal erythrocyte, the activity of GAL-i-PUT is 1/25that of 6-phosphogluconate dehydrogenase.Since the Km of the dehydrogenase for 6-phosphoglu-conate is relatively low (i8 imol/liter), a steady state(i.e., equal rates of production and of oxidation of 6-

U)

0U

mCl)

U-

0

‘Ii

U/g HbC

23.1 23.927.7 27.830.3 32.127.3 26.422.5 22.930.1 31.2

a Measured by the present assay, and calculated assumIng that2 mol ofNADPH isformed.

b ieea,ed by fog and bacterial glucose-6.c*ioephate dehy&ogenase

andcalculated,assumIng that 1mol of NADH Is formed.#{176}Mlcromoiesof galactose-1-phosphate consumed per hour.

O 5 I? 9 21 23 25 21 29 31 33

TRANSFERASE ACTIVITY u/gHb

Fig. 3. DIstrIbution of galactose-1-phosphate uridyiyltransferaseactivity for 205 children

Table 8. StabilIty of Galactose-1-phosphate Uridylyltransferase Activity In Blood at VariousTemperatures

Activity, U/g Hba

Temp Days#{176}C 0 i 2 3 7

Unwashed packed -20 22.7 21.0 21.3 20.3 18.9cells 4

Room temp.19.115.6

18.815.6

18.316.2

19.1

16.118.114.8

Packed cells -20 22.9 18.9 16.3 17.5 16.7washed wIth 4 21.5 18.9 15.0 17.9 17.4

saline Room temp. 20.0 16.7 18.2 17.3

Whole blood 4Room temp.

19.918.0

19.315.3

18.110.3

17.816.1

16.716.3

#{149}Micromoles of galactose-1-phosphate consumed per hour per gram of hemoglobIn.

CLINICALCHEMISTRY,Vol. 23. No. 9, 1977 1715

Table 7. Measurement of Galactose-1-phosphateUrldylyltransferase Activity by Our Assay and byUsing NAD and Bacterial Giucose-6-phosphate

DehydrogenaseGalactos.-1-phophat#{149} urldylyltran.feraa. actIvIty

A Bb

phosphogluconate) should be achieved after <6 s ofincubation under normal conditions. Furthermore, itcan be calculated that a 90% decrease in the activity of6-phosphogluconate dehydrogenase will delay theachievement of this steady state only by 2 mm. There-fore, even in cases of moderate 6-PGD deficiency thereshould be sufficient 6-PGD activity in the hemolysateto convert all the 6-phosphogluconic acid formed to ri-bulose-5-phosphate. To test the effect of moderate re-duction in 6-PGD activity in our assay, we prepared onehemolysate as stated in the Procedure by lysing onevolume of packed cells with iO volumes of distified waterand another hemolysate prepared by lysing one volumeof packed cells with 30 volumes of distilled water.GAL-i-PUT from yeast was added to both hemolysatesin activities comparable to normal values in erythro-cytes to determine if the GAL-i-PUT added could berecovered with the diluted 6-PGD hernolysate. Theresults (Table 6) indicate that 2 mol of NADPH isformed, even when the 6-PGD activity is a third ofnormal.

As a further proof that 2 mol of NADPH is formedwith our assay, the following experiment was performed.

GAL-i-PUT activity in hemolysates was measured intwo ways: with our assay and by using NAD and glu-cose-6-phosphate dehydrogenase obtained from Leu-conostoc mesenteroide8, all other reaction conditionsare the same as indicated in the Procedure. BecauseNAD does not react with 6-phosphogluconic acid de-hydrogenase only i mol of NADH is formed per moleof glucose-6-phosphate oxidized. If 2 mol of NADPHis formed in our assay, the GAL-i-PUT activity ob-tamed by both methods should be the same. The results(Table 7) indicate that 2 mol of NADPH is formed withour assay.

Most published methods for determining GAL-i-PUT activity in erythrocytes direct that the erythro-cytes be washed with isotonic saline to remove the leu-kocytes. In our studies, blood was collected in capillarytubes and the packed cells were cut off below the buffylayer to remove the leukocytes. To determine the ef-fectiveness of this method of removing leukocytes, themethods were compared by using blood samples col-lected simultaneously in evacuated blood-collectiontubes and in capillary tubes. In the saline-washederythrocyte preparation there were 850 leukocytes per

Pr.eant method

U/g Hb5

24.822.819.519.412.710.78.97.65.63-70.70.900

UDPG- method

U/g Hba

22.920.421.221.011.410.28.8

11.06.73.02.7

0.800

I !

iiv; .l

TT1I1

-U,.j..i_ :

---4. ti

1i

a Mlcromoles of substrateconsumed per hour per gram of hemoglobin.

i1-

r-t‘rri .

!_1: r

-N-t--1-rl-

ru:?#{247}±

+. tH-- f4-N±ftL: iii-j.t-L .

fl . j_4_ I ..f i4:

#{149} -‘- vt - I j:T . -:

-14 F4:E-4 1.- I #{149}r

Fig. 4. Reaction-rate curves mea&ting the change In absorbanceIn 9 mm for normal, heterozygote, and gaiactosemla samplesanalyzed In duplicateThe change In absorbance per minute for the normal sample was 0.005, for theheterozygote sample 0.0025

3 Galactose-1-phosphate uridylyltransferase activity is expressedas micromoles of substrate consumed per hour per gram of hemo-globin. To convert to micromoles of substrate consumed per minuteper kilogram of hemoglobin, multiply our activities by 16.66.

1716 CLINICALCHEMISTRY,Vol. 23, No. 9, 1977

Table 9. Comparison of Results for Galactose-1-phosphate Urldylyltransferase Activity with thePresent Method and by the UDPG-Consumptlon

Method

5 X i09 erythrocytes; in the capillary preparation therewere 530 leukocytes per 5 X i0 erythrocytes.

The stability of GAL-i-PUT in blood at differenttemperatures was investigated. Heparinized blood froma normal individual was collected and treated in threeways. Whole blood was stored at 4 #{176}Cand at roomtemperature. Packed cells from blood washed withisotonic saline and packed cells from blood collected incapillary tubes and not washed in isotonic saline werefrozen and stored at 4 #{176}Cor stored at room temperature.The GAL-i-PUT activities of these samples were de-termined over seven days. The greatest stability of thetransferase was observed in unwashed frozen packedcells (Table 8). After seven days, the GAL-i-PUT ac-tivity decreased by about i5% in the samples stored ateither room temperature or 4 #{176}C.This may present aserious problem if whole blood is to be transported bymail for analysis. Blood that cannot be immediatelyanalyzed for tranferase activity should be centrifugedand the packed erythrocytes frozen.

The precision of the assay was determined by mea-suing GAL-i-PUT activity in a pooled hemolysate. Fori i determinations the mean activity was 22 U/g of he-moglobin (±i.37, SD), and the CV was 6.i3%.

Values for erythrocyte GAL-i-PUT activity weredetermined on 205 patients at Babies Hospital. Themean and median were both 22.8 U/g of hemoglobin3(SD = 3.5). Figure 3 shows the distribution of GAL-i-PUT activity. The normal values for GAL-i-PUT ac-

tt.L.LL i

;- . I:I

± t- -

-

. .4.

-j

-.. +- .-

rl

i- .-.

zt-,#{149}

fI

--

.1

..1-. --

,

-:-

._.t

E

:jj::.L

tivity obtained by Colombo et al. (8 ) for newborns was34.6 U/g of hemoglobin (SD = 8.6). These values areconsiderably larger than would be expected, because incalculating the transferase activity they assumed thati mol of NADPH was produced per mole of glucose-6-phosphate oxidized, therefore overestimating theGAL- i-PUT activity.

The present assay was compared with the UDPGconsumption method by using samples from normal,heterozygous, and galactosemic subjects. Good agree-ment between the two methods was obtained (Table 9).Figure 4 shows the rates of change in absorbance thatwere obtained for normal, heterozygous, and galacto-semic individuals. The assay clearly distinguished theheterozygote from the normal sample.

With this procedure for determining GAL-i-PUTactivity, sensitivity has been maximized by performingthe determination at 37 #{176}Cand assuring that 2 mol of

CLINICALCHEMISTRY,Vol. 23, No. 9, 1977 1717

NADPH are formed per mole of glucose-6-phosphateoxidized. We have been using this assay routinely sinceJanuary 1975.

References1. Anderson, E. P., Kalckar, H. M., Kurahashi, K., eta!., A specificenzymatic assay for the diagnosis of congenital galactosemia. 1.Theconsumption test. J. Lab. Clin. Med. 50,469 (1957).2. Beutler, E., and Baluda, M. C., Improved method for measuringgalactose-1-phosphate uridyl transferase activity of erythrocytes.Clin.Chim. Acta 13,369 (1966).

3. Westwood, A., and Raine, D. N., Galactose-1-phosphate uridy!transferase: A new manometric assay specially suited to erythrocytestudies in galactosemia. Clin. Chim. Acta 49,449 (1973).4. Inouye, T., Nadler, H. L., and Hsia, Y. Y. D., Ga!actose-1-phos-phate uridyl transferase in red and white blood cells. Clin. Chim. Acta19, 169 (1968).5. Ng, U. G., Gergren, U. B., and Donnell, G. N., Galactose-1-phos-phate uridyl transferase assay by use of radioactive galactose-1-phosphate. Clin. Chim. Acta 10,337 (1964).

6. Beutler, E., and Mitchell, M., New rapid method for the estimationof red cell galactose-1-phosphate uridyl transferase activity. J. Lab.Clin. Med. 72,527 (1968).

7. Frazier, P. D. M., and Summer, G. K., Automated fluorometricmicromethod for detection of transferase-deficiency galactosemia.J. Lab. Clin. Med. 83, 334 (1974).8. Colombo, J. P., Moser, H., Rosin, S., et al., Bestimmung derGalakose-i-phosphate uridyl transferase im Capillarbiut bei derGulaktosamine. Kim. Wochenschr. 43, 1074 (1965).

9. Kanipen Von, E. J., and Zuistra, U. G., Standardization of hemo-globinometry 11. The hemoglobincyanide method. Clin. Chim. Acta6,538 (1961).

10. Tedesco, T. A., and Mellman, U. J., The UDP glu consumptionassay for gal-i-P uridyl transferase. In Galactosemia, Y. Y. D. Hsia,Ed. Charles C Thomas, Springfield, Ill., 1969, p66.

11. Beut!er, E., and Baluda, M.D., Biochemical properties of humanred cell galactose-1-phosphate uridyl transferase (UDPg!ucose:ga-lactose-i-phosphate uridyl transferase E.C. 2.7.7.12) from normal andmutant subjects. J. Lab. Clin. Med. 67, 947 (1966).12. Dern, R. J., Breuer, G. J., Tashan, R. E., eta!., Hereditary varia-tion of erythrocyte6-phosphogluconate dehydrogenase. J. Lab. Clin.Med. 67, 255 (1966).