uridylyltransferase activity is determined by mixing 25 ul of hemolysate with a reagent consisting of galactose-. 1-phosphate, uridine diphosphogiucose, NADP, ...
CLIN. CHEM.23/9, 1711-1717(1977)
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 galactose1-phosphate uridylyitransferase (EC 2.7.7. 12) activity in erythrocytes is described. BlOOd is collected in ammonium heparinized microhematocrit tubes, centrifuged and the erythrocytes are lysed with water. Galactose-1-phosphate uridylyltransferase activity is determined by mixing 25 ul of hemolysate with a reagent consisting of galactose1-phosphate, uridine diphosphogiucose, NADP, ethylenediaminetetraacetate (disodium salt), phosphoglucomutase, and glucose-6--phosphate dehydrogenase. The
reaction medium is maintained at 37 #{176}C. The increase in absorption of the NADPH formed (340 nm) is recorded for 9 mm. Under these conditions two moles of NADPH are produced per mole of glucose-6-phosphate oxidized. Activity is referred to hemoglobin, measured as cyanmethemoglobin. The chelator is added to activate the enzyme. Stability studies show that the transferase is stable for several days in frozen erythrocytes. Good comparisons were obtained when this assay was compared to the uridine diphosphogiucose consumption method. Because the method requires only a small amount of blood and is rapid, it can be used routinely to quantitate erythrocyte galactose-1 -phosphate uridylyltransf erase activity in newborn infants.
Addftlonal Keyphrases -
inheriteddlsorders nor, nal values
pediatric chem!sty screeningneonates
.
ga!actosemia sample stability
In galactosemia, there is marked reduction in activity of the enzyme galactose-1-phosphate uridylyltransferase (EC 2.7.7.12, UDPglucose:a-D-galactose1phosphateuridylyltransferase), resulting in the inability to convert galactose to glucose. The clinical disease produced by this deficiency has various manifestations: hepatic failure, cataracts, renal tubular dysfunction, and failure to thrive. While specific constellations of signs and systems are predominant at various ages in the growing child, the greatest threat to
Microchemistry
Laboratory,
Babies
Hospital,
Medical and Surgical
The
Children’s
Center of New York at Columbia-Presbyterian Medical 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.
central nervous system development, to the lens of the eye, and to life occurs when the disease is symptomatic in the neonatal period. For this reason there has been great interest in rapid and accurate laboratory methods for the diagnosis of galactosemia in the newborn infant. The assay most commonly used for quantitation of galactose-1-phosphate uridylyltransferase activity (GAL-i-PUT) is the uridine diphosphoglucose (UDPG) consumption assay (1, 2). Manometric (3) and radiochemical techniques (4, 5) have also been used to measure GAL-i-PUT activity. All of these methods are tedious and require large amounts of blood. Recently, fluorometric assays (6, 7) have been used to measure GAL-i-PUT activity. With these techniques, incubations of 30 mm to 1 h are required. A kinetic spectrophotometric assay for determining GAL-i-PUT activity was described by Colombo et al. (8). They measured the NADPH produced in 40 mm from the first three reactions shown in Table 1. With their procedure they assumed that 1 mol of NADPH was formed per mole of glucose-6-phosphate oxidized. However, since another enzyme, phosphogluconate dehydrogenase (decarboxylating)(EC 1.1.1.44)is present in hemolysates, more than 1 mol of NADPH is produced per mole of glucose-6-phosphate oxidized, as shown by reaction 4 in Table 1. Therefore with their method, GAL-i-PUT activity was overestimated. In this article, we present a kinetic micromethod for measuring GAL-i-PUT activity in erythrocytes. The assay is based on the reaction scheme shown in Table i. Under the conditions of the assay, 2 mol of NADPH isproducedper mole of galactose-1-phosphate reacting. Each sample is preincubated for 20 mm and then assayed, recording continuously for 9 mm the rate of increase in absorbance of NADPH. Because this proce-
‘Nonstandard abbreviations used: GAL-i-PUT, galactose-1phosphate uridylyltransferase; UDPG, uridine diphosphoglucose; EDTA, (disodium) ethylenediaminetetraacetate; GAL-i-P. galacthee-i-phosphate; and 6-PGD, 6-phosphogluconate dehydrogenase (EC 1.1.1.43).
CLINICALCHEMISTRY,Vol. 23, No. 9, 1977 1711
Table 1. Reaction Scheme for Analysis of Galactose-1-phosphate Urldylylti Galactose-1-phosphate
GAL-i-PUT
+ uridine diphosphoglucose
Activity
glucose-i-phosphate + uridine diphosphogalactose
phosploghicomutase
glucose-6-phosphate
Glucose-i-phosphate glucose-8-phospl’iate
Glucose-6-phosphate
+ NADP
6-Phosphogluconate
+ NADP
+ NADPH 6-phosphogluconate
dure is rapid and accurate, and can be performed small samples, it is suitable for use in surveying borns for GAL-i-PUT activity.
dehybogenase ‘
with new-
Materials and Methods An LKB Reaction Rate Analyzer (LKB Instruments Inc., Hicksville, N. Y. ii8Ol) was used in these studies. Reagents
Glycine (0.05 mol/liter, pH 8.6) with added ethylenediaminetetraacetate (EDTA) i mmol/liter. Dissolve 3.76 g of glycine (Sigma Chemical Co., St. Louis, Mo. 63i78) and 0.372 g of EDTA (Sigma) in 900 ml of distilled water. Adjust the pH to 8.6 and dilute to i liter. Magnesium chloride, 0.1 mol/liter. Dissolve 20.3 g of magnesium chloride in i liter of distilled water. NADP, disodium salt, 25 mmol/liter. Dissolve 20 mg of NADP (Boehringer Mannheim Corp., Indianapolis, md. 46250) in i ml of water. Galactose-1-phosphate, dipotassium salt, 80 mmol/liter. Dissolve 69 mg of galactose-1-phosphate (Boehringer Mannheim Corp.) in 2 ml of distilled water. 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 rabbit muscle, 2000 U/mi (Boehringer Mannheim). Glucose-6-phosphate dehydrogenase (EC 1.1.1.49), Grade I, from yeast, 700 kU/liter (Boehringer Mannheim). The working reagent is prepared by mixing 5 ml of buffer, 200 sl of magnesium chloride, 100 il of NADP, 200 il of galactose-1-phosphate, 20 il of PGM, and 20 l of glucose-6-phosphate dehydrogenase. Buffer: disodium
Procedure
Preparation of hemolysate. Whole blood iscollected in ammonium heparinized microhematocrit capillary tubes (i.d. i.i mm) and centrifuged. A 20-mm portion of the capillary tube (about 20 il of packed cells) is cut off below the plasma and buffy layer and the contents are lysed with 200 l of water. 1712 CLINICALCHEMISTRY,Vol. 23, No. 9, 1977
dehyogenase
+ NADPH
ribulose-5-phosphate
Analysis. To determine GAL-i-PUT activity, 800 l of working reagent, 25 ,l of hemolysate, and 50 il of UDPG are pipetted into disposable cuvets. The background 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}C for 20 mm, after which the increase in absorbance at 340 nm of the NADPH formed is recorded for 9 mm. The rate between 6 and 9 mm is divided by three, to determine #{163}4/mm. Pre-incubation of the samples is necessary because of a lag phase at the beginning of the reaction. Under the conditions of the assay, the reaction rate is linear for at least 25 mm after the incubation period. Hemoglobin assay. Hemoglobin isdeterminedby the cyanmethemoglobin method (9). Hemolysate, 5 gil, is mixed with 1.250 ml of Drabkin’s reagent. After 5 mm at room temperature, the absorbances are measured at 550 nm. The concentration of hemoglobin in the hemolysate is calculatedfrom cyanomethemoglobmn standards. GAL-i-PUT activity is calculated as follows:
U/g of hemoglobin
#{163}4 X TV =
EX
X
10 X 60
2 x SV x [Hb]
where #{163}4 is the change
in absorbance per minute, e is the molar absorptivity of NADPH at 340 nm (6.22 X i0) in liters/mole, TV is the total volume of the assay mixture (0.875 ml), SV is the sample volume (0.025 ml), 2 is the number of moles of NADPH produced, [HbJ is the hemoglobin concentration in the hemolysate in g/iOO ml, U is the activity of galactose-1-phosphate uridylyltransferase (in micromoles/hour).
Results and Discussion Because the activity of GAL-i-PUT in erythrocytes low and the absorbance of hemoglobin at 340 nm is rather high, instrumental requirements for accurate spectrophotometric kinetic assay of the enzyme in hemolysates are quite stringent. Under the conditions of this assay the hemoglobin concentration is between 25 and 30 g/liter, the initial absorbances range from i.3 to 1.7, and the observed changes in absorbances are 0.0005 to 0.006 per minute. Several currently available spectrophotometric systems have sufis relatively
010
Table 2. Comparison of Mlchaeils Constants Obtained by the Present Method and with Reported Assays Km GAL.1..P
Km UDPO
Procidur mmol/lft#{149}r
Present assay Colombo et al. ref. 8 UDPG consumption, ref. 11 UDPG consumptIon,ref. 10
5uO4tMMOL/L
AL-I-PO4
MMOL/L
Fig. 1. Llneweaver-Burk plot for galactose-1-phosphate, for three hemolysates
blanking ability and sensitivity to be used for enzyme assays under these conditions. We determined the optimal conditions for the assay on a pooled hemolysate at 37 #{176}C by varying one constituent while keeping all other conditions constant. Buffer. Maximum activity for the transferase was obtained using glycine, 50 mmolfliter, as buffer. As the molarity of the buffer increases, transferase activity decreases. When 1 mol of glycine was used per liter no transferase activity was recorded. When tris(hydroxymethyl)methylamine buffer was used, transferase activity was less than that obtained with glycine buffer. pH. When the buffer pH was 8.6, the pH of the working reagent was maintained in the range 7.8 to 8.0 and activity was maximum. If the pH of the working reagent was not in this range, considerably less activity was observed. Activators. With EDTA, 1 mmol/liter, in the buffer, activity of the transferase was maximum. At EDTA concentrations greater than 5 mmol/liter, less activity was observed, probably because of chelation of magnesium. Sulfhydryl compounds were also tested as activators of the transferase. Thioglycol and mercaptoethanol inhibited the enzyme system. Dithiothreitol at 0.1 mol/liter or cysteine at 0.2 mol/liter activated the enzyme, but less so than did EDTA, in either freshly drawn or stored blood samples. When 0.2 mol/liter dithiothreitol was used, spurious increases in absorbance were observed. A combination of EDTA and cysteine was less effective than EDTA alone in activating the enzyme. Magnesium chloride and NADP. With magnesium chloride 3.58 mmol/liter in the working reagent, the activity was highest. Higher concentrations of magnesium chloride resulted in lower activities. With NADP, 0.45 mmol/liter, in the working reagent, activity was maximum. Enzymes. Phosphoglucomutase, 7.2 kU/liter, was used in our working reagent to completely equilibrate glucose-i-phosphate with glucose-6-phosphate. Addition of glucose-i,6-diphosphate did not affect the rate ficient
0.455 0.192 0.39
0.56
0.164 not reported 0.16 0.111
of NADPH production. Glucose-6-phosphate dehydrogenase (2.5 kU/liter in the working reagent) was added to ensure that the oxidation of glucose-6-phosphate to 6-phosphogluconate would not be rate limiting. Substrates. Michaelis’ constant for GAL-1-P, and UDPG 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 0.65 mmolfliter 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-consumption assays (1 0, 1 1 but in disagreement with Colombo et al. (Table 2). Maximum activity was obtained with 2.64 mmol/liter GAL-i-P (5.8 X Km). Higher concentrations of GAL-i-P resulted in slower rates of reaction. The Km value for UDPG was obtained by using0.57,0.285,0.142,0.071, and 0.36 mmol of UDPG and 2.64 mmol of GAL-i-P per liter. By LineweaverBurk analysis (Figure 2) the Km for UDPG was 0.164 mmol/liter, in agreement with values previously reported (10, 11) (Table 2). The UDPG concentration used in our assay was i.26 mmol/liter (8 X Km). At UDPG concentrations between 8 and 12 X Km, trans ferase activity was maximum. Higher UDPG concentrations resulted in a loss of transferase activity. If UDPG was omitted from the working reagent, there was no reaction, indicating that the method specifically measures transferase activity. Table 3 shows the optimum conditions for measurement of GAL-i-PUT activity. The sensitivity and accuracy of this assay depends on )
0.06
0.05 V 0.04
0.03 KMOI64MM0L/L
002
CUOPO]
MMOL/L
Fig. 2. Lineweaver-Burk plot for uridine diphosphoglucose,
for
three hemolysates CLINICALCHEMISTRY,Vol. 23, No. 9, 1977 1713
Table 5. Analysis for Galactose-1-phosphate Urldylyltransferase Activity In the Presence of 6-Phosphogiuconate Dehydrogenase Added to Hemolysate
Table 3. Final Concentration of Constituents pH Glycine buffer Disodlum ethylenedlamlnetetraacetate
0.041 mol/Ilter 0.82 mmol/Ilter
MgCI2
3.3 mmol/Ilter
NADP+ Glucose-6-phosphate dehydrogenase Phosphoglucomutase
041 mmol/Iiter 2.3 kU/liter 66 kU/liter
Galactose-1-phosphate Uridlne-5-dlphosphoglucose
2.64
?‘
6 POD
addd
L-1-PU1 U/g Hbb
h.rnolys.t.5
Chang.
Hemolysate 1
mmol/llter 1.26 mmol/llter
-
21.1
320 500
20.0
1000 Hemolysate 11
17.6
-5
-6 -17
19.7
224
Table 4. AnalytIcal Recovery of Galactose-1phosphate Uridylyltransferase Added to Hemolysate GAL-i-PUT In h.molys.t.
10.0
8.82
9.8
10.39
a
Micromoles
Add.d
500 1000 1500
R.cov.ry,
GAL-i-PUT Found U/lIter of h.molys.t.a
3.66 7.32
13.52 17.01
14.63
b
hemoglobin.
13.66
96
23.90
17.32 24.63
96 95
2.56 3.85 10.24
11.20 12.32 17.92
11.38 12.67 19.06
93 96 89
3.78
13.30
13.85
93
7.56
18.20
17.36
111
9.45
20.30
19.25
111
5.6
15.86
15.45
108
10.12
21.16
20.51
106
20.24
28.87
30.63
98
of galactose-1-phosphate
Table 6. Effect of Low Phosphogluconate Dehydrogenase Activity on the Present Assay Galacto..-i-phosphat. In h.niolysat.
activity
of galactose-1-phosphate
Diluted hemolysate
consumed per minute.
uridylyltransferase
ob-
tamed was measured by mixing an aliquot of the aqueous transferase solution with our working reagent. The transferase activity was calculated by assuming that 1 mol of NADPH is formed per mole of glucose-6-phosphate oxidized.
1714 CLINICALCHEMISTRY,Vol. 23, No. 9, 1977
Added
Found5
ExpeCt.da
U/Ifterb Hemoiysate
of 2 mol of NADPH. One mole of NADPH is formed when glucose 6-phosphate is oxidized to 6-phosphogluconate by glucose-6-phosphate dehydrogenase. The second mole of NADPH is produced from the reaction of 6-phosphogluconate with NADP, catalyzed by 6-phosphogluconate dehydrogenase. We assume that the hemolysate contains sufficient 6-PGD to oxidize 6-phosphogluconate to ribulose-5phosphate. To test the validity of this assumption, we measured the recovery of GAL-i-PUT2 (from yeast, Sigma) added to hemolysates. When the activities were calculated assuming that 2 mol of NADPH is formed per mole of GAL-i-P consumed, recoveries between 89 and iii% were obtained (Table 4). Although these resuits indicate that there is usually sufficient 6-PGD in the hemolysate to account for complete conversion of 6-phosphogluconic acid to ribulose-5-phosphate with concurrent formation of NADPH, an inborn absence of
The
urldylyttr.nsfsrase
actIvity
the formation
2
-7
-21 -33
Micromoles of 6-phosphogluconate consumed per minute. Micromoles of galactose-1-phosphate consumed per hour per gram of
a
EXpCtId
20.9 17.8 15.1
10.69
3.81
14.21 14.21
25.04 17.59
a
Results calculated assuming that 2 mol of NADPH Is formed.
b
Micromoles
of galactose-i-phosphate
24.90 18.02
consumed per minute.
6-PGD would result in GAL-i-PUT activities cornpatible with heterozygosity for galactosemia. Addition of 6-PGD to the working reagent would assure that 2 mol of NADPH was formed per mole of glucose-6-phosphate oxidized. Hemolysates were analyzed for transferase activity by use of reagents with and without added 6-PGD (from yeast, Boehringer Mannheim). The results (Table 5) indicate that addition of 6-PGD inhibited transferase activity. This inhibition was not reversed by dialyzing the 6-PGD overnight at 4 #{176}C in glycylglycine buffer. 6-PGD also inhibited transferase from yeast (Sigma) or calf liver (Boehringer Mannheim). Because 6-PGD cannot be added to the system, individuals with moderate 6-PGD deficiency could exhibit falsely low transferase activity in our assay. However, in a survey of a large American population (12) it was shown that less than i% have reductions of erythrocyte 6-PGD to the range of 42 to 65% of normal; there was no instance of complete absence of 6-PGD. In the normal erythrocyte, the activity of GAL-iPUT is 1/25that of 6-phosphogluconate dehydrogenase. Since the Km of the dehydrogenase for 6-phosphogluconate is relatively low (i8 imol/liter), a steady state (i.e., equal rates of production and of oxidation of 6-
Table 7. Measurement of Galactose-1-phosphate Urldylyltransf erase Activity by Our Assay and by Using NAD and Bacterial Giucose-6-phosphate Dehydrogenase Galactos.-1-phophat#{149}
urldylyltran.feraa.
A
U) 0 U
m Cl) U-
actIvIty
0 ‘Ii
Bb U/g HbC
23.1
23.9 27.8
27.7 30.3 27.3
32.1 26.4
22.5 30.1 a
5
O
I?
9
21 23
TRANSFERASE
22.9 31.2
Fig. 3. DIstrIbution
25
ACTIVITY
21
29
31
33
u/gHb
of galactose-1-phosphate uridyiyltransferase
activity for 205 children
Measured by the present assay, and calculated assumIng that2 mol of
NADPH is formed. b ieea,ed by fog
and bacterial glucose-6.c*ioephate dehy&ogenase
andcalculated,assumIng that 1 mol of NADH Is formed. #{176}Mlcromoies of galactose-1-phosphate consumed per hour.
phosphogluconate) should be achieved after
