The assay of red cell galactokinase

BIOCHEMICAL

5, 325-332

MEDICINE

The Assay E. BEUTLER, City

of

(1917)

of Red

Cell

N. V. PANIKER,

Hope

Medical Duatie,

Center,

F. TRINIDAD

AND

1500

E. Duarte

Rd.,

California 91010

Received February Galactokinase phosphorylation

Galactokinase

17,

1971

catalyzes the first step of galactose by ATP to galactose-l-phosphate:

Galactose

+ ATP+Galactose-l-Phosphate

metabolism,

its

+ ADP.

A deficiency of this enzyme was first discovered by Gitzelman (1,2) in 1965. Several patients have been detected subsequently, and the defect has been found to be invariably associated with cataracts, unless a galactose-free diet has been instituted. Recently, we have presented evidence that not only the homozygous, completely enzyme-deficient patient is subject to the development of cataracts, but that mild galactokinase deficiency may also be associated with cataract formation (3, 4). Several methods for the assay of galactokinase using 14C-galactose have been presented previously (5-9). They depend upon incubation of radioactive galactose with ATP and the enzyme source with the subsequent separation of labeled galactose-l-phosphate. In applying such techniques to hemolysates from the red cells of galactokinase deficient and normal subjects we encountered various important technical problems. These concerned impurity of commercial galactose 14C solutions, the instability of solutions of unlabeled galactose, and the relative instability of galactokinase in stored blood. These problems have been investigated in some detail, and a procedure has been devised which permits relatively rapid assay of the enzyme in large numbers of blood samples. We also studied such factors as the effect of galactose intake of the patient and the effect of red cell age on enzyme activity, MATERIALS

AND

METHODS

‘*C-1-Galactose was obtained from several different commercial sources including Calbiochem, ICN, Volk Chemical Corp; and Nuclear-Chicago. Unlabeled n-galactose was obtained from Sigma Chemical Co. or Nutritional Biochemicals Corp. Blood from adult subjects was obtained from normal hospital employees or from hematologically normal subjects with325

out cataracts. Blood from children was obtaiucd from the children of hospital employees and from well children through the corutcas): of Dr. Darken Powars. 14C-Galactose from several suppliers has bet111 found to b(. contaminated with glucose or with other sugars which art’ phosphorylnted by hexokinasc and ATP. Since red cell hesokinasca lcvcls arc’ mor(‘ than 10 times as high as the lcvek of galactokiuasca. prcsencc of ~\YW small amounts of “C-glu~~~c produces a serious error in the procedure. Accordingly, the following purification proccdnrc \WS usc~l for all colnmcrcial A small column is prepared by- placing a 14C-galactose preparations. little glass wool into the bottom of ;I tuberculin syriugtl barrel and adding microgranular DEAE (Whatman DE-52) slurry to give a \~olumr~ of 0.2-0.3 ml. The column is washed with a fen1 drops of 3.8 mM galactosc solution followed by 5 ml of water. ‘Y-l-Calactosc~ is diluted to contain approximately 10 /~c/ml. Two microliters of 1 \I Tris-HCl h&r. pH 8, 5 ,.Ilitclrs of 0.1 M MgCl,, 100 hclitrrs of hcsokinnsc diluted in \\,ater to contain 2 units/ml and 10 Jiters of 60 mhr neutralized ATP ;W addrd to each milliliter of the I4C-galactosc solution. After 60 minutes incubation at 37” the reaction mixture is passed through the column which is then washed with sufficient additional distilled watrxr to civet S times thca original volume. The radioactive galnctosc solutiorl \\Thich has hecu collected contains approximately 2 ,IC~of ‘~C/ml and is stable whcbn frozen. A partial reaction mixture is preparcad 1,~ misil-tg thcl follolving rcagents in. the proportions shown:

?iaF,

100

111\1

MgCl,, 100 IllX Tris-HCI, I LI, pH 7.4 (hhc’to;;t’, 7.6 ElM ‘T-Galaclosc=, 2 pr/rnl ATP CpH 7‘1, .I” h,

IO0

100 1011 100

WI 1 100

The mixture is incubated at 37” for 4 hours and is then stored in the frozen state. The red blood cells were washed two times in 0.9% saline solution and then hemolyzed by the addition of 4 vol of a stabilizing solution containing 7 mhf of ,&mercaptoethanol, .Ol mM NADP, and 2.7 II~M EDTA. This solution is employed because we use it for other red cell enzymt assays (10). Equivalent results may bc obtained by hemolyzing the red cells in water or in 7 mhf p-mercaptoethanol solution. The enzyme activity is not as stable in a water hemolysatc, however. and therefore /Imercaptoethanol-containing solutions were used for hemolysis. The

ASSAY

OF

RED

CELL

GALACTOKINASE

327

hemolysate may be centrifuged at 5OOOg for 15 minutes, and the supernatant is used for assay, or the uncentrifuged hemolysate may be used with the same results. An aliquot of the reaction mixture is thawed, mixed thoroughly, and 100 pliters pipetted into a small test tube. The hemolysate (100 pliters) is mixed with the reaction mixture. TWO 50Jiter aliquots of the mixture are added to 20-pliter aliquots of a 1 M galactose solution. After mixing thoroughly, 50 ~1 of each aliquot is spotted on a circle of DE-81 paper, approximately 2.5 cm in diameter. One of the circles is allowed to dry (100% standard) while the other is placed into a small dish of distilled water ( 0 time sample). The remaining hemolysate-reaction mixture is incubated at 37” for 60 minutes, and another 50-pliter aliquot is treated in a fashion identical with the 0 time sample. This is designated as the 60-minute sample. The 0 time sample and the Ml-minute sample are placed, while wet, on a sintered glass funnel and are washed with 600-800 ml of distilled water which is drawn through the sintered glass with a vacuum. These two samples are now permitted to dry under a 150-200-w light bulb and are ready for counting, which is accomplished in a scintillating solution consisting of toluene with 0.3 gm POPOP and 5 gm PPO/liter. RESULTS

Linearity. Various dilutions of a normal hemolysate were made as shown in Fig. 1. The percentage of galactose phosphorylated increased in an approximately linear fashion. When fairly concentrated hemolysates are used, an appreciable degree of quenching is observed: there is approximately 55%quenching with a 50%hemolysate, 20%quenching with a 25% hemolysate, and little or no quenching with a 12% hemolysate. Little hemoglobin is removed from the DE-81 paper by washing, however, and in all but the most concentrated hemolysate the amount of quenching is very similar in the 100%sample, the 0 time sample and the CM-minute sample as shown by the channels ratio method. Therefore, it has no effect on the computed enzyme activity. In the case of hemolysates with hemoglobin concentrations considerably in excess of 5 gm %, however, quenching becomes significant, and there is sufficient difference in the quenching observed with the washed sample as compared with the 100%sample, so as to result in a slight over-estimate of enzyme activity. Drying samples at too high a temperature, e.g., a 142’ oven, produces an increase in the amount of quenching. Effect Of Time Of Zncubation. Aliquots were removed from the incubation mixture at Sminute intervals and the percentage of galactose phosphorylated measured. The phosphorylation of galactose was found to be linear with time over a 2.5hour period.

328

, S

0

,

!

0

5 Hb

1

.IO

15

CONCENTRATION

FIG. 1. ‘The relationship between hemoglobin wncentration of the lw~lolysatv assayed and the percentage of galactose phosphorylated after I hr of incubation. The hemolysates were prepared by freezing and thawing 1 vol of packed red cells with 2, 4, 8, 12, or 16 vol of hemolyzing solution. Each assay was carried out in triplicate.

The Stibility Of The Assay Mixture. The age of the unlabeled galactose solution and the time and temperature of its storage influenced the results of the assay. Freshly prepared galactose solutians resulted in lower assay values than solutions which had been stored for several days at 4’ or which had been incubated for several hours at 37”. This was interpreted as being a result of mutarotation of galactose from the (Yform to an equilibrium mixture of (Yand ,6 forms, a process which has been shown to occur in two phases, one rapid and one slow ( 11). It was found, however, that storage of galactose solutions at 4” for 24 hours or boiling for 10 minutes, which should bring the cy and /3 forms into equilibrium, did not, in point of fact, result in assay values as high as those obtained with solutions which had been permitted to age for longer periods of time. Furthermore, assay of the galactose content of such galactose solutions with galactose dehydrogenasc and NAD showed them to consist entirely of galactose. However, solutions which had been stored at 4’ for 4 days or 37” for 4 hours were more active in the

ASSAY

OF

RED

CELL

329

GALACTOKINASE

TABLE

1

STABILITY OF GALACTOKINASE IN BLOOD SAMPLES STORED AT 4 AND 25”

Time of storage (days) 25” Anticoagulant ACD (NIH formula B) Heparin (10 units/ml) EDTA (1 mg/ml) EDTA + glucose

1

2

35.2 31.9

0 29.9 31.0

29.6

32.4 30.5 33.5

28.3

34.7

29.3

30.2

4” 5

28.9 20.1 16.8 31.4

6

20

32.4 30.5 31.4 30.8

31.5 17.8 17.7 30.2

25"

0 ACD ACD + galactose EDTA EDTA + gala&se EDTA + glucose EDTA + glucose + gala&se

22.9 24.3 24.4

3

6

23.2 23.7 20.5

24.0 21.9 2.3

19.6 20.9 22.5

17.0 22.3 22.3

assay system and contained only approximately 88% galactose when assayed with galactose dehydrogenase. These findings suggest that a change was occurring in addition to mutarotation. Assay of galactose solutions for keto sugars (presumably tagatose) using the resorcinol reaction (12) showed that ketoses gradually accumulated reaching maximum levels at 4 hours at 37”, at 11 days at 25”, and at 20 days at 4”. Accordingly, assays should be carried out only with suitably aged galactose solutions. Reproductivity. Eight replicate determinations were carried out on separately washed red cells of a single donor. The enzyme activity was found to be 25.50 mUnits/gm Hb with a standard deviation of 2.90 mUnits/gm Hb. Galactokinase estimations were carried out 5 times on the blood of the same donor over a period of several months. The average of triplicate determinations was found to be 26.69 mUnits with a standard deviation of 1.34 mUnits. Efect of Anticoagulants clncl Storage. Identical galactokinase values were found when blood was drawn into ACD solution, EDTA, or heparin. On storage at 4” enzyme levels were well maintained for at least 20 days in ACD solution and 6 days in EDTA or heparin. The addition of sufficient glucose to give a final concentration of 26 mxq but galactokinase

5 1 OJ ml

I 01

05

1

2

5

10

20

%

40

60

80

90

993

OF SUBJECTS

FIG. 2. A probability plot of the galactokinase activities of normal adults and children aged 2-16. not of galactose to give a final concentration of 4 mu, had a considerable stabilizing effect on the enzyme (Table 1). Nomul Values. The distribution of galactokinase activities of the red cells of normal subjects is shown in Fig, 2. Blood samples from 32 children aged 2-16 are included in this series. Children of these ages were found not to have significantly higher enzyme activities than did adults [adult values: 29.33 +- 5.21 (mean -t 1 SD.) vs. children: 32.25 t lOI’%]. Only a few samples of blood from newborn infants have been tested, and these are not reported here. However. as previously found by Ng ( 7), galactokinase activity of infant red cells is approximately 2-3 times the level found in adults. To determine whether the activity of galactokinasc was influenced by gaIactosc intake, estimates of the galactose intake of 29 adults were made, and the activity of their red cell enzyme determined. As shown in Fig. 3, there was no correlation between the activity of this enzyme in the red cells and galactose intakca. Thawagcbof the red cell population did not appear to exert ;r major influence on red cell galactokinase activity, although rcticulocytcs prob-

ASSAY

OF

RED

CELL

GALACTOKINASE

DIETARY

GALACTOSE/WEEK

331

OA

200 qn

and

FIG. 3. The galactokinase

relationship between (GK) activity.

estimated

weekly

dietary

intake

of

galactose

ably contain slightly increased enzyme activity. The activity of the red cells of five subjects with elevated reticulocyte counts averaged 38.8 with a standard deviation of 10.4. A subject with aplastic anemia had 33.5 mUnits/ gm Hb galactokinase activity. DISCUSSION

The assay of red cell galactokinase activity appears to be clinically important in patients with cataracts, and the availability of a reliable method for the detection, not only of total enzyme deficiency, but also of reduced enzyme activity, is important. The procedure outlined here has been carried out on several hundred blood samples, both from humans and from rabbits. If proper care is taken to purify commercial galactosel-l% solutions, and if only properly aged unlabeled galactose solutions are employed, very satisfactory results are obtained. SUMMARY

A method for the assay of red cell galactokinase is presented. Comis puriiSed by incubating with ATP, mercially available W-galactose magnesium, and hexokinase and passed through a small DEAE column. Hemolysate is mixed with a reaction mixture, incubated, and the W-