An automated method for the determination of serum 5′-nucleotidase

CLfNICA

739

CHIMICA ACTA

AN AUTOMATED

METHOD

FOR THE DETERMINATION

OF SERUM

$-NUCLEOTIDASE P. G. HILL

AP*‘D 1%

G. SAMMONS

Departmentof Patholqy,

East Birmingham Hospital, Bordesley Green East, Birmingham, 9 (Great Britain) (Received

August

zgth,

1965)

SUMMARY

An automated procedure for the estimation of serum g’nucleotidase (EC 3.1.3.5, 5’-~bo~ucleotide phos~hohydroIase) for use with the Technicon Auto Analyzer is described. 5’-nucleotidase activity is measured as the nickel-sensitive hydrolysis of adenosine-5’-monophosphate at pH 7.4. The timed incubation period is terminated by the addition of ethylenediaminetetra-acetic acid. Results from the automated method are compared with the manual method for j’-nucleotidase, upon which the automated procedure is based.

INTRODUCTION

Serum levels of 5’-nucleotidase have been shown to be increased only in diseases affecting the liver and biliary system 1--3.It is thus a more specific test than is alkaline phosphatase (EC 3.1.3.1, orthophosphoric monoester phosphohydrolase) since serum levels of this latter enzyme are increased in both bone and liver diseases. It is also a more sensitive indicator of biliary obstruction than is alkaline phosphatase 4. In developing an automated method for the estimation of serum g’-nucleotidase our aim has been to produce a method suitable for the routine clinical laboratory since we feel that this is an enzyme which has been largely neglected as an aid to the diagnosis of hepatic disease. REAGENTS

Sodium diethylbarbiturate (VeronaI)-Hal buffer, pH 7.4 at 37”, containing 0.05~ M M&l, ~6 H,O (10.37 g/l). To I 1 0.04 &l Verona1 (8.25 g/l) was added 140 ml 0.2 N HCI. The M&l, was then added and dissolved, and the pH adjusted to 7.4. 2. Control-bu@er solution Nickel chloride was added to a portion of the test-buffer to give a nickel concentration of 0.0127 iM, (3.0188 g/l). The pH was then adjusted to 7.4. C&n.Chim. Acta,

13 (1966)

739-745

740

P. G. HILL, H. G. SAMMONS

3. Adenosine-5’-monophosplzate

(AMP)

AMP (347 mg) was dissolvedin with distilled water.

IO mM

18 ml 0.1 N NaOH, and made up to IOO ml

4. Ethylenediaminetetra-acetic acid, disodium salt (EDTA), 300 mM EDTA (55.8375 g/500 ml) was suspended in I N NaOH and dissolved by the addition of IO N NaOH, the pH of the solution was then adjusted to approximately 7.4 by the addition of hydrochloric acid. The solution was then diluted to volume with distilled water. 5. Amino-naphthol-s&phonic acid reagent I. Stock sol&ion. (a) Sodium metabisulphite 137 g. (b) Sodium sulphite (hy-

drated) IO g. (c) I-amino-z-naphthol-4-sulphonic acid 2.5 g. Reagents (a) and (b) were dissolved in about 800 ml distilled water, the solution was then warmed to approximately 50” and reagent (c) was added, and dissolved by stirring. The solution was then diluted to 1250 ml with distilled water and stored at 4” in a dark bottle. 2. Working solution. The working solution of amino-naphthol-sulphonic acid reagent was prepared by diluting IOOml of stock solution to I 1 with distilled water. 6. Ammonium

molybdate

reagent

Ammonium molybdate (15 g) was placed in a z-l volumetric flask; approximately I 1 of distilled water was added, followed by the careful addition of 107 ml concentrated sulphuric acid. The molybdate was dissolved and the solution was diluted to 2 1 with distilled water. This reagent was filtered before use. One small drop of “Quix” detergent was added to each litre of ammonium molybdate reagent and working solution of amino-naphthol-sulphonic acid. 7. Standard phosphate solutio+a I. Stock solution. Potassium dihydrogen orthophosphate (KH,PO,),

(4.381 g), was dissolved in I 1 of distilled water to give a stock solution containing 100 mg phosphorus per IOO ml. 2. Working standards were prepared from this stock solution to give phosphate standards equivalent to IO, 20, 30,40,60,80, IOO, 120, and 140 pugphosphorus per ml.

METHOD

The automated method presented in this paper is based upon the manual method of Campbell6 for the determination of serum 5’-nucleotidase. The method depends on the specific inhibition of 5’-nucleotidase by nickel ions. EDTA is used to stop the enzyme reaction at the end of the timed incubation period. The manifold for the automated procedure is shown in Fig. I. In this method it is necessary to analyse the samples twice, in the first determination the “Test”buffer is used to determine total phosphate liberated by alkaline phosphatase and 5’-nucleotidase at pH 7.4 together with any endogenous inorganic phosphate present in the sample. In the second determination, the nickel, included in the buffer to give a final concentration of IO mM, inhibits the 5’-nucleotidase and thus the phosphate Clin.

Chim.

Acta,

13 (1966)

739-745

SERUM 5’-NUCLEOTIDASE

741

DETERMINATION

which is measured, is inorganic phosphate plus phosphate released by alkaline phosphatase. Phosphate, in both cases, is measured calorimetrically by the method of Fiske and Subbarow 6. The difference between “Test” and “Control” represents the activity of 5’nucleotidase which is expressed as micromoles phosphate liberated per min per 1 of serum (I.U./l), as recommended in the Report of the Commission on Enzymes’.

15mm

colorlmeter tubular t/c

recorder

660 mp filter

Fig. I. Manifold for 5’-nucleotidase determination. symbols. Numbers to the right of the proportioning

SMC, DI, etc. are standard “Technicon” pump indicate millilitres pumped per min.

The sampler can be run satisfactorily at 60/h with a water wash between each sample cup. The result of “Test” and “Control” is converted to International Units per litre by the following formula, Test-Control 31

X--

IO00 t

=

International Units per litre.

The factor t is the incubation time for the enzyme estimation expressed in minutes. This is the time taken for a sample to pass from the point of addition of the substrate to the point of addition of EDTA. For the analyzer used in this laboratory t varied between 12.75 and 13.25 on different runs. This method has also been used for the determination of 5’-nucleotidase in hepatic bile.

Clin. Chim. Acta, 13 (1966) 739-745

P. G. HILL, H. G. SAMMONS

742 RESULTS

The reproducibility

of the method is demonstrated in Fig. 2 (a) for the IO and and in Fig. 2 (b), in which the seven peaks represent 60 lug/ml standards. Fig. 2 (a) also demonstrates that between the highest and lowest there is only slight contamination of the low standards by the high stan-

140pg/ml standards phosphate standards

dard. The greatest range normally encountered between serum specimens is not more than 100-20 ,q/ml, at which concentrations no contamination occurs. Fig. 2 (a) also shows the series of standards used for drawing up the calibration curve. A typical analysis of sera from eleven patients is shown in Fig. 3. The standards used were 20 ,ug/ml to 120 ,q/ml.

Fig. L. (a) Standards used for preparing calibration curve. (b) Reproducibility

Fig. 3. Analysis of sera from eleven patients, test and control series. Clin. Chim. Acta, 13 (1966) 739-745

of Oo,~g/ml standard.

SERUM $-NUCLEOTIDASE

5’-nucleotidase been compared

743

DETERMINATION

values obtained

by the manual

for both normal and pathological

and automated

methods

have

sera. For the normal sera (m = ZI),

the ranges obtained were 3.5-8.4 and 0.0-8.7 by the manual and automated methods respectively. The results for the pathological sera (n = 28) by the two methods are shown graphically in Fig. 4. Twelve analyses of the same sample gave a mean which gives an error for the method of 2.20/o.

of 55.8

Fig. 4. Comparison of Manual and Analyzer methods for 5’-nucleotidase. + = regression points.

I.U./l

(SD.

1.2),

o = exper?mentaipoints.

DISCUSSION

Although 5’-nucleotidase has been shown to be widely distributed in human tissues8, the serum level of this enzyme is raised only in post-hepatic or intra-hepatic obstruction. The raised serum level in these conditions has been attributed to a blockage of the normal excretory route of the enzyme from the liver in the bile, and indeed, 5’-nucleotidase is present in high concentrations in hepatic bile. In measuring the serum level of 5’-nucleotidase, the activity of non-specific phosphatases must be taken into consideration. Dixon and Purdom 2 estimated 5’nucleotidase by measuring the hydrolysis of AMP and p-glycerophosphate separately at pH 7.5. The difference gave a measure of 5’-nucleotidase activity. Ahmed and Reiss estimated the alkaline phosphatase contribution to the hydrolysis of AMP by measuring the hydrolysis of phenyl phosphate at pH 7.5. Both these methods suffer from the disadvantage that these three phosphate esters are not hydrolysed by alkaline phosphatase at the same rate. /&Glycerophosphate is hydrolysed by alkaline phosphatase more slowly than is AMP and phenyl phosphate more rapidly. Bardawill and Chang4 expressed as 5’-nucleotidase activity the hydrolysis of AMP at pH 7.3. The difficulty of interference by alkaline phosphatase was overcome by Youngl, who preincubated serum with 0.0015 M ethylenediaminetetra-acetic acid, which inactivates alkaline phosphatase but has no effect upon 5’-nucleotidase. The possiC&L Chim. Ada,

13 (1966) 739-745

P. G. HILL, H. G. SAMMONS

744

bility of using nickel to inhibit 5’-nucleotidase and thus obtain a measure of the hydrolysis of AMP by alkaline phosphatase at pH 7.5 was suggested by Ahmed and Reis O,and Campbell 6 developed a method based on this principle. It is this latter method that has been adapted for use with the AutoAnalyzer. In Campbell’s method the reaction is terminated by the addition of trichloracetic acid, however, this is unsuitable in an AutoAnalyzer procedure and therefore another enzyme inhibitor had to be used to stop the reaction at the end of the incubation period. We have found that the disodium salt of EDTA at a final concentration of 20-30 mM in the reaction mixture used in the method, gives complete inhibition of the enzyme, and also of alkaline phosphatase. This is demonstrated in Fig. 3, in which per cent activity of 5’-nucleotidase is plotted against EDTA concentration.

Fig. 5. Inhibition of 5’-nucleotidase automated method.

activity

by EDTA,

in the reaction

mixture used in the

The pH at which the reaction is carried out is critical in the automated method. It has been changed to 7.4 from 7.5 at 37’ since it was observed that at the latter pH the control standard peaks had a considerably lower absorbance than the test standard peaks although the control sera peaks were not proportionately decreased. It was also observed that in the control series the wash between the standards was very poor at this pH. These effects were shown to be due to the nickel in the control buffer. Presumably at pH 7.5 the low solubility product of the nickel phosphate complex, Ni,(P0J2. 8 H,O, is exceeded and precipitation occurs. At pH 7.7 the serum peaks are also decreased in the presence of nickel although still not so markedly as the standards. At pH 7.4 however, the nickel does not interfere with the control determinations. The method has been shown to be reproducible and good correlation has been obtained between this method and the manual method upon which it is based. In the development of this method and in its routine use a single calorimeter Clin.

Chim.

Acta,

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SERUM 5'-NUCLE~T~DASE DETERMINATION

and single pen recorder have been used. However with two calorimeters and a double pen recorder this method could easily be adapted for running “Test” and “Control” series together, as has been done for the automated determination of alkaline phosphatase lo. The method is being currently used as a test of liver function and also in the differentiation of elevated serum alkaline phosphatases due to liver and bone diseases. The method is also being used in a study of the properties of human 5’-nucleotidase. Results of these further studies are to be submitted for publication shortly. REFERENCES I 2 3 4 5 6 7

I.I.YOUNG, Ann. N.Y. Acad. Sci., 75 (1958) 357. T. F. DIXON AND M. PURDOM, J.Clin. Pathol., 7 (1954) 341. M. K. SCHWARTZ AND O.BODANSKY,GWWW, 18 (196.5) 886.

C. BARDAWILL AND C. GANG, Can. Med. Assoc. j., & (1963) 755. D. M. CAMPBELL, Biochem. .I., 82 (1962) 34P. C. H. FISKE AND Y. SUBBA~OW, j. kioi.-them., 66 (1925) 375. Refiort of the Commission on Enzymes, I.U.B. Symposium Series, Vol. 20, Pergamon Oxford, 1961, p. 8. 8 J. L. REIS, Biochem. J., 46 (1950) xxi. 9 2. AHMED AND J. L. REIS, Biochem. J., 6g (1958) 386. IO J. L. BELL AND M. COLLIER, J. C&z. Pathol., 17 (1964) 301. Clin. Chim. Acta,

Press,

13 (1966) 739-745