against adenosine monophosphate (Webster et al.,. 1978 .... OD obtained after 30 min incubation under the stan- .... Webster, A.P.B., North, M., Allsop, J., et al.
Journal oflmmunologicalMethods, 101 (1987) 73-78 Elsevier
73
JIM04396
A colorimetric assay for the determination of 5'-nucleotidase activity S. Perez 1, N. C o u r t i s
2, D.
K o k k i n o p o u l o s 1, M. P a p a m i c h a i l 1, C.M. Tsiapalis 2 a n d T. Trangas 2
Departments of l Immunology and 2Biochemistry, Hellenic A nticancer Institute, Papanicolaou Research Center, Athens, Greece (Received 21 November 1986, revised received 28 January 1987, accepted 6 March 1987)
A rapid, simple, quantitative and sensitive assay for the determination of 5'-nucleotidase has been developed. The method can be applied to both soluble and membrane bound forms of the leukocyte enzyme. Enzyme activity is determined by colorimetric estimation of N H 3 released from adenosine, the product of 5'-nucleotidase activity in the presence of adenosine deaminase. The assay may be performed in microtitre plates and read with an automatic multiscan spectrophotometer. Thus it can be applied to a large number of samples for routine medical and research purposes. Key words: 5'-Nucleotidase; Immunodeficiency; Adenosine deaminase
Introduction
Lymphocytes have a relatively high activity of membrane bound 5'-nucleotidase, which is active against adenosine monophosphate (Webster et al., 1978; Carson et al., 1981). However, it has been observed that the blood lymphocytes of patients with primary (variable) hypogammaglobulinaemia (CVH) have very low 5'-nucleotidase activity (Johnson et al., 1975). Since cord blood T and B cells have relatively low 5'-nucleotidase activity, it is possible that the reduced activity in the lymphocytes of CVH patients reflects a state of immaturity (Webster et al., 1979). It has also been demonstrated that 5'-nucleotidase activity is related to the stage of B cell maturation and, furthermore, that the enzyme is induced by phorbol esters (Hibi et al., 1984; Sen Correspondence to: T. Trangas, Department of Biochemistry, Hellenic Anticancer Institute, Papanicolaou Research Center, 171 Alexandras Av., Athens 11 522, Greece.
Gupta et al., 1984). Therefore, the low 5'nucleotidase detected in the lymphocytes of patients with severe combined immunodeficiency (Boss et al., 1981) may be attributed to lack of mature lymphocytes in the circulation. Measurement of the enzyme is of some value in distinguishing 'common' acute lymphoblastic leukaemia (cALL) from T acute lymphoblastic leukemia (T-ALL), since 5'-nucleotidase is lower in T-ALL while it is raised in cALL. 5'-Nucleotidase is significantly higher in lymphoblastic than myeloblastic transformation (Hoffbrand et al., 1982). 5'-Nucleotidase is low in B chronic lymphoblastic leukaemia (B-CLL) cells (Quagliata et al., 1976) and in other B cell disorders such as B-ALL, hairy cell leukaemia and WaldenstriSm macroglobulinaemia (Kramers et al., 1975). In order to study variations in 5'-nucleotidase activity in minor leukocyte populations we have developed a simple, sensitive and quantitative colorimetric microassay which can also be applied to measurement of serum 5'-nucleotidase activity.
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
74 Materials and methods
Reagents 14C-5'-AMP (507 m C i / m m o l ) was purchased from Amersham International, England; unlabelled 5 ' - A M P and adenosine were obtained from Boehringer-Mannheim, Tutzing, F.R.G.; Whatman no. 1 sheets were from Whatman products, Maidstone, Kent, England; 5'-ribonucleotide phosphorylase from Crotalus adamanteus venom, adenosine deaminase from calf intestinal mucosa and the phenol nitroprusside solution (reagent I) and alkaline hypochlorite solution (reagent II) were from Sigma Chemical Co., St. Louis, MO; all other reagents were of analytical grade; culture media were from Flow Laboratories, U.K.; fetal calf serum was a product of Filtron, Altona, Australia. Biological material The following cell lines were used: MOLT3, a T cell line originating from a 19-year-old patient with acute lymphoblastic leukaemia (Minowada et al., 1972); K562 cell line, which was originally established from the pleural effusion of a patient with chronic myelogenous leukaemia (Lozzio and Lozzio, 1975) and characterized as erythroleukaemic in nature (Anderson et al., 1979); KM3, a pre-B human leukaemic cell line established from a patient with ' c o m m o n ' acute lymphoid leukaemia (Schneider et al., 1977); Raji, a B cell line derived from African EBV-positive Burkitt lymphoma (Hinuma and Grace, 1967). Cells were routinely grown as exponentially proliferating cultures in suspension, in R P M I 1640 supplemented with 10% fetal calf serum, 2 m M k-glutamine and 50 ~ g / m l gentamycin and incubated at 37 ° C in a humidified atmosphere and 5% CO 2. Spleen and thymus cells were obtained from young ( 2 - 4 weeks) A K R mice. The tissues were removed, cut into pieces and the lymphocytes were subsequently isolated as previously described (Ford, 1979). Human peripheral blood leukocytes were isolated from the heparinized blood of healthy donors by the method of B/Syum (1968). Cells from all sources were washed three times with PBS (0.01 M phosphate-buffered saline, p H 7.3) prior to the assay procedures.
Viability was assessed by trypan blue dye exclusion and was always found greater than 98%.
Radiochemical 5 '-nucleotidase assay The assay measures the conversion of the radioactive substrate (14C-5'-AMP) to the corresponding nucleoside. The product is separated from the substrate by paper chromatography on Whatman no. 1 using a solvent system containing ethanol/1.0 M a m m o n i u m acetate p H 7.5 (7/3). The assay was performed with intact cells in PBS containing 0.5 m M 14C-5'-AMP (7-15 c p m / p m o l ) . The mixture was incubated at 3 7 ° C for 30 rain. The reaction was terminated by the addition of E D T A to give a final concentration of 1 mM. 10 /zl aliquots were spotted on the paper sheet. The substrate and products spots were detected with UV light. They were then excised and counted in a scintillation counter (Sen G u p t a et al., 1984). The enzyme activity was expressed as nmol of substrate hydrolysed per 30 min per 2 × 10 6 cells. Non-radioactive adenosine and A M P were used as markers. Colorimetric 5'-nucleotidase assay Adenosine, the product of 5'-nucleotidase action on 5'-AMP, is hydrolysed in the presence of an excess of adenosine deaminase (ADA) to ammonia and inosine. The amount of ammonia released can be quantitated using a modification of the Berthelot procedure (Chaney and Marbach, 1962). Under standard assay conditions, intact cells were added to the reaction mixture containing 0.5 mM 5'-AMP and 0.04 U//~I A D A in a final volume of 200 /~1 PBS. The reaction mixture was incubated at 3 7 ° C for 30 rain, unless otherwise indicated, and the reaction terminated by the addition of E D T A to give a final concentration of 1 raM. The samples were then centrifuged at 1 5 0 0 × g for 5 min and 60 ~1 of reagent I and subsequently 60 ~1 of reagent II were added to the supernatants followed by incubation at 3 7 ° C for 15 rain. The samples were then brought to a final volume of 1 ml by the addition of water and the O D measured at 630 nm. The same procedure was basically followed to assay the 5'-nucleotidase activity in smaller volumes using microtitre plates. An identical reaction mixture was used, except that, after centrifu-
75
gation, 50 /~1 of each of reagents I and II were added to 100/~1 of supernatant and the O D measured in an automatic microplate reader (Dynatech MR600) with a 630 nm filter.
10
~
o
5
mU/ml ~
05
Results
©
Standardization of the 5'-nucleotide assay Linear and reproducible relationships were found between O D and (1) the concentration of ammonia and (2) the amount of liberated ammonia when various amounts of commercial 5'nucleotidase were added to 5 ' - A M P in the presence of calf intestine adenosine deaminase (Fig. 1). Fig. 2 shows the time course of the reaction. Linear relationships were found between time and O D up to a 30 rain incubation period. A three-fold increase of the substrate concentration and of adenosine deaminase levels did not modify the initial velocity, indicating that under the assay conditions there is an excess of 5'-AMP and adenosine deaminase. Application of the ecto-5'-nucleotidase assay using intact cells Linear and reproducible relationships were obtained between O D and cell number per assay using both human peripheral blood leukocytes and the K562 cell line (Fig. 3). The difference in the slope of the lines reflects the difference in the m e m b r a n e 5'-nucleotidase activity, and, as expected, peripheral blood leukocytes had almost
i
0
30
i
i
i
60 90 time(min}
~
120
Fig. 2. OD at 630 nm as a function of incubation time using 5 m U / m l (D) and 25 m U / m l ( O ) commercial 5'-nucleotidase. Each point is the mean+_standard deviation of triplicate samples.
twice as much 5'-nucleotidase activity as K562 cells. The 5'-nucleotidase activity from the membrane of intact cells produced linear kinetics up to 30 min thereby mimicking the time course produced by the commercial enzyme (Fig. 4). Modification of the assay procedure for use under conditions suitable for the determination of 5'-nucleotidase activity in a large number of small leukocyte populations produced identical results.
0.7 0.6 0.5 o
up d
d m ~o
o.4
0.3 0.2-
©
0.1-
o5 0 o
10
20
30
40
50
rnU/ml Fig. I. OD at 630 nm as a function of 5'-nucleotidase per assay after a 30 min incubation period. Each point is the mean_+ standard deviation of triplicate samples.
1
2 3 4 cells (10"6)/ assay
T 5
Fig. 3. OD obtained after 30 min incubation under the standard assay procedure as a function of cell number using peripheral blood leukocytes (zx) and K562 cells (O). Each point is the mean +- standard deviation of triplicate samples.
76
04
80 c
o
03 ©
E c o
60
02 40
ila r
30
,
r
60
90
20
,
120
time(rnin)
Fig. 4. OD at 630 nm as a function of incubation time using the standard assay procedure with 2 × 106 K562 cells per assay. Each point is the mean_+ standard deviation of triplicate samples.
As shown in Fig. 5 the same linear relationship existed between OD and cell number under the conditions of the microassay within a range from 0.25 × 106 to at least 2 × 106 cells/well.
Comparison between radiochemical and colorimetric assays To compare the accuracy and sensitivity of the 5'-nucleotidase colorimetric assay to the widely
0
i
i
i
i
i
10
20
30
40
50
enzyme
concentration
L
(mU/ml)
Fig. 6. Hydrolysis of 5 ' - A M P as a function of 5'-nucleotidase c o n t e n t / a s s a y after 30 min incubation period, estimated by the radiochemical assay ( O ) and the standard colorimetric assay (zx). Each point is the mean-+ standard deviation of triplicate samples.
used radiochemical method, various concentrations of commercial 5'-nucleotidase were assayed by both methods. The OD values obtained by the colorimetric assay were converted to 5'-AMP degraded on the basis of a standard curve using various concentrations of ammonia, assuming that the amount of ammonia released per assay corresponds stoichiometrically to the adenosine converted from 5'-AMP by the action of 5'-nucleoti-
0.4
TABLE I C O M P A R A T I V E RESULTS OF C O L O R I M E T R I C A N D R A D I O C H E M I C A L ASSAYS IN V A R I O U S L Y M P H O I D POPU LATIONS
0.3
Source of cells 02
Enzyme activity ( n m o l / 3 0 m i n / 2 × 106 cells)
o
0.1
o
&
,% cell5
;s (1(5 6 )
2~o
/assay
Fig. 5. OD obtained after 30 min incubation under the microassay procedure as a function of cell number using peripheral blood leukocytes ( O ) and K562 cells (zx). Each point is the mean + standard deviation of triplicate samples.
Mouse spleen Mouse thymus H u m a n PBL K562 cell line MOLT3 cell line KM3 cell line Raji cell line
Colorimetric assay
Radiochemical assay
6.1 _+3.1 1.8_+0.6 29.4 +_7.6 16.0 _+5.9 3.5 + 0.2 8.7_+ 1.0 3.3 + 1.3
5.8 + 1.7 0.5 +0.1 24.3 + 4.9 13.5 + 7.7 3.4_+ 0.2 5.1 _+1.2 0
a (3) b (2) (6) (4) (3) (2) (2)
(2) (2) (5) (2) (2) (2) (2)
Mean + standard deviation. b N u m b e r in parentheses represents the number of independent determinations performed in triplicate.
77
dase. Fig. 6 shows that comparable curves were obtained by both assays. Furthermore, the radiochemical and colorimetric assays of 5'-nucleotidase were compared using various lymphocyte populations. The results presented in Table I indicate that the two methods produce estimates for the 5'-nucleotidase activity which are in agreement.
Discussion Several quantitative methods for the measurement of 5'-nucleotidase in blood serum and intact lymphocytes are available. Spectrophotometric assays are more commonly used for the quantitative diagnostic measurement of serum 5'-nucleotidase activity in hepatic diseases. There is a colorimetric assay which follows the release of inorganic phosphorus from the substrate by the Fiske and Subbarow method (1925). An alternative assay utilizes a sequence of enzymatic steps which lead to N A D formation monitored by a decrease in absorbance at 340 nm (Ellis et al., 1970). Both methods are relatively complicated and the latter requires a UV spectrophotometer. For the determination of ecto-5'-nucleotidase of intact lymphocytes the methods employed most commonly are either cytochemical (Misra et al., 1974) or radiochemical, in which case precipitation (Reaman et al., 1979) or chromatographic procedures (Sen G u p t a et al., 1984) are applied to achieve separation between substrate and product. This radiochemical assay with chromatographic detection is sensitive and highly specific but nevertheless time consuming and expensive. We have developed a colorimetric assay for the determination of 5'-nucleotidase activity. This assay is a modification of the method of Ellis et al. (1970) for the determination of serum 5'nucleotidase utilizing adenosine deaminase to convert the reaction products of the enzyme to inosine and ammonia. The latter is quantitatively measured by the Berthelot method, as previously described (Vielh and Castellazzi, 1984). The assay we have developed is simple and rapid, as it can be completed within 1 h. It has proved quantitative and produces linear kinetics for up to 30 min when used for both soluble and
membrane bound enzymes. The results can be monitored by a common laboratory spectrophotometer, and when a multiscan detection system is available the microassay procedure permits rapid screening of many samples. The assay gives the same results as the radiochemical assay when used with commercial enzyme and is as sensitive and reproducible for the determination of ecto-5'-nucleotidase activity of intact cells. As few as 2 × 10 s cells are required for an accurate estimate of the enzyme activity. It can also be used as an alternative method for the determination of serum 5'-nucleotidase when performed in the presence of excess /~-glycerophosphate to prevent the action of non specific phosphatases (Ellis et al., 1970). This assay can be used for the determination of ecto-5'-nucleotidase activity in leukocyte subpopulations for clinical purposes and to test variations of 5'-nucleotidase activity in lymphoid cell cultures. It may also be useful in studies of 5'nucleotidase levels in leukaemias and malignant lymphomas (Hoffbrand et al., 1982).
References Anderson, L.C., Nilsson, K. and Gahmberg, C.C. (1979) K562, a human erythroleukaemic cell line. Int. J. Cancer 23, 143. Boss, G.R., Thompson, LF., O'Connor, R.D., et al. (1981) Ecto-5'-nucleotidase deficiency association with adenosine deaminase deficiency and non-association with deoxyadenosine toxicity. Clin. Immunol. Immunopathol. 19, 1. B6yum, A. (1968) A separation of leukocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21 (suppl. 97), 1. Carson, D.A., Kaye, J. and Watson, D.B. (1981) The potential importance of deoxinucleotidase activity in mediating deoxyadenosine toxicity in human lymphoblasts. J. Immunol. 126, 348. Chaney, A . L and Marbach, E.P. (1962) Modified reagents for determination of urea and ammonia, Clin. Chem. 8, 130. Ellis, G., Belfield, A. and Goldberg, D.M. (1970) An NADHlinked kinetic 5'-nucleotidase assay. Clin. Enzymol. 2, 95. Fiske, C.H. and Subbarow, Y. (1925) The colorimetric determination of phosphorus. J. Biol. Chem. 66. 375. Ford, W . L (1979) In: D.M. Weir (Ed.), Handbook of Experimental Immunology, Vol. 2 (Blackwell Scientific Publications, Oxford) p. 23.1. Hibi, T., Sen Gupta, S., MacKay, A., Gelfand, E.W. and Chechik, B.E. (1984) Ecto-5'-nucleotidase. It. Effect of 12-O-Tetradecanoylphorbol 13-acetate on the expression of enzyme in normal and neoplastic B-cell lines. J. Natl. Cancer Inst. 73, 1081.
78 Hinuma, Y. and Grace, J.T. (1967) Cloning of immunoglobulin producing human leukemic and lymphoma cells in long term cultures. Proc. Soc. Exp. Biol. Med. 124, 107. Hoffbrand, A.V., Ma, D.D.F. and Webster, A.P.B (1982) Enzyme patterns in normal lymphocyte subpopulations, lymphoid leukaemias and immunodeficiency syndromes. Clin. Hematol. 11, 719. Johnson, S.M., North, M.E., Asherson, G.L. et al. (1977) Lymphocyte purine 5'-nucleotidase deficiency in primary hypogammaglobulinaemia. Lancet i, 168. Kramers, M.E.C., Catovsky, P., Fod, R., et al. (1975) 5'nucleotidase activity in leukaemic lymphocytes. Biomedicine 25, 362. Lozzio, C.B. and Lozzio, B.B. (1975) Human chronic myelogenous cell-line with positive Philadelphia chromosome. Blood 45, 321. Minowada, J., Ohnuma, T. and Moore, G.E. (1972) Rosetteforming human lymphoid cell lines. Establishment and evidence for thymus derived lymphocytes. J. Natl. Cancer Inst. 49, 891. Misra, D.N., Gill III, T.J. and Estes, LW. (1971) Lymphocyte plasma membranes. II. Cytochemical localization of 5'nucleotidase in rat lymphocytes. Biochim. Biophys. Acta 352, 455. Quagliata, F.D., Faig, D., Conklyn, M. and Silber, R. (1974) Studies on the lymphocyte 5'-nucleotidase in chronic lymphocytic leukemia, infectious mononucleosis, normal
subpopulations and phytohemagglutinin-stimulated cells. Cancer Res. 34, 3197. Reaman, G.H., Levin, N., Muchmore, A., Holiman, B.J. and Poplack, D.G. (1979) Diminished lymphoblast 5'-nucleotidase activity in acute lymphoblastic leukemia with T-cell characteristics. New Engl. J. Med. 300, 1374. Schneider, U., Schwenk, H.U. and Bornkamm, G. (1977) Characterization of EBV°genome negative 'Null' and 'T' cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non Hodgkin's lymphoma. Int. J. Cancer, 19, 621. Sen Gupta, S., Hibi, T., Mackay, A., Markovic, V., Stein, L.D., Sigal, N., Gelfand, E.W. and Chechik, B.E. (1984) Ecto-5'nucleotidase. I. Expression in human normal and neoplastic lymphoid cell lines representing sequential stages of B-cell differentiation. J. Natl. Cancer Inst. 73, 1075. Vielh, P. and Castellazzi, M. (1984) A colorimetric assay for serial determination of adenosine deaminase activity in small lymphocyte populations. J. Immunol. Methods 73, 313. Webster, A.P.B., North, M., Allsop, J., et al. (1978) Purine metabolism in lymphocytes from patients with primary hypogammaglobulinaemia. Clin. Exp. Immunol. 31,456. Webster, A.P.B., Rowe, M., Johnson, S., et aL (1979) Ecto-5'nucleotidase deficiency in primary hypogammaglobulinaemia. In: CIBA Foundation Symposium Series. Vol. 68 (Elsevier, Amsterdam) p. 135.
