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New optimized method for assaying 5'-Nucletidase in biological fluids.

Introduction

5'-Nucletidase(EC 3.1.3.5; 5'-ribonucleotide phosphohydrolase) is sensitive to superoxide anion, and acts as an indicator of oxidative stress in humans [1], its activity in serum is increased in diseases of the liver and biliary tract [2]. It hydrolyses nucleoside 5'-monophosphates to their corresponding nucleosides and releases inorganic phosphate.

Previous assays for 5'-ND employ one of three principles. In the first, adenosine deaminase (EC 3.5.4.4) is used as a linked enzyme reaction to form inosine and ammonia from adenosine. After that, the ammonia formed can be measured colorimetrically by the Berthelot reaction [3]. Also, glutamate dehydrogenase (EC 1.4.1.2) can be used as a further coupled-enzyme to form glutamic acid from ketoglutarate and ammonia; the reaction requires NADH and can be monitored at 340 nm [4]. These assays undergo certain disadvantages. The measurement of ammonia by Berthelot reaction needs additional coupled-enzymes, which raise the cost and intricacy of the assay [5]. Also, endogenous ammonia which produced by delay in separation or analysis affect the precision and sensitivity of the assay [6].

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In the second assay, 5'-Nucleotidase activity was determined using the [[sup.3]H]AMP as described by Avruch and Wallach [7] and modified by Newby et al. [8]. Enzyme is pre-incubated in Tris/HCl buffer (pH 7.5). The reaction was initiated by adding AMP containing a tracer [[sup.3]H] AMP (100000 cpm). Every 3min aliquots were taken and the reactions stopped by addition of ZnS[O.sub.4]. The following addition of Ba[(OH).sub.2] precipitated the non-hydrolyzed AMP. After centrifugation, the supernatant was added to scintillant and radioactivity was determined in a liquid scintillation spectrometer. The disadvantages of this assay involve the need for special technique and the reagents which are used in this assay have a limited shelf life.

The third assay involves colorimetric determination of the liberated phosphate [9] based on the complex reaction between phosphate and molybdate in an acid environment. After that, phosphomolybdate complex reduced to molybdenum blue, which can be measured colorimetrically at wavelength near 700 nm. The disadvantages of this assay involve the possibility of reduced excess molybdate to molybdenum blue. Also, hydrolysis labile organic phosphates (e.g., ATP, creatine-phosphate) affect the precision and sensitivity of the assay. Many authors have been concerned with the instability of molybdenum blue complex, {in the assaying of enzymes other than 5'-Nucletidase} and have added stabilizers to solution of reaction [10, 11]. Baginskiet al. [10] used sodium citrate in conjunction with sodium arsenite as stabilizer for the complex formed. Carianiet al.[11] eliminated the use of sodium arsenite (a hazard compound) and substituted it by bismuth citrate, which increase the sensitivity of the phosphate determination.

The present assay have been used another compound of bismuth (bismuth nitrate) to eliminate excess quantity of molybdenum and prevent the interaction with phosphate generated from the hydrolysis of labile organic phosphates (e.g., ATP, creatine-phosphate). The assay is simple and the reagents used are relatively stable.

Principle

Sample is incubated with Adenosine 5'-Monophosphate at pH = 7.5 and temperature 37[degrees]C, with and without nickel added. The amount of inorganic phosphate liberated is determine dafter 30 min. Phosphate produced in the absence of nickel shows the combined activities of alkaline phosphatase and 5'-Nucletidase, while the phosphate produced in the presence of nickel is due to alkaline phosphatase alone. Consequently, the difference between these two values for liberated phosphate corresponds to the activity of 5'-Nucletidasein the sample. Manganese ion usedto activate 5'- Nucletidase.

5-AMP+H2O [right arrow] 5'-Nucleotidase Adenosine + Pi

Pi +[molybdate.sup.reduction] [right arrow] Phosphomolybdate Complex ([lambda] [sub.max]at 714nm)

Abbreviations used

5'-AMP = Adenosine 5'-Monophosphate

Pi = Inorganic Phosphate

Reagents

All reagents prepared according to previous methods [5,9] to ensure a real comparison between this method and previous methods. Solutions for inorganic phosphate determination prepared according to previous method [11] except bismuth nitrate and sodium nitrate.

1. Barbiturate/HCl buffer (pH 7.5 at 37[degrees]C) dissolve 8.25 g of sodium diethyl barbiturate and 0.238 g of manganese sulfate in 1 L of water. Add 120 mL of 0.2 mol/L HCl(1.262 ml conc. HCl/100ml D.W) and adjust the pH (7.73 at 20[degrees]C), store at 4 [degrees]C.

2. Substrate (10 mmol of disodium adenosine 5'-monophosphate.6 [H.sub.2]O per liter. Dissolve 499.2mg of disodium AMP.6 [H.sub.2]O in 100 mL of water. Store 1.0-mL portions at -20 [degrees]C.

3. Stock phosphate solution (6 mmol/L): Dissolve 0.408 g of K[H.sub.2]P[O.sub.4] in 500 mL of de-ionized water. Prepare a working standard (300 [micro]mol/L) as required, by a 20-fold dilution.

4. Nickel chloride (0.1 mol/L): Dissolve 2.38 g of [NiCl.sub.2] .6 [H.sub.2]O in 100 mL of D.W.

5. Trichloroactic acid (5%).

6. Solutions for inorganic phosphate determination:

Solution I: 10% Ammonium molybdate.

Solution II: Dissolve 3 g ascorbic acid in 35 ml water, add 50 ml 1M HCl and cool to 0 [degrees]C ;when cool, add 5ml Solution I and 15 ml 20% (w/v)sodium dodecyl sulfate (SDS) with rapid stirring. The solution should be yellow, not blue-green. It can be kept for several hours on ice.

Solution III: dissolve 3 g bismuth nitrate in 100ml 1M HCl, then add 3 g sodium nitrate (stable when protected from light). This solution produce good results when be fresh.

Procedure

Table 1 shows the protocol. Standard and reagent blank tubes are prepared with each set of samples.

Calculation

5'-ND Activity = [A.sub.test] - [A.sub.cont]/[A.sub.STD] * 1/[vol.sub.of serum] * [vol.sub..STD]/1 * [conc.sub..STD]/1 * 1/time

= [A.sub.test] - [A.sub.cont]/[A.sub.STD] * 1/0.05 * 0.5/1 * 1/30

= [A.sub.test] - [A.sub.cont]/[A.sub.STD] * 100 U / L

(vol in ml, time in min)

Results and discussion

The spectrum for this assay has single peak at 714 nm (Fig. 1).

[FIGURE 1 OMITTED]

Results obtained by this method were compared with those by that of Campbell [9]. Identical sample, buffer, substrate, and nickel concentrations were used in both methods.

Accuracy of the entire assay protocol was measured by recovery of phosphate added to serum in the form of KH2PO4 detailed in table (3).

The precision of the assay was measured in-run on a single sample specimen and between-run over a period of three weeks with aliquots from the same sample pool but different reagents preparations. The results are shown in table (4).

Bi which is added as stabilizer reacts with excess Mo to form [[[Bi.sup.III][Mo.sub.6][O.sub.21][L.sub.3]].sup.3-], where L is an amino acid [11,12]. This reaction has two advantages. The first, its increase the intensity of the blue color via binding the excess of Mo [Excess Mo may affects the electronic distribution of phosphomolybdate complex and that decreases the intensity of blue color].The second advantage: [Bi.sup.III]-Mo compound prevents reaction of molybdate with further phosphate generated from the hydrolysis of labile organic phosphates (e.g., ATP, creatine-phosphate). Table (5) proofs this property because no significant change in the absorbance before and after addition of inorganic phosphate and labile organic phosphates following the addition of bismuth nitrate.

The assay presents a number of advantages more than existing methodologies. These advantages include, less quantity of sample is required (0.05 ml as compared to 0.4 ml),the reagents are relatively stable, instrumentals and apparatus are not complicated and available in most laboratories and the assay free from interference.

References

[1] Chalmers, A. H., 2000, "Blake-Mortimer J S and Winefield A H, Lymphocyte 5'-ecto-Nucletidase: an indicater of oxidative stress in humans?," Redox Rep., 5, pp.89-91.

[2] Dixon, T. M., and Purdom, M., 1954, "Serum 5'-nucleotidase," J. Clin. Pathol., 7, pp. 341-343.

[3] Beheld, A., Ellis, G., and Goldberg, P. M., 1970, "A specific colorimetric 5'nucleotidase assay utilizing the Berthelot reaction,". Clin. Chem., 16, pp.396-401.

[4] Arkesteijn, C.L.M., 1976, "A kinetic method for serum 5'-nucleotidase using stabilized glutamate dehydrogenase". J. Clin. Chem. Clin. Biochem., 15, pp.155-158.

[5] Wood R. J., and Williams D. G., 1981,"determination of Serum 5' Nucleotidase without Deproteinization". CLIN. CHEM., 27(3), pp.464-465.

[6] Ismail,A. A. A., and Williams, D. G., 1974, "Scope and limitations of a kinetic assay for serum 5'-nucleotidase activity,". Clin. Chim. Acta, 55, pp.211-216.

[7] Avruch, J. and Wallach, D. F. H. 1971,"Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells," Biochim. Biophys.Acta, 233, pp.334-347.

[8] Newby, A. C., Luzio, J. P. and Hales, C. M. 1975, "The properties and extracellular location of 5'-nucleotidase on the rat fat cell plasma membrane," Biochem. J., 146, pp.625-633.

[9] Campbell, D. M., 1962, "Determination of 5'-nucleotidase in blood serum, "Biochem. J., 82,34P. Abstract.

[10] Baginski, E.S., Foa, P.P., and Zak, B., 1969 "Determination of phosphate and phosphomonoesterases in biologic materials, " Am. J. Med. Technol., 35, pp.475-486.

[11] Cariani, L., Thomas, L., Brito., and del Castillo, J.R., 2004 "Bismuth citrate in the quantification of inorganic phosphate and its utility in the determination of membrane-bound phosphatases," Analytical Biochemistry, 324, pp.79-83.

[12] Kortz, U., Sauelieff, M.G., AbouGhali, F.Y., Khalil, etal., 2002, "Heteropolymolybdates of [As.sup.III], [Se.sup.IV] and [Te.sup.IV] functionalized by amino acids, "Angew. Chem. Int. Ed., 41, pp. 4070-4073.

Mahmoud H. Hadwan * (1), Lamia A. Almashhedy (1) and Abdul Razzaq S. Alsalman (2)

(1) Chemistry Dept., College of Science, Babylon University, Iraq

(2) Surgery Dept., College of Medicine, Babylon University, Iraq

* Corresponding Author E-mail: mahmoudhadwan@gmail.com
Table 1: Protocol for 5'-NT Assay

 Test Control Standard Blank
 ml ml ml ml

Buffer 0.4 0.4 -- --

Ni[Cl.sup.2] -- 0.05 -- --

Sample 0.05 0.05 -- --

Mix and incubate for
5 min at 37[degrees]C.

Substrate 0.05 0.05 -- --

Standard -- -- 0.5 --

De-ionized water -- -- -- 0.5

Mix and incubate for
exactly 30 min at
37[degrees]C, then add:

Ni[Cl.sub.2] 0.05 0.05 0.05

Trichloroacetic 0.5 0.5 0.5 0.5
acid (20%)

Mix by vortex, and centrifuge
for 15 minutes at 3000
xg, then remove
supernatant, and add:

Supernatant 0.5 0.5 0.5 0.5

Solution II 0.5 0.5 0.5 0.5

Mix and incubate
10 min on ice:

Solution III 0.5 0.5 0.5 0.5

Mix well, leave for 10 min at 37[degrees]C water
bath, and measure absorbance at 710nm vs.
the reagent blank.

Table 2: Statistical Analysis of The Values
Obtained for 5'-Nucletidase by Campbell's
Method and Present Method.

No. of Samples 20

Mean of Campelle method 11.7
Mean of test method 11.46
Mean of both methods 11.58
Regression coefficient B 0.9794
Regression coefficient A 0.0205
Correlation coefficient 0.9888

Table 3: Analytical Recovery of Phosphate
(P[O.sub.4]) Added to Sample

Present Equivalents Calculated Observed Recovery
in assay of activity activity %
 phosphate U/liter (a) U/
 added liter

Pooled sample -- -- 11.46 --
Pooled sample 3 14.46 14.32 99.03
 + phosphate
 (b)
Pooled sample 6 17.46 17.12 98.53
 + phosphate
Pooled sample 15 29.46 28.84 97.89
 + phosphate
Pooled sample 30 44.46 43.3 97.39
 + phosphate

(a) mean of triplicate determinations

(b) present as K[H.sub.2] P[O.sub.4]

Table 4: Precision of the Assay Procedure.

 N Mean CV%
 ([+ or -]SD)
 U/liter

Within-run 20 11.46 [+ or -] 0.82 7.1
Between-run 20 11.42 [+ or -] 1.04 9.15

Table 5: The Optical Absorbance before and after
Addition of 50 [micro]l of 50 mmol/l Inorganic
Phosphate and Labile Organic Phosphates Following
the Addition of Solution III.

 Absorbance

Control 0.329 [+ or -] 0.01
Pi 0.331 [+ or -] 0.01
ATP 0.320 [+ or -] 0.018
Boiling ATP 0.331 [+ or -] 0.021
Creatine-
 phosphate 0.317 [+ or -] 0.026
Boiling
 creatine-
 phosphate 0.335 [+ or -] 0.017
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Author:Hadwan, Mahmoud H.; Almashhedy, Lamia A.; Alsalman, Abdul Razzaq S.
Publication:International Journal of Biotechnology & Biochemistry
Date:Feb 1, 2012
Words:2047
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