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Simple, Direct Determination of Serum 5'-Nucleotidase

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Simple, Direct Determination of Serum 5'-Nucleotidase ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 7, No. 6 Copyright © 1977, Institute for Clinical Science Simple, Direct Determination of Serum 5'-Nucleotidase EUGENE S. BAGINSKI, Ph.D.,* SLAWA SUCHOCKA MARIE, M.S.,* EMANUEL EPSTEIN, Ph.D.f and BENNIE ZAK, Ph.D.* Department of Pathology, * St. Joseph Mercy Hospital, Pontiac, MI 48053 \William Beaumont Hospital, Royal Oak, MI 48072 tWayne State University School of Medicine, Detroit, MI 48201 ABSTRACT A simple determination of 5-Nucleotidase in blood serum without de- proteinization is described. The enzyme activity is distinguished from that of a nonspecific alkaline phosphatase by nickel inhibition and the inorganic phosphate released from adenosine monophosphate used as substrate is determined by a method previously described.3,8 Additional studies in­ clude the determination of optimal conditions for the reaction and nickel inhibition. Introduction One serious obstacle in serum 5'- nucleotidase (5N) determination is the The usefulness of serum 5'-nucle- presence of alkaline phosphatase (AP). otidase (5'-ribonucleotide phosphohy- The latter enzyme hydrolyses adenosine drolase, EC 3.1.3.5) determination is well monophosphate (AMP), a compound used established. The enzyme activity in­ as a substrate for 5N determination. As a creases in liver diseases, particularly result, when serum is incubated in the when the hepatobiliary tree is in­ presence of AMP, the degree of inorganic volved.31,32,42,50 A nonspecific alkaline phosphate released in the process is due phosphatase (orthophosphoric monoester to a collective action of both enzymes. In phosphohydrolase, EC 3.1.3.1) is also ele­ order to determine 5N in the presence of vated under these conditions, but in addi­ AP, it is often necessary to inhibit the ac­ tion, the enzyme may rise in bone dis­ tivity of one of the enzymes. eases and in non-pathological cir­ cumstances such as pregnancy or in­ Several methods have been described creased osteoblastic activity. In contrast, in which nickel is utilized as a selective 5'-nucleotidase is confined mainly to the inhibitor of 5N activity.21,54,62 Alkaline liver, making the enzyme a helpful tool in phosphatase, on the other hand, may be a differential diagnosis between bone and inhibited in the presence of ethyl- liver involvement. enediamine tetraacetic acid (EDTA)66 or 47 0 BAGINSKI, MARIE, EPSTEIN AND ZAK L-leucine.24,55 A technique called “en­ A simple method is proposed here zyme diversion” 15 has been used in which is based on nickel inhibition of 5N. which an excess of beta-glycero- The liberated inorganic phosphate is de­ phosphate11,14,16,23 or phenyl phos­ termined according to a method previ­ phate44,45 is utilized. While alkaline phos­ ously described.3,8 There are no problems phatase exhibits a high affinity for these associated with the enzyme stability or substrates, 5N shows no affinity. Under the presence of contaminants in tissue these conditions AP does not hydrolyze preparations containing ancillary en­ AMP to any appreciable degree, leaving zymes simply because such enzymes are the action to 5'-nucleotidase. It is obvious not employed in the method. Further­ that under these circumstances a product more, the method for inorganic phosphate of hydrolysis other than inorganic phos­ determination is very sensitive, making it phate must be determined. This is usually possible to detect small differences in 5N done by coupling the basic reaction with activity. other reactions by employing ancillary enzymes as outlined. Principle In the reaction, AMP is hydrolyzed by A mixture cu*.tahung the specimen, 5N to adenosine (A) and inorganic phos­ veronal buffer at pH 7.5, and adenosine phate (P) and the latter can then be monophosphate is incubated in duplicate measured.21,62 at 37° for 30 minutes. One of the dupli­ 5N cates contains in addition nickel (Ni) to 1. AMP— - > A + P inhibit 5N activity whereas the other con­ tains manganese (Mn) to enhance the ac­ Two ancillary enzymes are used to tivity. The amount of inorganic phosphate couple this step with two other reactions. liberated in the presence of Mn repre­ One of these enzymes, adenosine sents a combined activity of both 5' ■ deaminase (adenosine aminohydrolase, nucleotidase and alkaline phosphatase. EC 3.5.4.4, ADA), deaminates adenosine Phosphate found in the mixture contain­ to inosine (I) ing Ni is due to AP activity alone. The difference in phosphate concentration be­ tween the two is the measure of 5N. Inorganic phosphate is determined by and either the liberated ammonia is de­ complexing it with molybdate in the pres­ termined11,44,47 or a change in absorbance ence of ascorbic acid used as a reducing at 265 nm is measured.14,16 The other en­ agent. A heteropoly blue complex is zyme, glutamate dehydrogenase (L- formed which is stabilized by arsenite- glutamate: nicotinamide adenine di­ citrate reagent. Citrate also complexes ex­ nucleotide phosphate oxidoreductase, EC cess molybdate, a step necessary to pre­ 1.4.1.4, GDH), aminates alpha- vent molybdate from reacting with inor­ ketoglutarate (aKG) to L-glutamate, using ganic phosphate which may be generated ammonia produced in reaction 2. by subsequent hydrolysis of AMP in the acid medium.41,64- GDH 3. aKG + NH3 + NADH-----*> Methods and Materials L-glutamate + NAD R e a g e n t s and the concomitant oxidation of NADH Veronal buffer (0.05M). The solution is followed at either 334 nm20 or 340 nm.25 is prepared by dissolving 10.309 g of SIMPLE, DIRECT DETERMINATION OF 5-NUCLEOTIDASE 471 sodium diethylbarbiturate in about 800 Procedure ml of H20 and the pH is adjusted to 7.5 with dilute HC1. The solution is then di­ 1. Two 13 x 75 mm glass tubes are luted to one liter with H20. prepared for each specimen; one for sample blank (SB) and the other for sam­ Buffer-Ni (7.6 mM). The solution is ple (S). prepared by dissolving 950.8 mg of nickel 2. Buffer—Ni is pipeted in the amount chloride hexahydrate (NiCl2 • 6H20) and of 400 /ni to the tube marked SB and 400 diluting it to 5 dl with veronal buffer. /¿I Buffer—Mn to the one marked S, fol­ Buffer-Mn (2 mM). Exactly 169 mg of lowed by 75 ¡x\ of AMP which is added to manganese sulfate monohydrate (MnS04 both tubes. Following a thorough mixing, • H20) are dissolved and diluted to 5 dl the tubes are placed in a heating bath set with veronal.buffer. at 37° to equilibrate for about six min. AMP (13.3 mM). The solution is pre­ 3. An aliquot of serum equal to 25 is pared by dissolving 133.3 ¿(.moles of added to each tube, the content is mixed adenosine-5'-phosphate sodium salt in and the tubes are placed back in the bath the buffer and diluted to 10 ml with the to incubate for exactly 30 min. same. This reagent is kept in the re­ 4. In the meantime, a reagent blank frigerator at about 5°. and a standard are prepared by pipeting 500 /ni of H20 to a tube marked RB and Phosphate stock standard (0.1 M). The standard is prepared by dissolving 500 fil of the working phosphate standard 6.8045 g of potassium dihydrogen phos­ to another tube marked STD. From this point on these tubes are processed in the phate (KH2P04) and diluting it to 5 dl same fashion as those containing the with H 20. specimen. Phosphate working standard (0.2 5. Ascorbic acid in the amount of 0.5 mM). Precisely 2 ml of the stock phos­ ml is added to all tubes which are then phate standard are diluted to one liter vortexed to insure complete mixing. with H20. 6. Teepol in the amount of 0.25 ml is Ascorbic acid. Four g of ascorbic acid added to each tube, one at a time, and the are dissolved in H20, 2.0 ml of concen­ tube is vortexed to dissolve the turbidity trated sulfuric acid are added and the so­ before the reagent is added to the next lution is diluted to 1 dl with H20. Stabil­ tube. ity of this reagent is limited to three 7. Molybdate in the amount of 0.5 ml is weeks when kept in refrigerator. added to each tube and, again, vortexing Teepol. Teepol 610 (Shell Oil Co.) is follows. The reaction is allowed to attain diluted 1:1 v/v with H20. completion within one to two min before Molybdate. Five g of ammonium the next reagent is introduced. molybdate tetrahydrate are dissolved and 8. Exactly 1 ml of ACD reagent is diluted to 5 dl with H20. added to each tube and thoroughly vor­ ACD. This reagent is prepared by texed. dissolving 20.0 g of anhydrous sodium 9. After about 10 min, the absorbance arsenite and 20.0 g of sodium citrate di­ of each tube marked S is determined hydrate in about 300 ml of H20. Then against its sample blank (SB) using a 20.0 ml of glacial acetic acid are added spectrophotometer set at either 700 nm or followed by 400 ml of dimethyl sulfoxide 840 nm. The absorbance of the standard and the solution is diluted to one liter (STD) is determined against the reagent with H20. blank. 47 2 BAGINSKI, MARIE, EPSTEIN AND ZAK of AMP concentration. The curve is de­ A 0.6 -P i picted in figure 1 (open circles). In addition, inorganic phosphate was determined in separate mixtures contain­ ing a fixed amount of phosphate standard and increasing amounts of AMP. The lat­ ter results, represented by the curve (solid circles) in figure 1, clearly indicate that inorganic phosphate was recovered in the presence of AMP as long as the concentra­ AMP CONCENTRATION IN pM IN FINAL MIXTURE tion of AMP did not exceed 5 /nmoles in FIGURE 1.
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