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. Effect of substrate concentration on the final mixture. Above that concentra 5N activity and Pi determination. tion, AMP interfered in the color forma tion as indicated by a gradual decline in Calculations absorbance. However, in the actual en zyme determination (lower curve) the AS ■■ x °— x 40 x 1000 = U/L = mU/ml maximal activity, as represented by the ASTD 30 min plateau region, persisted up to AMP con AS 400 . . centration of about 2.0 /¿moles and then ---- x --- = mU/ml ASTD 3 decreased in a fashion similar to the one represented by the upper curve; an indi Where: cation that the enzyme activity decreased AS = absorbance of sample before AMP reached the concentration at ASTD = absorbance of standard which the interference with color forma 0.1 /xM = concentration of standard tion took place. 40 = conversion factor (25 fjd to 1.0 ml) It can be also seen that the true sub 30 min = time of incubation strate optimum lies between 0.75 and 2.0 /xmoles, a finding not obscured by an Results and Discussion AMP-molybdate interaction. If one con It has been reported that molybdate is siders that molybdate binds AMP in pref capable of complexing nucleotides in erence to phosphate,3,5 apparently there preference to inorganic phosphate.34,39 In must remain a sufficient amount of connection with this it is necessary to molybdate in the mixture to react with make a clear distinction between the en inorganic phosphate. Thus, the substrate zymatic and the analytical step and to concentration employed in the proposed show that the nucleotide used in the method is well within the plateau region former step does not interfere in the latter. and below the concentration at which the In order to demonstrate that the sub color formation between phosphate and strate concentration employed in the pro molybdate becomes affected. posed method is indeed optimal and that As shown in figure 2, there is a linear it does not interfere in the final color for relationship between (1) the enzyme ac mation, an experiment was set up in tivity and (2) the time of incubation and which 5N was determined in blood serum the amount of specimen used. These reac using increasing amounts of the substrate. tion characteristics are useful in actual Enzymes activities, expressed in terms of analysis because when large fluctuations differences in inorganic phosphate con in enzyme activities are expected, the time centration between the sample and the of incubation or the amount of specimen sample blank, were plotted as a function can be varied. The activity obtained under SIMPLE, DIRECT DETERMINATION OF 5-NUCLEOTIDASE 4 7 3 these circumstances can be then easily 100 interpolated to the standard time or vol ume. Since the inhibition of 5N activity by nickel was found to be dependent on var ious factors,1,55,61 it was necessary to de termine the concentration of Ni at which the enzyme was inhibited maximally in the proposed system. For this purpose 5N

obstructive liver disease. The values F ig u re 2. Relationship between 5N activity, listed in the horizontal column desig time of incubation and specimen concentration. nated as B represent the percent ac tivities based on a value of 247.0 mU per It can be seen that the activity toward ml obtained in the presence of 1.6 mM AMP increased first (tubes 3 and 4) and Mn but without Ni. The horizontal col then decreased. In view of some reports umn designated as A represents percent indicating that Ni inhibits 5N activ activities calculated on the basis of the ity,1’24,55 it may appear rather surprising activity obtained in the absence of both to find the metal acts also as an activator; Mn and Ni (tube #1). The values in col Aj similar observation was reported in umn B are higher than in column A be yeast61 and in human serum,37 but the pH cause Mn is known to enhance the en values employed were 6 and 10, respec zyme.10,21’25’37'56,60 It becomes obvious tively. The pH appears to be an important then that in the actual enzyme assay the factor in the degree of inhibition. How difference in activities is due to a com ever, there is a disagreement concerning bined effect of nickel inhibition and the optimal pH for 5N activity. The pH manganese activation. has been reported to be at '¡,5 ^ ^ 0,21,22

TABLE I

Effect of Nickel on 5'-Nucleotidase Activity

Tube 1 2 3 4 5 6 7 8 9 10 11

Ni++ Cone, in mM 0 0.008 0.04 0.08 0.4 0.8 4.0 8.0 16.0 24.0 32.0 Activity 113.1 113.1 130.4 133.7 112.5 83.3 44.6 28.6 21.4 18.8 20.6 A 0 0 +15 +18. -1 -26 -61 -75 -81 -83 -82 B 54 54 47 46 54 66 82 88 91 92 92

Nickel in mM concentration in final incubation mixture. Activity in mU per ml. A = percent loss(-) or gain(+) in activity owing to Ni++ based on activity of 100 obtained in absence of Ni++ (tube #1). B = percent .loss in activity owing to Ni++ based on activity of 247.0 mU per ml obtained in presence of 1.6 mM Mn++ but in absence of Ni++. 47 4 BAGINSKI, MARIE, EPSTEIN AND ZAK

6.8,33 7.0,37 7.2,25 7.4,35 7.8,1 7.9,11 8.5,30 cloudiness developed in the incubation 8.8,58 and ranges of 7 to 7.553 and 7.0 to mixture and progressively increased in 9.551 are also listed. intensity with Ni concentration. This The discrepancies are not surprising phenomenon was observed also by others because tissue homogenates from differ who attributed it to various factors, in ent organs as well as species have been cluding the formation of a nickel- employed for the studies. Furthermore, phosphate complex,31 metalophos- different buffers have been employed in phate-protein complex12 and nickel- the assay and there is a strong indication phosphate or hydroxide.55 that the optimal pH depends largely on The slight turbidity may be due to a the source of the enzyme and the buffer partial interaction between Ni and dieth- used.19 The degree of inhibition or activa ylbarbiturate, especially at pH above 7.5, tion will depend on the type of metal because at higher pH the turbidity forms used and its concentration. For example, in the absence of protein or phosphate. using 5N from purified bull seminal Whether or not the turbidity is in any way plasma and two different Ni concen related to 5N inhibition is difficult to trations, one group40 found the 20 percent demonstrate because at least some of the inhibition while another55 found the in inhibition takes place in the absence of hibition to the extent of 90 percent. It has turbidity. To avoid the turbidity, some been claimed that in human serum, man lowered the pH from 7.5 to 7.4.31 How ganese activates the enzyme more than ever, it was noticed by the present au magnesium.10 In bull seminal plasma, thors that at a pH as low as 7.2, a cloudi however, Mn could not replace mag ness still developed in the incubation nesium30 and in avian heart, magnesium mixture although the final solution was was more effective than Mn.29 clear in each instance. To avoid excessive The original Ni inhibition studies were turbidity a nickel concentration was performed using human aorta homoge employed which was responsible for nates in veronal buffer at pH 7.8 and the about 70 percent inhibition. The final so inhibition was almost total.1 However, lution used for spectrophotometric mea using purified 5N from human liver and 1 surements was always clear, however. mM Ni concentration, the inhibition was Although the proposed method is very found to be only 24 percent.60 However, simple, there are certain points which the latter studies were performed in Tris need to be outlined. Manganese and nic buffer which was shown to complex met kel salts may contain a certain amount of als,2 cause turbidity with nickel52 and to inorganic phosphate as impurities, there inhibit both 5N and AP at pH 9.5.24 It was fore the buffer reagents which contain also reported that Ni inhibits AP from these metals should be analyzed for human bone to the extent of 70 percent at phosphate and a correction applied. pH 7.5 while intestinal AP was not af The sequence of addition of reagents fected.55 should be followed as outlined in the Using human serum, the inhibition of procedure. When Teepol is added, tur AMP hydrolysis is approximately 70 per bidity develops but disappears momen cent at the Ni concentration employed in tarily upon mixing. Since each specimen our method. Whether or not the inhibi is run in duplicate with and without the tion is complete and the remaining activ presence of Ni and a difference in absor ity is due to AP is difficult to ascertain. It bances is used, there is no interference is interesting to note, however, that at the owing to lipemia, hemolysis or turbidity. point of the highest decrease in activity, a Furthermore, the presence of a detergent SIMPLE, DIRECT DETERMINATION OF 5-NUCLEOTIDASE 4 7 5 and a solvent in the medium appears to change in absorbance owing to conver be responsible for the final solution sion of NADH to NAD is measured at which is always clear. The color is stable either 334 nm20 or 340 nm,25 the enzyme for hours owing to the presence of preparations must be first purified.36 Also, arsenite-citrate reagent which also con the method at 340 nm is less sensitive than tributes to the high sensitivity of the the one at 265 nm13 and reagents are ex method. pensive.13 All reagents are stable except the acid- It is interesting to note that some au ascorbate which has a shelf life of about thors, being.critical of the Ni inhibition three weeks. However, it is prepared eas method, used it as a reference procedure ily from a stock sulfuric acid and weighed in the method they advocate and find an out ascorbic acid of high purity. For con excellent correlation between the two venience, all reagents can be kept in glass procedures.17,20,43,47 The difficulty ap repipetors from which they can be easily pears to be related to a particular research dispensed. approach by some workers and the There are several methods for 5N activ method employed for inorganic phos ity available, but only those in which 5N phate determination. Many18,31,55,56,63 still or AP are selectively inhibited appear use the Fiske-SubbaRow method28 which promising. The approach has drawbacks is unsuitable for inorganic phosphate de in which adenosine deaminase is used to termination in biological fluids3 because render ammonia and inosine, and sub of poor sensitivity and interference owing sequently NH3 is determined colorimet- to the presence of organophosphate com rically11,44 or a change in absorbance at pounds, including the substrate used for 265 nm is measured.14,16 For example, 5N determination. when NH3 is determined, it is necessary to purify commercial preparations of ADA Sources of Errors since they contain large quantities of NH3.47,48 Since serum may also contain As in the case of any other enzymatic NH3, it has to be removed prior to procedure, the one described here is sub analysis.36 Phenyl phosphate cannot be ject to similar errors which may arise used to suppress AP activity because it owing to poor control of temperature, pH, strongly absorbs at 265 nm,45 and its in time of incubation, etc. However, since hibition of AMP hydrolysis is limited to the time of incubation is directly related to the phosphatase of bone origin but not the the activity, the time may be altered and a liver.49 proper correction in calculation applied. The use of beta-glycerophosphate as AP Adenosine monophosphate may suppressant16 combined with the mea undergo a slow autocatalytic hydrolysis surement of the decrease in absorbance at even when kept at low temperature. As a 265 nm15 has also been criticized45,49 be result the blank will increase progres cause of poor sensitivity and a partial in sively. It may be wise to distribute the hibition of 5N by glycerophosphate. In freshly prepared reagent into small addition, the hydrolysis of AMP by serum aliquots and to freeze it, subsequently from patients with bone disease is largely thawing out each at a time as needed. reduced by glycerophosphate which is The optimal substrate concentration is not the case in patients with liver dis well within the plateau region and even a ease.16 substantial degree of hydrolysis should When the reaction is further coupled not affect the results. However, it should with glutamate dehydrogenase and the be clear that the hydrolysis may substan 4 7 6 BAGINSKI, MARIE, EPSTEIN AND ZAK tially increase the blank. The sample with the method, has been tentatively es blank should not exceed 0.8 in absor tablished at 0 to 12 mU per ml. bance, otherwise the absorbance due to the sample may be beyond the limits of Résumé of Clinical Interpretations accurate measurement by commonly used spectrophotometers. Ordinarily, the The most important value of 5-nucleo- blank due to AMP is negligible. Unless tidase activity determination in blood the patient has highly elevated serum in serum appears to be in distinguishing be organic phosphate, the sample blank tween liver and bone involvement.46,65,66 should be within limits of accurate This is because bone is not the source of analysis. Furthermore, since Beer’s law serum 5N activity23,38 and the enzyme is in the method applies to a very wide either never increased in bone disease42 range of phosphate concentrations, it is or increased very slightly.63 The activity possible to go beyond an absorbance of is increased in liver disease, particularly 1.0. when the hepatobiliary tree is in It is necessary to adhere to the se volved.32,42,30,65 The enzyme is more quence of addition or reagents. When markedly and persistently elevated than molybdate is added, the tube should be alkaline phosphatase in longstanding vortexed thoroughly in order for the reac biliary disease50 with intrahepatic31,32 or tion to go to completion. At least one min extrahepatic32 obstruction. should be allowed before arsenite-citrate Furthermore, the enzyme is elevated in is added. This is not usually a problem malignant infiltration of thè liver9 and is when several tubes are processed in suc useful to follow the course of carcinoma cession because there is always a lapse of metastatic to the liver.56 The enzyme ex time between the addition of a reagent to hibits greater sensitivity in the detection of the first and last tube. If the time be primary or metastatic hepatic malignancy tween the addition of molybdate and than does AP.59 In infancy ór childhood, arsenite-citrate is not allowed, the value when AP is usually elevated, the determi will be low because molybdate binds cit nation of5N is particularly useful.38,65 Dur rate in preference to inorganic phos ing the last trimester of pregnancy, when phate. On the other hand, if the time be serum alkaline phosphatase of placental tween the addition of the two reagents is origin is elevated, 5N determination is unduly prolonged (three min or more), very valuable in cases when liver disease is suspected.57 The enzyme activity may also some AMP may undergo acid-hydrolysis rise after surgery.26 and a cloudiness may develop. When the arsenite-citrate reagent is added, phos phate cannot react with molybdate to References form a color because the excess molyb date combines with citrate to form a col 1. A h m e d , Z. and R e is , J. L.: The activation and inhibition of 5'-nucleotidase. Biochem. J. orless complex.4,6,7 69:386-387, 1958. 2. A l l e n , D. E., Ba k e r , D. J., and G il l a r d , R. D.: Metal complexing by Tris buffer. Nature Normal Range 214:906-907, 1967. 3. B a g in s k i, E . S., E p s t e in , E ., and Z a k , B.: Re view of phosphate methodologies. Ann. Clin. There is a rather consistent range of nor Lab. Sei. .5:399-416, 1975. mal values reported in the literature,14, 4. Ba g in s k i, E. S., F o a , P. P., and Z a k , B.: Deter 17,20,32,47,50,66 starting from zero14 and ris mination of phosphate: study of labile organic phosphate interference. Clin. Chim. Acta ing as high as 17 mU per ml.50 Our normal 15:155-158, 1967. range, based on a three year experience 5. Ba g in s k i, E. S., F o a , P. P., and Z a k , B.: Deter SIMPLE, DIRECT DETERMINATION OF 5-NUCLEOTIDASE 4 7 7

mination of rat liver microsomal glucose-6 - 22. D i x o n , T. F . and P u r d o m , M.: Serum 5'- phosphatase activity: study of citrate and G-6 -P nucleotidase. Nature 170:500-501, 1952. inhibition. Anal. Biochem. 21:201-207, 1967. 23. D i x o n , T. F . and P u r d o m , M.: Serum 5'- 6 . Ba GINSKI, E. S., F o a , P. P. and Z a k , B.: Deter nucleotidase. J. Clin. Path. 7:341-343, 1954. mination of phosphate and phosphomonoester- 24. E l -a a s e r , A. A. and E l -m e r z a b a n i , M. M.: ases in biologic materials. Amer. J. Med. Tech. Simultaneous determination of 5'-nucleotidase 35:475-486, 1969. and alkaline phosphatase activities in serum. Z. 7. Ba g in s k i, E. S., F o a , P. P. and Z a k , B.: Micro- Klin. Chem. Klin. Biochem. 13:453—459, 1975. determination ofinorganic phosphate, phospho 25. E l l i s , G . and G o l d b e r g , D. M.: An improved lipids, and total phosphate in biologic materials. kinetic 5'-nucleotidase assay. Anal. Letters Clin. Chem. 13-,326-332, 1967. 5:65-73, 1972. 8 . Ba g in s k i, E. S., M a r ie , S. S., a n d Z a k , B.-. D i 26. E w e n , L. M. and G r if f i t h s , J.: Gamma-glu rect serum inorganic phosphate determination. tamyl transpeptidase: elevated activities in cer Microchem. J. 19:285-294, 1974. tain neurologic diseases. Amer. J. Clin. Path. 9. B a r d w i l l , C. and CHANG, C.: Serum lactic 59:2-9, 1973. dehydrogenase, leucine aminopeptidase and 27. F i o r e t t i , E., C a u l i n i , G ., M a g n i , G ., a n d 5'-nucleotidase activities. Canad. Med. Assoc. J. F e l i c i o l i , R. A.: Spectrophotometric assays for 89:755-761, 1963. 5'-nucleotidase, using IM P, GM P and CM P as 10. Be c k m a n n , J. v o n , L e y b o l d , K., and W e is - substrates. Ital. J. B io c h e m . 21:102-112, 1972. BECKER, L.: Zur bestimmung der 5'-nucleot- 28. F i s k e , C. and S u b b a r o w , Y.: The colorimetric idase im serum. Z. Klin. Chem. Klin. Biochem. determination of phosphorus. J. Biol. Chem. 7:18-24, 1969. 66:375, 1925. 11. B e l f i e l d , A., E l l i s , G., andGoLDBERG, D. M.: 29. G i b s o n , W . B. and D r u m m o n d , G . I.: Proper A specific colorimetric 5'-nucleotidase assay ties of 5'-nucleotidase from avian heart. utilizing the Berthelot reaction. Clin. Chem. Biochemistry 11 -.223-229, 1972. 16:396-401, 1970. 30. H e p p e l , L. A. and H i l m o e , R. J.: Purification 12. B e l f i e l d , A. andGOLDBERG, D. M.: Activation and properties of 5'-nucleotidase. J. Biol. of serum 5'-nucleotidase by magnesium ions Chem. 188:665-676, 1951. and its diagnostic applications. J. Clin. Path. 31. H i l l , P. G. and S a m m o n s , H . G.: An automated 22:144-151, 1969. method for the determination of serum 5'- 13. B e l f i e l d , A. and G o l d b e r g , D. M.: A note on nucleotidase. Clin. Chim. Acta 13:739-745, serum nucleotidase determinations. Z. Klin. 1966. Chem. Klin. Biochem. 9:197-200, 1971. 32. H i l l , P. G. and Sa m m o n s , H . G.: An assess 14. B e l f i e l d , A. and G o l d b e r g , D. M.: Applica ment of 5'-nucleotidase as a liver function test. tion ofacontinuous spectrophotometric assay of Quart J. Med. 36:457-468, 1967. 5'-nucleotidase activity in normal subjects and 33. H i l l , P. G. and Sa m m o n s , H. G.: The pH op patients with liver and bone disease. Clin. timum of human 5'-nucleotidases. Enzyme Chem. 15:931-939, 1969. 22:201-211, 1971. 15. Be l f i e l d , A. and G o l d b e r g , D. M.: Compari 34. I m s a n d e , J. and E p h r u s s i, B.: Réévaluation of son of sodium B-glycerophosphate and dis assays used to show RNA induction of odium phenyl phosphate as inhibitors of al glueose-6 -phosphatase in ascites cells. Science kaline phosphatase in determination of 5'- 144:854-856, 1964. nucleotidase activity of human serum. Clin. 35. I p a t a , P. L. : A coupled optical enzyme assay for Biochem. 3:105-110, 1970. 5'-nucleotidase. Anal. Biochem. 20:30-36, 16. Be l f i e l d , A. and G o l d b e r g , D. 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