alkaline phosphatases

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C LIN IC AL C HE MIS TR Y, Vol. 31, N o. 2, 1985 185

C LIN IC AL C H EM IS TRY, Vol. 3 1/2 , 1 85 -1 90 (1 98 5)

Effectsof Tem peratureon M easurem entof AlkalinePhosp hataseActivityWillia m H . C op ela nd , Daniel A . N ealo n, an d Robert Rej1

W e exam ined the effects of tem perature on the activity andsteady-state kinetic properties of alkaline phosphatase (EC3.1.3.1). Purified isoenzym es from hum an liver, intestine,and placenta were used, as w as hum an serum , and theenzym e from porcine kidney. Phosphatase activity was esti-m ated by tw o different assay techniques. For all isoenzym es,apparent M ichaelis constants for the substrate 4-nitrophenylphosphate decreased w ith increased tem perature; at37 {176}Cas typically half that determ ined at 25 {176}C.ll en-zym es of hum an origin exhibited sim ilar linear Arrheniusrelationships over the range exam ined, 20-37 {176}CE5 of 30-36 kJ m ol). The porcine kidney enzym e obeyed an Arrhe-nius relationship that w as slightly, but significantly, differentfrom the isoenzym es of hum an origin. Tem perature relation-ships based upon Arrhenius behavior and individual activitym easurem ents are presented. For hum an alkaline phospha-tases, they differed by no m ore than 10% .

A dd itio na l K eyp hra se s: enzym e assay kinetic analy-sis enzym e kinetics ch aracte ristics o f isoe nzym es A r-r he niu s re la tio ns hip s q ua lity c on tr ol

What assay temperature is most suitable formeasure-ment of enzyme activities has been the subject of consider-able discussion, with substantial differences in opinionevident 1-8 . Because of this lack of consensus regarding astandard tem perature, the use of tem perature-conversionfactors in the measurement of clinically useful enzymes hasbeen proposed (9,10 and observed in practice throughinterlabo ratory survey s 11, 12 . However, in many casescorrection factors are applied w ithout regard for the possibleisoenzym e heterogeneity of the specim en, even though som eisoenzym es may respond differently to changes in tempera-ture 13-15 . T hu s th e validity o f u sing tem pera ture-correc-tion factors has been questioned in at least some applica-tions 16-18 .

T he ca se o f alk aline p ho sph atase lo rtho pho sph oric-m on o-ester phosphohydrolase (alkaline optimum),. EC 3.1.3.1]

presents particular analytical problem s for accurate estim a-tion of total enzyme activity. N ot only may its isoenzym icform s in the hum an differ considerably in certain properties,but also the enzymes used in quality-control or calibrationfluids often are of nonhuman origin and may be very

Wadsw orth C enter for L aboratories andResearch, New YorkS tate D epartm ent of H ealth, A lbany, N Y12201.

Address c or re sp on de nc e to t hi s a ut ho r.P ortion s of this pap er w ere include d in p resentatio ns at th e 3 5th

n atio na l m ee tin g o f th e A A CC , N ew York , J uly 1 98 3 (a bs tr ac t:ClinChem 29. 1158, 1983) or at the 11th International Sym posium onE n zy m olo gy, R om e , F eb ru ar y1984.

R ec ei ve d O ct ob er 9 ,1984; a cc ep te d O cto be r 3 0,1984.

dissimilar to those of human serum 19,20 . Recent recom-mendations 2 1 from the Am erican A ssociation for ClinicalChemistry (A AC C) and 2 2 the International Federation ofClinical Chem istry (IFC C) propose sim ilar reference proce-dures for measurement of alkaline phosphatase activity at3 0 { 17 6} C .ecause these procedures probably will be used asreference techniques for routine measurements made atdifferent temperatures and under various modified assayconditions, we examined the effects of temperature on them easurem ent and steady-state kinetic properties of alkaline

phosphatase isoenzymes from several human sources andfrom porcine kidney, the latter representative of a nonhu-man enzyme used in quality-control fluids. Because theproposed reference method includes features presently un-common to routine alkaline phosphatase methods, such asaddition of zinc(ll) and metal chelator and use of lowerconcentrations of buffer, w e exam ined the effects of tem pera-ture, using both the reference assay 21,22 and one basedupon a routine procedure 2 3 .

M ate rials a nd M eth od s

Alkaline phosphatase was determined by the AACC/IF CC reference m ethod. We follow ed the published protocols 2 1, 22 exactly except for altering temperature as will be

described. Individual specimen-blank reactions were notdetermined. The magnitude of the reagent blank was as-sessed at each temperature; at most, it was equivalent to


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0

E

>

4-NITROPHENYLPHOSPHATE(mol L . 0)

0

E

>

6

4

2

- I 3 5 7 -I4 -N IT RO PH EN YL PH OS PH AT E (m ol L 1 0)

F ig . 1 . H an es p lo ts o f in itia l v elo city o f a lk alin e p ho sp ha ta se s w ith 4 -n it ro p ti en yl p h os p ha te a s t he v ar ie d s ub s tr at elsoenzymeswere:A,humanliver;B.humanintestine;C,humanplacenta;and0.porcinekidney.Enzymeactivitywas determinedby theAACC/IFCCprocedure 2 1, 2 . Curves ( topto bottom)are for 25 ,30 , ar id37 {176}C

186 C LIN IC AL C HE MISTR Y, Vol. 31, N o. 2, 1985

Arrhenius, and regression plots were calculated according toa robust linear regression technique based on the algorithmof Paulson and N icklin 2 6 . Activation energies and ther-modynamic quantities were estimated as previously de-scribed 2 7 .

Human liver and intestinal alkaline phosphatases wereprepared by the procedure of Sussman et al. 2 8 through thestage of Sephadex G-200 chromatography. Alkaline phos-phatase (B Grade) from human placenta was from Calbio-chem-Behring, San Diego, CA 92112. Porcine-kidney alka-line phosphatase was from M iles Laboratories, Elkhart, IN46515. The purified phosphatases were added to matrixes asd es cr ib ed p re vio us ly 2 9 and stored in 5.0-mL aliquots attemperatures below -20 {176}C.atients serum specim ens,selected to cover the normal and abnormal ranges of alka-line phosphatase activity, were obtained from a hospitalc lin ical labo ratory, stored at 4 {1 76 }C ,nd assayed within 24 h ofcollection.

2A2M 1P was lot number 1545, from Research Organics,Inc., C leveland, OH 44125. W e screened this material forchelating inhibitors of phosphatase activity, using bothc at aly tic a ct iv ity 2 2 and m etal binding procedures 3 0 ,and found it acceptable by both criteria. M agnesium acetateand zinc sulfate were from J. T. Baker Chemical Co.,P hil li ps bu rg , N J 0 88 65 . N -H y dr ox ye th yle th yle ne di am in e-

triacetic acid was from Aldrich Chemical Co., M ilwaukee,W I 53233. 4-Nitrophenyl phosphate-the disodium salt,hexahydrate-was from Sigma Chemical Co., St. Louis, M O63178.

Results

The effects of tem perature on the affinities of the alkalinephosphatase isoenzymes for 4-nitrophenyl phosphate weremeasured at three temperatures commonly used for theassay of the enzyme: 25, 30, and 37 { 176}C.epresentativeHanes plots are shown in Figure 1 for results obtained bythe A AC C/IFCC reference m ethod 21,22 and in Figure 2on using the method of Bowers et al. 2 3 . F or all pho sph a-tases we examined, the results show that as the tempera-ture increased, the apparent Km values for 4 -n it ropheny lphosphate decreased. Table 1 summarizes the average ap-parent Km values obtained on analyzing the original data by

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F ig . 2 . H an es p lo ts o f in itia l v elo city o f a lk alin e p ho sp ha ta se s w ith 4 -n itr op he ny l p ho sp ha te a s th e s ub str ate b ein g v an edIsoenzy me swere :A, humanlter;B,humanintestine;C,humanplacenta;an d D ,porcinekidney.Enzymeactivitywas determinedby the procedureof Bowersetal . (. Curves( topto bottom)are for 25 .30 , and37 C

six different estimation techniques. TheKm value deter-mined by any method agreed within 5 of the averageKmvalue reported in Table 1. The variation of apparentK mwith tem perature was log-linear with respect to the recipro-cal of the absolute tem perature. Apparent enthalpy changesfor substrate binding (.H8), calculated from the slopes ofsuch plots, are also presented in Table 1.

Arrhenius plots in Figure 3 show the effects of tempera-ture (20-37 C) on the activity of the various alkalinephosphatase isoenzymes, as measured by both assay meth-o ds. A lso shown in Figure 3 is the Arrhenius relationshipdetermined for patients sera over this temperature range.M ean and 1 SD are shown for individually determined

values. Thermodynamic parameters calculated from theseArrhenius relationships, activation enthalpy (H c), anda ct iv atio n e ne rg ie s (Fa) are shown in Table 2. A lso shown inTable 2 are the activation energies Ea calculated by usingdetermined values ofVmj, rather than measurements ofinitial velocity. Estim ates of each therm odynamic param e-ter are similar despite the differences in enzyme source. Inaddition, the average values for H37. an d Ea obtained for29 individual sera agree well w ith those obtained with thesem ipurifled alkaline phosphatase from the various humansources. These sera varied in enzyme activity from about 25to 550 U/L at 30 {176}C.able 2 also shows temperaturecoefficients, calculated from A rrhenius relationships in per-cent increase in activity per degree Celsius.

Table 3 gives tem perature/activity ratios calculated fromthe Arrhenius relationships for the different sources ofalkaline phosphatase and for the patients sera. D irectcom parison of alkaline phosphatase activities of 29 patientssera measured at 25, 30, and 37 {176}Cre shown in Figure 4.The slopes of the robust regression lines obtained for thesemeasurements, given in the legend to Figure 4, agree wellwith those calculated by the Arrhemus relationships for thepurified isoenzymes and the patients sera shown in Table 3.

Discussion

Evidently there are significant changes in apparent M i-chaelis constants for the human and porcine isoenzymes ofalkaline phosphatases in the range of temperatures usually


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K,,,-pp

K,,,-pp

ap aren t enthalpy change for substrate binding, in kJ.mol1

Enzyme source 25 C 30 C 37 {176}C H 25 C 30 C 37 {176}C HH um an liver 1.05 0.86 0.58 -38.7 1.22 1.03 0.76 -30.8H um an intestine 1.02 0.79 0.53 -41.4 1.28 0.93 0.61 -47.6H u m an p la ce nta 0.63 0.61 0.43 -24.4 0.62 0.53 0.39 -28.9Porcine k idney 1.11 0.74 0.47 -54.5 1.01 0.67 0.54 -39.6

C 37 3230 25 20 C 37 3230 25 20

I.,

CV

0

B

PAT IE NT S E RA

n29 4)10i420 PATIENTSERA

(n 1 9) 4 12 0 460LIVER4030

LIVER4180

INTESTINEINTESTINE3370

PORCINE KIDNEY PO RCINE KIDNEY930LACENTA PLACENTA)5043403330 3470

UL (30 {176}C(

Tab le 2 . A pp are nt E nth alp y C ha ng e H 43 7 c ) E nerg ie s o f A ctiv atio n E 5 a ndEa), and Tempera tu reC o effic ie nts fo r A lk alin e P h os ph a ta se s fr om Var io u s S o ur ce s

AACC/IFCCmethod 2 1, 22 M eth od o f B ow ers e tal . 2 3

Enzyme source Ht37 . E E Tem p. coeff., % . E E Tem p.c oe ff., %

Patients serum 31.6 34.2 - 5.9 31.7 34.3 - 5.9Hum an liver 31.1 33.7 28.1 5.8 32.2 34.8 28.9 6.0Hum an intestine 27.1 29.7 30.5 4.9 30.3 32.8 25.6 5.6Hum an placenta 33.5 36.1 30.9 6.3 31.9 34.5 33.3 5.9Porcine kidney 25.1 27.7 25.3 4.5 26.3 28.9 23.2 4.7

Va lu e s f o r e n th a lp y c ha n ge . E ,, , an d E , a re i n k J {1 4 9}ol. Al lva lues , except E, , ,were ca lcula ted by us ing ini tial ve loc ity measurements .Data for E, , werdeterminedby usingthe dependenceof apparentV, . withtemperature.Temperaturecoefficientsare givenin terms of averageincreasein activity per {176}Cvertherange 25-37 C (see text) . Relat ives tandarddeviat ionsfor repl ica teda ta were


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C LIN IC AL C HE MIS TR Y, Vol. 31, N o. 2, 1985 189

relationships: com pare the slopes w e obtained (Figure4)with the factors presented in Table 3. Particularly notable isth e c lo se a gre em en t between the tem pe rature relation ship sfor any single isoenzyme for the two methods studied (Table3). Heerspink et al. 1 0 , u sin g th e F re nc h- re co mm en de dprocedure 8 , found tem perature relationships in very closeagreement to those given for serum in Table 3. These dataindicate that temperature relationships for alkaline phos-phatase probably are identical among assay techniques thatare com parable in basic principles (e.g., identical substratesand nucleophilic acceptor) but that may differ in some

details. Furthermore, Heerspink et al. 1 0 f ou nd s ig nif i-cantly different factors for the procedure of theGermanSociety 3 , indicating that more profound changes in assaytechnique w ill not allowus e of factors derived for differentprocedures.

It is well known that the responses of enzymes in quality-control sera and calibrators to changes in assay conditionsdiffer from those of patients sera, and this is particularlytrue for alk aline p hosp hatases 19, 20, 45 . D ifferences inbehavior of alkaline phosphatases of quality-control fluidsin r es po ns e to changes in temperature have been described 10, 19 . Separate reports from our laboratory 20, 46 havesuggested that porcine-kidney phosphatase has characteris-tics resembling those of the enzyme from human liver andhuman serum. Thus we chose this nonhuman enzyme forthe present study, and our results show that the activity ofthis enzyme does in fact differ from those of human originwhen temperature is varied (Figure 3, Tables 2 and 3).Changes in activity with temperature were slightly butsignificantly lower than those found for any isoenzyme ofhuman origin. This observation was notexpected on thebasis ofou r earlier studies. Perhaps differences in specim enmatrix or in degree ofenzyme purity may account for thisobservation. A lternatively, the differences between the re-sponse of this nonhuman enzyme and that of human liver orserum origin are slight [


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190 C LIN IC AL C HEM ISTR Y, Vol. 31, N o. 2, 1985

19. M cCom b R, Bow ers GN Jr, Posen S.A lk alin e P ho sp ha ta se ,Plenum Press, New Y ork, NY , 1979, pp 229-372.20 . R ej R , Jenn y RW , Bretaudiere J -P. Q u ali ty c on tro l in c lin ic alchemistry: C ha ra cte riz atio n o f re fe re nc e m ate ria ls .Talanta 31 ,8 51 -8 62 (1 98 4).21. Tietz N W, B urtisCA, Duncan P,e t a l. A R efe re nc eMethod fo rmeasurement of alkaline phosphatase activity in hum an serum .C iin C /r em 2 9, 7 51-7 61 (1 98 3).22. Tiet.z NW , Rinker A D, Shaw LM . IFCC m ethods for them easurem ent of catalytic concentration of enzym es. Part 5. JFCCm etho d fo r alk alin e p hosp hatase (o rth oph osph oric-m ono ester ph os-phohydrolase, alkaline optim um , E C 3.1.3.1).J Clin C/rem Cimn

Biochem 2 1, 7 31-7 48 (1 98 3).23. Bowers GN Jr, M cCom b RB, K elley M -L. M easurem ent of t otalalkaline pho sph atase activity in hu ma n seru m.S ele cte d M e th od sC iin C /r em 8 , 31 -39 (1 977 ).24 . C le lan d W W. S tatistical analysis of enzym e kinetic data.M e th od s E n zy m oi 6 3, 1 03-1 38 (1 97 9).25 . Cornish-BowdenA, Eisenthal R . Statistical considerations inth e estim atio n o f en zym ek in et ic p ar am e te rs b y the d irect lin earplot an d other methods. Biochem J 1 39 , 7 21 -73 0 (19 74).26 . Paulson AS, N icklin EH . Integrated distance estim ators forlinear m odels applied to som e published data sets.Appi Statist 32 ,3 2-5 0 (1 98 3).

27. Rej R, V anderlinde RE. Effects of tem perature on the steady-state kinetics and m easurem ent of aspartateaminotransferases.C lin C /r em 2 7, 2 13-2 19 (19 81).

28. Sussm an HH , Sm all PA Jr, Cotlove E . Hum an alkaline phos-phat.ase. Im munochem ical identification of organ-specific isoen-zymes. J B io l C /r em 24 3, 1 60 -16 6 (19 68).29. Rej R, Bretaudiere J-P. Effects of m etal ions on the m easure-m ent of alkaline phosphatase activity.C iin C /rem 2 6, 4 23 -4 28(1980).30. Rej R , Bretaudiere J-P, Jenny RW , Jackson KY. M easurem entof alkaline phosphatase activity: Characterization and identifica-tio n o f a n in ac tiv ato r in 2 -am in o-2 -m eth yl-1 -p ro pa no l. C lin C hem2 7, 14 01-1 40 9 (1 981 ).

31. Hinberg I, Laidler KJ. The kinetics of reactions catalyzed byalkaline phosphatase: The effects of added nucleophiles.Can JBiochem 5 0, 1 36 0-1 368 (1 97 2).32 . Chappelet-Tordo D, Fosset M , Iwatsubo M , et al. Intestinalalkaline phosphatase. Catalytic properties andhalf of the sitesreactivity. Biochemistry 13 , 17 88-1 79 5 (1 97 4).33. Latner A L, H odson AW . H um an liver alkaline phosphatasepurified by affinity chrom atography, ultracentrifugation and poly-ac ry lain ide -ge l e l ec trophores is .Biochem J 1 59 , 69 7-7 05 (1 97 6).

3 4 . B r et au d ie re J-P, V assault A , Am sellem L,et al. C riteria forestablish in g a stand ard ized m eth odfo r determ ining alkaline phos-phatase activity in hum an serum .C iin C /r em 23,2263-2274(1977).35. D uncan PH, M cK neally SS, M acNeil M L, et al. D evelopm ent ofa R eferen ce M aterial fo r alk alin e p ho sph atase.C lin C /r em 3 0, 9 3-9 7 ( 19 84 ).

3 6. M a {2 38 }t re, C iesielski L , Cash C, M andel P. Purification ands tu dies o n s om e p ro perties o f th e 4 -a min ob uty ra te :2 -o xo glu t.ara tetransm inase from rat brain.Eur J Biochem 52 , 1 57-1 69 (1 97 5).

37. G ibson K D. True and apparent activation energies of enzym icreactions. B iochim Biophys Acta1 0, 2 21 -22 9 (1 95 3).38. H iw ada K , Wachsm uth E D. C atalytic properties of alkalinephosphatase from pig kidney.Biochem J 1 41 , 28 3-29 1 (1 974 ).39. B ador H, M orelis R ,Louisot P. Breaks in A rrhenius p lo ts o freact ions involvingmembrane-bound and solubil ized s ia lyl transfer-ases, due to tem perature dependence of kinetic param eters.Bio-c /rim B io ph ys A cta800, 75-86 (1984).

40. Bow ers G NJr, M cC om b R B. A co ntin uo us sp ectro ph oto metricm eth od for m easu ring th eactivity of s eru m a lk alin e p ho sp hata se.C lin C /r em 1 2, 70 -89 (1 96 6).41. Keleti T . Errors in the evaluation of A rrhenius and vant Hoffplots. Biochem J 2 09 , 2 77 -28 0 (1 98 3).

42. Bowers G N Jr, M cCom b RB, U pretti A . 4-Nitrophenyl phos-phate-characterization of high-purity m aterials for m easuringalkaline phosphatase activity in hum an serum .C lin C /r em 2 7, 1 35-1 43 ( 19 81 ).

43. Burtis CA, Seibert LE, Baird M A, Sam pson EJ. Tem peraturedependence of the absorbance ofa lk ali ne s olu tio ns o f4-nitrophenylphosphate-a potential source of error in the m easurem ent ofa lk alin e p ho sp ha ta se ac tiv ity .C lin C /r em 2 3, 15 41-1 547 (1 977 ).44. Lazdunski C ,L azdunski M . E tude cinetique de m ecanism edaction catalytique de la phosphatase alcalined E sc he ric hia c oli.B io chim B iop hys A cta1 13, 5 51 -56 6 (1 96 6).

45 . ODonnell NJ, Lott JA . Intralabo ratory survey o f alk alinep ho sp hata se m eth od s.Am J Clin Pathol 76 , 5 67 -5 74 (1 98 1).46. Bretaudiere J-P, Drake P, Rej B.. Influenceof th e origin ofa lk al in e p ho sp ha ta se on the behavior ofq ua li ty -c on tr ol s pe ci m en sfo r interlab oratory surv eys. Clin C /rem 26, 1014-1015 (1980).Abstract.

47. Bow ers G N Jr, M cCom b RB. A unifying reference system forclinical enzym ology: A spartate am inotransferase and the interna-tio nal clin ical en zym e scale.C lin C hem 3 0, 1 128 -11 36 (1 98 4).

48. T ietz N W , Rinker A D, Burtis C, et al. Transferability studiesfor the A ACC reference m ethod and the IFCC m ethod for m easure-m ent of alkaline phosphatase activity.C lin C /rem 30 , 704-706(1984).

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