J Clin Pthol 1988;41:202-206 Quntittive method for determining serum lkline phosphtse isoenzyme ctivity: estimtion of intestinl component M J PEAKE, M PEJAKOVIC, G H WHITE From the Deprtment ofbiochemistry nd Chemicl Pthology, Flinders Medicl Centre, Bedford Prk, South Austrli SUMMARY Intestinl lkline phosphtse ctivity ws mesured using levmisole inhibition, nd results were compred with previously reported method using L-phenyllnine. Sixty two per cent intestinl, 39% plcentl, nd 1-3% of either bone or liver lkline phosphtse ctivity remined when lkline phosphtse ctivity ws inhibited in 2-mino-2-methyl-l-propnol (AMP) buffer regent system with 10 mmol/l levmisole (finl ssy concentrtion 8-1 mmol/l). The ssy imprecision (SD) ws 0-6 U/l compred with 3-9 U/l using L-phenyllnine for specimens with totl lkline phosphtse ctivity less thn 250 U/l (reference rnge 30-120 U/l). In serum pools with rised totl lkline phosphtse ctivity errors in recovered intestinl ctivity were smll (usully less thn 3 U/1) when intestinl lkline phosphtse ws dded. Much lrger errors nd mny underestimted results were found using L-phenyllnine. For non-hemolysed specimens it is concluded tht n ssy bsed on levmisole inhibition provides better mesure of intestinl lkline phosphtse ctivity thn L-phenyllnine. A rised serum lkline phosphtse ctivity [EC 3.1.3.1] is common finding in the clinicl chemistry lbortory. In proportion of such cses either the tissue source of the enzyme ctivity is not identifible by the clinicin, or the contribution from one or more pthologicl processes is msked.' A method for seprtely quntifying bone, liver, intestinl nd plcentl lkline phosphtse isoenzyme ctivities hs, however, recently been described.2 This method determines the residul serum lkline phosphtse ctivity following vrious inhibitory tretments, with individul isoenzymes being ffected in predictble mnner. Ech isoenzyme ctivity present in the originl specimen is then quntified by insertion of the residul ctivities into n pproprite lgorithm. Becuse four seprte ctivity mesurements must be mde, it is importnt tht the precision nd ccurcy of ech quntittive step is optimised so tht the cumultive error on the clculted isoenzyme ctivities is minimised. One of the inhibitors used is L-phenyllnine, non-competitive inhibitor of intestinl lkline phosphtse. The ctivity of the intestinl component is computed from ctivities remining in the presence nd bsence of L- Accepted for publiction II August 1987 phenyllnine, with correction being mde for its prtil inhibition of the other isoenzymes. In routine use, however, we hve noticed tht the reported method my underestimte intestinl lkline phosphtse ctivity when totl lkline phosphtse ctivity is very high. Levmisole, phenylimidzothizole compound, hs been reported to ct in the opposite mnner to L- phenyllnine by inhibiting non-intestinl lkline phosphtse isoenzyme ctivity, while leving tht of the intestinl component lrgely unffected.3 We therefore investigted whether levmisole nd similr compound, bromotetrmisole,3 my be better regents thn L-phenyllnine for mesuring intestinl lkline phosphtse ctivity by permitting more direct mesuremient of the ctivity of the desired component. Mteril nd methods Totl, plcentl, nd intestinl lkline phosphtse ctivities (using L-phenyllnine) were mesured s previously reported.2 Levmisole (Sigm, moleculr weight 2408 dltons) ws ccurtely weighed out nd dissolved in 2-mino-2-methyl-l-propnol (AMP) buffer from single phil Monotest lkline phos- 202
Quntittive methodfor determining serum lkline phosphtse ctivity phtse kits (Boehringer, West Germny) t concentrtions of 5, 10, nd 20 mmol/l. Bromotetrmisole (Jnssen Phrmceutic Belgium moleculr weight 372-2 dltons nd Sigm moleculr weight 373-2 dltons) ws dissolved in AMP buffer t concentrtions of 0-27 mmol/l (0-01%) nd 1-34 mmol/l (005%). Assys were performed on Cobs Bio centrifugl nlyser (Roche Instruments, Bsel, Switzerlnd) using the instrument settings shown in tble 1. Extrcts of intestinl lkline phosphtse ctivity were prepred nd mesured s previously described.4 Results LEVAMISOLE OR BROMOTETRAMISOLE Inhibition of lkline phosphtse ctivity by levmisole nd bromotetrmisole hs previously been reported in ssy systems incorporting N-ethylminoethnol(NEAE) buffer-the plcentl nd intestinl isoenzymes being the most resistnt to inhibition.3 We tested these regents in our ssy system, using AMP buffer, t regent concentrtions designed to inhibit mximlly bone nd liver ctivity Tble I Rection conditions on Cobs Bio for mesuring intestinl lkline phosphtse isoenzyme ctivity using levmisole Instrument settings I Units U/I 2 Clcultion fctor 2688 3 Stndrd I concentrtion 0 4 Stndrd 2 concentrtion 0 5 Stndrd 3 concentrtion 0 6 Limit 15 7 Temperture (C) 37 8 Type of nlysis 2 9 Wvelength (nm) 405 10 Smple volume (il) 5 11 Diluent volume (jl) 30 12 Regent volume (jl) 150 13 Incubtion time (seconds) 120 14 Strt regent volume (j1) 0 15 Time of first reding (seconds) 30 16 Time intervl (seconds) 10 17 Number of redings 20 18 Blnking mode 1 19 Printout mode I Tble 2 Percentge ctivity remining for ech isoenzyme fter inhibition with levmisole or bromotetrmisole Percentge ctivity remining Levmisole Bromotetrmisole Isoenzyme 5 mmol/l 10 mmol/l 20 mmol/l 0-27 mmol/l Bone 2-0 1-3 0-7 4 Liver 2-0 1-3 0-7 - 4 Intestinl 75 62 47 90 Plcentl 53 39 25 -.90 203 while minimlly reducing intestinl ctivity. The mximum solubility of bromotetrmisole ws bout 0-27 mmol/l (0-0l %); the Sigm preprtion ws slightly more soluble. Bromotetrmisole ws dissolved in glss tubes becuse the regent prtilly dhered to plstic. Following inhibition with 0-27 mmol/l bromotetrmisole bout 90% of intestinl ctivity remined, nd 3-5% of bone nd liver lkline phosphtse ctivity. Unfortuntely, n ttempted five-fold increse in bromotetrmisole concentrtion minimlly incresed the inhibition of the bone nd liver isoenzyme ctivity, due minly to its low solubility. In contrst, levmisole ws redily soluble to t lest 20 mmol/l, the highest concentrtion used, nd inhibited more bone nd liver ctivity (tble 2). With specimens contining bone or liver lkline phosphtse ctivity s high s 1000 U/l, only 2-0%, 13%, nd 0 7% of ctivity remined fter tretment with 5, 10, nd 20 mmol/l levmisole, respectively. In contrst, 75%, 62%, nd 47% of intestinl ctivity remined under the sme conditions. The stbility of plcentl lkline phosphtse towrds levmisole ws investigted using serum from c c) -c CL U)1._ 0._ -E CL Fig 1 Intestinl lkline phosphtse - levmisole Comprison of intestinl lkline phosphtse ctivity in ptient specimens. L-phenyllnine or levmisole s inhibitor (totl lkline phosphtse S 250 Ull, n = 88, r = 0-922, y = 1 185x - 5-5). Points not shown: x, y; 97, 95; 53, 56.
204 Peke, Pejkovic, White Tble 3 Comprison of intestinl lkline phosphtse (AP) ctivity in ptient specimens using L-phenyllnine (y) or levmisole (x) s inhibitor Totl AP < 250 U/I Totl AP > 250 U/l L-Phenyllnine Levmisole L-Phenyllnine Levmisole Men intestinl ctivity (U/1) 2 6 6.8-17 6 3-3 Assy imprecision (SD) from duplictes 3-9 0-6 17 5 0-6 (U/l)* Regression eqution y = 1 185x -55 y = 3826x -32-4 r = 0922 r = 0-666 Sy.x = 4-991 Sy.x = 8 129 n = 88 n = 26 *s= d 2n ptient in the 34th week of pregnncy with totl lkline phosphtse of 940 U/l (885 U/l plcentl lkline phosphtse, het stble for 10 minutes t 65 C); 54%, 39% nd 26% of ctivity remined fter tretment with 5, 10, nd 20 mmol/l levmisole, respectively. Similr results were found for 15 other ser with plcentl ctivity rnging from 50-100 U/l. 4,.c c 0 0 c 'CI v} 4) -C C U) - - Q- Fig 2 30 20 10 / 10-20 -30 0 /10 20 30 Intestinl lkline phosphtose - levmisole Comprison ofintestinl lkline phosphtse ctivity in ptient specimens using L-phenyllnine or levmisole s inhibitor (totl lkline phosphtse > 250 Ull, n = 26, r = 0 666, y = 3 826x - 32-4. Points not shown: x, y; 51, 41; - 1, -54; 3, -55; 1, -56; -4, -67. Following these experiments levmisole ws used for further studies becuse of its better solubility, ese of preprtion, nd greter inhibition of non-intestinl components. LEVAMISOLE COMPARED WITH L- PHENYLALANINE Intestinl lkline phosphtse ctivity ws mesured in 114 selected specimens, inhibiting with either 10 mmol/l levmisole or 12 3 mmol/l L-phenyllnine (finl concentrtions in Cobs rotor 8 1 mmol/l nd 10 mmol/l, respectively). Totl lkline phosphtse ctivity exceeded 250 U/i in 26 specimens nd 750 U/l in five of these specimens. The highest ctivity ws 1850 U/l in ptient with biliry obstruction. All ssys were performed in duplicte in seprte nlyticl runs. With L-phenyllnine, intestinl lkline phosphtse ctivity ws clculted s previously described.2 With levmisole, intestinl lkline phosphtse ctivity ws clculted using n lgorithm derived from the dt shown in tble 2. I = 16474L - 0-0214T - 06210 P where I = intestinl ctivity; P = plcentl ctivity; T = totl ctivity; L = ctivity remining fter levmisole inhibition. Figs 1 nd 2 compre results, which re summrised in tble 3. For specimens with totl lkline phosphtse ctivity of less thn 250 U/l, the men intestinl ctivity ws 2-6 U/l using L-phenyllnine s inhibitor, nd 6-8 U/I using levmisole. Imprecision clculted from duplicte ssys ws significntly less using levmisole (SD = 0-6 U/l) thn with L-phenyllnine (SD = 3-9 U/i). For specimens with totl ctivity greter thn 250 U/i, mny negtive results were generted using L-phenyllnine s inhibitor. Additionlly, the reproducibility of some of the duplicte ssys ws poor. In contrst, the stndrd devition for intestinl ctivity clculted from duplicte ssys using levmisole ws similr to tht obtined when totl ctivity ws less thn 250 U/1.
Quntittive methodfor determining serum lkline phosphtse ctivity Tble 4 Specimens with intestinl lkline phosphtse (AP) exceeding 20 U/l (duplicte nlysis) Intestinl AP (U/l) Intestinl AP ( U/l) Totl AP (U/l) Levmisole L-Phenyllnine Clinicl notes 248/239 99(95) 95(95) Poorly controlled (non)insulin-dependent dibetes mellitus with metsttic crcinom of prostte 156/157 53(53) 53(59) Congestive crdic filure, chronic renl filure, multiple drug tretment 463/448 51(50) 47(34) Congestive crdic filure, nlgesic nephropthy, multiple drug tretment 161/155 31(32) 18(12) Chronic renl filure on continuous mbultory peritonel dilysis 105/103 29(29) 19(20) Insulin-dependent dibetes mellitus + renl trnsplnt + multiple drug tretment 228/221 24(23) 21(25) Non-Hodgkin's lymphom on chemotherpy 79/ 78 21(22) 21(21) Chronic renl filure 77/ 77 21(20) 14(16) Presumed cute severe multiple sclerosis Tble 5 Comprison of intestinl lkline phosphtse (AP) ctivity in serum pools supplemented with intestinl extrct using levmisole or L-phenyllnine s inhibitor Mesured intestinl AP (U/l) * (duplicte nlysis) Added Pool I Pool 2 Pool 3 Pool 4 Pool 5 Pool 6 intestinl Totl AP Plcentl AP Liver AP Liver AP Bone AP Bone AP AP (79 U/l) (179 U/l) (456 U/l) (896 U/l) (361 U/l) (877 U/l) Levmisole 176 174(173) 174(172) 184(179) 178(174) 183(185) 188(187) Phenyllnine 162(166) 185(180) 162(147) 178(198) 176(167) 152(209) Levmisole 88 83( 81) 94( 91) 88( 88) 87( 86) 92( 92) Phenyllnine 81( 76) 93( 94) 74( 88) 115( 59) 100( 97) Levmisole 44 45( 46) 44( 41) 43( 42) 44( 45) 44( 44) Phenyllnine 36( 43) 51( 45) 46( 42) 21( 32) 47( 40) Levmisole 22 23( 23) 22( 21) 22( 21) 19( 21) 23( 21) Phenyllnine 21( 20) 27( 20) 8( 12) -9 ( 2) 7( 14) Levmisole 1 1 12( 12) II( 7) 9( 10) 9( 11) II( 11) Phenyllnine 9( 10) 13( 13) 5(-7) -12(-4) 13( 15) Levmisole 0 2( 2) 1( 3) 0( 1) -2(-2) -1(-1) 0(- l) Phenyllnine -I( 2) 2( 6) 3(- 8) - 8(- 39) -6(-4) -2(- 3) *In ser 2 -. 6, stted isoenzyme ctivity represents t lest 90% of totl ctivity. Of the 114 specimens nlysed using levmisole, only eight hd intestinl lkline phosphtse bove 20 U/l. The highest ctivity ws 97 U/I in poorly controlled dibetic with metsttic crcinom of the prostte. Results from other ptients in this ctegory re summrised in tble 4. RECOVERY OF INTESTINAL ALKALINE PHOSPHATASE To investigte the recovery of intestinl lkline phosphtse ctivity intestinl extrcts with lkline phosphtse ctivities of 11, 22, 44, 88 nd 176 U/l were dded to ech of six pools of serum. One of these pools ws norml serum with totl lkline phosphtse ctivity of 80 U/l; two further pools contined liver lkline phosphtse ctivities of 455 nd 900 U/l; nother two pools hd bone ctivities of 360 nd 880 U/1; the remining pool hd plcentl ctivity of 190 U/l. The intestinl ctivity recovered from these 30 smples, using either levmisole or L- phenyllnine s inhibitor, is shown in tble 5. Duplicte results obtined for these prepred pools 205 using levmisole hd less imprecision thn those determined using L-phenyllnine, s ws lso the cse when estimting ptient specimens. The ltter inhibitor lso generted number of negtive results in smples contining low intestinl ctivities. The errors found using levmisole were in ll cses less thn 3 U/l when the dded intestinl ctivity ws below 22 U/1, nd less thn 6 U/I when the dded intestinl ctivity fell between 22 nd 88 U/1. EFFECT OF HAEMOLYSIS When levmisole ws used s the inhibitor it ws noticed tht the rection rtes of some hemolysed ser were less thn tht for the regent blnk. To investigte this, 10 serum smples with totl lkline phosphtse ctivity rnging from 40 to 325 U/I were supplemented with 300 mg/l of hemoglobin (visul hemolysis). The totl lkline phosphtse ctivity of ech smple uniformly fell by bout 8 U/l in the presence of hemoglobin. An identicl fll ws evident when L-phenyllnine ws used to inhibit intestinl lkline phosphtse in these specimens, lthough the
206 Peke, Pejkovic, White impct on clculted intestinl ctivity ws smll. ctivity nd produced positive vlues with cceptble With levmisole, however, the mount of precision. Levmisole therefore hs dvntges s n hemoglobin dded ws sufficient to render the inhibitor for determining intestinl lkline phosphtse ctivity in non-hemolysed serum, nd we mesured residul ctivity unrelible, vlues often being less thn tht of the regent blnk. conclude tht this is useful regent for the estimtion of intestinl lkline phosphtse ctivity. DETECTION LIMIT After levmisole inhibition residul serum lkline phosphtse ctivity is often less thn 20 U/l, even when totl ctivity is high. Depending on the nlyticl performnce of the vilble equipment, it my be desirble to increse the rection rte by djusting smple volume. Using 15 ser with totl ctivity rnging from 75 to 820 U/I, the lgorithm produced similr results for smple volumes of 5, 10, nd 25 gl. Thus smple volume cn be incresed without ffecting the clcultion of intestinl lkline phosphtse ctivity. Discussion Incresed intestinl lkline phosphtse ctivity hs been reported in certin diseses of the digestive trct,5 cirrhosis of the liver,6 nd in ptients receiving mintennce hemodilysis.7 Another study found tht rised serum intestinl lkline phosphtse ctivity in jundiced ptient suggested n intrheptic cuse.8 It hs lso been reported tht decresed mniotic fluid intestinl lkline phosphtse ctivity my be indictive of cystic fibrosis.9 The contribution to serum totl lkline phosphtse ctivity by the intestinl isoenzyme is reltively smll, even when the enzyme is pthologiclly incresed. It is therefore importnt tht such vlues re estimted with resonble ccurcy nd precision. Use of L-phenyllnine s previously described2 resulted in poor duplicte results, nd high incidence ofnegtive vlues for intestinl lkline phosphtse ctivity when totl ctivity ws high. The inhibitor levmisole, however, permitted more direct mesurement of intestinl lkline phosphtse We re grteful to Ms JR Burton for her expert preprtion of this mnuscript. References 1 Moss DW. Alkline phosphtse isoenzymes. Clin Chem 1982;28:2007-16. 2 Shephrd MDS, Peke MJ, Wlmsley RN. Quntittive method for determining serum lkline phosphtse isoenzyme ctivity II. Development nd clinicl ppliction of method for mesuring four serum lkline phosphtse isoenzymes. J Clin Pthol 1986;39:1031-8. 3 Vn Belle H. Alkline phosphtse. I. Kinetics nd inhibition by levmisole of purified isoenzymes from humns. Clin Chem 1976;22:972-6. 4 Shephrd MDS, Peke MJ. Quntittive method for determining serum lkline phosphtse isoenzyme ctivity I. Gunidine hydrochloride: new regent for selectively inhibiting mjor serum isoenzymes of lkline phosphtse. J Clin Pthol 1986;39:1025-30. 5 Dent CE, Norris TS, Smith R, Sutton RAL, Temperley JM. Stetorrhe with striking increse of plsm lkline phosphtse of intestinl origin. Lncet 1968;i: 1333-6. 6 Stolbch KL, Krnt MJ, Inglis NR, Fishmn WH. Correltion of serum L-phenyllnine-sensitive lkline phosphtse derived from intestine with ABO blood group of cirrhotics. Gstroenterology 1967;52:819-27. 7 Wlker AW. Intestinl lkline phosphtse in serum of ptients on mintennce hemodilysis. Clin Chim Act 1974;55: 399-405. 8 Wrnes TW, Hine P, Ky G. Intestinl lkline phosphtse in the dignosis of liver disese. Gut 1977;18:274-8. 9 Brock DJH, Bedgood D, Brron L, Hywood C. Prospective prentl dignosis of cystic fibrosis. Lncet 1985;i: 1175-8. Requests for reprints to: Dr GH White, Deprtment of Biochemistry nd Chemicl Pthology, Flinders Medicl Centre, Bedford Prk, South Austrli, 5042, Austrli. J Clin Pthol: first published s 10.1136/jcp.41.2.202 on 1 Februry 1988. Downloded from http://jcp.bmj.com/ on 30 April 2018 by guest. Protected by copyright.