Vol. 38, No. 12ANTIMICROBLAL AGENTS AND CHEMOTHERAPY, Dec. 1994, p. 2689-26940066-4804/94/$04.00+0Copyright X 1994, American Society for Microbiology

Evidence of Alkaline Phosphatase Interference ina Zidovudine Radioimmunoassay

ALICE M. O'DONNELL, DORCAS J. LE1TING, MARY F. DEREMER, AND GENE D. MORSE*Departments of Pharmacy Practice and Medicine, State University ofNew York at Buffalo, Buffalo, New York 14260

Received 14 February 1994/Returned for modification 31 May 1994/Accepted 26 August 1994

Phosphorylated zidovudine (ZDV) concentrations may provide a link between drug exposure and clinicalefficacy since these would include the active, intracellular form of the drug, ZDV triphosphate. Many groupsare investigating the optimal methodology that can be used to accomplish this goal. The initial purpose of thepresent studies was to examine the effect of the inclusion of cell wash steps on the quantitation of intracellularZDV. Ten milliliters of whole blood collected from healthy volunteers was spiked with increasing ZDVconcentrations (0.187, 0.375, 1.87, and 3.75 ,LM), allowed to equilibrate at room temperature for 1 h, andseparated into whole-blood components by a density gradient procedure. A mononuclear cell pellet wasobtained, reconstituted with 2 ml of phosphate-buffered saline (PBS), and split into two aliquots, one of whichwas not washed at all and the other ofwhich was washed four times with 1 ml of PBS. All samples were analyzedby ZDV radioimmunoassay (RIA) after a 1:1 dilution with either 1 mg of alkaline phosphatase (type 1-S;Sigma) per ml or PBS. Parent ZDV was measured in those samples which were not treated with the enzyme,while total ZDVwas measured in those samples which were exposed to alkaline phosphatase (21°C for 1 h). Theresult of the difference between the two samples is total phosphorylated ZDV. During the experiment, evidenceof alkaline phosphatase interference with the RIA became apparent, confusing interpretation of intracellularZDV concentrations. This evidence was based on three sets of data. First, wash samples showed increases inZDV concentrations of as great as 0.127 ,uM after exposure to alkaline phosphatase, even though onmicroscopic inspection the wash samples were acellular. Second, the sum of total ZDV recovered from the fourwash samples plus the washed cell pellet was as much as 14-fold greater than the total ZDV measured in theunwashed cell pellet. Theoretically, at least, these two entities should be equal. Finally, control samples ofalkaline phosphatase in PBS (0.5 mg/ml) run directly through the assay measured false ZDV levels rangingfrom 0.002 to 0.075 ,uM (0.6 to 20 ng/ml). Alkaline phosphatase is frequently used to measure phosphorylatedanabolites of ZDV in peripheral blood mononuclear cells. These data show that the particular form of alkalinephosphatase used may interfere with the ZDV RIA and may confuse the interpretation of phosphorylatedanabolite concentrations of ZDV.

Zidovudine (ZDV), the first of three nucleoside analogsapproved for the treatment of human immunodeficiency virus(HIV) disease, continues to be a mainstay of drug therapy,even though the optimal dosage regimen has yet to be deter-mined. Clinical correlates between plasma ZDV concentra-tions and drug efficacy remain elusive. Results from theoriginal placebo-controlled trial showed that ZDV prolongssurvival in patients with advanced AIDS (4). In subsequentclinical trials in asymptomatic and mildly symptomatic pa-tients, ZDV delayed the progression of disease (5, 20). In morerecent trials with longer periods of follow-up, however, resultshave demonstrated that ZDV does not prolong survival if it isused for an extended period of time as monotherapy (1, 7). Allof the nucleoside analogs may be thought of as prodrugs whichmust be activated intracellularly to the triphosphate form bycellular kinases. It is the triphosphate anabolite that binds toHIV reverse transcriptase and that results in proviral DNAchain termination (6, 8, 10, 13).The lack of correlation between plasma nucleoside analog

concentrations and clinical effect may be due to the fact thatthese methods do not measure the concentration of the activeintracellular moiety. Several investigators have attempted tomeasure intracellular ZDV concentrations. Those studies have

* Corresponding author. Mailing address: Center for Clinical Phar-macy Research, 247 Cooke Hall, State University of New York atBuffalo, Amherst, NY 14260. Phone: (716) 645-3635. Fax: (716)645-2886.

relied on three approaches: (i) in vitro cell culture methodswith human T-lymphoid lines (2, 3); (ii) ex vivo methods withhealthy or HIV-positive human peripheral blood mononuclearcells spiked with known concentrations of ZDV (15, 18, 19);and (iii) in vivo methods with isolated mononuclear cells fromHIV-positive patients receiving ZDV therapy (9, 11, 16).Results are typically reported either as total phosphorylatedZDV (mono-, di-, and triphosphate forms combined) or as theindividual phosphorylated anabolites. Quantification in vivo ofthe individual phosphorylated compounds requires a coupledhigh-performance liquid chromatography (HPLC)-radioim-munoassay (RIA) in which an HPLC column separates theZDV mono-, di-, and triphosphate peaks, which are subse-quently eluted, exposed to phosphatases, and analyzed by RIA(9, 11, 15). Although differences in methodology may partiallyexplain the variability in the data, several investigators havereported the total phosphorylated concentrations of ZDV invivo to be in the range of 0.3 to 3.5 pmol/106 mononuclear cells(9, 11, 16, 17).Those studies rest on three premises: (i) that exposure of

intracellular ZDV samples to some form of phosphataseresults in complete cleavage of all phosphate groups, leavingonly the parent compound to be assayed by RIA; (ii) ifphosphate bond cleavage is incomplete, that insignificant ZDVRIA interference occurs with any of the phosphorylatedanabolites at relevant intracellular concentrations or with anychemical or biologic reagents used in the laboratory; and (iii)that washing of the ZDV mononuclear cell pellet results in the

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minimal loss of intracellular anabolites because of an ion-trapping effect.The original purpose of the present study was to examine the

effect of the latter premise. An ex vivo method in which healthyhuman whole blood was spiked with known concentrations ofZDV is described. Isolated mononuclear cell pellets wereeither washed or not washed and were subsequently exposed toalkaline phosphatase to measure the total phosphorylatedZDV. During the course of these experiments, RIA assayinterference by alkaline phosphatase became apparent for anumber of reasons which are delineated, confusing interpreta-tion of the results. Subsequently, some experiments wererepeated with a more purified form of the enzyme, looking forsimilar evidence of assay interference. The second, morepurified form of alkaline phosphatase resulted in minimal, ifany, assay interference. These data may be particularly rele-vant in the overall search for an accurate methodology forquantifying phosphorylated ZDV concentrations. The partic-ular form of alkaline phosphatase used may influence theexperimental results; interference of the enzyme with the RIAshould be ruled out.

MATERUILS AND METHODS

Chemicals. ZDV was provided by Burroughs Wellcome(Research Triangle Park, N.C.). Bovine intestinal alkalinephosphatase (type 1-S; 6.2 U/mg, solid) and bovine intestinalalkaline phosphatase (type VII-NL; 1,550 U/mg, solid) werepurchased from Sigma Chemical Co. (St. Louis, Mo.). Leuco-prep tubes (10 ml) were provided by Becton-Dickinson (Lin-coln Park, N.J.). ZDV-Trac RIA kits were provided by INCstar(Stillwater, Minn.).Whole-blood sample collection, density gradient separation,

and mononuclear cell pellet isolation. Whole-blood samples(20 ml) were collected from healthy volunteers and placed inVacutainer tubes with EDTA. The tubes were spiked with0.187, 0.375, 1.87, or 3.75 ,uM ZDV. All spiked whole-bloodsamples were kept at room temperature for 1 h with gentleshaking until cell separation was performed. Each 20-mlsample of whole blood was then transferred to two 10-mlLeucoprep whole blood density gradient separation tubes.After centrifugation (1,600 x g for 20 min), the plasma wasaspirated and was stored at -20°C until analysis by RIA. Themononuclear cell layer was aspirated and was transferred to amicrocentrifuge tube, which was spun at 10,000 rpm for 10 min(Costar, Cambridge, Mass.). The extracellular fluid was de-canted and swabbed carefully without disturbing the cell pelletto remove as much plasma as possible. The mononuclear cellpellet was then resuspended in 2 ml of phosphate-bufferedsaline (PBS), and the mixture was divided into two aliquots,one that was washed four times with 1 ml of PBS and the otherthat was not washed at all. A summary of the experimentaldesign is shown in Fig. 1.Sample treatment with alkaline phosphatase. Bovine intes-

tinal alkaline phosphatase (type 1-S) was freshly dissolved inPBS to a concentration of 1 mg/ml (6.2 DEA U/ml). Washedmononuclear cell pellet (MNcpw) and unwashed mononuclearcell pellet (MNcp) samples as well as all four wash samples(samples Wl, W2, W3, and W4) were split into two aliquots,each sample was diluted 1:1 with either the solution of alkalinephosphatase described above or PBS, and the solutions wereleft at room temperature for 1 h. All alkaline phosphatase-exposed samples as well as all control samples were refrozen at-20°C until analysis by RIA. Prior to treatment with alkalinephosphatase, mononuclear cell pellet samples were vortexedfor approximately 30 s and were centrifuged at 5,000 rpm for 5

1 x 10ml LP (Done in duplicate) 1600g x 20 minutes

Il

PL MNC

l~

MNcp

LPsol RBC

10,000 rpm x 10 minutes

MNsup

qs to 2ml PBS

lml0> MNcp

AP

lml

qs to 2ml PBSV 0

Cen/Dec > Wash 1 <v AP

qs to iml PBSV 0

Cen/Dec i Wash 2 <v AP

qs to 1ml PBSV

Cen/Dec P

T

qs to lmI PBS

0Wash

AP

v 0Cen/Dec P Wash 4

I AP0

qs to 1 ml PBS MNcpw <'AP

FIG. 1. Summary of protocol for measuring ZDV concentration inunwashed versus washed mononuclear cell pellets. LP, Leucoprepseparation of whole blood; PL, plasma; AP, alkaline phosphatase;MNC, mononuclear cell layer; LPsol, dense Leucoprep solution; RBC,erythrocytes; MNsup, mononuclear supernatant; PBS, phosphate-buffered saline; MNcp, unwashed mononuclear cell pellet; MNcpw,washed mononuclear cell pellet; Cen/Dec, centrifuge and decant.

min to separate the cellular debris. Total ZDV (parent ZDVplus phosphorylated ZDV) was measured in the alkalinephosphatase-treated samples, and only parent ZDV was mea-sured in the PBS-treated samples. Fourteen Leucoprep sepa-rations were done with this form of alkaline phosphatase (twoat 0.187 ,uM, four at 0.375 ,uM, four at 1.87 ,uM, and four at3.75 ,uM). When assay interference became apparent with thisform of the enzyme (type 1-S), six more Leucoprep separations(two at 0.375 ,uM, two at 1.87 ,uM, and two at 3.75 jiM) wereperformed with a more purified form of alkaline phosphatase(type VII-NL).To confirm suspected interference with the RIA, alkaline

phosphatase solutions of both types in PBS were run directlythrough the assay. The type 1-S samples ranged from 0.75 to6.2 DEA U/ml, and the type VII-NL samples ranged from 2.5to 20 DEA U/ml. The range of the ZDV-Trac RIA is 0.25 to250 ng/ml (0.001 to 0.94 ,uM). Coefficients of variation for low-and high-quality controls are typically ± 15%. Usually, sampleswere first diluted 1:10; the exceptions were the mononuclearcell pellet and wash samples (pH 7.4), which were run undi-luted through the assay.

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0.15

0.12

0.09

0.06N

0.00 00 ~ ~ l . j

0.375 1.87 3.75

Whole Blood ZDV Spike ( uM

FIG. 2. Alkaline phosphatase type 1-S results: wash samples with and without enzyme treatment. EI1, without alkaline phosphatase; 1, withalkaline phosphatase. *, The first pair of bars in each group represents the means for wash 1; **, the second pair of bars in each group representsthe means for washes 2 through 4.

Recovery of ZDV. An erythrocyte sample and a densesolution sample were also obtained from each Leucoprepseparation. A dense solution layer is part of the Leucoprepdensity gradient whole blood separation system and restsabove the erythrocyte layer after centrifugation. All sampleswere frozen at -20°C until analysis by RIA, which also ensuredthe lysis of any cellular components. Cell lysis was confirmed bymicroscopic examination. In order to calculate the recovery ofthe original concentration of ZDV spiked in the tubes, on thebasis of 10 ml of whole blood, the volume of each densitygradient-separated layer was measured. These layers includedthe following: plasma, mononuclear supernatant (the volumeof extracellular fluid above the mononuclear cell pellet), adense Leucoprep solution layer, and an erythrocyte layer.These samples were diluted 1:10 prior to analysis by RIA.

RESULTSMost experimental protocols investigating intracellular nu-

cleoside analog concentrations include one or several cell washsteps to rid the cell pellet of contaminating plasma (9, 11, 16,17). Theoretically, this would not alter the intracellular con-centrations of phosphorylated anabolites because these com-pounds are presumed to be too polar to cross cell membranes.However, if exposure of the cell pellet to a drug-free mediumresults in an immediate equilibrium shift of parent ZDV out ofthe cell, it is conceivable that there is a simultaneous equilib-rium shift of the phosphorylated derivatives back to parentZDV and out of the cell. If the latter situation is true, theninclusion of a cell wash step(s) may decrease phosphorylatedZDV concentrations. It was the original intent of the protocoldescribed here to investigate this possibility. Washed andunwashed cell pellets were exposed to either PBS or alkalinephosphatase (type 1-S). As an additional control, wash sampleswere also exposed to PBS or alkaline phosphatase (type 1-S).Theoretically, the wash samples should contain only parentZDV, if any of the phosphorylated derivatives present are toopolar to escape from the cell. Therefore, there should be nosubstrate for alkaline phosphatase to act upon and no differ-ence in ZDV concentration between alkaline phosphatase-

treated and untreated wash samples. To the contrary, alkalinephosphatase-exposed wash samples in this series of experi-ments consistently displayed increases in their ZDV concen-trations (Fig. 2). These increases ranged from 0.005 to 0.127,uM for individual samples. For 75% of the Leucoprep sepa-rations, if ZDV was measurable in the first wash sample, itsconcentration was below the sensitivity of the assay for allsubsequent washes in untreated samples (not treated withalkaline phosphatase). Therefore, the mean increase in theZDV concentration after alkaline phosphatase treatment wascalculated for samples W2 to W4 as one group. The meanincrease in the concentration ZDV ranged from 0.011 FLM(3.75 ,uM spiked whole blood; W2 to W4 samples) to 0.054 ,uM(1.87 ,uM spiked whole blood; Wl samples). Upon microscopicexamination, before freezing, wash samples were always cell-free. These data suggested interference from alkaline phos-phatase (type 1-S) in the RIA. To confirm this hypothesis, twoLeucoprep separations were carried out with whole bloodspiked with ZDV at each of three concentrations (0.375, 1.87,and 3.75 ,uM), and a different form of bovine intestinal alkalinephosphatase, which is a more purified version of the sameenzyme, was used. When these wash samples were treated withalkaline phosphatase (type VII-NL), no increases beyond therange of assay variability (.15%) were observed comparedwith those in untreated samples (Fig. 3).No parent ZDV (no alkaline phosphatase treatment) was

measurable in either the unwashed or the washed cell pellet orany of the wash samples when whole blood was spiked withZDV at a concentration of 0.187 FiM. These samples alsoshowed increases in their ZDV concentrations when they wereexposed to type 1-S alkaline phosphatase; these increasesranged from 0.006 to 0.007 ,uM (data not shown). At the higherspike concentrations, parent ZDV was, without exception,measurable in the unwashed cell pellets and in 89% of Wlsamples (data not shown). The cell pellets (originally reconsti-tuted with 2 ml of PBS) resulting from whole blood spiked withZDV at 0.187 puM most likely fell beneath the lower limit ofthe assay's sensitivity. For this reason, no further experiments

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o0.9 0.0

0.375 1.87Whole Blood ZDV Spike ( uM )

FIG. 3. Alkaline phosphatase type VII-NL results: wash samples with and without enzyme treatment. =, without alkaline phosphatase; _,with alkaline phosphatase. *, The first pair of bars in each group represents the means for wash 1. **, The second pair of bars in each grouprepresents the means for washes 2 through 4.

were conducted with lower concentrations of spiked wholeblood.

Since the initial mononuclear cell pellet was reconstitutedwith 2 ml of PBS and was then split evenly into two samples,the total ZDV (alkaline phosphatase-treated samples) mea-sured in the first (unwashed) sample should equal the totalZDV measured in the second (washed) sample plus all fourwashed samples. When this comparison was made with type1-S alkaline phosphatase samples, however, the total ZDVrecovered in the washed pellet sample plus the four washedsamples ranged from 1.2- to 13.8-fold more than that measuredin the unwashed sample (Table 1). Eleven of 12 Leucoprepseparations showed recoveries beyond +15% assay variability(range, 145 to 1,383%). Only 1 of 12 Leucoprep separationsresulted in the similar recovery of total ZDV within thevariability of the assay (+ 15%) when MNcp was comparedwith MNcpw plus samples Wl to W4 (115%). When theexperiments were repeated with type VII-NL alkaline phos-

TABLE 1. Comparison of unwashed versus washed mononuclearcell pellet plus wash samples after type 1-S alkaline

phosphatase treatment

ZDV concn (,uM) in:

Spiked whole SmlsW eoeyblood MNcp MNcpw Samples W1 % Recoverya

0.375 0.029 0.055 0.064 1,2850.375 0.0315 0.057 0.076 1,3830.375 0.0195 0.015 0.030 8380.375 0.024 0.0195 0.021 5251.87 0.036 0.004 0.048 1451.87 0.036 0.004 0.0375 1151.87 0.071 0.097 0.57 9371.87 0.075 0.049 0.47 6953.75 0.082 0.013 0.14 1863.75 0.060 0.011 0.11 2083.75 0.056 0.006 0.085 1633.75 0.049 0.0135 0.092 217

a Percent recovery is defined as ratio of the ZDV concentrations in MNcpwplus those in samples Wl to W4/ZDV concentration in MNcp.

phatase, the same comparison resulted in a 67 to 100%recovery of total ZDV (Table 2). This appeared to be a clearindication that for the samples washed with type 1-S alkalinephosphatases, something other than ZDV was being measuredby the assay.

Next, control samples in PBS treated with type 1-S alkalinephosphatase were prepared at a variety of concentrationsranging from 0.75 to 6.2 DEA U/ml. The final concentrationused in the mononuclear cell pellet samples was 3.1 U/ml (0.5mg/ml). The control samples treated with type 1-S alkalinephosphatase were frozen at -20°C until analysis by RIA. Atotal of 46 samples were run on 7 different assay days. Thereappeared to be a crude correlation between the type 1-Salkaline phosphatase concentration and the mean false ZDVconcentration (r2 = 0.979), although for a given type 1-Salkaline phosphatase concentration there were considerableinterday (30- to 40-fold) and intraday (2.5- to 6-fold) assayvariabilities (Fig. 4). False ZDV concentrations ranged from0.002 to 0.075 ,uM (mean, 0.018 F.M) and from 0.006 to 0.258,uM (mean, 0.055 ,uM) for the type 1-S alkaline phosphataseconcentrations of 3.1 and 6.2 DEA U/ml, respectively (Fig. 4).Type VII-NL alkaline phosphatase (concentration range, 2.5to 20 DEA U/ml) samples were then prepared in PBS. These

TABLE 2. Comparison of unwashed versus washed mononuclearcell pellet plus wash samples after type VII-NL alkaline

phosphatase treatment

ZDV concn (,uM) in:

Spiked whole MNcpw + samples % Recovery'blood MNcp Wl to W4

0.375 0.0045 0.003 670.375 0.003 0.003 1001.87 0.027 0.0195 721.87 0.019 0.015 803.75 0.036 0.0255 713.75 0.036 0.030 83

a Percent recovery is defined as ratio of the ZDV concentration in MNcpwplus those in samples Wl to W4/ZDV concentration in MNcp.

0.05

0.04 1

0.03 1

0N-

0.02 -

0.01

0.00 1.0Io-oo-o3.75

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0.30

0.25 _

0.20

0.15 _

0.10 S

0.05S

0.00 . .i0 1 2 3 4 5 6 7

Type 1-S Alkaline Phosphatase units/mlFIG. 4. Type 1-S alkaline phosphatase samples in PBS analyzed by RIA for ZDV.

samples were also frozen at -20°C until analysis by RIA. Atotal of 28 samples were run on 5 different assay days.Borderline assay interference (0.00075 ,uM) was measured inonly 1 of 28 samples.The mean ZDV recovery from whole blood spiked with

ZDV at each of the four concentrations (0.187 to 3.75 ,iM)ranged from 86.4 to 92.4% (Table 3). Microscopic inspectionof erythrocytes showed uniform cell lysis after freezing over-night. The ZDV concentration measured in the mononuclearcell pellet was not included in these calculations because of theproblem encountered with alkaline phosphatase assay interfer-ence. This exclusion plus the fact that an erythrocyte extractionstep was not done may have contributed to the less than 100%recovery.

DISCUSSIONThe lack of correlation between plasma ZDV concentra-

tions and clinical efficacy has prompted several investigatorsto quantify intracellular, phosphorylated ZDV anabolites.Whether the ZDV mono-, di-, and triphosphate forms areindividually quantified by a coupled HPLC-RIA or whether allphosphorylated ZDV is pooled together, these studies ulti-mately depend on the fact that a phosphatase enzyme mustcleave off 5' phosphate groups so that the concentration of

TABLE 3. Mean ZDV recovery in Leucoprep separation fractions(10 ml of spiked whole blood)a

ZDV concn ZDV concn Recovery of ZDV (ng) in:(ILM) (ng/ml) %

spiked in in spiked Rcvrspike WBi PL MNsup LPsol RBC Total Recovery

0.187 50 279 44 10 126 459 91.80.375 100 520 125 14 205 864 86.41.87 500 2,598 713 167 869 4,347 86.93.75 1,000 5,575 1,663 233 1,764 9,235 92.4

aWB, whole blood; PL, plasma; MNsup, mononuclear supernatant; LPsol,dense Leucoprep solution; RBC, erythrocytes.

parent ZDV can be measured by RIA (9, 11, 16). Any form ofphosphorylated ZDV is presumed to be too polar to cross cellmembranes. For this reason it was anticipated that only parentZDV would be present in washed samples and, therefore, thatthe presence of alkaline phosphatase should not make adifference in the amount of parent ZDV measured by RIA. AsFig. 2 shows, more parent ZDV in type 1-S alkaline phos-phatase-treated wash samples than in untreated samples wasconsistently measured by RIA. Because fresh wash sampleswere examined microscopically and were found to be acellular,it was concluded that this particular form of bovine intestinalalkaline phosphatase may interfere with the RIA for ZDV.When type VII-NL bovine intestinal alkaline phosphatase wasused, this increase in parent ZDV concentration in alkalinephosphatase-treated wash samples disappeared, as shown inFig. 3, even though the second preparation undergoes a morerigorous purification process (11). Therefore, it may not be theenzyme itself but impurities in the manufacturer's product thatare responsible for assay interference. To rule out the possi-bility that the buffer system used was causing assay interfer-ence, samples containing PBS and nothing else were runundiluted through the assay, and the assay consistently mea-sured zero.Another approach to verifying that type 1-S alkaline phos-

phatase interfered with the RIA involved a comparison of thetotal ZDV (alkaline phosphatase-treated samples) recoveredin MNcp with the total ZDV measured in MNcpw plus the fourwash samples. Theoretically, the amount of ZDV in these twoentities should be equal; however, some loss of ZDV orphosphorylated anabolites would be expected in the cell washarm of the protocol from sheer manipulation. If anything, then,less total ZDV would be anticipated in the cell wash arm. Infact, just the opposite result materialized for the samplestreated with type 1-S alkaline phosphatase. In all but oneLeucoprep separation, the total ZDV measured in the cellwash arm was more than 115% of that measured in the MNcpsample. When these experiments were repeated with typeVII-NL alkaline phosphatase, the total ZDV measured in thecell wash arm ranged from 67 to 100% of that measured in the

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MNcp, which is consistent with some loss because of cell pelletmanipulation. These results confirm that assay interferencewas occurring with the type 1-S alkaline phosphatase prepara-tion.The third and most direct approach, using control samples in

PBS treated with either type of alkaline phosphatase, againverified that interference with the RIA was occurring above thelower limit of assay sensitivity (0.25 ng/ml or 0.001 ,uM). Only1 of 28 type VII-NL alkaline phosphatase-treated samplesshowed borderline assay interference (0.00075 ,uM), whereasall 46 type 1-S alkaline phosphatase-treated samples showedassay interference above 0.001 ,uM, with 2.5- to 6-fold intradayassay variabilities. The interday assay variabilities for the sameconcentration of the enzyme were even greater, ranging from30- to 40-fold. One explanation for this degree of assayvariability, which is at most ± 15% for plasma samples contain-ing ZDV, is that the type 1-S bovine intestinal alkalinephosphatase is a relatively impure product. These impurities,which may not be uniformly dispersed throughout the powder,could contribute to enhanced assay variability, particularly thegreat interday variability, since a fresh solution of alkalinephosphatase was prepared for each set of samples from thesame batch of powder. Lot-to-lot variation in the degree ofimpurities present could account for the fact that other inves-tigators, who used the same form of the enzyme, have notfound the same degree of assay interference (14). Anotherfactor that should be considered is the differences in the buffersystems used. We used PBS (pH 7.4), whereas others haveused a Tris buffer system at a higher pH (9, 18). Nevertheless,it is possible that if this form of alkaline phosphatase is used totreat mononuclear cell pellet samples containing ZDV, intra-and interday assay variabilities in enzyme interference couldlead to false conclusions regarding inter- and intraindividualvariabilities in phosphorylated ZDV concentrations in pa-tients.Measurement of intracellular ZDV concentrations typically

involves laboratory manipulation of a mononuclear cell pelletwhich includes dilution in some buffer system. These samplesmust be diluted further with some form of phosphatase enzymeto recover parent ZDV, which is the only form of ZDVmeasurable by RIA. For these reasons, mononuclear cell pelletsamples typically contain very low concentrations of ZDV, andtherefore, any assay interference above the lower limit ofsensitivity of the assay should be given attention. Acid (11) andalkaline phosphatase (9, 16) have been used by other investi-gators in the pursuit of an accurate methodology for measuringphosphorylated ZDV concentrations. The present series ofexperiments, which evolved to an investigation of possibleassay interference by one preparation of bovine intestinalalkaline phosphatase, may be viewed as a cautionary noteconcerning the choice of phosphatase enzyme to be used in thein vitro or in vivo quantification of phosphorylated ZDVconcentrations.

ACKNOWLEDGMENT

A. M. O'Donnell is supported by a grant from the AmericanFoundation for AIDS Research.

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