a direct ligand-binding radioassay for the measurement of … · of the enzyme dihydrofolate...

[CANCER RESEARCH 35, 2033-2038, August 1975]

SUMMARY

A direct ligand-binding radioassay for methotrexate(MTX) has been developed using dihydrofolate reductase,contained in the lysate of L12l0 leukemia cells, as thebinding determinant. The procedure is a two-phase reactionsystem where standard MTX concentrations or the samplebeing assayed is incubated with the reagent lysate in the firstphase, and [3HJMTX is then added in the second phase totitrate the remaining unoccupied binding sites on theenzyme. This method eliminates the need for measuring theresidual catalytic activity of the enzyme.

The sensitivity of the radioassay is limited only by thespecific activity of the [3H]MTX and now approximates 10pg of the drug. Folic acid, methyltetrahydrofolate, formyltetrahydrofolate, and dihydrofolate in concentrations thatare physiological do not interfere in the radioassay. Bothmercaptoethanol and reduced nicotinamide adenine dinucleotide phosphate increase the binding capacity of thelysate for MTX; but the reduced nucleotide also increasesthe affinity of the enzyme for the inhibitor.

MTX added to serum can be assayed without extractionif the concentration is greater than 500 pg/ml and recovery of the drug added to serum is about 92%. MTX hasbeen assayed in serum, spinal fluid, and urine of patientswho were treated with this drug. It has also been assayed inthe lysates of LI2IO cells from C57BL x DBA/2 F1 micetreated with MTX. The procedure is simple, rapid, andaccurate and should permit better correlation of the therapeutic and toxic effects of MTX with blood concentrationsover long-term treatment periods.

INTRODUCTION

The pharmacokinetics and tissue distribution of MTX8

1 This work was supported by Research Grants CA 08976 and AM

16220 from the NIH and by a grant from the National LeukemiaAssociation, Inc.

2 Medical student at New York Medical College.

3 Career Investigator of The New York City Health Research Council,

1-423. To whom request for reprints should be sent, at Division ofHematology-Oncology, Department of Medicine, New York MedicalCollege, 1249 Fifth Avenue, New York, N. Y. 10029.

4 Special fellow of the Leukemia Society of America.

5 Kresevich Foundation Cancer Research Scholar.

6 The abbreviations used are: MTX, methotrexate; FB, fraction

[3H]MTX bound; i.t., intrathecally.Received January 27, 1975; accepted April 22, 1975.

has been studied in animals and man (1, 7, 10, 12, 13, 15, 18,24) and has provided fundamental information aboutplasma clearance, tissue uptake, intestinal absorption, andurinary excretion of this drug. The experimental model formany of these studies has been the administration of a singleor a few multiple doses of the drug by a specific route afterwhich the pattern of excretion and distribution was measured over a finite time period. With respect to the clinicaluse of MTX, there is a dearth of information relating thelong-term low blood concentration with either therapeuticor toxic effects (10). Indeed, the importance of monitoringdrug concentration may be appreciated from a recent reportby Bleyer et a!. (5), which showed that the neurotoxicity ofintrathecal MTX could be correlated with a cerebrospinalfluid concentration higher than that observed in patientswithout neurotoxic complications.

One reason for the inadequate information correlatingconcentration with long-term administration of MTX hasbeen the lack of a sensitive assay method that could beadapted for routine clinical use. The fundamental principlefor the assay of these drugs by enzymatic physicochemical methods is based on the stoichiometric inhibitionof the enzyme dihydrofolate reductase (EC I .5. 1.3) (23).Whether the enzyme activity is measured by monitoring theoxidation of NADPH (3) or by radioenzymatic analysis(22), the assay for the antagonists has depended on 2

reactions: (a) the binding of the inhibitor to the enzyme; and(b) the reaction of the residual enzyme with the assaysubstrate (folic acid or dihydrofolic acid). Consequently,conditions that perturb either the inhibitor-enzyme reactionor the enzyme-substrate reaction may affect the results ofthe assay procedure. This report describes a radiometricassay for MTX that circumvents this disadvantage bydirectly measuring the binding of [3H]MTX, eliminating theneed to measure residual enzyme activity with a 2ndreaction. The absolute sensitivity of this radioassay islimited only by the specific activity ofthe [3H]MTX and canapproximate 10 pg of the drug.

MATERIALS AND METHODS

[3H]MTX with specific activity greater than 5 Ci/mmolewas purchased from Amersham/Searle Corp., ArlingtonHeights, Ill. The purity of this product was greater than95%, as was determined by descending paper chromatogra

AUGUST 1975 2033

A Direct Ligand-binding Radioassay for the Measurement ofMethotrexate in Tissues and Biological Fluids'

Elliot Arons,2 Sheldon P. Rothenberg,3 Maria da Costa,4 Craig Fischer,5 and M. Perwaiz lqbalDivision of Hematology-Oncology,DepartmentofMedicine, New York Medical College,New York, New York 10029

on June 20, 2021. © 1975 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

E. Arons et a!.

ficient time to run a number of reactions. This time periodis not critical, and shorter or longer periods do not alterthe results.

The reaction is stopped after the 2nd-phase incubation bythe addition of 0.4 ml of a suspension of 1% neutral Norit Acharcoal (Fisher Scientific Co., Springfield, N. J.) in 0.5%dextran of an average molecular weight of 10,000. After it isgently shaken for a few mm, the charcoal is precipitated bycentrifugation and the [3HJMTX remaining in the supernatant, which has bound to dihydrofolate reductase, is determined with a liquid scintillation counter. Usually, a 0.3-mialiquot of the supernatant solution is added to 15 ml of ascintillation solution containing 5 g of PPO and 75 ml ofBBS-3 solubilizer (Beckman Instruments, Inc., Palo Alto,Calif.) per liter of toluene, and sufficient counts areaccumulated for a counting error of 3% or less.

A reaction blank containing no binding lysate is simultaneously determined and the radioactivity not removed bythe charcoal is subtracted from the reactions containing thebinding lysate. This blank is usually 5% or less of the totalradioactivity used. The FB is calculated as the ratio of netcpm remaining in the supernatant to the total cpm used inthe reaction. For convenience, the same volume of[3H]MTX as was used in the assay reactions is diluted in atotal volume ofO.9 ml, and the radioactivity in 0.3 ml of thismixture represents the total radioactivity standard.

Standard Curve. A dose-response curve representing thesaturation of available binding sites by unlabeled MTX isobtained by preincubating standard concentrations of MTXwith the diluted binding lysate (Phase 1). The addition of[3H]MTX to the reaction mixture for Phase 2 then determines the remaining unoccupied binding sites on theenzyme. A standard curve is obtained by plotting l/FB as afunction of the pg of unlabeled MTX.

Assay of MTX in Biological Fluids or Cell Extracts. TheMTX concentration in serum, spinal fluid, or urine can beassayed directly in an aliquot of the sample withoutdeproteination. If MTX is to be measured in aliquots ofundiluted serum greater than 0.05 ml, a similar volume ofnormal serum should be added to the standard MTXreactions. Alternatively, the serum can be diluted 3-foldwith the phosphate buffer and boiled for 5 mm, and theMTX can be assayed in 0.2 to 0.3 ml of the supernatantafter centrifugation of the precipitate. This procedure isappropriate when the concentrations of MTX in the serumare expected to be very low (i.e., less than 0.5 ng/ml), and itavoids the necessity of adding normal serum to the standardreaction mixtures.

MTX can also be assayed in cells or tissues. For theseassays, the cell lysates or homogenized tissue should beboiled with the buffer in order to release bound drug and todenature any cellular dihydrofolate reductase that wouldinterfere in the assay by binding the [3H]MTX.

An aliquot of the samples to be assayed is incubated withthe binding lysate for the 1st-phase reaction and is followedby the 2nd-phase incubation with [3H]MTX as described forthe standard curve. The MTX concentration is then obtamed by referring the experimental 1/FB to the standardcurve determined simultaneously.

phy with unlabeled MTX using 0.1 M K2HPO4, pH 7.0,saturated with isoamyl alcohol. In addition, approximately98% of the radioactivity could be bound using an excess ofdihydrofolate reductase with the assay procedure to bedescribed. Folic acid and 5-methyltetrahydrofolate werepurchased from Sigma Chemical Co., St. Louis, Mo.Dihydrofolate was prepared by the method of Blakley (4).MTX and 5-formyltetrahydrofolate were obtained fromLederle Laboratories, Inc., Pearl River, N. Y.

The radioassay follows the scheme of a 2-phase sequentialnoncompetitive system. In the 1st phase, the standardconcentrations of unlabeled MTX or samples being assayedare incubated with binding ligand, and in the 2nd phase[3H]MTX is added to determine the remaining unoccupiedbinding sites on the ligand. The [3HJMTX bound isinversely related to the concentration of MTX in the standards or unknown samples. This type of radiometric assaygives the most sensitive dose-response curve.

Preparation of Ligand. The binding ligand is the enzymedihydrofolate reductase, obtained from the Ll2lO mouseascitic tumor. This tumor line is maintained in C57BL xDBA/2 F1 (hereafter called BD2F1) mice by i.p. transfer.For preparation of the binding reagent, the tumor cells areobtained by aspiration of the ascitic fluid and washed 3times with 0. 15 M NaCI. The red cells are lysed by diluting Ivolume of the 0. 15 M NaCI suspension of cells with 3volumes of distilled water. After 30 sec. 1 volume of 0.6 MNaCI is added and the cells are washed 2 more times with0. 15 M NaCI to remove the free hemoglobin. After the lastwash, the cells are packed by centrifugation, the supernatantNaCI solution is removed, and the cells are suspended in 3volumes of 0.05 M sodium citrate, pH 7.4. The suspension isfrozen in small aliquots at â€”70Â°.At this temperature theenzyme is stable for several months. For use in the radioassay, the lysate is thawed and the cell debris is precipitated bycentrifugation at 30,000 x g. The supernatant containingthe dihydrofolate reductase is the binding ligand reagent.

For each batch preparation of L12l0 lysate, it is necessary to determine the dilution that binds 50 to 60% of the[3H]MTX concentration to be used in the radioassay. TheMTX-binding capacity for most of the lysates prepared asdescribed approximates 6000 to 7000 pg/mi. For stabilization of the enzyme, the lysate is diluted in 5% bovinealbumin prepared in deionized water. The [3HJMTX concentration depends on the specific activity available and thedesired sensitivity of the dose-response curve. For this studywe have used a tracer concentration of either 130 or 330pg/reaction.

The 1st-phase reaction is in 0.05 M potassium phosphatebuffer, pH 6.0, and contains (in a volume of 0.4 ml) 0.01mmole KC1, 48 nmoles NADPH, 6.0 @moles2-mercaptoethanol, the unlabeled MTX standards or aliquot of samplematerial being assayed, and 0. 1 ml of the diluted lysate. After 30 mm incubation at room temperature, the 2nd phasecommences with the addition of 0. 1 ml of the [3H]MTXsolution (1 30 or 330 pg) and the incubation is continued foran additional 30 mm. The reaction between MTX and theenzyme is very rapid and maximal binding occurs by 5 mm.We have selected 30 mm incubation because it provides suf

2034 CANCER RESEARCH VOL. 35



â€”-- A@ = , 76+0092 (pgMTX)

::?t 208

I@ 6

0I@ 4__I@ 2

I'@-@0I z12Â°I@ â‚¬IL. 4

2

Ligand-binding Radioassay for MTX

RESULTS

The sensitivity of the dose-response curve is inverselyrelated to the concentration of both the [3HJMTX and thebinding lysate. Examples of 2 typical standard curves areshown in Chart 1. When the concentration of [3H]MTX wasI30 pg and the concentration of the lysate was adjusted tobind about 50% of the tracer, a dose-response curve with anabsolute sensitivity of approximately 10 pg of unlabeledMTX was obtained (Chart IA). This level of sensitivitycould be used to assay the low concentrations of MTX intissue or cell extracts, or in biological fluids up to 7 daysfollowing administration of the drug. Where greater concentrations of the drug are expected in the samples to beassayed, for example, in clearance studies, the less sensitivedose-response curve may be satisfactory. The standardcurve in Chart lB was obtained using 330 pg of [3H]MTX.In this instance, the absolute sensitivity approximates 25 pgof MTX.

Although it was anticipated that greater precision wouldbe obtained with the less sensitive assay, the experimentalplot for both assays resulted in a dose-response curveapproaching the plot obtained by the least-squares analyses(r = 0.98 and 0.97). Thus, just a few experimental pointscould be used to derive the linear standard curve using theleast-squares method for determining the equation of theline. We have also observed that linearity of the standardcurve ceases when the binding of the [3H]MTX decreasesbelow 12% (l/FB > 8).

The conditions necessary to stabilize the binding properties of the lysate and to obtain the maximum sensitivity ofthe dose-response curve were investigated. The data inChart 2 show the effects of NADPH and 2-mercaptoethanol. To appreciate better the comparative effects of theseadditions to the dose-response curve, the ratio of percentageof [3H]MTX bound to percentage of [3H]MTX free wasplotted as a function of the unlabeled MTX. This is a

pg MTX

Chart I . Standard dose-response curves for the inhibition of binding of'[3H]MTX to the Ll210 lysate by unlabeled MTX. In A, the [3H]MTXconcentration was I30 pg/reaction and the Iysate was appropriately dilutedto bind approximately 50%. The dose-response curve ranged from 10 to 160pg. In B, the [3H]MTX concentration was 330 pg/reaction and the lysatediluted to bind 35%. The dose-response curve here ranged from 25 to 400pg of unlabeled MTX. The experimental points (x) fall close to the linedetermined by the method of least squares (A).

standard method of expressing such data in ligand-bindingradioassays. Not only was maximum binding of the[3H]MTX observed when the reaction contained bothNADPH and 2-mercaptoethanol, but NADPH also in

creased the slope of the dose-response effect of the unlabeledMTX. It can be inferred from these observations that2-mercaptoethanol increases slightly the stability of theenzyme and that NADPH also increases the affinitybetween the drug and lysate-binding determinant(s).

Other buffer ions such as citrate or acetate were testedand in the range of pH 6.0 appeared to result in somewhatgreater variability of binding of the [3H]MTX. The mostconsistent results were obtained with potassium phosphateat pH 6.0, and variations less than Â±0.2unit did not appearto alter the dose-response curve. At pH greater than 7.0,there is lower affinity for the binding of MTX and thedose-response curve is consequently less sensitive.

The effect of physiological folate analogs on the bindingof [3HJMTX by the lysate was also studied since thesecompounds will be present in the biological fluids and tissueextracts to be assayed for this drug. The results are shown inChart 3. In the concentration range from 2.2 to 17.6pmoles/reaction mixture neither folic acid, nor 5-formyltetrahydrofolate, nor 5-methyltetrahydrofolate, nor dihy

01020 40 80

pg UNLABELED NIX

Chart 2. The effect of NADPH and 2-mercaptoethanol on the bindingof [3H]MTX by L12l0 lysate and the dose-response curve obtained withunlabeled MTX. These reaction mixtures contained 130 pg [3H]MTX.Reaction with both 6 .imoles 2-mercaptoethanol and 48 nmoles NADPH(A); reaction with 48 nmoles NADPH (â€¢); reaction with 6 j@moIes

2-mercaptoethanol (i); reaction with neither 2-mercaptoethanol norNADPH (0).

1@0LICACID50r _______@ a.a.-1CHOFH4

[@H3FH4

\.@NTX FH2

2 4 6 8 0 2 14 6 18p moles

Chart 3. Effect of physiological folates on the binding of [3HJMTX.CHO FH4, formyltetrahydrofolate;CH3 FH4, methyltetrahydrofolate;FH2; dihydrofolate. For this experiment the binding lysate was added to amixture of 330 pg [3H]MTX and folate analog.

Luz@ Lu0 I

@_J-@

3.0f

20 160B 7@8@â€˜268+00156(pgMTX)

r= 0 980

9.0

8.0

7.0

6.0

50

4.0

3012.0

I .0

40

@ 30

@20

@l0

40 80 20 60 00 200 300 400

AUGUST 1975 2035



SampleMTXadded

(pg/0. I ml)MTXrecovered

(pg/0. I ml)%recoveryâ€•I3203089621601601003808811044044110Sb320290906Â°240200837b160138868b80821039b402870lOb201470

3.0

B.A.ConcentrationConcentration/in

1:2dilutionaliquotassayedofaliquotSample(pg/mI)(pg/mI)B/Aâ€•Spinal

fluid6803300.48Spinalfluid2851600.56Ll2IOIysate3451500.43Ll2l0lysate3601850.51Serum100400.40Serum2701

300.48Urine126500.40

MTXSampleconcentrationRemarksSerum

(M. L.)[email protected]/ml410 pg/mI5

mm after 25 mg iv.7 days after 25 mgiv.Serum

(M. I.)I .3 ng/mI7 days after 25 mgiv.Serum(A. C.)2.4 ng/ml7 days after 25 mgiv.Spinalfluid (J. P.)28.5 ng/ml6 days after I5 mg i.t.

MTXSpinalfluid (F. P.)19.0 ng/ml3 days after 15mg it.

MTXUrine(J. M.)18.7 mg/24 hr24-hr excretion follow

ing25 mg MTXi.t.

ao

0 10 20 30 40 50 60 70 80MINUTESAFTERINJECTION

90

E. Arons et a!.

drofolate had any effect on the binding ofthe [3H]MTX. Inthe sample aliquot of biological material to be assayed forMTX, it is not likely that the concentration of physiologicalfolate will exceed this range (up to 17.6 pmoles/ml).

The recovery of MTX added to serum is shown in Table1. For this study the concentrations of MTX for thestandard curve were prepared in normal serum so that theconcentration of protein in all reaction mixtures (forstandard and assay reactions) was essentially similar. Therange ofrecovery was 70 to I 10% with a mean of9l.8%. Thegreatest errors were at the lowest concentration of MTXThus, values of MTX between 200 and 400 pg/mi should beconsidered as those measured with the greatest coefficient ofvariation.

Table 2 summarizes the MTX concentration in serum,spinal fluid, and urine of patients treated with this drug. Thetime each sample was taken following drug administrationis shown. It is evident that even 7 days followingthe administration of MTX i.v. or i.t. the drug could still be assayedin the serum and spinal fluid, respectively. The excretion of74% ofan i.t. dose ofMTX in the 1st 24hr following administration is consistent with the known rapid transit of thisdrug from brain to blood.

Chart 4 shows the progressive increase in concentrationof MTX in Ll210 leukemic cells following the s.c. administration of the drug at a dose of 3 mg/kg to BD2F1 mice with

Table I

the ascitic form ofthis tumor. The decrease in the activity offolate reductase in the cells measured by radioenzymaticassay (22) occurs simultaneously with the rise in concentration of drug.

An important quality control procedure with radiometricligand-binding radioassays is to assay 2 dilutions of asample. If there is no stoichiometric decrease in theconcentration of substrate assayed, then there.are interfering factors in the assay procedure. Table 3 shows samples ofserum, spinal fluid, urine, and leukemic cell lysates assayedat 2 dilutions. The mean concentration of the samplesassayed after a 2-fold dilution was 46% of the concentrationmeasured in the undiluted sample.

Chart 5 compares the sensitivity of this radioassay forMTX with the spectrophotometric method described byBertino and Fischer (3), which monitors the decrease inabsorbance at 340 nm as NADPH is oxidized to NADPas a consequence of the reduction of dihydrofolate totetrahydrofolate. Dihydrofolate reductase was prepared forthis assay from guinea pig liver (3). The enzyme concentration was adjusted to give the recommended maximum @Aof0.15 in 5 mm. With this assay, inhibition was not evident

Chart 4. The increase in MTX concentration in L12l0 leukemia cellsobtained from BD2FI mice given MTX at a dose of 3 mg/kg. The catalytic activity of the enzyme was measured in the lysates using a radioenzymatic assay (22) with [3H]folic acid as the substrate. Each valuerepresents the mean of duplicate determinations.

Table 3

120

110

. 100

. 90

z

@ 70

@ 40

I

5.0

4.0

Recovery of MTX added to normal human serum

1.0

Effect ofsample dilution on recovery of MTX

a Mean, 9 1.8%.

b MTX was added to a pool of normal serum.

Table 2MTX concentrationin serum,spinalfluid, andurineofpatientstreated

with this drug

C Mean, 46%.




Ligand-binding Radioassay for MTX

radioassay and has the advantage of greater sensitivity,particularly when there is little reversibility of the bindingreaction. In addition to the sensitivity of this radioassay,which could be adjusted to meet specific needs, the procedure can measure MTX in biological fluids without thenecessity of extracting the drug or deproteinating thesample. Where the MTX is bound, as in cells, thenextraction is necessary to free the drug and denature anybinding sites (e.g., dihydrofolate reductase).

It has generally been considered that the sole binding sitefor intracellular MTX is dihydrofolate reductase. With thisconsideration, and with the demonstrated stoichiometricinactivation of enzyme activity by MTX (2), we anticipatedthat a standard curve would not be necessary for thisradioassay. Under such circumstances, the [3H]MTX displaced from its binding determinant by a sample of materialcontaining this drug should be sufficient quantitative data tocalculate the unknown concentration of MTX, providingthat the specific activity (cpm/unit weight) of the [3HJMTXis accurately established. This has not been possible for theLl2lO lysate as prepared for this radioassay. We have foundthat the more MTX was added, the more it was bound overpart of the concentration range of the standard curve. Thenature of this phenomenon is now under investigation (9),but this information is presented now to stress the need for astandard curve with each assay.

The linearization of the standard curve obtained byplotting the l/FB as a function of the unlabeled MTX is adistinct advantage because only a few points are needed tocalculate the equation by the least squares method. Theequation can easily be programmed into a desk-top calculator for rapid calculation of results if many samples arebeing assayed. One caution is that the curve remains linearonly until about 12% of [3HJMTX is bound. With valuesbelow this, the slope of this plot (l/FB versus unlabeledMTX) increases sharply.

NADPH had a marked effect of increasing the binding of[3HJMTX by the lysate, but 2-mercaptoethanol had a lessereffect, and the combination of both agents appeared to besynergistic. It is known that NADPH binds to and stabilizesthe catalytic function ofdihydrofolate reductase (17, 19). Itis apparent from these studies that this cofactor alsoenhances binding of nonsubstrate folate analogs such asMTX. This enhanced binding is not simply a stabilization ofthe enzyme protein because the sharper slope of thedose-response curve obtained with unlabeled MTX andNADPH indicates an increased affinity for the analog,which would result primarily from some aliosteric conformational change in the reactive site of the enzyme. We arenow studying this phenomenon with purified enzyme todefine the mechanism better.

It should be appreciated that this radioassay procedurecould be adapted to measure any inhibitor of dihydrofolatereductase even with [3H]MTX as the tracer. It is necessaryonly to substitute standard concentrations of the inhibitorfor the MTX standards. What will vary with each inhibitoris the slope of the dose-response standard curve since this isa function of the relative affinity of the inhibitor and[3H]MTX for the enzyme. Inhibitors weaker than MTX will

0.05 0.1 0.2 0.3 G4 0.5 .â€”.2.0 5.0 0 20 30 4050 sâ€”s

ng MTXPERREACTION

AUGUST 1975 2037

S

S@ @90z

@ 80

4 @60

x! @50! @40

@ )( [email protected]@

U-5- 0

.0

Chart 5. Comparison of the sensitivity of the radioassay (â€¢)with thespectrophotometric method ( x ) for measuring MTX. By the spectrophoto

metric method, no change in the @Awas observed until the reaction mixture contained 5 ng of MTX. In contrast, with radioassay procedure using130 pg [3H]MTX and the appropriate dilutions of the lysate, inhibition ofthe binding of the tracer MTX was first evident with 15 pg of unlabeledMTX in the reaction mixture.

until the reaction mixture contained 5 ng of MTX. Incontrast, initial inhibition ofthe binding of[3H]MTX by theLl210 enzyme preparation was evident at approximately 15pg of unlabeled MTX.

With this dose-response sensitivity observed for thespectrophotometric assay, it would be difficult to measureMTX concentrations in the range of I to 5 ng/ml andvirtually impossible to measure levels in the pg/mi range.On the other hand, the spectrophotometric assay would besufficiently sensitive to measure the concentrations in blood,urine, and spinal fluid that occur within a few hr followingthe administration of MTX.

DISCUSSION

There is a dearth of information relating the concentration of MTX to therapeutic and toxic effects over prolongedperiods of treatment because of a lack of an assay procedureboth sensitive and easily adapted by clinical services.Consequently, the dosage schedules for the folate antagonists for most therapeutic protocols have, for the most part,been derived from empiric observation over short-termtreatment periods. The importance of knowing the concentration of the drug, however, has recently been demonstrated by Bleyer el a!. (5), who showed that toxicity to thecentral nervous system following the administration ofMTX could be correlated with high spinal fluid concentration of the drug.

Unlike previous methods for assaying MTX, whichdepend on monitoring the residual activity of the targetenzyme, dihydrofolate reductase, the method described inthis report measures directly the effect of unlabeled MTXon the binding of [3H]MTX. It is not truly a competitiveinhibition assay system because the binding lysate is notadded to a mixture of labeled and unlabeled MTX. Rather,this sequential system has been considered a noncompetitive



E. A roux el al.

yield curves with lesser slopes than equimolar concentrations of MTX.

Although we have not specifically studied the effect ofmetabolites of MTX on the assay system, it can generally beconcluded that if the metabolite binds to the enx.yme it willbe included in the assay. This is not likely to be a significantfactor since such metabolites will usually have a loweraffinity than the parent MTX compound for the binding siteon the enzyme, and the measured effect would be low. Onthe other hand, from a pharmacological consideration, ifsuch metabolites arc measurable, then it is best to have themincluded in the assay since they would be effective inhibitorsof the catalytic activity of dihydrofolate reducÃ-ase.

Most pharmacological studies to correlate clinical toxic-ity and dosage have been designed as acute experiments. Arecent report of such a study by Goldie et al. (14), confirmedthe clinical observation that acute toxicity (bone marrow,mucous membranes) was related to the duration of administration of an i.v. infusion and not to the dose. On the otherhand, there is good experimental evidence by Chabner andYoung (6) that shows that the suppression of DN A synthesisin L1210 leukemia and normal mouse tissues is a directfunction of the plasma concentration of MTX. Irrespectiveof the dose administered, de novo thymidylate synthesisfrom deoxyuridine recovered up to 50% of the treatmentvalues when the plasma concentration of MTX was IO"8 M

or less. This concentration is easily within the sensitivity ofthis radioassay, and a similar correlation of blood concentration and biochemical effect of MTX on target tissueswould be of interest in the treatment of various humanneoplasias.

Finally, closer observation of blood concentration andtissue accumulation of MTX may be of value in minimizingthe toxicity of MTX, particularly when the drug is administered over long treatment periods. Nonacute MTX toxicityhas been recognized in the brain (20). liver (16), kidney (8),lung (II), and bone (21 ). Although individual characteristicssuch as age, extent of malignant disease, and renal functioncan alter the metabolism of MTX, much may be learned bycorrelating these toxicities with the determination of bloodand, when possible, tissue concentration of this drug.

REFERENCES

1. Anderson. L. L.. Collins. G. J.. Ojima. Y.. and Sullivan, R. D. AStudy of the Distribution of Methotrexatc in Human Tissues and

Tumors. Cancer Res.. 30: 1344 1348, 1970.2. Berlino. J. R., Booth, A. B., Bieber. A. L., Cashmore, A., and

Sartorelli. A. C. Studies on the Inhibition of Dihydrofolate ReducÃ-aseby Ihe Folale Anlagonisls. J. Biol. Chem., 239: 479 485. 1964.

3. Berlino. J. R.. and Fischer. G. A. Techniques for Ihe Study ofResislance of Folie Acid Anlagonisls. Melhods Med. Res., 10:297-307, 1964.

4. Blakley. R. L. Crystalline Dihydropteroylglulamic Acid. Nalure. Â¡88:

231 232. 1960.5. Blever. W. A.. Drake. J. C.. and Chabner. B. A. Neuroloxicity and

Elevaled Cerebrospinal-Fluid Metholrexale Concenlralion in Menin

gea! Leukemia. New Engl. J. Med., 289: 770 773. 1973.6. Chabner. B. A., and Young. R. C. Threshold Melholrexale Concen

tration for in Vivo Inhibition of DNA Synthesis in Normal andTumorous Target Tissues. J. Clin. Invest.. 52. 1804 1811. 1973.

7. Charache, S.. Condii, P. T.. and Humphreys. S. R. Studies on IheFolie Acid Vilamins. IV. The Persislence of Amelhoplerin in Mammalian Tissues. Cancer, 13: 236 240. 1960.

8. Condii. P. T., Chames. R. E., and Joel. W. Renal Toxicity ofMelhotrexate. Cancer, 23: 126 131. 1969.

9. da Costa, M.. Rolhenberg. S. P., and Arons, E. Binding of|3H]-Mctholrexale (MTX) for the Radioassay of This Drug and Study

of Enzyme-Inhibitor Interaction. Proc. Am. Assoc. Cancer Res., 16:

88, 1975.10. Dedrick. R. L.. Bischoff. K. B., and Zaharteo. D. S. Inlerspecies

Correlation of Plasma Concentralion Hislory of Melholrexale. Cancer Chemotherapy Repl.. 54: 95 101, 1970.

11. Everts. C. S.. Westcoll. J. L.. and Bragg. D. G. Melholrexale Therapyand Pulmonary Disease. Radiology. 107: 539 543, 1973.

12. Founlain. J. R.. Hutchison. D. J.. Waring, P. B.. and Burchenal. J. H.Persislence of Amelhoplerin in Normal Mouse Tissues. Proc. Soc.Exptl. Biol. Med., 83: 369 373, 1953.

13. Fountain. J. R., Waring. G. B.. Hutchison. D. J.. and Burchenal. J. H.Dislribulion of Amelhopterin in Normal Mouse Tissues followingIntravenous Injection. Proc. Soc. Exptl. Biol. Med., 81: 193 196,1952.

14. Goldie, J. H., Price. L. A., and Harrap. K. R. Melholrexale Toxicily.Correlation with Duration of Administration. Plasma Levels. Dose,and Excretion Pattern. European J. Cancer. 8: 409 414. 1972.

15. Henderson. E. S., Adamson. R. H.. and Oliverio. V. T. The MetabolicFate of Tritiated Melholrexale. II. Absorplion and Excrelion in Man.Cancer Res., 25: 1018 1024, 1965.

16. Huiler. R. V. P., Shipkey. F. H.. Tan, C. T. C.. Murphy. M. L.. andChowdhury. M. Hepalic Fibrosis in Children wilh Acute Leukemia.Cancer, 13: 288 307, 1960.

17. Jackson. R. C.. and Huennekens, F. M. Turnover of DihydrofolateReducÃ-ase in Rapidi) Dividing Cells. Arch. Biochem. Biophys.. 154:192 198. 1973.

18. Johns, D. G., Hollingsworth. J. W., Cashmore, A. R.. Plenderleith, I.H., and Berlino, J. R. Metholrexate Displacement in Man. J. Clin.Invest., 43: 621 629, 1964.

19. Kaufman, B. T., and Gardiner. R. C. Studies on DihydrofolicReducÃ-ase. I. Purification and Properlies of Dihydrofolic ReducÃ-asefrom Chicken Liver. J. Biol. Chem., 241: 1319 1328, 1966.

20. Price. R. A.. Jamieson, P. A., and Pilner, S. E. Degeneralive Changesin Ihe Brain following Irradialion and Melholrexale Therapy forChildhood Lymphocylic Leukemia. Blood, 44: 929. 1974.

21. Ragab. A. H.. Freeh, R. S., and Vielti. T. J. Osteoporotic FracturesSecondary to Metholrexale Therapy of Acule Leukemia in Remission.Cancer, 25: 580 585, 1970.

22. Rolhenberg, S. P. A Rapid Radioassay for Folie Acid ReducÃ-ase andAmethopterin. Anal. Biochem., 16: 176 178, 1966.

23. Werkheiser, W. C. Specific Binding of 4-Amino Folie Acid Analogues

by Folie Acid ReducÃ-ase. J. Biol. Chem.. 236: 888 893, 1961.24. Werkheiser, W. C.. and Holland, J. F. Pharmacologie Studies of

Metholrexate (MTX) and 3',5'-Dichlorometholrexale (DCM) in

Man. Proc. Am. Assoc. Cancer Res., 3: 277, 1961.




1975;35:2033-2038. Cancer Res Elliot Arons, Sheldon P. Rothenberg, Maria da Costa, et al. Methotrexate in Tissues and Biological FluidsA Direct Ligand-binding Radioassay for the Measurement of

Updated version

http://cancerres.aacrjournals.org/content/35/8/2033

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/35/8/2033To request permission to re-use all or part of this article, use this link


http://cancerres.aacrjournals.org/content/35/8/2033http://cancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://cancerres.aacrjournals.org/content/35/8/2033http://cancerres.aacrjournals.org/

a direct ligand-binding radioassay for the measurement of … · of the enzyme dihydrofolate...

Documents