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A reagent kit was obtained from the New England Enzyme. Center, Boston, MA 02111. The reagents were reconstituted according to manufacturer's instructions.
CLIN. CHEM.27/3, 380-384(1981)

Enzyme Immunoassayand EnzymeInhibitionAssayof Methotrexate,with Use of the CentrifugalAnalyzer Michael A. Pesce and Selma H. Bodourlan Methotrexate

excretion

zyme

was determined by the homogeneous en(EMIT#{174}) with the Multistat and CentrifiChem centrifugal analyzers and by the enzyme inhibition assay with use of the Multistat centrifugal analyzer. With both methods, the standard curve extends from 0.2 to 2.0 mol/L and there is no Interference from bilirubin in concentrations up to 100 mg/L. Moderately lipemic samples do not interfere with the EMIT method, but lower the values obtained with the enzyme inhibition assay. Hemoglobin concentrations as great as 1 g/L do not affect results for methotrexate obtained by the enzyme inhibition assay. With the EMIT assay, methotrexate values are lowered in samples containing hemoglobin in concentrations exceeding 750 mg/L. With the EMIT assay, the following compounds in concentrations of 1 mmol/L do not interfere: leucovorin, 5-methyl-tetrahydrofolate, 5-fluorouracil, and 6-mercaptopurine. When folic acid (100 jmol/L) was added to a serum that did not contain methotrexate, a response equivalent to 0.15 mol/L was obtained. Methotrexate is stable for five days in serum stored at 23, 4, or -20 #{176}C. Within-run precision (CV) for the enzyme inhibition method ranged from 4.7 to 8.1% and for the EMIT assay from 2.5 to 6.2%. Methotrexateconcentrationsin

situation. symptoms

immunoassay

the serum

of children

receiving

high-dose

therapy

were

compared by three methods: competitive protein binding, EMIT, and enzyme inhibition assays. The correlatkn coefficients averaged 0.95. High-dose therapy with methotrexate has been used in the management of osteogenic sarcoma, acute lymphocytic leukemia, cancer of the head and neck, and other malignancies. Methotrexate acts as an antitumor agent by binding to the enzyme dihydrofolate reductase (EC 1.5.1.3) and preventing the formation

of reduced

folates

that are necessary

for many

of the biochemical reactions involving the transfer of onecarbon units. As a result, synthesis of nucleic acids and DNA is inhibited. Several hours after methotrexate infusion, leucovorin (5-formyltetrahydrofolate) is given to “rescue” the normal cells from the toxic effects of methotrexate (1). Leucovorin is converted to 5,10-methylenetetrahydrof#{244}late by a

pathway independent of the enzyme dihydrofolate reductase. The methylene group is the source of the one-carbon units, which are necessary for nucleic acid and DNA synthesis. High-dose therapy with methotrexate has been associated with myelosuppression, renal failure, vomiting, dermatitis, stomatitis, alopecia, and hepatotoxicity. It can also be fatal. In a survey of cancer treatment centers in the United States, fatalities occurred in 29 of 498 patients who were receiving high doses of methotrexate followed by leucovorin (2). Toxicity is related not only to the level of methotrexate attained but also to the duration of exposure to the drug. Renal failure is the underlying cause of toxicity; any factor that delays the Special Chemistry Laboratory, Columbia Presbyterian Medical Center, Box 253, 622 W. 168th St., New York, NY 10032; and Department of Pathology, Columbia University College of Physicians and Surgeons,

New York,

NY.

Received Aug. 25, 1980; accepted

380

CLINICAL

CHEMISTRY,Vol. 27, No.

3,

1981

of toxicity

are to be identified, methotrexate in patients being treated

centrations must be monitored high doses.

con-

with

has been measured by fluorometry (3), liquid (4), radioimmunoassay (5, 6), competitive (7), enzyme inhibition (8, 9), and homogeneous enzyme immunoassay (EMIT) (10) techniques.’ The EMIT and enzyme inhibition methods are suited for rapid monitoring of methotrexate concentrations in serum. We have

Methotrexate chromatographic protein binding

adapted the enzyme inhibition system to the Multistat centrifugal analyzer. A brief description of the EMIT assay was recently published (11). Here we present a detailed evaluation of the EMIT assay. The optimum conditions for the EMIT procedure were established and the assay adapted to the Multistat and CentrifiChem centrifugal analyzers. The stability of methotrexate in serum and the effects of bilirubin, hemoglobin, lipemia, and temperature with the assay were determined. Methotrexate values were compared by the EMIT, enzyme inhibition, and competitive protein-binding systems.

Materials and Methods Specimens

All patients in this study were children who were receiving methotrexate

in doses

exceeding

50 mg/kg

of body

weight,

followed by leucovorin “rescue.” Blood was collected immediately and at 6 to 72 h after methotrexate infusion. The blood was centrifuged cytes.

and the serum

separated

from the erythro-

Apparatus Two centrifugal analyzers, a “Multistat III” (Instrumentation Laboratories, Lexington, MA 02173) and a “CentrifiChem 400” (Union Carbide Corp Rye, NY 10580) were used in this study. Reagents A. For Enzyme Inhibition Technique Buffer. Tris(hydroxymethyl)methylamine hydrochloride (Tris) buffer, 1 mol/L. Dissolve 12.1 g of Tris (Sigma Chemical Co., St. Louis, MO 63178) in 100 mL of distilled water and adjust the pH to 7.5 with HCL. Disodium ethylenediaminetetraacetate (EDTA), 50 mmol/L. Dissolve 1.68 g of EDTA (Sigma) in 100 mL of distilled water. Mercaptoethanol (Sigma). Reaction buffer. Mix 0.7 mL of mercaptoethanol, 50 mL of Tris buffer, and 20 mL of EDTA solution, and dilute to 1 L

with distilled water. This mixture is stable for at least three months at 4 #{176}C. Dihydrofolate buffer. Mix 2.5 mL of Tris buffer and 5.3 mL of mercaptoethanol, and dilute to 250 mL with distilled water. This reagent is stable for at least two months at 4 #{176}C. EMIT

Dec. 5, 1980.

of the drug can produce a potentially hazardous If those individuals who are at risk of developing

94303.

is a registered

trademark

of the Syva Co., Palo Alto, CA

Dihydrofolate reductase from bovine liver, 3.5 kU/L (Sigma). Dissolve the contents of 1 vial in 1.3 mL of distilled water. Store 50-L aliquots at -20 #{176}C. NADPH, tetrasodium salt (Sigma). Dissolve 5 mg of

NADPH

in 1 mL of distilled water. Prepare this solution just

before use. Dihydrofolic acid (Grade 5, 25-mg ampules from Sigma). Wash the contents of one ampule into a small beaker with 10 mL of a solution of 5 mmol/L of HCL and 14 mmolfL of mercaptoethanol. Stir the suspension for 25 mm. Dilute an aliquot of this suspension 10-fold with the dihydrofolate

buffer. Add 20 uL of this solution, in 1 mL of reaction buffer. The absorbance of this suspension should be about 0.075 ± 0.005 at a wavelength of 340 nm. If necessary, the concentration of this suspension is adjusted to bring the absorbance within the proper range. Store this suspension in 100-L aliquots at -20 #{176}C. This preparation is stable for at least two months. Dihydrofolic acid solution. Prepare just before use by thawing one tube of dihydrofolic acid and diluting 10-fold with dihydrofolate buffer. Stock met hotrexate standard, 5.5 mmol/L, from Lederle Laboratories, Pearl River, NY 10965. Dilute the stock standard with distilled water to give standards with concentrations of 0.137, 0.275, 0.55, 1.1, and 2.2 smol/L. Working reagent for the enzyme inhibition method. Prepare working reagent for the Multistat by mixing 6 mL of reaction buffer, 125 itL of NADPH solution, and 30 tL of dihydrofolate reductase reagent. Add the dihydrofolic acid solution as a second reagent. B. For the EMIT Method A reagent kit obtained from the Syva Co.’ contains Reagents A and B, buffer, and calibrators. 1. Reagent A contains antibody to methotrexate, glucose-6-phosphate, and NAD. Dissolve the contents in 3.0 mL

of distilled water. When reconstituted, reagent A contains the antibody-substrate in 55 mmol/L Tris buffer (pH 5.2). This reagent is stable for at least two weeks if stored at 4 #{176}C. 2. Reagent B contains methotrexate chemically coupled to the enzyme glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Dissolve the contents in 3.0 mL of distilled water. When reconstituted, reagent B contains the enzyme-drug in a solution of 55 mmol/L of Tris buffer at pH 8.0. This reagent is stable for at least two weeks if stored at 4 #{176}C. 3. Tris buffer, 825 mmol/L, pH 8.0. 4. Tris buffer, 55 mmol/L. Dilute the stock buffer with distilled water to obtain a 55 mmolfL solution. 5. Standards and calibrators. Each kit contains five standards and a zero calibrator. When these are dissolved in 1 mL of distilled water, the following concentrations are obtained: 0.0,0.2,0.5, 1.0, 1.5, and 2.0 tmol/L. The reconstituted standards are stable for at least two weeks if stored at 4 #{176}C. 6. Working reagent for the Multi.stat centrifugal analyzer. Working reagent B is prepared by mixing together 400 iL of reagent B and 500 iL of Tris buffer (reagent 3) and 4.1 mL of distilled water. Reagent A, the antibody-substrate solution, is added as the second reagent and is used undiluted. 7. Working reagent for the Cent rifiChem centrifugal analyzer. Enzyme-drug reagent: mix together 800 iL of reagent B and 10.4 mL of Tris buffer (reagent 4). Antibody-substrate reagent: mix together 800 1L of reagent A with 800 tL of Tris

buffer (reagent

4). This reagent is manually

Procedures inhibition

method

with

pipet

3 iL of sample

or standard

and 48 iL of dihy-

drofolic acid solution together with 39 L of water into the sample compartment of the rotor. Pipet 225 1L of working reagent together with 10 ILL of water into the reagent compartment of the rotor. Pipet water into the reference position. Measure initial absorbance at 60s and the final absorbance at 120 s after the samples and reagents are mixed together. Measure the absorbances at a wavelength of 340 nm and at a temperature of 30 #{176}C. Use the change in absorbance between 60 and 120 s to calculate the concentration of methotrexate in the sample. Prepare a standard curve by plotting the changes in absorbances of the standards vs the concentrations on log-linear graph paper. Determine the methotrexate concentration in the samples from the standard curve. EMIT procedure with the Multistat. The pipetting sequence is as follows: Pipet zero calibrator into positions 2 and 3, standards in positions 4 through 8, control in position 9 and samples in positions 10 through 20. With the diluter, pipet into the appropriate sample compartments of the rotor 3.0 ILL of sample,

standard

and zero calibrator,

18 ILL of reagent

A, and

69 ILL of water. Pipet 225 ILL of working reagent B, together with 10 LL of water, into the reagent compartment of the rotor. Pipet water into the reagent compartment of position 1, which is the reference position. Measure an initial absorb-

ance at 30 s and the final absorbance

at 150 a. Measure

the

absorbances at a temperature of 30#{176}C and at a wavelength of 340 nm. The absorbance differences (.A) for the standards and samples are obtained as follows: = [A8

-

Ar]iis

-

[A8

-

Ar]308

=

[A0

-

Ar]is

-

[A0

-

Ar13

=

.A8

-

where A8 is absorbance

of the standards or samples, A0 is the absorbance of the zero calibrators, A,. is the absorbance of the water in the reference position, M8 is the change in absorbance of the standards or samples, and .Ao is the change in

absorbance

of the zero calibrators.

in absorbance (.A) are used to calculate the concentrations of methotrexate by use of a log-log program supplied with the Multistat. After each determination a print-out is obtained that contains the LA values and the concentrations of the standards and samples. EMIT procedure on the Cent rifiChem. Manually pipet 50 ILL of antibody substrate reagent into the sample compartment of the disc. With the diluter, pipet 5 1iL of sample, standard, and zero calibrator and 45 zL of water into the appropriate sample compartments of the disc, and 350 ILL of enzyme-drug solution into the reagent compartments of the disc. Pipet water into the reagent compartment of position 1, which is the reference position. Measure an initial absorbance at 30s, and the fmal absorbance at an interval of 60 s. Measure the absorbances at a temperature of 30#{176}C and at a wavelength The differences

of 340 nm. Obtain the i.A values as indicated in the previous procedure. The CentrifiChem 400 analyzer prints only the A8 and A0 values; the &4 values must be manually calculated. Prepare a standard curve by plotting the A values for the standards on the paper supplied by the Syva Co. or on

log-log graph

paper

methotrexate

in the samples

and determine

the concentrations

of

from the curve.

pipetted.

C. For the Competitive Binding Assay A reagent kit was obtained from the New England Enzyme Center, Boston, MA 02111. The reagents were reconstituted according to manufacturer’s instructions.

Enzyme

diluter,

the Multistat.

With the

Results and Discussion Enzyme

Inhibition

Method

Linearity. The linearity of the method when aqueous standards and those prepared in a 60 g/L albumin solution are used extends from 0.2 to 2.2 ILmol/L. Samples containing

CLINICALCHEMISTRY,Vol.

27, No. 3, 1981

381

Table 1. Precision of the Enzyme Inhibition Assay with the Multistat Centrifugal Analyzer A

Mean, jnnol/L SD, Itmol/L CV, %

Within-run (n = 17) Samples B

0.31 0.025 8.06

0.65 0.04 6.12

Between-run (n

=

0.15

0.10 0.08

C

1.90 0.09 4.74

0.05 0.03

5)

0.02

Samples

Mean, ILmol/L SD, ILmol/L CV, %

D

E

0.49 0.04 8.80

1.43 0.09 6.29

0.2

0.5

METHOTREXATE

1.0 1.5 2.0 STANDARDS,

1emol/L methotrexate at concentrations exceeding 2.2 ILmol/L are diluted with a 60 g/L solution of albumin. Recovery. We determined the accuracy of the method by adding methotrexate at concentrations ranging from 0.27 to 1.65 ILmol/L to three samples containing the drug. Analytical recoveries ranged between 99 and 115% (average 105%). Interference. There was no interference from bilirubin in serum at concentrations up to 100 mg/L. A 20% positive interference was observed at a bilirubin concentration of 150 mgfL. Hemoglobin added to serum at concentrations up to 1.0 g/L did not affect the assay, but at a concentration of 2.0 g/L there was a 12% positive interference. To determine the effect of lipemia, we added methotrexate at identical concentrations to lipemic and clear samples. There was no interference for lipemic samples with an absorbance of less than 0.45, measured at a wavelength of 690 nm. In very lipemic samples, with absorbances at 690 nm greater than 0.80,

Table 2. Analytical Recovery of Methotrexate with the EMIT Assay Methotrexate In the samPle

Found Added

of added Recovery,

pmOI,L

Serum samples A

B C

D E

F

G Cerebrospinal fluid

382

0.20 0.20 0.20 0.50 0.50 0.50 0.50 0.60 0.60 0.60 0.60 0.60 1.00 1.00 1.00 1.50

0.40 0.80 1.60 0.90 0.20 0.40 0.90 0.40 0.80 0.30 0.50 1.10 1.00 0.50 0.60 0.20

0.40 0.80 1.40 0.90 0.20 0.40 0.80 0.40 0.70 0.30 0.50 1.00 0.95 0.45 0.60 0.20

100 100 88 100 100 100 89 100 88 100 100 91 95 90 100 100

0 0 0 0

0.30

0.30 0.60 1.10 1.90

100 100 100 106

CLINICAL CHEMISTRY,

0.60

1.10 1.80

Vol. 27, No. 3, 1981

Fig. 1. Optimum concentration of the antibody-substrate antibody-substrate concentration as indicated in the procedure; twice the antIbody-substrate concentration; - - - one-half the antibody-substratecon-

cenfratlon

methotrexate pected. Precision.

concentrations

were about

50% lower than

ex-

precision

of the method

was

The between-run

determined from measurements made on five days. Withinrun precision of the method was determined by 17 replicate analysis of three serum samples. The results are shown in Table 1. EMIT Method Standard curve and optimum conditons. With either centrifugal analyzer the standard curve extends from 0.2 to 2.0 ILmol/L. Samples with methotrexate concentrations >2.0 imol/L can be diluted with either a 60 g/L albumin solution or with the 55 mmol/L Tris buffer. To measure methotrexate concentrations between 0.04 and 0.1 ILmoi/L with the Multistat increase the amount of sample to 24 ILL and calculate the results, using the appropriate factor. We did not measure methotrexate concentrations lower than 0.2 ILmol/L with the CentrifiChem because of the limitations of the pipettor. The effect of serum on the assay was determined by measuring fifteen sera that were drug free, using 3 and 24 ILL of sample. The E.A values obtained with either sample size were four- to

0.15 0.10 0.08

0.05 0.03#{149} 0.02

-

,0

,

,

,

,

I

0.2

I

I

0.5

1.0 1.5 2.0

METHOTREXATE mol/L

STANDARDS,

Fig. 2. Optimum concentration of the enzyme-drug enzyme-drug concentration as Indicated in the procedure; - - - twice the enzyme-drug concentration; one#.alf the enzyme-drug concentration

Table 3. Effect of Lipemia on Results with the EMIT Assay Methotrexate mnt/L

Ag

0.101 0.210 0.313 0.525 1.566

concn,

Table 6. StabIlity of Methotrexate Baseline value, pmol/L

Sample A

0.95 1.00 0.95 0.90 0.95

In Serum

Methotrex ate concn, imo1/L days Temp. #{176}C 1 2 3 4

0.5

Sample B 0.8 Sample C 1.3

Table 4. Effect of Hemoglobin on Results with the

5

23 4 -20

0.5 0.6 0.4

0.5 0.6 0.6

0.5 0.6 0.4

0.5 0.6 0.5

0.6 0.6 0.6

23 4 -20

0.7 0.8 0.7

0.8 0.8 0.9

0.9 0.9 0.7

0.7 0.6 0.7

0.6 0.8 0.9

23 4 -20

1.5 1.5 1.4

1.5 1.5 1.2

1.3 1.4 1.1

1.3 1.2 1.2

1.3 1.4 1.5

EMIT Assay Hemoglobin concn,

Methotrexate

mg/L

ounce, jmoI/L

0 370 750 1500 3000 6000

1.40 1.40 1.40 1.20 1.00 0.60

Interference,

14 29 57

five-fold lower than the iA values of the 0.2 ILmol/L standard, indicating that there is no reaction from serum itself. Optimum conditions for the assay were determined with the Multistat centrifugal analyzer by changing the amount of the antibody-substrate and enzyme-drug used while keeping all other conditions constant. As shown in Figure 1, by increasing the antibody-substrate concentration, sensitivity is decreased because there is less free enzyme-drug

Table 5. Effect of Folic Acid, Folate Metabolites, and Drugs on Results with the EMIT Assay Apparent methotrexate In the sample

Compound added imOl/L

Folic acid 0

0.0

10

0.05

100 1000

0.15 1.2

0 100 1000

0.0 0.0 0.0

0 100 1000

0.0 0.0 0.05

0 100 1000

0.0 0.0

0

0.0 0.0 0.0

Leucovorin

present in solution. If the antibody-substrate concentration is decreased the &4 values for the 1 and 2 ILmol/L standards are nearly identical (Figure 1). This may be the result of (a) a lack of available binding sites on the antibody, (b) decreased concentration of substrate, or (c) a combination of these two conditions. As shown in Figure 2, by increasing the enzymedrug concentration, the sensitivity of the assay is increased. By decreasing the concentration of enzyme-drug, sensitivity is decreased because of the small amount of free enzyme-drug present in solution. The proportions of antibody-substrate and enzyme-drug used in our procedure are those suggested by Syva. Temperature. To determine the effect of temperature on the assay we measured methotrexate standards at 30 and 37 #{176}C with use of the Multistat. At 37 #{176}C, the #{163}4 values were

significantly increased, resulting in a parallel shift of the standard curve when compared with that obtained at 30 #{176}C. At a methotrexate concentration of 0.2 ILmol/L the #{163}4 values are increased by 50%, at 2.0 ILmol/L by 30%. Recovery. We assessed the accuracy of the assay by adding methotrexate to seven serum samples and a cerebrospinal fluid specimen. The results are shown in Table 2. For sera, analytical recoveries ranged between 88 and 100%, and averaged 96%. For the cerebrospinal fluid specimen, recoveries ranged between 100 and 106%, and averaged 102%. Interferences. All interference studies were determined with the Multistat centrifugal analyzer. Bilirubin in final concentrations ranging from 12.5 to 100 mgfL was added to a serum containing 1.3 ILmol of methotrexate per liter. No interference was observed. The effect of lipemia was determined by serially diluting a lipemic sample with a serum containing a known quantity of methotrexate. As shown in Table 3, there was no interfer-

ence from lipemia. Hemoglobin in final concentrations ranging from 370 to 6000 mg/L was added to serum containing 1.4 Iimol of methotrexate per liter. The results are shown in Table 4. There was

5-Methyftetrahydrofolate

Table 7. Precision of the EMIT Method Within-run (n

5-Fluorouradil

0.0

Mean, fimol/L SD, tmol/L CV, %

6-Mercaptopurine

0.97 0.06 6.18

1000

=

1.20 0.03 2.50 Between-run (n

100

13) CentrifiChem Sample C Sample 0

Multlstat Sample A Sample B

1.56 0.05 3.20 =

1.91 0.09 4.71

6)

Muftistat

Mean, ILmol/L SD, zmol/L CV, %

0.80 0.06 7.91 CLINICAL CHEMISTRY,

1.50 0.06 4.22 Vol.

27, No.

3, 1981

383

Table 8. ComparIson of 36 Serum Samples Assayed with Competitive Inhibition and EMIT Assays x-axls

Competitive protein-binding Competitive protein-binding Competitive protein-binding Enzyme inhibition EMIT assay, Multistat

no interference from hemoglobin in concentrations up to 750 mg/L. At hemoglobin concentrations exceeding 750 mg/L, the methotrexate value for a sample is depressed, but interference by hemoglobin will be negligible if the sample has to be diluted. For example, a specimen that is drawn immediately or at 24 h after methotrexate infusion must be diluted 20- to 400fold with a 60 g/L albumin solution, or with the 55 mmol/L Tris buffer, to bring the methotrexate concentration into the linear range of the assay. At these dilutions, there will be no interference from hemoglobin. Specificity. The cross reactivity of folic acid, folate derivatives, and drugs with the assay was investigated with the Multistat centrifugal analyzer. The results (Table 5) indicate that at a folic acid concentration of 100 tmol/L there is a small positive interference. However, the concentration of folic acid in serum is usually