baled DHAP is not commercially avail- able, so it is generally prepared in a preincubation step by converting radiolabeled sn-glycerol. 3-phosphate. (3-GP) with.
Table 1. TypIcal Absorbance Differences for MIX and EDAM at 340nm t A/mm MIX or EDAM, nmol/L.
5.5 22 33
MIX 0.480 0.346 0.243
44
0.139
55
0.096
EDAM 0.518 0.377 0.296 0.229 0.179
activity of the enzyme [--12 kU/L; 1 U of DHFR will convert 7,8-dihydrofolate (F!!2) and NADPH to 5,6,7,8-tetrahydrofolate and NADP at 1.0 mmol/L per minute at pH 6.5 and 25#{176}C]; 1 mL of 0.18 mmol/L NADPH in 50 mmol/L K2HPO4-KH2PO4 buffer (pH 7.3); 19 mL of 50 mmol/L K2IWO4-KHPO4 buffer, pH 7.3, containing human albumin, 1 gfL and 3.6 mL of 1.0 mmol/L Tris-EIYFA buffer, pH 7.5. Reagent mixture B (start reagent B on the Cobas-Fara program) contained 0.15 mL of 10 moiJL FH2 (Sigma) solution in 6.0 mmol/L sodium acetate buffer, pH 4.7. F!!2 is photosensitive and must be stored in foil-covered tubes at -40#{176}C until used. To aid in the quantitative transfer of F!!2, rinse the storage tube with 0.4 mL of acetate buffer and add the rinse to the reaction mixture (9.5 mL of 50 mxnol/L K2HPO4-KH2PO4). Reagent mixtures A and B were freshly prepared before use. MTX standards were prepared by diluting 25 g/L solutions of MTX, EDAM, or TMTX with 50 mmol/L K2HPO4-KH2PO4 buffer to yield final concentrations of 0.11, 0.55, 1.1, 5.5, 22, and 33 nmol/L. The assay settings on the CobasFara were as follows: Measurement Reaction mode Calibration mode Reagent blank Wavelength Temp
ABS
Decimal position Sample vol
2 nmol/L 25 iL
Diluent Vol
25 L
Cleaner cycle
Off
Reagent vol A
100 &L
Unit
Time control Incubation Start reagent B Diluent vol Time control Wet cycle First reading
No. Interval
610
CLINICAL CHEMISTRY,
P-i-SRi-A Logit/log 4 No 340 nm
37#{176}C
H20
No 240 S
30 L 30 LL No
Off 108 10 30$
Interassay precision studies (n = 28) gave CVs of 6.4%, 6.3%, and 7.5% for MTX concentrations of 20, 75, and 600 nmol/L, respectively. The analytical recovery of MTX and EDAM added to a normal serum pool that contained no MTX or EDAM was acceptable over a concentration range of 0.5-55 nmol/L and averaged 99% and 96%, respectively (n = 2). The analogs of MTX (TMTX and EDAM) were analytically determined under the same assay conditions as for MTX. EDAM demonstrated 10-20% less inhibition of DHFR under the conditions of the MTX assay (Table 1). MTX and TMTX demonstrated equivalent DHFR inhibition. MTX and its analogs can be measured in cerebrospinal fluid, as well as in serum and urine specimens under the conditions indicated above. In conclusion,this application of a DHFR assay procedure allows the measurement of anti-folate analogs at an analytical detection limit of 0.5 nmol/L.
This
application allows for
rapid turnaround time, has acceptable accuracy and precision, permits the measurement of an entire family of drugs, and acts as a functional assay in that it reflects the in vivo effects of anti-folate activity. It shouldbe useful to the clinical chemistry laboratory charged with the routine monitoring of anti-folate chemotherapeutic treatment protocols.
References 1. Sirotnak FM, Donsbach RC, Dorick DM, Moccio DM. Tissue pharmacokinetics, inhibition of DNA synthesis, and tumor cell kifi after high-dose methotrexate in iramune tumor models. Cancer Res 1976; 36:4672-8.
2. Sirotnak FM, DeGraw JI, Moccio DM, Samuels LL, Goutas L,J. New folate analogs of the 1O-deazaaminopterin series. Basis for structural design and biochemical and pharmacologic properties. Cancer Chemother Pharmacol 1984;12:18-25. 3. Fanucchi MP, Walsh D, Fleisher M, et al. Phase land clinical pharmacology study of trimethotrexate administered weekly for three weeks. Cancer Res 1987;47:3303-8. 4. Yap HY, Blumenachein GR, Yap BS, et al. High-dose methotrexate for advanced breast cancer. Cancer Treat Rep 1979;63: 757-61.
5. Sternherg CN, Yagoda A, Bender NJ, et al. Phase I/il trial of intravesical methotrexate for superficial bladder tumors. Cancer Chemother Pharmacol 1986;18:265-9. 6. Bleyer WA, Drake JC, Chabner BA. Pharmacokinetics and neurotoxicity of intrathecal methotrexate therapy. N Engl J Med 1973;289:770-3. 7. Bleyer WA. The clinical pharmacology of methotrexate. Cancer 1978;46:36-51. 8. Shum KY, Kris MG, Gralla RJ, Burke MT, Marks LD, Heelan RT. Phase II study
Vol.38, No.4, 1992
of 10-ethyl-10-deazaaminopterin in patients with stage Ill and N non-small cell lung cancer. J Clin Oncol 1988;6:446-50. 9. Schwartz MK, Mehta B, Fleisher M. The application of centrifugal analysis to methotrexate determination compared to existing methodology [Abstract]. Clin Chem 1980;26:969. Martin
Fleisher
Morton K. Schwartz Dept. of Clin. Chem. Memorial Sloan-Kettering Center 1275 York Ave. New York, NY 10021
Cancer
Simplified Assay of AcyI-C0A:dlhydroxyacetonephosphate Acyltransferase To the Editor: The differential diagnosis of peroxisomal disorders requires several biochemical investigations (1), one of which is the assay of acyl-CoA:dthy-
droxyacetone-phosphate acyltransferase (DKAPAT, glycerone-phosphate acyltransferase, EC 2.3.1.42). This enzyme, located in the membrane of peroxisomes and involved in plasmalogen synthesis (2), is deficient in disorders that are characterized by a general dysfunction of peroxisomes or a deficiency of multiple peroxisomal enzymes, e.g., Zellweger syndrome and neonatal adrenoleukodystrophy (1,3). DHAPAT is routinely measured in fibroblasts by monitoring the incorporation of radiolabeled dihydroxyacetone phosphate (DRAP) into chloroform! methanol-extractable lipids. Radiolabaled DHAP is not commercially available, so it is generally prepared in a preincubation step by converting radiolabeled sn-glycerol 3-phosphate (3-GP) with 3-GP dehydrogenase (NAD) (EC 1.1.1.8) and an NADregenerating system (3, 4). Here we describe an alternative method for generating DHAP that greatly simplifies the assay of D}LAPAT. Radiolabeled DRAP is generated at room temperature in a reaction mixture containing, per liter, 114 mmol of 4-morpholinepropanesulfonic acid (MoPs) (pH 7.5), 9.1 mmol of NaF, 9.1 mmol of MgCl2, 11.4 g of fatty acidfree bovine serum albumin, 0.57 mmol of palmitoyl-CoA, 0.57 mmol of [IJ-14C]3-GP (specific activity 2 Cu mol), 5 mmol of N-ethylmaleimide (NEM), 1 g of taurocholate, and 4 kU of catalase(EC 1.11.1.6; Boehringer Mannheim, Mannheim, F.R.G.). Preincubation is started by adding 1 U of
3-GP oxidase (EC 1.1.3.21; Boehringer Mannheim) per milliliter of reaction mixture. Within 20 mm, [U-’4C]3-GP is quantitatively converted to [U-14C]DHAP, which remains stable for at least 1 h. During this preincubation, which routinely lasts 20 min, 175 L of the reaction mixture is pipetted into the
incubation vials. Incubation is started by adding 25 jL of fibroblast suspension (2 mg of protein per milliliter, suspended in, per liter, 50 mmol of NaC1, 5 mmol of NEM, 1 g of taurocholate, and 5 mmol of MOPS, pH 7.5) and transferring the stoppered vials to a shaking water bath at 37#{176}C. After 20 mm the reaction is stopped by adding 750 L of chloroform/methanol (1/2, by vol) and the samples are processed as described by Schutgens et al.
oxisomes isolated from cultured skin flbroblasts. Arch Biochem Biophys 1991;286: 277-83.
Hedwig K. Stals Peter
E. Dedercq’
Lab. voor Kim. Chemie Inst. voor Farmaceut. Wetenschappen E. Van Evenstraat 4 B -3000 Leuven, Belgium
1To whom correspondence should be addressed.
Metabolltes of Chiorpromazlne and Bromphenlramlne May Cause False-PosItIve Urine Amphetamine Results with Monoclonal EMIT#{174} d.a.u.T” Immunoassay
(3).
3-GP oxidase ible
oxidation
catalyzes
the irreversNo
of 3-GP to DHAP.
cofactors are needed other than molecoxygen, which is reduced to H202. H202 is converted to H20 by catalase. The oxidation of 3-GP is rapid and quantitative, even at low concentrations. Moreover, DHAP can be generated in the reaction mixture used for the subsequent assay of DHAPAT. The ular
medium we use includes NEM, which inhibits microsomal DHAPAT and makes the assay specific for the peroxisomal
activity (2), and taurocholate, causes complete disruption of the fibroblasts. Neither reagent affects the generation of DHAP. Catalase and 3-GP oxidase do not interfere with the DHAPAT assay and need not be inactivated after the preincubation. which
We thank B. Des for skillful technical support. This work was supported by grants 3.0060.89 and 9.0021.89 of the Fonds voor Geneeskundig Wetenschappelijk Onderzoek.
References 1. Wanders RJ, Van Roermund CW, Schutgens RB, et al. The inborn errors of peroxisomal /3-oxidation: a review. J Inherited Metab Dis 1990;13:4-36. 2. Declercq FE, Haagsman HP, Van Veldhoven P, Debeer U, Van Golde LM, Mannaerts GP. Rat liver dihydroxyacetonephosphate acyltransferases and their contribution to glycerolipid synthesis. J Biol Chem 1984;259:9064-75. 3. Schutgens RB, Romeyn GJ, Wanders RJ, van den Bosch H, Schrakamp G, Heymans HS. Deficiency of acyl-CoA:dihydroxyacetone phosphate acyltransferase in patients with Zeliweger (cerebro-hepatorenal) syndrome. Biochem Biophys Res Commun 1984;120:179-84. 4. Singh I, Lazo 0, Contreras M, Stanley W, Hashimoto T. Rhizomelic chondrodysplasia punctata: biochemical studies of per-
To the Editor:
The new monoclonal EMIT#{174} d.a.u.tM Amphetamine/Methamphetamine Immunoassay (Syva Co., Palo Alto, CA) specific for the detection of amphetamines was recently introduced (1). Interference from phenylisopropylamine derivatives
(e.g., phenylpropanolamine,
ephedrine, pseudoephedrine,
and phenylephrine) with the polyclona.l EMIT d.a.u. and with other immunoassays used for drug screening is well known (1). The monoclonal immunoassay, however, is reportedly highly stereospecific for amphetamines and does not cross-react with phenylpropanolamine derivatives (1). We (2) have used the polyclonal d.a.u. immunoassay for drug screening in blood and urine for several years. We reanalyze urine samEMIT
ples positive for amphetamine, using the EMIT immunoassay confirmatory test (EMIT d.a.u. Amphetamine Confirmation Kit; Syva) and adding sodium hydroxide and periodic acid to the urine sample to exclude phenylpropanolamine and other common derivatives. We further confirm positive amphetamine results from urine and blood with specific chromatographic methods (3). We recently evaluated the monoclenal EMIT d.a.u. immunoassay. We demonstrated interference with metabolites from chlorpromazine and possible interference from metabolites of the antthistamine bromphemramine. Product information from Syva indicated that therapeutic doses of chiorpromazine might produce positive results. Possible cross-reactivity caused by chlorpromazine metabolites was mentioned (1) but was not verified by specific chromatographic methods or sub-
stantiatedwith information about drug use. We therefore report two cases of false-positive amphetamine results obtained after screening with the monoclonal EMIT d.a.u. assay. Case 1: Three urine samples collected at different times from a patient being treated in a psychiatric department were screened for drugs in a local hospital laboratory. All samples were positive for amphetamine, but the patient denied taking this drug. Because the doctor relied on the laboratory results, he lost confidencein his patient, who was then discharged from treatment. The monoclonal EMIT d.a.u. assay was used for screening without confirmatory analyses, and the staff had no information on possible interference with other drugs or metabolites. The samples were sent to our institute for confirmatory analyses and were screened with monoclonal and polyclonal EMIT d.a.u. assays, followed by gas-chromatographic (GC) and thinlayer-chromatographic(TLC) analyses (3). Alkalinized urine extracts were also analyzed for antidepresaive and neuroleptic drugs (GC; SP-2250 column). Amphetamine was not detected by the polyclonal immunoassay, GC, or TLC but was detected by the monoclenal immunoassay (Table 1). Met.abolites of chlorpromazine were detected by GC and TLC. Only minor amounts of chlorpromazine were detected. The GC pattern was similar to patterns for samples from other patients taking chiorpromazine. The doctor said that the patient had been taking chlorpromazine medication for a long time. To test for interference, we added chlorpromazine to drug-free urine samplesto give concentrations of 100, 50, and 15 mg/L. All samples were negative by the monoclona.l ixnmunoassay, indicating that metabolites might be causing interference. Further evaluation of the monoclenal EMIT assay showed an additional 10 false-positive results for amphetamines in urine, all from different patients taking chlorpromazine. Amphetamine was not detected in any of these samples by the polyclonai EMIT d.a.u.
or by chromatographic
methods.
Case 2: A urine
sample from an employee with former drug problems was positive for amphetamine when screened in a local hospital laboratory by the monoclonal EMIT d.a.u. immunoassay. The employee denied use of
amphetamine, but was temporarily dismissed from his job. During the sampling period, he had been treated with a /3-receptor blocker and with a drug containing phenyipropanolamine and brompheniramine. The sam-
CLINICALCHEMISTRY, Vol. 38, No. 4, 1992 611
