Plasma methotrexate as determined by liquid chromatography, enzyme-inhibition assay, and radioimmunoassay after high-dose infusion.

1980 ◽  
Vol 26 (6) ◽  
pp. 734-737 ◽  
Author(s):  
S K Howell ◽  
Y M Wang ◽  
R Hosoya ◽  
W W Sutow

Abstract Three techniques for measuring methotrexate show various cross reactivities with methotrexate-related compounds: “high-pressure” liquid chromatography, by principle, is virtually specific for methotrexate; the enzyme-inhibition assay quantitates methotrexate, methotrexate diglutamate, and methotrexate triglutamate equally well, but has a 10% cross reactivity with 4-amino-4-deoxy-N10-methylpteroic acid and 1% with 7-hydroxymethotrexate; radioimmunoassay shows an equal cross reactivity with methotrexate, 4-amino-4-deoxy-N10-methylpteroic acid, methotrexate diglutamate and triglutamate, and a 5 to 10% cross reactivity with 7-hydroxymethotrexate. Radioimmunoassay almost always yielded the highest values for methotrexate, followed by enzyme-inhibition assay then liquid chromatography. The presence of two methotrexate-related compounds, 7-hydroxymethotrexate and 4-amino-4-deoxy-N10-methylpteroic acid, was confirmed in human urine samples and quantitated in patients’ plasma by liquid chromatography, the respective maximum plasma concentrations being 250 and 16 mumol/L. Materials cross reacting with methotrexate in radioimmunoassay of chromatographic fractions from plasma were also noted in fractions corresponding to methotrexate diglutamate and triglutamate peaks, in quantities estimated to be 47 and 30 nmol/L methotrexate equivalents, respectively. 7-Hydroxymethotrexate is eliminated more slowly than methotrexate and its production increases with dosages of methotrexate.

1980 ◽  
Vol 26 (6) ◽  
pp. 734-737 ◽  
Author(s):  
S K Howell ◽  
Y M Wang ◽  
R Hosoya ◽  
W W Sutow

Abstract Three techniques for measuring methotrexate show various cross reactivities with methotrexate-related compounds: “high-pressure” liquid chromatography, by principle, is virtually specific for methotrexate; the enzyme-inhibition assay quantitates methotrexate, methotrexate diglutamate, and methotrexate triglutamate equally well, but has a 10% cross reactivity with 4-amino-4-deoxy-N10-methylpteroic acid and 1% with 7-hydroxymethotrexate; radioimmunoassay shows an equal cross reactivity with methotrexate, 4-amino-4-deoxy-N10-methylpteroic acid, methotrexate diglutamate and triglutamate, and a 5 to 10% cross reactivity with 7-hydroxymethotrexate. Radioimmunoassay almost always yielded the highest values for methotrexate, followed by enzyme-inhibition assay then liquid chromatography. The presence of two methotrexate-related compounds, 7-hydroxymethotrexate and 4-amino-4-deoxy-N10-methylpteroic acid, was confirmed in human urine samples and quantitated in patients’ plasma by liquid chromatography, the respective maximum plasma concentrations being 250 and 16 mumol/L. Materials cross reacting with methotrexate in radioimmunoassay of chromatographic fractions from plasma were also noted in fractions corresponding to methotrexate diglutamate and triglutamate peaks, in quantities estimated to be 47 and 30 nmol/L methotrexate equivalents, respectively. 7-Hydroxymethotrexate is eliminated more slowly than methotrexate and its production increases with dosages of methotrexate.


1981 ◽  
Vol 27 (3) ◽  
pp. 380-384 ◽  
Author(s):  
M A Pesce ◽  
S H Bodourian

Abstract Methotrexate was determined by the homogeneous enzyme immunoassay (EMIT) 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 mumol/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 mumol/L) was added to a serum that did not contain methotrexate, a response equivalent to 0.15 mumol/L was obtained. Methotrexate is stable for five days in serum stored at 23, 4, or -20 degrees 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%. Methotrexate concentrations in the serum of children receiving high-dose therapy were compared by three methods: competitive protein binding, EMIT, and enzyme inhibition assays. The correlation coefficients averaged 0.95.


1999 ◽  
Vol 45 (2) ◽  
pp. 223-228 ◽  
Author(s):  
Brigitte C Widemann ◽  
Frank M Balis ◽  
Peter C Adamson

Abstract Microplate reader assays offer several advantages over conventional spectrophotometric assays. We adapted the dihydrofolate reductase (DHFR) enzyme inhibition assay for use in a 96-well microplate reader to measure plasma methotrexate (MTX) concentrations. The assay is linear from 0.01 to 0.1 μmol/L. The within-run CVs at 0.03 μmol/L and 0.08 μmol/L MTX were 4.0% and 2.7%, respectively, and the interday (total) CVs were 7.6% and 1.8%. Cross-reactivity with the inactive MTX metabolite 2,4-diamino-N10-methylpteroic acid (DAMPA) was 3.9%, significantly less than that described with commercial immunoassays; with 7-hydroxymethotrexate cross-reactivity was 1.7%. In addition to sensitivity and specificity, the advantages of this assay are small sample volumes, simultaneous analysis of multiple samples, and rapid turnaround. Because of its greater specificity, the DHFR enzyme inhibition assay may be useful when DAMPA is present in plasma samples and HPLC is not available.


1994 ◽  
Vol 12 (9) ◽  
pp. 1902-1909 ◽  
Author(s):  
D R Budman ◽  
L N Igwemezie ◽  
S Kaul ◽  
J Behr ◽  
S Lichtman ◽  
...  

PURPOSE To determine the toxicities, maximum-tolerated dose (MTD), and pharmacology of etoposide phosphate, a water-soluble etoposide derivative, administered as a 5-minute intravenous infusion on a schedule of days 1, 3, and 5 repeated every 21 days. PATIENTS AND METHODS Thirty-six solid tumor patients with a mean age of 63 years, performance status of 0 to 1, WBC count > or = 4,000/microL, and platelet count > or = 100,000/microL, with normal hepatic and renal function were studied. Doses evaluated in etoposide equivalents were 50, 75, 100, 125, 150, 175, and 200 mg/m2/d. Etoposide in plasma and urine and etoposide phosphate in plasma were measured by high-performance liquid chromatography (HPLC). Eleven of 36 patients were treated with concentrated etoposide phosphate at 150 mg/m2/d. RESULTS Grade I/II nausea, vomiting, alopecia, and fatigue were common. Leukopenia (mainly neutropenia) occurred at doses greater than 75 mg/m2, with the nadir occurring between days 15 and 19 posttreatment. All effects were reversible. Hypotension, bronchospasm, and allergic reactions were not observed in the first 25 patients. The MTD due to leukopenia was determined to be between 175 and 200 mg/m2/d. In 11 patients treated with concentrated etoposide phosphate, no local phlebitis was noted, but two patients did develop allergic phenomena. The conversion of etoposide phosphate to etoposide was not saturated in the dosages studied. Etoposide phosphate had peak plasma concentrations at 5 minutes, with a terminal half-life (t1/2) of 7 minutes. Etoposide reached peak concentrations at 7 to 8 minutes, with a t1/2 of 6 to 9 hours. Both etoposide phosphate and etoposide demonstrated dose-related linear increases in maximum plasma concentration (Cmax) and area under the curve (AUC). CONCLUSION Etoposide phosphate displays excellent patient tolerance in conventional dosages when administered as a 5-minute intravenous bolus. The suggested phase II dose is 150 mg/m2 on days 1, 3, and 5. The ability to administer etoposide phosphate as a concentrated, rapid infusion may prove of value both in the outpatient clinic and in high-dose regimens.


2008 ◽  
Vol 76 (10) ◽  
pp. 4546-4553 ◽  
Author(s):  
Maija Toropainen ◽  
Anna Raitolehto ◽  
Isabelle Henckaerts ◽  
Dominique Wauters ◽  
Jan Poolman ◽  
...  

ABSTRACT Haemophilus influenzae outer membrane protein D (PD) is a glycerophosphodiester phosphodiesterase (GlpQ) activity-possessing virulence factor and a promising vaccine antigen, providing 35.3% efficacy against acute otitis media caused by nontypeable H. influenzae (NTHI) when it was used as a carrier protein in a novel pneumococcal PD conjugate (Pnc-PD) vaccine. To study if PD-induced protection against NTHI could be due to antibodies that inhibit or neutralize its enzymatic activity, a GlpQ enzyme inhibition assay was developed, and serum samples collected from Finnish infants before and after Pnc-PD vaccination were analyzed for enzyme inhibition and anti-PD immunoglobulin G (IgG) antibody concentration. Before vaccination at age 2 months, the majority (84%) of infants (n = 69) had no detectable anti-PD IgG antibodies, and all were enzyme inhibition assay negative (inhibition index, <20). At age 13 to 16 months, all infants receiving three or four doses of Pnc-PD had detectable anti-PD IgG antibodies and 36% (8/22 infants) of the infants receiving three doses and 26% (6/23 infants) of the infants receiving four doses of Pnc-PD were inhibition assay positive (inhibition index, ≥20). No significant rise in anti-PD IgG antibodies or enzyme inhibition among control vaccinees (n = 24) receiving three doses of hepatitis B vaccine was detected. A modest correlation (r s , ∼0.66) between anti-PD IgG concentration and enzyme inhibition was detected; however, their kinetics were clearly different. These data suggest that measurement of antibody responses that inhibit PD's enzymatic activity could be a useful tool for assessing Pnc-PD vaccine-induced protective immunity against NTHI.


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