Methotrexate Polyglutamates

Test ID: 


CPT code:



MTX pgs

Clinical Use:

Methotrexate is an antimetabolite that combines with dihydrofolate reductase and interferes with the synthesis of tetrahydrofolic acid necessary for purines, DNA synthesis. The half-life in plasma is only several hours, while the methotrexate polyglutamates persist in cells for weeks or months.

Test Information:

Low-dose methotrexate (MTX) is used as a disease modifying antirheumatic drug (DMARD) alone or in combination with other DMARDs or biologic modifiers for long-term treatment of rheumatoid arthritis (RA), psoriasis, and other autoimmune diseases. Methotrexate is a first-line drug in RA treatment. One challenge in the use of the drug is that about 30% of patients do not respond to treatment or experience adverse effects, such as gastrointestinal symptoms, malaise, and psychological complaints. Because arthritic diseases can be steadily progressive diseases, the time to arrive at an effective treatment is important for each patient, many of whom will have different medication blood levels on the same methotrexate dosage schedule. Consequently, a test indicating whether or not a patient has achieved an expected therapeutic level on a specific dosage protocol can be useful in ongoing patient management.

After oral administration and variable intestinal absorption, there is a short-lived plasma peak of MTX concentration that returns to the zero-time baseline by 24 hours post dose. MTX is taken into cells through a variety of folate receptors and is modified by intracellular addition of glutamate residues producing MTX polyglutamates (MTXpgs). The MTXpgs accumulate in the cytoplasm of red blood cells and, presumably as well, in the cytoplasm of immune effector lymphocytes. The time to reach plateau levels of intracellular methotrexate polyglutamates is on the order of several months, similar to the time to achieve clinical response.While the effects of high-dose MTX in the treatment of malignancy appears to be related to the direct effect on tetrahydrofolate reductase and DNA synthesis, the long-term immune modulation of low-dose therapy appears to be related to multiple metabolic effects of the MTXpgs suggesting that whole blood or red blood cell total level of the variously lengthened polyglutamates can be a surrogate for intralymphocyte measurement. Indeed, some recent publications have found that MTXpgs levels do correlate with therapeutic response, though not all studies have found a significant relationship to exist between MTXpgs levels and clinical response.Toxic effects seem to correlate poorly with MTXpgs levels, and toxic levels have not been established. Safety monitoring recommendations are published by the American College of Rheumatology.

The potential utility of MTXpgs is suggested to be as a therapeutic drug monitor related to patient compliance, individual pharmacodynamics, and clinical response. A recent publication6 found strong correlation of MTXpgs to response in a multisite clinical trial with nearly 400 patients. On stable dosage schedules, the six-month and nine-month levels were similar; however, three-month levels were somewhat lower. Nonresponders with thtal MTXpgs concentration below 74 nmol/L by three months of treatment and no toxicity had dosage increased before responding to MTX treatment.

• The minimal concentrations of MTX-polyglutamates associated with a significantly decreased disease activity score (DAS28) at three months were:

− 20 nmol/L MTX-PG3

− 50 nmol/L Total-PGS (MTX-PG 1−5)

• 85% of patients having a significant reduction (-2) grades of their DAS did so prior to reaching a

− Total MTX-PG (1−5) of 150 nmol/L

− MTX-PG2 of 22 nmol/L

− MTX-PG3 of 60 nmol/L

− 15% of eventual responders required higher levels.

Specimen Type:

Whole blood

Requested Volume: 

4 mL

Minimum Volume: 

1 mL

Container Type: 

Lavender-top (EDTA) tube or green-top (heparin) tube


Refrigerate immediately after collection.

Storage Instructions:

Refrigerate. Stable refrigerated for seven days.

Rejection Criteria

Hemolysis; frozen specimen; whole blood not submitted.

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