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Methotrexate Polyglutamates

CPT: 80204
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  • MTX pgs

Test Includes

Serial monitoring

Special Instructions

Collection must be performed at least 36 hours after last methotrexate dose.

Expected Turnaround Time

7 - 15 days

Related Documents

For more information, please view the literature below.

Rheumatoid Arthritis

    Specimen Requirements


    Whole blood


    4 mL

    Minimum Volume

    1 mL (Note: This volume does not allow for repeat testing.)


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


    Refrigerate immediately after collection.

    Storage Instructions

    Refrigerate. Stable refrigerated for seven days.

    Causes for Rejection

    Hemolysis; frozen specimen; whole blood not submitted

    Test Details


    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.


    This test was developed and its performance characteristics determined by Labcorp. It has not been cleared or approved by the Food and Drug Administration.


    High-pressure liquid chromatography/tandem mass spectrometry (HPLC/MS-MS)

    Additional 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.1 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.1 MTX is taken into cells through a variety of folate receptors2,3 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.4 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.1,5 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,1,3,5 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 publications3-8 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.1,9 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.10,11

    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.


    1. Danila MI, Hughes LB, Brown EE, et al. Measurement of erythrocyte methotrexate polyglutamate levels: Ready for clinical use in rheumatoid arthritis? Curr Rheumatol Rep. 2010 Oct; 12(5):342-347. 20665136
    2. Hendel J, Nyfors A. Pharmakokinetics of methotrexate in erythrocytes in psoriasis. Eur J Clin Pharmacol. 1984; 27(5):607-610. 6519167
    3. Kremer JK. Toward a better understanding of methotrexate. Arthritis Rheum. 2004 May; 50(5):1370-1382. 15146406
    4. Fairbanks LD, Rückemann K, Qiu Y, et al. Methotrexate inhibits the first committed step of purine biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for efficacy in rheumatoid arthritis? Biochem J. 1999 Aug 15; 342(Pt 1):143-152. 22309614
    5. Angelis-Stoforidis P, Vajda FJ, Christophidis N. Methotrexate polyglutamate levels in circulating erythrocytes and polymorphs correlate with clinical efficacy in rheumatoid arthritis. Clin Exp Rheumatol. 1999 May-Jun; 17(3):313-320. 10410264
    6. de Rotte MC, den Boer E, de Jong PH, et al. Methotrexate polyglutamates in erythrocytes are associated with lower disease activity in patients with rheumatoid arthritis. Ann Rheum Dis. 2015 Feb; 74(2):408-414. 24297383
    7. Woolf RT, West SL, Arenas-Hernández M, et al. Methotrexate polyglutamates as a marker of patient compliance and clinical response in psoriasis: a single-centre prospective study. Br J Dermatol. 2012 Jul 1; 67(1):165-173. 22309614
    8. Hobl EL, Jilma B, Erlacher L, et al. A short-chain methotrexate polyglutamate as outcome parameter in rheumatoid arthritis patients receiving methotrexate. Clin Exp Rheumatol. 2012 Mar-Apr; 30(2):156-163. 22152098
    9. Stamp LK, O'Donnell JL, Chapman PT, et al. Methotrexate polyglutamate concentrations are not associated with disease control in rheumatoid arthritis patients receiving long-term methotrexate therapy. Arthritis Rheum. 2010 Feb; 62(2):359-368. 20112376
    10. Singh Ja, Furst DE, Bharat A, et al. 2012 update of the 2008 American College of Rheumatology recommendations for the use of disease-modifying antirheumatic drugs and biologic agents in the treatment of rheumatoid arthritis. Arthritis Care Res. 2012 May; 64(5):625-639. 22473917
    11. Saag KG, Teng GG, Patkar NM, et al. American College of Rheumatology 2008 recommendations for the use of nonbiologic and biologic disease-modifying antirheumatic drugs in rheumatoid arthritis. Arthritis Rheum. 2008 Jun 15; 59(6):762-784. 18512708


    Hornung N, Ellingsen T, Attermann J, Stengaard-Pedersen K, Poulsen JH. Patients with rheumatoid arthritis treated with methotrexate (MTX): Concentrations of steady-state erythrocyte MTX correlate to plasma concentrations and clinical efficacy. J Rheumatol. 2008 Sep; 35(9):1709-1715. 18634162
    National Institute of Arthritis and Musculoskeletal and Skin Diseases. Handout on Health: Rheumatoid Arthritis. Bethesda, Md: National Institutes of Health, NIAMS; 2014.

    LOINC® Map

    Order Code Order Code Name Order Loinc Result Code Result Code Name UofM Result LOINC
    504104 Methotrexate Polyglutamates 81630-6 504048 Methotrexate PG Total nmol/L RBC 76351-6
    504104 Methotrexate Polyglutamates 81630-6 504049 Methotrexate PG1 (parent drug) nmol/L RBC 81628-0
    504104 Methotrexate Polyglutamates 81630-6 504051 Methotrexate PG2 nmol/L RBC 81627-2
    504104 Methotrexate Polyglutamates 81630-6 504052 Methotrexate PG3 nmol/L RBC 81626-4
    504104 Methotrexate Polyglutamates 81630-6 504053 Methotrexate PG4 nmol/L RBC 81625-6
    504104 Methotrexate Polyglutamates 81630-6 504054 Methotrexate PG5 nmol/L RBC 81624-9
    504104 Methotrexate Polyglutamates 81630-6 504055 Interpretation 81625-6
    Reflex Table for Interpretation
    Order Code Order Name Result Code Result Name UofM Result LOINC
    Reflex 1 000000 Serial Monitoring 000000 Serial Monitoring n/a

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