Leon van Haandel

The effect of genotype on methotrexate polyglutamate variability in juvenile idiopathic arthritis and association with drug response

OBJECTIVE The response to and toxicity of methotrexate (MTX) are unpredictable in patients with juvenile idiopathic arthritis (JIA). Intracellular polyglutamation of MTX, assessed by measuring concentrations of MTX polyglutamates (MTXGlu), has been demonstrated to be a promising predictor of drug response. Therefore, this study was aimed at investigating the genetic predictors of MTXGlu variability and associations between MTXGlu and drug response in JIA. METHODS The study was designed as a single-center cross-sectional analysis of patients with JIA who were receiving stable doses of MTX at a tertiary care childrens hospital. After informed consent was obtained from the 104 patients with JIA, blood was withdrawn during routine MTX-screening laboratory testing. Clinical data were collected by chart review. Genotyping for 34 single-nucleotide polymorphisms (SNPs) in 18 genes within the MTX metabolic pathway was performed. An ion-pair chromatographic procedure with mass spectrometric detection was used to measure MTXGlu1-7. RESULTS Analysis and genotyping of MTXGlu was completed in the 104 patients. K-means clustering resulted in 3 distinct patterns of MTX polyglutamation. Cluster 1 had low red blood cell (RBC) MTXGlu concentrations, cluster 2 had moderately high RBC MTXGlu1+2 concentrations, and cluster 3 had high concentrations of MTXGlu, specifically MTXGlu3-5. SNPs in the purine and pyrimidine synthesis pathways, as well as the adenosine pathway, were significantly associated with cluster subtype. The cluster with high concentrations of MTXGlu3-5 was associated with elevated liver enzyme levels on liver function tests (LFTs), and there were higher concentrations of MTXGlu3-5 in children who reported gastrointestinal side effects and had abnormal findings on LFTs. No association was noted between MTXGlu and active arthritis. CONCLUSION MTXGlu remains a potentially useful tool for determining outcomes in patients with JIA being treated with MTX. The genetic predictors of MTXGlu variability may also contribute to a better understanding of the intracellular biotransformation of MTX in these patients.

Analysis of intracellular methotrexate polyglutamates in patients with juvenile idiopathic arthritis: Effect of route of administration on variability in intracellular methotrexate polyglutamate concentrations

OBJECTIVE Intracellular methotrexate (MTX) polyglutamates (MTXGlu) have been shown to be potentially useful biomarkers of clinical response in adult patients with rheumatoid arthritis. The present study was undertaken to measure intracellular MTXGlu concentrations in a cohort of patients with juvenile idiopathic arthritis (JIA) to determine the predictors of MTXGlu variability in these patients. METHODS Blood samples were obtained from patients with JIA who were being treated with a stable dose of MTX for >or=3 months. Clinical data were collected by chart review. Concentrations of MTXGlu(1-7) in red blood cell lysates were quantitated using an innovative ion-pairing chromatography procedure, with detection by mass spectrometry. RESULTS Patients with JIA from a single center (n = 99; mean +/- SD age 117.8 +/- 56.5 months, 69 female) were included in the analysis. The mean +/- SD dose of MTX was 0.51 +/- 0.25 mg/kg per week, with a median treatment duration of 18 months (interquartile range 3-156 months). MTX was administered subcutaneously in 66 patients (67%). Fifty-six patients (57%) had active arthritis at the time of the clinic visit. Total intracellular MTXGlu (MTXGlu(TOT)) concentrations varied 40-fold, with a mean +/- SD total concentration of 85.8 +/- 48.4 nmoles/liter. Concentrations of each MTXGlu subtype (MTXGlu(1-7)) were measured individually and as a percentage of MTXGlu(TOT) in each patient. MTXGlu(3) was the most prominent subtype identified, comprising 42% of MTXGlu(TOT), and the interindividual variability in the concentration of MTXGlu(3) was the most highly correlated with that of MTXGlu(TOT) (r = 0.96). The route of MTX administration was significantly associated with MTXGlu(1-5) subtypes; higher concentrations of MTXGlu(1 + 2) were observed in patients receiving oral doses of MTX, whereas higher concentrations of MTXGlu(3-5) were observed in patients receiving subcutaneous doses of MTX (P < 0.0001). CONCLUSION In this cohort of patients with JIA, the MTXGlu(TOT) concentration varied 40-fold. Individual MTXGlu metabolites (MTXGlu(1-7)), which have, until now, not been previously reported in patients with JIA, were detected. The route of MTX administration contributed to the variability in concentrations of MTXGlu(1-5).

A novel high‐performance liquid chromatography/mass spectrometry method for improved selective and sensitive measurement of methotrexate polyglutamation status in human red blood cells

The folate antagonist methotrexate is commonly used in low dose for treatment of rheumatoid arthritis and juvenile idiopathic arthritis. Therapeutic effects are attributed to intracellular levels of various methotrexate polyglutamates. The present methodology, combining a simple preparation step with ion-pairing reversed-phase liquid chromatography and electrospray ionization mass spectrometry, is suitable for the measurement of methotrexate and its polyglutamates(2-7), in human red blood cells. Sample preparation consists of perchloric acid protein precipitation followed by solid-phase extraction. Baseline separation of all analytes was achieved within 10 min using a Phenomenex Synergy C18 column together with a gradient solvent program obtained from blending acetonitrile with pH 7.5, 5 mM aqueous dimethylhexylamine. Seven methotrexate polyglutamates were detected using multiple reaction monitoring, with the mass spectrometer operating in positive ion mode. Using 20 microL injection volumes, limits of detection were 2.5 nM for individual methotrexate polyglutamates, while large volume (100 microL) injections led to detection limits of 0.5 nM and linear calibration from 0.5 to 100 nM for individual analytes. Finally, the presented methodology was applied for the analysis of methotrexate and its polyglutamates in red blood cells obtained from patients being treated for juvenile idiopathic arthritis with methotrexate. Significantly, the methodology proved suitable for determination of long-chain methotrexate polyglutamates(5-7) and further, appears to be superior with respect to sensitivity, selectivity and speed as compared to all previously described approaches.

Red blood cell folate concentrations and polyglutamate distribution in juvenile arthritis: predictors of folate variability.

Objective Methotrexate (MTX) has several enzymatic targets in the folate pathway. To better understand the variability in response to MTX, we characterized the interindividual variability of intracellular folate pools in children with juvenile arthritis (JA) and determined clinical and genetic contributors to this variability. Study design This exploratory single-center cross-sectional study evaluated 93 patients with JA not currently receiving MTX. Whole blood, plasma, and erythrocyte folate concentrations were determined after deconjugation and analyzed through reversed-phase separation and stable isotope dilution tandem mass spectrometry. Folate polyglutamates were measured in red blood cell lysates using an ion-pair reversed phase chromatography tandem mass spectrometry method. Results Intracellular concentrations of 5-methyl-tetrahydrofolate (5-CH3-THF) and 5,10-methenyl-tetrahydrofolate varied approximately 20-fold and 80-fold, respectively. The polyglutamated forms of 5-CH3-THF as a percentage of total 5-CH3-THF (5-CH3-THFGlun) were also measured. Hierarchical clustering of 5-CH3-THFGlun revealed two groups, each with two distinct clusters. There was an inverse relationship between 5-CH3-THFGlun chain length and plasma 5-CH3-THF concentrations. A subgroup of patients with a historical intolerance to MTX had significantly lower cellular folate concentrations (P<0.0001). In univariate analyses, clinical variables including sex, age, and folate supplementation in addition to variations in MTHFR, MTR, and SLC25A32 were associated with differential intracellular folate redox concentrations. Multivariate analysis further supported the association of single nucleotide polymorphisms in SLC25A32, MTHFR, and MTR with variability in intracellular 5-CH3-THF and 5,10-methenyl-tetrahydrofolate concentrations, respectively. Conclusion Measurement of intracellular folate isoforms may contribute toward a better understanding of individual MTX effects in JA. Clinical variables in addition to genotypic differences beyond MTHFR may additionally explain differential intracellular folate concentrations and variable responses to MTX.

Measurement of methotrexate polyglutamates in human erythrocytes by ion-pair UPLC-MS/MS.

BACKGROUND Low-dose methotrexate is used for the treatment of rheumatoid arthritis and juvenile idiopathic arthritis, but its effectiveness greatly varies between individuals. Therapeutic drug monitoring of intracellular methotrexate metabolites, the γ-polyglutamates (MTXGlu(n)), in human erythrocytes has shown promise in providing a basis for individualization of therapy. RESULTS This work presents expedient methodology for the analysis of MTXGlu(1-7) in human erythrocytes by ion-pair UPLC with detection by tandem MS (UPLC-ESI-MS/MS). The use of N,N-dimethylheptylamine as an ion-pair agent was found to be favorable over others. Thermal extraction of erythrocyte lysates provides a simple one-step extraction procedure. The entire chromatographic run time is 6 min and the assay was validated within the therapeutic range of these metabolites CONCLUSION The developed sample preparation procedure in combination with ion-pair UPLC-ESI-MS/MS analysis allowed for expedient quantitation of MTXGlu(1-7) in human erythrocytes. The rapid analysis time would enable therapeutic drug monitoring of MTXGlu(1-7) in the clinic.

Metabolic and molecular insights into an essential role of nicotinamide phosphoribosyltransferase

Nicotinamide phosphoribosyltransferase (NAMPT) is a pleiotropic protein implicated in the pathogenesis of acute respiratory distress syndrome, aging, cancer, coronary heart diseases, diabetes, nonalcoholic fatty liver disease, obesity, rheumatoid arthritis, and sepsis. However, the underlying molecular mechanisms of NAMPT in these physiological and pathological processes are not fully understood. Here, we provide experimental evidence that a Nampt gene homozygous knockout (Nampt−/−) resulted in lethality at an early stage of mouse embryonic development and death within 5–10 days in adult mice accompanied by a 25.24±2.22% body weight loss, after the tamoxifen induction of NamptF/F × Cre mice. These results substantiate that Nampt is an essential gene for life. In Nampt−/− mice versus Nampt+/+ mice, biochemical assays indicated that liver and intestinal tissue NAD levels were decreased significantly; histological examination showed that mouse intestinal villi were atrophic and disrupted, and visceral fat was depleted; mass spectrometry detected unusual higher serum polyunsaturated fatty acid containing triglycerides. RNA-seq analyses of both mouse and human pediatric liver transcriptomes have convergently revealed that NAMPT is involved in key basic cellular functions such as transcription, translation, cell signaling, and fundamental metabolism. Notably, the expression of all eight enzymes in the tricarboxylic acid cycle were decreased significantly in the Nampt−/− mice. These findings prompt us to posit that adult Nampt−/− mouse lethality is a result of a short supply of ATP from compromised intestinal absorption of nutrients from digested food, which leads to the exhaustion of body fat stores.

Comprehensive quantitative measurement of folate polyglutamates in human erythrocytes by ion pairing ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE The erythrocyte folate pool is reflective of an individuals long-term folate status; however, comprehensive quantitative determination of the various folate isoforms including polyglutamation (Glu(n)) status has posed an analytical problem. Factors complicating such analysis are the absence of authentic (isotope-labeled) standards and the large number of potential analytes. The present work presents high-throughput analytical methodology for the indirect comprehensive quantitation of the erythrocyte folate pool with commercially available standards. METHODS The erythrocyte folate pool was determined comprehensively by utilizing a cascade of three complementary ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (MS/MS) assays. In a first assay utilizing ion-pairing UPLC/MS/MS the relative (%) polyglutamation distribution (Glu(3-10)) of 5-methyltetrahydrofolate, tetrahydrofolate and 5-formyltetrahydrofolate is determined in a thermal extract obtained from packed erythrocytes, not requiring analytical standards. Quantitation of the erythrocyte folate pool was accomplished by performing two additional stable isotope dilution UPLC/MS/MS assays to determine whole blood and plasma folate content, utilizing commercially available [(13)C(5)]-labeled analogs of the Glu(1) analytes. Based on the values provided by each individual assay the comprehensive erythrocyte folate content could be calculated. RESULTS The various assays have been validated for intra- and inter-run precision, accuracy, linearity and are robust. The method was sensitive enough to measure the comprehensive erythrocyte folate distribution in a Downs syndrome patient with extremely low folate, bearing the C677T mutation in the gene encoding for methylenetetrahydrofolate reductase. CONCLUSIONS The erythrocyte folate pool can be comprehensively quantitated by running three complementary UPLC/MS/MS assays. The present assays are robust and allow for high-throughput analysis. The method can be utilized to support larger investigations that investigate the relationship between folate isoform and polyglutamation distribution and disease pathogenesis.

Urinary biomarkers of trimethoprim bioactivation in vivo following therapeutic dosing in children.

The antimicrobial trimethoprim-sulfamethoxazole (TMP-SMX) is widely used for the treatment of skin and soft-tissue infections in the outpatient setting. Despite its therapeutic benefits, TMP-SMX has been associated with a number of adverse drug reactions, which have been primarily attributed to the formation of reactive metabolites from SMX. Recently, in vitro experiments have demonstrated that TMP may form reactive intermediates as well. However, evidence of TMP bioactivation in patients has not yet been demonstrated. In this study, we performed in vitro trapping experiments with N-acetyl-l-cysteine (NAC) to determine stable markers of reactive TMP intermediates, focusing on eight potential markers (NAC-TMP adducts), some of which were previously identified in vitro. We developed a specific and sensitive assay involving liquid chromatography followed by tandem mass spectrometry for measurement of these adducts in human liver microsomal samples and expanded the methodology toward the detection of these analytes in human urine. Urine samples from four patients receiving TMP-SMX treatment were analyzed, and all samples demonstrated the presence of six NAC-TMP adducts, which were also detected in vitro. These adducts are consistent with the formation of imino-quinone-methide and para-quinone-methide reactive intermediates in vivo. As a result, the TMP component of TMP-SMX should be considered as well when evaluating adverse drug reactions to TMP-SMX.

Folate Depletion and Increased Glutamation in Juvenile Idiopathic Arthritis Patients Treated with Methotrexate

Folates exist as a fluctuating pool of polyglutamated metabolites that may serve as a clinical marker of methotrexate (MTX) activity. This study was undertaken to evaluate circulating folate content and folate polyglutamate distribution in juvenile idiopathic arthritis (JIA) patients and in a cell culture model based on MTX exposure and folate supply.

Expedient methodology for total methotrexate polyglutamation pool determination in human erythrocytes

The measurement of methotrexate polyglutamate metabolites in red blood cells has potential to aid in individualization of methotrexate therapy in rheumatoid arthritis and juvenile idiopathic arthritis. In this report a method is presented for rapid analysis of these metabolites in human red blood cells. The analytical procedure is a simple “one pot” pre-column reaction involving the addition of sodium dithionite as a reducing agent followed by 15 minutes of boiling. After centrifugation the supernatant is introduced into a conventional HPLC system equipped with a fluorescence detector, without the need for further workup. By performing the derivatization reaction pre-column, a time consuming (6–14 h) commonly used deglutamation procedure utilizing blank human plasma, becomes obsolete. Using the described procedure the total sample preparation time for a 50 sample run should not exceed 1–1.5 hours. The chromatographic run time per sample is 7 minutes using isocratic elution conditions. The method was found to be linear over the clinical relevant concentration range of 10–500 nM of intra-cellular methotrexate polyglutamates. The intra-run mean accuracy of the target value was between 98.1% and 106.0%. The intra-run precision was between 1.2% and 8.8%.