Pharmacokinetics of oral and subcutaneous methotrexate in red and white blood cells in patients with early rheumatoid arthritis: the methotrexate monitoring trial.

OBJECTIVE: To investigate the pharmacokinetics of methotrexate polyglutamate (MTX-PG) accumulation in red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) in patients with early rheumatoid arthritis (RA) after oral and subcutaneous MTX treatment. METHODS: In a clinical prospective cohort study (Methotrexate Monitoring study), newly diagnosed patients with RA were randomised for oral or subcutaneous MTX. At 1, 2, 3 and 6 months after therapy initiation, blood was collected and RBCs and PBMCs were isolated. MTX-PG1-6 concentrations were determined by mass spectrometry methods using stable isotopes of MTX-PG1-6 as internal standards. RESULTS: 43 patients (mean age: 58.5 years, 77% female) were included. PBMCs and RBCs revealed disparate pharmacokinetic profiles in both absolute MTX-PG accumulation levels and distribution profiles. Intracellular MTX-PG accumulation in PBMCs was significantly (p<0.001) 10-fold to 20-fold higher than RBCs at all time points, regardless of the administration route. MTX-PG distribution in PBMCs was composed of mostly MTX-PG1 (PG1>PG2>PG3). Remarkably, the distribution profile in PBMCs remained constant over 6 months. RBCs accumulated mainly MTX-PG1 and lower levels of MTX-PG2-5 at t=1 month. After 3 months, MTX-PG3 was the main PG-moiety in RBCs, a profile retained after 6 months of MTX therapy. Subcutaneous MTX administration results in higher RBC drug levels than after oral administration, especially shortly after treatment initiation. CONCLUSIONS: This is the first study reporting disparate MTX-PG accumulation profiles in RBCs versus PBMCs in newly diagnosed patients with RA during 6 months oral or subcutaneous MTX administration. This analysis can contribute to improved MTX therapeutic drug monitoring for patients with RA. TRIAL REGISTRATION NUMBER: NTR 7149.

Associations of Early Pregnancy and Neonatal Circulating Folate, Vitamin B-12, and Homocysteine Concentrations with Cardiometabolic Risk Factors in Children at 10 y of Age.

BACKGROUND: Higher circulating folate and vitamin B-12 concentrations and lower circulating homocysteine concentrations during pregnancy seem to be associated with fetal development. These micronutrients may also be associated with cardiometabolic health. OBJECTIVE: We examined the associations of circulating folate, vitamin B-12, and homocysteine concentrations during pregnancy and in neonates with childhood cardiometabolic outcomes. METHODS: This study was embedded in the Generation R Study, a population-based prospective cohort study from early pregnancy onward. We sampled blood in early pregnancy and cord blood. We measured cardiometabolic outcomes in the children at school age. Among 4449 children aged 10 y (median: 9.7; 95% range: 9.3, 10.7), we examined associations of plasma folate, serum vitamin B-12, and plasma homocysteine concentrations in early pregnancy and at birth with BMI, body fat distribution, heart rate, blood pressure, and insulin, glucose, and lipid concentrations, using linear regression models. Using logistic models, we examined the associations of these micronutrients with risks of overweight/obesity and clustering of cardiovascular risk factors. RESULTS: One standard deviation score (SDS) higher maternal plasma folate concentration was associated with lower BMI (-0.04 SDS; 95% CI: -0.08, -0.01), android-to-gynoid fat ratio (-0.04 SDS; 95% CI: -0.07, -0.01), systolic blood pressure (-0.06 SDS; 95% CI: -0.10, -0.03), risk of overweight (OR: 0.87; 95% CI: 0.78, 0.96), and clustering of cardiovascular risk factors (OR: 0.79; 95% CI: 0.68, 0.91). One SDS higher maternal serum total B-12 concentration was associated with lower glucose (-0.06 SDS; 95% CI: -0.10, -0.02) and higher HDL cholesterol concentrations (0.04 SDS; 95% CI: 0.00, 0.08). Cord blood folate, vitamin B-12, and homocysteine concentrations were not consistently associated with cardiometabolic outcomes. CONCLUSIONS: Subtle differences in circulating folate and vitamin B-12 concentrations in early pregnancy may be associated with child cardiometabolic health at age 10 y. The causality and mechanisms underlying these associations need further study.

Interaction between plasma homocysteine and the MTHFR c.677C > T polymorphism is associated with site-specific changes in DNA methylation in humans.

One-carbon metabolism provides a direct link among dietary folate/vitamin B12 exposure, the activity of the enzyme methylenetetrahydrofolate reductase (MTHFR), and epigenetic regulation of the genome via DNA methylation. Previously, it has been shown that the common c.677C > T polymorphism in MTHFR influences global DNA methylation status through a direct interaction with folate status and (indirectly) with total homocysteine (tHcy) levels. To build on that and other more recent observations that have further highlighted associations among MTHFR c.677C > T, tHcy, and aberrations in DNA methylation, we investigated whether the interaction between mildly elevated plasma tHcy and the c.677C > T polymorphism is associated with site-specific changes in DNA methylation in humans. We used data on plasma tHcy levels, c.677C > T polymorphism, and site-specific DNA methylation levels for a total of 915 white women and 335 men from the TwinsUK registry ( n = 610) and the Rotterdam study ( n = 670). We performed methylome-wide association analyses in each cohort to model the interaction between levels of tHcy and c.677C > T genotypes on DNA methylation ß values. Our meta-analysis identified 13 probes significantly associated with rs1801133 × tHcy levels [false-discovery rate (FDR) < 0.05]. The most significant associations were with a cluster of probes at the AGTRAP-MTHFR-NPPA/B gene locus on chromosome 1 (FDR = 1.3E-04), with additional probes on chromosomes 2, 3, 4, 7, 12, 16, and 19. Our top 2 hits on chromosome 1 were functionally associated with variability in expression of the TNF receptor superfamily member 8 ( TNFRSF8) gene/locus on that chromosome. This is the first study, to our knowledge, to provide a direct link between perturbations in 1-carbon metabolism, through an interaction of tHcy and the activity of MTHFR enzyme on epigenetic regulation of the genome via DNA methylation.-Nash, A. J., Mandaviya, P. R., Dib, M.-J., Uitterlinden, A. G., van Meurs, J., Heil, S. G., Andrew, T., Ahmadi, K. R. Interaction between plasma homocysteine and the MTHFR c.677C>T polymorphism is associated with site-specific changes in DNA methylation in humans.

Intakes of Vitamin B-12 from Dairy Food, Meat, and Fish and Shellfish Are Independently and Positively Associated with Vitamin B-12 Biomarker Status in Pregnant Dutch Women.

Background: The effect of vitamin B-12 from different animal foods on vitamin B-12 biomarker status has not previously been evaluated in pregnant women. Objective: We examined the association of vitamin B-12 intake from dairy, meat, fish (including shellfish), and eggs with circulating concentrations of vitamin B-12 biomarkers and with the presence of vitamin B-12 deficiency in 1266 pregnant women participating in the KOALA Birth Cohort Study. Methods: Blood samples were collected in weeks 34-36 of pregnancy, and vitamin B-12 intake from foods and supplements was estimated with a semiquantitative food-frequency questionnaire (FFQ). Total vitamin B-12, holotranscobalamin (holoTC), and methylmalonic acid (MMA) were determined in plasma. Vitamin B-12 deficiency was defined as holoTC <35 pmol/L and MMA >0.45 µmol/L. Associations were evaluated with linear and logistic regression analyses, adjusting for potential confounders. Results: Significant dose-response relations were observed between vitamin B-12 intake from dairy, meat, and fish and plasma vitamin B-12, holoTC, and MMA [P-trend for (shell)fish with MMA = 0.002; P-trend for dairy, meat, and fish with all other markers < 0.001]. The OR (95% CI) of vitamin B-12 deficiency in the third compared with the first tertile of dairy-derived vitamin B-12 was 0.13 (0.04, 0.49), and the ORs for vitamin B-12 from meat and fish were 0.33 (0.11, 0.97) and 0.25 (0.08, 0.82), respectively. Egg-derived vitamin B-12 was only associated with holoTC. Additional analyses showed that self-defined vegetarians and FFQ-defined lacto-ovo-vegetarians had lower median total dietary vitamin B-12 intake and considerably worse vitamin B-12 biomarker status than omnivores and pescatarians. Conclusions: In pregnant Dutch women, higher intakes of vitamin B-12 from dairy, meat, and fish were positively associated with vitamin B-12 status, suggesting that dairy, meat, and fish are good sources of bioactive vitamin B-12 in pregnancy. Nevertheless, for (lacto-)vegetarians, vitamin B-12 supplementation is recommended.

The role of genetic polymorphisms in the thymidylate synthase (TYMS) gene in methotrexate-induced oral mucositis in children with acute lymphoblastic leukemia.

OBJECTIVE: Methotrexate (MTX) is an important drug in the treatment of pediatric acute lymphoblastic leukemia (ALL). MTX is cytotoxic as it impairs DNA and RNA synthesis by inhibiting the enzymes dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS). The association between genetic variants within the TYMS gene and MTX-induced toxicity has been studied, but results are inconsistent. We determined the role of three previously described variants within the TYMS gene and MTX-induced oral mucositis in a prospective cohort of Dutch children with ALL and performed a meta-analysis of the previous results. MATERIALS AND METHODS: We analyzed the presence of a 28-base pair tandem repeat (rs34743033; 2R3R), a single nucleotide polymorphism present within the 28-base pair repeat on the 3R allele (rs2853542; 3RG>C) and a 6-base pair deletion (rs15126436; TTAAAG) within the TYMS gene in germline DNA of 117 pediatric patients with ALL. Oral mucositis was defined as grade≥3 according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) v.3.0. Data were analyzed for the individual rs34743033 (2R3R) and rs151264360 (6 bp deletion) polymorphisms, whereas rs2853542 (3RG>C) was combined with rs34743033 (2R3R) and analyzed according to predicted expression levels of TYMS: low expression (2R/2R, 2R/3RC and 3RC/3RC), median expression (2R/3RG and 3RC/3RG) and high expression (3RG/3RG). We performed a meta-analysis of the current literature on these polymorphisms in relation to oral mucositis using a fixed effects model. RESULTS: The 2R2R genotype (rs34743033) was not significantly associated with developing MTX-induced oral mucositis compared with the 2R3R/3R3R genotypes, which was confirmed in a meta-analysis [odds ratio (OR): 1.17 (0.62-2.19)]. Patients carrying the low-expression TYMS genotype (2R2R, 2R3RC, 3RC3RC) had an increased odds of developing MTX-induced oral mucositis [OR: 2.42 (0.86-6.80)], which did not reach statistical significance. The 6-bp deletion [rs151264360, OR: 0.79 (0.20-3.19)] was not associated with the development of MTX-induced oral mucositis. CONCLUSION: The TYMS 6-bp deletion and 2R3R polymorphism were not associated with MTX-induced oral mucositis. Validation studies in prospective cohorts are necessary to assess the possible role of the low-expression TYMS genotypes in relation to MTX-induced oral mucositis.

The miR-1206 microRNA variant is associated with methotrexate-induced oral mucositis in pediatric acute lymphoblastic leukemia.

Five-year survival rates of pediatric acute lymphoblastic leukemia (ALL) have reached 90% in the developed countries. However, toxicity because of methotrexate (MTX) occurs frequently. Variety in the occurrence of toxicity is partly determined by single nucleotide polymorphisms (SNPs) in coding regions. Recently, five SNPs in non-coding pre-microRNAs and microRNA processing (miRNA) genes were identified in association with MTX-induced oral mucositis. This study aimed to replicate the association of these miRNA variants in relation to MTX-induced oral mucositis in a prospective childhood ALL cohort. Three out of five SNPs with a minor allele frequency more than 0.15 [CCR4-NOT transcription complex (CNOT4) rs3812265, miR-1206 rs2114358, miR-2053 rs10505168] were analyzed in 117 pediatric ALL patients treated with 5 g/m MTX (DCOG ALL-10). Oral mucositis was defined as grade more than or equal to 3 according to the National Cancer Institute criteria. rs2114358 in miR-1206 was associated with oral mucositis [odds ratio (OR): 3.6; 95% confidence interval (CI): 1.1-11.5], whereas we did not confirm the association of CNOT4 rs3812265 (OR: 0.69; 95% CI: 0.27-1.80) and miR-2053 rs10505168 (OR: 2.50; 95% CI: 0.76-8.24). Our results replicate the association between rs2114358 in miR-1206 and MTX-induced oral mucositis in childhood ALL. Genetic variation in miR-1206 has potential as a novel biomarker to predict MTX-induced toxicity.

Cetuximab Prevents Methotrexate-Induced Cytotoxicity in Vitro through Epidermal Growth Factor Dependent Regulation of Renal Drug Transporters.

The combination of methotrexate with epidermal growth factor receptor (EGFR) recombinant antibody, cetuximab, is currently being investigated in treatment of head and neck carcinoma. As methotrexate is cleared by renal excretion, we studied the effect of cetuximab on renal methotrexate handling. We used human conditionally immortalized proximal tubule epithelial cells overexpressing either organic anion transporter 1 or 3 (ciPTEC-OAT1/ciPTEC-OAT3) to examine OAT1 and OAT3, and the efflux pumps breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp) in methotrexate handling upon EGF or cetuximab treatment. Protein kinase microarrays and knowledge-based pathway analysis were used to predict EGFR-mediated transporter regulation. Cytotoxic effects of methotrexate were evaluated using the dimethylthiazol bromide (MTT) viability assay. Methotrexate inhibited OAT-mediated fluorescein uptake and decreased efflux of Hoechst33342 and glutathione-methylfluorescein (GS-MF), which suggested involvement of OAT1/3, BCRP, and MRP4 in transepithelial transport, respectively. Cetuximab reversed the EGF-increased expression of OAT1 and BCRP as well as their membrane expressions and transport activities, while MRP4 and P-gp were increased. Pathway analysis predicted cetuximab-induced modulation of PKC and PI3K pathways downstream EGFR/ERBB2/PLCg. Pharmacological inhibition of ERK decreased expression of OAT1 and BCRP, while P-gp and MRP4 were increased. AKT inhibition reduced all transporters. Exposure to methotrexate for 24 h led to a decreased viability, an effect that was reversed by cetuximab. In conclusion, cetuximab downregulates OAT1 and BCRP while upregulating P-gp and MRP4 through an EGFR-mediated regulation of PI3K-AKT and MAPKK-ERK pathways. Consequently, cetuximab attenuates methotrexate-induced cytotoxicity, which opens possibilities for further research into nephroprotective comedication therapies.

Mitochondrial dysfunction in primary human fibroblasts triggers an adaptive cell survival program that requires AMPK-α.

Dysfunction of complex I (CI) of the mitochondrial electron transport chain (ETC) features prominently in human pathology. Cell models of ETC dysfunction display adaptive survival responses that still are poorly understood but of relevance for therapy development. Here we comprehensively examined how primary human skin fibroblasts adapt to chronic CI inhibition. CI inhibition triggered transient and sustained changes in metabolism, redox homeostasis and mitochondrial (ultra)structure but no cell senescence/death. CI-inhibited cells consumed no oxygen and displayed minor mitochondrial depolarization, reverse-mode action of complex V, a slower proliferation rate and futile mitochondrial biogenesis. Adaptation was neither prevented by antioxidants nor associated with increased PGC1-α/SIRT1/mTOR levels. Survival of CI-inhibited cells was strictly glucose-dependent and accompanied by increased AMPK-α phosphorylation, which occurred without changes in ATP or cytosolic calcium levels. Conversely, cells devoid of AMPK-α died upon CI inhibition. Chronic CI inhibition did not increase mitochondrial superoxide levels or cellular lipid peroxidation and was paralleled by a specific increase in SOD2/GR, whereas SOD1/CAT/Gpx1/Gpx2/Gpx5 levels remained unchanged. Upon hormone stimulation, fully adapted cells displayed aberrant cytosolic and ER calcium handling due to hampered ATP fueling of ER calcium pumps. It is concluded that CI dysfunction triggers an adaptive program that depends on extracellular glucose and AMPK-α. This response avoids cell death by suppressing energy crisis, oxidative stress induction and substantial mitochondrial depolarization.

Homocysteine and DNA methylation: a review of animal and human literature.

Homocysteine (Hcy) is a sulfur-containing non-protein forming amino acid, which is synthesized from methionine as an important intermediate in the one-carbon pathway. High concentrations of Hcy in a condition called hyperhomocysteinemia (HHcy) are an independent risk factor for several disorders including cardiovascular diseases and osteoporotic fractures. Since Hcy is produced as a byproduct of the methyltransferase reaction, alteration in DNA methylation is studied as one of the underlying mechanisms of HHcy-associated disorders. In animal models, elevated Hcy concentrations are induced either by diet (high methionine, low B-vitamins, or both), gene knockouts (Mthfr, Cbs, Mtrr or Mtr) or combination of both to investigate their effects on DNA methylation or its markers. In humans, most of the literature involves case-control studies concerning patients. The focus of this review is to study existing literature on HHcy and its role in relation to DNA methylation. Apart from this, a few studies investigated the effect of Hcy-lowering trials on restoring DNA methylation patterns, by giving a folic acid or B-vitamin supplemented diet. These studies which were conducted in animal models as well as humans were included in this review.

Methotrexate Polyglutamates Exposure - Response Modeling in a Large Cohort of Rheumatoid Arthritis Patients Starting Methotrexate.

Methotrexate polyglutamates (MTX-PG) concentrations in red blood cells (RBCs) have been suggested as a biomarker of response in patients with rheumatoid arthritis (RA) receiving low-dose MTX therapy. We investigated the association and interpatient variability between RBC-MTX-PG3-5 -exposure and response in patients with RA starting MTX. Data of three prospective cohorts were available. The relationship between exposure and Disease Activity Score in 28 joints (DAS28) was analyzed using a population pharmacokinetic-pharmacodynamic model. Relevant covariates were tested using full covariate modeling and backward elimination. From 395 patients, 3,401 MTX-PG concentrations and 1,337 DAS28 measurements were available between 0 and 300 days after MTX treatment onset. The developed model adequately described the time course of MTX-PG3-5 and DAS28. The median MTX-PG3-5 level at month 1 was 30.9 nmol/L (interquartile range (IQR): 23.6-43.7; n = 41) and at month 3: 69.3 nmol/L (IQR: 17.9-41.2; n = 351). Clearance of MTX-PG3-5 from RBCs was 28% lower (95% confidence interval (CI): 23.6-32.8%) in a woman and 10% lower (95% CI: 7.7-12.4%) in a 65-year-old compared with a 35-year-old patient. MTX-PG3-5 concentrations associated with DAS28: half-maximal effective concentration (EC50 ) was 9.14 nmol/L (95% CI: 4.2 nmol/L-14.1 nmol/L). EF at 80% (EC80 ) above 47 nmol/L was regarded as the optimal response. Independent of the MTX-PG 3-5 - response association, co-administration of disease-modifying antirheumatic drugs and corticosteroids improved response (additive effect on maximum effect (Emax )), whereas smoking, high body mass index and low albumin decreased Emax . In patients with RA starting MTX, RBC-MTX-PG3-5 was associated with clinical response. A dose increase is suggested when MTX-PG3-5 at month 1 is below 9.15 nmol/L, continued with the same dose when the concentration is above 47 nmol/L, and consider other treatment options above 78 nmol/L from 3 months onwards.

A meta-analysis of epigenome-wide association studies on pregnancy vitamin B12 concentrations and offspring DNA methylation.

Circulating vitamin B12 concentrations during pregnancy are associated with offspring health. Foetal DNA methylation changes could underlie these associations. Within the Pregnancy And Childhood Epigenetics Consortium, we meta-analysed epigenome-wide associations of circulating vitamin B12 concentrations in mothers during pregnancy (n = 2,420) or cord blood (n = 1,029), with cord blood DNA methylation. Maternal and newborn vitamin B12 concentrations were associated with DNA methylation at 109 and 7 CpGs, respectively (False Discovery Rate P-value <0.05). Persistent associations with DNA methylation in the peripheral blood of up to 482 children aged 4-10 y were observed for 40.7% of CpGs associated with maternal vitamin B12 and 57.1% of CpGs associated with newborn vitamin B12. Of the CpGs identified in the maternal meta-analyses, 4.6% were associated with either birth weight or gestational age in a previous work. For the newborn meta-analysis, this was the case for 14.3% of the identified CpGs. Also, of the CpGs identified in the newborn meta-analysis, 14.3% and 28.6%, respectively, were associated with childhood cognitive skills and nonverbal IQ. Of the 109 CpGs associated with maternal vitamin B12, 18.3% were associated with nearby gene expression. In this study, we showed that maternal and newborn vitamin B12 concentrations are associated with DNA methylation at multiple CpGs in offspring blood (PFDR<0.05). Whether this differential DNA methylation underlies associations of vitamin B12 concentrations with child health outcomes, such as birth weight, gestational age, and childhood cognition, should be further examined in future studies.

Associations between ABCB1, CYP2A6, CYP2B6, CYP2D6, and CYP3A5 alleles in relation to efavirenz and nevirapine pharmacokinetics in HIV-infected individuals.

BACKGROUND: Human immunodeficiency virus (HIV)-infected individuals show large interindividual variation in response to antiretroviral therapy. Efavirenz (EFV) and nevirapine (NVP) are nonnucleoside reverse transcriptase inhibitors, which are prescribed in combination with other antiretroviral therapy in so-called highly active antiretroviral therapy. Recent studies provide evidence for the role of cytochrome P450 (CYP) genes, in particular CYP2B6, in relation to EFV and NVP pharmacokinetics. In this study, the authors investigated whether common ABCB1, CYP2A6, CYP2B6, CYP2D6, and CYP3A5 alleles are associated with plasma concentrations of EFV and NVP in HIV-infected individuals. METHODS: Plasma drug concentrations were quantified by high-performance liquid chromatography in 143 HIV-infected individuals receiving either EFV or NVP. Genotyping for common alleles was performed by restriction fragment length polymorphism and Taqman assays. Individuals were genotyped for 11 single-nucleotide polymorphisms in 5 genes. CYP2B6 haplotypes were reconstructed by PHASE. RESULTS: Plasma EFV concentrations were positively associated with CYP2B6 c.516G>T, c.785A>G, and c.983A>G single-nucleotide polymorphisms in HIV-infected individuals. Increased plasma concentrations of EFV and NVP were present in individuals with the CYP2B6*6/*6 or *6/*18 haplotype compared with CYP2B6*1/*1 [increase of 62% (95% confidence interval, 44.0-80.1) and 24% (95% confidence interval, 7.0-40.0), respectively, P < 0.01]. No significant association with other genes in relation to EFV or NVP concentrations was found. CONCLUSIONS: In this study, a strong association of CYP2B6*6 and CYP2B6*18 alleles in relation to EFV and NVP plasma concentrations was found, which confirmed previous studies.

A U-HPLC-ESI-MS/MS-based stable isotope dilution method for the detection and quantitation of methotrexate in plasma.

INTRODUCTION: High-dose methotrexate (MTX) is used in the treatment of proliferative diseases such as acute lymphoblastic leukemia. Therapeutic drug monitoring of plasma MTX is important to monitor efficacy and adverse events. The authors aimed to develop a liquid chromatography, electrospray ionization, tandem mass spectrometry (LC-ESI-MS/MS)-based method to determine MTX in plasma for therapeutic drug monitoring and pharmacokinetic studies. METHODS: Samples were analyzed using a Waters Acquity UPLC and Quattro Premier XE. A Waters Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm) was used running an isocratic mobile phase of 21% methanol and 10 mM ammonium bicarbonate. The electrospray was operated in the positive ionization mode monitoring the following mass transitions: m/z 455.2 > 308.2 for MTX and m/z 458.2 > 311.2 for MTXd3. The analysis combined straightforward sample preparation, consisting of dilution and protein precipitation, with a 3-minute run time. RESULTS: The method was linear up to 50 µM (r > 0.99), and the coefficient of variation was <6% for intraday and <10% for interday precision. Average recovery was 99%. There were no significant matrix effects. The lower limit of quantitation, defined as the lowest concentration at which the coefficient of variation <20% and S/N > 1:10, was 5 nM. Method comparison with the Abbott TDx fluorescent polarization immunoassay (FPIA) showed excellent agreement, and a small but significant negative constant bias was detected (LC-MS/MS = 0.98 × FPIA - 7.3). CONCLUSIONS: [corrected] The authors developed a specific and sensitive stable isotope dilution LC-ESI-MS/MS method to monitor MTX concentrations in plasma within the clinically relevant range. The method can be easily applied in clinical laboratories because it combines straightforward sample pretreatment with LC-MS/MS.

"Macro transcobalamin causing raised vitamin B12: Case-based laboratory investigation".

Determination of plasma vitamin B12 (B12) is a frequently requested laboratory analysis, mainly employed to establish B12 deficiency. However, an increased level of B12 is a common unexpected finding that may be related to an increased concentration of one of the B12 binding proteins, haptocorrin or transcobalamin. This paper describes the extensive laboratory evaluation of a patient with an elevated level of plasma B12 with various well-established assays. Initial studies suggested the presence of a macromolecule consisting of haptocorrin bound B12. Specific determinations of the B12-binding proteins revealed normal amounts of haptocorrin but a markedly increase in both total and B12 saturated transcobalamin (holo-TC). The results are in accord with the presence of macro-transcobalamin. These experiments reveal that determination of the nature of the B12-macromolecules is troublesome due to differences in assays applied to measure these proteins. In addition, this publication creates awareness of macro-holo-TC as a cause of an unexplained increased B12 level.

Knockout of the Bcmo1 gene results in an inflammatory response in female lung, which is suppressed by dietary beta-carotene.

Beta-carotene 15,15'-monooxygenase 1 knockout (Bcmo1 (-/-)) mice accumulate beta-carotene (BC) similarly to humans, whereas wild-type (Bcmo1 (+/+)) mice efficiently cleave BC. Bcmo1 (-/-) mice are therefore suitable to investigate BC-induced alterations in gene expression in lung, assessed by microarray analysis. Bcmo1 (-/-) mice receiving control diet had increased expression of inflammatory genes as compared to BC-supplemented Bcmo1 (-/-) mice and Bcmo1 (+/+) mice that received either control or BC-supplemented diets. Differential gene expression in Bcmo1 (-/-) mice was confirmed by real-time quantitative PCR. Histochemical analysis indeed showed an increase in inflammatory cells in lungs of control Bcmo1 (-/-) mice. Supported by metabolite and gene-expression data, we hypothesize that the increased inflammatory response is due to an altered BC metabolism, resulting in an increased vitamin A requirement in Bcmo1 (-/-) mice. This suggests that effects of BC may depend on inter-individual variations in BC-metabolizing enzymes, such as the frequently occurring human polymorphisms in BCMO1.

Associations of circulating folate, vitamin B12 and homocysteine concentrations in early pregnancy and cord blood with epigenetic gestational age: the Generation R Study.

BACKGROUND: Circulating folate, vitamin B12 and homocysteine concentrations during fetal development have been associated with health outcomes in childhood. Changes in fetal DNA methylation may be an underlying mechanism. This may be reflected in altered epigenetic aging of the fetus, as compared to chronological aging. The difference between gestational age derived in clinical practice and gestational age predicted from neonatal DNA methylation data is referred to as gestational age acceleration. Differences in circulating folate, vitamin B12 and homocysteine concentrations during fetal development may be associated with gestational age acceleration. RESULTS: Up to 1346 newborns participating in the Generation R Study, a population-based prospective cohort study, had both cord blood DNA methylation data available and information on plasma folate, serum total and active B12 and plasma homocysteine concentrations, measured in early pregnancy and/or in cord blood. A subgroup of 380 newborns had mothers with optimal pregnancy dating based on a regular menstrual cycle and a known date of last menstrual period. For comparison, gestational age acceleration was calculated based the method of both Bohlin and Knight. In the total study population, which was more similar to Bohlin's training population, one standard deviation score (SDS) higher maternal plasma homocysteine concentrations was nominally associated with positive gestational age acceleration [0.07 weeks, 95% confidence interval (CI) 0.02, 0.13] by Bohlin's method. In the subgroup with pregnancy dating based on last menstrual period, the method that was also used in Knight's training population, one SDS higher cord serum total and active B12 concentrations were nominally associated with negative gestational age acceleration [(- 0.16 weeks, 95% CI - 0.30, - 0.02) and (- 0.15 weeks, 95% CI - 0.29, - 0.01), respectively] by Knight's method. CONCLUSIONS: We found some evidence to support associations of higher maternal plasma homocysteine concentrations with positive gestational age acceleration, suggesting faster epigenetic than clinical gestational aging. Cord serum vitamin B12 concentrations may be associated with negative gestational age acceleration, indicating slower epigenetic than clinical gestational aging. Future studies could examine whether altered fetal epigenetic aging underlies the associations of circulating homocysteine and vitamin B12 blood concentrations during fetal development with long-term health outcomes.

Vitamin B12, folate and homocysteine concentrations during pregnancy and early signs of atherosclerosis at school-age.

BACKGROUND & AIMS: Suboptimal circulating vitamin B12, folate and homocysteine concentrations during fetal life seem to be associated with cardiometabolic health at school-age. We examined whether fetal exposure to lower circulating vitamin B12 and folate concentrations and higher circulating homocysteine concentrations is also associated with early signs of atherosclerosis at school-age. METHODS: This study among 3826 school-age children and their mothers was embedded in the Generation R Study, a population-based prospective cohort study from early pregnancy onwards. We examined the associations of early-pregnancy and cord blood serum total and active B12 and plasma folate and homocysteine concentrations with common carotid artery intima-media thickness and distensibility in the children aged ten years. RESULTS: As compared to normal early-pregnancy serum total B12 concentrations (≥145 pmol/L), low serum total B12 concentrations (<145 pmol/L) were associated with higher carotid intima-media thickness in the children at school-age (difference 0.09 standard deviations score (SDS); 95% confidence interval (CI): 0.01, 0.16). As compared to normal early-pregnancy plasma folate concentrations (≥8 nmol/L), low plasma folate concentrations (<8 nmol/L) were associated with lower carotid distensibility in the children at school-age (difference -0.16 SDS; 95% CI: -0.28, -0.04). Early-pregnancy circulating total and active B12, folate and homocysteine concentrations measured continuously were not associated with carotid intima-media thickness or carotid distensibility in the children at school-age. One SDS higher plasma homocysteine concentrations measured in cord blood at birth was associated with a -0.05 SDS (95% CI: -0.09, -0.02) lower carotid distensibility at school-age. Cord blood total and active B12 and folate concentrations were not associated with carotid intima-media thickness or carotid distensibility at school-age. CONCLUSIONS: Circulating total B12, folate and homocysteine concentrations during fetal life seem to be associated with markers of subclinical atherosclerosis at school-age. Further studies need to examine the causality and mechanisms underlying these associations.

Global DNA (hydroxy)methylation is stable over time under several storage conditions and temperatures.

BACKGROUND: Epigenetic markers are often quantified and related to disease in stored samples. While, effects of storage on stability of these markers have not been thoroughly examined. In this longitudinal study, we investigated the influence of storage time, material, temperature, and freeze-thaw cycles on stability of global DNA (hydroxy)methylation. METHODS: EDTA blood was collected from 90 individuals. Blood (n = 30, group 1) and extracted DNA (n = 30, group 2) were stored at 4°C, -20°C and -80°C for 0, 1 (endpoint blood 4°C), 6, 12 or 18 months. Additionally, freeze-thaw cycles of blood and DNA samples (n = 30, group 3) were performed over three days. Global DNA methylation and hydroxymethylation (mean ± SD) were quantified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with between-run precision of 2.8% (methylation) and 6.3% (hydroxymethylation). Effects on stability were assessed using linear mixed models. RESULTS: global DNA methylation was stable over 18 months in blood at -20°C and -80°C and DNA at 4°C and -80°C. However, at 18 months DNA methylation from DNA stored at -20°C relatively decreased -6.1% compared to baseline. Global DNA hydroxymethylation was more stable in DNA samples compared to blood, independent of temperature (p = 0.0131). Stability of global DNA methylation and hydroxymethylation was not affected up to three freeze - thaw cycles. CONCLUSION: Global DNA methylation and hydroxymethylation stored as blood and DNA can be used for epigenetic studies. The relevance of  small differences occuring during storage depend on the expected effect size and research question.

Epigenome wide association study of response to methotrexate in early rheumatoid arthritis patients.

AIM: To identify differentially methylated positions (DMPs) and regions (DMRs) that predict response to Methotrexate (MTX) in early rheumatoid arthritis (RA) patients. MATERIALS AND METHODS: DNA from baseline peripheral blood mononuclear cells was extracted from 72 RA patients. DNA methylation, quantified using the Infinium MethylationEPIC, was assessed in relation to response to MTX (combination) therapy over the first 3 months. RESULTS: Baseline DMPs associated with response were identified; including hits previously described in RA. Additionally, 1309 DMR regions were observed. However, none of these findings were genome-wide significant. Likewise, no specific pathways were related to response, nor could we replicate associations with previously identified DMPs. CONCLUSION: No baseline genome-wide significant differences were identified as biomarker for MTX (combination) therapy response; hence meta-analyses are required.

A Hypomethylating Ketogenic Diet in Apolipoprotein E-Deficient Mice: A Pilot Study on Vascular Effects and Specific Epigenetic Changes.

Hyperhomocysteneinemia (HHcy) is common in the general population and is a risk factor for atherosclerosis by mechanisms that are still elusive. A hypomethylated status of epigenetically relevant targets may contribute to the vascular toxicity associated with HHcy. Ketogenic diets (KD) are diets with a severely restricted amount of carbohydrates that are being widely used, mainly for weight-loss purposes. However, studies associating nutritional ketosis and HHcy are lacking. This pilot study investigates the effects of mild HHcy induced by nutritional manipulation of the methionine metabolism in the absence of dietary carbohydrates on disease progression and specific epigenetic changes in the apolipoprotein-E deficient (apoE-/-) mouse model. ApoE-/- mice were either fed a KD, a diet with the same macronutrient composition but low in methyl donors (low methyl KD, LMKD), or control diet. After 4, 8 or 12 weeks plasma was collected for the quantification of: (1) nutritional ketosis, (i.e., the ketone body beta-hydroxybutyrate using a colorimetric assay); (2) homocysteine by HPLC; (3) the methylating potential S-adenosylmethionine to S-adenosylhomocysteine ratio (AdoHcy/AdoMet) by LC-MS/MS; and (4) the inflammatory cytokine monocyte chemoattractant protein 1 (MCP1) by ELISA. After 12 weeks, aortas were collected to assess: (1) the vascular AdoHcy/AdoMet ratio; (2) the volume of atherosclerotic lesions by high-field magnetic resonance imaging (14T-MRI); and (3) the content of specific epigenetic tags (H3K27me3 and H3K27ac) by immunofluorescence. The results confirmed the presence of nutritional ketosis in KD and LMKD mice but not in the control mice. As expected, mild HHcy was only detected in the LMKD-fed mice. Significantly decreased MCP1 plasma levels and plaque burden were observed in control mice versus the other two groups, together with an increased content of one of the investigated epigenetic tags (H3K27me3) but not of the other (H3K27ac). Moreover, we are unable to detect any significant differences at the p < 0.05 level for MCP1 plasma levels, vascular AdoMet:AdoHcy ratio levels, plaque burden, and specific epigenetic content between the latter two groups. Nevertheless, the systemic methylating index was significantly decreased in LMKD mice versus the other two groups, reinforcing the possibility that the levels of accumulated homocysteine were insufficient to affect vascular transmethylation reactions. Further studies addressing nutritional ketosis in the presence of mild HHcy should use a higher number of animals and are warranted to confirm these preliminary observations.