The evaluation of red blood cell folate and methotrexate levels during protocol M in childhood acute lymphoblastic leukemia.

BACKGROUND: After High-Dose Methotrexate (HD-MTX), folinic acid rescue therapy (Leucovorin) is administered to reduce side effects in pediatric acute lymphoblastic leukemia (ALL) patients. Leucovorin and MTX are structural analogues, possibly competing for cellular transport and intracellular metabolism. We hypothesize that Leucovorin accumulates during consecutive courses, which might result in a lower MTX uptake. METHODS: We prospectively measured red blood cell (RBC) folate and MTX levels during four HD-MTX and Leucovorin courses in 43 patients treated according the DCOG ALL-11 protocol with 2-weekly HD-MTX (5 g/m2/dose) and Leucovorin (15 mg/m2/dose) using LC-MS/MS. We estimated a linear mixed model to assess the relationship between these variables over time. RESULTS: Both RBC MTX-PG and folate levels increased significantly during protocol M. MTX-PG2-5 levels increased most substantially after the first two HD-MTX courses (until median 113.0 nmol/L, IQR 76.8-165.2) after which levels plateaued during the 3d and 4th course (until median 141.3 nmol/L, IQR 100.2-190.2). In parallel, folate levels increased most substantially after the first two HD-MTX courses (until median 401.6 nmol/L, IQR 163.3-594.2) after which levels plateaued during the 3d and 4th course (until median 411.5 nmol/L, IQR 240.3-665.6). The ratio folate/MTX-PG decreased significantly over time, which was mostly due to the relatively higher increase (delta) of MTX-PG. CONCLUSION: These results suggest that the increase in RBC folate levels does not seem to have a large effect on RBC MTX levels. Future studies, assessing competition of Leucovorin and MTX on other cellular mechanisms which might negatively affect treatment efficacy, are necessary.

[Simultaneous determination of leucovorin and 5-methyl-tetrahydrofolate diastereoisomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry coupled with ultrafiltration centrifugation-based pretreatment and its application to a pharmacokinetic study].

A simple, sensitive, and stable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the simultaneous determination of leucovorin and 5-methyltetrahydrofolate diastereomers in human plasma using methotrexate as the internal standard. The analytes and the internal standard were extracted from plasma samples by simple ultrafiltration centrifugation-based extraction. The separation was achieved on a chiral HSA column (150 mm×4 mm, 5 µm) using mobile phases containing 10 mmol pH 8.0 ammonium acetate and acetonitrile in gradient mode. The method showed good linearities in the ranges of 25-5000 µg/L and 12.5-3000 µg/L for leucovorin and 5-methyltetrahydrofolate diastereoisomers, respectively. The method was fully validated with respect to sensitivity, precision, accuracy, matrix effect, extraction recovery, and stability of analytes under various conditions. The method was successfully applied to a pharmacokinetic study of 125 mg/m2 6R,S-leucovorin and 62.5 mg/m2 6S-leucovorin. The results showed that the maximum observed concentrations (Cmax) of 6S-leucovorin and L-5-methyltetrahydrofolate were (3137.917±408.837) and (1679.633±244.132) µg/L, respectively, and the areas under the curve from the time of dosing to the last measurable concentration (AUC0-t) were (7504.883±1185.101) and (14001.214±2868.949) µg/L in the 125 mg/m2 6R,S-leucovorin dose group. The Cmax values of 6S-leucovorin and L-5-methyltetrahydrofolate were (3187.917±387.298) and (1739.204±224.755) µg/L, respectively, and AUC0-t values were (7426.664±854.825) and (14884.331±1843.353) µg/L in the 62.5 mg/m2 6S-leucovorin dose group. There were no significant diffe-rences in the main pharmacokinetic parameters between the two dose groups, and the pharmacokinetic characteristics as well as the rate and extent of absorption were consistent. This method can provide technical support for future bioequivalence studies of sodium leucovorin.

Relationship between folate concentration and expression of folate-associated genes in tissue and plasma after intraoperative administration of leucovorin in patients with colorectal cancer.

PURPOSE: The aim of study was to investigate the relationship between folate concentration and expression of folate-associated genes in tumour, mucosa and plasma of patients with colorectal cancer, after intraoperative administration of bolus leucovorin (LV). METHODS: Eighty patients were randomized into four groups to receive 0, 60, 200, or 500 mg/m2 LV, respectively. Tissue and plasma folate concentrations were assessed by LC-MS/MS. Gene expression of ABCC3/MRP3, FPGS, GGH, MTHFD1L, SLC46A1/PCFT, and SLC19A1/RFC-1 was determined using quantitative PCR. RESULTS: The folate concentration in tumour increased with increasing dosage of LV. Half of the patients treated with 60 mg/m2 did not reach a level above the levels of untreated patients. A significant correlation between folate concentration in tumour and mucosa was found in untreated patients, and in the group treated with 60 mg/m2 LV. The 5-MTHF/LV ratio correlated negatively with folate concentration in mucosa, whereas a positive correlation was found in tumour of patients who received 200 or 500 mg/m2 LV. A positive correlation was found between folate concentration and expression of all genes, except MTHFD1L, in patients who received LV. There was a negative correlation between 5-MTHF concentration in plasma of untreated patients and expression of GGH and SLC46A1/PCFT in tumour. CONCLUSIONS: The results indicate the possibility of using the individual plasma 5-MTHF/LV ratio after LV injection as a surrogate marker for tissue folate concentration. Expression of several folate-associated genes is associated with folate concentration in tissue and plasma and may become useful when predicting response to LV treatment.

Folate status and concentrations of serum folate forms in the US population: National Health and Nutrition Examination Survey 2011-2.

Serum and erythrocyte (RBC) total folate are indicators of folate status. No nationally representative population data exist for folate forms. We measured the serum folate forms (5-methyltetrahydrofolate (5-methylTHF), unmetabolised folic acid (UMFA), non-methyl folate (sum of tetrahydrofolate (THF), 5-formyltetrahydrofolate (5-formylTHF), 5,10-methenyltetrahydrofolate (5,10-methenylTHF)) and MeFox (5-methylTHF oxidation product)) by HPLC-MS/MS and RBC total folate by microbiologic assay in US population ≥ 1 year (n approximately 7500) participating in the National Health and Nutrition Examination Survey 2011-2. Data analysis for serum total folate was conducted including and excluding MeFox. Concentrations (geometric mean; detection rate) of 5-methylTHF (37·5 nmol/l; 100 %), UMFA (1·21 nmol/l; 99·9 %), MeFox (1·53 nmol/l; 98·8 %), and THF (1·01 nmol/l; 85·2 %) were mostly detectable. 5-FormylTHF (3·6 %) and 5,10-methenylTHF (4·4 %) were rarely detected. The biggest contributor to serum total folate was 5-methylTHF (86·7 %); UMFA (4·0 %), non-methyl folate (4·7 %) and MeFox (4·5 %) contributed smaller amounts. Age was positively related to MeFox, but showed a U-shaped pattern for other folates. We generally noted sex and race/ethnic biomarker differences and weak (Spearman's r< 0·4) but significant (P< 0·05) correlations with physiological and lifestyle variables. Fasting, kidney function, smoking and alcohol intake showed negative associations. BMI and body surface area showed positive associations with MeFox but negative associations with other folates. All biomarkers showed significantly higher concentrations with recent folic acid-containing dietary supplement use. These first-time population data for serum folate forms generally show similar associations with demographic, physiological and lifestyle variables as serum total folate. Patterns observed for MeFox may suggest altered folate metabolism dependent on biological characteristics.

Tolerability and pharmacokinetics of disodium folinate following single intravenous doses in healthy Chinese subjects: an open-label, randomized, single-center study.

The tolerability and pharmacokinetics of disodium folinate may vary with different races, and these variations might result in different outcomes. This study assessed the tolerability and pharmacokinetics of disodium folinate following single intravenous doses in healthy Chinese subjects, with gender factor also taken into account. Subjects were randomized to receive a single dose of disodium folinate at 20, 200, or 300 mg/m(2) administered intravenously over a time period of 10 min. Sequential blood samples were collected at regular intervals over 24 h after dosing and were analyzed using a validated high-performance liquid chromatography (HPLC) method. Pharmacokinetic parameters, including C max, AUC0-t, t 1/2, V d, and CL, were calculated using non-compartmental models. Tolerability was assessed by collecting adverse events (AEs) and monitoring vital signs, physical examinations, laboratory tests, and electrocardiograms. Following a single intravenous administration of disodium folinate 20, 200, and 300 mg/m(2), the mean (standard deviation) pharmacokinetic parameters were as follows: C max = 5.18 (0.58), 47.80 (10.10), and 69.93 (9.72) µg/mL; AUC0-t = 25.85 (3.36), 194.53 (30.18), and 355.26 (35.31) µg h/mL; AUC0-∞ = 30.24 (6.19), 215.43 (27.34), and 417.88 (54.81) µg h/mL; t 1/2 = 8.77 (2.57), 7.64 (1.81), and 9.08 (1.64) h; CL = 1.12 (0.18), 1.55(0.25), and 0.78 (0.09) L/h; V d = 13.75 (2.61), 17.38 (6.44), and 10.05 (1.49) L, respectively. The mean C max, AUC0-t, and AUC0-∞ increased in a dose-proportional manner. No significant differences in pharmacokinetic parameters were noted by gender. The most common AEs reported were mild redness at the injection site and neurological symptoms (headache, dizziness, and fatigue).

Folate is absorbed across the human colon: evidence by using enteric-coated caplets containing 13C-labeled [6S]-5-formyltetrahydrofolate.

BACKGROUND: Folate intakes that do not meet or greatly exceed requirements may be associated with negative health outcomes. A better understanding of contributors that influence the input side will help establish dietary guidance that ensures health benefits without associated risks. Colonic microbiota produce large quantities of folate, and [(13)C5]5-formyltetrahydrofolate infused during colonoscopy is absorbed. However, it is unclear if significant quantities of folate are absorbed in an intact microbiome. OBJECTIVE: We determined whether and how much of a physiologic dose of [(13)C5]5-formyltetrahydrofolate delivered in a pH-sensitive enteric caplet to an intact colonic microbiome is absorbed. DESIGN: Healthy adults ingested a specially designed pH-sensitive acrylic copolymer-coated barium sulfate caplet that contained 855 nmol (400 µg) [(13)C5]5-formyltetrahydrofolate. After a washout period ≥ 4 wk, subjects received an intravenous injection of the same compound (214 nmol). Serially collected blood samples before and after each test dose were analyzed by using a microbiological assay and liquid chromatography-tandem mass spectrometry. RESULTS: Caplet disintegration in the colon was observed by fluoroscopic imaging for 6 subjects with a mean (± SD) complete disintegration time of 284 ± 155 min. The mean (± SEM) rate of appearance of [(13)C5]5-methyltetrahydrofolate in plasma was 0.33 ± 0.09 (caplet) and 5.8 ± 1.2 (intravenous) nmol/h. Likely because of the significant time in the colon, the mean apparent absorption across the colon was 46%. CONCLUSIONS: Folate is absorbed across the colon in humans with an undisturbed microbiome. This finding and previous observations of the size of the colonic depot of folate and its potential for manipulation by diet (eg, dietary fiber, oligosaccharides, and probiotics) suggest that an individual's dietary folate requirement may differ depending on the consumption of dietary constituents that affect the size and composition of their gastrointestinal microbiota. In addition, a systematic investigation of the role of colonic folate on gastrointestinal development and the prevention of colorectal cancer is warranted. This trial was registered at clinicaltrials.gov as NCT00941174.

Differential behavior of serum and red blood cell folate during a treatment with levofolinic acid. Gender differences.

BACKGROUND: Folates are essential nutrients that maintain nucleotide synthesis and methylation reactions. Folate levels depend essentially on the diet. In the present work, the changes in the folate-homocysteine (Hcy) metabolic axis were studied in response to treatment with levofolinic acid. METHODS: 49 college students (23 men and 26 women) underwent a treatment voluntarily with 5 mg/day levofolinic acid for one month. Serum and red blood cell folate, vitamin B12, and Hcy levels were determined on days 2, 5, 10, and 30 during treatment and 30 days after completion of treatment. RESULTS: Serum folate and Hcy levels showed a plateau beginning on day 10, while red blood cell folate increased towards treatment completion. Gender differences were found in basal levels of Hcy, these differences remaining until the 10th day of treatment and reappearing 30 days after the treatment was finished. Between gender differences in treatment evolution were found only in percentage changes in red blood cell folate in women and men at day 30 of treatment. CONCLUSIONS: There is a compartmentalization of folates in the body that presents a plateau in serum and an erythrocyte reservoir. Folate metabolism presents differential features between genders. The greater physiological need for folate in women of childbearing age could be the determining factor in this difference.

Quantification of folate metabolites in serum using ultraperformance liquid chromatography tandem mass spectrometry.

Folate deficiency is considered a risk factor for many diseases such as cancer, congenital heart disease and neural tube defects (NTDs). There is a pressing need for more methods of detecting folate and its main metabolites in the human body. Here, we developed a simple, fast and sensitive ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method for the simultaneous quantifications of folate metabolites including folic acid, 5-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF), homocysteine (Hcy), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). The method was validated by determining the linearity (r(2)>0.998), sensitivity (limit of detection ranged from 0.05 to 0.200ng/mL), intra- and inter-day precision (both CV<6%) and recovery (each analyte was >90%). The total analysis time was 7min. Serum samples of NTD-affected pregnancies and controls from a NTD high-risk area in China were analyzed by this method, the NTD serum samples showed lower concentrations of 5-MeTHF (P<0.05) and 5-FoTHF (P<0.05), and higher concentrations of Hcy (P<0.05) and SAH (P<0.05) compared with serum samples from controls, consistent with a previous study. These results showed that the method is sensitive and reliable for simultaneous determination of six metabolites, which might indicate potential risk factors for NTDs, aid early diagnosis and provide more insights into the pathogenesis of NTDs.

Folic acid handling by the human gut: implications for food fortification and supplementation.

BACKGROUND: Current thinking, which is based mainly on rodent studies, is that physiologic doses of folic acid (pterylmonoglutamic acid), such as dietary vitamin folates, are biotransformed in the intestinal mucosa and transferred to the portal vein as the natural circulating plasma folate, 5-methyltetrahydrofolic acid (5-MTHF) before entering the liver and the wider systemic blood supply. OBJECTIVE: We tested the assumption that, in humans, folic acid is biotransformed (reduced and methylated) to 5-MTHF in the intestinal mucosa. DESIGN: We conducted a crossover study in which we sampled portal and peripheral veins for labeled folate concentrations after oral ingestion with physiologic doses of stable-isotope-labeled folic acid or the reduced folate 5-formyltetrahydrofolic acid (5-FormylTHF) in 6 subjects with a transjugular intrahepatic porto systemic shunt (TIPSS) in situ. The TIPSS allowed blood samples to be taken from the portal vein. RESULTS: Fifteen minutes after a dose of folic acid, 80 ± 12% of labeled folate in the hepatic portal vein was unmodified folic acid. In contrast, after a dose of labeled 5-FormylTHF, only 4 ± 18% of labeled folate in the portal vein was unmodified 5-FormylTHF, and the rest had been converted to 5-MTHF after 15 min (postdose). CONCLUSIONS: The human gut appears to have a very efficient capacity to convert reduced dietary folates to 5-MTHF but limited ability to reduce folic acid. Therefore, large amounts of unmodified folic acid in the portal vein are probably attributable to an extremely limited mucosal cell dihydrofolate reductase (DHFR) capacity that is necessary to produce tetrahydrofolic acid before sequential methylation to 5-MTHF. This process would suggest that humans are reliant on the liver for folic acid reduction even though it has a low and highly variable DHFR activity. Therefore, chronic liver exposure to folic acid in humans may induce saturation, which would possibly explain reports of systemic circulation of unmetabolized folic acid.

Use of short chain alkyl imidazolium ionic liquids for on-line stacking and sweeping of methotrexate, flinic acid and folic acid: their application to biological fluids.

Methotrexate (MTX) is widely used for the treatment of many types of cancer. Folinic acid (FNA) and folic acid (FA) were usually simultaneously supplemented with MTX to reduce the side effects of a folate deficiency. This study, for the first time, included on-line sample preconcentration by stacking and sweeping techniques under reduced or enhanced electric conductivity in the sample region using short chain alkyl imidazolium ionic liquids (ILs) as micelle forming agents for analyte focusing. Both analyte focusing by micelle collapse (AFMC) and sweeping-MEKC had been investigated for the comparison of their effectiveness to examine simultaneously MTX, FNA and FA in plasma and urine under physiological conditions. In sweeping-MEKC, the sample solution without micelles was hydrodynamically injected as a long plug into a fused-silica capillary pre-filled with phosphate buffer containing 3.0 mol/L of 1-butyl-3-methylimidazolium bromide (BMIMBr). Using AFMC, the analytes were prepared in BMIMBr micellar matrix and hydrodynamically injected into the phosphate buffer without IL micelles. The conductivity ratio between BGE and sample (γ, BGE/sample) was optimized to be 3.0 in sweeping-MEKC and 0.33 in AFMC resulting the adequate separation of analytes within 4.0 min. To reduce the possibility of BMIMBr adsorption, an appropriate rinsing protocol was used. The limits of detection were calculated as 0.1 ng/mL MTX, 0.05 ng/mL FNA and 0.05 ng/mL FA by sweeping-MEKC and 0.5 ng/mL MTX, 0.3 ng/mL FNA and 0.3 ng/mL FA by AFMC. The accuracy was tested by recovery in plasma and urine matrices giving values ranging between 90 and 110%. Both stacking and sweeping by BMIMBr could be successfully used for the rapid, selective and sensitive determination of pharmaceuticals in complex matrices due to its fascinating properties, including high conductivity, good thermal stability and ability to form different types of interactions by electrostatic, hydrophobic, hydrogen bonding and π-π interactions. In sweeping-MEKC, the using of BMIMBr enhanced the γ factor, k retention factor and the injected amount of sample. Consequently, this technique offers particular potential for higher sensitivity by giving 22- and 5-fold sensitivity enhancement factors (SEFs) of MTX compared to CZE and AFMC, respectively.

Analyses of copy number variation reveal putative susceptibility loci in MTX-induced mouse neural tube defects.

Copy number variations (CNVs) are thought to act as an important genetic mechanism underlying phenotypic heterogeneity. Impaired folate metabolism can result in neural tube defects (NTDs). However, the precise nature of the relationship between low folate status and NTDs remains unclear. Using an array-comparative genomic hybridization (aCGH) assay, we investigated whether CNVs could be detected in the NTD embryonic neural tissues of methotrexate (MTX)-induced folate dysmetabolism pregnant C57BL/6 mice and confirmed the findings with quantitative real-time PCR (qPCR). The CNVs were then comprehensively investigated using bioinformatics methods to prioritize candidate genes. We measured dihydrofolate reductase (DHFR) activity and concentrations of folate and relevant metabolites in maternal serum using enzymologic method and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Three high confidence CNVs on XqA1.1, XqA1.1-qA2, and XqE3 were found in the NTD embryonic neural tissues. Twelve putative genes and three microRNAs were identified as potential susceptibility candidates in MTX-induced NTDs and possible roles in NTD pathogenesis. DHFR activity and 5-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF), and S-adenosylmethionine (SAM) concentrations of maternal serum decreased significantly after MTX injection. These findings suggest that CNVs caused by defects in folate metabolism lead to NTD, and further support the hypothesis that folate dysmetabolism is a direct cause for CNVs in MTX-induced NTDs.

Phase 1 study of efatutazone, a novel oral peroxisome proliferator-activated receptor gamma agonist, in combination with FOLFIRI as second-line therapy in patients with metastatic colorectal cancer.

BACKGROUND: Efatutazone, a novel oral highly-selective peroxisome proliferator-activated receptor gamma (PPARγ) agonist, has demonstrated some inhibitory effects on disease stabilization in patients with metastatic colorectal cancer (mCRC) enrolled in previous phase I studies. Here, we evaluate the safety and pharmacokinetics of efatutazone combined with FOLFIRI (5-fluorouracil, levo-leucovorin, and irinotecan) as second-line chemotherapy in Japanese patients with mCRC. METHODS: Dose-limiting toxicities (DLTs) were evaluated at 2 efatutazone dose levels of 0.25 and 0.50 mg (the recommended dose [RD] of efatutazone monotherapy) twice daily in combination with FOLFIRI in a 3-9 patient cohort. Furthermore, tolerability at the RD level was assessed in additional patients, up to 12 in total. Blood samples for pharmacokinetics and biomarkers and tumor samples for archival tissues were collected from all patients. RESULTS: Fifteen patients (0.25 mg, 3; 0.5 mg, 12) were enrolled. No DLTs were observed. Most patients experienced weight increase (100 %) and edema (80.0 %), which were manageable with diuretics. Common grade 3/4 toxicities were neutropenia (93.3 %), leukopenia (46.7 %), and anemia (33.3 %). Stable disease was observed in 8 of the 14 patients evaluable for tumor response. The plasma adiponectin levels increased over time and increased dose. No clear relationship was detected between treatment efficacies and plasma levels of adiponectin as well as the expression levels of PPARγ and the retinoid X receptor in tumor tissues. CONCLUSIONS: Efatutazone combined with FOLFIRI demonstrates an acceptable safety profile and evidence of disease stabilization in Japanese patients with mCRC. The RD for efatutazone monotherapy can be used in combination with FOLFIRI.

A high-throughput LC-MS/MS method suitable for population biomonitoring measures five serum folate vitamers and one oxidation product.

Small specimen volume and high sample throughput are key features needed for routine methods used for population biomonitoring. We modified our routine eight-probe solid phase extraction (SPE) LC-MS/MS method for the measurement of five folate vitamers [5-methyltetrahydrofolate (5-methylTHF), folic acid (FA), plus three minor forms: THF, 5-formylTHF, 5,10-methenylTHF] and one oxidation product of 5-methylTHF (MeFox) to require less serum volume (150 µL instead of 275 µL) by using 96-well SPE plates with 50 mg instead of 100 mg phenyl sorbent and to provide faster throughput by using a 96-probe SPE system. Total imprecision (10 days, two replicates/day) for three serum quality control pools was 2.8-3.6% for 5-methylTHF (19.5-51.1 nmol/L), 6.6-8.7% for FA (0.72-11.4 nmol/L), and ≤11.4% for the minor folate forms (<1-5 nmol/L). The mean (±SE) recoveries of folates spiked into serum (3 days, four levels, two replicates/level) were: 5-methylTHF, 99.4 ± 3.6%; FA, 100 ± 1.8%; minor folates, 91.7-108%. SPE extraction efficiencies were ≥85%, except for THF (78%). Limits of detection were ≤0.3 nmol/L. The new method correlated well with our routine method [n = 150, r = 0.99 for 5-methylTHF, FA, and total folate (tFOL, sum of folate forms)] and produced slightly higher tFOL (5.6%) and 5-methylTHF (7.3%) concentrations, likely due to the faster 96-probe SPE process (1 vs. 5 h), resulting in improved SPE efficiency and recovery compared to the eight-probe SPE method. With this improved LC-MS/MS method, 96 samples can be processed in ~2 h, and all relevant folate forms can be accurately measured using a small serum volume.

Accounting for an isobaric interference allows correct determination of folate vitamers in serum by isotope dilution-liquid chromatography-tandem MS.

Mild and prolonged oxidative degradation of 5-methyltetrahydrofolate (5-methylTHF) leads to the biologically inactive pyrazino-s-triazine derivative of 4α-hydroxy-5-methylTHF (MeFox). MeFox and the biologically active 5-formyltetrahydrofolate (5-formylTHF) are isobaric compounds that behave similarly during chromatographic and mass separation, making coelution and misidentification likely. Our published routine liquid chromatography-tandem MS (LC-MS/MS) method did not discern between 5-formylTHF and MeFox, measuring the sum of these compounds at a mass to charge ratio (m/z) of 474→327 as 5-formylTHF. We modified this method to separate MeFox and 5-formylTHF by either chromatography or unique mass transitions and then applied the 2 methods to serum specimens to determine typical concentrations of these compounds. The 2 unique transitions (m/z: 5-formylTHF, 474→299; MeFox, 474→284) showed good sensitivity [limit of detection (nmol/L): 5-formylTHF, 0.21; MeFox, 0.34], selectivity (no interfering peaks), spiking recovery (mean ± SD: 5-formylTHF, 103 ± 3.4%; MeFox, 94 ± 10%), and low imprecision (CV: 5-formylTHF, 3.9% at 2.4 nmol/L; MeFox, 5.1% at 2.9 nmol/L). The mass separation method detected 5-formylTHF in the same specimens as the chromatographic separation method. Analysis of several thousand serum specimens showed that the majority (∼85%) contained MeFox at <3 nmol/L but no detectable 5-formylTHF concentrations, some (∼14%) contained 5-formylTHF at <0.5 nmol/L, and a few specimens contained 5-formylTHF at >1 nmol/L and MeFox at >10 nmol/L. In summary, serum can contain 5-formylTHF high enough to contribute to total folate and contains MeFox that will bias total folate if not appropriately separated. Including measurements of MeFox and 5-formylTHF along with the other folate vitamers will enhance assessments of the association between biologically active folate and health effects.

Predicting 5-fluorouracil toxicity in colorectal cancer patients from peripheral blood cell telomere length: a multivariate analysis.

BACKGROUND: Identifying various pretreatment factors that predict chemotherapy-induced toxicity in colorectal cancer (CRC) patients undergoing treatment for their disease is crucial to optimising patient care. METHODS: Seventy-three patients received adjuvant 5-fluorouracil (5FU)/leucovorin using either the Mayo Clinic (n=42) or a weekly schedule (n=31) and evaluated for clinical toxicity. Pretreatment blood analysis included measures of plasma uracil and dihydrouracil, peripheral blood mononuclear cell (PBMNC) telomere length (TL), standard biochemistry and cell differential analysis. On the first day of treatment 5FU-pharmacokinetic variables of area under the curve, half life and clearance were also measured. These variables together with age and gender were used in univariate and multivariate analysis as predictors of clinical toxicity. RESULTS: For the Mayo schedule the primary toxicities were neutropenia (69%), mucositis (58%) and leukopenia (46%), with 70% of patients presenting with haematological toxicity ≥grade 1 (neutropenia and/or leukopenia). Multivariate analysis showed that haematological toxicity was predicted by short TL, high platelet lymphocyte ratio (PLR) and low neutrophil count (R(2)=0.38, P<0.0006), whereas mucositis was predicted by age, TL and PLR (R(2)=0.34, P<0.001). For the weekly schedule diarrhoea predominated (16%), with female gender as the only predictive factor. Although measures of uracil metabolism correlated well with 5FU metabolism (r=0.45-0.49), they did not indicate abnormal pyrimidine metabolism in this cohort and not surprisingly failed to predict for 5FU toxicity. CONCLUSION: Short TL of PBMNC and an increased PLR were strong predictors of mucositis and haematological toxicity in CRC patients undergoing 5FU treatment in the adjuvant setting.

Phase I results from a two-part Phase I/II study of cediranib in combination with mFOLFOX6 in Japanese patients with metastatic colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the second most common malignancy in Japan. Inhibition of vascular endothelial growth factor (VEGF) signaling is a clinically validated therapeutic strategy in patients with metastatic CRC. Cediranib is an oral, highly potent VEGF signaling inhibitor of all three VEGF receptors. METHODS: This Phase I study investigated the safety, tolerability and pharmacokinetics of cediranib (20 or 30 mg) in combination with mFOLFOX6 in Japanese patients with previously untreated metastatic CRC. If the safety of the 20 mg dose was confirmed, a second cohort of patients was to be recruited to receive cediranib 30 mg + mFOLFOX6. RESULTS: Six patients received cediranib 20 mg + mFOLFOX6 and seven received cediranib 30 mg + mFOLFOX6. One patient in the initial cediranib 20 mg cohort experienced a dose-limiting toxicity (DLT; grade 3 bilirubin increase); no DLTs were observed in the 30 mg cohort. The most commonly reported adverse events were diarrhea, decreased appetite, peripheral neuropathy, hypertension and fatigue. Two patients in the 20 mg cohort and three in the 30 mg cohort experienced serious adverse events during all treatment courses. Cediranib was generally well tolerated in this patient population with no evidence to suggest any significant pharmacokinetic interactions between cediranib and fluorouracil or oxaliplatin. A preliminary evaluation showed that five of nine evaluable patients achieved a best response of partial response. CONCLUSION: Cediranib (20 or 30 mg) in combination with mFOLFOX6 was considered tolerable according to the protocol-defined criteria, providing justification for the Phase II part of this study.

Maternal serum folate species in early pregnancy and lower genital tract inflammatory milieu.

OBJECTIVE: We previously reported that elevated antiinflammatory cervical cytokines in early pregnancy were associated with spontaneous preterm birth. Our objective was to explore the relation between serum folate vitamers and the lower genital tract inflammatory milieu. STUDY DESIGN: Pregnant women (n = 417) at <16 weeks' gestation had serum samples that were analyzed for folate species 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and cervical fluid that was assayed for cytokine concentrations. Patterns in proinflammatory cytokines (interleukin [IL]-1ß, -6, -8, and -10; monocyte chemotactic protein-1) and antiinflammatory cytokines (IL-4, IL-10, IL-13) were identified with factor analysis. RESULTS: After confounder adjustment, maternal serum 5-methyltetrahydrofolate concentrations had a strong negative association with elevated antiinflammatory scores; serum 5-formyltetrahydrofolate concentrations were associated positively with elevated antiinflammatory scores (both P < .05). Maternal folate was not associated with proinflammatory scores. CONCLUSION: Maternal serum folate vitamers are associated with cervical cytokine concentrations, which suggests a possible mechanistic link between folate and preterm birth risk.

Pharmacokinetics of oxaliplatin in gastrointestinal cancer patients with malignant ascites.

The pharmacokinetics of oxaliplatin in plasma and ascitic fluid was investigated in 5 gastrointestinal cancer patients with malignant ascites. Oxaliplatin was administered at 85 mg/m² by 2-hour infusion in the FOLFOX4 regimen, and the concentrations of total and free platinum were measured. There was a trend of lower plasma C(max) values of total platinum in patients with a larger volume of ascitic fluid. The AUC(0-t) values of mean concentration curves of total plasma platinum, total ascites platinum, free plasma platinum, and free ascites platinum were 31.15, 7.96, 4.93 and 2.93 mg•h/ml, respectively. The concentrations of free ascites platinum were similar to those of free plasma platinum at the last sampling time of 26 h in each patient. The decrease or disappearance of ascitic fluid was observed in 4 patients. These results suggest that oxaliplatin exerted a beneficial effect in gastrointestinal cancer patients with malignant ascites, even when administered intravenously.

Pharmacokinetics of oxaliplatin in a hemodialytic patient treated with modified FOLFOX-6 plus bevacizumab therapy.

PURPOSE: To establish an appropriate administration schedule for oxaliplatin in FOLFOX plus bevacizumab therapy for a hemodialytic patient. METHODS: A 50-year-old man on chronic hemodialysis was treated for colon cancer and synchronous hepatic metastasis with modified FOLFOX-6 plus bevacizumab therapy every 3 weeks. The plasma concentration of free platinum was measured at eight points, before and within the first 50 h after oxaliplatin administration. A dose escalation study of oxaliplatin was performed at doses of 60, 70, and 85 mg/m(2). A 4-h dialysis session was begun at the end of the oxaliplatin treatment. RESULTS: The pharmacokinetics of free platinum showed a bimodal pattern at each dose: The serum concentration decreased rapidly soon after dialysis, then increased, and remained at a high level for 24 h. The areas under the curves (AUC) for free platinum were 17.6, 23.6, and 32.6 µg h/mL after doses of 60, 70, and 85 mg/m(2) oxaliplatin, respectively. These exceeded the AUC when 90 mg/m(2) was given to a patient with normal renal function (7.9 µg h/mL). Treatment was safely continued for 6 months without severe toxicity. CONCLUSION: FOLFOX plus bevacizumab therapy can be given safely to hemodialytic patients with no reduction in the dose of oxaliplatin if hemodialysis is performed soon after the administration of oxaliplatin and the dosing interval is extended to 3 weeks.

Maternal serum folate species in early pregnancy and risk of preterm birth.

BACKGROUND: Poor maternal folate status has been associated with an increased risk of preterm birth. However, major gaps remain in our understanding of how individual folate species relate to preterm birth. OBJECTIVE: Our objective was to assess the association between maternal folate status as measured by 5-methyltetrahydrofolate (5MeTHF), 5-formyltetrahydrofolate (5FoTHF), and folic acid concentrations, which are the 3 primary folate species in serum, and the risk of preterm birth and spontaneous preterm birth (sPTB). DESIGN: A cohort of 313 pregnant women who received care at resident antepartum clinics at Magee-Womens Hospital (Pittsburgh, PA) (2003-2007) was enrolled at <16 wk gestation. We analyzed nonfasting blood samples that were drawn from subjects at enrollment for the 3 folate species by using HPLC-tandem mass spectrometry. RESULTS: Serum 5MeTHF and 5FoTHF concentrations comprised 65% and 33% of total folate concentrations, respectively. In confounder-adjusted, multivariable, log-binomial regression models, 1-SD increases in serum total folate and serum 5MeTHF concentrations were associated with significant reductions in the risk of sPTB (P < 0.05). There was a significant interaction between serum 5MeTHF and 5FoTHF concentrations and risk of preterm birth (P = 0.01). When serum 5MeTHF concentrations were low, there was a positive linear relation between 5FoTHF and risk of preterm birth. When 5MeTHF concentrations were high, there was a strong negative relation between 5FoTHF and preterm birth. CONCLUSIONS: Our results imply that the relative concentrations of folate species may be more critical than total folate in preventing preterm birth. An improved understanding of folate metabolism during pregnancy may lead to targeted intervention strategies that decrease the rate of preterm birth.