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.

Cardiac troponin I is a sensitive, specific biomarker of cardiac injury in laboratory animals.

This study directly demonstrates that cardiac troponin I (cTnI) is a sensitive, specific, and persistent biomarker in laboratory animals. Histopathological and pathophysiological cardiac changes in dogs, rats and mice correlated with increased serum cTnI with various cardiac inotropic agents, and cardiotoxic drugs and with cardiac arrhythmias, tachycardia, cardiac effusion with dyspnoea, and ageing. A comparison of six immunoassays for cTnI and cardiac troponin T (cTnT) to detect and monitor cardiac injury in a rodent model indicated that enzyme-linked immunosorbent (Life Diagnostics Inc and TriChem Resources Inc, West Chester, Philadelphia, USA) and Immulite (Diagnostic Products Corporation, Llanberis, UK) assays had low sensitivity and less than 1% of the dynamic range of Centaur (Bayer Healthcare Diagnostics, Newbury, UK) cTnI and Elecsys (Roche Diagnostics, Basel, Switzerland) and M8 (Bioveris Europe, Whitney, UK) cTnT assays. In dogs, however, the Immulite assay was effective and correlated with the Centaur. Serum concentrations were highly correlated but 10-fold lower for cTnT compared with cTnI with cardiac injury. Centaur assay also detected cTnI in myocardium from marmosets, swine, cattle, and guinea pigs, indicating it to be candidate cardiac biomarker for these species as well. Purified rat cTnI was 50% more reactive than purified human cTnI in the Centaur assay. In the rat, an age- and gender-dependent variation in serum cTnI was found. Male rats aged six and eight months had a 10-fold greater serum cTnI than age-matched females and three-month-old rats. These increases correlated with minimal histopathological change. Isoproterenol-induced serum cTnI increased up to 760-fold the minimal detectable concentration of 0.07 microg/L, within 4-6 h and decreased with a half-life of 6 h, with an expected return to baseline of 60 h. Severity of histopathological change correlated with serum cTnI during the ongoing injury.

[6S]5-methyltetrahydrofolate or folic acid supplementation and absorption and initial elimination of folate in young and middle-aged adults.

OBJECTIVES: To assess the effects of supplementation with the diastereoisomer of 5-methyltetrahydrofolate ([6S]5-methylTHF), as an alternative supplement for folic acid, on folate absorption and elimination, in two age groups. DESIGN: A randomized, double-blind intervention study. SUBJECTS: A total of 12 young (<30 y) and 12 middle-aged (> or =50 y) healthy volunteers were recruited. METHODS: Volunteers were randomized to receive daily supplementation with 400 mug folic acid or equimolar amounts of [6S]5-methylTHF during 5 weeks. Before and after supplementation, absorption and initial elimination were calculated following oral [(2)H(2)]folic acid test doses using isotope kinetics in plasma. RESULTS: Folic acid absorption was lower in the middle-aged as compared to the young adults, both before (P = 0.03) and after (P = 0.05) supplementation. In the young adults, absorption decreased by 22% after [6S]5-methylTHF and increased by 21% after folic acid (P = 0.02). In the other age group, no such changes were found. The folate rate constant of elimination increased after folic acid supplementation in the young (+50%; P = 0.05) but not in the middle-aged (+18%; P = 0.5) adults. CONCLUSIONS: Young adults show increased folate turnover after folic acid supplementation relative to the effect of [6S]5-methylTHF supplementation. Similar differences are not observed in middle-aged adults, in whom folic acid absorption was found to be lower as compared to the young adults. SPONSORSHIP: Financial support was received from the European Union 5th Framework Programme (Grant QLRT-1999-00576).

[6S]-5-methyltetrahydrofolate increases plasma folate more effectively than folic acid in women with the homozygous or wild-type 677C-->T polymorphism of methylenetetrahydrofolate reductase.

BACKGROUND AND PURPOSE: 5,10-Methylenetetrahydrofolate reductase (MTHFR) is responsible for the synthesis of 5-methyltetrahydrofolate (5-MTHF). The 677C-->T mutation of MTHFR reduces the activity of this enzyme. The aim of this study was, first, to compare pharmacokinetic parameters of [6S]-5-MTHF and folic acid (FA) in women with the homozygous (TT) and wild-type (CC) 677C-->T mutation, and second, to explore genotype differences. The metabolism of [6S]-5-MTHF and FA was evaluated by measuring plasma folate derivatives. EXPERIMENTAL APPROACH: Healthy females (TT, n= 16; CC, n= 8) received a single oral dose of FA (400 microg) and [6S]-5-MTHF (416 microg) in a randomized crossover design. Plasma folate was measured up to 8 h after supplementation. Concentration-time-profile [area under the curve of the plasma folate concentration vs. time (AUC)], maximum concentration (C(max)) and time-to-reach-maximum (t(max)) were calculated. KEY RESULTS: AUC and C(max) were significantly higher, and t(max) significantly shorter for [6S]-5-MTHF compared with FA in both genotypes. A significant difference between the genotypes was observed for t(max) after FA only (P < 0.05). Plasma folate consisted essentially of 5-MTHF irrespective of the folate form given. Unmetabolized FA in plasma occurs regularly following FA supplementation, but rarely with [6S]-5-MTHF. CONCLUSIONS AND IMPLICATIONS: These data suggest that [6S]-5-MTHF increases plasma folate more effectively than FA irrespective of the 677C-->T mutation of the MTHFR. This natural form of folate could be an alternative to FA supplementation or fortification.

Cellular folate vitamer distribution during and after correction of vitamin B12 deficiency: a case for the methylfolate trap.

Haematological sequellae of vitamin B12 deficiency are attributed to disturbed DNA synthesis, but vitamin B12 itself plays no role in DNA biosynthesis. A proposed explanation for this is the methylfolate trap hypothesis. This hypothesis states that B12 deficiency impairs overall folate metabolism because 5-methyltetrahydrofolate (5MTHF) becomes metabolically trapped. This trap results from the fact that 5MTHF can neither be metabolised via the methionine synthase pathway, nor can it be reconverted to its precursor, methylenetetrahydrofolate. Other manifestations of the methylfolate trap include cellular folate loss because of shorter 5MTHF polyglutamate chains and global hypomethylation. The methylfolate trap has never been demonstrated in humans. We describe a patient with B12 deficiency who was homozygous for the common methylenetetrahydrofolate reductase (MTHFR) C677T mutation. We analysed red blood cell (RBC) folate vitamers and global DNA methylation by liquid chromatography (LC) in combination with tandem mass spectrometry, and 5MTHF polyglutamate length by LC-electrochemical detection. Compared to post-B12 supplementation values, homocysteine was higher (52.9 micromol/l vs. 16.8 micromol/l), RBC folate was lower (268.92 nmol/l vs. 501.2 nmol/l), the 5MTHF fraction of RBC folate was much higher (94.5% vs. 67.4%), polyglutamate chain length was shorter (more tetra- and pentaglutamates), and global DNA methylation was 22% lower. This is the first time that virtually all features of the methylfolate trap hypothesis have been demonstrated in a human with vitamin B12 deficiency.

Analysis of polyols in urine by liquid chromatography-tandem mass spectrometry: a useful tool for recognition of inborn errors affecting polyol metabolism.

Several inborn errors of metabolism with abnormal polyol concentrations in body fluids are known to date. Most of these defects can be diagnosed by the assessment of urinary concentrations of polyols. We present two methods using tandem mass spectrometry for screening for inborn errors affecting polyol metabolism. Urine samples supplemented with internal standards ([13C4]erythritol, [13C2]arabitol and [2H3]sorbitol) were desalted by a mixed-bed ion-exchange resin. Separation was achieved by two different columns. Sugar isomers could not be separated using a Prevail Carbohydrate ES 54 column (method 1), whereas with the other column (Aminex HPX-87C) separation of the isomers was achieved (method 2). Multiple reaction monitoring polyol detection was achieved by tandem mass spectrometry with an electron ion-spray source operating in the negative mode. Age-related reference ranges of polyols (erythritol, treitol, arabitol, ribitol, xylitol, galactitol, mannitol, sorbitol, sedoheptitol and perseitol) in urine were established. The applicability of the method was demonstrated by the abnormal polyol concentrations observed in patients with transaldolase deficiency, ribose-5-phosphate isomerase deficiency and classical galactosaemia. This paper describes two methods for the analysis of urinary polyols by liquid chromatography-tandem mass spectrometry. Method 1 is a fast screening method with the quantification of total isomers and method 2 is a more selective method with the separate quantification of the polyols. Both methods can be used for diagnosing inborn errors of metabolism affecting polyol metabolism.

5-Methyltetrahydrofolic acid and folic acid measured in plasma with liquid chromatography tandem mass spectrometry: applications to folate absorption and metabolism.

We describe a liquid chromatography (LC) tandem mass spectrometry (MS-MS) method for the determination of 5-methyltetrahydrofolic acid (5-methylTHF) and folic acid concentrations and enrichments in human plasma. It was used to study absorption and initial metabolism in five volunteers with two simultaneously administered oral test doses ([(13)C(6)]folic acid in capsules and [(2)H(2)]folic acid in a drink). [(13)C(5)]5-methylTHF and [(2)H(4)]folic acid were used as internal standards. Plasma samples (2 ml) were purified using folate binding protein affinity columns, followed by a concentration step. After LC separation, folates were detected using positive electrospray ionization MS-MS under multiple reaction monitoring conditions. Calibrations were linear for 5-methylTHF over the range 1.2 x 10(-11) (=limit of detection) to 3.2 x 10(-7)mol/L and for folic acid over the range 5 x 10(-10) (=limit of detection) to 4.5 x 10(-8)mol/L. For 5-methylTHF concentration in plasma, intraassay coefficient of variation was within 8.6% (and for unlabeled 5-methylTHF it was within 2.8%) and interassay coefficient of variation was within 9.0%. For folic acid concentrations these coefficient of variations were within 7.5% and within 6.5%, respectively. The [(13)C(6)] and [(2)H(2)] isotopomers of folic acid and 5-methylTHF were measured in the plasma of each volunteer for 8h. After accounting for the time delay due to capsule opening, the modeling results showed no significant differences in absorption time, first pass effect, and elimination rate in the folic acid test doses in capsule or drink. We conclude that LC-MS-MS offers increased sensitivity for quantification of plasma concentrations and enrichments of 5-methylTHF and folic acid and is applicable to stable-isotope studies in humans.