levoleucovorin and 5-methyltetrahydrofolate

1. Folate, an essential micronutrient, is a critical cofactor in one-carbon metabolism. Mammals cannot synthesize folate and depend on supplementation to maintain normal levels. Low folate status may be caused by low dietary intake, poor absorption of ingested folate and alteration of folate metabolism due to genetic defects or drug interactions. 2. Folate deficiency has been linked with an increased risk of neural tube defects, cardiovascular disease, cancer and cognitive dysfunction. Most countries have established recommended intakes of folate through folic acid supplements or fortified foods. External supplementation of folate may occur as folic acid, folinic acid or 5-methyltetrahydrofolate (5-MTHF). 3. Naturally occurring 5-MTHF has important advantages over synthetic folic acid - it is well absorbed even when gastrointestinal pH is altered and its bioavailability is not affected by metabolic defects. Using 5-MTHF instead of folic acid reduces the potential for masking haematological symptoms of vitamin B12 deficiency, reduces interactions with drugs that inhibit dihydrofolate reductase and overcomes metabolic defects caused by methylenetetrahydrofolate reductase polymorphism. Use of 5-MTHF also prevents the potential negative effects of unconverted folic acid in the peripheral circulation. 4. We review the evidence for the use of 5-MTHF in preventing folate deficiency.

Topics: Animals; Dietary Supplements; Folic Acid; Folic Acid Deficiency; Food, Fortified; Humans; Leucovorin; Methylenetetrahydrofolate Reductase (NADPH2); Polymorphism, Genetic; Tetrahydrofolates

The use of two antidepressants from the initiation of treatment in major depressive disorder has been investigated in several recent studies and forms a paradigm shift in the pharmacotherapy of the condition. Several, but not all, trials have claimed improved response and remission rates with the combinations as opposed to monotherapy. The use of folate preparations (folic and folinic acid and l-meth-ylfolate) have shown effective augmentation of antidepressant response in a variety of controlled and open-label settings in patients with normo- and hypofolatemic status. Several recent trials using L-methylfolate, the active and more bioavailable form of folic acid, have shown promising adjunctive use with a well-tolerated adverse event profile.

Topics: Antidepressive Agents; Depressive Disorder, Major; Drug Therapy, Combination; Folic Acid; Folic Acid Deficiency; Humans; Leucovorin; Tetrahydrofolates; Treatment Outcome

Over the past decade, a syndrome consisting of low folate values in the cerebrospinal fluid (CSF) has been described. The syndrome has been associated with both genetic and acquired conditions that affect folate transport and metabolism and can result in severe neurological disorders. There is a wide range of underlying pathophysiological mechanisms, but a common feature in most patients is a good clinical response to folate therapy, especially when the syndrome is diagnosed early. In this review, we focus our attention on the genetic diseases leading to profound cerebral folate deficiency (CFD) and review current clinical, metabolic and therapeutic approaches.

Topics: Betaine; Cerebral Cortex; Folic Acid; Folic Acid Deficiency; Genetic Predisposition to Disease; Humans; Intestinal Absorption; Leucovorin; Nervous System Diseases; Tetrahydrofolates

Cerebral folate deficiency (CFD) is defined as any neurological syndrome associated with a low cerebrospinal fluid (CSF) concentration of 5-methyltetrahydrofolate (5MTHF) in the presence of normal peripheral folate status. CFD has a wide clinical presentation, with reported signs and symptoms generally beginning at around 4 months of age with irritability and sleep disturbances. These can be followed by psychomotor retardation, dyskinesia, cerebellar ataxia and spastic diplegia. Other signs may include deceleration of head growth, visual disturbances and sensorineural hearing loss. Identification of CFD is achieved by determining 5MTHF concentration in CSF. Once identified, CFD can in many cases be treated by administering oral folinic acid. Supplementation with folic acid is contraindicated and, if used, may exacerbate the CSF 5MTHF deficiency. Generation of autoantibodies against the folate receptor required to transport 5MTHF into CSF and mutations in the folate receptor 1 (FOLR1) gene have been reported to be causes of CFD. However, other mechanisms are probably also involved, as CFD has been reported in Aicardi-Goutiere's and Rett syndromes and in mitochondriopathies. Several metabolic conditions and a number of widely used drugs can also lead to a decrease in the concentration of CSF 5MTHF, and these should be considered in the differential diagnosis if a low concentration of 5MTHF is found following CSF analysis.

Topics: Administration, Oral; Autoantibodies; Brain Diseases; Dietary Supplements; Folate Receptor 1; Folic Acid Deficiency; Folic Acid Transporters; Genetic Predisposition to Disease; Humans; Leucovorin; Mutation; Risk Factors; Tetrahydrofolates; Treatment Outcome

Supplementation with folate may help reduce depressive symptoms. Folate, a naturally occurring B vitamin, is needed in the brain for the synthesis of norepinephrine, serotonin, and dopamine. Three forms of folate are commonly used: folic acid, 5-methyltetrahydrofolate (5-MTHF) (also known as methylfolate and L-methylfolate), and folinic acid. Some forms may be more bioavailable than others in patients with a genetic polymorphism and in those who take particular medications or use alcohol. Folic acid augmentation in depressed patients may reduce residual symptoms. The 5-MTHF formulation indicated efficacy as adjunctive therapy or monotherapy in reducing depressive symptoms in patients with normal and low folate levels, improving cognitive function and reducing depressive symptoms in elderly patients with dementia and folate deficiency, and reducing depressive and somatic symptoms in patients with depression and alcoholism. Adjunctive folinic acid reduced depressive symptoms in patients who were partially responsive or nonresponsive to a selective serotonin reuptake inhibitor. Evidence for the efficacy of folate in improving cognitive symptoms is equivocal, but most studies used folic acid. Although the studies reviewed have limitations and, historically, concerns have been raised about the role of folate in increasing cancer risk, masking B(12) deficiency, and worsening depressive symptoms, folate is generally well tolerated, and 5-MTHF may be less likely to incur some of these risks. Several forms of folate appear to be safe and efficacious in some individuals with major depressive disorder, but more information is needed about dosage and populations most suited to folate therapy.

Topics: Aged; Chemistry, Pharmaceutical; Clinical Trials as Topic; Dementia; Depressive Disorder, Major; Dietary Supplements; Dosage Forms; Female; Folic Acid; Folic Acid Deficiency; Humans; Leucovorin; Male; Tetrahydrofolates; Treatment Outcome

Pharmacologic rescue methods used in combination with intermediate and high-dose methotrexate therapy are reviewed, with special emphasis on rescue with nucleosides and folinic acid. The mechanism of action, pharmacokinetics, and clinical applications of the rescue agents are described in detail in view of the literature and also of the own findings of the authors. In spite of the promising results of the in vitro studies and in vivo experiments in animal models, the clinical value of thymidine as a rescue agent remains to be determined. Currently, the only indication to use thymidine instead of folinic acid following high-dose methotrexate is to prevent toxicity related to extremely high methotrexate levels in patients with delayed elimination of methotrexate. In spite of the widespread application of folinic acid rescue, the exact mechanism of its action is not fully understood. The rescue dose and schedule in the majority of clinical protocols is empirical, and the start of the rescue administration is too early, allowing less than 36 to 42 hours of exposure to methotrexate. Clinical and laboratory findings indicate that while the early start of FA administration is unnecessary for protecting normal cells, it is potentially dangerous in terms of reduction of the antitumor effect of methotrexate. Our findings suggest that less than the most widely used 12-15 mg/m2 per dose rescue may be sufficient in preventing methotrexate related toxicity in patients with normal elimination of the drug. In addition, reducing the dose of the rescue may be beneficial to achieve better therapeutic results with high-dose methotrexate. Due to methodological problems, the pharmacokinetics of folinic acid rescue has not been excessively studied in humans. Recent data indicate that the pharmacokinetics of folinic acid in children is characterized by great intra- and interpatient variability. The effect of food on the bioavailability of folinic acid has not yet been studied, though it is most frequently administered orally. The introduction of the pure l-stereoisomer of the rescue agent in the clinical practice may eliminate potential interactions with the d-isomer, and may also simplify the introduction of therapeutic drug monitoring for folinic acid as well. This could lead to more rational clinical use of folinic acid as a rescue agent following intermediate and high-dose methotrexate therapy.

Topics: Cell Survival; Drug Therapy, Combination; Humans; Leucovorin; Methotrexate; Nucleosides; Tetrahydrofolates

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.. We tested the assumption that, in humans, folic acid is biotransformed (reduced and methylated) to 5-MTHF in the intestinal mucosa.. 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.. 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).. 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.

Topics: Administration, Oral; Adult; Biotransformation; Carbon Radioisotopes; Cohort Studies; Cross-Over Studies; Dietary Supplements; Female; Folic Acid; Food, Fortified; Humans; Intestinal Mucosa; Kinetics; Leucovorin; Male; Methylation; Middle Aged; Portal Vein; Portasystemic Shunt, Transjugular Intrahepatic; Tetrahydrofolates

Calcium folinate (leucovorin), which is converted in vivo into biologically active folate, enhances the potency of 5-fluorouracil (5-FU)-based chemotherapy in colorectal cancer. A common dosage of leucovorin in adjuvant and palliative settings is 60 mg/m(2). The aim was to determine the levels of tetrahydrofolate (THF), 5,10-methylenetetrahydrofolate (methyleneTHF), and 5-methyltetrahydrofolate (methylTHF) in tumour and mucosa of colorectal cancer patients who received different dosages of leucovorin intravenously at time of surgery.. Eighty patients scheduled for colorectal resection with indication of colorectal cancer were randomised into four groups to receive leucovorin at 0, 60, 200, or 500 mg/m(2), respectively. Blood samples were taken 10 and 30 min after leucovorin administration. Biopsy samples from tumour and mucosa were collected and snap-frozen at surgery. The levels of THF, methyleneTHF, and methylTHF in tumour and mucosa were assessed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-MS/MS) and the results were related to clinical diagnosis and therapeutic regimens.. The folate levels in tissue revealed extensive inter-individual variability. The mean methyleneTHF value for the four treatment groups were 880, 1,769, 3,024 and 3,723 pmol/gww. Only half of the patients who received 60 mg/m(2) leucovorin had higher levels of methyleneTHF in tumour than patients who received 0 mg/m(2) leucovorin. Rectal cancer patients had significantly lower levels of methyleneTHF compared with colon cancer patients.. There was a large inter-patient variability of tissue folate levels in colorectal cancer patients after supplementation with leucovorin at standardised dosage. High leucovorin doses were needed to exceed baseline methyleneTHF values, especially in rectal cancer patients. The results indicate that the standardised leucovorin dose may be insufficient to attain the full antitumour effect of 5-FU. Further studies are needed to establish whether higher dosage yields a better treatment response.

Topics: Adult; Aged; Aged, 80 and over; Chromatography, Liquid; Colorectal Neoplasms; Dose-Response Relationship, Drug; Female; Humans; Leucovorin; Male; Middle Aged; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Tetrahydrofolates; Vitamin B Complex

Cerebral folate deficiency (CFD) syndrome is a neurodevelopmental disorder typically caused by folate receptor autoantibodies (FRAs) that interfere with folate transport across the blood-brain barrier. Autism spectrum disorders (ASDs) and improvements in ASD symptoms with leucovorin (folinic acid) treatment have been reported in some children with CFD. In children with ASD, the prevalence of FRAs and the response to leucovorin in FRA-positive children has not been systematically investigated. In this study, serum FRA concentrations were measured in 93 children with ASD and a high prevalence (75.3%) of FRAs was found. In 16 children, the concentration of blocking FRA significantly correlated with cerebrospinal fluid 5-methyltetrahydrofolate concentrations, which were below the normative mean in every case. Children with FRAs were treated with oral leucovorin calcium (2 mg kg(-1) per day; maximum 50 mg per day). Treatment response was measured and compared with a wait-list control group. Compared with controls, significantly higher improvement ratings were observed in treated children over a mean period of 4 months in verbal communication, receptive and expressive language, attention and stereotypical behavior. Approximately one-third of treated children demonstrated moderate to much improvement. The incidence of adverse effects was low. This study suggests that FRAs may be important in ASD and that FRA-positive children with ASD may benefit from leucovorin calcium treatment. Given these results, empirical treatment with leucovorin calcium may be a reasonable and non-invasive approach in FRA-positive children with ASD. Additional studies of folate receptor autoimmunity and leucovorin calcium treatment in children with ASD are warranted.

Topics: Adolescent; Autoantibodies; Child; Child Development Disorders, Pervasive; Child, Preschool; Female; Folate Receptor 1; Humans; Leucovorin; Male; Tetrahydrofolates; Vitamin B Complex

Rett syndrome is a neurodevelopmental disorder in girls, related to mutations in MECP2 gene. It has been postulated that low 5-methyltetrahydrofolate (5-MTHF) levels are present in cerebrospinal fluid. Folinic acid demonstrated clinical improvement. However, because studies have produced conflicting results, we performed a randomized, double-blind crossover, long-term, follow-up study on folinic acid. Eight Rett syndrome patients received both folinic acid and placebo, for 1 year each. Measurements included plasma folate, 5-MTHF, and clinical outcome scores like Rett Syndrome Motor Behavioral Assessment, Hand Apraxia Scale, and the parental Overall Well-Being Index. In 2 patients, low 5-MTHF levels were present. Folinic acid supplementation increased cerebrospinal fluid 5-MTHF levels, but with no objective evidence of clinical improvement. The Overall Well-Being Index showed a significant difference in favor of folinic acid, not confirmed objectively. In our double-blind randomized study, folinic acid supplementation resulted in increased 5-MTHF levels, but with no objective signs of clinical improvement.

Topics: Adolescent; Adult; Child; Child, Preschool; Cross-Over Studies; Dietary Supplements; Double-Blind Method; Female; Folic Acid; Humans; Leucovorin; Linear Models; Longitudinal Studies; Methyl-CpG-Binding Protein 2; Mutation; Neurologic Examination; Rett Syndrome; Tetrahydrofolates; Time Factors; Treatment Outcome; Young Adult

UFT is composed of tegafur (FT), a prodrug of 5-fluorouracil (5-FU), and uracil in a fixed combination (1:4). In conjunction with leucovorin, UFT is being developed for the first-line oral treatment of metastatic colorectal cancer. The effect of food on the oral bioavailability of UFT (2 x 100 mg capsules; dose in terms of FT) and leucovorin (2 x 15 mg tablets) was evaluated in a single-dose, randomized, two-way crossover study. Patients (n = 25) were assigned to receive both drugs after an overnight fast or 5 min after completion of a high-fat meal (721 calories) with a 3-day washout period between treatments; then they were permitted to continue on oral UFT/leucovorin therapy for safety assessment. UFT (300 mg/m2/day as three divided doses) and leucovorin (90 mg/day as three divided doses) were given for 28 days. After a 7-day rest, the 28-day cycle was repeated. Pharmacokinetics (n = 22 patients) were determined for FT, 5-FU, uracil, leucovorin, and 5-methyltetrahydrofolate (an active metabolite of leucovorin). The absence of food-effect on peak plasma concentration (CMAX) and the area under the curve (AUC) was concluded if the 90% confidence interval for the ratio of the treatment means was entirely contained in 0.75-1.33. Administration of UFT with food resulted in a 34% decrease in CMAX of FT, whereas the AUC of FT remained unchanged. Food decreased the CMAX and AUC values of uracil and 5-FU by 37-76%. On the contrary, the CMAX and AUC values of leucovorin and 5-methyltetrahydrofolate were increased by 14-60% with food. Time to reach CMAX for all analytes was significantly (P < or = 0.001) delayed by food. Except for the AUCs of FT, the statistical criterion for concluding a lack of food-effect was not met. These data suggest that UFT/leucovorin should not be dosed simultaneously with food. It is recommended that food should not be consumed for 1 h before and after an oral dose of UFT and leucovorin in a manner similar to pivotal Phase III trials. The 28-day oral regimen of UFT and leucovorin was generally well tolerated in the population studied.

Topics: Administration, Oral; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Biological Availability; Colorectal Neoplasms; Cross-Over Studies; Dose-Response Relationship, Drug; Drug Combinations; Fasting; Female; Fluorouracil; Food; Humans; Leucovorin; Male; Middle Aged; Tegafur; Tetrahydrofolates; Time Factors; Uracil

Effective correction of hyperhomocysteinemia in hemodialysis patients by intravenous folinic acid and pyridoxine therapy.. Folic acid supplementation is only partially efficacious in correcting moderate elevation of plasma total homocysteine (tHcy) concentrations observed in hemodialysis (HD) patients. Experimental and clinical data have suggested that this partial efficacy may be due to impairment of folic acid metabolism to 5-methyltetrahydrofolate (MTHF) and of MTHF transmembrane transport as well. To bypass these difficulties, we assessed the efficacy of intravenous (i.v.) folinic acid, a ready precursor of MTHF, on reducing plasma tHcy concentrations in HD patients.. In a cohort of 37 patients on intermittent HD treatment, plasma tHcy concentrations were determined before and during i.v. supplementation of folinic acid (50 mg once per week), together with i.v. pyridoxine (250 mg 3 times per week), to prevent vitamin deficiency, particularly in those treated by recombinant erythropoietin.. Folinic acid and pyridoxine i.v. supplementation was given for 11.2 +/- 2.45 months (range 7.5 to 17 months). The mean plasma tHcy levels decreased significantly from 37. 3 +/- 5.8 microM at baseline to 12.3 +/- 5.4 microM on folinic acid treatment (P < 0.001). Moreover, 29 of the 37 patients (78%) had normal plasma tHcy levels at the end of follow-up (that is, <14.1 microM, mean 9.8 microM, range 6.2 to 13 microM). No adverse effects attributable to folinic acid treatment were observed during this time.. Intravenous folinic acid therapy (50 mg) once per week associated with pyridoxine supplementation appears to be an effective and safe strategy to normalize plasma tHcy levels in the majority of chronic HD patients.

Topics: Adult; Aged; Erythrocytes; Female; Humans; Hyperhomocysteinemia; Injections, Intravenous; Kidney Failure, Chronic; Leucovorin; Male; Middle Aged; Pyridoxine; Renal Dialysis; Tetrahydrofolates; Vitamin B 12

To investigate the hypothesis that methotrexate causes demyelination due to a deficiency in S-adenosylmethionine (SAM) during the treatment of acute lymphoblastic leukemia (ALL).. Twenty-four patients treated on the Medical Research Council United Kingdom ALL trial no. 11 (MRC UKALL XI) were studied. The trial randomized patients at the presymptomatic CNS treatment (PCNS) phase to receive (1) intrathecal methotrexate and cranial radiotherapy (CRTX); (2) high-dose intravenous methotrexate with folinic acid rescue and continuing intrathecal methotrexate (HDMTX); and (3) continuing intrathecal methotrexate alone (ITMTX). Serial CSF samples were collected throughout treatment and concentrations of 5-methyltetrahydrofolate (MTF), methionine (MET), SAM, and myelin basic protein (MBP) were measured. The results were grouped into treatment milestones and compared with an age-matched reference population.. There was a highly significant effect of both treatment milestones and trial arm on the metabolite and MBP concentrations. CSF MTF reached a nadir during the induction phase of treatment, while SAM and MET reached their nadir during the consolidation phase. CSF MBP mirrored SAM concentration and there was a significant inverse relationship between the two. MTF, SAM, and MBP returned to normal values by the end of treatment, while MET was increased significantly. The effect of treatment was decremental across the ITMTX, HDMTX, and CRTX groups.. Treatment of ALL causes marked abnormalities in the single-carbon transfer pathway and subclinical demyelination. Methotrexate is one cause of this. Whether these abnormalities contribute to the late cognitive deficits requires further study.

Topics: Antidotes; Antimetabolites, Antineoplastic; Central Nervous System; Child; Child, Preschool; Combined Modality Therapy; Demyelinating Diseases; Drug Interactions; Humans; Infant; Leucovorin; Methionine; Methotrexate; Myelin Basic Protein; Precursor Cell Lymphoblastic Leukemia-Lymphoma; S-Adenosylmethionine; Tetrahydrofolates

The phase I development program of tegafur and uracil (UFT) in the United States has included evaluation of the drug as a single agent and subsequent studies of its biochemical modulation by oral leucovorin. Phase I trials of single-agent UFT examined both a 5-day schedule repeated every 21 days and a 28-day schedule repeated every 35 days. In all of the trials the total dose was divided by three and administered three times daily at 8-hour intervals. Like intravenous schedules of fluorouracil (5-FU), UFT has schedule-dependent toxicity, with granulocytopenia being the dose-limiting toxicity for the 5-day regimen and diarrhea being the dose-limiting toxicity for the 28-day regimen. The suggested phase II doses for UFT administered without leucovorin were 800 mg/m2/day for the 5-day schedule and 360 mg/m2/day for the 28-day schedule. Subsequent phase I studies combining UFT with oral leucovorin used a 28-day schedule repeated every 35 days. Diarrhea was the dose-limiting toxicity, and the recommended phase II dose was UFT, 300 mg/m2/day, plus leucovorin, 90 mg/day. Pharmacokinetic evaluation showed that single-dose UFT results in maximum plasma levels and an area under the concentration-time curve that increased with escalating UFT doses. In addition, 5-FU levels were detectable throughout the 28-day dosing period; however, there was no evidence of significant accumulation of uracil, tegafur, or 5-FU. The administration of leucovorin in this trial provided continuous exposure of d,l-leucovorin and 5-methyltetrahydrofolate with little variation between doses or days.

Topics: Administration, Oral; Antidotes; Antineoplastic Combined Chemotherapy Protocols; Clinical Trials, Phase I as Topic; Drug Combinations; Fluorouracil; Humans; Leucovorin; Neoplasms; Tegafur; Tetrahydrofolates; Uracil

A phase I and pharmacokinetics study of oral uracil, ftorafur, and leucovorin was performed in patients with advanced cancer. Uracil plus ftorafur (UFT) was given in a 4:1 molar ratio in three divided doses for 28 consecutive days. Patient cohorts were treated at 200, 250, 300, and 350 mg/m2 of UFT daily. For all patients, 150 mg of leucovorin was given daily in three oral doses. A 1-week rest period followed each 28-day treatment course. Gastrointestinal toxicity, characterized by diarrhea, nausea, and vomiting, was dose-limiting at 350 mg/m2 UFT in patients who had received prior chemotherapy. Mild fatigue and transient hyperbilirubinemia were also common. In previously untreated patients, UFT at 350 mg/m2 was well-tolerated, suggesting this as an acceptable phase II dose in this schedule with leucovorin. Two of eight previously untreated patients with advanced colorectal cancer had partial responses with UFT (350 mg/m2) plus leucovorin. Pharmacokinetic parameters [ftorafur, uracil, 5-fluorouracil (5-FU), 5-methyltetrahydrofolate] showed wide interpatient variations. Plasma levels of 5-FU (Cmax 1.4 +/- 1.9 microM) were comparable to those achieved with protracted venous infusions, and folate levels (Cmax 6.1 +/- 3.6 microM) were sufficient for biochemical modulation. Ongoing study will determine if this convenient oral regimen will compare favorably in terms of efficacy, toxicity, and cost with intravenous fluoropyrimidine programs.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Female; Humans; Hyperbilirubinemia; Leucovorin; Male; Middle Aged; Tegafur; Tetrahydrofolates; Uracil

The purpose of this study was to test the hypothesis that 5-methyltetrahydrofolate (Me-THF), a source of reduced folates alternative to leucovorin, could effectively modulate 5-fluorouracil's (5-FU) cytotoxic activity in patients with advanced colon cancer. A total of 23 patients were enrolled in a phase 11 trial; they received 5-FU as a 30-min infusion at a dose of 370 mg/m2 following a rapid i.v. push of 200 mg/m2 Me-THF, both drugs being given for 5 consecutive days. Cycles were repeated every 4 weeks until disease progression. No patient achieved a complete response. In all, 4 patients showed a partial response (17.4%), 7 developed stable disease (30.4%), and the remaining 12 (52.2%) progressed. Toxicity was acceptable and never exceeded WHO grade III intensity. According to our experience, the MeTHF/5-FU combination does not appear to be an effective treatment for advanced colon cancer. Despite its low toxic profile, in our opinion its wider use should be discouraged.

Topics: Adult; Aged; Antidotes; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cell Death; Colonic Neoplasms; Female; Fluorouracil; Humans; Leucovorin; Male; Middle Aged; Neoplasm Metastasis; Tetrahydrofolates

To optimize the biochemical modulation of fluorouracil (5-FU), we administered the pure I-stereoisomer of leucovorin (LV) as a 132-hour continuous intravenous infusion (CIV) with cisplatin 100 mg/m2, 5-FU 640 mg/m2/d as a 120-hour CIV, and interferon alfa-2b (IFN) at 2 MU/m2/d for 6 days for three cycles (I-PFL-IFN). Pharmacologic parameters included morning (AM) and afternoon (PM) plasma concentrations of 5-FU, LV and its active metabolite 5-methyl tetrahydrofolate (MTHF), and dihydropyrimidine dehydrogenase (DPD) activity in peripheral mononuclear cells.. Eighty-nine patients were treated (86 stage IV). Neutropenia and mucositis were the most common toxicities. Sixty-six percent achieved a complete remission (CR). There was a trend for higher PM versus AM 5-FU concentrations (median, 1.64 v 1.51 mumoles/L; P = .08), but not for LV plus MTHF (P = .66). The mean +/- SD DPD activity was 0.21 +/- 0.14 nmol/min/mg and did not correlate with plasma concentrations of 5-FU or LV plus MTHF or clinical toxicities. Higher PM 5-FU concentrations correlated with worse leukopenia (P = .04) and severity of mucositis (P = .04). PM 5-FU concentration was higher in women than in men (P = .07), with no apparent difference in severity of toxicities. The maximum 5-FU concentration was higher in CR than non-CR patients (median, 2.01 v 1.54 mumoles/L; P = .02) and higher in women than men who achieved a CR (median, 2.77 v 1.91 mumoles/L; P = .03). No correlation of CR with dose-intensity was found.. L-PFL-IFN is active in stage IV head and neck cancer. 5-FU concentration is a significant predictor of toxicity. In women, optimization of response outcome requires a higher 5-FU concentration. Individualized 5-FU dosing to obtain higher 5-FU plasma concentrations may be indicated.

Topics: Adolescent; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cisplatin; Female; Fluorouracil; Head and Neck Neoplasms; Humans; Interferon alpha-2; Interferon-alpha; Leucovorin; Male; Middle Aged; Neoplasms, Squamous Cell; Recombinant Proteins; Stereoisomerism; Tetrahydrofolates

To analyze clinical and pharmacokinetic data of cisplatin (CP)/fluorouracil (FU)/l folinic acid (l FA) chemotherapy administered as first-line treatment to locally advanced head and neck cancer patients.. Thirty-nine patients (35 men and four women; median age, 60 years; six stage III and 33 stage IV) received CP on day 1 (100 mg/m2) followed by l FA (200 mg/m2/d x 5) plus FU (500 mg/m2/d x 5) administered by continuous venous infusion (three cycles planned). Mean plasma concentrations of FU, l FA, and 5-methyltetrahydrofolate (5MTHF) over the cycle were computed.. Clinical response was assessable for 33 patients. Response rates on the primary tumor site (n = 33) were 63.7% complete responses (CRs), 24.2% partial responses (PRs), and 12.1% treatment failures. Response rates on lymph nodes (n = 27) were 40.7% CRs, 37.1% PRs, and 22.2% treatment failures. The most frequent toxicity was mucositis (36.2% of cycles grade 3 to 4). Grade 3 to 4 nausea-vomiting, diarrhea, neutropenia, and thrombocytopenia occurred in 6.7%, 1.9%, 13.3%, and 1% of cycles, respectively. Pharmacokinetic analysis showed a wide interpatient variability for both FU (mean, 1.01 mumol/L; range, 0.16 to 2.09), l FA (mean, 1.89, mumol/L; range, 0.52 to 7.88) and 5MTHF plasma concentrations (mean, 3.85 mumol/L; range, 1.30 to 8.11). A significant correlation was demonstrated between FU concentration and hematologic toxicity grade, mucositis grade, and nausea-vomiting/diarrhea grade. Regarding tumor response, patients who failed to respond significantly exhibited lower FU and total folate concentrations than patients with a CR or PR.. This study highlights the efficacy of CP/FU/l FA in head and neck carcinoma and establishes the clinical importance of coupled FU/FA pharmacokinetics to predict pharmacodynamic variability.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cisplatin; Female; Fluorouracil; Humans; Hypopharyngeal Neoplasms; Laryngeal Neoplasms; Leucovorin; Male; Middle Aged; Nose Neoplasms; Oropharyngeal Neoplasms; Tetrahydrofolates

Pharmacokinetics of total platinum, 5-fluorouracil, l-folinic and d-folinic acid, and 5-methyltetrahydrofolate were studied in plasma from nine patients with advanced colorectal cancer treated with oxaliplatin (20 mg/m2/day), 5-fluorouracil (600 mg/m2/day), and folinic acid (300 mg/m2/day). Drugs were administered with a programmable-in-time pump by continuous infusion for 5 days. We compared two drug delivery schedules: constant rate versus chronomodulated rate with peak of oxaliplatin at 4 pm and peak of 5-fluorouracil and folinic acid at 4 am. In the chronomodulated schedule, plasma concentrations of the drugs paralleled the pump functioning: maximum platinum concentration near 4 pm, and maximum 5-fluorouracil and folate concentrations near 4 am. When drugs were administered at a constant rate, mean plasma concentration of 5-fluorouracil varied in a circadian manner each treatment day, that is, a peak at 4 am (approximately 800 ng/ml) and a trough at 1 pm (approximately 100 ng/ml). Mean plasma levels of total platinum and folate compounds increased over the first 24 hours. Total platinum mean level and that of the inactive d-folinic acid isomer reached a constant plasma concentration, whereas biologically active folates exhibited circadian variation in their plasma concentrations (peak around 7 am, trough near 6 pm, and amplitude approximately 10%). Severe mucositis was exhibited by all four patients on the flat schedule, but only by one on the chronomodulated schedule (p < 0.008). Individual pharmacokinetic and toxicity data showed that patients with circadian rhythms in 5-fluorouracil concentrations were least sensitive to 5-fluorouracil-related toxicity. Thus amplification or induction of such rhythm in 5-fluorouracil exposure may permit dose escalation.

Topics: Adult; Aged; Analysis of Variance; Antineoplastic Combined Chemotherapy Protocols; Circadian Rhythm; Colorectal Neoplasms; Female; Fluorouracil; Folic Acid; Humans; Infusion Pumps; Leucovorin; Male; Middle Aged; Mouth Mucosa; Organoplatinum Compounds; Oxaliplatin; Stereoisomerism; Stomatitis; Tetrahydrofolates

1. The pharmacokinetics of [R]-leucovorin ([R]-LV), [S]-leucovorin ([S]-LV) and the circulating metabolite [S]-5-methyltetrahydrofolate ([S]-5-MTHF) were studied after administration of racemic LV and [S]-LV in 21 subjects. 2. After intravenous infusion of 600 mg m-2 rac-LV (group 1, n = 7) or 300 mg m-2 [S]-LV (group 3, n = 7), the decay of [S]-LV in plasma was biexponential with a distribution half-life of 0.8 to 1 h and an elimination half-life of 11 to 23 h. When rac-LV was administered as a 2 h i.v. infusion (400 mg m-2) following a loading dose of 200 mg m-2 (group 2, n = 7), the plasma concentrations of [R]-LV and [S]-5-MTHF decayed monoexponentially with mean (+/- s.d.) half-lives of 10 +/- 3 h and 7 +/- 2 h, respectively. 3. The AUC of [S]-5-MTHF was significantly higher after infusion of 300 mg m-2 [S]-LV than after infusion of 600 mg m-2 rac-LV (83 +/- 22 micrograms ml-1 h vs 53 +/- 22 micrograms ml-1 h; P = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Aged; Female; Gastrointestinal Neoplasms; Half-Life; Hepatic Artery; Humans; Infusions, Intra-Arterial; Infusions, Intravenous; Injections, Intravenous; Leucovorin; Male; Middle Aged; Stereoisomerism; Tetrahydrofolates

The bioequivalence and bioavailability of oral and intravenous formulations of levoleucovorin and leucovorin were studied, and the absolute bioavailabilities of levoleucovorin and leucovorin tablet formulations were determined. Healthy male volunteers participated in two randomized, single-dose, four-way crossover studies. The treatment groups were as follows: A = five 2.5-mg levoleucovorin tablets, B = five 5-mg leucovorin tablets, C = one 12.5-mg levoleucovorin tablet, D = one 25-mg leucovorin tablet, in study 1; A = 15-mg levoleucovorin injection, B = two 7.5-mg levoleucovorin tablets, C = 30-mg leucovorin injection, D = two 15-mg leucovorin tablets, in study 2. Serum concentrations of N-5-methyltetrahydrofolate (the primary metabolite and circulating form of reduced folate after leucovorin administration) and total tetrahydrofolate were measured over 24 hours after dose administration. Pharmacokinetic values were calculated for N-5-methyltetrahydrofolate and total tetrahydrofolates; values were compared for A versus B, C versus D, A versus C, and B versus D in study 1 and A versus C and B versus D in study 2. Results from 35 men in study 1 and 33 in study 2 showed that 12.5-mg oral doses and 15-mg intravenous doses of levoleucovorin are bioequivalent to 25-mg oral doses and 30-mg intravenous doses of leucovorin, respectively. Equivalence was observed after oral and intravenous administration. The absolute bioavailability of levoleucovorin (74%) was not significantly different from that of leucovorin (65%).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Administration, Oral; Adult; Biological Availability; Humans; Injections; Leucovorin; Male; Stereoisomerism; Tetrahydrofolates; Therapeutic Equivalency

Studies have shown that conversion of leucovorin to the metabolite 5,10-methylenetetrahydrofolate (5,10-CH2FH4) is responsible for enhancement of the antitumor effects of fluorouracil given in combination with leucovorin, but the biochemical basis of this conversion in humans is not fully understood. To determine a possible sequence of metabolic steps, we studied the pharmacokinetics of leucovorin and its reduced folate metabolites in plasma in healthy volunteers. Groups of five subjects were given two equal doses of 10, 25, 125, 250, or 500 mg/m2 leucovorin, one orally and one intravenously at a 30-day interval. A sensitive radioenzymatic method that we developed previously was used to measure plasma concentrations of [S]5-formyltetrahydrofolate, 10-formyltetrahydrofolate (10-CHOFH4), 5-methyltetrahydrofolate (5-CH3FH4), and the combined 5,10-CH2FH4 plus tetrahydrofolate (FH4) pools. Intravenous administration of leucovorin resulted in dose-dependent accumulation of 5,10-CH2FH4 + FH4 exceeding 2 microM at peak levels. After oral and intravenous administration, 10-CHOFH4 and 5,10-CH2FH4 + FH4 exhibited peak levels earlier and were eliminated more rapidly than 5-CH3FH4. Accumulation of all metabolites after intravenous administration was linearly dose dependent, while oral administration appeared to result in saturation. We propose that the host activation of leucovorin suggested by these findings could be responsible for elevation of intratumor 5,10-CH2FH4 levels, thus enhancing the antitumor effects of fluorouracil. These results also suggest that 10-CHOFH4, 5,10-CH2FH4, and FH4 are intermediate metabolites and that 5-CH3FH4 is the terminal metabolite. In addition, our results indicate that attainment of high plasma levels of the metabolites active in modulation of the therapeutic effects of fluorouracil is best achieved through intravenous administration of high doses of leucovorin. Our future studies will address the proposed sequential conversion pathway and, thus, the mechanism by which pharmacologically relevant reduced folates accumulate in plasma after leucovorin administration.

Topics: Administration, Oral; Adult; Female; Humans; Injections, Intravenous; Leucovorin; Male; Random Allocation; Tetrahydrofolates; Time Factors

Because of the synergy seen in adult trials when 5-fluorouracil is combined with leucovorin, we initiated a Phase I trial of this combination in children's refractory cancer. Leucovorin, an equal mixture of the (6R,S)-diastereoisomers, was administered p.o. for 6 consecutive days as 4 equal doses at 0, 1, 2, and 3 h totaling 500 mg/m2/day. 5-Fluorouracil was given daily on days 2 to 6 as an i.v. bolus immediately following the last dose of leucovorin. The leucovorin dose was held constant while the 5-fluorouracil dose was escalated in cohorts of patients from 300 mg/m2/day to its maximally tolerated dose. Thirty-five patients (19 with acute leukemia and 16 with solid tumors) were evaluable for toxicity. The maximally tolerated dose of FUra was 450 mg/m2/day for 5 treatments for patients with solid tumors and 650 mg/m2/day for 5 treatments for the children with leukemia. The dose-limiting toxicities were myelosuppression and stomatitis. Other side effects included transient, mild elevations of serum transaminases, mild nausea, vomiting, and diarrhea. The pharmacokinetics of high-dose p.o. leucovorin was studied in 23 children. There was considerable interpatient variability in the plasma concentrations of total bioactive folates (TBAF), (6S)-leucovorin, and (6S)-5-methyltetrahydrofolic acid. The maximum plasma concentration (Cmax) of TBAF was 821 +/- 97 (SE) nM, occurring at a median of 8 h; the Cmax of (6S)-leucovorin was 77 +/- 11 nM, occurring at 4 h. The TBAF concentration fell to 146 +/- 42 nM by 24 h. (6S)-5-Methyltetrahydrofolic acid accounted for 90 +/- 7% of the TBAF at the Cmax. The plasma concentration of (6R)-leucovorin, the unnatural isomer, was equal to that of TBAF. Thus, p.o. leucovorin reduced the 5-fold excess of (6R)-leucovorin over TBAF seen after i.v. doses. The relative amounts of the three major plasma species were approximately the same as in adults, even though the Cmax of each compound was lower.

Topics: Administration, Oral; Adolescent; Adult; Child; Child, Preschool; Drug Administration Schedule; Drug Evaluation; Drug Synergism; Drug Therapy, Combination; Female; Fluorouracil; Humans; Infant; Leucovorin; Male; Neoplasms; Stereoisomerism; Tetrahydrofolates

Eleven patients treated with a 5.5-day continuous i.v. infusion of 500 mg/m2/day of (6R,S)-folinic acid in combination with daily bolus 5-fluorouracil had a median steady-state plasma concentration of 3.25 microM (6S)-folinic acid (the bioactive diastereoisomer). The bioactive metabolite (6S)-5-methyltetrahydrofolic acid, analyzed in six patients, reached a median steady-state plasma concentration of 5.7 microM. The lowest plasma concentrations at steady-state were 1.86 microM (6S)-folinic acid and 3.12 microM (6S)-5-methyltetrahydrofolic acid. These concentrations are above the minimum concentrations shown by other investigators to produce synergism between (6R,S)-folinic acid and 5-fluorouracil in vitro. The median steady-state plasma concentration of (6R)-folinic acid was 38.2 microM, more than 10 times the concentration of (6S)-folinic acid. Along with other plasma pharmacokinetic parameters, terminal half-lives were estimated for (6S)-folinic acid (median, 45.4 min), (6R)-folinic acid (median, 388 min), and (6S)-5-methyltetrahydrofolic acid (median, 446 min). Investigation of the renal pharmacokinetics confirmed the marked difference in the renal clearance of the two diastereoisomers of folinic acid which had been observed after low doses of (6R,S)-folinic acid (J. A. Straw, D. Szapary, and W. T. Wynn, Cancer Res., 44: 3114-3119, 1984). However, the low renal clearance of (6R)-folinic acid (median, 8.2 ml/min/m2) was attributable to the extensive binding of (6R)-folinic acid to plasma proteins (median, 8.7% free), not to reabsorption in the kidney.

Topics: Clinical Trials as Topic; Dose-Response Relationship, Drug; Fluorouracil; Humans; Infusions, Intravenous; Kidney; Leucovorin; Metabolic Clearance Rate; Protein Binding; Stereoisomerism; Tetrahydrofolates

In this study, 50 patients were randomly assigned to treatment with 5-fluorouracil (FUra) or FUra plus high-dose continuous-infusion folinic acid. Five of 27 evaluable patients in the FUra group versus 10 of 21 patients in the FUra plus folinic acid arm of the study had objective partial remissions, P = 0.02. Time to progression was 3.9 months for FUra and 8.0 months for FUra and folinic acid, P = 0.006; however, median survivals (11.9 versus 14.5 months) were not different in this crossover study. Toxicity in both treatment arms was mild, although patients receiving FUra plus folinic acid experienced significantly more stomatitis than patients treated with FUra alone. This study suggests that high-dose, continuous-infusion folinic acid, which produces a steady-state level of biologically active folates of 10 microM, significantly increases the therapeutic activity of FUra.

Topics: Adenocarcinoma; Biological Availability; Colonic Neoplasms; Fluorouracil; Hematopoiesis; Humans; Infusions, Intravenous; Leucovorin; Rectal Neoplasms; Tetrahydrofolates

Hyperhomocysteinemia is recognized as a risk factor for several diseases and conditions. The aim of this study was to investigate and compare the efficacy of two total homocysteine (tHcy)-lowering treatments including folinic acid or l-methylfolate in healthy Greek adults.. Two hundred and seventy-two healthy Greek adults (143 men, 129 women; mean age±SD: 43.0 ± 15.3 years), with serum tHcy levels ≥10 μmol/L received randomized folinic acid ("Folinic acid Group") or l-methylfolate ("l-methylfolate Group") orally for three months. All subjects with serum cobalamin (Cbl) levels <300 pg/mL additionally received 1 mg hydroxycobalamine intramuscularly twice a week for the first month only. Serum folate, Cbl and tHcy levels were determined using immunoassays methods at the beginning and the end of the study period. The MTHFR C677T and MTHFR A1298C gene polymorphisms were genotyped using polymerase chain reaction and reverse hybridization.. At the end of the 3-month intervention period, the levels of serum folate and Cbl increased significantly, whereas the levels of serum tHcy decreased significantly in the two groups. The individuals with MTHFR 677TT genotype had a significantly higher reduction in serum tHcy levels than the individuals with the MTHFR 677CC or MTHFR 677CT genotypes. Although the "Folinic acid Group" had a considerably higher increase in their serum folate levels (but not Cbl) than the "l-methylfolate Group", the reduction of serum tHcy levels between the two groups was not substantially different. The individuals with MTHFR 677CT genotype had a statistically significant higher reduction in serum tHcy levels when supplemented with folinic acid rather than l-methylfolate.. The administration of folinic acid compared to l-methylfolate caused a higher increase of serum folate levels but no difference in the reduction of serum tHcy levels. The reduction of serum tHcy levels was influenced by the existence of MTHFR C677T and not MTHFR A1298C gene polymorphisms. The individuals with MTHFR 677CT genotype appear to benefit more by folinic acid than l-methylfolate supplementation.

Topics: Adult; Dietary Supplements; Female; Folic Acid; Homocysteine; Humans; Leucovorin; Male; Methylenetetrahydrofolate Reductase (NADPH2); Middle Aged

Folate (vitamin B

Topics: Animals; Antioxidants; Epithelial Cells; Folic Acid; Glucose; Inflammation; Leucovorin; Oxidative Stress; Tetrahydrofolates

Red, white, blue, green, and yellow lights were applied to investigate their effects on folate accumulation in wheat seedlings. The different lights, especially red light, significantly increased the total folate content. Total folate showed maximum accumulation under 30 μmol/(m

Topics: Folic Acid; Germination; GTP Cyclohydrolase; Leucovorin; Light; Seedlings; Tetrahydrofolates; Transaminases; Triticum

Topics: Cadherins; Child; Epileptic Syndromes; Female; Folic Acid Deficiency; Humans; Leucovorin; Pedigree; Protocadherins; Tetrahydrofolates; Treatment Outcome; Vitamin B Complex

Topics: Chromatography, Thin Layer; Fabaceae; Folic Acid; Food Analysis; Germination; Image Processing, Computer-Assisted; Lens Plant; Leucovorin; Mass Spectrometry; Molecular Imaging; Multivariate Analysis; Reproducibility of Results; Seeds; Tetrahydrofolates

Cerebral folate deficiency (CFD) is a neurological disease, hallmarked by remarkable low concentrations of 5-methyltetrahydrofolic acid (5-MTHF) in cerebrospinal fluid (CSF). The primary causes of CFD include the presence of folate receptor (FR) autoantibodies, defects of FR encoding gene FOLR1, mitochondrial diseases and congenital abnormalities in folate metabolism.. Here we first present a Chinese male CFD patient whose seizure onset at 2 years old with convulsive status epilepticus. Magnetic Resonance Imaging (MRI) revealed the development of encephalomalacia, laminar necrosis in multiple lobes of the brain and cerebellar atrophy. Whole Exome Sequencing (WES) uncovered a homozygous missense variant of c.524G > T (p.C175F) in FOLR1 gene. Further laboratory tests demonstrated the extremely low level of 5-MTHF in the CSF from this patient, which was attributed to cerebral folate transport deficiency. Following the intravenous and oral treatment of calcium folinate, the concentrations of 5-MTHF in CSF were recovered to the normal range and seizure symptoms were relieved as well.. One novel variation of FOLR1 was firstly identified from a Chinese male patient with tonic-clonic seizures, developmental delay, and ataxia. The WES and laboratory results elucidated the etiology of the symptoms. Clinical outcomes were improved by early diagnosis and proper treatment.

Topics: Age of Onset; Cerebral Cortex; Child; Encephalomalacia; Exome Sequencing; Folate Receptor 1; Folic Acid Deficiency; Homozygote; Humans; Leucovorin; Magnetic Resonance Imaging; Male; Seizures; Status Epilepticus; Tetrahydrofolates

The cofactor tetrahydrofolate (THF) is used to reduce, oxidize, and transfer one-carbon (1C) units required for the synthesis of nucleotides, glycine, and methionine. Measurement of intracellular THF species is complicated by their chemical instability, signal dilution caused by variable polyglutamation, and the potential for interconversion among these species. Here, we describe a method using negative mode liquid chromatography-mass spectrometry (LC-MS) to measure intracellular folate species from mammalian cells. Application of this method with isotope-labeled substrates revealed abiotic interconversion of THF and methylene-THF, which renders their separate quantitation particularly challenging. Chemical reduction of methylene-THF using deuterated sodium cyanoborohydride traps methylene-THF, which is unstable, as deuterated 5-methyl-THF, which is stable. Together with proper sample handling and LC-MS, this enables effective measurements of five active folate pools (THF, 5-methyl-THF, methylene-THF, methenyl-THF/10-formyl-THF, and 5-formyl-THF) representing the biologically important 1C oxidation states of THF in mammalian cells. Graphical abstract Chemical derivatization with deuterated cyanoborohydride traps unstable methylene-THF as isotope-labeled 5-methyl-THF, enabling accurate quantification by LC-MS.

Topics: Cell Culture Techniques; Chromatography, Liquid; Folic Acid Antagonists; HEK293 Cells; Humans; Leucovorin; Mass Spectrometry; Methotrexate; Tetrahydrofolates

Cerebral folate transporter deficiency caused by FOLR-1 mutations has been described in 2009. This condition is characterized by a 5MTHF level <5 nmol/l in the CSF, along with regression of acquisition in the second year of life, ataxia, and refractory myoclonic epilepsy. Oral or intravenous folinic acid (5-formyltetrahydrofolate) treatment has been shown to improve clinical status.. We present the cases of two sisters with cerebral folate transport deficiency caused by mutation in the folate receptor 1 (FOLR1) gene (MIM *136430). Following recommendations, we administered oral folinic acid at 5 mg/kg/day, resulting in some initial clinical improvement, yet severe epilepsy persisted. During treatment, cerebrospinal fluid (CSF) analysis revealed normal 5-methyltetrahydrofolate (5MTHF) levels (60.1 nmol/l; normal range: 53-182 nmol/l). Epilepsy proved difficult to control and the younger patient exhibited neurological regression. We then administered high-dose folinic acid intravenously over 3 days (6 mg/kg/day for 24 h, then 12 mg/kg/day for 48 h), which significantly improved clinical status and epilepsy. CSF analysis revealed high 5MTHF levels following intravenous infusion (180 nmol/l). Treatment continued with monthly intravenous administrations of 20-25 mg/kg folinic acid. At 2 years post-treatment, clinical improvement was confirmed.. This report illustrates that cerebral folate transporter deficiency caused by FOLR-1 mutations is a treatable condition and can potentially be cured by folinic acid treatment. As already reported, early effective treatment is known to improve outcomes in affected children. In our study, intravenous high-dose folinic acid infusions appeared to optimize clinical response.

Topics: Child; Child, Preschool; Female; Folate Receptor 1; Humans; Infusions, Intravenous; Leucovorin; Mutation; Neuroaxonal Dystrophies; Siblings; Tetrahydrofolates

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.

Topics: Adolescent; Adult; Biomarkers; Body Mass Index; Child; Child, Preschool; Chromatography, High Pressure Liquid; Erythrocytes; Ethnicity; Female; Folic Acid; Humans; Infant; Leucovorin; Life Style; Male; Middle Aged; Nutrition Surveys; Nutritional Status; Reference Values; Sex Factors; Tandem Mass Spectrometry; Tetrahydrofolates; United States; Young Adult

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.

Topics: Animals; Chromatography, Liquid; Comparative Genomic Hybridization; Disease Models, Animal; DNA Copy Number Variations; Folic Acid; Gene Expression Profiling; Genetic Predisposition to Disease; Leucovorin; Methotrexate; Mice, Inbred C57BL; MicroRNAs; Neural Tube Defects; Real-Time Polymerase Chain Reaction; S-Adenosylmethionine; Tandem Mass Spectrometry; Tetrahydrofolate Dehydrogenase; Tetrahydrofolates

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.

Topics: Chromatography, High Pressure Liquid; Female; Folic Acid; Homocysteine; Humans; Leucovorin; Limit of Detection; Neural Tube Defects; Pregnancy; S-Adenosylhomocysteine; S-Adenosylmethionine; Tandem Mass Spectrometry; Tetrahydrofolates

Folinic acid-responsive seizures (FARS) are a rare treatable cause of neonatal epilepsy. They have characteristic peaks on CSF monoamine metabolite analysis, and have mutations in the ALDH7A1 gene, characteristically found in pyridoxine-dependent epilepsy. There are case reports of patients presenting with seizures at a later age, and with folate deficiency due to different mechanisms with variable response to folinic acid supplementation. Here, we report 2 siblings who presented with global developmental delay and intractable seizures who responded clinically to folinic acid therapy. Their work-up included metabolic and genetic testing. The DNA sequencing was carried out for the ALDH7A1 gene, and the folate receptor 1 (FOLR1) gene. They had very low 5-methyltetrahydrofolate (5-MTHF) in CSF with no systemic folate deficiency and no characteristic peaks on neurotransmitter metabolite chromatogram. A novel mutation in the FOLR1 gene was found. The mutation in this gene is shown to affect CSF folate transport leading to cerebral folate deficiency. The response to treatment with folinic acid was dramatic with improvement in social interaction, mobility, and complete seizure control. We should consider the possibility of this treatable condition in appropriate clinical circumstances early, as diagnosis with favorable outcome depends on the specialized tests.

Topics: Atrophy; Brain; Brain Diseases, Metabolic, Inborn; Child Development Disorders, Pervasive; Child, Preschool; Consanguinity; Developmental Disabilities; Early Diagnosis; Electroencephalography; Epilepsies, Myoclonic; Female; Folate Receptor 1; Folic Acid Deficiency; Humans; Leucovorin; Magnetic Resonance Imaging; Male; Mutation, Missense; Point Mutation; Pyridoxine; Siblings; Tetrahydrofolates

The present study aimed to determine the effects of different traditional cooking methods on folate (tetrahydrofolate - THF, 5-methyltetrahydrofolate - 5- MTHF and 5-formyltetrahydrofolate - 5-FTHF) retention in leafy vegetables. The analysis of folates was carried out by high performance liquid chromatography (HPLC), with detection by fluorescence, using gradient elution, mobile phase of acetonitrile and phosphate buf- fer solution. The retention of isomers in vegetables after cooking ranged from 17.0 % to 87.2 % for THF, 53.4 - 94.1% for 5-MTHF and 39.0 - 107.9% for 5-FTHF. The retention of folates depended on the food matrix, the kind of isomer, and the cooking methods used. It is recommended that one should have more control over the choices for methods and time of cooking and the amount of water used at home and at foodservice as well.

Topics: Brassica; Brazil; Chromatography, High Pressure Liquid; Cooking; Leucovorin; Spinacia oleracea; Tetrahydrofolates; Time Factors

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.

Topics: Chromatography, Liquid; Folic Acid; High-Throughput Screening Assays; Humans; Leucovorin; Limit of Detection; Oxidation-Reduction; Solid Phase Extraction; Tandem Mass Spectrometry; Tetrahydrofolates

Folate is a nutrient crucial for rapidly growing tissues, including developing embryos and cancer cells. Folate participates in the biosynthesis of nucleic acids, proteins, amino acids, S-adenosylmethionine, many neurotransmitters, and some vitamins. The intracellular folate pool consists of different folate adducts, which carry one-carbon units at three different oxidative states and participate in distinct biochemical reactions. Therefore, the content and dynamics of folate adducts will affect the homeostasis of the metabolites generated in these folate-mediated reactions. Currently, the knowledge on the level of each individual folate adduct in developing embryos is limited. With an improved high-performance liquid chromatography protocol, we found that tetrahydrofolate (THF), the backbone of one-carbon carrier, gradually increased and became dominant in developing zebrafish embryos. 5-methyl-tetrahydrofolate (5-CH3-THF) was abundant in unfertilized eggs but decreased rapidly when embryos started to proliferate and differentiate. 10-formyltetrahydrofolate at first increased after fertilization, and then dropped dramatically before reaching a sustained level at later stages. Dihydrofolate (DHF) slightly decreased initially and remained low throughout embryogenesis. Exposure to methotrexate significantly decreased 5-CH3-THF levels and increased DHF pools, besides causing brain ventricle anomaly. Rescuing with leucovorin partly reversed the abnormal phenotype. Unexpectedly, the level of 5-CH3-THF remained low even when leucovorin was added for rescue. Our results show that different folate adducts fluctuated significantly and differentially in concert with the physiological requirement specific for the corresponding developmental stages. Furthermore, methotrexate lowered the level of 5-CH3-THF in developing embryos, which could not be reversed with folate supplementation and might be more substantial to cellular methylation potential and epigenetic control than to nucleotide synthesis.

Topics: Animals; Embryo, Nonmammalian; Embryonic Development; Leucovorin; Methotrexate; Tetrahydrofolates; Zebrafish

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.. 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]-1β, -6, -8, and -10; monocyte chemotactic protein-1) and antiinflammatory cytokines (IL-4, IL-10, IL-13) were identified with factor analysis.. 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.. Maternal serum folate vitamers are associated with cervical cytokine concentrations, which suggests a possible mechanistic link between folate and preterm birth risk.

Topics: Cytokines; Female; Folic Acid; Humans; Incidence; Inflammation; Leucovorin; Pregnancy; Pregnancy Complications, Infectious; Premature Birth; Prevalence; Reproductive Tract Infections; Tetrahydrofolates; Vaginosis, Bacterial

This study investigated the role of an ABC transporter, Mrp3/Abcc3 in intestinal folate absorption.. Plasma concentrations of folic acid and leucovorin, given orally, were determined in wild-type and Mrp3 ( -/- ) mice. Mucosal-to-serosal transport was determined in the everted intestinal sacs. The plasma concentrations of endogenous 5-methyltetrahydrofolic acid, homocysteine and vitamin B(12), and mRNA levels of hepatic and intestinal folate metabolizing enzymes were compared between wild-type and Mrp3 ( -/- ) mice.. C ( max ) and area-under plasma concentration-time curve of folic acid were 3.0- and 2.3-fold lower in Mrp3 ( -/- ) mice compared with wild-type mice, whereas the total body clearance was unchanged. Absorption of leucovorin was significantly delayed in Mrp3 ( -/- ) mice. Mucosal-to-serosal transport of folic acid and leucovorin was significantly decreased in the duodenum of Mrp3 ( -/- ) mice, where their PS ( serosal ) was decreased to 6.3 and 22% of that in wild-type mice, respectively. PS ( serosal ) of 5-methyltetrahydrofolic acid was moderately decreased in Mrp3 ( -/- ) mice. There was no obvious abnormality in folate homeostasis in Mrp3 ( -/- ) mice.. Mrp3 accounts for the serosal efflux of folic acid and leucovorin, while it makes a moderate contribution to the serosal efflux of 5-methyltetrahydrofolic acid in mice. Mrp3 dysfunction does not disrupt folate homeostasis in mouse.

Topics: Animals; Folic Acid; Gene Deletion; Homocysteine; Intestinal Absorption; Leucovorin; Male; Mice; Multidrug Resistance-Associated Proteins; Tetrahydrofolates; Vitamin B 12

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.. 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).. 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.. 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.. 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.

Topics: Black People; Chromatography, High Pressure Liquid; Female; Folic Acid; Humans; Infant, Newborn; Infant, Premature; Leucovorin; Pennsylvania; Pregnancy; Pregnancy Trimester, First; Pregnancy Trimester, Second; Prospective Studies; Risk Factors; Surveys and Questionnaires; Tetrahydrofolates; White People

A selective and rapid method was developed and validated for determination of 6R-leucovorin (LV), 6S-leucovorin and 5-methyltetrahydrofolate (5-MeTHF) in human plasma using stereoselective liquid chromatography-tandem mass spectrometry. All analytes and the internal standard were extracted from plasma by solid phase extraction using Oasis HLB C(18) cartridges. A macrocyclic glycopeptide teicoplanin column was used for chiral separation of LV and 5-MeTHF isomers with NH(4)TFA or NH(4)OAc in methanol as mobile phase. Detection was performed on an API 4000 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The calibration curves were linear in the range of 0.050-20.0microg/mL for 6R-LV and 6S-LV, and 0.025-10.0microg/mL for 5-MeTHF. The intra- and inter-assay precision was 3.6-13.2%, 3.4-12.9% and 5.3-9.3% for 6R-LV, 6S-LV and 5-MeTHF, respectively. The accuracy was 99.4-102.4%, 95.3-96.8% and 93.0-110% for 6R-LV, 6S-LV and 5-MeTHF, respectively. The lower limit of quantification (LLOQ) was 0.050microg/mL for each LV isomer and 0.025microg/mL for 5-MeTHF. The method was successfully applied to a comparative pharmacokinetic study between leucovorin calcium and levoleucovorin calcium in 10 volunteers. No significant differences between levoleucovorin and leucovorin in pharmacokinetic parameters of 6S-LV and 5-MeTHF were found in volunteers.

Topics: Chromatography, Liquid; Humans; Leucovorin; Solid Phase Extraction; Tandem Mass Spectrometry; Tetrahydrofolates

Topics: Base Sequence; Brain; Carrier Proteins; Child, Preschool; Disease Progression; DNA Mutational Analysis; Dose-Response Relationship, Drug; Folate Receptor 1; Folate Receptors, GPI-Anchored; Humans; Leucovorin; Male; Mutation; Nervous System Diseases; Receptors, Cell Surface; RNA; Tetrahydrofolates; Treatment Outcome

Topics: Endothelium, Vascular; Humans; Hyperhomocysteinemia; Kidney Diseases; Leucovorin; Renal Dialysis; Stroke; Tetrahydrofolates; Vitamin B 12; Vitamin B 6

The folate derivatives folic acid (FA) and folinic acid (FNA) decrease the in vivo and in vitro activities of antifolate drugs in Plasmodium falciparum. However, the effects of 5-methyl-tetrahydrofolate (5-Me-THF) and tetrahydrofolate (THF), the two dominant circulating folate forms in humans, have not been explored yet. We have investigated the effects of FA, FNA, 5-Me-THF, and THF on the in vitro activity of the antimalarial antifolates pyrimethamine and chlorcycloguanil and the anticancer antifolates methotrexate (MTX), aminopterin, and trimetrexate (TMX), against P. falciparum. The results indicate that these anticancers are potent against P. falciparum, with IC50 < 50 nM. 5-Me-THF does not significantly decrease the activity of all tested drugs, and none of the tested folate derivatives significantly decrease the activity of these anticancers. Thus, malaria folate metabolism has features different from those in human, and the exploitation of this difference could lead to the discovery of new drugs to treat malaria. For instance, the combination of 5-Me-THF with a low dose of TMX could be used to treat malaria. In addition, the safety of a low dose of MTX in the treatment of arthritis indicates that this drug could be used alone to treat malaria.

Topics: Aminopterin; Animals; Antimalarials; Antineoplastic Agents; Folic Acid; Folic Acid Antagonists; Inhibitory Concentration 50; Leucovorin; Methotrexate; Molecular Structure; Plasmodium falciparum; Proguanil; Pyrimethamine; Tetrahydrofolates; Triazines; Trimetrexate

Topics: Antiparkinson Agents; Atrophy; Basal Ganglia; Brain Diseases, Metabolic; Carbidopa; Cerebellum; Demyelinating Diseases; Dystonia; Female; Folic Acid Deficiency; Humans; Leucovorin; Levodopa; Male; Syndrome; Tetrahydrofolates; Treatment Outcome; Vitamin B Complex

Cerebral folate deficiency is characterized by low cerebrospinal fluid (CSF) concentrations of 5-methyltetrahydrofolate and a broad spectrum of clinical signs and symptoms. A patient with progressive spasticity, gait disturbance, speech difficulties, initially diagnosed as a recessive spastic paraplegia recovered on folinic acid (15-30 mg/day) and her 5-methyltetrahydrofolate in CSF normalized. This report demonstrates the importance of CSF investigation in the diagnosis of cerebral folate deficiency and efficiency of folinic acid (5-formyltetrahydrofolate) supplementation.

Topics: Brain Diseases; Child; Child, Preschool; Dietary Supplements; Dose-Response Relationship, Drug; Female; Folic Acid Deficiency; Gait; Humans; Leucovorin; Life Style; Movement Disorders; Paraplegia; Pregnancy; Speech Disorders; Tetrahydrofolates

The authors describe a 6-year-old girl with developmental delay, psychomotor regression, seizures, mental retardation, and autistic features associated with low CSF levels of 5-methyltetrahydrofolate, the biologically active form of folates in CSF and blood. Folate and B12 levels were normal in peripheral tissues, suggesting cerebral folate deficiency. Treatment with folinic acid corrected CSF abnormalities and improved motor skills.

Topics: Adaptation, Physiological; Autistic Disorder; Cerebral Cortex; Child; Developmental Disabilities; Disease Progression; Female; Folic Acid; Folic Acid Deficiency; Genetic Predisposition to Disease; Humans; Intellectual Disability; Leucovorin; Mutation; Recovery of Function; Reduced Folate Carrier Protein; Seizures; Tetrahydrofolates; Transcription Factors; Treatment Outcome

Topics: Adolescent; Adult; Biomarkers; Blood-Brain Barrier; Brain; Cerebrospinal Fluid; Child; Child, Preschool; Female; Folic Acid; Folic Acid Deficiency; Humans; Leucovorin; Predictive Value of Tests; Reference Values; Rett Syndrome; Tetrahydrofolates; Treatment Outcome

Current clinical methods for folate give different results and cannot measure the various forms of folate. We developed an isotope-dilution tandem mass spectrometric method coupled to liquid chromatography (LC/MS/MS) as a candidate reference method for 5-methyltetrahydrofolic acid (5MeTHF), 5-formyltetrahydrofolic acid (5FoTHF), and folic acid (FA) in human serum.. We quantitatively isolated folates from 275 microL of serum with a phenyl solid-phase extraction cartridge, then detected and quantified them in stabilized serum extracts by positive-ion electrospray ionization LC/MS/MS. We used an isocratic mobile phase of acetic acid in organic solvent on a C(8) analytical column. (13)C-labeled folates were used as internal standards.. Limits of detection in serum were 0.13 (5MeTHF), 0.05 (5FoTHF), and 0.07 (FA) nmol/L. Within- and between-run imprecision (CV) was <7% for 5MeTHF and <10% for 5FoTHF at concentrations >0.5 nmol/L, and <10% for FA at concentrations >2.0 nmol/L. Total folate (TFOL) concentrations determined by competitive protein binding radioassay were approximately 9% lower than results obtained with LC/MS/MS. The microbiologic assay gave approximately 15% higher TFOL results with FA calibrator and no difference with 5MeTHF calibrator. The mean (SD) [range] TFOL in 42 sera was 35.5 (17.8) [6.5-75.6] nmol/L. Thirty-two samples with TFOL <50 nmol/L had, on average, 93.3% 5MeTHF, 2.3% FA, and 4.4% 5FoTHF. Ten samples with TFOL >50 nmol/L had, on average, 81.7% 5MeTHF, 15.7% FA, and 2.5% 5FoTHF.. This stable-isotope-dilution LC/MS/MS method can quantify 5MeTHF, 5FoTHF, and FA in serum. Currently used clinical assays agree with this candidate reference method.

Topics: Autoanalysis; Chromatography, Liquid; Folic Acid; Humans; Indicator Dilution Techniques; Leucovorin; Microbiological Techniques; Radioligand Assay; Sensitivity and Specificity; Serum; Spectrometry, Mass, Electrospray Ionization; Tetrahydrofolates

Topics: Chromatography, High Pressure Liquid; Drug Stability; Folic Acid; Hydrogen-Ion Concentration; Leucovorin; Tetrahydrofolates; Thermodynamics

Transport of 5-formyltetrahydrofolate (5-FTHF) into primary cultured rat astrocytes was studied. Uptake of 5-FTHF into astrocytes was linear in the first 60 min and is saturable with K(m)=3.3 microM and V(max)=27.5 pmol/mg protein/45 min in pH 7.4 medium. Uptake of 5-FTHF displayed the characteristics of countertransport. Uptake of 5-FTHF was inhibited by the structural analogs 5-methyltetrahydrofolate, methtrexate, and folic acid (K(i)=3.8, 2.7, and 18.4 microM, respectively). Uptake was significantly decreased by sodium azide but was increased by high concentration of sodium cyanide and low concentration of sodium arsenate. Uptake was also inhibited by p-chloromercuriphenylsulfonate and by the anions probenecid and 4,4(')-diisothiocyanostilbene-2,2(')-disulfonic acid. Acute exposure of the cells to ethanol (100mM) inhibited the uptake for 90 min of the experimental duration. It is concluded that astrocytes have a system for the uptake of 5-FTHF and folate analogs which is carrier mediated, this system is sensitive to energy inhibitors and alcohol exposure.

Topics: 4-Chloromercuribenzenesulfonate; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Arsenates; Astrocytes; Biological Transport; Buffers; Cells, Cultured; Enzyme Inhibitors; Ethanol; Folic Acid; Hydrogen-Ion Concentration; Leucovorin; Methotrexate; Probenecid; Rats; Rats, Sprague-Dawley; Sodium Cyanide; Tetrahydrofolates

The newborns of mammals have a high folate demand, yet obtain adequate folate nutrition solely from their mothers' milk despite its low folate content. Milk folate is entirely bound by an excess of folate-binding protein (FBP), prompting speculation that FBP may affect the bioavailability of the limited folate supply. Previous research has shown that FBP-bound folic acid is more gradually absorbed, thereby reducing the peak plasma folate concentration and preventing loss into the urine. Natural folates are reduced derivatives of folic acid, with milk predominantly containing 5-methyltetrahydrofolate, yet little research has been carried out to determine the role of FBP in the bioavailability of reduced folates. We studied the effect of FBP on folate nutrition of rats in both single-dose and 4-wk feeding experiments. The effect of FBP was influenced by the presence of other milk components. FBP increased bioavailability of dietary folate when it was consumed with other whey proteins or with soluble casein. However, in the presence of acid-precipitated casein or a whey preparation enriched in lipids, bioavailability was decreased. These results highlight the difficulties of extrapolating from experimental results obtained using purified diets alone and of studying interactions among dietary components. They suggest that the addition of FBP-rich foods to folate-rich foods could enhance the bioavailability of natural folates, but that the outcome of such a combination would depend on interactions with other components of the diet.

Topics: Animals; Biological Availability; Carrier Proteins; Cattle; Diet; Folate Receptors, GPI-Anchored; Intestinal Absorption; Kidney; Leucovorin; Liver; Male; Rats; Rats, Wistar; Receptors, Cell Surface; Tetrahydrofolates; Tissue Distribution

Previous CSF studies in Rett syndrome suggest reduced turnover of the biogenic monoamines serotonin and dopamine. Because diminished turnover may result from CNS folate depletion, the authors studied transport of folate across the blood-brain barrier.. In four patients with Rett syndrome, the authors measured CSF values of 5-methyltetrahydrofolate (5MTHF), biogenic monoamine end-metabolites, and pterins together with serum and red blood cell folate. In CSF, the overall folate binding capacity by the two soluble folate-binding proteins FBP1 and FBP2 (sFBP) was measured using a radioligand binding method for H3-labeled folate. A specific immunoreactive test (ELISA) detected sFBP1, which normally contributes to 30 to 35% of the total folate binding capacity. Genetic analysis included DNA sequencing of the MECP2, FBP1, and FBP2 genes. Empirical treatment with oral folinic acid was evaluated.. Two patients without and two with mutations of the MECP2 gene had normal values for red blood cell folate, serum folate, homocysteine, and methionine. In CSF, all patients had low values for 5MTHF, neopterin, and the serotonin end-metabolite 5-hydroxyindoleacetic acid (5-HIAA). Genetic analysis of FBP1 and FBP2 genes had normal results. Compared to controls, patients with Rett syndrome had normal immunoreactive sFBP1 in CSF, whereas the total folate binding capacity was disproportionately lowered. Empirical treatment with oral folinic acid normalized 5-MHTF and 5-HIAA levels in CSF, and led to partial clinical improvement.. Irrespective of the MECP2 genotype, 5MTHF transfer to the CNS is reduced in Rett syndrome. Folinic acid supplementation restores 5MTHF levels and serotoninergic turnover. The lowered folate binding capacity of FBP is not explained by a defect of the FBP1 or FBP2 gene, but most likely occurs as a secondary phenomenon in Rett syndrome.

Topics: Biogenic Monoamines; Biomarkers; Blood-Brain Barrier; Carrier Proteins; Central Nervous System; Child, Preschool; Female; Folate Receptors, GPI-Anchored; Folic Acid; Humans; Leucovorin; Protein Isoforms; Pterins; Receptors, Cell Surface; Rett Syndrome; Sequence Analysis, DNA; Tetrahydrofolates

Uptake of 5-methyltetrahydrofolate into the PC-3 human prostate cancer cells was linear for the first 60 min. There was no difference in the initial rate of uptake in cells incubated in folate-free medium for 24 or 48 hr compared to control cells grown in folate-containing medium. The initial rate of 5-methyltetrahydrofolate uptake showed little dependence on extracellular pH and it was independent of extracellular sodium ions. Transport of 5-methyltetrahydrofolate into PC-3 cells was saturable - K(m) = 0.74 micro M and V(max) = 7.78 nmol/10(9)cells/min and these kinetic constants were not different in cells incubated for 24 hr in folate-free medium (K(m) = 0.80 +/- 0.22, V(max) = 8.52 +/- 0.50; P = 0.09, N = 3). Uptake of 5-methyltetrahydrofolate was inhibited by structural analogs with the K(i) values being 0.50, 1.79, and 31.8 micro M for 5-formyltetrahydrofolate, methotrexate, and folic acid, respectively. Uptake of 5-methyltetrahydrofolate was inhibited by the energy poisons, sodium cyanide, sodium arsenate, p-chloromercuriphenylsulfonate, and sodium azide. Uptake was inhibited by increasing concentrations of sulfate and phosphate ions, suggesting that 5-methyltetrahydrofolate may be transported by an anion-exchange mechanism. These results show that 5-methyltetrahydrofolate is transported into PC-3 prostate cancer cells by a carrier-mediated process.

Topics: Arsenates; Biological Transport; Carrier Proteins; Folic Acid; Humans; Hydrogen-Ion Concentration; Leucovorin; Male; Methotrexate; Phosphates; Prostatic Neoplasms; Sodium; Sodium Azide; Sodium Cyanide; Sulfates; Tetrahydrofolates; Tumor Cells, Cultured

[2H4]Folic acid was synthesized by deuterating p-aminobenzoic acid, which was then coupled to glutamic acid and 6-formylpterin. Using [2H4]folic acid as starting component enabled the preparation of labeled vitamers tetrahydrofolate, 5-formyltetrahydrofolate, 5-methyltetrahydrofolate, and 10-formylfolate which were characterized by electrospray mass spectrometry and collision-induced dissociation. The mass spectrometric studies confirmed that the compounds could be used as internal standards in stable isotope dilution assays.

Topics: 4-Aminobenzoic Acid; Chromatography, High Pressure Liquid; Deuterium; Folic Acid; Glutamic Acid; Isotope Labeling; Leucovorin; Magnetic Resonance Spectroscopy; Pteridines; Pterins; Spectrometry, Mass, Electrospray Ionization; Tetrahydrofolates

Topics: Chromatography, High Pressure Liquid; Drug Monitoring; Folic Acid Antagonists; Humans; Leucovorin; Methotrexate; Neoplasms; Tetrahydrofolates

Normal brain development and function depend on the active transport of folates across the blood-brain barrier. The folate receptor-1 (FR 1) protein is localized at the basolateral surface of the choroid plexus, which is characterized by a high binding affinity for circulating 5-methyltetrahydrofolate (5-MTHF).. We report on the clinical and metabolic findings among five children with normal neurodevelopmental progress during the first four to six months followed by the acquisition of a neurological condition which includes marked irritability, decelerating head growth, psychomotor retardation, cerebellar ataxia, dyskinesias (choreoathetosis, ballism), pyramidal signs in the lower limbs and occasional seizures. After the age of six years the two oldest patients also manifested a central visual disorder. Known disorders have been ruled out by extensive investigations. Cerebrospinal fluid (CSF) analysis included determination of biogenic monoamines, pterins and 5-MTHF.. Despite normal folate levels in serum and red blood cells with normal homocysteine, analysis of CSF revealed a decline towards very low values for 5-methyltetrahydrofolate (5-MTHF), which suggested disturbed transport of folates across the blood-brain barrier. Genetic analysis of the FR 1 gene revealed normal coding sequences. Oral treatment with doses of the stable compound folinic acid (0.5-1 mg/kg/day Leucovorin(R)) resulted in clinical amelioration and normalization of 5-MTHF values in CSF.. Our findings identified a new condition manifesting after the age of 6 months which was accompanied by low 5-MTHF in cerebrospinal fluid and responded to oral supplements with folinic acid. However, the cause of disturbed folate transfer across the blood-brain barrier remains unknown.

Topics: Blood-Brain Barrier; Brain Diseases, Metabolic, Inborn; Carrier Proteins; Child; Child, Preschool; DNA-Binding Proteins; Erythrocytes; Female; Folate Receptor 1; Folate Receptors, GPI-Anchored; Humans; Infant; Intellectual Disability; Leucovorin; Male; Membrane Proteins; Membrane Transport Proteins; Movement Disorders; Neurologic Examination; Paraplegia; Psychomotor Disorders; Receptors, Cell Surface; Replication Protein C; Spinocerebellar Degenerations; Tetrahydrofolates; Transcription Factors

In previous reports, an E45K mutation in reduced folate carrier (RFC1) resulted in marked substrate-specific changes in folate binding and the induction of an obligatory inorganic anion requirement for carrier function. In this study, site-directed mutagenesis was employed to further characterize the role of glutamate-45 in carrier function by replacement with glutamine, arginine, aspartate, leucine, or tryptophan followed by tranfection of the mutated cDNAs into the MTX(r)A line, which lacks a functional endogenous carrier. Alterations in transport function with amino acid substitutions at this residue were not charge related. Hence, E45Q, E45R, and E45K all 1) increased carrier affinity for 5-formyltetrahydrofolate approximately 4-fold, 2) increased affinity for folic acid approximately 6- to 10-fold, 3) did not change affinity for 5-methyltetrahydrofolate, and 4) except for E45R decreased affinity for methotrexate (2- to 3-fold). In contrast, mutations E45D, E45L, and E45W generally reduced affinity for all these folates except for folic acid. Finally, chloride-dependent influx was only noted in the E45R mutant. These data further substantiate the important role that glutamate-45 plays in the selectivity of binding of folates to RFC1 and establish that it is the addition of a positive charge at this site and not the loss of a negative charge that results in the induced anion dependence. These and other studies indicate that mutations in the first transmembrane domain can have a markedly selective impact on the affinity of RFC1 for folate compounds and in particularly a highly salutary effect on binding of the oxidized folate, folic acid.

Topics: Amino Acid Substitution; Animals; Anions; Antimetabolites, Antineoplastic; Biological Transport; Carrier Proteins; Folic Acid; Glutamic Acid; Leucovorin; Leukemia L1210; Membrane Proteins; Membrane Transport Proteins; Methotrexate; Mice; Mutagenesis, Site-Directed; Reduced Folate Carrier Protein; Tetrahydrofolates; Transfection; Tumor Cells, Cultured

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolic acid (5-CH(3)-H(4) folic acid), the methyl donor for the formation of methionine from homocysteine. A common C677T transition in the MTHFR gene results in a variant with a lower specific activity and a greater sensitivity to heat than the normal enzyme, as measured in vitro. This study was undertaken to determine the capacity of homozygotes for the MTHFR C677T transition to convert 5-formyltetrahydrofolic acid (5-HCO-H(4) folic acid) to 5-CH(3)-H(4) folic acid, a process that requires the action of MTHFR. Six subjects homozygous for the C677T transition (T/T) and 6 subjects with wild-type MTHFR (C/C) were given a 5-mg oral dose of (6R:,S:)-5-HCO-H(4) folic acid. Plasma and urine were analyzed for 5-CH(3)-H(4) folic acid concentrations using affinity/HPLC coupled with fluorescence or UV detection. The mean areas under the curves created by the rise and fall of plasma 5-CH(3)-H(4) folic acid after the oral dose did not differ between the two genotypes, 424.5 +/- 140.3 (T/T) vs. 424.1+/- 202.4 h.nmol/L (C/C). There also was no significant difference in the mean cumulative 7-h urinary excretion of 5-CH(3)-H(4) folic acid between the T/T (2.5 +/- 1.4 micromol) and C/C (1.9 +/- 1.0 micromol) genotypes. Under the conditions employed, the conversion of oral 5-HCO-H(4) folic acid to 5-CH(3)-H(4) folic acid is not impaired in persons with the T/T MTHFR genotype. Possible reasons for these findings are discussed.

Topics: Administration, Oral; Adult; Area Under Curve; Female; Folic Acid; Genotype; Homocysteine; Humans; Leucovorin; Male; Methylenetetrahydrofolate Reductase (NADPH2); Middle Aged; Mutation; Oxidoreductases Acting on CH-NH Group Donors; Polymorphism, Genetic; Tetrahydrofolates

The use of high-performance liquid chromatography for the determination of folates is well documented. The methods used are based on reversed-phase chromatography with UV and/or fluorescence detection. In many instances it is difficult to reach the required detection limits and many of the methods lack specificity. High-performance liquid chromatography-mass spectrometry (LC-MS) has become a well established analytical tool in modern laboratories. It can offer superior specificity and often lower detection limits than conventional LC detection methods and thus is ideally suited to the analysis of folates. A system capable of separating the four main folates [folic acid (pteroylglutamic acid, PGA)], 5-methyltetrahydrofolic acid, terahydrofolic acid and 5- and/or 10-formyltetrahydrofolic acid [folinic acid (CHOTHF)] using LC-MS with negative ion electrospray has been developed. After optimisation, a system using a 12.5 cm x 3 mm, 3 microm Hypersil ODS column and a mobile phase of 2.5 mM acetic acid-acetonitrile (88:12, v/v) was developed which was capable of separating the four folates in under 10 min without the use of a gradient system. Extraction of the folates is by heat treatment and sample clean-up is by solid-phase extraction (C18). On column limits of confirmation are 0.6 ng for PGA and CHOTHF.

Topics: Acetic Acid; Acetonitriles; Chromatography, High Pressure Liquid; Dietary Supplements; Edible Grain; Folic Acid; Leucovorin; Mass Spectrometry; Meat; Tetrahydrofolates; Vegetables; Vitamins

Although reabsorption across the apical (AP) membrane of the renal proximal tubule cell plays a vital role in the conservation of plasma 5-methyltetrahydrofolate, basolateral (BL) membrane-directed secretory pathways may also be important in regulating the urinary excretion of folate. Folate transport proteins, folate receptor and the reduced folate carrier have been implicated in the renal conservation of folate across the AP membrane, but their role in BL membrane-directed folate transport has not been studied. 5-Methyltetrahydrofolate transport across the AP and BL membranes of human proximal tubule cells was studied in cells grown on membrane inserts to allow optimum differentiation of AP and BL domains. Colchicine, an inhibitor of vesicular-mediated endocytosis, inhibited AP binding and AP-directed transport without affecting BL transport. Probenecid, an inhibitor of anion exchange, did not affect binding, but inhibited both AP and BL-directed transport with a greater effect on BL transport. Folic acid abolished AP binding of 5-methyltetrahydrofolate, but diminished AP-mediated transport by only 50%. These data suggest that both the folate receptor and the reduced folate carrier participate in AP uptake of folates by human kidney cells, but that BL-mediated uptake occurs primarily by the reduced folate carrier. Folate transport from the secretory direction occurred as readily as that from the reabsorptive direction, indicating that altered secretion could mediate excess urinary folate excretion.

Topics: Antidotes; Basilar Membrane; Biological Transport; Carrier Proteins; Cells, Cultured; Folate Receptors, GPI-Anchored; Folic Acid; Humans; Kidney Tubules, Proximal; Leucovorin; Receptors, Cell Surface; Tetrahydrofolates

Topics: Leucovorin; Oxidation-Reduction; Pteroylpolyglutamic Acids; Stereoisomerism; Tetrahydrofolates

Topics: Blotting, Southern; Carbon-Nitrogen Ligases; Cloning, Molecular; Cytosol; DNA Primers; Enzyme Inhibitors; Escherichia coli; Humans; Kinetics; Leucovorin; Ligases; Liver; Mitochondria, Liver; Molecular Sequence Data; Recombinant Proteins; RNA, Messenger; Sequence Analysis, DNA; Spectrophotometry; Tetrahydrofolates

Prolonged cell culture of human leukemia cells at folate concentrations in the (sub)physiological range (1-5 nM) rather than at 'standard' supraphysiological concentrations of 2-10 microM folic acid elicited a number of regulatory aspects of the reduced folate carrier (RFC), the membrane transport protein for natural reduced folate cofactors and folate-based chemotherapeutic drugs such as methotrexate (MTX). One subline of human CCRF-CEM leukemia cells grown under folate-restricted conditions (CEM-7A) exhibited a 95-fold increased Vmax for uptake of [3H]-MTX. The increased uptake of MTX in CEM-7A cells is based on at least two factors: (a) a constitutive 10-fold overexpression of the RFC1 gene and RFC1 message; and (b) a 7-9-fold up-regulation of RFC transport activity under low intracellular reduced folate concentrations. This second component appeared to be regulatable by changes in the cellular folate, purine and methylation status as judged from a 7-9 fold down-regulation of RFC transport activity after short term (1-2 hr) incubation of CEM-7A cells with reduced folate cofactors (25 nM LV), purines (100 microM adenosine) or S-adenosylmethionine (100 microM), respectively. Gradual folate restriction in the cell culture medium of CEM/MTX cells, a subline of CCRF-CEM resistant to MTX due to defective transport via the RFC, revealed the up-regulated expression of an altered RFC protein that is characterized by a 35-fold decreased Km for folic acid and a 10-fold decreased Km for the reduced folate cofactor LV compared to the RFC expressed in CCRF-CEM and CEM-7A cells. As a result of the markedly increased efficiency of folic acid uptake in CEM/MTX cells, intracellular folate pools were 7-fold higher than in CCRF-CEM cells when both cell lines were incubated in the presence of 2 microM folic acid. The high intracellular folate pools in CEM/MTX cells appeared to impair the polyglutamylation of antifolates and confer resistance to ZD1694, an antifolate drug that depends on polyglutamylation for its biological activity. Collectively, these studies provide a better insight into the basic regulation of RFC-mediated membrane transport of clinically active antifolates. In addition, these studies may also provide an opportunity to exploit the transport system as a target for biochemical modulation by which it may contribute to an improved efficacy of folate-based chemotherapy in a clinical setting.

Topics: Adenosine; Biological Transport; Carrier Proteins; Cell Division; Drug Resistance, Neoplasm; Enzyme Inhibitors; Folic Acid; Folic Acid Antagonists; Humans; Kinetics; Leucovorin; Membrane Proteins; Membrane Transport Proteins; Methotrexate; Reduced Folate Carrier Protein; S-Adenosylmethionine; Tetrahydrofolates; Thymidylate Synthase; Tumor Cells, Cultured

Rat placenta contains virtually no unsaturated (i.e., apo-form) folate binding protein. However, by lowering the pH of a solubilized membrane preparation of this tissue to 3.5, the endogenous bound folate was dissociated from the protein and adsorbed to charcoal. The apo-form of the folate binding protein thus obtained was purified by affinity chromatography using pteroylglutamic acid covalently coupled to Sepharose 4B. A single protein band with an apparent M(r) of 36,000 was observed by SDS-polyacrylamide gel electrophoresis of the eluate from the affinity matrix. Western blot of this preparation using a rabbit antiserum raised with the affinity eluate also identified a single 36 kDa protein band. However, peptide sequencing of the N-terminal region of the proteins in the affinity eluate established that it contained two homologous proteins. Computer alignment of the first 22 N-terminal amino acids of each rat placental protein with human, bovine milk and mouse folate binding proteins showed 50-64% identical homology and 27% homology when the eight proteins were aligned together. The affinity of both rat proteins is highest for pteroylglutamic acid (Ka = 1.6.10(9) l/mol) lower for N5-methyltetrahydrofolate and substantially lower for N5-formyltetrahydrofolate. In the dose-response range studied there was no apparent affinity for methotrexate. The folate binding proteins could be released from a preparation of placental membranes using phospholipase C indicating that these proteins belong to the class of proteins anchored to the plasma membrane by a glycosyl phosphatidylinositol adduct.

Topics: Amino Acid Sequence; Animals; Apoproteins; Binding, Competitive; Blotting, Western; Carrier Proteins; Cattle; Chromatography, Affinity; Female; Folate Receptors, GPI-Anchored; Folic Acid; Humans; Leucovorin; Mice; Molecular Sequence Data; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoric Diester Hydrolases; Placenta; Pregnancy; Rats; Rats, Sprague-Dawley; Receptors, Cell Surface; Sequence Alignment; Species Specificity; Tetrahydrofolates

Mild hyperhomocysteinaemia is associated with increased risk for vascular disease. We studied homocysteine export from human umbilical vein endothelial cells (HUVECs) by measuring total homocysteine (tHcy) concentrations in the culture medium. Under standard culture conditions tHcy concentrations in the HUVEC culture medium increased by constant amounts after 24, 48 and 72 h [mean = 2.5 (SD +/- 0.7) mumol L-1 homocysteine every 24 h]. As the cells are the only source of homocysteine increase in the culture medium, we designate this as homocysteine export from HUVEC. Folic acid supplementation to the culture medium lowered the homocysteine export in a dose-dependent manner. Methyl-tetrahydrofolate (MeTHF) and folinic acid (a stable precursor of MeTHF) were in this respect about 10 times more effective than folic acid. A 50% reduction in the homocysteine export was seen with 10-30 nmol L-1 MeTHF supplementation; reduction to almost zero was seen with 100-300 nmol L-1 MeTHF. Additions to the culture medium of the other vitamins involved in the homocysteine metabolism, such as vitamin B12, vitamin B6 and flavin adenine dinucleotide, did not show any effect on homocysteine export. Because homocysteine export reflects an imbalance in the homocysteine metabolism, our observations showed a susceptible dependency of this metabolism on folic acid in endothelial cells.

Topics: Cells, Cultured; Endothelium, Vascular; Folic Acid; Homocysteine; Humans; Kinetics; Leucovorin; Tetrahydrofolates; Time Factors; Umbilical Veins

Two variant sublines of murine L1210 leukemia cells (L1210A and L1210JF) overexpress the cell surface folate receptor (FR). The membrane bound FR in L1210A cells exhibited significantly (up to 17-fold) greater relative affinities for (6S)-N5-methyltetrahydrofolate, (6S)-N5-formyltetrahydrofolate and methotrexate compared to the FR in L1210JF cells. Furthermore, receptor-mediated transport of [3H]-(6S)-N5-methyltetrahydrofolate was much more efficient in L1210A cells compared to L1210JF cells. When solubilized with Triton X-100, the ligand binding characteristics of FR from both sublines resembled those of the receptor associated with L1210 JF cell membranes. N-terminal amino acid sequence analysis as well as RT-PCR analysis of the entire coding region revealed a single species of FR in both cells, identical to murine FR-alpha. The FR in L1210JF cells was sensitive to phosphatidylinositol specific phospholipase C (PI-PLC) indicating the presence of a glycosyl-phosphatidylinositol (GPI) membrane anchor while the FR in L1210A cells was resistant to PI-PLC; however, the FR in L1210A cells was released from plasma membranes by nitrous acid, as expected for GPI and its PI-PLC resistant structural variants. Treatment of L1210A cell membranes with mild base rendered the protein PI-PLC sensitive as expected for GPI anchors acylated in the inositol ring and also decreased the affinities of the membrane associated FR for reduced folates. When the cDNA for murine FR-alpha was expressed in parental L1210 cells the protein was PI-PLC resistant but was sensitive to PI-PLC when the cDNA was expressed in human 293 fibroblasts. In L1210JF, L1210A, and parental L1210 cells, several cell surface proteins, including FR, incorporated [3H]ethanolamine, a component of the GPI membrane anchor; however, the labeled proteins were released by PI-PLC only in L1210JF cells. The above results preclude any peculiarity of the FR polypeptide in either L1210 subline as the basis for the observed differences in PI-PLC sensitivity and membrane-associated functions of FR. Partial deglycosylation of membrane associated FR from either cell with N-glycanase did not influence its ligand binding characteristics. The results of this study lead to the hypothesis that variant GPI structures may modulate the function of a protein by influencing its conformation/topography in the membrane. Such effects may be identified by their disappearance/reduction upon detergent solubilization or mild base treatme

Topics: Amino Acid Sequence; Animals; Base Sequence; Biological Transport; Carrier Proteins; Cell Line; Cell Membrane; DNA Primers; Ethanolamine; Ethanolamines; Folate Receptors, GPI-Anchored; Glycosylphosphatidylinositols; Humans; Kidney; Leucovorin; Leukemia L1210; Methotrexate; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide Phospholipase C; Phosphoric Diester Hydrolases; Polymerase Chain Reaction; Radioligand Assay; Receptors, Cell Surface; Recombinant Proteins; Sequence Homology, Amino Acid; Tetrahydrofolates; Transfection; Tritium; Tumor Cells, Cultured

The amount of biologically active folate excreted in the urine corresponds to a small fraction of the recommended dietary allowance, suggesting that a large amount of folate is excreted as its catabolites. A strategy of assessing folate requirement by measuring the daily urinary levels of products of folate catabolism depends on the demonstration of an exclusive mechanism of breakdown as well as a suitable marker of the catabolic process. Rats were given [3H] and [14C]folic acid by gastric intubation daily for 10 d to simulate normal dietary intake of the vitamin. Total urine was collected throughout this period as well as for the following 10 d. Reverse-phase HPLC of the radiolabeled urinary products revealed the presence of a variety of intact folates as well as products of C9-N10 scission of the folate molecule, pteridines, para-aminobenzoylglutamate and para-acetamidobenzoylglutamate. We detected no other N10-containing catabolites, nor did we find the oxidized folate derivative '4 alpha-hydroxy-5-methyltetrahydrofolate'. Of all the urinary folate metabolites, only para-acetamidobenzoylglutmate persisted at high levels up to 10 d after radiolabel treatment was withdrawn. We conclude that folate catabolism occurs exclusively through C9-N10 cleavage and that measurement of urinary para-acetamidobenzoylglutmate provides a suitable indicator of daily folate turnover.

Topics: 4-Aminobenzoic Acid; Animals; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Female; Folic Acid; Intubation, Gastrointestinal; Leucovorin; para-Aminobenzoates; Rats; Rats, Wistar; Tetrahydrofolates; Tritium

A column-switching chiral HPLC assay was developed that allows the separation and quantitation of the diastereomers of leucovorin (LV, 5-formyltetrahydrofolic acid) and its metabolite 5-methyltetrahydrofolate (METHF) in serum and urine by means of fluorescence detection. The analysis procedure consists of an on-line concentration of the folates in the HPLC system which is followed by the elution and separation of folates on an achiral 3-microns Microbore C18 column in (6R,S)-LV and (6R,S)-METHF. (6R,S)-LV and (6R,S)-METHF are subsequently transferred on-line onto a chiral 7-microns bovine serum albumin column through a Rheodyne valve system and are separated into their diastereometers. Time of analysis is 70 min. Detection limit is 5 ng/ml for each diastereometer. The within-day variation ranges between 3.2 and 15.8% in relation to the measured concentration. Between-day variation is 4.4-12.1% for a concentration of 100 ng/ml for each diastereometer. (6R,S)-LV and (6S)-LV pharmacokinetics were assessed by analyzing serum and urine samples of four-healthy volunteers.

Topics: Antineoplastic Agents; Chromatography, High Pressure Liquid; Humans; Leucovorin; Reference Values; Reproducibility of Results; Spectrometry, Fluorescence; Stereoisomerism; Tetrahydrofolates

The folic acid content of total daily diet was determined by means of high-performance liquid chromatography (HPLC). The contents of tetrahydrofolic acid (THF), 5-methyl-THF and 5-formyl-THF were differentiated. The mean of the folic acid content of the total daily diet samples determined analytically was 205 +/- 60 micrograms and the mean of the individual ingredients of the samples was 401 +/- 78 micrograms, which implies that about 50% of folic acid is destroyed by common household food preparation methods. If the contents of pteroylglutamic acid (PteGlu) and 10-formyl-PteGlu (which cannot be determined analytically) are added, it can be assumed that the folic acid content with only be reduced by about 40%. THF and 5-methyl-THF proved to be less stable than 5-formyl-THF. The monoglutamate portion of the total folat content was higher in the total diet samples than in the individual foodstuffs as a consequence of the action of the enzyme "deconjugase" which is released when the matrix of food-stuffs is destroyed.

Topics: Chromatography, High Pressure Liquid; Diet; Folic Acid; Food Analysis; Food Handling; Humans; Leucovorin; Tetrahydrofolates

10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) was previously identified as a folate-binding protein in rat liver cytosol (R.J. Cook and C. Wagner, Biochemistry 21, 4427-4434, 1982) by virtue of the tetrahydrofolate polyglutamate tightly bound to the partially purified enzyme. In this current study we provide evidence to show that when liver cytosol was rapidly processed to identify the protein bound folate, large amounts of both 10-formyl- and 5-formyltetrahydrofolate were present. After overnight storage of the cytosol at 5 degrees C before processing, almost no formylfolates were present and the major protein-bound form was tetrahydrofolate. This suggests that 10-formyltetrahydrofolate polyglutamates are tightly bound to the enzyme in vivo and are converted to tetrahydrofolate forms during isolation by the hydrolase activity associated with the enzyme. Covalent binding of the stable folate analogue, 5-formyltetrahydrofolate, to the purified enzyme resulted in 2 mol bound per mole of enzyme subunit. This is consistent with earlier reports suggesting the enzyme is capable of carrying out both oxidative and hydrolytic conversion of 10-formyltetrahydrofolate to tetrahydrofolate at the same time. Partial tryptic digestion of the purified enzyme selectively inhibited dehydrogenase activity of the enzyme but did not affect the hydrolase or aldehyde dehydrogenase activities.

Topics: Animals; Cytosol; Folic Acid; Leucovorin; Ligands; Liver; Oxidoreductases Acting on CH-NH Group Donors; Pteroylpolyglutamic Acids; Rats; Tetrahydrofolates; Trypsin

This report describes the intracellular metabolism of 5-methyltetrahydrofolate (5-methyl-H4PteGlu) and 5-formyltetrahydrofolate (5-formyl-H4PteGlu) to the various folate forms and their respective polyglutamated states in the MCF-7 human breast-cancer cell line. The intracellular folate distribution observed in MCF-7 cells treated with 5-methyl-H4PteGlu was similar to that seen in cells treated with 5-formyl-H4PteGlu. In cells exposed to 5-formyl-H4PteGlu for 24 h, the folate pool consisted of 103 +/- 10 pmol/mg 10-formyl-H4PteGlu, 120 +/- 18 pmol/mg H4PteGlu, and 71 +/- 18 pmol/mg 5-methyl-H4PteGlu versus 88 +/- 5, 54 +/- 20 and 87 +/- 10 pmol/mg, respectively, for cells exposed to 5-methyl-H4PteGlu. Only the difference seen in H4PteGlu levels between cells exposed to either 5-methyl-H4PteGlu or 5-formyl-H4PteGlu reached statistical significance (P < 0.05). In the absence of vitamin B12, exposure to 5-methyl-H4PteGlu resulted in 154 +/- 17 pmol/mg 5-methyl-H4PteGlu along with only 8 +/- 5 pmol/mg 10-formyl-H4PteGlu and 4 +/- 2 pmol/mg H4PteGlu, thus demonstrating the marked dependence on vitamin B12 for the metabolism of 5-methyl-H4PteGlu to the other intracellular folates. 5-10-Methylene- H4PteGlu (2 +/- 1.3 pmol/mg) was detected only in cells exposed to 5-formyl-H4PteGlu for 24 h, not in cells treated with 5-methyl-H4PteGlu. The profile of polyglutamates detected in cells treated with either 5-formyl-H4PteGlu or 5-methyl-H4PteGlu for 24 h was not significantly different, although cells treated with 5-methyl-H4PteGlu tended to have less conversion to the higher polyglutamates (Glu3-Glu5) as compared with those treated with 5-formyl-H4PteGlu. In 5-methyl-H4PteGlu-treated cells grown in the absence of vitamin B12, the pentaglutamate was the only polyglutamate form detected, accounting for only 11% of the total folate pool. Since there does not appear to be a greater formation of the optimal reduced-folate forms necessary to achieve enhanced thymidylate synthase (TS) inhibition through ternary-complex formation in cells exposed to 5-methyl-H4PteGlu versus 5-formyl-H4PteGlu, these studies suggest that the use of 5-methyl-H4PteGlu would not be advantageous over that of 5-formyl-H4PteGlu in combination regimens with the fluoropyrimidines.

Topics: Breast Neoplasms; Humans; Leucovorin; Polyglutamic Acid; Tetrahydrofolates; Tumor Cells, Cultured

A method for the simultaneous determination of the antifolates methotrexate and 7-hydroxymethotrexate as well as the folates 5-methyltetrahydrofolic acid and folinic acid (5-formyltetrahydrofolic acid) in serum and cerebrospinal fluid (CSF) is described. High-performance liquid chromatography with gradient elution and dual detection (ultraviolet absorption and fluorescence) was used to separate and quantitate the analytes. Serum samples containing high levels of the substances of interest and CSF samples were injected directly onto the HPLC column. For determination of low concentrations, serum samples were subjected to a solid-phase extraction method for clean-up and concentration purposes. The determination limits were 10 ng/ml for both antifolates, 100 ng/ml for folinic acid, and 0.1 ng/ml for the physiologically occurring methylated folate which is about 1/100 the serum concentration in healthy children. The suitability of the method for pharmacokinetic monitoring of high-dose methotrexate therapy combined with leucovorin rescue administered to children with acute lymphoblastic leukemia was demonstrated. Minimum values of the serum folate during treatment ranged from 0.2 to 3.1 ng/ml. Even those very low concentrations could be reliably measured.

Topics: Calibration; Child; Chromatography, High Pressure Liquid; Folic Acid Antagonists; Humans; Leucovorin; Methotrexate; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Specimen Handling; Tetrahydrofolates

The pharmacokinetic profiles of folinic acid (FA) and its active metabolite, 5-methyltetrahydrofolic acid, were studied after oral administration of decreasing doses of calcium folinate during 37 courses of high and intermediate dose methotrexate treatment in 25 lymphoma patients. FA was administered at a dose of 6 x 50 mg in 15 courses, 6 x 25 mg in seven courses, 6 x 15 mg in 10 courses and 6 x 7.5 mg in 5 courses. FA, 5-methyltetrahydrofolic acid, methotrexate and 70H-methotrexate were assayed simultaneously by high performance liquid chromatography. When FA was administered at doses between 50 and 15 mg, maximum concentrations of both the drug and its metabolite were always obtained after 1 to 2 h and remained stable. The same was true for the equilibrium concentration of the two products at doses over 15 mg. These findings suggest saturation of absorption and metabolism of folinic acid at doses over 15 mg.

Topics: Administration, Oral; Adult; Female; Humans; Leucovorin; Lymphoma, Non-Hodgkin; Male; Methotrexate; Middle Aged; Tetrahydrofolates; Time Factors

This report describes an analytical method for the measurement of tetrahydrofolate (H4PteGlu), 10-formyltetrahydrofolate (10-HCO-H4PteGlu) and 5-methyltetrahydrofolate (5-CH3-H4-PteGlu) in rat bile by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). After diluting the bile sample with 0.2% sodium ascorbate solution, the sample was analyzed under the following conditions; (a) phenyl bonded phase column as an analytical column; (b) mobile phase consisting of 20 mM acetate buffer (pH 5.0) containing 0.1 mM EDTA; (c) an applied ECD potential of +300 mV; (d) 0.8 ml/min of flow rate. Under the above conditions, peaks of H4PteGlu, 10-HCO-H4PteGlu and 5-CH3-H4PteGlu in rat bile were well separated on the ECD-chromatogram. Detection limits of H4PteGlu, 10-HCO-H4PteGlu and 5-CH3-H4PteGlu were 0.13, 0.11 and 0.10 ng/ml, respectively, at S/N = 3. Bile excretion rates for H4PteGlu, 10-HCO-H4PteGlu and 5-CH3-H4PteGlu, which were analyzed by this method in rats, were 314 +/- 181, 321 +/- 179 and 449 +/- 198 ng/hr, respectively. Bile concentrations of the folates were more than 5,000 times higher than the detection limits for this method. This HPLC-ECD method is, therefore, a useful tool for bile folate analysis.

Topics: Animals; Bile; Chromatography, High Pressure Liquid; Electrochemistry; Evaluation Studies as Topic; Female; Leucovorin; Rats; Rats, Sprague-Dawley; Tetrahydrofolates

A pharmacokinetic study of folic acid and its metabolites was conducted to provide a basis to consider folic acid as a therapeutic alternative to leucovorin. Leucovorin has been used in various folate antagonist rescue regimens and to modulate fluorouracil activity in the treatment of solid tumors. Although leucovorin is typically administered intravenously in fluorouracil modulation therapy, limited oral administration trials have yielded equivalent responses. Because metabolites rather than leucovorin are the predominant circulating species after oral administration, these clinical results indicate that metabolites themselves can be modulating agents. Folic acid could be an attractive alternative to leucovorin provided it effectively elevates the same plasma metabolites. Hence, folic acid at doses of 25 and 125 mg/m2 was administered orally and intravenously to normal volunteers. Plasma folic acid and its reduced folate metabolites were monitored over a 24-hour period by use of a previously developed radioenzymatic method. The metabolites that accumulated--5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate, tetrahydrofolate, and 10-formyltetrahydrofolate--were the same metabolites that were observed previously after leucovorin administration. Folic acid metabolites accumulated more slowly and persisted longer than leucovorin metabolites, which can be attributed to slower metabolism of the fully oxidized vitamin. Based on these results, it is concluded that folic acid could be an attractive therapeutic alternative to leucovorin for fluorouracil modulation.

Topics: Adult; Female; Folic Acid; Humans; Leucovorin; Male; Reference Values; Tetrahydrofolates

Reduced folate derivatives in rat bile were examined using high-performance liquid chromatography with electrochemical detection (HPLC-ED). Three peaks of folate compounds were observed on the chromatograms. From the retention-time profiles and hydrodynamic voltammograms, and the profiles of ultraviolet (UV) absorbance spectra obtained by HPLC with photodiode array detection, these 3 peaks were identified as 10-formyltetrahydrofolate (10-HCO-H4PteGlu), tetrahydrofolate (H4PteGlu) and 5-methyltetrahydrofolate (5-CH3-H4PteGlu). The rates of bile secretion of 10-HCO-H4PteGlu, H4PteGlu and 5-CH3H4PteGlu were 314 +/- 181, 321 +/- 179 and 449 +/- 198 ng/h (mean +/- S.D.), respectively. 10-HCO-H4PteGlu and H4PteGlu together wtih 5-CH3-H4-PteGlu are found to be the major folate derivatives in rat bile. The nonmethylated folates, 10-HCO-H4PteGlu and H4PteGlu, may also play an important role in folate homeostasis.

Topics: Animals; Bile; Chromatography, High Pressure Liquid; Female; Folic Acid; Hydrogen-Ion Concentration; Leucovorin; Rats; Rats, Sprague-Dawley; Spectrophotometry, Ultraviolet; Tetrahydrofolates; Tritium

Highly purified 5-l-methyltetrahydrofolate (m-THF) and 5-l-formyl-THF (f-THF) preparations were compared for rescuing from methotrexate (MTX) toxicity in DBA2 mice transplanted with L1210 leukemia. Mice received two doses of reduced folates (2 mg/kg, s.c.) 16 and 24 h after a single s.c. MTX dose. f-THF was 1.8 time more effective than m-THF in protecting tumor cells from MTX (800 mg/kg). This MTX dose caused a 57% fall in circulating polymorphonucleates, which was prevented by both reduced folates. Treatment with 800 mg/kg of MTX plus m-THF was 1.5 fold more effective than the same MTX dose plus f-THF in increasing survival time of tumor-bearing mice. These data suggest a higher selectivity and efficacy of l-m-THF with respect to l-f-THF in rescuing from MTX toxicity.

Topics: Animals; Leucovorin; Leukemia L1210; Methotrexate; Mice; Mice, Inbred DBA; Neoplasm Transplantation; Tetrahydrofolates

Folinic acid (dlFA) is increasingly used in clinical oncology. The active isomer lFA is intensively metabolized into l5-methyltetrahydrofolate (l5MTHF), the relative proportions of lFA, dFA and l5MTHF in blood varying considerably between oral and i.v. FA administration. The purpose of the study was to compare the in vitro activities of pure lFA and pure l5MTHF at equivalent drug exposure [area under curve (AUC)], taking into account their respective chemical stability in the culture medium. The in vitro growth inhibition [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) test] was evaluated on five human tumor cell lines after methotrexate (MTX)-folate or 5-fluorouracil (5FU)-folate exposures. Not only were the activities of lFA and l5MTHF compared, but also clinically relevant mixtures of lFA + dFA + l5MTHF corresponding to the proportions found at steady state during oral (PO mixture, 4, 39 and 57%, respectively) and i.v. administrations (i.v. mixture, 7, 81 and 12%, respectively). Measurement of folates demonstrated the marked lability of l5MTHF (65.8% loss over 5 days in the culture medium) as compared to lFA (2.6% loss). Whatever the pharmacological model tested (MTX-folate or 5FU-folate), comparison of the folate effects at equivalent drug exposure taking into account their relative stability showed that l5MTHF was never more potent than lFA. Moreover, a higher efficiency of lFA was demonstrated for the cell line most sensitive to 5FU; in this case, as expected, the i.v. mixture was more potent than the PO mixture. This study shows that depending on the tumor, lFA can be more potent than its main circulating metabolite l5MTHF. Along with the limited capacity of oral absorption, the choice between oral and i.v. route for FA administration in patients should take into consideration the different pharmacological activities between lFA and l5MTHF which suggest that the oral route is potentially detrimental to the optimal activity of the 5FU-FA combination as compared to i.v. administration.

Topics: Antineoplastic Agents; Cell Division; Drug Stability; Drug Synergism; Fluorouracil; Humans; Leucovorin; Methotrexate; Oxidation-Reduction; Stereoisomerism; Tetrahydrofolates; Tumor Cells, Cultured

The folic acid content of grain, cereal products (including beer), bakery products and legumes was determined by means of high-performance liquid chromatography (HPLC). Free folate (monoglutamate forms) and total folate (monoglutamate + polyglutamate forms) were differentiated. Of the grain analysed, rye, with a mean value of 143 micrograms/100 g, contained more total folate than wheat (mean = 91 micrograms/100 g). The total folate content of bakery products ranged from 14 micrograms/100 g (whole grain rye bread) to 88 micrograms/100 g (crispbread). Beer had a very low total folate content (mean = 3 micrograms/100 ml). The mean of the free folate portion was 76.5% in grain and cereal products and 65.6% in bakery products. Of the legumes analysed, beans (mean = 128 micrograms/100 g) had the highest content of total folate, followed by lentils (103 micrograms/100 g) and peas (57 micrograms/100 g). The mean value of the free folate portion in legumes (73.1%) was comparable with the values of grain, cereal products and bakery products. In addition to tetrahydrofolic acid (THF), 5-methyl-THF and 5-formyl-THF, pteroylglutamic acid (PteGlu) and 10-formyl-PteGlu were determined in all products (except beer). Their proportion (PteGlu + 10-formyl-PteGlu) of the total folate content ranged from 23.5% to 44.4%.

Topics: Beer; Bread; Edible Grain; Fabaceae; Folic Acid; Leucovorin; Plants, Medicinal; Secale; Tetrahydrofolates; Triticum

Topics: Electrochemistry; Leucovorin; Magnetic Resonance Spectroscopy; Molecular Conformation; Oxidation-Reduction; Tetrahydrofolates

The folic acid content of food of animal origin was determined by means of high-performance liquid chromatography (HPLC). Free folate (monoglutamate forms) and total folate (monoglutamate + polyglutamate forms) were differentiated. Liver (bovine 963, hog 136 micrograms/100 g) and kidney (bovine 410, hog 93 micrograms/100 g) contained the highest content of total folate, followed by egg (67 micrograms/100 g), soft cheese (44-62 micrograms/100 g), milk and other dairy products (3-28 micrograms/100 g). Chicken, fish, beef and pork contained only a low content of total folate (1-12 micrograms/100 g). The free folate portion ranged from 14.7 to 100% (mean 69.2%). The main folate derivatives tetrahydrofolic acid (THF), 5-methyl-THF and 5-formyl-THF showed the following distribution. In milk and milk products the mean was 70% for 5-formyl-THF, 20% for 5-methyl-THF and 10% for THF; in the other products the three derivatives were distributed about equally but the range of variation was high.

Topics: Animals; Cattle; Cheese; Chickens; Chromatography, High Pressure Liquid; Dairy Products; Eggs; Fishes; Folic Acid; Kidney; Leucovorin; Liver; Meat; Milk; Swine; Tetrahydrofolates

The pharmacokinetic values of d,l-leucovorin and l-leucovorin were compared in eight healthy volunteers following oral administration of 25 mg d,l-leucovorin and 12.5 mg l-leucovorin. Serum levels of l-5-formyltetrahydrofolate, l-5-methyltetrahydrofolate, and total reduced folates were measured by an established microbiological method. Pharmacokinetic data for both preparations were consistent with those previously reported for d,l-leucovorin, with essentially complete first pass metabolism to l-5-methyltetrahydrofolate, the active metabolite. No differences were found between the two preparations in serum concentrations of active folate fractions, AUC, or Cmax, or in clearance and volume of distribution estimates. These data suggest that after administration of 25 mg of d,l-leucovorin, the d-diastereoisomer has no significant effect on the standard pharmacokinetic measurements of the active l-folates.

Topics: Adolescent; Adult; Humans; Leucovorin; Male; Middle Aged; Stereoisomerism; Tetrahydrofolates

We present a rapid, sensitive, and automated HPLC method with direct resolution of l-folinic acid (l-FA), d-folinic acid (d-FA), and 5-methyltetrahydrofolate (5MTHF) from plasma samples. Plasma (500 microL) is first extracted on solid phase (RP-18 cartridge). The dI-FA peak is collected on-line from a reversed-phase column (RP-8, 119 x 2 mm, 4 microns: HPLC 1) and then automatically loaded onto a chiral stationary phase (human serum albumin, 150 x 4.6 mm, 7 microns: HPLC 2). The same mobile phase flows in both systems (0.2 mol/L Na2HPO4:1-propanol, 98:2, pH 6.2). HPLC 1 allows quantification of 5MTHF by absorption at 313 nm; HPLC 2, the quantification of l-FA and d-FA by electrochemical detection in the oxidation mode (total run time 18 min). Recoveries are > 80%. CVs for intra- and interassay reproducibilities are < 5% and 15%, respectively. Linearity of the response (0.1-1 mumol/L and 1-50 mumol/L, r = 0.99, P < 0.01) is satisfactory. The sensitivity limit is 50 nmol/L for 5MTHF and 20 nmol/L for l-FA and d-FA. This assay is substantially improved over existing methods regarding feasibility and is being used in pharmacokinetic investigations in cancer patients.

Topics: Acetaminophen; Aspirin; Chromatography, High Pressure Liquid; Fluorouracil; Humans; Leucovorin; Quality Control; Stereoisomerism; Tetrahydrofolates

The critical disturbance of folate metabolism caused by vitamin B12 deficiency which results in megaloblastic anaemia remains controversial. Vitamin B12 is required in the methionine synthase reaction in which homocysteine is converted to methionine and methyl tetrahydrofolate (methyl THF) to THF. The 'methyl-folate trap' hypothesis suggested that failure of demethylation of methyl THF with consequent deficiency of folate co-enzymes derived from THF is the crucial lesion caused by vitamin B12 deficiency. A more recent theory suggested that reduced supply of methionine leads to reduced availability of 'activated formate' and hence of formyl THF and it is this defect that results in failure of folate co-enzyme synthesis. The present results, based on deoxyuridine suppression tests on 103 cases of megaloblastic anaemia, show that THF itself is equally capable of correcting the failure of thymidylate synthesis in vitamin B12 deficiency as in folate deficiency. Although not as effective as formyl THF in correcting the dU blocking test in vitamin B12 deficiency, this is equally so for the correction of the test by THF compared with formyl THF in folate deficiency. The results therefore favour the theory that it is in the supply of THF and not of 'active formate' or formyl THF that vitamin B12 plays a critical role in folate metabolism.

Topics: Anemia, Megaloblastic; Bone Marrow; Cells, Cultured; Deoxyuridine; DNA; Female; Humans; Leucovorin; Male; Megaloblasts; Tetrahydrofolates; Thymidine Monophosphate; Vitamin B 12; Vitamin B 12 Deficiency

The therapeutic activity of FUra alone or combined with [6RS]LV doses ranging from 50 to 1,000 mg/m2 was examined in eight colon adenocarcinoma xenografts, of which five were established from adult neoplasms (HxELC2, HxGC3, HxVRC5, HxHC1, and HxGC3/c1TK-c3 selected for TK deficiency) and three were derived from adolescent tumors (HxSJC3A, HxSJC3B, and HxSJC2). The growth-inhibitory effects of FUra were potentiated by higher doses of [6RS]LV (500-1,000 mg/m2) in three lines (HxGC3/c1TK-c3, HxSJC3A, and HxSJC3B) and by a low dose of [6RS]LV in only one tumor (HxVRC5). Expansion of pools of CH2-H4PteGlun+H4PteGlun (greater than or equal to 2.4-fold) in response to higher doses of [6RS]LV was obtained in all lines except HxHC1. Metabolism of [6RS]LV was high in HxVRC5, with high levels of 5-CH3-H4PteGlu being detected, but not in HxHC1, in which levels of 5-CH3-H4PteGlu and CH = H4PteGlu+10-CHO-H4PteGlu remained relatively low. In the adolescent tumors, levels of CH = H4PteGlu+10-CHO-H4PteGlu were consistently higher than those of 5-CH3-H4PteGlu following [6RS]LV administration, and in HxSJC3A, in which pools of CH2-H4PteGlun+H4PteGlun were significantly expanded, 5-CH3-H4PteGlu concentrations were lower than those observed in the other two lines. The sensitivity of tumors to FUra +/- [6RS]LV and the characteristics of [6S]LV metabolism did not correlate with the activity of CH = H4PteGlu synthetase, the enzyme responsible for the initial cellular metabolism of [6S]LV to CH = H4PteGlu. Thus, no single metabolic phenotype correlated with the [6RS]LV-induced expansion of CH2-H4PteGlun+H4PteGlun pools. Potentiation of the therapeutic efficacy of FUra by [6RS]LV was observed in HxGC3/c1TK-c3 xenografts but not in parent HxGC3 tumors, demonstrating the influence of dThd salvage capability in the response to FUra-[6RS]LV combinations. Plasma dThd concentrations in CBA/CaJ mice were high (1.1 microM). The present data therefore demonstrate the importance of (1) higher doses of [6RS]LV, (2) expansion of pools of CH2-H4PteGlun+H4PteGlun, and (3) dThd salvage capability in potentiation of the therapeutic efficacy of FUra in colon adenocarcinoma xenografts. The plasma levels of FUra achieved in mice are presented.

Topics: Adenocarcinoma; Animals; Antineoplastic Combined Chemotherapy Protocols; Colonic Neoplasms; Fluorouracil; Humans; Leucovorin; Mice; Mice, Inbred CBA; Neoplasm Transplantation; Tetrahydrofolates

We have characterized the uptake of 5-formyltetrahydrofolate into isolated rat liver mitochondria. Uptake of 5-formyltetrahydrofolate, 5-methyltetrahydrofolate and folic acid was linear for the first 2 min of incubation. 5-Methyltetrahydrofolate and 5-formyltetrahydrofolate were accumulated in mitochondria to 3.7- and 4.3-fold, respectively. 5-Formyltetrahydrofolate transport showed a pH optimum at 5.5 and was saturable with an apparent Km = 2.80 mumol.L-1 and Vmax = 3.49 pmol.min-1.mg protein-1. Uptake of 5-formyltetrahydrofolate was inhibited by the structural analog 5-methyltetrahydrofolate and, to a much lesser extent, folic acid and methotrexate. Uptake was not inhibited by various substrates of known mitochondrial transporters, e.g., succinate, malate, citrate, glutamate and phosphate, nor was uptake inhibited by the sulfhydryl reagent, N-ethylmaleimide. Uptake was not stimulated by preloading of mitochondria with malate or aspartate. Uptake of 5-formyltetrahydrofolate was not affected by the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenyl-hydrazone nor by collapsing the electrical gradient with valinomycin. These results indicate that 5-formyltetrahydrofolate uptake into mitochondria via a carrier-mediated system specific for the reduced, naturally occurring folates. The folates, once in the mitochondria, may be converted to the polyglutamylated forms, which will not readily diffuse out of the mitochondria.

Topics: Animals; Biological Transport, Active; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Carrier Proteins; Cells, Cultured; Diffusion; Folic Acid; Hydrogen-Ion Concentration; Leucovorin; Methotrexate; Mitochondria, Liver; Rats; Rats, Sprague-Dawley; Tetrahydrofolates; Valinomycin

Two membrane folate receptor (MFR) isoforms are present in human tissues i.e. MFR-1 (e.g. placenta) and MFR-2 (e.g. placenta, KB cells, CaCo-2 cells). MFR-1 was expressed in COS-1 cells and the resulting protein had the same polypeptide molecular weight as the native protein. The affinities of (6S) and (6R) diastereoisomers of N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, and 5,10-dideazatetrahydrofolate as well as folic acid and methotrexate to MFR-1, MFR-2 and placental MFR (MFR-1 plus MFR-2) were determined in terms of the Ki values for their competitive inhibition of the binding of [3H]folic acid to these proteins. The results indicated a striking difference in the stereospecificity of MFR-1 and MFR-2 for reduced folate coenzymes; MFR-2 preferentially bound to the physiological (6S) diastereoisomers and MFR-1 bound preferentially to the unphysiological (6R) diastereoisomers, while dideazatetrahydrofolate did not show significant stereospecificity for MFR-1. Furthermore, MFR-2 displayed significantly (2- to 100-fold) greater affinities for all the compounds tested compared to MFR-1. Purified placental MFR, a natural source of MFR-1 which contains variable amounts of MFR-2, showed intermediate Ki values for the compounds tested compared with MFR-1 and MFR-2 and stereospecificities similar to MFR-1. These observations demonstrate striking differences in the ligand binding sites of MFR-1 and MFR-2 which could potentially be exploited in the design of MFR isoform specific antifolates.

Topics: Animals; Binding, Competitive; Carrier Proteins; DNA; Female; Folate Receptors, GPI-Anchored; Folic Acid; Folic Acid Antagonists; Haplorhini; Humans; KB Cells; Kinetics; Leucovorin; Methotrexate; Placenta; Receptors, Cell Surface; Stereoisomerism; Tetrahydrofolates

The intestinal absorption and in vivo kinetics of (6S)-[3H]-5-methyl-tetrahydrofolate (5-methyl-H4folate), (6S)-[3H]-5-formyl-H4folate and [3H]folic acid were investigated to determine whether inherent differences exist in the overall bioavailability of these folates in rats. Adult rats (n = 9 per group) were given an intragastric dose of the appropriate folate (50 pmol/100 g body wt) in 50 mmol/L ascorbate (pH 7). Each compound underwent nearly complete absorption within 8 h, and there was no significant difference in the excretion kinetics in relation to the form of folate administered. A biphasic pattern of excretion was observed over the following 8 d. Both urine and feces were important excretory routes. The rapid phase of total isotopic excretion (urinary and fecal) exhibited a half time (t1/2) of 0.11-0.12 d, whereas the t1/2 of the slower phase was 13.4-15.9 d. Isotopic distributions and the pattern of labeled folates in urine and tissues were similar regardless of the form administered. These results indicate that the bioavailability of orally administered folic acid, 5-methyl-H4folate and 5-formyl-H4folate is equivalent in rats under the conditions of this study.

Topics: Animals; Biological Availability; Feces; Folic Acid; Intestinal Absorption; Kinetics; Leucovorin; Male; Rats; Tetrahydrofolates; Tritium

A method for the determination of low levels of the stereoisomers of leucovorin and 5-methyltetrahydrofolate has been developed and validated. The assay involved initial chromatography on a bovine serum albumin (BSA)-based high-performance liquid chromatography chiral stationary phase (CSP) followed by post-column peak compression and elution on two C18 columns. In this manner, the poor efficiency of the BSA-CSP was overcome and sub-microgram quantities of the target solutes could be detected. The BSA-CSP separated the leucovorin and 5-methyletrahydrofolate from interfering plasma components and from each other and achieved the stereochemical resolution of the diastereomeric (6S)-and (6R)-leucovorin. The eluent containing (6S)-leucovorin was directed onto one C18 column and the eluent containing (6R)-leucovorin and 5-methyltetrahydrofolate was directed onto the other. This was followed by sequential rapid gradient elution of the target compounds from the respective C18 columns. The method was validated for plasma levels ranging from 15 to 500 ng/ml and was able to detect leucovorin concentrations of as low as 5 ng/ml.

Topics: Animals; Chromatography, High Pressure Liquid; Dogs; Humans; Kinetics; Leucovorin; Microchemistry; Serum Albumin, Bovine; Stereoisomerism; Tetrahydrofolates

[6RS]Leucovorin (5-formyltetrahydrofolate; 5-CHO-H4PteGlu) administered in different regimens in combination with 5-fluorouracil (FUra) has increased the response rates to FUra in patients with colon adenocarcinoma. Using preclinical models of human colon adenocarcinomas as xenografts in immune-deprived mice, the effect of the rate of administration of racemic [6RS]leucovorin on the concentration-time profile of reduced folates in plasma, size of intratumor pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun), and the distribution of their polyglutamate species have been examined. Bolus injection i.v., or 4-h or 24-h infusion of [6RS]leucovorin (500 mg/m2) yielded similar concentration profiles of the biologically active [6S] and inactive [6R] isomers of 5-CHO-H4-PteGlu and 5-methyltetrahydrofolate (5-CH3-H4PteGlu) in mouse plasma to those previously reported in humans, but with more rapid elimination half-lives (t1/2 = 11 to 16 min, 23 to 41 min, and 30 to 35 min, respectively). Thus, reduced folates remained elevated in plasma during the period of [6RS]leucovorin administration. In HxELC2 and HxGC3 tumors, pools of CH2-H4PteGlun and H4PteGlun were increased from 350% to 700% of control, but only during [6RS]leucovorin infusion. Intracellular levels subsequently declined rapidly, similar to the loss of reduced folates from plasma. Increasing the rate of [6RS]leucovorin delivery by decreasing the time for administration from a 24-h to a 4-h infusion did not further increase the intratumor pools of CH2-H4PteGlun and H4PteGlun, suggesting saturation in the cellular metabolism of [6RS]leucovorin. In HxGC3 tumors, CH2-H4PteGlu4-5 were elevated more rapidly than in line HxELC2, which accumulated predominantly a shorter chain length species following i.v. bolus injection. During the 4-h infusion schedule, di- and triglutamate species in particular accumulated in both tumors with no elevation in CH2-H4PteGlu5 until the infusion was discontinued, when this species increased as the shorter chain length forms were declining. However, during the 24-h infusion of [6RS]leucovorin, CH2-H4PteGlu3-5 were elevated in tumors. Since these species have been reported to increase the binding affinity of [6-3H]5-fluorodeoxyuridine monophosphate ([6-3H]FdUMP) to thymidylate synthase, and intratumor pools of CH2-H4PteGlun and H4PteGlun were elevated during the 24-h infusion of [6RS]leucovorin, this was considered to be the preferred schedule

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Female; Floxuridine; Half-Life; Humans; Injections, Intravenous; Leucovorin; Mice; Mice, Inbred CBA; Tetrahydrofolates; Thymidylate Synthase; Time Factors

To study the activity of continuous infusion cisplatin, 5-fluorouracil, and high-dose leucovorin (PFL) as induction chemotherapy in patients with previously untreated, advanced squamous cell carcinoma of the head and neck.. Nonrandomized, prospective trial.. A comprehensive cancer center.. Thirty-five patients (4 patients [11%], stage III; 31 patients [89%], stage IV [MO]), all evaluable for response and toxicity.. Two to three cycles of PFL before definitive, local-regional therapy (surgery and radiation therapy or radiation therapy alone). Chemotherapy included continuous intravenous infusion of cisplatin (25 mg/m2 body surface area daily, days 1 through 5); 5-fluorouracil (800 mg/m2 body surface area daily, days 2 through 6); and leucovorin (500 mg/m2 body surface area daily, days 1 through 6) administered once every 28 days. Pathologic response was evaluated by surgical resection or biopsy. Serum-reduced folates were measured before and 18 hours after the initiation of chemotherapy.. A clinical response to PFL was achieved in 28 of 35 (80%) patients: 23 (66%) patients had a complete response (90% CI, 50% to 79%) and 5 (14%) patients, a partial response. A complete response was confirmed pathologically in 14 of 19 (74%) patients. The most common toxicity was mucositis (grade 2 to 3; 94% of patients). Dose reduction for toxicity was necessary in 11 (31%) patients. There were no treatment-related deaths. Serum levels of leucovorin and (6S)5-methyltetrahydrofolate were measured in 7 patients. After 18 hours, the mean leucovorin level (+/- SD) was 34.3 +/- 1.5 mumol/L, of which only 8.0 +/- 0.5% was the active 6S isomer. The mean serum (6S)5-methyltetrahydrofolate was 9.2 +/- 0.6 mumol/L.. Continuous infusion cisplatin, 5-fluorouracil, and high-dose leucovorin is a new and highly active chemotherapy regimen that can achieve clinical and pathologically confirmed complete responses in a substantial proportion of patients with advanced, local-regional squamous cell carcinoma of the head and neck. Further studies are needed to confirm the activity of PFL and to determine its potential impact on local tumor control and disease-free and overall survival.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cisplatin; Drug Administration Schedule; Drug Evaluation; Female; Fluorouracil; Head and Neck Neoplasms; Humans; Infusions, Intravenous; Leucovorin; Male; Middle Aged; Neoplasm Staging; Tetrahydrofolates

After the use of d,1-folinic acid (d,1-CHO-THF), pharmacokinetic measurements should take into account 1-CHO-THF and its metabolite 1-methyltetrahydrofolic acid (1-CH3-THF) as well as d-CHO-THF. For this purpose, we developed a simple and rapid assay by combining reversed-phase HPLC to determine total levels of d,1-CHO-THF and CH3-THF and chiral HPLC to separate the biologically active 1-CHO-THF from the inactive d-CHO-THF. We investigated the pharmacokinetics after short-term infusion of 300 mg d,1-CHO-THF in ten healthy volunteers. With a mean of 56.5 min, 1-CHO-THF exhibits a rapid body clearance of 222 ml/min, about 60% of which is caused by metabolism to CH3-THF and 40%, by renal excretion. CH3-THF has a terminal half-life of 208 min and a total body clearance of 88.9 ml/min, which is essentially the same as the renal clearance. Due to the lower clearance of CH3-THF, its AUC (2,132 microM x min) exceeds that of 1-CHO-THF (1445 microM x min) by approximately 50%. In contrast to that of the reduced 1-folates, the total body and renal clearance of d-CHO-THF is very low, with values of 13.2 and 12.9 ml/min, respectively. This results in a very high AUC of 24, 269 microM x min, which is higher by factors of 17 and 11 than those of 1-CHO-THF and CH3-THF, respectively. The implications of the distinct kinetics of the reduced 1-folates and d-CHO-THF for the efficacy of folinic acid/5-fluorouracil therapy and adequate protocols for the treatment of advanced colorectal cancer are discussed.

Topics: Adult; Chromatography, High Pressure Liquid; Female; Half-Life; Humans; Infusions, Intravenous; Kidney; Leucovorin; Male; Metabolic Clearance Rate; Molecular Conformation; Oxidation-Reduction; Tetrahydrofolates; Time Factors

Although folate deficiency and increased requirements for folate are observed in most alcoholics, the possibility that acetaldehyde generated from ethanol metabolism may increase folate catabolism has not been previously demonstrated. Folate cleavage was studied in vitro during the metabolism of acetaldehyde by xanthine oxidase, measured as the production of p-aminobenzoylglutamate from folate using h.p.l.c. Acetaldehyde/xanthine oxidase generated superoxide, which cleaved folates (5-methyltetrahydrofolate greater than folinic acid greater than folate) and was inhibited by superoxide dismutase. Cleavage was increased by addition of ferritin and inhibited by desferrioxamine (a tight chelator of iron), suggesting the importance of catalytic iron. Superoxide generated from the metabolism of ethanol to acetaldehyde in the presence of xanthine oxidase in vivo may contribute to the severity of folate deficiency in the alcoholic.

Topics: Acetaldehyde; Ethanol; Ferritins; Folic Acid; Leucovorin; Superoxides; Tetrahydrofolates; Xanthine Oxidase

Patients with advanced gastric cancer were treated with methotrexate-5-fluorouracil sequential therapy. In order to rescue of methotrexate toxicity on the normal proliferating tissue, leucovorin was administered orally to outpatients. As the comparative study, we examined plasma kinetics following oral administration of leucovorin (15 mg) in 6 patients with gastric cancer (received subtotal gastrectomy) and 5 normal subjects. In the patients, peak plasma concentrations of d-1-formyltetrahydrofolate and its main metabolite 5-methyltetrahydrofolate were found after 2 hrs. (7.35 X 10(-7) M, 3.09 X 10(-7) M), and in normal controls also both after 2 hrs. (1.01 X 10(-6) M, 4.89 X 10(-7) M). The active folate metabolite persisted for more than 6 hrs. at over 1 X 10(-7) M that could rescue normal tissue from methotrexate toxicity after leucovorin oral administration in the patients received subtotal gastrectomy.

Topics: Administration, Oral; Adult; Biological Assay; Chromatography, High Pressure Liquid; Female; Formyltetrahydrofolates; Gastrectomy; Humans; Leucovorin; Male; Middle Aged; Stomach Neoplasms; Tablets; Tetrahydrofolates

As part of a clinical trial of cisplatin, 5-fluorouracil (5-FU), and leucovorin (LV) for treatment of patients with advanced head and neck cancer, patients received 100 mg of LV (d,l-5-formyltetrahydrofolate) orally every 4 hours for 5 days. On days 2 and 4 of treatment, plasma samples were obtained 2 hours after (peak) and 30 minutes before (trough) a dose of LV. Total LV and 5-methyltetrahydrofolate (THF) concentrations were measured with high-performance liquid chromatography (HPLC) analysis. LV stereoisomer concentrations were determined by chiral HPLC on a bovine serum albumin-bonded silica column. Thus far, plasma folate levels have been analyzed for ten cycles of treatment administered to 7 patients (40 samples). Administration of LV in divided oral doses approximates a plasma steady state with no significant differences noted between peak and trough concentrations. Mean (+/- SD) plasma concentrations for all samples were (mumol): LV, 3.2 +/- 1.3; l-LV, 0.28 +/- 0.21; d-LV, 2.9 +/- 1.2; and THF, 4.25 +/- 2.5. Plasma levels of d-LV and THF tended to be approximately 10% higher on day 4 than day 2, although mean differences were not significantly different due to substantial interpatient variability. Of note was that the sum of THF and l-LV exceeds that of d-LV which was consistent with selective absorption of the l-isomer of LV. Mean ratios of d-LV/l-LV and d-LV/l-LV and THF were 13.7 +/- 10 and 0.88 +/- 0.68, respectively. The authors conclude that oral administration of LV in divided dose (1) simulates a continuous intravenous infusion; (2) produces plasma levels of l-reduced folates in a range known to potentiate 5-FU cytotoxicity; and (3) results in low ratios of d/l-reduced folates that may be important in maximizing the effectiveness of 5-FU-LV chemotherapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cisplatin; Drug Administration Schedule; Fluorouracil; Head and Neck Neoplasms; Humans; Leucovorin; Stereoisomerism; Tetrahydrofolates

Two trials of leucovorin (LV) and 5-fluorouracil (5-FU) in patients with metastatic colorectal cancer were done, both using a 3-day loading dose and then weekly doses to minimize toxicity. The first trial used LV administered by intravenous infusion with a constant dose of 5-FU 400 mg/m2, and the second trail used oral LV with increasing doses of 5-FU. In the first trail, 45 eligible patients (20 with and 25 without previous therapy) were treated. Toxicity usually consisted of diarrhea or weakness and was controlled by delaying or decreasing the 5-FU dose. Subjective responses occurred in 75% of patients but did not correlate with antineoplastic effect. Objective responses were seen in 36% and stabilization of disease in 31% of patients; these correlated with prolonged survival. Median survival was 8 months for patients with previous treatment and 10 for those without. Twelve-month survival was 32% and 40%, respectively. There was no correlation between the development of toxicity and response or survival. The second trial was conducted recently in cooperation with Duke University to determine toxicity and efficacy of oral LV with intravenous 5-FU before a randomized trial of this combination versus placebo with intravenous 5-FU. Eighteen patients were treated, and serum levels of folates were obtained on ten. First toxicity occurred at 5-FU doses ranging from 375 to 850 mg/m2, and consisted of diarrhea in nine, lethargy in seven, nausea/vomiting in four, dermatitis in four, conjunctivitis in two, hypersalivation in two, stomatitis in one, and profound granulocytopenia in one. Response rate was 35%, and stabilization was 35% with median survival of 14 months. Twelve-month survival was 56%.

Topics: Adenocarcinoma; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Carcinoembryonic Antigen; Colorectal Neoplasms; Drug Administration Schedule; Female; Fluorouracil; Humans; Leucovorin; Male; Middle Aged; Prognosis; Tetrahydrofolates

The carcinogenic effects of methyl-deficient, amino acid-defined diets have been attributed to alterations in cellular methylation reactions. These diets contain no choline, and methionine is replaced by homocysteine. Hence, all methyl groups needed for methionine biosynthesis with subsequent formation of S-adenosylmethionine and polyamines must be formed de novo utilizing folate-dependent reduction of one-carbon units. In rats fed the methyl-deficient diet, there was a marked decrease in total liver folate levels. This decrease was apparent in the levels of the individual forms of folate: 10-HCO-H4folate, 5-HCO-H4folate, 5-CH3-H4folate and H4folate. The percent of the total folate pool made up by 5-CH3-H4folate did not change, however, until after the rats had been fed the methyl-deficient diet for 4 wk, and then an increase was seen. After the methyl-deficient rats were switched to a nutritionally adequate control diet containing methionine and choline, all values rapidly reversed. Increased use of folate for methyl group biosynthesis may be responsible for the loss of folates from the liver.

Topics: Amino Acids; Animals; Choline Deficiency; Chromatography, High Pressure Liquid; Diet; Folic Acid; Leucovorin; Liver; Male; Methionine; Methylation; Rats; Rats, Inbred F344; Tetrahydrofolates

In the first part of this study the availability of folinic acid (FA) and its main active circulating metabolite, 5-methyltetrahydrofolate (5-MTHF), were studied in plasma and cerebrospinal fluid (CSF) from normal subjects after i.v. administration of 100 and 250 mg of FA. 5-MTHF rapidly appeared in plasma, the maximum value being reached at the first observation time point (1 h). FA was eliminated in plasma more slowly than 5-MTHF. Between the two doses, there was no evidence of modification in pharmacokinetic parameters (terminal half-life, clearance) for either FA or 5-MTHF in plasma and CSF; 5-MTHF was the only product detectable in CSF. Considering FA plus 5-MTHF together, the AUC (area under the curve) ratios between CSF and plasma were close to 1%. 5-MTHF was cleared very slowly from CSF (t 1/2 = 85 h). This finding suggested possible accumulation of 5-MTHF in CSF during repeated administration of FA combined with medium or high dose MTX. In the second part of the study, dealing with a group of eight children treated by such protocols, an increase in CSF 5-MTHF was detected from cycle to cycle in five (r = 0.91, P less than 0.01) with a maximum at 5 x 10(-8) M. This progressive accumulation of 5-MTHF in CSF may have a negative effect on the local action of MTX and should be taken into account for therapeutic strategies designed for the management of meningeal leukaemia.

Topics: Child; Child, Preschool; Female; Humans; Leucovorin; Male; Methotrexate; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Tetrahydrofolates

A high-performance liquid chromatographic method for the determination of N5-methyltetrahydrofolic acid, leucovorin, methotrexate and 7-hydroxymethotrexate in plasma and liquor samples is presented. Gradient elution is used to increase the sensitivity. Four sample preparation methods were compared with respect to the stability of the injectable sample. Samples can be pretreated with a simple deproteinization method. For enhanced selectivity a solid-phase extraction procedure is described.

Topics: Chromatography, High Pressure Liquid; Humans; Leucovorin; Methotrexate; Spectrophotometry, Ultraviolet; Tetrahydrofolates

Topics: Humans; Leucovorin; Methotrexate; Tetrahydrofolates

Several chemotherapeutic protocols for the treatment of malignancies include administration of methotrexate (MTX) during or shortly after total anesthesia. Clinical observations in patients treated for breast carcinoma or childhood cancer have shown unexpected myelosuppression and mucosal damage. This phenomenon may be attributed to the synergistic effects of nitrous oxide, which inactivates the cobalamin coenzyme of methionine synthase, and MTX, which inhibits dihydrofolate reductase, on folate metabolism. However, no quantitative data on dose-effect relationships are available regarding the combined toxicity of MTX and N2O. We investigated the effect of exposure to N2O on the toxicity of MTX. Groups of male Wistar rats were exposed to either 50% N2O/50% O2 or air for 12-48 h. Subsequently, a single i.p. injection of 10, 20, 40, or 80 mg MTX/kg body weight was given. Gastrointestinal toxicity resulted in diarrhea and weight loss in all groups for 5 days after MTX administration. Concomitantly, bone marrow depression with leukocytopenia and thrombocytopenia occurred. Exposure to N2O did not alter the plasma clearance of MTX. No substantial liver or kidney toxicity could be detected, but the 50% lethal dose for MTX was reduced from 60 mg/kg to 10 mg/kg if rats had been exposed to N2O for 48 h; the main causes of death were dehydration and bleeding. The administration of 5-formyl-tetrahydrofolate (4 x 10 mg i.p.) but not 5-methyltetrahydrofolate protected completely against the lethal effect of the drug combination. Altogether, cytotoxic effects of MTX on proliferating cells are potentiated by N2O. Therefore, the use of this anesthetic shortly before or during MTX administration should be avoided.

Topics: Animals; Digestive System; Drug Interactions; Kidney; Lethal Dose 50; Leucovorin; Leukocyte Count; Liver; Male; Metabolic Clearance Rate; Methotrexate; Nitrous Oxide; Platelet Count; Rats; Rats, Inbred Strains; Tetrahydrofolates

Leucovorin (5-formyltetrahydrofolate, LV) is a reduced folate that has been in clinical use for many years as a rescue agent following methotrexate (MTX) therapy. Commercially available LV is a 1:1 mixture of [6R]-and [6S]-isomers. Due to the lack of a specific method for directly separating and quantitating the stereoisomers of LV, it has been difficult to precisely define the pharmacokinetic and biological characteristics of each stereoisomer. We have now developed a novel HPLC method to completely separate [6S]-LV and [6S]-5-methyltetrahydrofolate (MeTHF) from their respective [6R]-isomers using bovine serum albumin (BSA)-bonded silica as the chiral stationary phase. Baseline separation was achieved using 5 and 25 mM sodium phosphate buffers (pH 7.4) as the mobile phase with resolution factors of 1.65 for LV and 2.31 for MeTHF, respectively. The purity of each isomer prepared by this HPLC method is greater than 99%. The stereoisomers were identified by examining their ability to protect CEM cells from MTX (0.04 microM)-induced inhibition of growth. In the LV chromatogram, the first eluted peak provided complete protection from MTX growth inhibition when LV concentrations of 0.1 microM and above were used, whereas the last eluted peak failed to reverse MTX toxicity at concentrations up to 1.0 microM. Chemically pure synthetic [6R]-and [6S]-LV standards confirmed that the first eluted, biologically active peak is the [6S]-isomer. For MeTHF, only the last eluted peak effectively protects cells from MTX growth inhibition and is therefore believed to be the [6S]-isomer. This new HPLC method will serve as a useful tool to elucidate the clinical and cellular pharmacology of the stereoisomers of LV and MeTHF.

Topics: Chromatography, High Pressure Liquid; Leucovorin; Serum Albumin, Bovine; Silicon Dioxide; Stereoisomerism; Tetrahydrofolates

Topics: Animals; Cattle; Chromatography, High Pressure Liquid; Leucovorin; Serum Albumin, Bovine; Spectrophotometry, Ultraviolet; Stereoisomerism; Tetrahydrofolates

Plasma pharmacokinetics of high-dose (500 mg/m2) leucovorin calcium (dl-5-formyltetrahydrofolic acid [dl-CF]) and fluorouracil (FUra) have been evaluated in patients with advanced colorectal cancer treated with the combination of FUra and dl-CF by two different intravenous (IV) schedules: (A) In patients with no prior chemotherapy, dl-CF was administered by a two-hour IV infusion and FUra by rapid IV injection one hour after the start of the dl-CF infusion and (B) in previously treated patients, dl-CF and FUra were administered by five-day continuous IV infusion (CI). Following the two-hour infusion of dl-CF, mean peak plasma concentration and elimination half-life of I-5-formyltetrahydrofolic acid (I-CF) were 24 +/- 6 mumol/L and 0.8 +/- 0.1 hour, respectively. CI of dl-CF over five days yielded a mean steady-state plasma level of I-CF of only 1.2 +/- 0.5 mumol/L. Peak and steady-state plasma concentrations of the metabolite 5-methyl tetrahydrofolic acid were comparable in the two schedules (17 +/- 8 mumol/L for the two-hour infusion and 12 +/- 5 mumol/L for the CI). Areas under the concentration v time curve (AUC) of total reduced folates were significantly greater under conditions of CI: 89.0 v 16.7 mmol/L/min for the two-hour infusion. In tumor tissue, 5,10-methylenetetrahydrofolate increased eight-fold two to four hours following the two-hour infusion and two-fold during the CI of dl-CF and FUra. Inhibition of thymidylate synthase (dTMP-S) by the two-hour and CI infusion schedules were 66% v 39%, respectively. The observed differences in the intracellular dTMP-S folate cofactor pools and the degree of inhibition of dTMP-S achieved in patients treated by two different schedules may be due to differences in the biochemical properties and/or to differences in the modulation of FUra metabolism by folate of tumor tissues obtained from newly diagnosed and previously treated patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Chromatography, High Pressure Liquid; Colonic Neoplasms; Fluorouracil; Half-Life; Humans; Infusions, Intravenous; Injections, Intravenous; Leucovorin; Rectal Neoplasms; Tetrahydrofolates

This article documents the case of a patient with severe renal failure immediately after having been given high-dose methotrexate; the patient was effectively treated with repeated hemodialysis, charcoal hemoperfusion, leucovorin, and thymidine. The methotrexate plasma concentration was reduced from 390 mumol/L to 7 mumol/L as a result of 24.5 hours of hemodialysis along with 39.5 hours of hemoperfusion. Although a rebound in the plasma methotrexate concentration occurred the first three times that hemodialysis and/or hemoperfusion was stopped, reinstitution of the procedure was always effective in further lowering methotrexate concentrations. The patient was subsequently managed with leucovorin and thymidine rescue. Simultaneous measurements before and after the hemodialysis-hemoperfusion apparatus and before and after the hemoperfusion device alone revealed a percent decrease in the concentration of d-leucovorin of 36% and 79%; 1-leucovorin, 82% and 75%; 5-methyltetrahydrofolate, 52% and 64%; methotrexate, 73% and 37%; and 7-hydroxymethotrexate, 21% and 24%, respectively. Gastrointestinal and hematologic toxicities were completely prevented, and serum creatinine normalized within 24 days.

Topics: Adolescent; Female; Hemoperfusion; Humans; Leucovorin; Methotrexate; Osteosarcoma; Renal Dialysis; Tetrahydrofolates

Topics: Antineoplastic Combined Chemotherapy Protocols; Drug Administration Schedule; Drug Evaluation; Fluorouracil; Humans; Infusions, Intravenous; Leucovorin; Neoplasms; Stereoisomerism; Tetrahydrofolates

A reversed-phase HPLC method is described for the simultaneous determination of folinic acid, MTX, and their plasma metabolites 5-CH3-FH4 and 7-OH-MTX respectively. In addition, this technique allows the separation of FA another naturally occurring folate, and of AMT, used as internal standard. Separation of these compounds was achieved on a Waters Spherical C18 column at a flow rate of 0.8 ml.min-1. Elution was carried out with 0.1 M sodium acetate buffer (pH 5.5) as solvent A and 7.5% acetonitrile 92.5% bidistilled water as solvent B. UV detection was performed at 280 nm. This method was applied in a pharmacokinetic study of folinic acid and its plasma metabolite 5-CH3-FH4 following two different protocols: (1) i.v. bolus injection of 50 mg calcium folinate in six healthy volunteers and (2) simultaneous i.v. bolus injections of 50 mg/m2 MTX and 50 mg/m2 folinic acid in four cancer patients. Mean apparent half-life values for folinic acid and its metabolite were 7.02 +/- 1.81 h and 3.90 +/- 0.86 respectively in the first protocol, 4.80 +/- 1.48 h and 4.74 +/- 1.47 h in the second protocol. MTX and 7-OH-MTX were also quantified in the second protocol and were found not to affect the pharmacokinetics of folinic acid and 5-CH3-FH4. Since in vitro studies on metabolism of folinic acid might be of great interest in trying to assess the mechanism of action of the folates and the potential interaction of MTX and 7-OH-MTX in this mechanism via the metabolism, the chromatographic method we describe here has been adapted for the separation of all the potential intracellular monoglutamyl metabolites of folinic acid.

Topics: Adult; Aged; Chromatography, High Pressure Liquid; Female; Half-Life; Humans; Kinetics; Leucovorin; Male; Methotrexate; Middle Aged; Neoplasms; Tetrahydrofolates

The pharmacokinetics of the active and inactive diastereoisomers of leucovorin and its active metabolite, 5-methyltetrahydrofolate (5-CH3-THF) were studied after iv injection of leucovorin in normal human subjects at a dose of 28 mg/m2, and in patients given 500 mg/m2 daily by constant iv infusion for a 5.5 day period. In both studies the plasma half-life (t1/2) of the active isomer, L-formyltetrahydrofolate (CHO-THF), was only 32 to 35 minutes, whereas the inactive isomer, D-CHO-THF had a plasma t 1/2 of 352 to 485 minutes. During constant infusion, the plasma levels reached plateaus of 2.33 and 37.5 microM for L-CHO-THF and D-CHO-THF, respectively. The inactive isomer was cleared from plasma only by urinary excretion of the unchanged drug. The active isomer was also excreted unchanged in the urine but in addition was extensively metabolized to the active metabolite L-5-CH3-THF. The active metabolite achieved a plasma level of 4.85 microM during constant infusion and appeared to have a longer t 1/2 after constant infusion than was observed after iv injection. Furthermore a larger apparent volume of distribution (Vd) of 5-CH3-THF was obtained in the constant infusion study. These findings suggest that constant iv infusion of large doses of leucovorin can considerably expand the intracellular pools of active folate. The consequence of the extensive accumulation of the inactive isomer, D-CHO-THF, is not known. However, the small Vd of D-CHO-THF suggests that it does not extensively accumulate in tissues.

Topics: Biotransformation; Humans; Infusions, Intravenous; Injections, Intravenous; Leucovorin; Stereoisomerism; Tetrahydrofolates

Topics: Adult; Animals; Chromatography, High Pressure Liquid; Electrochemistry; Folic Acid; Humans; Leucovorin; Male; Rats; Rats, Inbred Strains; Species Specificity; Tetrahydrofolates

Human lymphoid cell lines were studied for leucovorin requirements to protect from methotrexate (MTX)-induced growth suppression. Over a 72h continuous exposure leucovorin provided better protection to the cell lines LAZ-007 and RAJI than to the cell lines CCRF-CEM and MOLT-4. The lower leucovorin requirement for LAZ-007 protection versus CCRF-CEM was also seen over a 3h exposure period in which leucovorin protection was assessed by measuring its effect on MTX-induced suppression of 3H-deoxyuridine incorporation into acid-precipitable material. Growth experiments with addition of hypoxanthine or thymidine did not abolish differential protection, suggesting that the phenomenon is not related to selective differences in the tolerance of these cells to an MTX-induced purineless or thymineless state. Preloading of cells with calcium leucovorin caused an identical shift of the CCRF-CEM and LAZ-007 MTX dose - response curves, suggesting that differential catabolism of leucovorin does not contribute to differential protection. The same degree of differential protection was observed for 5-methyltetrahydrofolate as for leucovorin, suggesting that differences in the metabolism of leucovorin do not contribute to differential protection. To elucidate the mechanism of differential protection the influence of leucovorin on [3H]MTX transport and polyglutamylation were studied. Although the Km(MTX) influx and the Ki(leucovorin) for MTX uptake were lower in CCRF-CEM compared with LAZ-007 cells, the size of the difference does not seem adequate to explain differential protection. The extent of MTX polyglutamylation in CCRF-CEM and LAZ-007 cells was identical and the influence of leucovorin on MTX polyglutamylation was the same in both cell lines.

Topics: Cell Division; Cell Line; Drug Antagonism; Humans; Hypoxanthine; Hypoxanthines; Leucovorin; Lymphoid Tissue; Methotrexate; Polyglutamic Acid; Tetrahydrofolates; Thymidine; Uridine

Leucovorin requirements for protection of the T cell line CCRF-CEM and the B cell line LAZ-007 against the cytotoxic effects of a variety of antifolates were studied. Differential leucovorin protection for DDMP-induced growth suppression occurred in the opposite direction to that for MTX, with CCRF-CEM requiring less leucovorin than LAZ-007 for equivalent protection. A pattern of differential protection from DDMP different from that of protection from MTX was also seen for the cell lines RAJI and MOLT-4. Differential leucovorin protection was observed for the chain-extended MTX analogue PT441. The degree of differential protection was similar to that seen for MTX, and transport studies showed that PT441 was a weak inhibitor of tritiated MTX uptake into CCRF-CEM cells. Differential leucovorin protection was observed for the lipophilic antifolate trimetrexate glucoronate (TMQ) but the degree of differential protection was smaller than that seen for PT441 or for MTX. Since TMQ is not transported into cells by the reduced folate system, while PT441 is a weak competitive inhibitor of [3H]MTX transport, and since neither is polyglutamylated, these results support the conclusion reached in previous experiments that differential leucovorin protection of MTX is unlikely to be a transport-related phenomenon and is not due to an effect on polyglutamylation. In addition, the different patterns of relative leucovorin requirements for DDMP and MTX protection suggest that differential metabolism or catabolism of leucovorin does not account for differential protection.

Topics: Biological Transport; Cell Division; Cell Line; Drug Antagonism; Folic Acid; Folic Acid Antagonists; Humans; Leucovorin; Lymphoid Tissue; Methotrexate; Tetrahydrofolates; Uridine

Serum and cerebrospinal fluid (CSF) concentrations of citrovorum factor (CF) and 5-methyltetrahydrofolic acid (5-MTHFA) were measured after i.v. infusion of leucovorin (50 or 100 mg/sq m) in seven patients undergoing treatment for meningeal carcinomatosis by intra-Ommaya reservoir injection of methotrexate (MTX). Serum CF levels rapidly rose after leucovorin administration as did 5-MTHFA, its conversion product. A small amount of CF entered the CSF, but peak CSF 5-MTHFA increased about 10-fold. The concentration X time (C X t) of additional 5-MTHFA in the CSF was greater [114.4 +/- 36.1 (S.E.) microgram/ml X min] after 100-mg/sq m doses of leucovorin than after 50 mg/sq m [14.2 +/- 4.3 micrograms/ml X min] (p less than 0.05). The CSF MTX concentration exceeded CSF 5-MTHFA by 2 to 3 logs throughout the 48 hr of observation, while serum 5-MTHFA and CF exceeded serum MTX by 0.5 to 2 logs. This study demonstrates that leucovorin administered i.v. to patients receiving intra-Ommaya MTX does not increase CSF concentrations of "rescue" folate above those of CSF MTX and are unlikely to interfere with MTX action against meningeal tumor. On the other hand, i.v. leucovorin does permit serum "rescue" folate to operate, thus reducing the systemic toxicity that may follow intraventricular administration of MTX.

Topics: Aged; Carcinoma; Female; Humans; Injections, Intravenous; Injections, Intraventricular; Leucovorin; Male; Meningeal Neoplasms; Methotrexate; Middle Aged; Tetrahydrofolates; Time Factors

Topics: Animals; Drug Administration Schedule; Drug Therapy, Combination; Leucovorin; Leukemia L1210; Life Expectancy; Methotrexate; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Tetrahydrofolates

Osteosarcoma patients free of CNS metastases are at risk for acquiring leukoencephalopathy after receiving multiple courses of high dose intravenous methotrexate followed by oral leucovorin rescue (MTX-LV). A prospective study of the adequacy of CNS rescue of MTX biochemical toxicity by oral leucovorin was undertaken in newly diagnosed neurologically normal osteosarcoma patients. Prior to surgical resection of the primary tumor, ten patients received 4 weekly courses of MTX-LV. During the fourth weekly MTX-LV treatment, 0 and 72 hr serum and CSF determinations of MTX, 5-methyl-tetrahydrofolate (5-MTHF) and LV were made. No CSF MTX was detectable at 0 hr in any patient, but a significant elevation in CSF MTX occurred in 9/9 patients at 72 hr (mean 47.2 +/- 31.8 ng/ml or 1.04 +/- 0.7 X 10(-7) M). There was no significant change in mean CSF 5-MTHF over 72 hr despite a rise in serum 5-MTHF. MTX exceeded 5-MTHF in 6/9 patients in CSF, whereas only 3/8 patients had higher MTX in the serum at 72 hr. No acute systemic or neurotoxicity was seen. The failure of oral leucovorin to consistently elevate CSF 5-MTHF levels at 72 hr in the context of significant levels of CSF MTX may result in intermittent CNS folate deficiency. The clinical and pathological syndrome of leukoencephalopathy may be related to this phenomenon and may evolve after repeated MTX-LV treatments.

Topics: Adolescent; Adult; Child; Drug Therapy, Combination; Female; Humans; Leucovorin; Male; Methotrexate; Osteosarcoma; Tetrahydrofolates

The rescue of lymphocytes from methotrexate (MTX) growth inhibition by 5-methyltetrahydrofolate (5-methyl-THF) and 5-formyltetrahydrofolate (5-formyl-THF) has been studied. Rescue by 5-methyl-THF is selective for cells with high levels of homocysteine:5-methyl-THF methyl-transferase (methyltransferase). At MTX concentrations which inhibited growth greater than or equal to 85% in both leukemic T-lymphocytes (CCRF-CEM) and Epstein-Barr-transformed B-lymphocytes (LAZ-007), 5 micro M 5-formyl-THF rescued more effectively than did 5-methyl-THF, in either the presence or absence of the methyltransferase inhibitor, nitrous oxide. At less inhibitory MTX concentrations, both reduced folates rescued equally, except when methyltransferase was inhibited by nitrous oxide in which case 5-formyl-THF was clearly superior. In the absence of nitrous oxide, both cell lines contained approximately equal amounts of methyltransferase. Some apparent differences in the rescue of these cell lines with 5-methyl-THF were attributable to their different sensitivity to MTX. When metabolism of reduced folates was severely impaired by MTX and nitrous oxide, lymphocytes were rescued with 5-[methyl-14C]methyl-THF, and the uptake of 14C into DNA was measured. In corporation was very low, indicating that cellular oxidation of 5-methyl-THF to 5,10-methylene-tetrahydrofolate is minimal even under forcing conditions. MTX selectively in vivo will be influenced by the level of methyltransferase in tumor and normal tissues.

Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Ascorbic Acid; Cell Line; DNA; Humans; Leucovorin; Lymphocytes; Methotrexate; Nitrous Oxide; Oxidation-Reduction; Tetrahydrofolates

The role of vitamin B12 in the folate dependent biosynthesis of thymidine nucleotides is controversial. In an attempt to clarify this, three methods have been used to assess the relative efficacy of vitamin B12 (hydroxocobalamin) and various folate analogues in titrated concentrations at correcting 'de novo' thymidylate synthesis by megaloblastic human marrow cells: (1) The deoxyuridine (dU) suppression test which analyses the reduction in (3H)-thymidine labeling of DNA by unlabeled dU. Marrow cells were also labeled with (6-3H)-dU with assessment of (2) its incorporation into DNA and (3) the accumulation of (6-3H)-deoxyuridine monophosphate (3H-dUMP). The three methods gave similar results. In both, N6-formyl tetrahydrofolate (formyl-FH4) was the most effective agent at correcting thymidylate synthesis in megaloblastic anemia due to vitamin B12 or folate deficiency. Vitamin B12 corrected the lesion in vitamin B12 deficiency but not in folate deficiency. Tetrahydrofolate (FH4) and folic acid were effective in deficiency of vitamin B12 or folate, although in both deficiencies they were less effective than formyl-FH4. Methyl-FH4 was effective in folate deficiency but not in vitamin B12 deficiency. These results confirm the failure of methyl-FH4 utilisation in vitamin B12 deficiency. They suggest that if vitamin B12 is needed in the formylation of FH4, this is a minor role in provision of the correct coenzyme for thymidylate synthesis compared with its major role of provision of FH4 from methyl-FH4.

Topics: Anemia, Macrocytic; Anemia, Megaloblastic; Bone Marrow; Deoxyuridine; DNA; Folic Acid; Folic Acid Deficiency; Humans; Leucovorin; Tetrahydrofolates; Thymine Nucleotides; Vitamin B 12; Vitamin B 12 Deficiency

Topics: Animals; Ascorbic Acid; Biopterins; Child, Preschool; Humans; Hydroxocobalamin; In Vitro Techniques; Infant; Leucovorin; Male; NADH, NADPH Oxidoreductases; Phenylketonurias; Rats; Tetrahydrofolates

Topics: Animals; Cerebral Cortex; Evoked Potentials; Folic Acid; In Vitro Techniques; Kainic Acid; Leucovorin; Pyrrolidines; Rats; Tetrahydrofolates

Topics: Animals; Chemical Phenomena; Chemistry; Goats; Haptens; Humans; Leucovorin; Methotrexate; Neoplasms; Rabbits; Radioimmunoassay; Sheep; Tetrahydrofolates

Topics: Animals; Cattle; Dose-Response Relationship, Drug; Female; Folic Acid; Leucovorin; Milk; Radioligand Assay; Tetrahydrofolates

Topics: Animals; Catalysis; Folic Acid; Intestine, Small; Kidney; Leucovorin; Liver; Methotrexate; Mice; Tetrahydrofolate Dehydrogenase; Tetrahydrofolates

The activities of 5-methyltetrahydrofolate (5-CH3THF) related enzymes and DNA polymerase alpha were determined in bone marrow cells obtained from patients with vitamin B12 deficient megaloblastic anemia and compared with those from healthy volunteers and patients with hemolytic anemia. 5-CH3THF homocysteine methyltransferase activity was significantly lower than that in the control subjects. 5,10-methylenetetrahydrofolate reductase activity was only slightly elevated to that in the control subjects. DNA polymerase alpha activity was significantly higher than that in the control. High deoxyuridine suppression test values in vitamin B12 deficient bone marrow cells were improved by tetrahydrofolate, but not by 5-CH3THF. These data indicate that, even though the reverse reaction catalyzed by 5,10-methylenetetrahydrofolate reductase may be operative in vitamin B12 deficiency, it is not sufficient to correct the disturbance in folate metabolism in vitamin B12 deficiency. Increased DNA polymerase alpha activity may be due to compensation for disarranged DNA synthesis.

Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; 5,10-Methylenetetrahydrofolate Reductase (FADH2); Adult; Aged; Alcohol Oxidoreductases; Anemia, Macrocytic; Anemia, Megaloblastic; Bone Marrow; Bone Marrow Cells; Deoxyuridine; DNA Polymerase II; DNA-Directed DNA Polymerase; Female; Folic Acid; Humans; Hydroxocobalamin; Leucovorin; Male; Methylenetetrahydrofolate Reductase (NADPH2); Middle Aged; Tetrahydrofolates; Thymidine; Vitamin B 12; Vitamin B 12 Deficiency

The properties of the folate transport system in H35 hepatoma cells have been studied by measuring the transport of (+)-5-methyltetrahydrofolate. Using initial rates of uptake, it has been demonstrated that the uptake is saturable, carrier mediated, and shared by methotrexate. The accumulation of (+)-5-methyltetrahydrofolate is concentrative, demonstrating the presence of an active transport process. A previous study suggested that methotrexate-resistant sublines (H35R) acquired methotrexate insensitivity because of an impaired capacity for transport. This postulate was substantiated in the present investigation by several observations. The initial uptake and steady-state level of (+)-5-methyltetrahydrofolate were markedly reduced in the resistant sublines as was the case with methotrexate. Triazinate (2-(chloro-4-[4,6-diamino-2,2-dimethyl-S-triazine-1(2H)-ylphenoxyl])-N,N-dimethyl-m-toluamide . ethanesulfonic acid) an inhibitor of dihydrofolate reductase which enters the cells by a pathway independent of the folate coenzyme, was equally toxic to H35 cells and to an H35 subline resistant to 0.3 microM methotrexate. Resistant sublines that are insensitive to methotrexate up to 1 microM display a transport defect but have normal levels of dihydrofolate reductase. Sublines resistant to higher levels of methotrexate showed not only defective transport but also commensurate increases in dihydrofolate reductase. Attempts to demonstrate carried-dependent transport of (+)-5-methyltetrahydrofolate or methotrexate in resistant sublines were negative, suggesting the lack of a functional carrier. These properties were readily demonstrated in H35 cells and included temperature dependence, competition for uptake with analogs, and transstimulation.

Topics: Animals; Antineoplastic Agents; Biological Transport; Drug Resistance; Folic Acid Antagonists; Leucovorin; Liver Neoplasms, Experimental; Methotrexate; Rats; Tetrahydrofolate Dehydrogenase; Tetrahydrofolates; Triazines

Topics: Amygdala; Animals; Cerebral Cortex; Folic Acid; Kainic Acid; Leucovorin; Neurotoxins; Pyrrolidines; Rats; Structure-Activity Relationship; Tetrahydrofolates

The liquid-chromatographic measurement of 5-methyltetrahydrofolate in biological fluids is described. The sensitivity of the spectrophotofluorometric detector used allows direct evaluation of basal concentrations of the compound in plasma. Because it is resolved from the other common folates and from methotrexate, the procedure is suitable for monitoring it in plasma of patients receiving high-dose therapy with methotrexate.

Topics: Adult; Animals; Chromatography, High Pressure Liquid; Dogs; Folic Acid; Humans; Leucovorin; Leukemia, Lymphoid; Lymphoma; Male; Middle Aged; Spectrophotometry, Ultraviolet; Tetrahydrofolates; Time Factors

Serum and urinary distributions of following oral and parenteral administration of leucovorin (3-15 mg) were examined in normal adult volunteers microbiologically using lactobacillus casei, Streptococcus faecalis and Pediococcus cerevisiae as test organisms. By the parenteral route, nearly one-third of the folate in the serum and urine was in the form of folinic acid and the remainder as 5-methyltetrahydrofolic acid. Almost all the folate in serum and urine was in the form of 5-methyltetrahydrofolic acid after oral administration. Peak serum folate was observed 3 hr after oral administration, later than that seen after parenteral administration (30 min). Elevation of serum folate was achieved by the increase of the methyl form of folate following repeated administration of leucovorin orally and parenterally. As the form of folate actually rescuing normal cells in a high-dose methotrexate regimen was thought to be methyl, use of the oral route as a principal means of administration of leucovorin in a rescue program was looked into.

Topics: Administration, Oral; Humans; Injections, Intramuscular; Injections, Intravenous; Leucovorin; Methotrexate; Tetrahydrofolates

The ability of CF to protect cells from methotrexate toxicity was measured in a series of murine lymphoma cells of varying degrees of sensitivity to methotrexate. The cells that were the most resistant to methotrexate showed the least degree of protection from methotrexate cytotoxicity. The extent of protection afforded by CF was found to correlate with the capacity of the cells to take up C14-5-methyltetrahydrofolate. That is, the cells with the lowest extent of uptake of reduced folate were afforded the least degree of protection from methotrexate by CF.

Topics: Animals; Biological Transport; Cell Line; Cell Survival; Kinetics; Leucovorin; Leukemia L1210; Leukemia L5178; Leukemia, Experimental; Methotrexate; Mice; Tetrahydrofolates

An enzyme that catalyzes the hydrolysis of folic acid and the antifolate methotrexate nearly 20 times more rapidly than the hydrolysis of 5-methyltetrahydrofolate was extraced from a gram-negative bacterium tentatively identified as a Flavobacterium sp. The enzyme was purified 500-fold and found to have a molecular weight of about 53,000. Apparently a metallo-enzyme, it is inhibited by citrate and ethylenediaminetetraacetic acid (EDTA). Ca2+, Co2+, Mg2+, and Zn2+ reverse inhibition by EDTA, whereas Ca2+ and Zn2+ are weak activators in the absence of EDTA. The enzymatic reaction releases the carboxy-terminal glutamyl moiety of derivatives of pteroyl-mono-L-glutamic acid. Substituents on N5 of the pteridine ring decrease the velocity of hydrolysis. Some non-specificity for the terminal amino acid is expressed. The strikingly different rates of hydrolysis of methotrexate and 5-methyltetrahydrofolate have stimulated interest in this enzyme for its potential clinical value in improving the therapeutic index of methotrexate.

Topics: Carboxypeptidases; Flavobacterium; Hydrogen-Ion Concentration; Kinetics; Leucovorin; Metals; Methotrexate; Molecular Weight; Temperature; Tetrahydrofolates

Topics: Folic Acid; Leucovorin; Tetrahydrofolates