RESUMEN
BACKGROUND: The number of erectile dysfunction (ED) patients is increasing annually. How to improve the effectiveness of ED treatment is an important issue for the field of andrology. OBJECTIVES: To investigate whether low androgen status impairs the erectile function of rats by regulated endothelial nitric oxide synthase (eNOS) uncoupling. MATERIALS AND METHODS: Thirty-six 8-week-old male Sprague Dawley (SD) rats were randomly divided into six groups as follows: 4-week sham-operated group (4w-sham), 4-week castration group (4w-cast), 4-week castration + testosterone (T) group (4w-cast + T), 8-week sham-operated group (8w-sham), 8-week castration group (8w-cast), and 8-week castration + T group (8w-cast + T). Three mg/kg of T was subcutaneously injected every other day in castration + T groups. The ratio of the maximum intracavernous pressure/the mean arterial pressure (ICPmax/MAP), the level of serum T, dihydrobiopterin(BH2 ), tetrahydrobiopterin (BH4 ), nitric oxygen(NO), 3-nitrotyrosine(3NT), dihydrofolate reductase (DHFR), guanosine triphosphate cyclohydrolase 1 (GTPCH1), nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), and eNOS monomers/dimers in the corpus cavernosum were detected. RESULTS: The ratio of ICPmax/MAP and BH4 /BH2 , the level of serum T, NO, and GTPCH1 decreased significantly in castration groups compared with sham-operated groups and castration + T groups (P < .05) and decreased significantly in 8w-cast group compared with 4w-cast group (P < .05). The expression of 3NT and NOX2 and the ratio of eNOS monomers/dimers increased significantly in castration groups compared with sham-operated groups and castration + T groups (P < .01) and increased significantly in 8w-cast group compared with 4w-cast group (P < .01). The expression of DHFR in 4w-cast group was significantly higher than that in 4w-sham group and 4w-cast + T group (P < .01) and in 8w-cast group was significantly lower than that in 8w-sham group and 8w-cast + T group (P < .01). DISCUSSION AND CONCLUSION: Low androgen status induces eNOS uncoupling by reducing BH4 /BH2 and increasing 3NT. Due to the decreased NO production, the erectile function of the rats was impaired.
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Disfunción Eréctil/fisiopatología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Erección Peniana/fisiología , Pene/irrigación sanguínea , Testosterona/sangre , Animales , Presión Arterial/fisiología , Castración , GMP Cíclico/metabolismo , Disfunción Eréctil/terapia , Masculino , NADPH Oxidasa 2/sangre , Óxido Nítrico/sangre , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Testosterona/uso terapéutico , Tetrahidrofolato Deshidrogenasa/sangre , Tirosina/análogos & derivados , Tirosina/sangreRESUMEN
PURPOSE: We examined the association of plasma B-vitamins and metabolites, and related genetic variants, with risk of breast cancer among predominantly premenopausal women. METHODS: We conducted a nested case-control study within the Nurses' Health Study II. From blood samples collected in 1996-1999 and follow-up through 2007, plasma measures were available for 610 cases and 1207 controls. Unconditional multivariable logistic regression was used to estimate relative risks (RR) of breast cancer and 95% confidence intervals (CIs). We examined whether associations varied by methylenetetrahydrofolate reductase (MTHFR) and dihydrofolate reductase polymorphisms, breast cancer risk factors, or tumor characteristics. RESULTS: Plasma vitamin B12 was associated with a 64% higher risk of breast cancer comparing the highest versus lowest quintile (95% CI 1.17-2.29, p-trend = 0.02). Plasma folate (comparable RR = 1.18, 95% CI 0.84-1.66), pyridoxal 5'-phosphate (RR = 1.18, 95% CI 0.85-1.64), homocysteine (RR = 0.93, 95% CI 0.67-1.28), cysteine (RR = 1.14, 95% CI 0.81-1.62), and cysteinylglycine (RR = 0.93, 95% CI 0.66-1.31) were not associated with overall breast cancer risk. Folate was significantly positively associated with invasive and estrogen receptor-positive/progesterone receptor-positive breast cancer, and this association was suggestively stronger for bloods collected post-fortification. Several nutrient/breast cancer associations varied across subgroups defined by age, smoking, alcohol, multivitamin use, and MTHFR status (p-interaction < 0.05). CONCLUSIONS: Overall, plasma B-vitamins and metabolites were not associated with lower breast cancer risk. Plasma vitamin B-12 was positively associated with higher risk of overall breast cancer, and plasma folate was positively associated with risk of invasive breast cancer. Additionally, there may be associations in subgroups defined by related genetic variants, breast cancer risk factors, and tumor factors. Further studies in younger women and in the post-fortification era are needed to confirm these findings.
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Biomarcadores , Neoplasias de la Mama/etiología , Neoplasias de la Mama/metabolismo , Carbono/metabolismo , Susceptibilidad a Enfermedades , Complejo Vitamínico B/sangre , Adulto , Factores de Edad , Biomarcadores de Tumor , Neoplasias de la Mama/sangre , Neoplasias de la Mama/epidemiología , Estudios de Casos y Controles , Dieta , Femenino , Humanos , Redes y Vías Metabólicas , Metilenotetrahidrofolato Reductasa (NADPH2)/sangre , Persona de Mediana Edad , Vigilancia en Salud Pública , Medición de Riesgo , Factores de Riesgo , Tetrahidrofolato Deshidrogenasa/sangreRESUMEN
BACKGROUND: Pralatrexate (PDX) is an inhibitor of dihydrofolate reductase that was rationally designed to improve cellular uptake and retention of the drug. Preclinical data have shown synergy with the sequential administration of a dihydrofolate reductase inhibitor followed 24 hours later by 5-fluorouracil (5-FU). METHODS: Twenty-seven patients were enrolled at 1 of 5 PDX dose levels from 75 to 185 mg/m(2) on day 1 followed 24 hours later by 5-FU at a dose of 3000 mg/m(2) /48 hours every 2 weeks with folic acid and vitamin B12 supplementation. Baseline blood was collected for pharmacogenetic analysis of polymorphisms of methylenetetrahydrofolate reductase and thymidylate synthase. RESULTS: Mucositis was the most common dose-limiting toxicity. When the worst toxicities across all cycles were considered, grade 3 to 4 neutropenia, anemia, and thrombocytopenia were found to have occurred in 14.8%, 14.8%, and 0% of patients, respectively. Grade 2 to 3 toxicities included mucositis (66.6%), dehydration (33.3%), fatigue (25.9%), and diarrhea (22.2%). Version 3.0 of the National Cancer Institute Common Toxicity Criteria was used to grade toxicities The median progression-free survival (PFS) was 112 days (range, 28-588 days). Seven patients (26%) had a PFS of >180 days (5 patients with colorectal cancer, 1 patient with pancreatic cancer, and 1 patient with non-small cell lung cancer). Polymorphisms in methylenetetrahydrofolate reductase and thymidylate synthase did not correlate with toxicity. CONCLUSIONS: The recommended dose of PDX was 148 mg/m(2) . A subset of heavily pretreated patients had PFS durations of ≥6 months with this regimen.
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Aminopterina/análogos & derivados , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Colorrectales/tratamiento farmacológico , Fluorouracilo/administración & dosificación , Fluorouracilo/uso terapéutico , Neoplasias Gastrointestinales/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Aminopterina/administración & dosificación , Aminopterina/efectos adversos , Aminopterina/uso terapéutico , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Quimioterapia Combinada , Femenino , Antagonistas del Ácido Fólico/administración & dosificación , Antagonistas del Ácido Fólico/efectos adversos , Antagonistas del Ácido Fólico/uso terapéutico , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Mucositis/inducido químicamente , Mucositis/epidemiología , Polimorfismo Genético/genética , Tetrahidrofolato Deshidrogenasa/sangre , Tetrahidrofolato Deshidrogenasa/genética , Timidilato Sintasa/sangre , Timidilato Sintasa/genética , Factores de Tiempo , Resultado del TratamientoRESUMEN
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.
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Variaciones en el Número de Copia de ADN , Defectos del Tubo Neural/genética , Animales , Cromatografía Liquida , Hibridación Genómica Comparativa , Modelos Animales de Enfermedad , Ácido Fólico/sangre , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Leucovorina/sangre , Metotrexato , Ratones Endogámicos C57BL , MicroARNs , Defectos del Tubo Neural/patología , Defectos del Tubo Neural/fisiopatología , Reacción en Cadena en Tiempo Real de la Polimerasa , S-Adenosilmetionina/sangre , Espectrometría de Masas en Tándem , Tetrahidrofolato Deshidrogenasa/sangre , Tetrahidrofolatos/sangreRESUMEN
Rapid diagnostics tests (RDTs) detect malaria specific antigen(s) in the circulation, even when parasites are sequestered in the placenta and not visible by microscopy. However, research on their diagnostic accuracy during pregnancy is limited. Pregnant women (n = 418) were screened for malaria during routine antenatal care by using two RDTs that detect histidine-rich protein 2 (HRP2) or Plasmodium lactate dehydrogenase, and enzyme-linked immunosorbent assays with antibodies that detect dihydrofolate reductase-thymidylate synthase or heme-detoxification protein, and compared with real-time polymerase chain reaction (RT-PCR) and microscopy for evaluation of their diagnostic accuracy. Prevalence of malaria infection was high (53% by PCR). The RT-PCR and the HRP2 RDT detected most cases of malaria during pregnancy, whereas microscopy, the Plasmodium lactate dehydrogenase RDT, and enzyme-linked immunosorbent assays for dihydrofolate reductase-thymidylate synthase and heme-detoxification protein antibodies did not detect several low-density infections. Therefore, the HRP2 RDT could be a useful tool in high-transmission areas for diagnosis of malaria in asymptomatic pregnant women.
Asunto(s)
Antígenos de Protozoos/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , L-Lactato Deshidrogenasa/sangre , Malaria Falciparum/diagnóstico , Complejos Multienzimáticos/sangre , Plasmodium falciparum/aislamiento & purificación , Complicaciones Parasitarias del Embarazo/diagnóstico , Proteínas Protozoarias/sangre , Tetrahidrofolato Deshidrogenasa/sangre , Timidilato Sintasa/sangre , Adolescente , Animales , Burkina Faso/epidemiología , Femenino , Humanos , Malaria Falciparum/sangre , Malaria Falciparum/epidemiología , Malaria Falciparum/parasitología , Plasmodium falciparum/genética , Embarazo , Complicaciones Parasitarias del Embarazo/sangre , Complicaciones Parasitarias del Embarazo/epidemiología , Complicaciones Parasitarias del Embarazo/parasitología , Prevalencia , ARN Protozoario/química , ARN Protozoario/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Adulto JovenRESUMEN
OBJECTIVES: To evaluate persistence of several Plasmodium antigens in pregnant women after treatment and compare diagnostics during treatment follow-up. METHODS: Thirty-two pregnant women (N = 32) with confirmed malaria infection by a histidine-rich protein 2 (HRP2)-based rapid diagnostic test (RDT) and microscopy were followed for 28 days after artemisinin-based combination therapy (ACT). A Plasmodium lactate dehydrogenase (pLDH)-based RDT and two ELISAs based on the detection of dihydrofolate reductase-thymidylate synthase (DHFR-TS) and haeme detoxification protein (HDP) were compared with each other and to RT-PCR at each visit. RESULTS: The mean visit number (95% confidence interval) on which the HRP2-based RDT was still positive after treatment was 3.4 (2.7-4.1) visits with some patients still positive at day 28. This is significantly later than the pLDH-based RDT [0.84 (0.55-1.1)], microscopy (median 1, range 1-3), DHFR-TS-ELISA [1.7 (1.1-2.3)] and RT-PCR (median 2, range 1-5) (P < 0.05), but not significantly later than HDP-ELISA [2.1 (1.6-2.7)]. Lower gravidity and higher parasite density at day 0 resulted in significantly longer positive results with most tests (P < 0.05). CONCLUSIONS: HRP2 can persist up to 28 days after ACT treatment; therefore, this test is not suitable for treatment follow-up in pregnant women and can generate problems when using this test during intermittent preventive treatment (IPTp). DHFR-TS is less persistent than HRP2, making it a potentially interesting target for diagnosis.
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Antígenos de Protozoos/sangre , Malaria Falciparum/diagnóstico , Malaria Falciparum/inmunología , Complicaciones Parasitarias del Embarazo/diagnóstico , Complicaciones Parasitarias del Embarazo/inmunología , Adulto , Análisis de Varianza , Antiinfecciosos/uso terapéutico , Artemisininas/uso terapéutico , Biomarcadores/sangre , Burkina Faso , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Estudios de Seguimiento , Humanos , L-Lactato Deshidrogenasa/sangre , Malaria Falciparum/tratamiento farmacológico , Complejos Multienzimáticos/sangre , Plasmodium falciparum/parasitología , Embarazo , Complicaciones Parasitarias del Embarazo/tratamiento farmacológico , Proteínas Protozoarias/sangre , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Tetrahidrofolato Deshidrogenasa/sangre , Timidilato Sintasa/sangre , Resultado del TratamientoRESUMEN
A low serum folate and high homocysteine phenotype is associated with an increased risk of neural tube defects (NTDs), cardiovascular diseases and other pathologies. Thus defining both genetic and non-genetic factors that may impact folate/homocysteine metabolism will enhance our understanding of the etiologic mechanisms underlying these conditions and facilitate risk assessment. Dihydrofolate reductase catalyzes the reduction of folic acid to dihydrofolate and thereafter to tetrahydrofolate. The impact of the dihydrofolate reductase (DHFR) c.86 + 60_78 insertion/deletion (ins/del) polymorphism on folate and homocysteine concentrations was analyzed using data from healthy young adults from Northern Ireland, collected as part of visit three of the Young Hearts Project. Among men the DHFR c.86 + 60_78 polymorphism was not significantly associated with serum or red blood cell folate concentrations, or with homocysteine concentrations. Among women the DHFR c.86 + 60_78 polymorphism explained 2% of the variation in RBC folate levels and 5% of the variation in serum folate levels, but did not appear to have an independent effect on homocysteine. Relative to women with the DHFR c.86 + 60_78 ins/ins and ins/del genotypes, del/del homozygotes had increased serum and red blood cell folate concentrations and may therefore be at decreased risk of having offspring affected by NTDs and of other adverse reproductive and health outcomes attributable to low folate.
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Eritrocitos/metabolismo , Ácido Fólico/sangre , Polimorfismo Genético/genética , Tetrahidrofolato Deshidrogenasa/sangre , Tetrahidrofolato Deshidrogenasa/genética , Adolescente , Estudios de Casos y Controles , Niño , Cromatografía Líquida de Alta Presión , Femenino , Ácido Fólico/genética , Genotipo , Homocisteína/sangre , Humanos , Estudios Longitudinales , Masculino , Irlanda del Norte , Reacción en Cadena de la Polimerasa , Factores de Riesgo , Complejo Vitamínico B/sangreRESUMEN
Microplate reader assays offer several advantages over conventional spectrophotometric assays. We adapted the dihydrofolate reductase (DHFR) enzyme inhibition assay for use in a 96-well microplate reader to measure plasma methotrexate (MTX) concentrations. The assay is linear from 0.01 to 0.1 micromol/L. The within-run CVs at 0.03 micromol/L and 0.08 micromol/L MTX were 4.0% and 2.7%, respectively, and the interday (total) CVs were 7.6% and 1.8%. Cross-reactivity with the inactive MTX metabolite 2, 4-diamino-N10-methylpteroic acid (DAMPA) was 3.9%, significantly less than that described with commercial immunoassays; with 7-hydroxymethotrexate cross-reactivity was 1.7%. In addition to sensitivity and specificity, the advantages of this assay are small sample volumes, simultaneous analysis of multiple samples, and rapid turnaround. Because of its greater specificity, the DHFR enzyme inhibition assay may be useful when DAMPA is present in plasma samples and HPLC is not available.
Asunto(s)
Metotrexato/sangre , Tetrahidrofolato Deshidrogenasa/sangre , Antagonistas del Ácido Fólico/sangre , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , TemperaturaRESUMEN
The efficacy of sulphadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Africa is increasingly compromised by development of resistance. The occurrence of mutations associated with the active site sequence in the Plasmodium falciparum genes coding for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) is associated with in vitro resistance to pyrimethamine and sulphadoxine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results show that alleles of DHPS (436-alanine, 437-alanine and 540-lysine) were significantly reduced in prevalence on day 7 after S/P treatment. In this area, a DHPS with 436-serine, 437-glycine and 540-glutamate appears to play a major role in resistance to S/P in vivo. Evidence for the influence of mutations in the DHFR gene in this investigation is not clear, probably because of the high prevalence of 'resistance-related' mutations at day 0 in the local parasite population. For apparently the same reason, it was not possible to show a statistical association between S/P resistance and the presence of particular polymorphisms in the DHFR and DHPS genes before treatment.
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Antimaláricos/uso terapéutico , Dihidropteroato Sintasa/genética , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Plasmodium falciparum/enzimología , Pirimetamina/uso terapéutico , Sulfadoxina/uso terapéutico , Tetrahidrofolato Deshidrogenasa/genética , Animales , Niño , Preescolar , Cartilla de ADN , Dihidropteroato Sintasa/sangre , Farmacorresistencia Microbiana , Femenino , Humanos , Lactante , Malaria Falciparum/sangre , Masculino , Plasmodium falciparum/genética , Reacción en Cadena de la Polimerasa , Tanzanía , Tetrahidrofolato Deshidrogenasa/sangreAsunto(s)
Malaria Falciparum/parasitología , Complejos Multienzimáticos/genética , Plasmodium falciparum/genética , Mutación Puntual , Tetrahidrofolato Deshidrogenasa/genética , Timidilato Sintasa/genética , Animales , Antimaláricos/uso terapéutico , Resistencia a Medicamentos/genética , Humanos , Malaria Falciparum/tratamiento farmacológico , Complejos Multienzimáticos/sangre , Plasmodium falciparum/enzimología , Plasmodium falciparum/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Tetrahidrofolato Deshidrogenasa/sangre , Tailandia , Timidilato Sintasa/sangreRESUMEN
The relationships between dihydrofolate reductase (DHFR) levels or methotrexate membrane transport and acute lymphoblastic leukemia (ALL) immunophenotype were evaluated on 51 T-cell and 44 B-precursor ALL specimens from 90 pediatric ALL patients at diagnosis and relapse, using a fluorescent methotrexate analog (PT430) and flow cytometry assay (Matherly et al, Blood 85:500, 1995). For T-cell ALL, 35 of 45 (78%) of newly diagnosed patients' specimens exhibited elevated DHFR relative to DHFR levels in ALL blasts from methotrexate-responsive patients. For 30 of 45 diagnostic T-ALL specimens, DHFR expression was heterogeneous, with up to 3 separate subpopulations covering a 44-fold range; the DHFR-overproducing fractions comprised 10% to 88% of the total blasts. Elevated DHFR was less common in B-precursor ALL at diagnosis, being detected in only 17 of 36 specimens (47%); 11 of these samples exhibited DHFR heterogeneity. Median maximal DHFR levels were fourfold higher in T-cell than B-precursor ALL at diagnosis. Within a particular phenotypic group, there were no correlations between DHFR levels and patient prognostic features, including age, sex, chromosomal abnormalities, white blood cell counts (WBCs), and percentage of S-phase. However, for B-precursor ALL, there was a notably higher fraction of African-American than white patients with elevated DHFR. For patients (both phenotypes) with low WBCs (<50,000/ microL), event-free survival times were significantly shorter for those expressing DHFR above a threshold level than for patients expressing DHFR below this level (P < .016); this relationship was not seen for patients with high WBCs (>50,000/microL). Elevated DHFR was detected in 11 of 14 relapse specimens (5 of 6 T-cell and 6 of 8 B-precursor). Two of five paired relapse specimens (both T-cell) from patients treated with methotrexate exhibited increased DHFR levels over those at diagnosis (2.5- to 5-fold); the fraction of DHFR-overproducing blasts was also increased in 4 of 5 paired relapse specimens (2 B-precursor and 2 T-cell). In contrast to the variations in DHFR, highly impaired methotrexate transport was detected in only 6 of 95 ALL specimens, including both diagnosis and relapse. There was no correlation between phenotype and impaired transport. These data provide further rationale for the use of mechanistically based prognostic factors to complement known biologic or disease-based prognostic indicators in the design of ALL therapy including methotrexate, particularly with patients presenting with low WBCs. The finding of a markedly increased frequency of DHFR overexpression in T-cell over B-precursor ALL suggests that this difference is associated with the poorer prognosis of patients with T-cell ALL treated with standard-dose antimetabolite therapy and implies that higher-dose methotrexate (> or = 1 g/m2) during consolidation therapy may be useful in the treatment of this disease.
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Transportadoras de Casetes de Unión a ATP , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfoma de Burkitt/enzimología , Aberraciones Cromosómicas , Trastornos de los Cromosomas , Proteínas de Neoplasias , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimología , Tetrahidrofolato Deshidrogenasa/sangre , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Adolescente , Crisis Blástica , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/genética , Linfoma de Burkitt/patología , Proteínas Portadoras/análisis , Niño , Preescolar , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos , Femenino , Humanos , Recuento de Leucocitos , Masculino , Metotrexato/metabolismo , Metotrexato/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Recurrencia , Tetrahidrofolato Deshidrogenasa/biosíntesisRESUMEN
1. Dietary folic acid requirements of broilers were studied in three experiments using wheat- and maize-based practical diets. Requirements were assessed on the basis of performance and metabolic criteria. 2. Growth and food conversion efficiencies were optimised with supplements of 1.5 mg folic acid/kg added to basal mash starter diets. The dietary folic acid requirement of broilers was estimated to be in the range of 1.7 to 2.0 mg/kg. 3. Red blood cell phosphoribosylpyrophosphate concentrations and dihydrofolate reductase activities did not show consistent changes over the range of dietary folate concentrations studied but plasma folate concentrations responded markedly to dietary folate supplementation. 4. Adding choline to diets in amounts greater than the normal requirement did not spare the requirement for folic acid. 5. It is suggested that minimum folic acid supplements for pelleted practical diets should be in the order of 2.5 to 3 mg/kg.
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Pollos/crecimiento & desarrollo , Ácido Fólico/fisiología , Animales , Biomarcadores , Pollos/sangre , Femenino , Masculino , Necesidades Nutricionales , Fosforribosil Pirofosfato/sangre , Tetrahidrofolato Deshidrogenasa/sangreRESUMEN
The effects of folate sufficiency and deficiency in three pathways of folate metabolism were studied in 2-and 3-week-old broiler chicks. Erythrocyte phosphoribosylpyrophosphate concentrations and dihydrofolate reductase (EC 1.5.1.3) activity were significantly elevated in folate deficiency. Percentage incorporation of deoxyuridine into bone marrow DNA was reduced in folate deficiency. There was a trend towards reduced liver dihydrofolate reductase activity in deficient chicks. These studies identify further biochemical criteria that can be used to assess folate status of chicks.
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Pollos/metabolismo , Deficiencia de Ácido Fólico/metabolismo , Ácido Fólico/metabolismo , Animales , Médula Ósea/metabolismo , ADN/metabolismo , Desoxiuridina/metabolismo , Eritrocitos/metabolismo , Hígado/enzimología , Masculino , Estado Nutricional/fisiología , Fosforribosil Pirofosfato/sangre , Tetrahidrofolato Deshidrogenasa/sangreRESUMEN
The disposition of [14C]piritrexim ([14C]PTX) in male dogs after iv and po doses of 1.8 mg/kg was examined. After either route of administration, greater than 90% of the dose was recovered in the exreta within 72 hr; approximately 20% was recovered in urine and 70% in feces. [14C]PTX was extensively metabolized by dogs; unchanged drug accounted for less than 15% of the dose in the excreta. The O-demethylated metabolites, 2'- and 5'-demethyl PTX, the glucuronide conjugate of 2'-demethyl PTX, and the sulfate conjugate of 5'-demethyl PTX were the major metabolites. Unchanged drug accounted for a large proportion of the drug-related radiocarbon in plasma. The average plasma half-life of PTX after iv administration was 2.6 +/- 0.3 hr, and the average total body clearance was 0.33 +/- 0.13 liter/hr/kg. After po administration, peak plasma concentrations of 0.9 +/- 0.3 micrograms/ml occurred about 1.1 hr after the dose; the absolute oral bioavailability of PTX was 0.63 +/- 0.14. Because the O-demethyl metabolites were active dihydrofolate reductase inhibitors, 2'- and 5'-demethyl PTX were synthesized, and the pharmacokinetics and bioavailability of these compounds in dogs after iv and po administration (5 mg/kg) were examined. The plasma concentration-time data for both compounds after iv doses were described by a two-compartment model, with t1/2 beta = 1.3 and 0.8 hr for the 2'- and 5'- demethyl compounds, respectively. Neither compound showed significant advantages over PTX in terms of pharmacokinetics or bioavailability.
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Antineoplásicos/farmacocinética , Pirimidinas/farmacocinética , Administración Oral , Animales , Antineoplásicos/sangre , Antineoplásicos/metabolismo , Disponibilidad Biológica , Radioisótopos de Carbono , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Perros , Heces/química , Inyecciones Intravenosas , Masculino , Piridinas/farmacocinética , Pirimidinas/sangre , Pirimidinas/metabolismo , Tetrahidrofolato Deshidrogenasa/sangreRESUMEN
Because of a previously observed plateau of clinical response after long-term methotrexate (MTX) therapy for rheumatoid arthritis (RA), we investigated whether such treatment might lead to acquired resistance to the drug. We studied the activity of dihydrofolate reductase (DHFR) (the target enzyme of MTX) in peripheral blood mononuclear cells of 11 RA patients who had been treated with MTX for a median of 43 months. The enzyme levels were markedly increased compared with levels found in the cells of 6 RA patients treated with other slow-acting drugs. Quantitative dot-blot analysis of DNA from 7 of these patients showed no evidence of DHFR gene amplification. No correlation was observed between increased levels of DHFR and either response to therapy or to the weekly MTX dosage. Phytohemagglutinin-stimulated peripheral blood lymphocytes from 6 patients with increased levels of DHFR showed no evidence of MTX resistance in vitro. The increased DHFR levels may result from binding of MTX to the enzyme, which may block the normal degradation pathways; they do not appear to be a marker of impending drug resistance.
Asunto(s)
Artritis Reumatoide/enzimología , Linfocitos/enzimología , Metotrexato/uso terapéutico , Tetrahidrofolato Deshidrogenasa/sangre , Adulto , Anciano , Artritis Reumatoide/tratamiento farmacológico , ADN/aislamiento & purificación , Resistencia a Medicamentos , Tolerancia a Medicamentos , Amplificación de Genes , Humanos , Linfocitos/efectos de los fármacos , Persona de Mediana Edad , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
This report describes the effect of intermediate methotrexate (MTX) doses on dihydrofolate reductase (DHFR) activity in vivo in the leukocytes of 16 children with malignant diseases. The authors used a cytochemical technique, and the enzyme was studied in intact cells. The treatment protocols included MTX 500 mg-2 g/m2 weekly with leucovorin rescue. The above doses of MTX partially inhibit DHFR. The reduction of enzyme activity was observed in leukocytes within 24 h after MTX infusion, and it was more obvious in the polymorphonucleas and the monocytes. Complete inhibition of enzyme activity was not observed. These results do not agree with those of previous reports using biochemical techniques, which showed that small amounts of MTX inhibit DHFR activity. Even the large doses of MTX used in this study do not completely inhibit enzyme activity. It would be worthwhile to test the effect of even larger doses of MTX to find out if DHFR activity is inhibited.
Asunto(s)
Antagonistas del Ácido Fólico , Metotrexato/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/enzimología , Niño , Humanos , Leucocitos/enzimología , Linfoma no Hodgkin/tratamiento farmacológico , Linfoma no Hodgkin/enzimología , Metotrexato/administración & dosificación , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/sangre , Neoplasias/enzimología , Sarcoma/tratamiento farmacológico , Sarcoma/enzimología , Tetrahidrofolato Deshidrogenasa/sangreRESUMEN
In the serum of patients receiving high-dose methotrexate (MTX), concentrations of the drug were monitored using both high-performance liquid chromatographic (HPLC) and enzyme-inhibition assays. In samples obtained greater than or equal to 24 hours after drug application, the HPLC assay measured higher MTX concentrations than the enzyme-inhibition assay. In 72-hour samples, the increase was usually greater than 200%. This difference was not observed in serum spiked with MTX. While the HPLC assay needs sample cleanup, ie, protein precipitation, the enzyme-inhibition assay routinely employs native serum. When MTX was measured in the supernatant with the enzyme inhibition assay, the results equaled those obtained with the HPLC procedure. In patient serum, MTX eluted from a Sephadex G-75 column in two fractions. One had the same retention volume as free MTX and could be assayed directly. The other had a retention volume like dihydrofolate reductase. In this fraction, MTX could only be determined after denaturing proteins. Only small amounts of tightly bound MTX were found in the serum of patients on intermediate-dose therapy (500-600 mg/m2). The observation that after high-dose MTX therapy part of the MTX in patient serum is strongly bound to a protein which could be DHFR raises the question as to the source and fate of this complex. Furthermore, the present finding has clinical relevance to the extent that the accepted limits for risk of MTX toxicity are based on methods using native serum. However, any procedure employing protein precipitation for sample cleanup may grossly overestimate active MTX serum concentrations, especially in the 72-hour serum samples.
Asunto(s)
Antagonistas del Ácido Fólico , Metotrexato/sangre , Adulto , Animales , Bioensayo , Bovinos , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Diálisis , Humanos , Infusiones Parenterales , Hígado/enzimología , Metotrexato/administración & dosificación , Metotrexato/farmacología , Unión Proteica , Ratas , Tetrahidrofolato Deshidrogenasa/sangreAsunto(s)
Pruebas Enzimáticas Clínicas , Leucemia/diagnóstico , Tetrahidrofolato Deshidrogenasa/análisis , Enfermedad Aguda , Médula Ósea/enzimología , Histocitoquímica , Humanos , Leucemia/clasificación , Leucemia Linfoide/diagnóstico , Leucemia Mieloide Aguda/diagnóstico , Linfocitos/enzimología , Tetrahidrofolato Deshidrogenasa/sangreRESUMEN
The inhibitory effect of triamterene and its metabolites on human leucocyte dihydrofolate reductase has been studied. Under test conditions with dihydrofolic acid 0.5 X 10(-5) M, triamterene 7 X 10(-5) M produced total enzyme inhibition, whereas the metabolites hydroxytriamterene and the sulphate ester of hydroxytriamterene were less effective inhibitors; at their maximum attainable concentration of 5 X 10(-5) M, dihydrofolate reductase was inhibited by 80% and 50%, respectively. Cultures of the BJAB-B95-8 human lymphoma cell line were incubated with various concentrations of triamterene. Because of their increased specific activity of dihydrofolate reductase, the cells were able to maintain normal DNA metabolism, as measured by the ratio of the incorporation rates of 3H-deoxyuridine and 3H-thymidine, as well as normal cell growth at 1 X 10(-6) M, and in some cases at 1 X 10(-5) M triamterene. At 8 X 10(-5) M triamterene, the strong inhibitory effect caused severe impairment of DNA metabolism and subsequently dissolution of the cell culture. The results are discussed in relation to the possible toxic side effects of long-term triamterene treatment in patients suffering from alcoholic cirrhosis, who may have impaired metabolism of triamterene and a concomitant severe folate deficiency.