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1.
Clin Biochem ; 42(12): 1275-81, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19427846

RESUMEN

OBJECTIVES: A low folate/high homocysteine phenotype is associated with several pathologies, including spina bifida and cardiovascular disease. Folate and total homocysteine (tHcy) measurements are used clinically to assess risk and the need for folic acid supplementation and in research to investigate the metabolic basis of disease. Red blood cell (RBC) folate, the best indicator of long-term folate status, is usually measured as "total" folate. However, different folate derivatives support distinct biochemical functions, suggesting a need to develop more precise methods. This study was designed to evaluate a method based on stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS). DESIGN AND METHODS: We used LC-MRM/MS to quantify the RBC folate derivatives 5-methyltetrahydrofolate (5-CH(3)-THF), tetrahydrofolate (THF), and 5,10-methenyltetrahydrofolate (5,10-methenylTHF) in pre-menopausal women. The concentration of each folate derivative was assessed for utility in predicting tHcy levels, and compared to folate and tHcy measurements derived by routine clinical laboratory methods. RESULTS: LC-MRM/MS was qualitatively and quantitatively superior to routine clinical laboratory methods for determining folate and tHcy concentrations. RBC 5-CH(3)-THF had a reciprocal relationship with tHcy (p=0.0003), whereas RBC THF and RBC 5,10-methenylTHF had direct relationships (p=0.01, 0.04 respectively). In combination, these three variables accounted for 42% of the variation in tHcy. CONCLUSIONS: Robust methods for measuring RBC 5-CH(3)-THF would improve the utility of folate/homocysteine phenotyping in patient management. The use of LC-MRM/MS would allow studies of hyperhomocysteinemia and diseases associated with a low folate/high homocysteine phenotype to be performed with less measurement error and greater statistical power to generate data with the potential to elucidate the etiologic mechanisms of complex diseases and traits.


Asunto(s)
Cromatografía Liquida/métodos , Ácido Fólico/sangre , Homocisteína/sangre , Espectrometría de Masas/métodos , Fenotipo , Adulto , Cromatografía Liquida/normas , Eritrocitos/química , Femenino , Ácido Fólico/química , Humanos , Espectrometría de Masas/normas , Persona de Mediana Edad , Embarazo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Adulto Joven
2.
Rapid Commun Mass Spectrom ; 22(16): 2403-12, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18634122

RESUMEN

Red blood cell (RBC) folate levels are established at the time of erythropoiesis and therefore provide a surrogate biomarker for the average folate status of an individual over the preceding four months. Folates are present as folylpolyglutamates, highly polar molecules that cannot be secreted from the RBCs, and must be converted into their monoglutamate forms prior to analysis. This was accomplished using an individual's plasma pteroylpolyglutamate hydrolase by lysing the RBCs in whole blood at pH 5 in the presence of ascorbic acid. Quantitative conversion of formylated tetrahydrofolate derivatives into the stable 5,10-methenyltetrahydrofolate (5,10-MTHF) form was conducted at pH 1.5 in the presence of [(13)C(5)]-5-formyltetrahydrofolate. The resulting [(13)C(5)]-5,10-MTHF was then used as an internal standard for the formylated forms of tetrahydrofolate that had been converted into 5,10-MTHF as well any 5,10-MTHF that had been present in the original sample. A stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry method was validated and then used for the accurate and precise quantification of RBC folic acid, 5-methyltetrahydrofolate (5-MTHF), tetrahydrofolate (THF), and 5,10-MTHF. The method was sensitive and robust and was used to assess the relationship between different methylenetetrahydrofolate reductase (MTHFR) 677C>T genotypes and RBC folate phenotypes. Four distinct RBC folate phenotypes could be identified. These were classified according to the relative amounts of individual RBC folates as type I (5-MTHF >95%; THF <5%; 5,10-MTHF <5%), type II (5-MTHF <95%; THF 5% to 20%; 5,10-MTHF <5%), type III (5-MTHF >55%; THF >20%; 5,10-MTHF >5%), and type IV (5-MTHF <55%; THF >20%; 5,10-MTHF >5%).


Asunto(s)
Eritrocitos/química , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Polimorfismo de Nucleótido Simple , Ácidos Pteroilpoliglutámicos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Complejo Vitamínico B/análisis , Adulto , Biomarcadores/análisis , Cromatografía Líquida de Alta Presión , ADN/química , Femenino , Deficiencia de Ácido Fólico/sangre , Genotipo , Humanos , Técnicas de Dilución del Indicador , Metilenotetrahidrofolato Reductasa (NADPH2)/sangre , Persona de Mediana Edad
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