Characterization and Interrelations of One-Carbon Metabolites in Tissues, Erythrocytes, and Plasma in Mice with Dietary Induced Folate Deficiency

Abstract

Studies on one-carbon metabolism for the assessment of folate deficiency have focused on either metabolites of folate metabolism or methionine cycle. To bridge the gap between deficiency markers in these pathways we designed a dietary induced folate deficiency study using male C57BL/6N mice. After weaning (3 weeks) mice were fed a defined control diet (1 week) before being fed a folate deficient diet (n = 6 mice) and the control diet (n = 6 mice) for 12 additional weeks. Thereafter, we determined total homocysteine in plasma and folate in erythrocytes as well as S-adenosylmethionine, S-adenosylhomocysteine, and six folate vitamers in tissues including 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, tetrahydrofolate, 10-formylfolic acid, and folic acid by means of stable isotope dilution assays coupled with liquid chromatography tandem mass spectrometry. In all organs, except heart (mainly 5-mehtyltetrahydrofolate), tetrahydrofolate constitutes the main vitamer. Moreover, in liver tetrahydrofolate was most abundant followed by 5-methyltetrahydrofolate (heart: tetrahydrofolate), 5-formyltetrahydrofolate, and 5,10-methenyltetrahydrofolate. Because of the significant decrease (p < 0.05) of folate status and S-adenosylmethionine/S-adenosylhomocysteine ratio accompanied with increasing S-adenosylhomocysteine (p < 0.05), hepatocytes are most susceptible to folate deficiency. To the best of our knowledge, we herein present the first method for simultaneous quantitation of eight metabolites for both folate and methionine cycle in one tissue sample, tHcy in plasma, and erythrocyte folate to shed light on physiological interrelations of one-carbon metabolism.

Keywords: S-adenosylhomocysteine; S-adenosylmethionine; folate deficiency; folate metabolism; homocysteine; methionine metabolism.

Figure 1

Analytes determined by stable isotope dilution assays coupled with liquid chromatography tandem mass spectrometry (SIDA-LC-MS/MS) in selected tissue samples. Ox: Oxidation, GSH: Glutathione, dTMP: Deoxythymidine monophosphate, dUMP: Deoxyuridine monophosphate. Analytes under study are framed in bold lines.

Figure 2

Body weight of controls (n = 6, dashed line) and mice fed a folate-deficient diet (n = 6, straight line), * significant difference (p < 0.05).

Figure 3

Total homocysteine (tHcy) (a) in plasma and 5-CH₃-H₄folate in lyophilized erythrocytes (b). dw: Dry weight, cd: Control diet (n = 6), fdd: Folate-deficient diet (n = 6), * significant difference (p < 0.05).

Figure 4

Percental decrease of total tissue folate in folate-deficient mice after 12 weeks. cd: Control diet (n = 6), fdd: Folate-deficient diet (n = 6).

Figure 5

Tissue-specific concentrations of folate vitamers depending on diet in (a) liver and (b) kidney. dw: Dry weight, cd: Control diet (n = 6), fdd: Folate-deficient diet (n = 6), * significant difference (p < 0.05).

Figure 6

Tissue-specific concentrations of folate vitamers depending on diet in (a) brain and (b) heart. dw: Dry weight, cd: Control diet (n = 6), fdd: Folate-deficient diet (n = 6), * significant difference (p < 0.05).

Figure 7

Tissue-specific concentrations of S-adenosylhomocysteine (AdoHcy) (a) and S-adenosylmethionine (AdoMet) (b) depending on diet. dw: dry weight, cd: Control diet (n = 6), fdd: Folate-deficient diet (n = 6), * significant difference (p < 0.05).

Figure 8

Tissue-specific AdoMet/AdoHcy ratio depending on diet. cd: Control diet (n = 6), fdd: Folate-deficient diet (n = 6), * significant difference (p < 0.05).