Riboflavin has been suggested to act with folate to lower homocysteine (Hcy). However, these interactions may differ among the several known forms of folate. Therefore, we examined the effects of riboflavin interactions with 5-methyltetrahydrofolate (5-MTHF) and tetrahydrofolate (THF) on changes in Hcy and folate derivative levels, under conditions with and without methionine addition. Rat hepatocytes were cultured for 48 hours in medium with or without 2.64 μM riboflavin addition, under the following conditions: 1) without the addition of either methionine or folate; 2) with addition of 2 μM folate derivatives [(A): 5-MTHF, (B): THF]; 3) with addition of both 5 mM methionine and 2 μM folate derivatives [(A): 5-MTHF, (B): THF]. The supernatants were collected at 0, 24, and 48 hours for Hcy and folate derivative measurements. The Hcy lowering effect of 5-MTHF, as well as inhibition of 5-MTHF reduction and THF elevation, appeared to be enhanced by riboflavin addition. THF addition did not lower the Hcy level, regardless of the presence of riboflavin and/or methionine, while THF and 5,10-methenyl THF levels were maintained. Further examination is needed to elucidate the interactive effects of riboflavin and folate derivatives on Hcy and folate metabolism.
Folate plays an important role in the remethylation of homocysteine (Hcy), and the synthesis of thymidylate and purine which are precursors for DNA and RNA synthesis [
However, in our previous short-term supplementation study, serum 5-MTHF levels were increased by folic acid supplementation, but not by folic acid plus riboflavin supplementation, in healthy young Japanese men. Furthermore, no significant Hcy reduction was observed during the intervention in the group given folic acid combined with riboflavin as compared to the group given folic acid alone [
5-MTHF and lower Hcy in the healthy subject group, while the plasma 5-MTHF level was increased by riboflavin supplementation in colorectal polyp patients with the CT or the TT genotype [
It is difficult to demonstrate the effects of riboflavin on folate and Hcy metabolism in a human study because numerous interrelated factors influence these metabolic processes. Thus, a simple cellular system is needed to clarify the effects of riboflavin. Earlier studies demonstrating the combined effects of folic acid and riboflavin were performed using lymphocytes [8,9]. The liver is reportedly the major site of methionine catabolism [
Among folate derivatives, 5-MTHF and THF are the two dominant circulating forms in mammals [
Thus, changes in THF and 5,10-methylene THF levels are useful for estimating the effects of riboflavin on 5- MTHF production. However, 5,10-methylene THF cannot be measured because of its instability [
Therefore, we examined the effects of riboflavin interactions with 5-MTHF and THF on changes in Hcy, 5- MTHF, THF and 5,10-methenyl THF levels in rat hepatocytes, under conditions with and without methionine addition.
Medium 199 was obtained from Invitrogen (Netherlands). Riboflavin was obtained from Sigma Aldrich Japan (Japan). 6S-5-metyltetrahydrofolate, 6S-tetrahydrofolate, and 6S-5,10-methenyltetrahydrofolate were obtained from Shircks Laboratories (Switzerland).
In this experiment, hepatocytes (Culture Kit P-4 of Primary Cell, Japan) were used. This kit consisted of hepatocytes isolated from Sprague-Dawley rat livers, and seeded at 2 × 105 cells/well in 0.5 ml of Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum. The cultures were kept in a humidified incubator with 5% CO2 and maintained at 37˚C. After 2 hours of incubation to allow adherence, the medium was gently removed. The cells were then pre-incubated for 24 hours in the basal medium; Medium 199 (containing 26 nM riboflavin, 20 nM folic acid and 0.1 mM methionine) supplemented with 10% fetal calf serum and 1.5 μM cyanocobalamin according to a slight modification of the culture conditions described by Christensen et al. [
The supernatants were collected at 0, 24 and 48 hours, centrifuged to remove cells, and the medium was frozen at −80˚C until determination of folate derivatives and Hcy.
The 5-MTHF, THF, and 5,10-methenyl THF levels in supernatants were determined using high-performance liquid chromatography (HPLC) with the JASCO HPLC system (Jasco, Japan). Each sample was placed in a glove bag filled with nitrogen gas shielded from light, and applied to a COSMOSIL (R) 5PE-MS Packed Column (4.6 mm I.D. × 150 mm) (Nacalai Tesque, Japan). The mobile phase was a solution of 50 mM potassium dihydrogen phosphate (pH3.5) containing 0.1 mM ethylene diamine tetraacetic acid disodium salt, dehydrated in 5% methanol, with the flow rate set at 1.0 mL/min. Detection was performed using a UV 970 (Jasco, Japan) at 280 nm.
The Hcy levels in supernatants were determined by reverse-phase HPLC and fluorescence detection with a Shimadzu HPLC system (Shimadzu, Japan). HPLC was performed on a CAPCEL PAK C18 UG120 (4.6 mm I.D. × 100 mm) (Shiseido, Japan) using 0.1 M potassium dihydrogen phosphate with 5% methanol, adjusted to pH 2.7 with phosphoric acid as a mobile phase with a flow rate of 1.0 mL/min. Detection was performed at an excitation wavelength of 385 nm and an emission wavelength of 515 nm. As a reducing reagent, tris-(2-carboxy-ethyl)- phosphine was used, with 4-fluoro-7-sulfobenzo-furazan, ammonium salt serving as the derivatization agent.
All results are expressed as means ± SD. Statistical significance was judged employing Student’s t test for unpaired data and significance was defined as p < 0.05. Analyses were conducted using SPSS for Windows version 16.0J for Windows (IBM, USA).
As shown in
Under methionine-deficient but folate-supplemented conditions, low Hcy levels were maintained during the incubation period, regardless of whether or not riboflavin was added to the incubation medium (
With the addition of 5-MTHF, the 5-MTHF level was markedly decreased, showing severe depletion, after 48 hours, while the THF level remained unchanged during the incubation period. These results were similar regardless of whether or not riboflavin was added to the incubation medium. The 5,10-methenyl THF level was increased at 24 hours followed by a decrease at 48 hours in the medium with only 5-MTHF added, while higher levels were maintained by riboflavin addition (p < 0.01) (
With the addition of THF, 5-MTHF remained at an extremely low level. THF decreased markedly to one-third of the initial level at 24 hours, and this level was maintained at 48 hours regardless of whether or not riboflavin was added to the incubation medium. The 5,10-methenyl
THF level was increased at 24 hours and this level was maintained at 48 hours, with the levels being lower in the riboflavin-added medium than in that without riboflavin at both 24 and 48 hours (p < 0.05) (
Under methionine-loaded conditions, the Hcy level was increased with a concomitant reduction in the 5-MTHF level throughout the incubation period in the riboflavindeficient state. However, with the addition of riboflavin, the Hcy level did not increase and was actually lower than under the riboflavin deficient condition (p < 0.05). The 5-MTHF level decreased to half of the initial level and was maintained above this level under riboflavin-added condition at 48 hours (p < 0.05). The THF level was increased after 24 hours and this level was maintained at 48 hours, while in the medium with riboflavin the THF level was lower than that without riboflavin at 24 hours (p < 0.05). The 5,10-methenyl THF level did not change regardless of whether or not riboflavin was added to the incubation medium (
With the addition of THF, the Hcy level was markedly increased after 24 hours and continued to rise during the entire incubation period. The 5-MTHF level showed no change, remaining at an extremely low level. The THF level showed a marked decrease to one-third of the initial level at 24 hours, and was maintained at this level through 48 hours. The 5,10-methenyl THF level continued to increase throughout the incubation period. Changes in Hcy, 5-MTHF, THF, and 5,10-methenyl THF levels did not differ between the incubation conditions with and without riboflavin (
MTHFR is a flavoprotein enzyme requiring a FAD, a derivative of riboflavin. Riboflavin would be expected to enhance the Hcy-lowering effect of folate. Thus, we examined the effects of riboflavin interactions with THF or 5-MTHF on changes in the levels of Hcy and folate derivatives in the present study.
To our knowledge, this is the first study to examine the effects of riboflavin alone and combined with 5-MTHF or THF on changes in the levels of Hcy and folate derivatives in rat primary hepatocytes. In prior studies using rat hepatocytes, significant amounts of methionine were added to EMEM so that Hcy could be recovered [
The elevation of Hcy with methionine loading wasconfirmed in the present study (
An association between elevated Hcy and the risk of developing colorectal polyps [
However, with addition of THF under conditions of methionine loading, there were no differences, between the presence and absence of riboflavin, in either folate derivative or Hcy levels. Hcy levels were markedly increased, to the same level as under 5-MTHF-added condition with methionine loading (
THF is metabolized via two pathways. One involves direct synthesis of 5,10-methylene THF by serine hydroxymethyltransferase, and the other is conversion to 5,10-methylene THF via 10-formyl THF and 5,10-methenyl THF. The 10-formyl THF can be converted back into THF and used for the formylation of 5-amino-4-imidazole carboxamide ribonucleotide to 5-formamidoimidazole-4-carboxamide ribonucleotide (FAICAR). The FAICAR is then catalyzed to inosine monophosphate, which is involved in purine and pyrimidine metabolism [
Under conditions of THF addition, regardless of methionine loading, the THF level was decreased to some extent followed by a maintained the level while 5,10- methenyl THF was increased (
However, with the addition of 5-MTHF, the 5,10- methenyl THF level increase was suppressed to approximately half that under conditions with THF addition, while THF and 5,10-methenyl THF levels did not increase. It was not possible to ascertain, in the present study, whether regenerated THF from 5-MTHF is stored as other folate derivatives, such as 10-formyl THF, 5,10- metyleneTHF and dihydrofolate, or used for purine synthesis.
In the complete absence of methionine, when supplemental folate was provided in the form of THF, storage of 5,10-methenyl THF was suggested to be suppressed by the addition of riboflavin, while it increased when 5-MTHF and riboflavin were both added to the medium. We can offer no explanation for the effects of riboflavin on the 5,10-methenyl THF level, which apparently differ depending on the form of added folate (
A limitation of this study is that we examined only a single amount of each additive, such that dose-dependent effects and those related to the relative amounts of additives could not be clarified. Furthermore, we examined only the levels of change in Hcy and folate derivat ives in the supernatant. The intracellular levels of Hcy and folate derivatives should also have been measured. It is not possible to assess changes in the levels of riboflavin, SAM, and folate derivatives under the conditions used in the present study. In addition, expressions of the genes for enzymes associated with folate, Hcy and nucleic acid metabolism should be studied. Thus, elucidation of the mechanisms underlying our results requires further study.
Although much research remains to be conducted, the present results raise the following possibilities.
• Under conditions of methionine loading, supplementation with both 5-MTHF and riboflavin might enhance Hcy remethylation. However, this effect is less marked with THF supplementation.
• THF can be metabolized flexibly to 5,10-methylene THF and used in purine synthesis.
• The effects of riboflavin on 5,10-methenyl THF levels might differ between 5-MTHF and THF.
Appropriate folate intake is essential to health, but excess folic acid is reportedly associated with an increased risk of cancer [