Letter to the editor Received: 22 November 2013,
Revised: 28 February 2014,
Accepted: 14 March 2014
Published online in Wiley Online Library: 22 April 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3214
5,10-Methylene-tetrahydrofolate dissociates into tetrahydrofolate and formaldehyde at physiological pH and acidic pH, typical conditions used during sample extraction and LC-MS/MS analysis of biological samples To the Editor
Biomed. Chromatogr. 2014; 28: 1041–1042
acidic mobile phase. However, stability experiments are an important part of the bioanalytical method validation (Tiwari and Tiwari, 2010). The lack of such experiments makes the reliability of the analytical method developed by Odin and coworkers highly questionable. Moreover, there are no data in the literature on stability of 5,10-CH2-H4folate under similar conditions and in similar concentration ranges. There are only limited stability data in some patents regarding the brand Modufolin, which is 5,10-CH2-H4folate containing trisodium citrate and ascorbic acid delivered in lyophilized form (US Patents 2009/0221594A1 and 2007/009866 A1). The patents present stability data for Modufolin in concentrations >0.5 mg/mL at pH 5–7 and storage in room temperature up to 24 h. Data presented indicate good stability in the presence of trisodium citrate and the analyses are based on HPLC, but no information is given on the conditions of the HPLC analysis or validation data for the methods used. Therefore, Odin et al. (2013) should address specifically the conversion of 5,10-CH2-H4folate into H4folate during sample preparation, analysis and storage. This could be accomplished by monitoring the formation of H4folate from 5,10-CH2-H4folate standard over time and under various conditions. The authors could also have made it clearer that 5,10-CH2-H4folate is stable against oxidation and needs stabilization against dissociation, whereas the other two reduced folates, H4-folate and 5-CH3-H4folate, need antioxidants for their stabilization against oxidative degradation (Osborn et al., 1960; Blakley, 1969; Hawkes and Villotta, 1989). Hence, the stability of Modufolin may be a result of sodium citrate’s capacity to prevent its dissociation into H4folate and formaldehyde. If we accept the data presented in the patents and the stability work for Modufolin, shown by Odin et al. (2013), these are new findings that citrate, similar to formaldehyde, could stabilize 5,10-CH2-H4folate by preventing dissociation into formaldehyde and H4folate. Then it is, however, difficult to understand how the native 5,10-CH2-H4folate analyzed in all the tissue extracts shown in Tables 5 and 6 have been stabilized because no citrate has been added during tissue extraction and LC-MS/MS determination of these samples.
* Correspondence to: M. Jägerstad, Department of Food Science, Swedish University of Agricultural Sciences (SLU), PO Box 7051, SE-750 07 Uppsala, Sweden. Email: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
1041
In a paper by Odin and coworkers entitled ‘Determination of reduced folates in tumor and adjacent mucosa of colorectal cancer patients using LC-MS/MS’, published in Biomedical Chromatography, 27, pp. 485–495, 2013, the authors present data on three forms of reduced folates, tetrahydrofolate, 5-methyltetrahydrofolate and 5,10-methylene-tetrahydrofolate (5,10-CH2-H4folate) in colorectal mucosa and corresponding tumors. The most surprising outcome of this paper is the data on 5,10-CH2-H4folate in colorectal mucosa and corresponding tumors. It is well recognized from the literature that 5,10-CH2-H4folate dissociates rapidly to tetrahydrofolate and formaldehyde at physiological pH, and heating enhances this dissociation. In fact, 5,10-CH2-H4folate is only stable towards dissociation into tetrahydrofolate at pH >8 or in the presence of huge molar excess of formaldehyde (Osborn et al., 1960; Blakley, 1969; Shin et al., 1994; Horne, 2001; Quinlivan et al., 2006; De Brouwer et al., 2007). Quinlivan et al. (2006), for example, states: ‘At physiological pH values, 5,10-CH2-H4folate dissociates to formaldehyde and H4folate. Using typical extraction conditions (e.g. extraction at pH 7.85), this causes total 5,10-CH2-H4folate depletion and a corresponding overestimation of H4folate concentration. However, homogenizing samples at pH 10 prevents dissociation of 5,10-CH2-H4folate into formaldehyde and H4folate.’ Moreover, one recent study by Ramos-Parra et al. (2013) presents H4folate as the sum of H4folate and 5,10-CH2-H4folate, referring to the fact that acidic mobile phase was used for HPLC determination with electrochemical detection. This should be the correct way to present these two reduced folate forms in biological samples. Otherwise there is a risk of false results by underestimating 5,10-CH2-H4folate and/or overestimating H4folate owing to 5,10-CH2-H4folate dissociation into H4folate and formaldehyde. The extraction in the work by Odin et al. (2013) was performed at pH 7 and the LC-MS/MS analyses were carried out at acidic pH, approximately 3–3.5. Excess of formaldehyde was not used for stabilization of 5,10-CH2-H4folate. These analytical conditions are highly favorable for the fast dissociation of 5,10-CH2-H4folate into H4folate and formaldehyde according to all previous work. Therefore it should be reasonable to expect that 5,10-CH2-H4folate will be completely dissociated to H4folate and formaldehyde during the passage of the chromatographic column. Surprisingly, Odin and co-workers did not performed stability study of 5,10-CH2-H4folate during various stages of analysis, including stability of 5,10-CH2-H4folate in real samples, sample extracts or
Letter to the Editor Odin and coworkers discuss their results by referring to previous work (Liu et al., 2011; Kim et al., 1998; Powers et al., 2007; Elmore et al., 2007; Horne, 2001). Only two of the referred papers present data on 5,10-CH2-H4 folate. Horne (2001) reported 5,10-CH2-H4folate in rat liver. His figures on 5,10-CH2-H4 folate were calculated as the difference between samples treated with borohydride at pH 8-9, converting 5,10-CH2-H4folate into 5-CH3-H4folate minus the original concentration of 5-CH3-H4folate. The paper of Elmore et al. (2007) presented 5,10-CH2-H4folate as the sum of H4folate and 5,10-CH2-H4folate, without being able to separate these forms. Instead they concluded that the eventually occurring 5,10-CH2-H4folate was interconverted into H4folate during sample preparation and analysis. We have recently published a review on formyl folates where we present data on folate profiling in different food items (Jägerstad and Jastrebova, 2013). We were not able to find any study reporting occurrence of 5,10-CH2-H4folate in foods. In conclusion, we argue that the H4folate concentrations reported by Odin et al. correspond to the sum of H4folate and 5,10-CH2-H4folate. The figures, claimed to represent native 5,10-CH2-H4folate, need to be more specifically explained and discussed, considering present knowledge of pH-dependent dissociation of native 5,10-CH2-H4folate at physiological and acidic pH. It is of utmost importance to present reliable data on 5,10-CH2-H4folate in tissues in order to make correct interpretations in normal vs tumor tissues. H4folate is a general receiver of C1 groups within the cell folate metabolism whereas 5,10-CH2-H4folate has two specific tasks as co-enzyme: first in donating its C1 group in a key step of the DNA synthesis, that is, in the methylation of uridylate into thymidylate. Second, 5,10-CH2H4folate is also a precursor of 5-CH3-H4folate, the C1 donor and co-enzyme in remethylation of homocysteine into methionine.
References Blakley RL. The biochemistry of folic acid and related pteridines. In Frontiers of Biology. North Holland: Amsterdam, 1969; 1–99. De Brouwer V, Zhang G-F, Storozhenko S, van der Straeten D and Lambert W. pH stability of individual folates during critical sample preparation steps in prevision of the analysis of plant folates. Phytochemical Analysis 2007; 18: 496–508. Elmore CL, Wu X, Leclerc D, Watson ED, Bottiglieri T, Krupenkoe NI, Krupenkoe SA, Cross JC, Rozen R, Gravel RA and Matthews RG. Metabolic derangement of methionine and folate metabolism in mice
deficient in methionine synthase reductase. Molecular Genetics and Metabolism 2007; 91(1): 85–97. Hawkes JG and Villota R. Folates in foods: reactivity, stability during processing, and nutritional implications. Critical Reviews in Food Science and Nutrition 1989; 28: 439–538. Horne DW. High performance liquid chromatographic measurement of 5,10-methylenetetrahydrofolate in liver. Analytical Biochemistry 2001; 297: 154–159. Jägerstad M and Jastrebova J. Occurrence, stability and determination of formyl folates in foods. Review. Journal of Agricultural Food and Chemistry 2013; 61: 9758–9768. Kim YI, Fawaz K, Knox T, Lee YM, Norton R, Arora S, Paiva L and Mason JB. Colonic mucosal concentrations of folate correlate well with blood measurements of folate status in persons with colorectal polyps. American Journal of Clinical Nutrition 1998; 68: 866–872. Liu J, Pickford R, Meagher AP and Ward RL. Quantitative analysis of tissue folate using ultra high-performance liquid chromatography tandem mass spectrometry. Analytical Biochemistry 2011; 411: 210–217. Odin E, Wettergren Y, Carlsson G and Gustavsson B. Determination of reduced folates in tumor and adjacent mucosa of colorectal cancer patients using LC-MS/MS. Biomedical Chromatography 2013; 27: 487–495. Osborn MJ, Talbert PT and Huennekens FM. The structure of ‘active form5 10 aldehyde’ (N ,N -methylene tetrahydrofolic acid. Journal of American Chemical Society 1960; 82: 4921–4927. Powers HJ, Hill MH, Welfare M, Spiers A, Bal W, Russell J, Duckworth Y, Gibney E, Williams EA and Mathers JC. Responses of biomarkers of folate and riboflavin status to folate and riboflavin supplementation in healthy and colorectal polyp patients (the FAB2 Study). Cancer Epidemiology, Biomarkers and Prevention 2007; 16: 2128–2135. Quinlivan EP, Hanson AD and Gregory JF. The analysis of folate and its metabolic precursors in biological samples. Analytical Biochemistry 2006; 348: 163–184. Ramos-Parra PA, Garcia-Salinas C, Hernández-Brenes C and Diaz de la Garza RI. Folate levels and polyglutamylation profiles of Papaya (Carica papaya cv Maradol) during fruit development and ripening. Journal of Agricultural Food and Chemistry 2013; 61: 3949–3956. Shin HC, Shimoda M and Kokue E. Identification of 5,10methylenetetrahydrofolate in rat bile. Journal of Chromatrography B 1994; 661: 237–244. Tiwari G and Tiwari R. Bioanalytical method validation: an updated review. Pharmaceutical Methods 2010; 1(1): 25–38.
Margaretha Jägerstad Jelena Jastrebova Department of Food Science, Swedish University of Agricultural Sciences (SLU), PO Box 7051, SE-750 07 Uppsala, Sweden [email protected]
1042 wileyonlinelibrary.com/journal/bmc
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014; 28: 1041–1042
Revised: 28 February 2014,
Accepted: 14 March 2014
Published online in Wiley Online Library: 22 April 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3214
5,10-Methylene-tetrahydrofolate dissociates into tetrahydrofolate and formaldehyde at physiological pH and acidic pH, typical conditions used during sample extraction and LC-MS/MS analysis of biological samples To the Editor
Biomed. Chromatogr. 2014; 28: 1041–1042
acidic mobile phase. However, stability experiments are an important part of the bioanalytical method validation (Tiwari and Tiwari, 2010). The lack of such experiments makes the reliability of the analytical method developed by Odin and coworkers highly questionable. Moreover, there are no data in the literature on stability of 5,10-CH2-H4folate under similar conditions and in similar concentration ranges. There are only limited stability data in some patents regarding the brand Modufolin, which is 5,10-CH2-H4folate containing trisodium citrate and ascorbic acid delivered in lyophilized form (US Patents 2009/0221594A1 and 2007/009866 A1). The patents present stability data for Modufolin in concentrations >0.5 mg/mL at pH 5–7 and storage in room temperature up to 24 h. Data presented indicate good stability in the presence of trisodium citrate and the analyses are based on HPLC, but no information is given on the conditions of the HPLC analysis or validation data for the methods used. Therefore, Odin et al. (2013) should address specifically the conversion of 5,10-CH2-H4folate into H4folate during sample preparation, analysis and storage. This could be accomplished by monitoring the formation of H4folate from 5,10-CH2-H4folate standard over time and under various conditions. The authors could also have made it clearer that 5,10-CH2-H4folate is stable against oxidation and needs stabilization against dissociation, whereas the other two reduced folates, H4-folate and 5-CH3-H4folate, need antioxidants for their stabilization against oxidative degradation (Osborn et al., 1960; Blakley, 1969; Hawkes and Villotta, 1989). Hence, the stability of Modufolin may be a result of sodium citrate’s capacity to prevent its dissociation into H4folate and formaldehyde. If we accept the data presented in the patents and the stability work for Modufolin, shown by Odin et al. (2013), these are new findings that citrate, similar to formaldehyde, could stabilize 5,10-CH2-H4folate by preventing dissociation into formaldehyde and H4folate. Then it is, however, difficult to understand how the native 5,10-CH2-H4folate analyzed in all the tissue extracts shown in Tables 5 and 6 have been stabilized because no citrate has been added during tissue extraction and LC-MS/MS determination of these samples.
* Correspondence to: M. Jägerstad, Department of Food Science, Swedish University of Agricultural Sciences (SLU), PO Box 7051, SE-750 07 Uppsala, Sweden. Email: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
1041
In a paper by Odin and coworkers entitled ‘Determination of reduced folates in tumor and adjacent mucosa of colorectal cancer patients using LC-MS/MS’, published in Biomedical Chromatography, 27, pp. 485–495, 2013, the authors present data on three forms of reduced folates, tetrahydrofolate, 5-methyltetrahydrofolate and 5,10-methylene-tetrahydrofolate (5,10-CH2-H4folate) in colorectal mucosa and corresponding tumors. The most surprising outcome of this paper is the data on 5,10-CH2-H4folate in colorectal mucosa and corresponding tumors. It is well recognized from the literature that 5,10-CH2-H4folate dissociates rapidly to tetrahydrofolate and formaldehyde at physiological pH, and heating enhances this dissociation. In fact, 5,10-CH2-H4folate is only stable towards dissociation into tetrahydrofolate at pH >8 or in the presence of huge molar excess of formaldehyde (Osborn et al., 1960; Blakley, 1969; Shin et al., 1994; Horne, 2001; Quinlivan et al., 2006; De Brouwer et al., 2007). Quinlivan et al. (2006), for example, states: ‘At physiological pH values, 5,10-CH2-H4folate dissociates to formaldehyde and H4folate. Using typical extraction conditions (e.g. extraction at pH 7.85), this causes total 5,10-CH2-H4folate depletion and a corresponding overestimation of H4folate concentration. However, homogenizing samples at pH 10 prevents dissociation of 5,10-CH2-H4folate into formaldehyde and H4folate.’ Moreover, one recent study by Ramos-Parra et al. (2013) presents H4folate as the sum of H4folate and 5,10-CH2-H4folate, referring to the fact that acidic mobile phase was used for HPLC determination with electrochemical detection. This should be the correct way to present these two reduced folate forms in biological samples. Otherwise there is a risk of false results by underestimating 5,10-CH2-H4folate and/or overestimating H4folate owing to 5,10-CH2-H4folate dissociation into H4folate and formaldehyde. The extraction in the work by Odin et al. (2013) was performed at pH 7 and the LC-MS/MS analyses were carried out at acidic pH, approximately 3–3.5. Excess of formaldehyde was not used for stabilization of 5,10-CH2-H4folate. These analytical conditions are highly favorable for the fast dissociation of 5,10-CH2-H4folate into H4folate and formaldehyde according to all previous work. Therefore it should be reasonable to expect that 5,10-CH2-H4folate will be completely dissociated to H4folate and formaldehyde during the passage of the chromatographic column. Surprisingly, Odin and co-workers did not performed stability study of 5,10-CH2-H4folate during various stages of analysis, including stability of 5,10-CH2-H4folate in real samples, sample extracts or
Letter to the Editor Odin and coworkers discuss their results by referring to previous work (Liu et al., 2011; Kim et al., 1998; Powers et al., 2007; Elmore et al., 2007; Horne, 2001). Only two of the referred papers present data on 5,10-CH2-H4 folate. Horne (2001) reported 5,10-CH2-H4folate in rat liver. His figures on 5,10-CH2-H4 folate were calculated as the difference between samples treated with borohydride at pH 8-9, converting 5,10-CH2-H4folate into 5-CH3-H4folate minus the original concentration of 5-CH3-H4folate. The paper of Elmore et al. (2007) presented 5,10-CH2-H4folate as the sum of H4folate and 5,10-CH2-H4folate, without being able to separate these forms. Instead they concluded that the eventually occurring 5,10-CH2-H4folate was interconverted into H4folate during sample preparation and analysis. We have recently published a review on formyl folates where we present data on folate profiling in different food items (Jägerstad and Jastrebova, 2013). We were not able to find any study reporting occurrence of 5,10-CH2-H4folate in foods. In conclusion, we argue that the H4folate concentrations reported by Odin et al. correspond to the sum of H4folate and 5,10-CH2-H4folate. The figures, claimed to represent native 5,10-CH2-H4folate, need to be more specifically explained and discussed, considering present knowledge of pH-dependent dissociation of native 5,10-CH2-H4folate at physiological and acidic pH. It is of utmost importance to present reliable data on 5,10-CH2-H4folate in tissues in order to make correct interpretations in normal vs tumor tissues. H4folate is a general receiver of C1 groups within the cell folate metabolism whereas 5,10-CH2-H4folate has two specific tasks as co-enzyme: first in donating its C1 group in a key step of the DNA synthesis, that is, in the methylation of uridylate into thymidylate. Second, 5,10-CH2H4folate is also a precursor of 5-CH3-H4folate, the C1 donor and co-enzyme in remethylation of homocysteine into methionine.
References Blakley RL. The biochemistry of folic acid and related pteridines. In Frontiers of Biology. North Holland: Amsterdam, 1969; 1–99. De Brouwer V, Zhang G-F, Storozhenko S, van der Straeten D and Lambert W. pH stability of individual folates during critical sample preparation steps in prevision of the analysis of plant folates. Phytochemical Analysis 2007; 18: 496–508. Elmore CL, Wu X, Leclerc D, Watson ED, Bottiglieri T, Krupenkoe NI, Krupenkoe SA, Cross JC, Rozen R, Gravel RA and Matthews RG. Metabolic derangement of methionine and folate metabolism in mice
deficient in methionine synthase reductase. Molecular Genetics and Metabolism 2007; 91(1): 85–97. Hawkes JG and Villota R. Folates in foods: reactivity, stability during processing, and nutritional implications. Critical Reviews in Food Science and Nutrition 1989; 28: 439–538. Horne DW. High performance liquid chromatographic measurement of 5,10-methylenetetrahydrofolate in liver. Analytical Biochemistry 2001; 297: 154–159. Jägerstad M and Jastrebova J. Occurrence, stability and determination of formyl folates in foods. Review. Journal of Agricultural Food and Chemistry 2013; 61: 9758–9768. Kim YI, Fawaz K, Knox T, Lee YM, Norton R, Arora S, Paiva L and Mason JB. Colonic mucosal concentrations of folate correlate well with blood measurements of folate status in persons with colorectal polyps. American Journal of Clinical Nutrition 1998; 68: 866–872. Liu J, Pickford R, Meagher AP and Ward RL. Quantitative analysis of tissue folate using ultra high-performance liquid chromatography tandem mass spectrometry. Analytical Biochemistry 2011; 411: 210–217. Odin E, Wettergren Y, Carlsson G and Gustavsson B. Determination of reduced folates in tumor and adjacent mucosa of colorectal cancer patients using LC-MS/MS. Biomedical Chromatography 2013; 27: 487–495. Osborn MJ, Talbert PT and Huennekens FM. The structure of ‘active form5 10 aldehyde’ (N ,N -methylene tetrahydrofolic acid. Journal of American Chemical Society 1960; 82: 4921–4927. Powers HJ, Hill MH, Welfare M, Spiers A, Bal W, Russell J, Duckworth Y, Gibney E, Williams EA and Mathers JC. Responses of biomarkers of folate and riboflavin status to folate and riboflavin supplementation in healthy and colorectal polyp patients (the FAB2 Study). Cancer Epidemiology, Biomarkers and Prevention 2007; 16: 2128–2135. Quinlivan EP, Hanson AD and Gregory JF. The analysis of folate and its metabolic precursors in biological samples. Analytical Biochemistry 2006; 348: 163–184. Ramos-Parra PA, Garcia-Salinas C, Hernández-Brenes C and Diaz de la Garza RI. Folate levels and polyglutamylation profiles of Papaya (Carica papaya cv Maradol) during fruit development and ripening. Journal of Agricultural Food and Chemistry 2013; 61: 3949–3956. Shin HC, Shimoda M and Kokue E. Identification of 5,10methylenetetrahydrofolate in rat bile. Journal of Chromatrography B 1994; 661: 237–244. Tiwari G and Tiwari R. Bioanalytical method validation: an updated review. Pharmaceutical Methods 2010; 1(1): 25–38.
Margaretha Jägerstad Jelena Jastrebova Department of Food Science, Swedish University of Agricultural Sciences (SLU), PO Box 7051, SE-750 07 Uppsala, Sweden [email protected]
1042 wileyonlinelibrary.com/journal/bmc
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014; 28: 1041–1042
