We tested whether homocysteine is formed from methionine and other thioethers in vitro and in vivo, because methionine can be chemically demethylated to homocysteine.
In in vitro studies, chemical conversions of thioethers (methionine, S-adenosylhomocysteine and cystathionine) into homocysteine were measured under various aerobic conditions. In humans, oral methionine (0.17 mmol/kg body weight) loading tests with and without an oral iron dose (ferrous sulfate, 13 μmol/kg) were performed.
A university setting in Birmingham, AL, USA.
A total of five healthy adult subjects volunteered.
The in vitro incubation of methionine, S-adenosylhomocysteine or cystathionine with chelated iron resulted in the formation of homocysteine. These conversions were iron- and pH-dependent (pH optima between 5.0 and 6.0) and it was also chelator-dependent. In humans, oral methionine loading tests resulted in a 45% increase in the area-under-the-curve for plasma total homocysteine concentrations, when iron was given together with methionine.
Our data suggest that iron-dependent chemical formation of homocysteine can occur in vivo, and contribute to the plasma total homocysteine pool, since this formation can occur ceaselessly. We hypothesize that plasma total homocysteine concentrations reflect, in part, non-protein-bound iron in the body.
In humans, homocysteine (Hcy) is derived solely from methionine through the transmethylation pathway and is remethylated to methionine by methionine synthase or is metabolized to cysteine via the transsulfuration pathway (Refsum et al., 1998). A blockage in these pathways leads to elevated plasma total homocysteine (tHcy) concentration (hyperhomocysteinemia). Mild to moderate hyperhomocysteinemia has been implicated as an independent risk factor for occlusive-vascular disease (OVD). However, a mechanism by which hyperhomocysteinemia results in this pathology remains unidentified about 15 years after the initial report on the association (Clarke et al., 1991). The majority of recent clinical trials failed to demonstrate that the supplementation of B vitamins, including folic acid, reduces the risk of OVD despite a reduction in plasma tHcy (Liem et al., 2003; Lange et al., 2004; Toole et al., 2004; Bønna et al., 2006; HOPE 2 Investigators 2006). These findings suggest that it is over due to reevaluate the cause and effect of elevated Hcy in relation to the risk for OVD.
We hypothesized that Hcy is formed in vitro from methionine and other thioethers (S-adenosylhomocysteine and cystathionine) under various conditions in the presence of iron. This hypothesis was based on the facts that methionine is chemically demethylated to Hcy in acid (Stekol, 1957) and that Lewis acids, such as iron, are known to catalyze the cleavage of oxygen ethers (March, 1968). Furthermore, we postulated that non-protein-bound iron (“free iron”) is exposed to methionine (free or in peptides or proteins) or other thioethers at in vivo acidic sites including the endosome and lysosome, and in the stomach (Nalini et al., 1992; Harrison and Arosio, 1996; Ponka et al., 1998; Petrat et al., 2001) and tested whether this chemical conversion occurs in humans. In the study presented here, we describe the in vitro iron-dependent chemical conversions of these thioethers into Hcy under mild or moderate acidic conditions, and the results of oral methionine loading tests with and without an oral iron dose in humans.
In vitro experiments
All reagents including methionine, S-adenosylhomocysteine, and L-cystathionine were purchased from Sigma (St Louis, MO, USA). L-[1-14C]methionine (56 mCi/mmol) was obtained from Moravek Biochemicals (Brea, CA, USA). Distilled-deionized water was used for all experiments.
Methionine, S-adenosylhomocysteine or cystathionine in citrate buffer or ethylene diamine tetraacetic acid (EDTA) solution (5.0 mM) was incubated for 1–2 h at 23 or 37°C under aerobic conditions. Ferrous sulfate (FeSO4) and ascorbic acid were added immediately before the initiation of the incubation in rotating tubes containing 1.0–5.0 ml of the mixture. The pH of each mixture was measured at the beginning of the incubation. The incubation of 10 μCi of [1-14C]methionine (adjusted to 3.0 mM with cold methionine) with FeSO4 (5.0 mM) and EDTA (5.0 mM, pH 5.0) took place in a total volume of 0.4 ml for 30 min at 37°C.
The Institutional Review Board for Human Use of the University of Alabama at Birmingham approved the protocol, and informed consent was obtained from each subject. Five healthy adults (two females and three males with a mean age of 41 years old) participated in the study. Their folate, vitamin B12 and vitamin B6 status, assessed by the analyses of plasma and red-cell folate and plasma vitamin B12 and pyridoxal-5′-phosphate, was normal (data not shown), and their fasting plasma tHcy concentrations were lower than 10 μ M. After overnight fasting, they ingested methionine (0.17 mmol (25 mg)/kg of body weight) with 100 ml of water. Blood samples (< 4.0 ml each time) were obtained using tubes containing sodium heparin (Beckton Dickinson, Rutherford, NJ, USA) immediately before and 3, 6, 9 and 24 h after the methionine ingestion, and were kept at 4°C until the separation of plasma. Plasma was stored at −70°C until analyses. Subjects ate a low-methionine meal (300 g of canned boiled corn and 270 g of vegetable soup) shortly after the 3-h blood collection (around noon). After the 9-h blood collection, they were asked to eat an evening meal low in folate and iron (no green vegetables and meat). A 24-hour blood sample was obtained after overnight fasting. After at least a 7-day washout period, the protocol was repeated using the identical dose of methionine immediately followed by an independent dose of FeSO4 (13 μmol (3.7 mg)/kg of body weight) with 50 ml of water.
The study design permitted each subject to serve as her/his own control when evaluating the effect of iron on the area-under-the-curve (AUC) of tHcy concentrations (above baseline (0 h)) after the methionine ingestion. Such a design also minimized individual variations in vitamin and mineral nutritional status, including iron status, at the time of the test and precluded the necessity of the assessment of iron status of each subject. Mean AUCs with and without iron were compared by the paired-Student's t-test.
An high-pressure liquid chromatography (HPLC)-fluorescent detection method was used to measure Hcy concentration in each incubation mixture or plasma, and the coefficient of variation for the Hcy assay using pooled human plasma is 8% in our laboratory (Tamura et al., 1996, 1998). The assay procedure for [1-14C]Hcy was similar to the HPLC-fluorescence method for non-radioactive Hcy with the exception of the use of proportionally smaller amounts of reagents with higher concentrations to avoid excess dilutions, which would have interfered with radioactivity detection. The fluorescence of the eluent was monitored and fractions were collected for radioactivity counting.
The in vitro chemical conversion of methionine into Hcy occurred and was iron-dependent, and the pH optimum was between 4.5 and 6.0 (Figure 1). The conversion of S-adenosylhomocysteine and cystathionine into Hcy also required iron with a pH optimum of about 6.0 (Figure 2). The formation of Hcy from cystathionine was greater than that from S-adenosylhomocysteine under identical conditions. The conversion into Hcy was low for all thioethers at pH 2–3, and no Hcy was detected, when iron was eliminated from the reactions (Figures 1 and 2). The detection limit for the assay was about 0.05 μ M Hcy.
The addition of 1.0 mM ascorbate to 5 mM citrate with 1.0 mM FeSO4 resulted in a 23% increase in the amount of Hcy formed from 5.0 mM methionine at pH 5.5 (2 h at 37°C). Under identical conditions, but substituting EDTA for citrate, there was a 103% increase in the amount of Hcy formed, suggesting that iron-redox cycling is involved in this reaction (Aisen et al., 1990). Without the addition of ascorbate, the formation of Hcy from methionine was not different between citrate and EDTA solutions.
In the experiment to confirm that the fluorescent peak represents the derivative of Hcy, [1-14C]-methionine was used. The peaks of radioactivity and the fluorescent derivative of Hcy coeluted from the HPLC. Both radioactivity and fluorescence analyses indicated that methionine was converted into Hcy at the rate of 0.3–0.4% per hour at pH 5.0.
The changes in plasma tHcy concentrations after the oral methionine loading with and without a dose of iron in humans were measured (Figure 3). The mean AUC after the loading of methionine and iron together [90±19 (s.d.) μ M × h] was 45% greater than that after the loading of methionine alone (62±16 μ M × h) in five subjects, and this difference was significant (P< 0.025, paired-Student's t-test).
The in vitro Hcy formation from thioethers was dependent upon the iron concentration, the pH, the type of chelator and thioethers used (Figures 1 and 2). No detectable Hcy was formed without the addition of iron. Although the mechanism of the chemical reactions presented here, including the pH dependency, is unknown and may be quite complicated, this is the first documentation of iron-dependent Hcy formation from thioethers. One weakness of our in vitro experiments is that the concentrations of iron and thioethers are higher than those found in vivo. However, we had to use these concentrations due to the low sensitivity of our Hcy-detection method.
Our findings of the formation of Hcy from methionine may be analogous to cleavage of oxygen ethers catalyzed by Lewis acids (March, 1968) or may involve the hydroxyl free radical generated by iron-redox cycling, which is stimulated by ascorbate and EDTA (Aisen et al., 1990). Ubbink et al. (1992) reported that plasma samples separated from EDTA-containing whole blood stored at room temperature showed a greater increase in plasma tHcy than samples containing sodium fluoride. Although Hcy is exported from erythrocytes if whole blood samples are left at room temperature, the difference obtained with EDTA and sodium fluoride cannot be explained by this mechanism. Therefore, the findings of Ubbink et al. (1992) are consistent with our data that the iron-dependent Hcy formation more readily occurs in the presence of EDTA, which stimulates iron-redox cycling than with a non-iron chelator such as sodium fluoride.
In the human study, loading of methionine and iron together resulted in a significantly greater AUC for plasma tHcy than that of methionine alone (Figure 3). We used only 25 mg of methionine/kg body weight, which is one-fourth the oral doses that are generally used for methionine (100 mg/kg of body weight) loading test (Ditschield et al., 2005). This dose was equivalent to the methionine content of 200–300 g of meat, and this amount of daily intake is not uncommon (Pennington, 1998). Although the amount of iron used is 20–200 times greater than that found in the same portions of meat (Pennington, 1998), this level of iron intake could readily be achieved by taking commercial iron or multivitamin/mineral tablets.
To our knowledge, there have been only a limited number of human studies evaluating the relationship between iron status and plasma tHcy concentrations, and these provided conflicting findings. Daher and Van Lente (1995) reported a positive association between plasma tHcy and serum iron concentrations. Recently, Mattioli et al. (2005) also reported a significant positive correlation between serum iron and plasma tHcy concentrations in young males with acute myocardial infarction. These findings support the idea that plasma tHcy may in part reflect the amount of iron available for iron-dependent Hcy formation. Shimakawa et al. (1997) reported a negative association between plasma tHcy and dietary iron intake, although they did not examine this association using blood indices of iron status. Recently, Zheng et al. (2005) reported that plasma tHcy concentrations were similar in high- and low-frequency blood donors, whereas serum ferritin concentrations were significantly lower in high-frequency donors. Zheng et al. (2006) also showed a slight higher increase in plasma tHcy from baseline after an iron sucrose plus methionine load compared to methionine load alone. In contrast, Facchini and Saylor (2002) reported iron depletion in patients with diabetes or impaired glucose tolerance did not reduce fasting plasma tHcy.
The following in vivo site(s) can be considered for the formation of Hcy from thioethers with an iron dose. A large quantity of free iron is generated by the degradation of hemoglobin, myoglobin and ferritin in the reticuloendothelial system (Bothwell et al., 1995). For example, a high amount of free iron is produced by the lysosomal digestion of hemoglobin and ferritin (Harrison and Arosio, 1996; Ponka et al., 1998). Petrat et al. (2001) found the highest concentrations (about 16 μ M) in the endosomal/lysosomal compartment of intact cells. The acidic environment of the stomach also has a possibility for generating free iron (Nalini et al., 1992), and the concentration could be high when ingesting foods rich in iron, such as red meat. We postulate that such free iron is constantly exposed to thioethers, and the iron-dependent chemical reactions occur ceaselessly throughout the body. Thus, we believe that the amount of Hcy formed by such reactions and transported out of cells into the circulation is significant, even though the yield of Hcy from thioethers was relatively low as shown in our in vitro study.
It is worth noting that findings in the literature suggest, although controversial, that an association exists between increased body iron stores and increased risk for cardiovascular disease (Roest et al., 1999), similar to the association between hyperhomocysteinemia and this disease. Sullivan (2006) postulated that iron stores influence vascular endothelial function and that elevated plasma tHcy is an iron-dependent phenomenon, although his proposed mechanism is different from that proposed us. On the basis of our findings, it may be reasonable to postulate that plasma tHcy concentration is affected by the amount of iron available to react with thioethers, and reflects, in part, body iron stores that are generally positively associated with the amount of free iron. The association between hyperhomocysteinemia and cardiovascular disease stems largely from epidemiological studies (Clarke et al., 1991; Refsum et al., 1998), and it is disappointing to see that the mechanism of the association remains unsolved even after about 15 years of extensive research. In the last several years, skepticism concerning the causal association to hyperhomocysteinemia for cardiovascular disease has surfaced (Brattström and Wilcken, 2000), and there is even dampened enthusiasm that lowering Hcy will decrease the disease risk (Davey Smith and Ebrahim, 2005). Furthermore, the majority of recent clinical trials have failed to demonstrate that the supplementation of B vitamins, including folic acid, reduces the risk of cardiovascular disease despite a reduction in plasma tHcy (Liem et al., 2003; Toole et al., 2004; Lange et al., 2004; Bønna et al., 2006; HOPE 2 Investigators, 2006). Elevated tHcy may be a surrogate for yet-to-be-identified clinical conditions or a reaction to pre-existing conditions, and lowering circulating tHcy with B-vitamin supplementation appears to have no significant effect on the clinical course. Perhaps we should carefully examine the cause of elevated Hcy in patients with OVD. We propose a hypothesis that plasma tHcy represents, to a certain extent, free iron in the body. Further investigations are warranted to evaluate critically the association between plasma tHcy and the amount of free iron or body iron stores in a large population.
Aisen P, Cohen G, Kang JO (1990). Iron toxicosis. Inter Rev Exp Pathol 31, 1–46.
Bønna KH, Njølstad I, Ueland PM, Schirmer H, Tverdal A, Steigen T et al. (2006). Homocysteine lowering and cardiovascular events after acute myocardial infarction. N Engl J Med 354, 1578–1588.
Bothwell TH, Charlton RW, Motulsky AG (1995). Hemochromatosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Disease, 7th edn, New York: McGraw-Hill. pp 2237–2269.
Brattström L, Wilcken DEL (2000). Homocysteine and cardiovascular disease: cause or effect? Am J Clin Nutr 72, 315–323.
Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B et al. (1991). Hyperhomocysteinemia: an independent risk factor for vascular disease. N Eng J Med 324, 1149–1155.
Daher R, Van Lente F (1995). Relationship of increased homocysteine with copper, iron, and zinc concentrations in serum. Irish J Med Sci 164 (suppl. 15), 21 (abstract).
Davey Smith G, Ebrahim S (2005). Folate supplementation and cardiovascular disease. Lancet 366, 1679–1681.
Ditschield B, Fünfstück R, Busch M, Schubert R, Gerth J, Jahreis G (2005). Effect of L-methionine supplementation on plasma homocysteine and other free amino acids: a placebo-controlled double-blind cross-over study. Eur J Clin Nutr 59, 768–775.
Facchini FS, Saylor KL (2002). Effect of iron depletion on cardiovascular risk factors. Studies in carbohydrate-intolerant patients. Ann NY Acad Sci 967, 342–351.
Harrison PM, Arosio P (1996). The ferritins: molecular properties, iron storage function and cellular regulation. Biochim Biophys Acta 1275, 161–203.
Heart Outcomes Prevention Evaluation (HOPE) 2 Investigators (2006). Homocysteine lowering with folic acid and B vitamins in vascular disease. N Engl J Med 354, 1567–1577.
Lange H, Suryapranata H, De Luca G, Böner C, Dille J, Kallmayer K et al. (2004). Folate therapy and in-stent restenosis after coronary stenting. N Eng J Med 350, 2673–2681.
Liem A, Reynierse-Buitenwerf GH, Zwinderman AH, Jukema JW, van Veldhuisen DJ (2003). Secondary prevention with folic acid: effects on clinical outcomes. J Am Coll Cardiol 41, 2105–2113.
March J (1968). Advanced organic chemistry: reactions, mechanisms, and structure. New York: McGraw-Hill. pp 344–345.
Mattioli AV, Bonetti L, Zennaro M, Bertoncelli P, Mattioli G (2005). Acute myocardial infarction in young patients: nutritional status and biochemical factors. Int J Cardiol 101, 185–190.
Nalini S, Ramakrishna BS, Mohanty A, Balasubramanian KA (1992). Hydroxyl radical formation in human gastric juice. J Gastroenterol Hepatol 7, 497–501.
Pennington JAT (1998). Bowes & Church's food values of portion commonly used. Philadelphia, PA: Lippincott-Raven. pp 105–125.
Petrat F, de Groot H, Rauen U (2001). Subcellular distribution of chelatable iron: a laser scanning microscopic study in isolated hepatocytes and liver endotherial cells. Biochem J 356, 61–69.
Ponka P, Beaumont C, Richardson DR (1998). Function and regulation of transferrin and ferritin. Sem Hematol 35, 35–54.
Refsum H, Ueland PM, Nygård O, Vollset SE (1998). Homocysteine and cardiovascular disease. Annu Rev Med 49, 31–62.
Roest M, van der Schouw YT, de Valk B, Marx JJM, Templeman MJ, de Groot PG et al. (1999). Heterozygosity for a hereditary hemochromatosis gene is associated with cardiovascular death in women. Circulation 100, 1268–1273.
Shimakawa T, Nieto FJ, Malinow MR, Chambless LE, Schreiner PJ, Szklo M (1997). Vitamin intake: a possible determinant of plasma homocyst(e)ine among middle-aged adults. Ann Epidemiol 7, 285–293.
Stekol JA (1957). Preparation and determination of sulfur amino acids and related compounds. Method Enzymol 3, 578–600.
Sullivan JL (2006). Is homocysteine an iron-dependent cardiovascular risk factor? Kidney Int 69, 642–644.
Tamura T, Bergman SM, Morgan SL (1998). Homocysteine, B vitamins, and vascular-access thrombosis in patients treated with hemodialysis. Am J Kidney Dis 32, 475–481.
Tamura T, Johnston KE, Bergman SM (1996). Homocysteine and folate concentrations in blood from patients treated with hemodialysis. J Am Soc Nephrol 7, 2414–2418.
Toole JF, Malinow MR, Chambless LE, Spence JD, Pettigrew LC, Howard VJ et al. (2004). Lowering homocysteine in patients with ischemic stroke to prevent recurrent stroke, myocardial infarction, and death. The Vitamin Intervention for Stroke Prevention (VISP) randomized controlled trial. JAMA 291, 565–575.
Ubbink JB, Vermaak WJH, van der Merwe A, Becker PJ (1992). The effect of blood sample aging and food consumption on plasma total homocysteine levels. Clin Chim Acta 207, 119–128.
Zheng H, Cable R, Spencer B, Votto N, Katz SD (2005). Iron stores and vascular function in voluntary blood donors. Arterioscler Thromb Vasc Biol 25, 1577–1583.
Zheng H, Huang X, Zhang Q, Katz SD (2006). Iron sucrose augments homocysteine-induced endothelial dysfunction in normal subjects. Kidney Int 69, 679–684.
Contributors: Both authors (JEB and TT) contributed in designing and performing the experiments and writing the manuscript.
About this article
Cite this article
Baggott, J., Tamura, T. Iron-dependent formation of homocysteine from methionine and other thioethers. Eur J Clin Nutr 61, 1359–1363 (2007) doi:10.1038/sj.ejcn.1602665
- iron-dependent chemical reactions
Vitamin D Supplementation and Nordic Walking Training Decreases Serum Homocysteine and Ferritin in Elderly Women
International Journal of Environmental Research and Public Health (2018)
Amino Acids (2018)
FEBS Letters (2016)