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Increased homocysteine and S-adenosylhomocysteine concentrations and DNA hypomethylation in vascular disease.

DNA methylation is an important epigenetic feature of DNA that plays a critical role in gene regulation (1) and has recently been implicated in chronic disease states, including atherosclerosis (2, 3). DNA methylation patterns are maintained by DNA methyltransferases (4-7), which use S-adenosylmethione (AdoMet) [5] as the methyl group donor. AdoMet is also the methyl donor in other cellular methyltransferase reactions, including in RNA and protein methylation, synthesis, and detoxification processes. As a result of the transfer of the methyl group, AdoMet is converted to S-adenosylhomocysteine (AdoHcy) and a decrease in the AdoMet/AdoHcy ratio is often taken as an indicator of reduced cellular methylation capacity (8-10). Under physiologic conditions, AdoHcy is hydrolyzed to Hcy and adenosine. This reaction is reversible, with a dynamic equilibrium that strongly favors AdoHcy synthesis rather than hydrolysis (8). For this reason, the removal of Hcy and adenosine is crucial, and disturbed metabolic pathways that lead to increased intracellular Hcy concentrations will also increase AdoHcy (10). AdoHcy binds to methyltransferases with higher affinity than does AdoMet, and as a potent inhibitor of most methylation reactions, including those of DNA methyltransferases, it potentially affects cell homeostasis (11).

Hyperhomocysteinemia is a well-established risk factor for vascular disease, but the underlying pathogenic mechanism remains poorly understood, although thoroughly studied. Most studies have been focused on the direct effect of Hcy, but it was recently suggested that a chronic increase in AdoHcy (as a result of the Hcy-mediated reversal of the AdoHcy hydrolase reaction) might have significant pathologic consequences (12). Furthermore, the role of epigenetic alterations has emerged as a crucial mechanism for regulating genes responsible for cell proliferation in atherosclerosis (3). The present study was undertaken to assess the possible association between increased Hcy and epigenetic alterations in vascular disease. Plasma total homocysteine (tHcy), AdoMet, and AdoHcy concentrations, as well as leukocyte DNA methylation status, were evaluated in vascular disease and control groups. The results support the hypothesis that the pathogenic role of hyperhomocysteinemia in vascular disease might be mediated by AdoHcy accumulation and DNA hypomethylation.

Materials and Methods


We studied 17 male atherosclerotic vascular patients [mean (SD) age, 54 (5.8) years] and 15 healthy male controls [mean (SD) age, 53 (6.9) years]. Cases were recruited among patients who had been admitted to hospital with a diagnosis of stroke (n = 8) or angiographically confirmed myocardial infarction (n = 9); samples were collected at least 3 months after the occurrence. Controls were selected among hospital employees whose lifestyle details (i.e., smoking, alcohol consumption, medication, physical exercise, and personal and family histories) and baseline laboratory measurements were established by use of standardized questionnaires and protocols. The criteria for inclusion in the control group were: normal hematology and liver/renal function tests and no history of vascular pathology. Exclusion criteria for both groups were metabolic, hepatic, or renal pathology; cancer; alcohol or drug abuse; and use of vitamin B6, vitamin B12, or folic acid supplements within the 2 previous months. Written informed consent was obtained from all participants, and the Local Ethical Committee approved the study.

Blood samples were collected from all participants after overnight fasting by venipuncture into EDTA-containing tubes. One tube was immediately placed on ice and centrifuged at 4000g for 10 min at 4[degrees]C. One plasma aliquot was stored at -20[degrees]C until tHcy quantification, whereas 1000 [micro]L was immediately deproteinized by adding 624 [micro]L of 100 g/L perchloric acid and then stored at -20[degrees]C until AdoMet and AdoHcy determinations. Hematologic analysis revealed no significant differences in the mononuclear vs polymorphonuclear cells ratios between the individuals of both groups. Genomic DNA was extracted from white blood cells according to standard phenol-chloroform procedures (13, 14) and then stored at 4[degrees]C until quantification of global DNA methylation.

tHcy, AdoMet, and AdoHcy measurements Plasma tHcy was quantified in the thawed aliquots by a specific immunoassay (IMx; Abbott Laboratories). For plasma AdoMet and AdoHcy quantification, the deproteinized samples were thawed and centrifuged, and the obtained supernatant was analyzed by stable-isotopedilution tandem mass spectrometry, as described previously in detail (15).


The global DNA methylation status was assessed by use of the cytosine extension assay (16). Briefly, 1 [micro]g of genomic DNA was digested overnight with a methylationsensitive endonuclease (HpaII), which left a single guanine overhang at unmethylated CpG sites. A second DNA aliquot was incubated without HpaII and served as background control. A single nucleotide extension with [[sup.3]H]dCTP (Amersham Biosciences) was then performed. Duplicate 10-[micro]L aliquots from each reaction were applied on Whatman DE-81 ion-exchange filters, dried, and washed three times with sodium phosphate buffer (pH 7.0) at room temperature. Filters were dried and processed for scintillation counting. Background radiolabel incorporation was subtracted from enzyme-treated samples, and the results were expressed as relative [[sup.3]H]dCTP incorporation/[micro]g of DNA. Incorporation of the radiolabel was considered proportional to the number of unmethylated (cleaved) sites in DNA. All samples were analyzed at the same time to minimize variation. The within-assay imprecision of the cytosine extension assay was <6%.


Data are presented as medians and interquartile ranges. The distributions of plasma tHcy, AdoMet, and AdoHcy and the relative [[sup.3]H]dCTP incorporation/[micro]g of DNA were positively skewed and logarithmically transformed before further statistical analysis. Differences between patient and control groups were evaluated with the independent-samples Student t-test. Correlations were determined with the Spearman method; P values were two-tailed. Statistical significance was set at P <0.05.


The median plasma tHcy, AdoMet, and AdoHcy concentrations, the AdoMet/AdoHcy ratios, and the global DNA methylation status in the studied population are presented in Table 1. Compared with controls, patients had significantly higher (P <0.01) plasma tHcy and AdoHcy concentrations, decreased (P <0.01) plasma AdoMet/ AdoHcy ratios, and lower (P <0.05) global DNA methylation status. We observed no significant differences in plasma AdoMet concentrations between the two groups. The variables displaying statistical difference were subdivided into quartiles, and the patients and controls were distributed accordingly, as shown in Fig. 1. More than 50% of the patients fell into the highest quartiles of plasma tHcy and AdoHcy concentrations and [[sup.3]H]dCTP incorporation/[micro]g of DNA (i.e., the lowest quartile of DNA methylation status) and into the lowest quartile of plasma AdoMet/AdoHcy ratios for the controls. When we considered the patients and controls together, we observed that the increase in plasma tHcy was significantly correlated with a parallel increase in plasma AdoHcy (r = 0.81; P <0.0001) and with a decrease in plasma AdoMet/AdoHcy ratios (r = -0.68; P <0.0001; Fig. 2).

We found no apparent association between plasma tHcy and AdoMet concentrations (data not shown). In addition, there was a significant correlation between DNA methylation status and plasma tHcy (r = 0.47, P <0.01) and plasma AdoHcy concentrations (r = 0.54; P <0.01; Fig. 3), but no correlation with plasma AdoMet/AdoHcy ratios (data not shown).


A growing body of evidence has documented the role of hyperhomocysteinemia as an independent vascular risk factor. However, the mechanisms through which increased plasma Hcy concentrations cause endothelial dysfunction and atherosclerosis remain elusive. At present, atherogenesis is believed to involve mainly gene function, such as epigenetic changes leading to alterations in gene expression and genomic integrity (2). Methylation is an important epigenetic feature of DNA, which is catalyzed by DNA methyltransferases with AdoMet as a methyl donor. Recently, it has been suggested that the pivotal biomarker of the association between hyperhomocysteinemia and vascular disease would not be Hcy itself, but increases in AdoHcy, secondary to the Hcy-mediated reversal of the AdoHcy hydrolase reaction. An increase in AdoHcy causes feedback inhibition of AdoMet-dependent methyltransferases, including the DNA methyltransferases (12, 17). The tissue-specific metabolic pathways are crucial in determining the relative sensitivity to alterations in AdoHcy concentrations (18). The demonstrated lack of cystathionine [beta]-synthase (19) and betaine-homocysteine methyltransferase (17) activity in endothelial cells may enhance their sensitivity to AdoHcy accumulation and, consequently, to DNA hypomethylation. Our findings are in line with the hypothesis that the pathogenic role of hyperhomocysteinemia in vascular disease might be caused by AdoHcy accumulation and DNA hypomethylation. However, because of the small size of the study sample, further larger studies are needed to confirm these findings.



In the present report, patients with vascular disease had significantly higher plasma tHcy and AdoHcy concentrations, decreased plasma AdoMet/AdoHcy ratios, and lower global DNA methylation status compared with controls. Significantly, >50% of the patients fell into the highest quartiles for plasma tHcy and AdoHcy concentrations and into the lowest quartiles for plasma AdoMet/AdoHcy ratios and DNA methylation status for the controls (Fig. 1). In addition, when patients and controls were considered together, the increase in plasma tHcy was highly correlated with a parallel increase in plasma AdoHcy (Fig. 2A) and with a decrease in plasma AdoMet/AdoHcy ratios (Fig. 2B), but not with plasma AdoMet. Moreover, plasma tHcy and AdoHcy were significantly correlated with the global DNA methylation status (Fig. 3). However, we observed no correlation between plasma AdoMet concentrations or AdoMet/AdoHcy ratios and the global DNA methylation status. Similar observations have also been reported by Yi et al. (20).

The intracellular AdoMet/AdoHcy ratio has been used as a predictor of cellular methylation capacity. A significant correlation between plasma and lymphocyte AdoMet/AdoHcy ratios has been reported (12). This observation suggests that the plasma AdoMet/AdoHcy ratio may be used as a reasonable predictor of the cellular methylation status. In the present study, the lack of correlation between the plasma AdoMet/AdoHcy ratio and DNA methylation status could seem unexpected. However, the authors of two recent studies in humans (20) and animals (18) claimed that intracellular AdoHcy is a more reliable biomarker for cellular methylation status. Our findings are in accordance with this observation.

In conclusion, the present study shows that patients with vascular disease have disturbed global DNA methylation status associated with increased plasma tHcy and AdoHcy concentrations. Whether these observations contribute to the pathogenic role of hyperhomocysteinemia in vascular disease needs to be further ascertained.

We gratefully acknowledge support from Dr. Adelaide Belo (Hospital Distrital de Beja). We also thank Fernanda Ramalho, Elisa Alves, and Amelia Pereira for expert sampling and routine biochemical determinations. This study was supported in part by a grant (Praxis XXI/BD/11383/97) from the F.C.T. (Fundacao para a Ciencia e Tecnologia) to Rita Azevedo e Castro. Henk Blom is supported by the Netherlands Heart Foundation (D97.021).

Received February 28, 2003; accepted May 23, 2003.


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Rita Castro, [1] Isabel Rivera, [1] Eduard A. Struys, [2] Erwin E.W. Jansen, [2] Paula Ravasco, [3] Maria Ermelinda Camilo, [3] Henk J. Blom, [4] Cornelis Jakobs, [2] and Isabel Tavares de Almeida [1] *

[1] Centro de Patogenese Molecular, Faculdade de Farmacia da Universidade de Lisboa, and 3 Centro de Nutricao e Metabolismo, Faculdade de Medicina da Universidade de Lisboa, 1649-039 Lisboa, Portugal.

[2] Metabolic Unit, Department of Clinical Chemistry, VU University Medical Center, 1081 HV Amsterdam, The Netherlands.

[4] Department of Paediatrics, University Medical Center St. Radboud, 6500 HB Nijmegen, The Netherlands.

[5] Nonstandard abbreviations: AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; and tHcy, total homocysteine.

* Address correspondence to this author at: Centro de Patogenese Molecular, Faculdade de Farmacia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-039 Lisboa, Portugal. Fax 351-217946491; e-mail
Table 1. Plasma tHcy, AdoMet, and AdoHcy concentrations and
global DNA methylation status in the studied population. (a)

 Patients (n = 17) Controls (n = 15)

tHcy, [micro]mol/L 10.4 (8.8-12.7) (b) 7.5 (6.5-8.9)
AdoMet, nmol/L 81.1 (63.0-107) 77.5 (67.0-96.0)
AdoHcy, nmol/L 16.9 (12.7-19.6) (b) 12.2 (10.2-16.9)
AdoMet/AdoHcy 4.8 (4.3-6.2) (b) 5.8 (5.3-6.9)
DNA methylation, 14 900 (9993-19 130) (c) 10 155 (6843-11 518)
 dpm/[micro]g (a)
 of DNA

(a) Values are medians and interquartile ranges.

(b) Statistically different (Student t-test) between patients
and controls: (b) P <0.01; (c) P <0.05.

(c) dpm, disintegrations per minute.
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Title Annotation:Molecular Diagnostics and Genetics
Author:Castro, Rita; Rivera, Isabel; Struys, Eduard A.; Jansen, Erwin E.W.; Ravasco, Paula; Camilo, Maria E
Publication:Clinical Chemistry
Date:Aug 1, 2003
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