Reduction in plasma total homocysteine through increasing folate

European Journal of Clinical Nutrition (2003) 57, 483–489 ß 2003 Nature Publishing Group All rights reserved 0954–3007/03 $25.00 www.nature.com/ejcn

ORIGINAL COMMUNICATION Reduction in plasma total homocysteine through increasing folate intake in healthy individuals is not associated with changes in measures of antioxidant activity or oxidant damage SJ Moat1,2, MH Hill1, IFW McDowell2, CH Pullin2, PAL Ashfield-Watt2, ZE Clark2, JM Whiting2, RG Newcombe3, MJ Lewis2 and HJ Powers1* 1 The Centre for Human Nutrition, University of Sheffield, The Northern General Hospital, UK; 2Cardiovascular Sciences Research Group, Wales Heart Research Institute, University of Wales College of Medicine, Cardiff, Wales, UK; and 3Department of Computing and Medical Statistics, University of Wales College of Medicine, Cardiff, Wales, UK

Background: Various mechanisms have been proposed to explain the association between plasma total homocysteine (tHcy) and risk of cardiovascular disease, including oxidative activity of homocysteine. Objective: To explore the putative role of reactive oxygen species in the association between plasma tHcy and risk of cardiovascular disease in healthy individuals. Design: A double-blind, placebo-controlled crossover intervention to increase folate intake through diet (increased consumption of folate-rich foods) and supplement (400 mg folic acid) was carried out in 126 healthy men and women. Measurements were made of antioxidant activity in red blood cells and plasma, and products of oxidant damage in plasma. Results: Diet and supplement-based interventions led to an increase in measures of folate status and a reduction in plasma tHcy. This was not associated with any significant change in measures of antioxidant activity (plasma and red blood cell glutathione peroxidase activity and red blood cell superoxide dismutase activity) or oxidant damage (plasma malondialdehyde), although an improvement in plasma total antioxidant capacity just failed to reach significance. Conclusions: In healthy individuals lowering plasma tHcy does not have any functional implications regarding oxidative damage. European Journal of Clinical Nutrition (2003) 57, 483 – 489. doi:10.1038=sj.ejcn.1601554 Keywords: homocysteine; folate; oxidant damage; antioxidant activity; cardiovascular disease

Introduction Risk factors for cardiovascular disease extend beyond those conventionally considered to be significant. Plasma total homocysteine (tHcy) has been suggested as a risk factor independent of hyperlipidaemia, diabetes, hypertension and smoking. The risk increases progressively with increased

*Correspondence: HJ Powers, The Centre for Human Nutrition, The University of Sheffield, The Northern General Hospital, Sheffield S5 7AU, UK. E-mail: [email protected] Received 20 November 2001; revised 14 June 2002; accepted 18 June 2002

homocysteine levels (Boushey et al, 1995; Boston et al, 1997). Hyperhomocysteinaemia can result from genetic and dietary factors which influence the transsulphuration or remethylation pathways of homocysteine metabolism (Mudd et al, 1995; Verhoef et al, 1996). Of these, the most important to the general population are homozygosity for a relatively common mutation of 5,10-methylene tetrahydrofolate reductase (C677T MTHFR; Todesco et al, 1999), and inadequate intake of folate. Folate supplementation has been shown to be effective in reducing plasma total homocysteine (tHcy) concentrations (Homocysteine Lowering Trialists Collaboration, 1998). Even plasma tHcy concentrations falling in current reference ranges for normality are responsive to folate supplementation (Jacques et al, 1999). As there appears to be a continuum of risk this is of considerable relevance to the general population. However, whether the epidemiolo-

Plasma total homocysteine, oxidative damage and antioxidant activity SJ Moat et al

484 gical evidence for a link between elevated plasma homocysteine and risk of cardiovascular disease has any causative basis is not yet understood. Various mechanisms have been proposed for the association between plasma tHcy and risk of cardiovascular disease. One such mechanism invokes the potentially pro-oxidative potential of homocysteine, and argues that the increasing plasma concentrations of this amino acid are associated with increased oxidative stress and ensuing tissue damage (Bellamy & McDowell, 1997). Results from in vitro studies are generally supportive of a pro-oxidative role of homocysteine (Hirano et al, 1994; Starkebaum & Harlan, 1986), but the relevance of these observations to conditions in vivo are not known. Relatively few studies have explored this role for homocysteine in vivo and these have concentrated almost exclusively on events associated with greatly elevated plasma tHcy concentrations in patients with genetically determined homocystinuria, or with a transient acute elevation of plasma tHcy following an oral load of methionine (Moat et al, 2000, 2001; Wilcken et al, 2000). As concentrations of plasma tHcy increase to within the range seen in patients with homocystinuria, there is an associated increase in the activity and protein concentration of the plasma isoform of the antioxidant enzyme glutathione peroxidase (Moat et al, 2000). In the same study a similar but less striking relationship was observed between the activity of the red blood cell antioxidant enzyme superoxide dismutase and plasma tHcy, although this failed to reach significance (Moat et al, 2000). Wilcken et al (2000) recently confirmed a positive association between extracellular SOD activity and plasma tHcy in patients with homocystinuria. These results may reflect an adaptive response to an oxidative challenge posed by elevated plasma tHcy. An inverse relationship between a measure of plasma antioxidant activity and plasma tHcy has also been reported (Moat et al, 2001). An impaired flow-mediated dilatation following hyperaemia is a feature of homocystinuria, also of acute elevations in plasma tHcy following methionine loading. The fact that this impaired function can be corrected by the antioxidant ascorbic acid (Bonham et al, 2000) is consistent with the proposal that the effect of homocysteine on vascular function may be mediated by reactive oxygen species (ROS). This study explores the putative role for ROS in the association between plasma tHcy and cardiovascular risk in apparently healthy individuals. Specifically, the study sought to evaluate the effect of homocysteine-lowering, through increased folate intake, on a range of measures of oxidant stress and antioxidant activity in relation to plasma tHcy and vascular function in adults carrying different genotypes for C677T MTHFR.

Subjects and methods Protocol Healthy men and women between 18 and 65 y of age were recruited from workplaces and blood donor sessions European Journal of Clinical Nutrition

and screened for C677T MTHFR genotype (Clark et al, 1998). Exclusion criteria were hypertension (diastolic > 100 mmHg), diabetes, smoking, supplementation with folic acid, vitamins B6 or B12 and pregnancy. Ethical approval was obtained from the local Research Ethics Committee, Bro Taf Health Authority. Forty-two subjects of each genotype (CC, CT or TT), were recruited to a Latin Square crossover 34-month intervention study, with informed consent. Details of the interventions to lower homocysteine are reported elsewhere (Ashfield-Watt et al, 2002). The focus of this paper is the relationship between homocysteine and measures of oxidant stress and antioxidant activity. Briefly, each subject undertook three interventions of 4 months, in random order, being: 1

2

3

Normal diet þ placebo tablet daily ¼ ‘control’. Subjects were asked to consume their usual diet but to avoid folate-fortified foods and to take a placebo capsule daily. Naturally folate-enriched diet ¼ ‘enriched diet’. Subjects were encouraged to increase their consumption of specific folate-rich foods to achieve a total intake of 400 mg folate=day. Subjects were advised to consume a bowl of folate-fortified cereal plus three slices of folate-fortified bread, daily, as well as increasing their intakes of naturally folate-rich foods. Normal diet þ 400 mg folic acid ¼ ‘supplement’. Subjects were asked to consume their normal diet but avoiding folate-fortified foods, in addition to taking a capsule containing 400 mg folic acid, daily.

The study was double-blind for interventions 1 and 3 but single-blind for intervention 2, which just involved a change of diet. On entry into the study (baseline) and after each of the three dietary periods subjects visited the study clinic. On each occasion a fasting blood sample was collected, processed immediately and stored appropriately for biochemical measurements to be made later. Vascular endothelial function was assessed by measuring flow mediated dilatation of the brachial artery following hand hyperaemia. Only the data relating to measures of antioxidant activity and oxidant stress are reported here. Details of the dietary methodology and effects of interventions on endothelial function are reported elsewhere (Ashfield-Watt et al, 2002; Pullin et al, 2002).

Blood sampling Following an overnight fast, venous blood was sampled into vacutainers containing EDTA (for plasma tHcy and 5-methyltetrahydrofolate (5 MeTHF) and lithium=heparin (for plasma folate, total antioxidant capacity (TAOC), glutathione peroxidase activity, malondialdehyde (MDA), retinol and alpha tocopherol, b-carotene, ascorbic acid and erythrocyte glutathione peroxidase and superoxide dismutase activities). Samples were centrifuged within 10 min and the plasma removed. Plasma samples for analysis of ascorbic acid were


485 treated with 9 vol of 5% metaphosphoric acid to produce a deproteinized extract. Deproteinized plasma extract and plasma were stored at 7 70 C until assayed. Erythrocytes from the lithium heparin tubes were washed three times with an equal volume of saline (0.9%), resuspended in an equal volume of distilled water and stored at 7 70 C

Biochemical analyses Plasma tHcy was measured by immunoassay using an Abbott IMX instrument (Donnelly & Pronovost, 2000). Plasma 5MeTHF was measured by HPLC with fluorescence detection (Loehrer et al, 1996). Plasma MDA was measured by HPLC according to the method described by Nielsen et al (1997). Plasma a-tocopherol, retinol and b-carotene were analysed by HPLC with UV detection following extraction with heptane containing BHT (Thurnham et al, 1988). Plasma total ascorbic acid was measured spectrofluorometrically using a Cobas Bio Autoanalyser, by the method of Vuilleumier and Keck (1989). Plasma TAOC (Miller et al, 1993) and both plasma and erythrocyte GSHPx activities were measured using the Cobas Bio-Autoanalyser (Roche Diagnostica); Paglia et al, 1967) with commercially available kits (Randox Laboratories). Determination of erythrocyte SOD activity was performed using a modification of the method of Marklund and Marklund (1974) that was adapted for use on the Cobas Bio-Autoanalyser. Standard calibration curves were prepared from purified bovine erythrocyte SOD (Sigma Chemical Co). Erythrocyte GSHPx and SOD activity was expressed as a function of haemoglobin (Hb) concentration (U=g Hb). Hb was determined using the cyanmethaemoglobin method (Crosby & Houchin, 1957), which was adapted for the Cobas Bio Auto-Analyser using cyanmethaemoglobin standards (57.2 mg=100 ml). Interbatch coefficients of variation were 5.3% (tHcy), 5.8% (5-MeTHF), 2.7% (TAOC), 8.8% (MDA), 5.5% (plasma GSHPx), 3.3% (vitamin C), 8.2% (retinal), 7.3% (a-tocopherol), 23%(b-carotene) 3.4% (erythrocyte SOD) and 6.3% (erythrocyte GSHPx).

Data handling Baseline biochemical data were analysed according to genotype, using a one-way ANOVA. Response to the intervention was explored using a three-way ANOVA, using data from each of the three treatment phases of the trial but excluding baseline data. The analysis was modelled on subject, visit and treatment and combined data for all genotypes.

Results Baseline characteristics of subjects recruited to the study Forty-two subjects from each genotype group were randomised to dietary intervention. Twenty subjects dropped out of the study before completion. Mean age ( þ s.d.) at recruitment according to genotype were 40 (11), 36 (12) and 42 (12)

for TT, CT and CC respectively. The numbers of males recruited were 21, 23 and 29 for TT, CT and CC respectively.

Baseline antioxidant biochemistry Table 1 shows baseline biochemistry for all subjects recruited to the study, according to genotype. There were no significant effects of genotype on measures of antioxidant activity or oxidant damage at baseline. Rank correlations between flow-mediated dilatation and all biochemical variables at baseline were carried out and none approached statistical significance. One way ANOVA revealed the predicted gradient effect of genotype on baseline plasma tHcy (P < 0.0001), and 5-Me THF (P ¼ 0.006).

Effects of the interventions A three-way ANOVA was applied to each variable, using data from the three treatment phases of the trial, excluding baseline data (Table 2). The analysis models on subject, visit and treatment and includes subjects of all genotypes together. The effect of treatment was highly significant for 5-Me THF (P < 0.001) and plasma tHcy (P < 0.0001), significant for a-tocopherol (P ¼ 0.019), and approached significance for plasma total antioxidant capacity (P ¼ 0.055). 5-Me THF concentrations were doubled following the folate-rich diet (P < 0.001) and the folate supplement (P < 0.001). Concentrations of a-tocopherol were higher at baseline than at any other time (P < 0.05). Mean plasma TAOC was higher following both the folate-rich diet and the folate supplement but the difference did not reach statistical significance (P ¼ 0.055). There was no evidence that genotype influenced the response to intervention of measures of antioxidant activity or oxidant damage.

Discussion Diet and supplement-based interventions to increase folate intake led, as predicted, to both an increase in folate status and a significant reduction in plasma homocysteine. This was not associated with any significant change in measures of either antioxidant activity or oxidative damage, although an improvement in plasma total antioxidant capacity just failed to reach statistical significance. Effects of the intervention on folate status and homocysteine levels are discussed in depth elsewhere (Ashfield-Watt et al, 2002).

Effect on antioxidant activity The reduction in plasma total homocysteine in response to the folate intervention was associated with a small but not significant increase in plasma total antioxidant capacity. This may or may not be causal. The measure of antioxidant capacity used in this study is a measure of the ability of plasma constituents to act in synergy to quench a radical species generated in vitro. Several plasma components will European Journal of Clinical Nutrition


486 Table 1 Baseline biochemistry for all subjects recruited. There were no significant effects of genotype on measures of antioxidant activity or damage. One-way ANOVA showed the predicted gradient of genotype on baseline plasma tHcy (P < 0.0001) and 5-MeTHF (P ¼ 0.006) Variable Plasma total homocysteine (mmol=l)

Plasma 5-Me THF (nmol=l)

Plasma total antioxidant capacity (nmol=l)

Plasma malondialdehyde (mmol=l)

Plasma glutathione peroxidase (U=l)

RBC glutathione peroxidase (U=gHb)

RBC superoxide dismutase (U=gHb)

Plasma a-tocopherol (mmol=l)

Plasma ascorbic acid (mmol=l)

Plasma retinol (mmol=l)

Plasma b-carotene (mmol=l)

n

MTHFR genotype

Mean

Standard deviation

42 42 42 126 41 41 39 121 41 41 40 122 41 41 40 122 41 40 40 121 41 41 39 121 40 40 38 118 38 41 36 115 40 39 38 117 38 41 36 115 38 41 36 115

TT CT CC All TT CT CC All TT CT CC All TT CT CC All TT CT CC All TT CT CC All TT CT CC All TT CT CC All TT CT CC All TT CT CC All TT CT CC All

12.5 9.3 8.8 10.19 17.44 21.27 28.79 22.40 1.49 1.50 1.52 1.50 2.54 2.40 2.39 2.44

5.7 2.5 2.4 4.19 12.85 14.71 19.09 16.27 0.10 0.10 0.11 0.10 0.79 0.76 0.52 0.70 146.3 122.8 144.6 138.4 16.07 16.79 11.31 14.87 185 222 202 202 7.9 10.1 8.6 8.9 17.49 35.12 19.98 25.44 0.45 0.56 0.53 0.52 0.186 0.341 0.332 0.293

contribute to this activity including uric acid, albumin, thiols and a-tocopherol (Miller et al, 1993). Plasma thiols have been shown to contribute to various measures of antioxidant activity (Moat et al, 2001; Miller et al, 1993; Wayner et al, 1987) and there may have been changes in the redox state of plasma thiols following a reduction in plasma total homocysteine. There is current interest in the potential antioxidant role for folate, but a doubling in plasma folate was associated with such a small increase in TAOC that it is unlikely that there is a direct causative association. The effect on TAOC was small and may not be functionally significant. The folate supplementation was associated with an increase in plasma a-tocopherol relative to the other two treatment regimens. a-tocopherol was actually lower than European Journal of Clinical Nutrition

901.7 915.4 920.7 46.32 46.75 45.16 46.09 1082 1067 1049 1066 29.8 29.9 28.8 29.6 52.78 61.20 55.10 56.31 1.91 1.85 1.81 1.85 0.288 0.293 0.288 0.289

baseline following all intervention periods. It is difficult to explain these observations other than in terms of a small modification of the dietary intake of vitamin E or lipid. Three-way ANOVA showed no consistent effect of the treatments on antioxidant concentrations in the plasma. There are therefore no grounds for concern that changes in dietary antioxidants might be masking any observed treatment effects on dilatation. There was no evidence for a fall in plasma malondialdehyde in association with a reduction in plasma tHcy, suggesting that at the levels of homocysteine observed in this study homocysteine-related oxidative damage to lipid did not occur. It has been hypothesized that elevated plasma homocysteine increases the risk of cardiovascular disease


487 Table 2 Effects of treatment. 5-Me THF concentrations were doubled following the folate rich diet (P < 0.001) and the folate supplement (P < 0.001). Plasma tHcy fell following the folate-rich diet and the folate supplement (P < 0.001). Concentrations of a-tocopherol were higher at baseline than at any other time (P < 0.05). Mean plasma TAOC was higher following both the folate-rich diet and the folate supplement but the difference did not reach statistical significance (P ¼ 0.055) Treatment in preceding period Variable Plasma total homocysteine (mmol=l)

Plasma 5-Me THF (nmol=l)

Plasma total antioxidant capacity (mmol=l)

Plasma malondialdehyde (mmol=l)

Plasma glutathione peroxidase (U=l)

RBC glutathione peroxidase (U=gHb)

RBC superoxide dismutase (U=gHb)

Plasma ascorbic acid (mmol=l)

Plasma retinol (mmol=l)

Plasma a-tocopherol (mmol=l)

Plasma b-carotene (mmol=l)

n Mean s.d. n Mean s.d. n Mean s.d. n Mean s.d. n Mean s.d. n mean s.d. n Mean s.d. n Mean s.d. n Mean s.d. n Mean s.d. n Mean s.d.

through promoting the oxidative modification of low density lipoproteins (LDLs). There is however a lack of consensus regarding the potential pro-oxidative role of homocysteine towards LDL either in vitro or in vivo (Voutilainen et al, 1999, Blom et al, 1995). One group of workers (Halvorsen et al, 1996) has shown that at concentrations of homocysteine considered to increase CVD risk homocysteine may protect LDL against oxidative damage in vitro. We have recently confirmed this (Nakano, 2000). Similarly, no changes in the activities of circulating antioxidant enzymes were observed in association with a reduction in plasma homocysteine. We have previously reported a strong linear relationship between plasma glutathione peroxidase activity and plasma total homocysteine in samples from patients with homocystinuria. The increase in activity was explained at least in part by an increase in the concentration of circulating enzyme. We suggested that this reflected an adaptive response to an oxidant stress posed by elevated plasma homocysteine (Moat et al, 2000). Other groups have reported

Baseline

Enriched diet

Control

Supplement

126 10.19 4.19 121 22.40 16.27 122 1.50 0.10 122 2.44 0.70 121 920.74 138.39 121 46.09 14.9 118 1066 202.4 117 56.31 25.44 115 1.85 0.52 115 29.55 8.91 115 0.290 0.293

108 8.73 3.33 105 42.59 24.40 104 1.53 0.11 107 2.40 0.89 108 844.4 151.53 102 47.04 15.3 104 1100 200.1 105 62.31 20.46 108 1.86 0.43 108 25.62 7.15 108 0.318 0.320

110 10.85 6.93 109 24.11 14.73 107 1.50 0.11 109 2.29 0.85 108 847.6 125.3 109 45.93 13.85 106 1070 253.6 106 58.32 21.06 108 1.86 0.44 108 25.52 6.84 108 0.330 0.282

108 8.55 3.08 107 50.34 25.42 106 1.53 0.11 105 2.44 0.71 108 863.7 138.5 107 46.87 16.7 107 1089 251.4 104 61.44 21.57 104 1.84 0.48 104 26.93 8.71 104 0.315 0.272

an association between activity of extracellular SOD in vivo and plasma homocysteine, in patients with coronary artery disease (Wang et al, 1998) and erythrocyte SOD following methionine loading in obligate heterozygotes for CbS deficiency (Clarke et al, 1992). In each of these studies plasma tHcy reached levels substantially higher than observed in the study described here. The pro-oxidant hypothesis for homocysteine is based largely on results from in vitro studies. When exposed to free homocysteine at high (supraphysiological) concentrations, endothelial cells in culture show a loss of structural and functional integrity that can be mitigated by the presence of additional antioxidants (Starkebaum & Harlan, 1986; Blundell et al, 1996). The deleterious effects of homocysteine on endothelial cells in culture may be mediated by the increased production of superoxide radicals resulting from the autooxidation of homocysteine (Lang et al, 2000). However, these observations may not be relevant to events in vivo, where plasma total homocysteine concentrations are European Journal of Clinical Nutrition


488 much lower than those showing effects in vitro. In particular, the concentration of the reduced form of homocysteine, which autooxidizes to generate ROS, and which therefore clearly does have pro-oxidant potential, usually represents only about 1% of plasma total homocysteine (Andersson et al, 1993). In subjects with homocystinuria, however, among whom there is evidence of homocysteine-related oxidant stress and vascular damage, levels of the reduced form of free homocysteine may increase significantly (Moat et al, 1999). Plasma tHcy is reported to be a graded risk factor for cardiovascular disease, therefore, if homocysteine per se is the causative agent, any reduction in plasma tHcy, however modest, should reduce risk. We hypothesized that homocysteine exerts pro-oxidative effects in vivo and that lowering plasma homocysteine would cause a reduction in these effects which would be measurable as a change in anitoxidant activity and=or a fall in a measure of oxidatively damaged lipid. The modest nature of the increase in plasma antoxidant activity associated with homocysteinelowering may reflect the relatively low plasma mean tHcy concentration in the cohort at baseline. It has been argued that an impaired endothelial function associated with transient elevation in plasma tHcy is mediated by oxidative mechanisms (Kanani et al, 1999; Chambers et al, 1999). The fall in plasma tHcy in response to folate intervention in the study reported here, did not elicit any improvement in endothelial function (to be reported elsewhere) which is consistent with the failure to observe any convincing effects of homocysteine-lowering on antioxidant activity or oxidative damage. There has been recent interest in a potential antioxidant role for folate independent of its homocysteine-lowering effect. This has been demonstrated in experimental systems in vitro (Usui et al, 1999), and at a very high concentration in vivo (Wilmink et al, 2000; Verhaar et al, 1998; Nakano et al, 2002). There is no evidence from this study that physiological intakes of folate have a measurable antioxidant effect in vivo.

Conclusions The results of this study show that in healthy individuals lowering plasma tHcy does not have any functional implications regarding oxidative damage. There may be subtle changes in measures of oxidative damage not explored in this study, which might include damage to protein.

Acknowledgements This study was supported by a project grant from the Food Standards Agency of the UK (ANO506 and N05006).

European Journal of Clinical Nutrition

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