Effect of fish oil on LDL oxidation and plasma homocysteine concentrations in health

Effect of fish oil on LDL oxidation and plasma homocysteine concentrations in health ALAIN PIOLOT, DENIS BLACHE, LUCIE BOULET, LOUIS JACQUES FORTIN, DENISE DUBREUIL, CAROLINE MARCOUX, JEAN DAVIGNON, and SUZANNE LUSSIER-CACAN MONTREAL, QUEBEC, CANADA, and DIJON, FRANCE

Oxidation of low-density lipoprotein (LDL) and hyperhomocysteinemia are believed to play a role in atherogenesis. Whether n-3 polyunsaturated fatty acids increase LDL susceptibility to oxidation or influence homocysteine (Hcy) metabolism has long been a subject of controversy. In this study, we evaluated the effect of 8 weeks of dietary supplementation with 6 g/day of fish oil (FO; 3 g of n-3 fatty acids) on plasma lipoproteins, in vitro LDL peroxidation, antioxidant status, and plasma Hcy concentrations in 16 normolipidemic subjects. FO rapidly and significantly (P < .01) decreased plasma total and very low density lipoprotein triglyceride concentrations and had no effect on LDL or high-density–lipoprotein cholesterol. The mean lag time before onset of Cu2ⴙ-induced LDL oxidation, as well as plasma and LDL ␣-tocopherol and ␤-carotene concentrations, was unchanged. However, changes in plasma aminothiol concentrations occurred during the study. Specifically, a progressive and significant increase in total Hcy plasma concentrations was observed (13.4% and 20% after 4 and 8 weeks, respectively; P < .01). Total glutathione concentrations were significantly higher after 8 weeks (P < .05). The tHcy increase was not associated with changes in plasma folate or vitamin B12 concentrations. However, concentrations of plasma nitric oxide metabolites (NOx ⴝ NO2 ⴙ NO3) were significantly higher than at baseline after 8 weeks of FO intake (74%; P < .01). Further, the changes in total Hcy and NOx plasma concentrations observed after 8 weeks of FO were found to be significantly correlated (r ⴝ .78, P < .001). With this study, we report for the first time the apparent interaction of n-3 fatty acids and nitric oxide on Hcy metabolism. (J Lab Clin Med 2003;141:41-9) Abbreviations: ANOVA ⫽ analysis of variance; CHD ⫽ coronary heart disease; DHA ⫽ docosahexaenoic acid; EDTA ⫽ ethylenediaminetetraacetate; EPA ⫽ eicosapentaenoic acid; FO ⫽ fish oil; Hcy ⫽ homocysteine; HDL ⫽ high density lipoprotein; HPLC ⫽ high-pressure liquid chromatography; LDL ⫽ low-density lipoprotein; NAME ⫽ NG-nitro-L-arginine-methyl ester; NO ⫽ nitric oxide; NOx ⫽ NO circulating oxidation products (nitrites and nitrates: NO2 ⫹ NO3); PBS ⫽ phosphate-buffered saline solution; PUFA ⫽ polyunsaturated fatty acid; SBD-F ⫽ ammonium-7-fluoro-benzo-2-oxa-1,3-diazole-4-sulfonate; TBARS ⫽ thiobarbituric acid–reactive substances; VLDL ⫽ very low density lipoprotein

he consumption of diets rich in fish or of FO supplements has been associated with a reduced risk of CHD and fatal myocardial infarction.1-4 This phenomenon has been attributed to the long-chain n-3 PUFAs in fish lipids. Not all studies, however, have

T

shown the beneficial effect of fish intake on death resulting from CHD or on the risk of coronary disease, and it may be that fish consumption is beneficial only in high-risk populations.5-8 Impressive evidence indicates that the peroxidation

From the Hyperlipidemia and Atherosclerosis Research Group of the Clinical Research Institute of Montreal and INSERM-U498 —Faculte´ de Me´decine, Universite´ de Bourgogne, Dijon. Supported in part by a grant from the Heart and Stroke Foundation of Quebec, a joint grant within the Canadian Institutes of Health Research/Research and Development program and Pfizer Canada (DOP40844), and by La Succession J. A. DeSe`ve. Alain Piolot was the recipient of a scholarship from Le Comite´ Franc¸ais de Coordination des Recherches sur l’Athe´roscle´rose et le Choleste´rol. Denis Blache was the recipient of a fellowship from Le Fonds de la Recherche en Sante´ du Que´bec as a visiting scientist.

Submitted for publication December 19, 2001; revision submitted September 13, 2002; accepted September 24, 2002. Reprint requests: S. Lussier-Cacan, PhD, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec, H2W 1R7, Canada; e-mail: [email protected]. Copyright © 2003 by Mosby, Inc. All rights reserved. 0022-2143/2003 $30.00 ⫹ 0 doi:10.1067/mlc.2003.3

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of LDL plays an important role in atherogenesis.9-11 The susceptibility of LDL to oxidation is influenced by its composition and structure.12 Because fatty acids of dietary origin are incorporated in LDL, intake of marine lipids that contain large amounts of long-chain n-3 PUFAs could have deleterious consequences as a result of the LDL enrichment with molecules that are highly susceptible to lipid peroxidation.13-15 However, the susceptibility of LDL to oxidation is dependent not only on its fatty-acid composition but on plasma and lipoprotein antioxidant concentrations. Among mechanisms involved in LDL oxidation, a role for sulfur-containing molecules has been reported in several studies.16-18 One such molecule is Hcy, and hyperhomocysteinemia is a well-known risk factor for CHD.19 We previously reported that moderately increased doses of FO (12 g/day ⫽ 6 g n-3 fatty acids), given as a dietary supplement in the treatment of subjects with type IV hypertriglyceridemia, enhanced the susceptibility of their LDL to oxidation.20 This effect was counteracted by the addition of the antioxidant probucol. We also found that although 12 weeks’ treatment with FO was associated in hypertriglyceridemic subjects with decreased plasma total Hcy concentrations, a trend toward increase was observed when probucol was added to FO.21 These results motivated the study being reported here, which was designed to investigate, this time in normolipidemic subjects, the effects of a lower dose of FO (6 g/day) on plasma lipid, lipoprotein, and total Hcy concentrations, as well as on ex vivo lipidperoxidation parameters. METHODS Subjects. Twenty healthy normolipidemic subjects (10 women) were originally recruited for a 4-week study. Of these, 16 accepted continued participation in a 4-week extension of the experimental period. In this article, we report the results obtained over 8 weeks in 16 subjects (the treated group, comprising 7 women and 9 men). The mean ⫾ SD age was 35 ⫾ 7 years (range 23-47). Selection criteria included reliability, regular eating habits, normal weight, and nonsmoking status. None of the selected subjects used any vitamins or dietary supplements and was taking no medication for at least 8 weeks before the start of and during the entire experimental period. A dietary evaluation was conducted by a dietitian at the beginning of the study, and participants were encouraged to maintain constant dietary habits and to pursue their normal activities throughout the study period. Food intake was verified with a 3-day self-report diary comprising two weekdays and one weekend day. The diary was completed during the experiment. The study was conducted between March and November of the same year. A control group of 16 healthy, normolipidemic, and untreated subjects (11 women), ranging in age from 24 to 56 years (38 ⫾ 12 years, mean ⫾ SD), who were advised not to make any

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lifestyle changes, contributed three consecutive monthly blood samples, which served as controls for a possible time effect on plasma lipoprotein and aminothiol concentrations. All subjects gave their informed consent, and the protocols were approved by the Ethics Committee of the Clinical Research Institute of Montreal. Procedure. The 16 treated subjects received 6 g/day of FO in the form of six capsules of SuperEPA (Bronson Pharmaceuticals, St Louis, Mo) over a period of 8 weeks. Each 1-g capsule provided 0.671 mg (1 IU) of RRR-␣-tocopherol, 300 mg of EPA, and 200 mg of DHA. This dose had been shown previously to significantly decrease triglyceride levels in hyperlipidemic individuals.22 Compliance with the drug regimen was verified through a count of unused capsules and confirmed with the plasma fatty-acid profile. Blood sampling. Fasting venous blood samples were drawn immediately before the study and after 2, 4, and 8 weeks of FO supplementation. Blood was collected in EDTAcontaining (1.5 mg/mL) evacuated tubes and cooled immediately; plasma and blood cells were separated by means of low-speed centrifugation (1800g for 15 minutes at 4°C). Plasma lipid and lipoprotein analyses were performed within 3 days of sampling. Several portions of plasma were stored at ⫺70°C for future analyses. Plasma lipid and lipoprotein determinations. Plasma lipoproteins were separated under standard conditions by means of a combination of ultracentrifugation (320,000g for 8 hours at 4°C) to isolate VLDL at d ⫽ 1.006 g/mL and heparin-manganese precipitation of the apolipoprotein B– containing lipoproteins in the d ⫽ 1.006 g/mL infranatant for measurement of LDL- and HDL-cholesterol concentrations in accordance with the Lipid Research Clinics protocol.23 Plasma and lipoprotein cholesterol and triglycerides were measured enzymatically with an automated autoanalyzer (Cobas Mira S; F. Hoffman-La Roche, Ltd, Diagnostica, Basel, Switzerland). For the oxidation study, we isolated LDL by means of sequential ultracentrifugation of plasma between d ⫽ 1.019 and d ⫽ 1.050 g/mL. Density solutions were prepared with potassium bromide and contained 1 mmol/L EDTA. The isolated LDL fraction was subjected to dialysis against PBS (pH 7.4) and 0.3 mmol/L EDTA at 4°C for 24 hours with at least three changes of the dialysis solution. LDL was aliquoted and kept under nitrogen at 4°C in the dark until analysis. Fatty-acid analyses. After extraction with chloroform/ methanol (2:1, vol/vol), the thin-layer chromatography–isolated plasma phospholipids or total LDL lipids were transmethylated with 14% boron trifluoride in methanol at 100°C for 90 minutes under a N2 atmosphere in accordance with the protocol of Morrison and Smith.24 The fatty-acid methyl esters were injected into a gas chromatograph (HP-5880A; Hewlett-Packard, Palo Alto, Calif) equipped with a 0.53 mm ⫻ 30 m Supelcowax 10 capillary column (Supelco, Mississauga, Ontario, Canada) and a flame ionization detector, with helium as the carrier gas. Peaks were identified against reference fatty acids (Supelco), and results are expressed as percentages of the sum of all identified peaks.

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Lipid-oxidation study. TBARS were measured with a spectrophotometric assay25; results are expressed as nanomoles per liter of malondialdehyde equivalents per milliliter of plasma after use of the molar-extinction coefficient for malondialdehyde of ⑀234 ⫽ 1.56 ⫻ 105 M⫺1 cm⫺1. After determining its protein content in accordance with the protocol of Lowry et al,26 with bovine serum albumin as the standard, we filtered LDL through a disposable 14-cm desalting column (Econo-Pac 10 DG; Bio-Rad) to remove the EDTA just before the oxidation experiments. EDTA-free LDL (100 ␮g protein/mL PBS) was oxidized with 2 ␮mol/L CuSO4. We monitored the production of conjugated dienes by recording absorbance at 234 nm at 5-minute intervals for 800 minutes in a thermostat-equipped cuvette maintained at 37°C (DU64-Spectrophotometer; Beckman). The lag time and propagation rate were determined essentially as described by Esterbauer et al.27 The propagation rate (expressed as total dienes formed per minute per milligram of LDL protein) was calculated with the use of the molar-extinction coefficient for conjugated dienes of (⑀234 nm ⫽ 2.95 ⫻ 104 M⫺1 cm⫺1. Plasma and LDL (␣-tocopherol and ␤-carotene) concentrations were determined with HPLC.28,29 Aminothiols and related molecules. Concentrations of plasma tHcy (the sum of Hcy, homocystine, and Hcy-cysteine mixed disulfides, free and protein-bound) and other thiols were measured in frozen plasma samples (⫺70°C) by means of HPLC as described.30 In brief, after the addition of the internal standard acetylcysteine, the sample was treated with tri-n-butylphosphine in dimethylformamide to reduce thiols and release proteins. After precipitation of protein and centrifugation, the supernatant was derivatized with SBD-F and injected into an analytical reverse-phase column (C18 ODS, 150 ⫻ 4.6 mm; Beckman Instruments, Inc, Palo Alto, Calif). The HPLC system was a Beckman System Gold equipped with a pump, an autosampler, and a fluorescence detector. The intra- and interassay coefficients of variation for tHcy are 3.0% (n ⫽ 12) and 3.3% (n ⫽ 50), respectively. We determined plasma folate concentrations by means of radioassay using a commercial kit (ICN Simultrac; ICN Pharmaceuticals Inc, Costa Mesa, Calif) and plasma vitamin B12 (cobalamin) on an automated immunoanalyzer (ADVIA Centaur; Bayer Diagnostics, Toronto, Ontario, Canada). The interassay coefficients of variation for folate are 4.7% (n ⫽ 11) and 6.9% (n ⫽ 31) for vitamin B12. All folate and B12 determinations were performed within the same assays. NOx were determined in plasma with a commercial kit (catalog number 1756281; Boehringer Mannheim, Laval, Quebec, Canada). The samples were first incubated with the enzyme nitrate reductase and cofactors to convert NO3 to NO2, then treated with the Griess reagent. After color development, the samples were deproteinized by means of trichloroacetic acid precipitation, after which absorbance was read at 540 nm. Statistical analyses. Data were analyzed with SAS software (version 6.12; SAS Institute Inc, Cary, NC). We performed two-factor (group, time) ANOVA for repeated measures when comparing Hcy concentrations between control and experimental subjects. One-factor (time) contrast ANOVA for repeated measures was used to evaluate the

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effect of FO intake on all measured variables. Both types of analyses were followed with Bonferroni’s procedure for multiple comparisons.31 Alternatively, paired t tests were used to compare differences between baseline and end-of-study data. Associations between variables were evaluated with simple linear regression, and Pearson’s r coefficient was used. In all analyses, we considered a P value of .05 the threshold of significance.

RESULTS Treatment outcome. The FO supplement was well tolerated and caused no significant changes in body weight (66.9 ⫾ 13.5 and 67.1 ⫾ 13.7 kg at baseline and after 8 wk, respectively). Subjects generally adhered to their usual eating habits, as assessed with a dietary interview at each visit, and compliance with the treatment was excellent, as confirmed by the phospholipid fatty-acid profile (see below). Influence of sex. Comparison of the changes observed in men and women on two-factor ANOVA for repeated measures showed no significant sex-related differences in response to FO intake. All data are therefore presented for the pooled sample. Lipids and lipoproteins. The FO long-chain n-3 fatty acids were incorporated into plasma phospholipids, with C20:5 (EPA) and C22:6 (DHA) showing four- and twofold increases, respectively, whereas the concentration of C20:4n-6 (arachidonic acid) was significantly lower after 4 and 8 weeks of FO than at baseline (Table I). This amounted to a significant decrease in the ratio of n-6 to n-3 PUFAs. These changes were associated with significant reductions in total and VLDL triglycerides, which occurred within 2 weeks of supplementation and were 21% and 29%, respectively, after 8 weeks of FO supplementation (P ⬍ .05; Table I). VLDL cholesterol was also significantly reduced, but we noted no changes in LDL or HDL cholesterol (not shown). Nor did we note significant plasma lipid and lipoprotein changes over 2 months in the untreatedcontrol group (not shown). Lipid peroxidation. Lipid-peroxidation parameters are presented in Table II. No significant changes were observed in the concentrations of plasma TBARS. An important heterogeneity in the lag time of the in vitro LDL oxidation course was observed; however, mean values were not significantly changed during the study (Table II), whereas a significant decrease in the dienepropagation rate was observed after 4 weeks of FO supplementation (Table II). LDL-oxidation parameters were unrelated to LDL concentrations of ␣-tocopherol and ␤-carotene or to LDL fatty acids, taken individually or as classes (n-6 or n-3 PUFAs). The latter were, however, significantly modified during this study (data

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Table I. Influence of FO on phospholipid long-chain polyunsaturated fatty acids and plasma total triglycerides and VLDL in normolipidemic subjects Variable

C20:4n-6 C20:5n-3 C22:6n-3 Ratio of n-6/n-3 Total triglycerides VLDL triglycerides

Baseline

2 Wk

4 Wk

8 Wk

8.01 ⫾ 1.40 0.88 ⫾ 0.38 2.31 ⫾ 0.84 9.03 ⫾ 3.26 1.05 ⫾ 0.43 0.75 ⫾ 0.38

7.25 ⫾ 0.81 4.15 ⫾ 0.92* 3.70 ⫾ 0.94* 3.25 ⫾ 0.83* 0.80 ⫾ 0.30† 0.50 ⫾ 0.25†

6.48 ⫾ 1.09* 4.20 ⫾ 1.00* 3.38 ⫾ 1.13† 3.29 ⫾ 0.90* 0.80 ⫾ 0.29‡ 0.48 ⫾ 0.23‡

6.56 ⫾ 1.00* 4.67 ⫾ 1.53* 3.89 ⫾ 1.20* 2.93 ⫾ 0.60* 0.83 ⫾ 0.54‡ 0.53 ⫾ 0.42‡

Data expressed as mean ⫾ SD in nanomoles per liter (triglycerides) or in percentage of total fatty acids (n ⫽ 16; 9 men). Significantly different from baseline (one-factor ANOVA for repeated measures followed by Bonferroni’s procedure for multiple comparisons): *P ⬍ .001. †P ⬍ .01. ‡P ⬍ .05.

Table II. Plasma and LDL lipid-oxidation parameters during FO study FO supplementation Variable

Plasma TBARS (nmol MDA/mL) LDL oxidation Lag time (min) Diene-propagation rate (nmol/mg LDL protein/min)

Baseline

2 Wk

4 Wk

8 Wk

2.67 ⫾ 0.97

2.89 ⫾ 0.97

2.89 ⫾ 0.94

2.63 ⫾ 1.21

58.0 ⫾ 18.2 9.36 ⫾ 1.60

47.4 ⫾ 18.1 8.23 ⫾ 2.43

51.6 ⫾ 17.5 7.43 ⫾ 1.43*

51.1 ⫾ 18.5 8.77 ⫾ 2.27

Data expressed as mean ⫾ SD (n ⫽ 16). Significantly different from baseline (one-factor ANOVA for repeated measures followed by Bonferroni’s correction for multiple comparisons): *P ⬍ .01. TBARS expressed as malondialdehyde equivalents.

not shown) and reflected changes observed in total plasma (Table I). Antioxidants. Baseline concentrations of ␣-tocopherol and ␤-carotene, which were 27.0 ⫾ 6.6 and 0.58 ⫾ 0.22 ␮mol/L (mean ⫾ SD) in total plasma and 12.6 ⫾ 3.2 and 0.57 ⫾ 0.28 nmol/mg protein in LDL, were unchanged after 8 weeks of FO intake (28.1 ⫾ 6.7 and 0.64 ⫾ 0.31 ␮mol/L, and 14.4 ⫾ 5.2 and 0.63 ⫾ 0.28 nmol/mg protein in total plasma and LDL, respectively). Plasma tHcy and related molecules. Results are presented in Table III for the FO-treated and untreated healthy subjects. Two-way ANOVA showed the significantly different response of aminothiol concentrations in the two groups. The overall baseline values for tHcy, cysteine, and cysteinylglycine were significantly lower in the untreated group than in the treated group (P ⬍ .05 for cysteine; P ⬍ .001 for tHcy and cysteinylglycine). No significant changes occurred in any of the measured aminothiols in the subjects who did not take FO, whereas significant changes were observed in the treated group after 4 and 8 weeks of FO for tHcy and after 8 weeks for glutathione (P ⬍ .05). The increase in

tHcy was progressive, and mean increases of 9.7%, 13.4% (P ⬍ .01), and 20.1% (P ⬍ .01) were observed after 2, 4, and 8 weeks, respectively. Increases occurred in all subjects but one as can be seen in Fig 1, which shows individual differences in absolute values and in percentages between baseline and 8-week measurements. We noted no significant changes in cysteine or cysteinylglycine or in the ratio of tHcy to cysteine. Table IV shows the results of plasma folate, vitamin B12, and NOx measurements performed at baseline and after 4 and 8 weeks of FO. We found no consistent or statisticially significant changes in plasma folate and B12 concentrations during the study. Conversely, NOx plasma concentrations increased progressively, and the difference was significant (74%, P ⬍ .01) after 8 weeks of supplementation. Fig 2 shows absolute and percentage NOx changes from baseline and illustrates the important increase that occurred in most subjects. Individual increases at 8 weeks varied from 12% to 112%, with an outlier at 327%. Correlates of tHcy. The negative correlation observed in the 16 subjects between tHcy and folate concentrations at baseline (r ⫽ ⫺.51, P ⫽ .04) and after 2 weeks

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Table III. Plasma total aminothiol concentrations in FO treated and untreated control subjects Weeks Variable

tHcy (␮mol/L) Cysteine (␮mol/L) Ratio tHcy/cysteine Cysteinylglycine (␮mol/L) Glutathione (␮mol/L)

Group

0

2

4

8

Treated Untreated Treated Untreated Treated Untreated Treated Untreated Treated Untreated

8.8 ⫾ 1.4 6.7 ⫾ 1.8 216 ⫾ 16 200 ⫾ 28 0.041 ⫾ 0.008 0.033 ⫾ 0.005 32.0 ⫾ 5.5 27.2 ⫾ 4.3 5.7 ⫾ 1.5 5.4 ⫾ 1.5

9.6 ⫾ 1.9 — 219 ⫾ 16 — 0.044 ⫾ 0.008 — 32.4 ⫾ 4.4 — 6.1 ⫾ 1.6 —

10.0 ⫾ 1.8* 6.7 ⫾ 1.9 224 ⫾ 16 203 ⫾ 32 0.045 ⫾ 0.007 0.033 ⫾ 0.005 33.2 ⫾ 5.4 26.9 ⫾ 3.7 5.9 ⫾ 1.6 5.2 ⫾ 1.4

10.5 ⫾ 2.2* 6.8 ⫾ 1.6 225 ⫾ 18 201 ⫾ 26 0.047 ⫾ 0.010 0.034 ⫾ 0.005 33.3 ⫾ 4.8 26.9 ⫾ 3.9 7.0 ⫾ 1.8† 5.5 ⫾ 1.3

Data expressed as mean ⫾ SD (n ⫽ 16 in each group). Significantly different from baseline (one-way ANOVA for repeated measures followed by Bonferroni’s correction for multiple comparisons): *P ⬍ 0.01. †P ⬍ .05.

of FO supplementation (r ⫽ ⫺.53, P ⫽ .03) was no longer significant after 4 weeks (r ⫽ ⫺.10, NS) and 8 weeks (r ⫽ ⫺.38, P ⫽ .15). A similar pattern was observed for vitamin B12, with a baseline correlation between tHcy and B12 of ⫺.48 (P ⫽ .07) and of ⫺.42 (P ⫽ .12) after 8 weeks. The opposite was seen for the relationship between tHcy and NOx (n ⫽ 15), which showed a negative trend at baseline (r ⫽ ⫺.42, P ⫽ .11) and was nonsignificant after 4 weeks (r ⫽ .17, NS) but was positive and near significance (r ⫽ .49, P ⫽ .06) after 8 weeks of FO supplementation. To further scrutinize the tHcy increases observed during this intervention study and to focus on the actual changes, we calculated correlations between the differences in potential predictors and the tHcy levels and differences obtained after 8 weeks of FO supplementation (Table V). We found no correlation between tHcy (levels or differences) and the n-3 PUFAs or plasma folate and B12 concentrations. However, the tHcy changes (increases) were remarkably dependent on the extent of the increase in circulating NO (assessed as its metabolites, nitrates ⫹ nitrites). This important, quantitative association is illustrated in Fig 3. DISCUSSION

In this study, intake of 6 g/day of fish oil (3 g n-3 PUFAs) significantly modified the plasma fatty acid profile and caused significant reductions in plasma total and VLDL triglyceride concentrations, consistent with observations made in other studies in healthy individuals.32 Because of the normotriglyceridemic status of the participants, no other significant changes in the lipoprotein profile were expected, and none was observed. Whether FO or isolated n-3 fatty acids increase LDL

susceptibility to oxidation remains a matter of controversy.33 Results from human studies that have involved different populations or different FO or n-3 FA dosages and assay conditions are contradictory. Though heterogeneous, our current results in healthy, normolipidemic subjects taking a FO dose half that used in our earlier study20 did not show overt, sustained changes in LDL-oxidation parameters or in plasma TBARS concentrations. Moreover, we recently reported that the n-3 fatty-acid supplementation protected these subjects’ erythrocytes against free radical–induced hemolysis.34 It is thus concluded that moderate quantities of FO did not cause an oxidative stress, given the healthy status of the participants, their regular food intake, and their stable plasma concentrations of antioxidants. In the absence of increased susceptibility to oxidation, the progressive and significant increase in plasma tHcy concentrations was unexpected. Earlier studies, including our own, have reported either no significant changes,35-38 an increase,39 or a decrease21,40 in Hcy levels after supplementation with n-3 fatty acids. However, the current study sample and the dosage of FO used represent entirely different conditions from those in these reports and, specifically, in our earlier study.20,21 The reduced Hcy concentrations observed in the latter study were attributed to possible FO-induced oxidative stress and stimulation of the oxidative catabolism of Hcy.19 Because no overt changes in susceptibility to enhanced lipid peroxidation were seen in this study, other explanations for the increase in Hcy plasma concentrations had to be sought. Several pathways and many enzymes and vitamin cofactors are active in Hcy metabolism.19 The other measured aminothiols that are structurally or metabolically related (eg, cysteine, cysteinylglycine) showed

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Fig 1. Individual changes in plasma tHcy concentrations between baseline (0) and after 8 weeks of FO supplementation: A, absolute values; B, percent changes from baseline.

Table IV. Plasma folate, vitamin B12, and NOx concentrations during FO supplementation Variable

Folates (nmol/L) Vitamin B12 (pmol/L)* NOx (␮mol/L)*

Baseline

4 wk FO (⌬) (⌬%)

8 wk FO (⌬) (⌬%)

13.0 ⫾ 4.4 299 ⫾ 68 8.1 ⫾ 2.9

11.9 ⫾ 3.6 (–1.1) (–5.3) 294 ⫾ 75 (–4.8) (–1.4) 10.4 ⫾ 8.1 (2.3) (41.4)

12.0 ⫾ 3.8 (–1.0) (–5.3) 306 ⫾ 62 (6.5) (4.2) 13.7 ⫾ 6.9† (5.6) (74.0)

Data expressed as mean ⫾ SD. *n ⫽ 15, one missing sample. ⌬ ⫽ mean difference from baseline in absolute values; ⌬% ⫽ mean difference in percent. Significantly different from baseline (one-way ANOVA for repeated measures followed by Bonferroni’s correction for multiple comparisons): †P ⬍ .01.

no significant change in this study. Further, the Hcy/ cysteine ratios remained similar over the 8-week period, suggesting that the transsulfuration pathway of Hcy metabolism was not negatively affected by FO intake.41 The remethylation pathway of Hcy metabolism was next considered. Folate and vitamin B12 are key elements in this pathway41,42 because folate, in the form of N-5-methyl tetrahydrofolate, provides a methyl group for the remethylation of Hcy, in a reaction catalyzed by the vitamin B12– containing enzyme methionine synthase (N-5-methyltetrahydrofolate:Hcy methyltransferase).41 The question naturally arose as to the availability of these two vitamins during our FO study. However, it does not appear that these were responsible for the Hcy increase. First, lack of significant dietary modifications during the study, apart from the FO supplementation, was documented by dietetic control and follow-up. Second, there was no indication of changes in vitamin absorption; our subjects’ plasma folate and vitamin B12 concentrations were not significantly modified during the study period. Furthermore, increases in Hcy were not correlated with decreases in folate or vitamin B12.

Recent observations on the diverse effects of n-3 fatty acids led us to consider another option. Dietary FO has been shown to enhance the production and the release of NO43-45 and the creatinine-adjusted urinary excretion of NO metabolites.46 In the last study, the serum nitrate concentrations of healthy subjects tended to be greater, though not significantly so, after 3 weeks’ supplementation with 3 g of n-3 fatty acids (EPA ⫹ DHA.46 Studies that have used purified single n-3 fatty acids have provided contradictory results.46-49 In our 8-week study employing a FO concentrate (EPA ⫹ DHA, wt/wt 3:2), we were able to show a progressive increase in NOx that was not statistically significant after 4 weeks, as in the study reported in reference 46, but was after 8 weeks. The exact mechanisms for the nitrate-increasing effect of n-3 fatty acids have not been completely elucidated,46 and our study does not attempt to shed any light on this question. Enhanced production and release of NO are usually associated with favorable effects on vascular reactivity. However, NO is a highly reactive molecule that is involved in different systems. For instance, the interactions of Hcy and NO are the focus of intense re-

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Fig 2. Individual changes in plasma NOx concentrations between baseline (0) and after 8 weeks of FO supplementation: A, absolute values; B, percent changes from baseline.

Table V. Correlates of plasma tHcy concentrations and changes after 8 weeks of FO supplementation in 15 subjects tHcy (␮mol/L) ⌬ Variable

C20:5n-3 (EPA) C22:6n-3 (DHA) Plasma folates Plasma vitamin B12 Plasma NOx

⌬ tHcy

r

P

r

P

–.18 –.12 .24 –.18 .55

NS NS NS NS .03

–.10 .12 –.25 .25 .78

NS NS NS NS .0006

⌬ ⫽ difference in absolute values; NS ⫽ not significant.

search.50-54 The main purpose of these studies has been to evaluate the influence of Hcy on NO function. One of the mechanisms whereby Hcy is believed to be involved in atherothrombotic disease is through the induction of oxidative stress.19 Oxidative products can inactivate NO and compromise its vasodilation and antiaggregation activities.52 However, NO has been shown to modulate the potentially adverse vascular effects of Hcy.50,53 The association of Hcy with NO leads to the formation of S-NO Hcy. This S-nitrosation of Hcy does not support H2O2 generation; the absence of an oxidative stress could therefore preserve NO antiatherogenic potential.50 One less-studied phenomenon is the influence exerted by NO on Hcy metabolism. However, it should be emphasized that the inhibition by NO of the enzyme methionine synthase has been documented under various in vitro and in vivo conditions.55-58 It has been shown to occur by way of an interaction of NO and cobalamins (B12), resulting in the latter having dimin-

ished ability to act as a cofactor for methionine synthase.57,58 One study conducted in cultured mammalian cells58 showed convincingly how NO bound to cobalamin and modified its structure, resulting in the in vivo inhibition of methionine synthase activity. The latter phenomenon was documented by means of observation of disruption of the carbon flow through the folate pathway and decreased synthesis of methionine, serine, and de novo purine nucleotide synthesis. Treatment of cells with NAME, a NO synthase inhibitor, increased the rates of methionine, serine, and de novo purine nucleotide synthesis.58 Hcy concentrations were not measured in that study, but the authors asserted that NO-mediated inhibition of methionine synthesis could increase intracellular (and thus the serum) concentrations of Hcy. Although this study does not include documentation of the mechanisms underlying the effects reported, we conclude that our novel observation of a strong correlation between increased plasma NOx and Hcy concentrations occurring during FO supplementation in a small group of healthy individuals is consistent with the demonstrations cited above. Considering that methionine synthase is the main enzyme responsible for the remethylation and conversion of Hcy to methionine, we hypothesize that the increase in plasma Hcy concentrations that occurred during our FO study is due, at least in part, to the concurrent increase in NOx. It is possible that the increase in Hcy levels represents, in healthy subjects, an induction of NO production and a physiologic regulation of Hcy metabolism in response to dietary supplementation with modest doses of n-3 fatty acids. Furthermore, because of its association with increased NOx levels and in the absence of an oxidative stress, the

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Fig 3. Correlation between absolute changes (⌬; difference between 8-week and baseline values) in plasma tHcy concentrations and absolute changes (⌬) in NO metabolites (NO2 ⫹ NO3) that occurred with intake of FO.

Hcy increase observed in this study is probably not deleterious. We thank Claudia Rodriguez and Nancy Doyle for technical assistance. We are grateful to Dr Roger Sansfac¸ on for the vitamin B12 measurements and to Dr Philippe Durand for insightful and helpful comments. REFERENCES

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