REVIEW ARTICLE

METABOLIC SYNDROME AND RELATED DISORDERS Volume 12, Number 8, 2014  Mary Ann Liebert, Inc. Pp. 403–415 DOI: 10.1089/met.2014.0045

Do n-3 Polyunsaturated Fatty Acids Increase or Decrease Lipid Peroxidation in Humans? Nirvair S. Kelley, MS,1 Yasukazu Yoshida, PhD,2 and Kent L. Erickson, PhD 3

Abstract

Background: Despite many known health benefits of n-3 polyunsaturated fatty acids (PUFA), there is a concern that their high degree of unsaturation may actually increase oxidative stress, lipid peroxidation (LPO), and chronic inflammatory diseases. Methods: In this review, we have analyzed results from published human studies regarding the effects of n-3 PUFA supplementation on markers of lipid peroxidation. Results: Of the 22 published human studies, nine found no change, eight a decrease, and five an increase in markers of LPO. These inconsistencies may be due to methods, subject characteristics, dose, duration, fatty acid and antioxidant composition of supplements, and basal diets. Methods used for analysis seem to be the most significant factor. Six of eight studies with a decrease in LPO determined F2-isoprostanes produced in vivo, and two determined plasma antioxidant capacity or hydroperoxides. n-3 PUFA can serve as scavengers for free radicals and also modulate expression of genes that determine the balance between oxidative and antioxidative status. Recent studies that monitored oxidation products of cholesterol and fatty acids support the hypothesis that n-3 PUFA decrease LPO. Most of the studies showing no change or increase in LPO determined markers that involved ex vivo sample preparation or oxidation (malondialdehyde, low-density lipoprotein oxidation, lipid hydroperoxides). Conclusion: A majority of studies do not indicate that n-3 PUFA increased LPO. Future studies need to investigate the effects of dose, duration, and composition of n-3 PUFA with standardized diets and methods on concentrations and types of LPO products produced.

Introduction

D

ietary n-3 polyunsaturated fatty acids (PUFA) have numerous health benefits and help counteract symptoms of several chronic diseases. Diets rich in n-3 PUFA decreased platelet aggregation, plasma triglycerides, inflammation, and the number of total as well as atherogenic small dense low-density lipoprotein (LDL) particles.1–3 Despite health benefits, there is concern that n-3 PUFA may increase oxidative stress due to their high degree of unsaturation. However, this is debatable because studies have shown that not only unsaturation but also the position of double bonds are factors.4 Thus, it is not entirely clear whether n-3 PUFA reduce or increase oxidative stress. A main consequence of oxidative stress is lipid peroxidation (LPO), which increases inflammation and the development and progression of a number of chronic inflammatory 1 2 3

diseases, including some components of metabolic syndrome (insulin resistance, hypertension, nonalcoholic fatty liver disease), cardiovascular diseases, and diabetes.5–8 LPO is a term used to describe the oxidative degradation of lipids typically found in biological membranes such as phospholipids and cholesterol. This process can be achieved via enzymatic, nonenzymatic, nonradical, or nonenzymatic free radical-mediated peroxidation. LPO-mediated degradation compromises membrane integrity and can lead to changes in permeability and fluidity. It also propagates free radicals, which initially act locally with short half-lives. These local insults are followed by formation of more stable secondary breakdown products of lipid peroxides, such as alkanes, aldehydes, ketones, alcohols, and furans. Those products can then disseminate from their site of formation and diffuse across cellular membranes. Most secondary breakdown products of LPO are reactive due to their electrophilic nature.

Arizona College of Osteopathic Medicine, Midwestern University, Glendale, Arizona. Health Technology Research Center, National Institute of Advanced Industrial Science and Technology, Osaka, Japan. Department of Cell Biology and Human Anatomy, University of California, School of Medicine, Davis, California.

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They play powerful roles in cell signaling, mainly in cell cycle regulation.9–12 The concentration of the secondary breakdown products can be quantified to assess the degree of in vivo lipid peroxidation. Some of those compounds include malondialdehyde (MDA), 4-hydroxynonenal (HNE), 4-hydroxyhexenal (HHE), F2-isoprostanes (F2-IsoPs)/8-isoprostaglandin F2a, acrolein, hexenal, hydroxyoctadenaoic acid (HODE), hydroxyeicosatetraenoic acid (HETE), 7a-hydroxycholesterol, and 7b-hydroxycholesterol. The in vivo concentrations of those specific LPO biomarkers vary greatly as does the sensitivity of their assay methods. Although some LPO biomarkers indicate the specific lipid substrate used to generate the measured breakdown product, others serve as an overall assessment of LPO. Because of discrepancies among published results, concerns have been raised regarding whether n-3 PUFA reduce or induce oxidative stress and LPO. From all the publications in English that have focused on the effects of n-3 PUFA on LPO in humans, 27% reported an increase,13–18 32% a decrease,19–25 and 41% no change in LPO.26–34 Several other studies showed that n-3 PUFA decreased LPO in cultured cells. However, those results from in vitro studies may not be representative of the in vivo milieu and should be interpreted with caution.35–39 The discrepancies of published studies need to be reconciled because n-3 PUFA are being recommended as components of healthy diets. The aim of this review was to analyze results from published human studies that investigated effects of n-3 PUFA on LPO. For that, we compared dose, duration, and form of n-3 PUFA supplemented; antioxidants supplementation; health status of participants; and methods used to evaluate LPO. We analyzed studies published in past 23 years.

Methods PubMed was used as the primary search engine to obtain publications for this review. The following criteria were used to determine exclusion or inclusion of n-3 PUFA and LPO human studies. All subjects received n-3 PUFA as combined or individual supplements of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) or a fish oil mixture. Studies selected were published in 1990–2014.

Results Biomarkers of LPO F2-isoprostanes. F2-IsoPs refer to a collection of compounds created by nonenzymatic, free radical attack of the arachidonic acid (AA) components in lipid membranes. Most AA present in animal cells is esterified to phospholipid components of lipid membranes. F2-IsoPs are hydrolyzed and released by the catalytic action of phospholipase A2 and platelet activating factor acetylhydrolase. F2-IsoPs are usually measured from samples of urine, plasma, serum, and, less commonly, other body fluids. Temporarily, plasma levels of F2-IsoP peak quicker than urinary levels. Hence, urine and plasma samples should not be viewed interchangeably. It is desirable to include 24-hr pooled urine or at least several time points for measurement.40 Specimens undergo various purification procedures and then are measured by gas chromatography/mass spectrometry (GC/MS), gas chromatography/tandem mass spectrometry (GC-MS/MS), liquid chromatography/tandem mass

KELLEY, YOSHIDA, AND ERICKSON

spectrometry (LC-MS/MS), or enzyme-linked immunosorbent assay (ELISA).40 Mass spectrometry techniques do not measure F2-IsoP directly, but its metabolites. Usually, the metabolite quantified is 8-isoprostaglandin F2.40 However, there are other isomers and various mass spectrometry techniques to assay their concentrations. F2-IsoP is usually considered as the ‘‘gold standard’’ biomarker of lipid peroxidation in vivo and is increasingly used to examine n-3 PUFA and LPO. However, using F2-IsoP as a marker for LPO has several limitations. For example, detection methods such as high-performance liquid chromatography (HPLC)-MS/MS are expensive, time consuming and technically challenging. Currently available ELISA kits to determine F2-IsoP have poor reproducibility. Additionally, F2-IsoP is present in the picomole range in body fluids, which further complicates their quantification and use as biomarkers. Malondialdehyde. The bulk of LPO-derived MDA is produced by nonenzymatic free radical peroxidation. However, it can also be produced enzymatically. The free radical degradation of membrane PUFA creates highly unstable and reactive lipid hydroperoxides, which decompose to form more stable aldehyde products, specifically MDA.41 MDA is generated by peroxidation of PUFA containing at least three double bonds, such as AA or DHA, and serves as a biomarker for both n-3 and n-6 PUFA LPO.41 MDA levels are most commonly quantified by the thiobarbituric reactive species (TBARS) assay. The TBARS assay is conducted with acidic conditions at high temperatures (95–100C), and involves the reaction of MDA with thiobarbituric acid. TBARS assay measures not only the quantity of MDA, but also lipid hydroperoxides that yield MDA. Decomposition of those lipid hydroperoxides into MDA can be induced by transition metal ions, such as Fe3 + or Cu2 + , included in the assay. Specificity of the TBARS assay is questionable due to the production of reactive aldehyde species from non-LPO sources, and cross-reactivity resulting from acidic and high temperature conditions of the assay. MDA concentrations assayed by those methods are normally in the nanomole range. MDA concentrations can also be quantified by HPLC; however, it is not commonly used. While it has high reproducibility and precision, because of high volatility of MDA, the concentrations obtained by this method are much lower than those obtained by TBARS. Thus, HPLC analysis under estimates and TBARS over estimates the MDA concentrations. 4-Hydroxy-2(E)-nonenal and 4-hydroxy-2(E)-hexenal. MDA and TBARS can be generated from peroxidation of both n-6 and n-3 PUFA. In contrast, HNE is generated by autooxidation of n-6 PUFA42 and HHE is produced from autooxidation of n-3 PUFA.43 Alkenal species such as HHE and HNE are reactive substances and can be assayed by the unique adducts formed. For example, HNE readily reacts with cellular proteins forming adducts with cysteine, histidine, lysine, and serine.42,44–46 In addition, glutathione (GSH) conjugation is a key step in the metabolism of secondary LPO products such as HNE. The GSH moiety of these by products can be reduced to mercapturic acid, which is the major metabolite of HNE production found in urine.41 Because multiple metabolites can be formed, there are numerous methods to quantifying these byproducts. Some immunochemical methods, such as ELISA, can be used for quantification because of adducts formed between aldehydes and specific peptides.47 Nonimmunological methods are based on the

n-3 PUFA EFFECTS ON LIPID PEROXIDATION IN HUMANS

reaction of the aldehyde moiety with dinitrophenylhydrazine to form hydrazone compounds. They can be measured by spectrophotometric methods.48 Those methodologies are rather nonspecific, because many carbonyl-modified proteins can be identified along with LPO alkenal byproducts.41 Thus, quantification methods were developed to determine specific adducts formed with peptides. Those include methods for quantifying carbonyl groups associated with sulfhydryl groups of LPO products that can be used to identify cysteine adducts.49 Other peptide adducts formed with histidine and lysine can be quantified with HPLC when a strong reducing agent such as sodium borohydride (NaBH4) is used.50 The advantage of using HHE, HNE, or HHE as LPO biomarkers is the specificity of substrate. However, quantification of HNE and HHE is less sensitive than those of F2-IsoP or MDA.

Degree of LDL oxidation The degree of LDL oxidation is of particular interest in LPO studies because of its direct link to atherosclerosis. Numerous studies have investigated the ex vivo, metal ioninduced oxidation of LDL lipoproteins, in addition to the quantification of other LPO biomarkers. Inducing agents include metal ions, such as copper and iron, and also other agents such as carbon tetrachloride and 2,2¢-azobis(-amidinopropane) dihydrochloride. The time elapsed between introduction of oxidizing agents and the actual oxidation of LDL lipoproteins indicates the susceptibility of the lipoprotein to oxidation. A longer time lapse to oxidation indicates less susceptibility of the LDL to oxidation, and vice versa. Therefore, lag time is a commonly employed technique to assess LPO. However, the ex vivo measurement of LDL oxidizability may not correlate with in vivo oxidation of LDL. Incubation of LDL with several cell types such as monocytes, macrophages, or fibroblasts or metal ions such as copper in the absence of cells can result in the oxidative modification of LDL. Although both routes have similar properties, there are a number of differences, such as sensitivity to inhibition by superoxide dismutase and lipoxygenase inhibitors as well as levels of TBAR produced. Oxidation products of individual fatty acids, such as HODE from linoleic acid and HETE from arachidonic acid, or a- and b-oxidation products of cholesterol, have also been used to assess lipid peroxidation. Other methods assess oxidative stress by a determination of oxygen radical absorbance capacity, Trolox equivalent antioxidant capacity, total antioxidant status, or enzyme activity, such as catalase and glutathione peroxidase. Because most of the surveyed studies in this review used the methods described above, our discussion will be limited to those methods.

Effects of n-3 PUFA on LPO We found a total of 22 published reports that investigated the effects of feeding fish, or supplementing diets with fish oil, purified EPA or DHA. Nine studies reported no change,26–34 eight a decrease,19–25 and five an increase13–18 in markers of LPO. No change in LPO. Studies showing no change in LPO after n-3 PUFA supplementation are listed in Table 1. The amounts of n-3 PUFA varied from 529 mg/day27 to 5.0 grams/ day.27,29 Intervention time ranged from a single administration to 5 months of supplementation.32,34 One of those studies used purified DHA (2.14 grams/day)33 and none used purified

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EPA. The remaining eight studies used a combined supplement of both EPA and DHA, with one study including docosapentaenoic acid (DPA) as well.30 In most of these studies, concentrations of EPA were greater than DHA, except in one where the DHA concentration was more than three times that of EPA32; three studies did not indicate the concentrations of individual fatty acids. Because n-3 PUFA are susceptible to oxidation prior to ingestion, a number of different antioxidants were included. Four studies used a-tocopherol ranging from 3.0 to 20 mg/day.27,31–33 One study incorporated 0.9 mg/day DLa-tocopherol.28 Mixtures of other antioxidants were used (Table 1). Two studies did not use any antioxidants.26,34 The ages of men and women studied ranged from 18 to 75 years. Six studies involved one gender exclusively. Study participants included normolipidemic, obese, hypertriglyercidemic men and women, and postmenopausal and pregnant women. The methods used for assessing lipid peroxidation varied among these studies: Total anti-oxidant status (two studies31,34); oxidized LDL and lipid hydroperoxides (seven studies26–,34); MDA (two studies31,32); F2-IsoP (two studies31,33); and 8-hydroxy-2-deoxyguanosine (one study32). Those biomarkers were assayed from serum, plasma, and urine samples. Considering the wide range of subject characteristics, dose and duration of n-3 PUFA, antioxidants, and methods used, it is difficult to determine why n-3 PUFA failed to increase or decrease LPO in these studies. Other confounding variables such as anti-oxidant nutrients and health status of participants may have contributed to the results reported. Decrease in LPO. Eight studies demonstrated a decrease in LPO biomarkers after intervention with n-3 PUFA (Table 2). The administered n-3 PUFA dosages varied from 0.2 to 4.8 grams/day.23,24 Intervention time ranged from 6 weeks to 5 months.20,21 Two of these studies administered both purified DHA and EPA to separate groups20,25; one used only purified DHA.24 Remaining studies provided a mixture of EPA and DHA at different ratios with no rationale for specific ratios selected. Various antioxidants were included. Three studies utilized 6.4 to 32 mg/day a-tocopherol20,23,25; two also included an additional 3.6 mg/day of g-tocopherol.20,25 One study used 0.125 mg of synthetic DL-a-tocopherol and 0.125 mg of ascorbic palmitate per 200-mg capsule of DHA.24 Three studies did not use antioxidants.20–22 The study participants were men and women who ranged from 18 to 75 years of age and were healthy, overweight, dyslipidemic, hypertensive type 2 diabetic, postmenopausal, or pregnant (Table 2). Markers of LPO investigated were plasma or urine F2IsoP20–22,24 in all studies except for two, which examined plasma oxidized LDL, lipid hydroperoxides, MDA, HHE, and HNE.23,24 While other factors may have contributed to the observed decreases in markers of LPO in these studies, the most likely reason was the assay system used to quantify LPO products. Plasma or urinary F2-IsoP was monitored in six of eight studies. The concentrations of the antioxidant nutrients used overlapped among the studies showing no change or decrease in markers of LPO following supplements with n-3 PUFA. Generally their concentrations were higher in those showing decrease than those showing no change. Increase in LPO. Five studies reported an increase of LPO biomarkers after administration of n-3 PUFA for 1–6 months (Table 3). The amount of n-3 PUFA administered ranged from 0.2 to 10 grams/day. All studies used a mixture of DHA and EPA in supplement form, except one, which

406

Hypertriglycemic men and women; n = 9

Hypertriacylglycemic hemodialyzed men and women (mean = 64 – 2); n = 12 Male smokers (40–60); n = 9–11/group

Healthy men (29–60), n = 21–24/group

Postmenopausal women (50–75); n (treatment) = 15

Frankel et al.27

Bonanome et al.28

Sorensen et al.30

Higdon et al.31

Brude et al.29

Normolipidemic men and women (27–63); n = 11–12

Participants (age), n

Nenseter et al.26

Authors

2.0 grams EPA + 1.4 grams DHA/ day; 5 weeks; 15 grams/day sunflower or safflower oil; three periods, three treatments, doubleblinded crossover trial

2.5 grams EPA + DHA/day; 2 months, no control; two-phase sequential intervention, supplementation followed by washout Groups: (1) 5 grams EPA + DHA/ day, (2) n-3 + antioxidants, (3) antioxidants, (4) control oil; no EPA or DHA; 6 weeks; 8 grams oil; randomized, double-blind, placebo-controlled trial 0.91 gram EPA, DPA, and DHA/ day; 7 week, 4 grams sunflower oil, two-phase randomized design

2.83 grams EPA + 1.78 grams DHA/ day; 4 months; 6 grams corn oil; randomized placebo; controlled trial 375 mg EPA + 154 mg DHA/day; 12 weeks; no placebo; nutritional intervention

Plasma; F2-IsoP and MDA; @ 0 and 5 weeks; blood; LOOH, ox-LDL, total antioxidant activity; @ 0 and 5 weeks

Plasma; ox-LDL; @ 3 weeks (after run in period) and 7 weeks

Plasma; LOOH, oxLDL, conjugated diene formation; @ 0 and 6 weeks

Blood; propanal, hexenal, and oxLDL; @ 6 and 12 weeks Plasma; ox-LDL; @ 0, 1, and 2 months

Plasma; ox-LDL; @ 0 and 4 months

Sample type; biomarker; time of measurement

20 mg a-tocopherol/day

FO margarine contained propylgallate (20%), citric acid monoglyceride ester (80%), and 0.1 gram EDTA/kg

75 mg vitamin E/day, 150 mg vitamin C/day, 15 mg bcarotene/day, and 30 mg coenzyme Q10/day

0.9 mg DL-a-tocopherol/day

16.75 mg a-tocopherol/day

None indicated

Antioxidant

Human Studies in Which n-3 PUFA Did Not Change Lipid Peroxidation

n-3 PUFA dose; intervention time; placebo/comparison; study design

Table 1.

(continued)

All subjects healthy and nonsmokers except 8 active smokers; broad age range; low-sensitivity nonspecific LPO quantification methods Plasma F2-IsoP decreased, differences eliminated after adjusting for AA concentrations; broad age range

Low-sensitivity nonspecific LPO quantification methods; broad age range

Low-sensitivity nonspecific LPO quantification methods

Low-sensitivity nonspecific LPO quantification methods

Low-sensitivity nonspecific LPO quantification methods; broad age range

Comments

407

2.14 grams DHA/day, 42 days, 6 grams corn oil; single-blind, randomized, placebo-controlled trial 2.8 grams EPA and 1.4 grams DHA with small amounts of LA, OA, PA, and ALA; acute admininstration; 7 grams corn oil; randomized double-blind, placebo-controlled crossover 8 grams/day oxidized or nonoxidized fish oil (1.6 grams EPA/DHA) or 8 grams/day higholeic sunflower oil; randomized double-blind, placebo-controlled

500 mg DHA and 150 mg EPA/day; week 20 gestation to delivery; negligible amounts of DHA and EPA; double-blind randomized control trial

Urinary 8-iso PGF2a2, plasma 4-hydroxy-2hexenal, 4-hytroxy-2nonenal @ 3 and 7 weeks

Plasma and urine; MDA, 8-hydroxy-2deoxyguanosine, atocopherol; @ 20, 30 weeks and @ delivery Plasma and urine; F2isoP and atocopherol; @ 0 and 42 days Serum; LOOH, oxLDL, total antioxidant status; @ 2, 4, and 6 hr postprandially

Sample type; biomarker; time of measurement

Tocopherols measured and standardized within all diets

None indicated

4 mg a-tocopherol acetate/day

3.0 mg a-tocopherol/day

Antioxidant

Oxidized or nonoxidized fish oil did not altered markers of oxidative stress. Whether 4HNE is a reliable marker for in vivo LPO needs to be confirmed

Lack of effect may be due to acute administration of n-3 PUFA; low-sensitivity nonspecific LPO quantification methods

NC in markers of oxidative stress

Low-sensitivity nonspecific LPO quantification methods; broad age range

Comments

n-3 PUFA, n-3 polyunsaturated fatty acids; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; ox-LDL, oxidized low-density lipoprotein; LPO, lipid peroxidation; LOOH, lipid hydroperoxides; FO, fish oil; F2-IsoPs, F2-isoprostanes; MDA, malondialdehyde; AA, arachidonic acid; NC, no change; LA, linoleic acid; OA, oleic acid; PA, palmitic acid; ALA, a-linolenic acid; PGF2a2, prostaglandin F2 alpha 2; 4HNE, 4-hydroxynonenal.

Ottestad et al.63

Healthy men and women 18–50; n = 58

Postmenopausal women vegetarians ( < 60 years); n = 11–14/ group Hypertriglycemic overweight and obese men ( > 45); n = 10

Wu et al.33

Hanwell et al.34

Healthy pregnant women (18–40); n = 23/ group

Participants (age), n

Shoji et al.32

Authors

n-3 PUFA dose; intervention time; placebo/comparison; study design

Table 1. (Continued)

408

Nonsmoking men and women with NIDDM (30–65); n = 11–14

Nonsmoking, treated hypertensive, type 2 diabetic men and postmenopausal women (40–75); n = 16–17/ group

Pregnant women (mean = 31); n = 40–43/ group

Healthy men and women (30–65); n = 39–40/ group

Healthy young (18–42 years) and old (53–70) males; n = 93 and 62

Mori et al.20

Barden et al.21

Nalsen et al.22

Cazzola et al.23

Participants (age); n

Mori et al.64

Authors

Groups: (1) 2.4 grams/day EPA + DHA + SFA diet, (2) n3 + MUFA, (3) SFA + placebo, (4) MUFA + placebo; 3 months; 3.6 grams/day OO; randomized placebo controlled Treatment groups: (1) 1.35 grams EPA + 0.27 gram DHA/day, (2) 2.7 grams EPA + 0.54 gram DHA/day, (3) 4.05 EPA + 0.81 DHA/day, (4) placebo; 12 weeks; (1) 6 grams/day, (2) 3 grams/day, (3) 0 grams/day, (4) 9 grams/day corn oil; successive randomized placebo-controlled intervention

1.1 grams EPA + 2.2 grams DHA/day; week 20 gestation to term; 4 grams/day OO; double-blind placebocontrolled parallel intervention

Treatment groups: (1) 3.6 grams EPA + DHA/day (1 fish meal/ day), 2) n-3 + moderate exercise, (3) low-fat diet, (4) low-fat diet + moderate exercise; 8 weeks; no placebo; two-way factorial intervention, parallel design 4 grams/day of EPA or DHA; 6 weeks; 4 grams/day OO placebo; double-blind placebocontrolled

Plasma; LOOH, oxLDL, GSSH; @ baseline and 12 weeks

Plasma; F2-IsoP @ baseline and 3 months

Cord blood, plasma and urine F2-IsoP @ delivery and 1 week postpartum

Urine; F2-IsoP @ baseline and at 6 weeks

Urine; F2-IsoP @ baseline and at 8 weeks

Sample type; biomarker; time of measurement

32 mg a-tocopherol/day

None indicated

EPA: 6.8 mg a-tocopherol/ day, 3.6 mg gtocopherol/day; DHA: 6.4 mg a-tocopherol/day, 3.6 mg g-tocopherol/ day; OO: 4.0 mg atocopherol/day, 3.6 mg g-tocopherol/day None indicated

None indicated

Antioxidant

Results

Plasma lipid hydroperoxides decreased by *10% in both young and older subjects in EPA group

Plasma F2-IsoP decreased significantly in n-3 group

Both plasma and urinary F2-IsoP decreased by n3 PUFA, but significant only for plasma

F2-IsoP decreased 20% by DHA and 19% by EPA relative to OO

F2-IsoPs decreased by 20% in fish fed and fish fed + moderate exercise groups

Human Studies in Which n-3 PUFA Decreased Lipid Peroxidation

n-3 PUFA dose; intervention duration; placebo/ comparison; study design

Table 2.

(continued)

Broad age range; lowsensitivity nonspecific LPO quantification methods

Potentially increased oxidative stress due to pregnancy; high-sensitivity LPO detection methods, may have contributed to the results Decrease detected despite healthy subjects; may be due to the high sensitivity of LPO detection methods

Increased oxidative stress due to HT and NIDDM; highsensitivity LPO detection methods may have contributed to the results

Increased oxidative stress due to NIDDM; high sensitivity LPO detection methods may have contributed to the results

Comments

409

18

Study A: Overweight, dyslipidemic men (20– 65 years); n = 17–20/ group. Study B: Hypertensive type 2 diabetic and postmenopausal women (40–75); n = 16–18

Healthy males (53–65); n = 12

Healthy men and women (20–40 years); n = 67– 74/group

Participants (age); n

Groups: (1) 4 grams/day EPA, (2) 4 grams/day DHA, (3) placebo; 6 week; 4 grams/day OO; both studies, the same three groups

EPA + DHA/day intake: (1) Cod diet = 227 – 29 mg/day; (2) salmon diet = 1418 – 34 mg/ day; (3) fish oil supplement = 3003 – 128 mg/ day; 8 weeks; daily EPA + DHA intake for SFA placebo: 5.6 – 0.2 mg/day; parallel randomized placebocontrolled, intervention 200, 400, 800, and 1600 mg/day DHA; 2 weeks each dose; no placebo; successive dose intervention

Plasma; F2-IsoP @ baseline and at 6 weeks

Urine; F2-IsoP @ baseline and end of each 2-week dose, and 8 weeks after end of supplement. Plasma; ox-LDL, MDA, 4HHE, and 4HNE @ baseline and at end of each 2week dose, and 8 weeks after end of supplement

Plasma; MDA and total antioxidant capacity (colorimetric assay); @ baseline and 56 days

Sample type; biomarker; time of measurement Antioxidant

EPA: 6.8 mg a-tocopherol/ day, 3.6 mg gtocopherol/day DHA: 6.4 mg atocopherol/day, 3.6 mg g-tocopherol/day

Each capsule contained 200 mg DHA and 0.125 mg DL-atocopherol and 0.125 mg ascorbic palmitate (1–8 capsules/ day)

None indicated

Table 2. (Continued)

Urinary F2-IsoP significantly lower after 200 mg/day DHA, increased significantly in the 1600 mg/day group. MDA: Concentrations significantly decreased after 200,400, and 800, not 1600 mg DHA. 4HHE: No changes after 200 and 400 mg DHA, but increased with 800 and 1600 mg DHA 4HNE: No change. ox-LDL: Significant increased lag time to oxidation in 200- to 800-mg group Study A: Plasma F2-IsoP decreased 24% by EPA and 14% by DHA relative to OO. Study B: Plasma F2IsoP decreased 23% by DHA and 19% by EPA relative to OO

Total plasma antioxidant capacity increased in all dietary groups except control, but increase was significant only in the cod-based diet. MDA significantly decreased only in cod based diet

Results

Dyslipidemia and HT may have compromised oxidative stress; LPO was decreased by n-3 PUFA and not by OO in both studies. Potentially increased oxidative stress prior to joining the study, high concentration of antioxidants, and highsensitivity. LPO quantification method may have contributed to the results

Effect observed despite healthy subjects, short duration and high concentrations of antioxidants. Because the dose was increased every 2 weeks, effects seen at higher doses may be due to the dose and increased duration. Study used highsensitivity specific LPO quantification methods. Increase in HHE expected as longer supplementation should lead to inclusion of n-3 PUFA in membranes

Source of dietary n-3 PUFA impacted oxidative or antioxidative effects. This may be due to the interaction of components of diet other than n-3 PUFA

Comments

n-3 PUFA, n-3 polyunsaturated fatty acids; NIDDM, non-insulin-dependent diabetes mellitus; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; F2-IsoP, F2- isoprostanes; LPO, lipid peroxidation; OO, olive oil; HT, hypertension; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; LOOH, lipid hydroperoxides; ox-LDL, oxidized low-density lipoprotein; GSSH, oxidized glutathione; MDA, malondialdehyde; HHE, 4-hydroxyhexenal; 4HNE, 4-hydroxynonenal.

Mas et al.25

Guillot et al.24

Parra et al.

Authors

n-3 PUFA dose; intervention duration; placebo/ comparison; study design

410

13

Palozza et al.16

Suzukawa et al.15

Harats et al.14

Meydani et al.

Authors

Stable uncomplicated hypertensive adults treated with blood pressure–lowering medication (mean = 60 – 10); n = 10/group Healthy men and women (25–46 years); n = 10/group

Young females (22–35 years) and older females (51–71 years); n = 14 (young) and 9 (older) Male and female smokers and nonsmokers; 3 study groups: mean age(s): Group A (smokers) = 37.4, Group B (smokers) = 29.1 and 35.2, Group C (nonsmokers) = 41.0 and 38.8); Group A: n = 5–6, Group B: n = 3–4 Group C: n = 6–8

Participants (Age); n

Groups: (1) 1.4 grams EPA + 1.1 grams DHA/day, (2) 2.7 grams EPA + 2.4 grams DHA/day, (3) 4.1 grams EPA + 3.6 grams DHA/day; 180 days; placebo, 7.2 grams blend of 25% saturated, 65% oleic and 10% LA; placebocontrolled parallel study

1680 mg EPA + 720 mg DHA/ day (600 mg other fatty acids); 3 months; no placebo; randomized intervention with before and after comparison Groups: A = FO, B = FO + antioxidants, C = FO and FO + antioxidants; Group A: 10 grams FO/day, Group B: same as A + 400 mg/day a-tocopherol, Group C: 10 grams/day FO and 10 grams/day FO + 400 mg/ day a-tocopherol (FO supplement contained 16.9% EPA + 11 % DHA); no placebo; 4 weeks; randomized controlled intervention 1.642 grams EPA + 1.210 grams DHA/day; 6 weeks; 3.4 grams corn oil/day; randomized double-blind crossover Plasma; LOOH and MDA @ baseline, 30 days, and 180 days

Plasma; MDA and ox-LDL;@ baseline and at 6 weeks

Plasma; MDA and LOOH; @ baseline and 4 weeks

Plasma; MDA and LOOH @ baseline & 1, 2, and 3 months

Sample type; biomarker; time of measurement

0 .9 mg DL-atocopherol, 1.5 mg DL-atocopherol, 2.1 mg DL-atocopherol/day. No atocopherol included in placebo capsules

2.0 mg atocopherol/mL FO and 2.2 mg a-tocopherol / mL CO

Indicated in the n-3 PUFA dosage; groups were created based partially on antioxidants

4 mg atocopherol/day

Antioxidant

30 days slight, dosedependent decrease in AAPH-induced lipid peroxidation in n-3 PUFA groups. At 180 days, n-3 PUFA treatment = dosedependent increase in MDA

Lag time of LDL oxidation significantly reduced compared to baseline. TBARS production from LDL significantly increased

TBARS; Study A: 50% increase; Study B: 38% increase; Study C: 33%. Individuals receiving atocopherol, no increase in plasma TBARS

Plasma MDA increased in both groups up to 2 months of supplementation then declined, but remained higher than baseline levels

Results

Human Studies in Which n-3 PUFA Increased Lipid Peroxidation

n-3 PUFA dose; intervention duration; placebo/ comparison; study design

Table 3.

(continued)

LPO increased with increased doses of n-3 PUFA; effect was biphasic with an initial decrease and a later increase in LPO

LPO significantly increased by n-3 PUFA despite increased intake of antioxidants; may be due to the health status of subjects

Low-sensitivity nonspecific LPO quantification methods; intervention includes lifestyles characterized by elevated levels of oxidative stress

Effect seemed to be biphasic

Comments

411

Maragine with 10% ALA (4.4 grams/day), EPA (2.2 grams/day), or DHA (2.3 grams/day) ethyl esters; randomized

Groups: (1) 2.8 grams/day EPA – 0.6 gram; (2) 2.9 grams/day DHA – 0.5 gram; 5 weeks; 6.0 grams/ day ALA – 1.1 gram; parallel randomized controlled dietary intervention 0.6 EPA + 0.4 DHA grams/ day alone or with additional antioxidants; placebocontrolled parallel study Plasmaconjugated dienes, MDA, LOOH; baseline and 6 weels, pre- and postexercise Plasma and RBC LPO, MDA and antioxidant capacity; baseline and 6 weeks

Plasma; ox-LDL; @ baseline and 5 weeks

Sample type; biomarker; time of measurement

Mixed tocopherols added

30 mg vitamin E/ day

2.0 mg atocopherol/day

Antioxidant

No change in antioxidant capacity; MDA increased in plasma but not RBCs

n-3 FA increased oxidative stress; did not reduce exercise-induced oxidative stress

Both EPA and DHA decreased lag time for LDL peroxidation compared to ALA group; both also increased the amounts of conjugated dienes

Results

No placebo group; DHA superior to EPA with EPA + DHA tissue incorporation

Antioxidants + n-3 FA decreased some LPO after judo training

Both EPA and DHA increased LPO compared to ALA

Comments

n-3 PUFA, n-3 polyunsaturated fatty acids; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; MDA, malondialdehyde; LOOH, lipid hydroperoxides; FO, fish oil; TBARS, thiobarbituric reactive species; LPO, lipid peroxidation; ox-LDL, oxidized low-density lipoprotein; CO, corn oil; AAPH, 2,2¢-azobis-(2-amidinopropane); ALA, a-linolenic acid; FA, fatty acid; RBC, red blood cells.

Healthy men and women (19–45 years); n = 24–25/ group

Egert et al.66

Healthy men and women (18–45 years); n = 15–17/ group

Participants (Age); n

Male judo Competitors (22–23 years); n = 8–10/ group

17

Filaire et al.65

Egert et al.

Authors

n-3 PUFA dose; intervention duration; placebo/ comparison; study design

Table 3. (Continued)

412

compared supplements versus diets containing salmon and cod.18 All studies used a-tocopherol, ranging from 2 to 400 mg/day as an antioxidant.13–15,17 Thus, the increase in LPO in these studies could not be attributed to the lack of antioxidant nutrients because the concentrations of atocopherol supplemented were comparable or higher than used in studies that showed no change or a decrease in LPO. The subjects studied were both men and women and ranged in age from 18 to 71 years. Only one study used a single gender with young and elderly women.13 Other studies used both genders with subjects who were cigarette smokers, hypertensive, or healthy. All studies showing an increase in LPO following n-3 PUFA administration utilized combinations of MDA, lipid hydroperoxides, and oxidation of LDL to assess LPO.13–18 Because all of these methods involve ex vivo sample preparation, oxidation, or both, it is possible that the increases in LPO were introduced ex vivo and did not reflect in vivo LPO.

Reasons for the discrepancies Whereas all 22 studies examined the effects of n-3 PUFA on markers of lipid peroxidation, there were differences in individual characteristics of each study, and therefore, the results obtained. Some possible explanations center on the quantification methods of LPO, study subject characteristics, n-3 PUFA composition, dosages and duration, and the concentrations of other pro- or anti-inflammatory nutrients in diet. Biomarkers and quantification methods. The most notable differences among all studies were the biomarkers and quantification methods used. Most studies demonstrating no change or an increase in LPO relied on nonspecific quantification methods of LPO, such as MDA, lipid hydroperoxides, and oxidized LDL. The use of thiobarbituric acid to assay MDA and lipid hydroperoxides presents specificity issues because some of these species can result from nonlipid peroxidation origins. Unwarranted increases in reactive aldehyde species alone are not the only concern. Chemical influences that perturb TBAR values can result in either a perceived increase or decrease in quantified MDA. Due to the complex milieu of biological samples and large number of reactive species, the reliability of MDA as an indicator of lipid peroxidation is questionable. Some investigators have taken this into account when interpreting their results. It is difficult to exclude all reactive species that appear naturally in biological samples and could influence the results of MDA quantification. In addition, studies using HPLC to quantify MDA may also lack reliability due to the volatility of MDA, which can impede accurate detection. Of the studies surveyed in this review that used MDA as an LPO biomarker, only three24,31,32 quantified MDA via HPLC, whereas the remainder used the TBARS method for analysis. HPLC methods were used in studies that found no change or a decrease in LPO. However, more studies that incorporate HPLC detection methods need to be published before a definitive conclusion can be made. Other classical methods used to assess lipid peroxidation, such as LDL oxidation susceptibility, reduced GSH levels, and total antioxidant status, produced results that are questionable due to concerns regarding specificity and sensitivity. Each of those methods is better classified as a general indicator of oxidative stress and not necessarily as specific

KELLEY, YOSHIDA, AND ERICKSON

assessments of lipid peroxidation. Therefore, it is preferable to use biomarkers when investigators can be certain of the lipid substrate precursors. For example, AA containing phospholipids are common membrane components, and they can be modified via oxidative and enzymatic pathways. F2IsoP is specifically generated from the free radical–mediated oxidative degradation of membrane-bound AA. Thus, F2IsoP is a valuable biomarker because it only represents the oxidative degradation of a specific membrane component in vivo. Of the eight studies incorporating F2-IsoPs as LPO biomarkers after n-3 PUFA administration, six reported a decrease and two reported no change. This high level of consistency among the studies was unique. All other biomarkers produced variable results, including no change, an increase, or a decrease. Besides of their high level of consistency, F2-IsoP measurements were much more sensitive than classical methods of LPO detection. Study participants. The age and health status of subjects studied varied greatly. The age could have influenced results because older subjects are expected to have reduced antioxidant capacity. Also, several studies investigated subjects with either lifestyles or disease states characterized by elevated levels of oxidative stress. These studies included cigarette smokers, a lifestyle known to increase oxidative stress and directly promote the oxidation of LDL. In terms of the health status, studies incorporated individuals that were hypertensive, type 2 diabetic, pregnant, postmenopausal, hypertriglyceridemic, or obese; some were also healthy. Thus, the extensive subject variety and respective levels of oxidative stress could have contributed to the discrepancy in the results. Administered dosages. Large variations in the concentration of n-3 PUFA were used; their specific fatty acid composition and the duration of the supplements may have also influenced results. All studies included in this review used less than 5 grams of EPA + DHA per day. Although one study14 used 10 grams/day fish oil, the EPA (16.9%) and DHA (11%) amounts were less than 5 grams/day. Most studies used fish oil supplements containing both EPA and DHA with varying amounts and ratios of each fatty acid. One study used purified samples of DHA or EPA. A few studies administered n-3 PUFA as part of the diet, mainly through fish or enriched foodstuffs such as margarine. The composition and amounts of fatty acids may vary extensively among different types of fish oil. Moreover, the major n-3 fatty acids have different protective effects. To compare results among studies with extensive variation of fatty acid sources, we reported the EPA and DHA concentrations. One study tested DHA doses of 200, 400, 800, and 1600 mg/day, and different methods to determine markers of LPO.24 Urinary F2-IsoP concentrations decreased with 200 mg/day DHA but increased with 1600 mg/day, whereas plasma MDA decreased with 200–800 mg/day. Thus, it appears that the overall fatty acid and antioxidant composition of n-3 PUFA supplements and diets may have influenced the generation of markers of LPO. Duration of supplementation. There was also considerable variation in the duration of supplementation of n-3 PUFA. One study used single administration, whereas others spanned 5 months. An acute administration of EPA and DHA did not alter total antioxidant status, oxidized LDL, or lipid hydroperoxides.34 Alternatively, the apparent rationale for using much longer periods of n-3 PUFA supplementation

n-3 PUFA EFFECTS ON LIPID PEROXIDATION IN HUMANS

in many of the other studies was to allow time for the incorporation of n-3 PUFA into membrane phospholipids. One study incorporated a stepwise increased concentration of n-3 PUFA every 2 weeks.24 Thus, differences in a number of factors probably contributed to the discrepancies in results among different studies.

Mechanisms by which n-3 may decrease lipid peroxidation n-3 PUFA can serve as antioxidants either as scavengers for reactive oxygen species (ROS), or by modulating the metabolism of n-6 PUFA. The latter would be through the expression of a variety of enzymes that regulate a balance between the oxidative and antioxidative forces. Superoxide scavenging capacity depends on the number of double bonds in a fatty acid.36 Because the most common n-3 PUFA (ALA, EPA, DHA) have more double bonds than the most common n-6 PUFA [linoleic acid (LA) and AA], the n-3 PUFA would have a greater scavenging capacity than n-6 PUFA of the same chain length. In addition to the number of double bonds, the position of double bonds seems to be important in determining peroxidation of PUFA. Thus, in HT-29 human colorectal tumor cells cultured with different 22C fatty acids and less than 2% oxygen, delta 7, 10, 13, and 16 were the most sensitive to peroxidation, whereas delta 4 and 19 were involved in assimilation of those 22C fatty acids into phospholipids.51 Thus, both n-6 and n-3 PUFA can act as scavenger molecules, but n-3 seems to be more effective than n-6 PUFA. EPA and DHA are incorporated into cellular membranes by replacing AA. This fatty acid replacement in the phoshopholipids can occur during cellular maturation and also by direct plasma exchange via transfer of serum albuminassociated DHA and EPA containing lysophosphatidylcholine.52 This decreases the amount of membrane AA, which serves as a precursor for the F2-IsoPs. Thus, in a human study, supplementing diets with DHA (3 grams/day for 90 days) decreased the red blood cell-AA content from 14.7% to 10.3%, and increased DHA from 2.4% to 8.1% and EPA from 0.44% to 0.96%.53 Such replacement of AA with n-3 PUFA decreased the plasma F2-IsoPs, but this may simply be due to the decreased availability of AA and the percent of oxidation of AA may not have decreased. Most of the published data are plasma or urinary isoprostanes that are expressed on the basis of milligrams of lipids or milliliters of plasma or urine, not on the basis of AA concentration. To have a better understanding of the LPO, results should be expressed on the basis of AA; LPO products of EPA and DHA should also be monitored.54,55 In addition to limiting the incorporation of AA into cell membranes, DHA also inhibits phospholipase A2 (PLA2), which releases free AA to be used by the cyclooxygenase, lipoxygenase, and P450 enzymes. Furthermore, EPA competes with AA for these enzymes.52 The oxygenation products of AA are much more proinflammatory than those produced from EPA and DHA. Thus, a decrease in inflammation may be another mechanism by which n-3 PUFA reduce LPO. EPA and DHA also increased cellular concentrations of reduced glutathione, glutathione peroxidase, and superoxide dismutase and decreased that of catalase, which improved the antioxidant status.56–59 Studies conducted in mice and HepG2-8 cells indicated that DHA decreased the expression of NRF2 which increased expression of the above enzymes that in turn reduced oxidative stress.60,61 Global gene arrays

413

of the white blood cells from human subjects who supplemented their diets with DHA revealed that DHA supplementation decreased the expression of several genes found in white blood cells that are involved in lipid peroxidation, including cytochrome p450, oxidized LDL receptor 1, prostaglandin E synthase, cathepsin L, and several others.62 These findings support the conclusion that DHA decreased LPO. This is further supported by the decrease in plasma concentration of oxidation products of cholesterol and fatty acids caused by DHA. Collectively, these findings suggest that n-3 PUFA decrease lipid peroxidation; however, this remains controversial because of the effects of a number of confounding factors.

Conclusion and Future Research Directions On the basis of the relevant literature, the main cause for variations in assessments of lipid peroxidation was the methods used. Studies incorporating nonspecific methods that measured MDA, lipid hydroperoxides, or oxidized (ox)LDL resulted in extensive variation, including no change as well as increased or decreased LPO. Most investigators who used methods specific for in vivo LPO products, such as F2IsoP, concluded that n-3 PUFA supplementation decreased LPO. On the basis of the results with F2-IsoP, n-3 PUFA supplementation did not increase LPO and could be considered as safe for human consumption. It is possible that doses greater than 5 grams/day may increase LPO; thus, it is hard to justify doses of EPA + DHA higher than 5 grams/day at this time. While appearing safe, additional work must still be conducted to definitively demonstrate that n-3 PUFA supplementation does not increase LPO. For those studies, specific improvements in the assessment of LPO must be incorporated. For example, instead of nonspecific methods, investigators should employ a panel of substrate-specific, high-sensitivity tests that determine the in vivo concentrations of LPO products. Similarly, tests for other metabolites produced should be specific for the oxidative degradation products from membrane components. Examples of metabolites that should be considered in future studies include HODE, produced from LA, HETE from AA, as well as a and b LPO products from cholesterol. Those metabolites, as part of a comprehensive panel including biomarkers such as F2-IsoP, HNE, and HHE, should provide a more thorough and specific assessment of in vivo lipid peroxidation. The basal diet is likely to play a critical role in the production of LPO products before and after n-3 PUFA supplementation. Several nutrients, including minerals (such as Cu, Zn, Se, and Fe), vitamins (like E, C, and A), amino acids, as well as fatty acids, can all affect the oxidative status in the cells and the resulting LPO. Therefore, to compare results among different studies, it is important that standardized diets be used with a defined fatty acid and antioxidant composition. It is debatable whether DHA is more protective than EPA, therefore studies should be done with the two fatty acids individually as well as mixtures to determine their additive, synergistic, or antagonistic effects. Dose responses, which have generally not been done, are necessary to determine the efficacy, adverse effects, or the duration of the alterations observed. Study subject heterogeneity has made results difficult to interpret. Mixing subjects with a broad range of ages and health status adds a confounding variable and may skew the results for individual groups.

414

Author Disclosure Statement No conflicting financial interests exist. N.S.K. and K.L.E. designed the project; N.S.K. collected the data; N.S.K., Y.Y., and K.L.E. analyzed the data; N.S.K., Y.Y., and K.L.E. wrote the paper; K.L.E. had primary responsibility for final content. All authors read and approved the final manuscript

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Address correspondence to: Kent L. Erickson, PhD Department of Cell Biology and Human Anatomy University of California, Davis School of Medicine One Shields Avenue Davis, CA 95616 E-mail: [email protected]