E m e r g i n g T r e a t m e n t s a n d T e c h n o l o g i e s
`B R I E F
`R E P O R T
`
`Purified Eicosapentaenoic Acid Reduces
`Small Dense LDL, Remnant Lipoprotein
`Particles, and C-Reactive Protein in
`Metabolic Syndrome
`
`1
`NORIKO SATOH, MD, PHD
`1
`AKIRA SHIMATSU, MD, PHD
`KAZUHIKO KOTANI, MD, PHD
`1
`NAOKI SAKANE, MD, PHD
`
`1
`
`2
`KAZUNORI YAMADA, MD, PHD
`TAKAYOSHI SUGANAMI, MD, PHD
`2
`HIDESHI KUZUYA, MD, PHD
`YOSHIHIRO OGAWA, MD, PHD
`
`3,4
`
`3
`
`E icosapentaenoic acid (EPA), one rep-
`
`drome, a precursor of CVD. Furthermore,
`sdLDL has been reported to synergisti-
`cally interact with inflammation in patho-
`physiologic processes leading to CVD (8).
`Therefore, we simultaneously measured
`effects of EPA on C-reactive protein
`(CRP), a marker of inflammation, and ex-
`amined how alteration of lipoprotein pro-
`fi l e s b y E P A a f f e c t s
`s y s t e m i c
`inflammation.
`
`resentative of n-3 unsaturated fatty
`acids (n-3 PUFAs), is clinically used
`for its lipid-lowering effects (1). n-3
`PUFAs were shown to exert various phys-
`iological functions such as antiplatelet ac-
`tions (by antagonizing effects of
`arachidonic acid) and plaque stabilization
`(2,3). Several epidemiological studies
`have explored antiatherogenic and car-
`dioprotective effects of n-3 PUFA that are
`RESEARCH DESIGN AND
`abundantly contained in fish oil (4). Dys-
`METHODS — A total of 44 Japanese
`lipidemia accompanying the metabolic
`syndrome is often associated with ele-
`obese type 2 diabetic patients were re-
`vated levels of remnant lipoprotein parti-
`cruited in our clinics (Table 1). All pa-
`cles and small dense LDL (sdLDL), which
`tients satisfied the definition and
`are newly recognized risk factors for car-
`diagnostic criteria of the metabolic syn-
`diovascular disease (CVD) (5). It was re-
`drome proposed by the National Meta-
`ported that fish oil improved lipoprotein
`bolic Syndrome Criteria Study Group of
`subclass profiles in subjects with an
`Japan in 2005 (9). Accordingly, an indi-
`atherogenic lipoprotein phenotype (6).
`vidual is diagnosed with metabolic syn-
`Besides EPA, docosahexaenoic acid and
`drome if he or she has central adiposity
`cholesterol are present in fish oil (7), but
`plus two or more of the following three
`it is not clear whether purified EPA inde-
`factors: 1) raised concentration of triglyc-
`erides (ⱖ150 mg/dl) or reduced concen-
`pendently affects lipoprotein subclass
`tration of HDL cholesterol (⬍40 mg/dl);
`profiles. Therefore, we used purified EPA
`ethyl ester and examined effects of EPA on
`2) raised blood pressure: systolic blood
`pressure (ⱖ130 mmHg) or diastolic
`atherogenic sdLDL particles and remnant
`blood pressure (ⱖ85 mmHg) or treat-
`lipoprotein particles in the metabolic syn-
`● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
`
`From the 1Clinical Research Institute for Endocrine Metabolic Diseases, National Hospital Organization,
`Kyoto Medical Center, Kyoto, Japan; the 2Diabetes Center, National Hospital Organization, Kyoto Medical
`Center, Kyoto, Japan; the 3Department of Molecular Medicine and Metabolism, Medical Research Institute,
`Tokyo Medical and Dental University, Tokyo, Japan; and the 4Center of Excellence Program for Frontier
`Research on Molecular Destruction and Reconstitution of Tooth and Bone, Tokyo Medical and Dental
`University, Tokyo, Japan.
`Address correspondence and reprint requests to Noriko Satoh MD, PhD, Clinical Research Institute for
`Endocrine Metabolic Diseases, National Hospital Organization, Kyoto Medical Center, 1-1 Fukakusa Mu-
`kaihata-cho, Fushimi-ku, Kyoto 612-8555, Japan. E-mail: nsato@kyotolan.hosp.go.jp.
`Received for publication 7 June 2006 and accepted in revised form 6 October 2006.
`Abbreviations: CETP, cholesteryl ester transfer protein; CRP, C-reactive protein; CVD, cardiovascular
`disease; EPA, eicosapentaenoic acid; n-3 PUFA, n-3 unsaturated fatty acid; RLP, remnant lipoprotein parti-
`cle; sdLDL, small dense LDL.
`A table elsewhere in this issue shows conventional and Syste`me International (SI) units and conversion
`factors for many substances.
`DOI: 10.2337/dc06-1179
`© 2007 by the American Diabetes Association.
`The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby
`marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`
`ment of previously diagnosed hyperten-
`sion; and 3) raised fasting plasma glucose
`concentration (ⱖ110 mg/dl). The thresh-
`olds for waist circumference to define
`central adiposity were ⱖ85 cm for men
`and ⱖ90 cm for women. The study pro-
`tocol was approved by the ethical com-
`mittee on human research of the Kyoto
`Medical Center, and all participants gave
`written informed consent. Patients were
`assigned to one of the following treatment
`groups (a single-blind and a run-in period
`randomization, which patients received):
`1) treated for 3 months with either diet
`alone (the control group) (8 men and 14
`women; mean ⫾ SE age 51.6 ⫾ 3.2 years)
`or 2) diet plus EPA (1.8 g daily) (the EPA
`group) (8 men and 14 women; mean age
`51.6 ⫾ 2.8 years). The subjects in the EPA
`group received an EPA capsule containing
`highly purified (⬎98%) EPA ethyl ester.
`Patient’s diets are based on the Japan Ath-
`erosclerosis Society Guidelines for Diag-
`nosis and Treatment of Atherosclerotic
`Cardiovascular Diseases, consisting of 25
`kcal/kg of ideal body weight per day (60%
`of total energy as carbohydrates, 15–20%
`as protein, and 20 –25% as fat with the
`ratio of polyunsaturated, monounsatu-
`rated, and saturated fatty acids being 3:4:
`3). Lipid-lowering medications such as
`statins and fibrates were excluded.
`At the beginning and at the end of the
`study, we measured BMI, serum levels of
`EPA and arachidonic acid, and glycolipid
`parameters according to standard proce-
`dures. Remnant lipoprotein particle
`(RLP) cholesterol and RLP triglycerides
`were measured using an assay kit (Japan
`Immunoresearch Laboratories, Takasaki,
`Japan) (10). Plasma cholesteryl ester
`transfer protein (CETP) activity was mea-
`sured using an assay kit (BioVision,
`Mountain View, CA) (11). LDL choles-
`terol subfractions were separated using
`the Quantimetrix Lipoprotein LDL sys-
`tem (12). According to the specific sub-
`fractions of LDL cholesterol obtained by
`this system (LDL3–7:sdLDL), the sdLDL
`proportion was defined as the percentage
`of sdLDL over the whole amount of LDL
`(13). Plasma level of CRP was measured
`
`144
`
`DIABETES CARE, VOLUME 30, NUMBER 1, JANUARY 2007
`
`Hikma Pharmaceuticals
`
`IPR2022-00215
`
`Ex. 1006, p. 1 of 3
`
`

`

`Satoh and Associates
`
`Table 1—Baseline characteristics and effects of EPA on metabolic parameters, lipoprotein profiles, and CRP
`
`Control
`
`EPA
`
`3 months
`Before
`3 months
`Before
`30.8 ⫾ 1.26
`31.0 ⫾ 1.22
`29.1 ⫾ 0.92
`29.2 ⫾ 0.91
`BMI (kg/m2)
`125 ⫾ 12.3†‡
`77.2 ⫾ 11.5
`64.6 ⫾ 11.6*
`79.2 ⫾ 17.8
`EPA (␮g/ml)
`152 ⫾ 9.72†
`165 ⫾ 7.34
`148 ⫾ 8.08
`145 ⫾ 12.1
`Arachidonic acid (␮g/ml)
`0.88 ⫾ 0.11†‡
`0.48 ⫾ 0.07
`0.44 ⫾ 0.08*
`0.52 ⫾ 0.10
`EPA/arachidonic acid
`6.00 ⫾ 0.52
`6.16 ⫾ 0.57
`7.16 ⫾ 0.62
`6.88 ⫾ 0.47
`FPG (mmol/l)
`6.02 ⫾ 0.24
`6.11 ⫾ 0.20
`6.47 ⫾ 0.26
`6.57 ⫾ 0.25
`A1C (%)
`95.4 ⫾ 15.1
`104 ⫾ 23.1
`100 ⫾ 12.0
`101 ⫾ 14.5
`Insulin concentration (pmol/l)
`5.15 ⫾ 0.16
`5.46 ⫾ 0.19
`5.30 ⫾ 0.24
`5.38 ⫾ 0.21
`Total cholesterol (mmol/l)
`3.18 ⫾ 0.15
`3.44 ⫾ 0.15
`2.97 ⫾ 0.19
`3.18 ⫾ 0.16
`LDL cholesterol (mmol/l)
`1.39 ⫾ 0.07
`1.42 ⫾ 0.08
`1.38 ⫾ 0.11
`1.46 ⫾ 0.07
`HDL cholesterol (mmol/l)
`1.57 ⫾ 0.11
`1.93 ⫾ 0.20
`1.62 ⫾ 0.21
`1.89 ⫾ 0.19
`Triglyceride (mmol/l)
`0.15 ⫾ 0.01
`0.19 ⫾ 0.02
`0.16 ⫾ 0.03
`0.19 ⫾ 0.02
`RLP cholesterol (mmol/l)
`0.26 ⫾ 0.02*§
`0.43 ⫾ 0.09
`0.58 ⫾ 0.16
`0.44 ⫾ 0.06
`RLP triglyceride (mmol/l)
`CETP activity (nmol 䡠 ml⫺1 䡠 h⫺1)
`200 ⫾ 1.75*
`205 ⫾ 3.05
`202 ⫾ 2.26
`203 ⫾ 1.47
`0.18 ⫾ 0.04†
`0.23 ⫾ 0.04
`0.23 ⫾ 0.08
`0.26 ⫾ 0.08
`sdLDL (mmol/l)
`5.51 ⫾ 1.09†
`6.78 ⫾ 1.28
`6.82 ⫾ 2.36
`7.33 ⫾ 2.18
`sdLDL proportion (%)
`0.08 ⫾ 0.02*
`0.22 ⫾ 0.08
`0.10 ⫾ 0.03
`0.11 ⫾ 0.03
`CRP (mg/dl)
`Data are means ⫾ SE. *P ⬍ 0.05, †P ⬍ 0.01 vs. before determined by two-tailed, paired t test. ‡P ⬍ 0.01, §P ⬍ 0.05 vs. control determined by Student’s t test. FPG,
`fasting plasma glucose.
`
`by the latex-enhanced assay using the
`particle-enhanced technology performed
`on the Behring BN nephelometer (Dade
`Behring, Marburg, Germany) (14). Data
`are presented as mean ⫾ SE, and P ⬍ 0.05
`was considered statistically significant.
`Repeated-measures ANOVA (control and
`EPA groups ⫻ before and after treatment)
`was used to access the comparative effects
`of EPA treatment on the measured vari-
`ables. A two-tailed, paired t test was ap-
`plied for the evaluation of changes from
`baseline conditions to those at 3 months.
`Comparisons of the means between the
`two groups at baseline or posttreatment
`were performed by Student’s t test. All sta-
`tistical analyses were performed using the
`Stat View program version 5.0 for Win-
`dows (SAS Institute, Cary, NC).
`
`RESULTS — There were no significant
`differences between the control and EPA
`groups for all measured variables at baseline
`(Table 1). Treatment with EPA significantly
`increased EPA and EPA/arachidonic acid
`levels, while it decreased arachidonic acid
`levels compared with baseline levels (P ⬍
`0.01). Differences of EPA and EPA/
`arachidonic acid levels at 3 month were ob-
`served between the control and EPA groups
`(P ⬍ 0.01). EPA also caused significant
`overall effects on RLP triglyceride, CETP ac-
`tivity, sdLDL, and the proportion of sdLDL
`and CRP by repeated-measures ANOVA.
`There were also significant reductions in
`values compared with baseline by paired t
`
`test, despite no changes in BMI, fasting
`plasma glucose, A1C, insulin concentra-
`tion, and HDL cholesterol in both groups.
`Significant reductions of total cholesterol,
`LDL cholesterol, triglycerides, and RLP cho-
`lesterol in the EPA group was observed (P ⫽
`0.035, 0.004, 0.047, and 0.035, respec-
`tively) by two-tailed, paired t test, although
`there were no significant overall effects on
`those parameters by repeated-measures
`ANOVA. Increases in EPA/arachidonic acid
`for 3 months inversely correlated with de-
`creases in RLP cholesterol, sdLDL, and CRP
`for 3 months (P ⫽ 0.0379, 0.0479, and
`0.0467, respectively). Furthermore, reduc-
`tion in CRP with EPA treatment for 3
`months showed a significant positive corre-
`lation with reductions in RLP cholesterol
`and sdLDL for 3 months (P ⫽ 0.0075 and
`0.0142, respectively).
`
`CONCLUSIONS — This study is the
`first to demonstrate that EPA significantly
`reduces serum sdLDL and CRP in the
`metabolic syndrome. Reduction of sdLDL
`by EPA treatment in this study is believed
`to be due to a suppression of triglycerides
`production in the liver by EPA. In addi-
`tion, since CETP is an important enzyme
`in cholesterol metabolism—responsible
`for the transfer of cholesteryl esters from
`HDL to LDLs (11)— degradation of CETP
`activity by EPA treatment may also have
`contributed to the decrease in the gener-
`ation of remnants and sdLDL. Further-
`more, we detected that reductions in RLP
`
`cholesterol and sdLDL also correlated
`with a decrease in CRP by EPA, which was
`consistent with a previous report (8)
`showing that LDL particle size had a
`strong inverse association with CRP.
`Atherogenic sdLDL particles are suscepti-
`ble to oxidative modifications; then, oxi-
`dized LDL is easily taken into macrophages
`through damaged endothelial cells, thereby
`inducing inflammation and early athero-
`sclerotic lesions (5,15). On the other hand,
`CRP has also been shown to accelerate LDL
`modifications during inflammatory pro-
`cesses (8). These findings suggest that EPA
`may be capable of preventing the progres-
`sion of atherosclerosis in the metabolic
`syndrome by suppressing reciprocal in-
`teractions of atherogenic lipoproteins and
`inflammation. There are several reports
`demonstrating that n-3 PUFA does not
`decrease CETP protein mass and CRP
`(16,17). This may be caused by the differ-
`ences outlined in the research designs and
`methods of each report.
`Recently, the Japan EPA Lipid Inter-
`vention Study reported that EPA pro-
`vided further benefits in preventing major
`coronary events without changing reduc-
`tions in LDL cholesterol levels (18). Con-
`sidering the improvements in lipoprotein
`profiles by EPA in this study, EPA may
`exert cardioprotective effects not by
`changing the quantity but by improving
`the quality of LDL cholesterol.
`Collectively, the present study is the
`first to demonstrate that purified EPA re-
`
`DIABETES CARE, VOLUME 30, NUMBER 1, JANUARY 2007
`
`145
`
`Hikma Pharmaceuticals
`
`IPR2022-00215
`
`Ex. 1006, p. 2 of 3
`
`

`

`Effect of EPA on lipoprotein and CRP
`
`duces sdLDL, remnants, and CRP,
`thereby potentially leading to the reduc-
`tion in development of atherosclerosis
`and CVD in the metabolic syndrome.
`
`Acknowledgments — This work was sup-
`ported in part by a Grant-in-Aid for Scientific
`Research from the Ministry of Education, Cul-
`ture, Sports, Science, and Technology of Japan
`and research grants for cardiovascular diseases
`(16C-2 and 17C-5 to N.S.) from the Ministry
`of Health, Labor, and Welfare, the Smoking
`Research Foundation.
`We thank Seiho Kouno and Hajime Yamak-
`age for their valuable discussions, and Kokoro
`Tsuzaki for her excellent technical assistance.
`
`References
`1. Mizuguchi K, Yano T, Tanaka Y, Ishibashi
`M, Masada A, Mizota M, Fukutake K,
`Saito Y: Mechanism of the lipid-lowering
`effect of ethyl all-cis-5,8,11,14,17- icosa-
`pentaenoate. Eur J Pharmacol 231:121–
`127, 1993
`2. Hirai A, Terano T, Hamazaki T, Sajiki J,
`Kondo S, Ozawa A, Fujita T, Miyamoto T,
`Tamura Y, Kumagai A: The effects of the
`oral administration of fish oil concentrate
`on the release and the metabolism of
`[14C]arachidonic acid and [14C]eicosap-
`entaenoic
`acid by human platelets.
`Thromb Res 28:285–298, 1982
`3. Thies F, Garry JM, Yaqoob P, Rerkasem K,
`Williams J, Shearman CP, Gallagher PJ,
`Calder PC, Grimble RF: Association of n-3
`polyunsaturated fatty acids with stability
`of atherosclerotic plaques: a randomised
`controlled trial. Lancet 361:477– 485,
`2003
`4. Hu FB, Bronner L, Willett WC, Stampfer
`MJ, Rexrode KM, Albert CM, Hunter D,
`
`Manson JE: Fish and omega-3 fatty acid
`intake and risk of coronary heart disease
`in women. JAMA 287:1815–1821, 2002
`5. Krauss RM: Lipids and lipoproteins in pa-
`tients with type 2 diabetes. Diabetes Care
`27:1496 –1504, 2004
`6. Wilkinson P, Leach C, Ah-Sing EE, Hus-
`sain N, Miller GJ, Millward DJ, Griffin BA:
`Influence of alpha-linolenic acid and fish-
`oil on markers of cardiovascular risk in
`subjects with an atherogenic lipoprotein
`phenotype. Atherosclerosis 181:115–124,
`2005
`7. Nozaki S, Matsuzawa Y, Hirano K, Sakai
`N, Kubo M, Tarui S: Effects of purified
`eicosapentaenoic acid ethyl ester on
`plasma lipoproteins in primary hypercho-
`lesterolemia. Int J Vitam Nutr Res 62:256 –
`260, 1992
`8. St-Pierre AC, Cantin B, Dagenais GR,
`Mauriege P, Bernard PM, Despres JP,
`Lamarche B: Low-density lipoprotein
`subfractions and the long-term risk of
`ischemic heart disease in men: 13-year
`follow-up data from the Quebec Cardio-
`vascular Study. Arterioscler Thromb Vasc
`Biol 25:553–559, 2005
`9. Matsuzawa Y: Metabolic syndrome: defi-
`nition and diagnostic criteria in Japan. J
`Atheroscler Thromb 12:301, 2005
`10. Kugiyama K, Doi H, Takazoe K, Kawano
`H, Soejima H, Mizuno Y, Tsunoda R,
`Sakamoto T, Nakano T, Nakajima K,
`Ogawa H, Sugiyama S, Yoshimura M, Ya-
`sue H: Remnant lipoprotein levels in fast-
`ing serum predict coronary events in
`patients with coronary artery disease. Cir-
`culation 99:2858 –2860, 1999
`11. Bisgaier CL, Minton LL, Essenburg AD,
`White A, Homan R: Use of fluorescent
`cholesteryl ester microemulsions in cho-
`lesteryl ester transfer protein assays. J
`Lipid Res 34:1625–1634, 1993
`
`12. Hoefner DM, Hodel SD, O’Brien JF, Bra-
`num EL, Sun D, Meissner I, McConnell
`JP: Development of a rapid, quantitative
`method for LDL subfractionation with use
`of the Quantimetrix Lipoprint LDL Sys-
`tem. Clin Chem 47:266 –274, 2001
`13. Lee W, Min WK, Chun S, Jang S, Kim JQ,
`Lee do H, Park JY, Park H, Son JE: Low-
`density lipoprotein subclass and its corre-
`lating factors in diabetics. Clin Biochem 36:
`657– 661, 2003
`14. Satoh N, Ogawa Y, Usui T, Tagami T,
`Kono S, Uesugi H, Sugiyama H, Sugawara
`A, Yamada K, Shimatsu A, Kuzuya H, Na-
`kao K: Antiatherigenic effect of pioglita-
`zone in type 2 diabetic patients irrespective
`of the responsiveness to its antidiabetic
`effect. Diabetes Care 26:2493–2499, 2003
`15. Zhu H, Xia M, Hou M, Tang Z, Li Y, Ma J,
`Ling W: Ox-LDL plays dual effect in mod-
`ulating expression of inflammatory mole-
`cules through LOX-1 pathway in human
`umbilical vein endothelial cells. Front Bio-
`sci 10:2585–2594, 2005
`16. Calabresi L, Villa B, Canavesi M, Sirtori
`CR, James RW, Bernini F, Franceschini G:
`An omega-3 polyunsaturated fatty acid
`concentrate increases plasma high-den-
`sity lipoprotein 2 cholesterol and para-
`oxonase levels in patients with familial
`combined hyperlipidemia. Metabolism
`53:153–158, 2004
`17. Madsen T, Christensen JH, Blom M,
`Schmidt EB: The effect of dietary n-3 fatty
`acids on serum concentrations of C-reac-
`tive protein: a dose-response study. Br J
`Nutr 89:517–522, 2003
`18. Yokoyama M: Effects of eicosapentaenoic
`acid (EPA) on major cardiovascular
`events in hypercholesterolemic patients:
`the Japan EPA Lipid Intervention Study
`(JELIS) (AHA late-breaking clinical trial
`abstract). Circulation 112:3362, 2005
`
`146
`
`DIABETES CARE, VOLUME 30, NUMBER 1, JANUARY 2007
`
`Hikma Pharmaceuticals
`
`IPR2022-00215
`
`Ex. 1006, p. 3 of 3
`
`