Journal of Atherosclerosis and Thrombosis
Online ISSN : 1880-3873
Print ISSN : 1340-3478
ISSN-L : 1340-3478
Original Article
Effects of Nutrition Education Program for the Japan Diet on Serum Phospholipid Fatty Acid Compositions in Patients with Dyslipidemia: Re-analysis of Data from a Previous Randomized Controlled Trial
Chizuko Maruyama, Aisa Sato, Yui Nishikata, Mariko Nakazawa, Yuri Shijo, Noriko Kameyama, Ariko Umezawa, Makoto Ayaori, Masako Waki, Katsunori Ikewaki, Ai Nishitani, Tamio Teramoto
著者情報
キーワード: Nutrition education, Diet, Dyslipidemia, Fatty acids, Japanese
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2023 年 30 巻 12 号 p. 1849-1869

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Abstract

Aim: We investigated changes in serum phospholipid fatty acid compositions with intake of the Japan Diet (JD) (higher consumption of fish, soybeans, vegetables, seaweed/mushrooms/konjak, and unrefined cereals with reduced consumption of animal fat, meat and poultry with fat, sweets and alcoholic drinks) recommended by the Japan Atherosclerosis Society.

Methods: A randomized parallel controlled clinical trial on JD intake was conducted on Japanese patients with dyslipidemia. Nutrition education, based on the JD or partial JD (PJD) at baseline and at 3 months, was provided and the participants were followed up for 6 months. Fatty acids comprising serum phospholipids were measured in the JD (n=44) and PJD (n=44) groups.

Results: Fatty acid intakes of C20:4, C20:5 and C22:6 increased in the JD group as compared with the PJD group. The percentages of serum phospholipid, C22:1 and C20:5 increased, while those of C18:1, C20:3(n-6) and C20:4(n-6) decreased in the JD as compared with the PJD group at 3 months. Changes in the phospholipid concentrations of C20:5, C22:5 and C22:6 reflected those intake volumes. Serum phospholipid C20:5 and C22:6 showed inverse correlations with C18:1, C18:2, and C20:3(n-6) at baseline and the changes at 3 and 6 months. In contrast, no correlation was observed between C20:4(n-6) and those n-3 fatty acids. The ratios of fatty acid concentrations, C16:1/C16:0 and C18:1/C18:0, decreased, but the ratio of C20:4(n-6)/C20:3(n-6) increased in the JD group.

Conclusion: Nutrition education on the JD changed serum phospholipid fatty acid profiles in favor to prevent against cardiovascular risk factors in patients with dyslipidemia.

Introduction

The Japanese dietary style in the 1960s to 1970s, which was rich in fish, with relatively low consumptions of meat, poultry and animal fats, was recognized as cardioprotective1, 2). However, increased Westernization and diversification had resulted in greater consumption of meat and poultry than fish by 2009 and, even thereafter, meat and poultry and fat intakes continued to rise with concurrent declines in fish and vegetable intakes. The Japanese population has thus not maintained healthy eating habits in recent decades3, 4). These dietary changes have been regarded as being among the reasons for rising blood cholesterol levels in Japanese people, which in turn has produced an increased incidence of cardiovascular disease5-8).

The Japan Atherosclerosis Society (JAS) has recommended the Japan Diet (JD) (higher consumption of fish, soybeans and soy products, vegetables, seaweed/mushrooms/konjak, and unrefined cereals with reduced consumption of animal fat, fatty meat and poultry, sweets including desserts and snacks, and alcoholic drinks, along with reducing salt intake)9). The JD has a characteristic fatty acid content derived from seafood and plant sources. Recently, combinations of fatty acids have been recognized as being important in the etiologies of several diseases10) and combining foods as well as modifying volumes are now considered to be important in practice to improve the mix of fatty acids and to exert a protective effect against coronary heart disease11). We reported that JD intake changed the fatty acids to an antiatherogenic profile, e.g., increasing serum phospholipid eicosapentaenoic acid (C20:5), docosahexaenoic acid (C22:6) and odd-chain fatty acids, with an increase in the estimated delta-5 desaturase (D5D) activity in healthy volunteers12). However, the study design was single arm, with a short duration of only 6 weeks, and we studied only in healthy men.

We conducted a randomized controlled trial (RCT) on patients with dyslipidemia receiving a nutrition education program to clarify the effect of the JD, as described above, for comparison with the Partial Japan Diet (PJD). Recommendations for both groups included decreasing consumption of animal fats, such as fatty meats, confectionaries and alcohol. Furthermore, we reported reduced serum low density lipoprotein-cholesterol (LDL-C) and triglyceride (TG) concentrations in the JD as compared with the PJD group13). In order to investigate the long-term effect of JD on the fatty acid profile, we studied the fatty acid compositions of phospholipids.

Aim: We aimed to confirm beneficial changes in phospholipid fatty acid profiles in response to the JD as compared with the PJD in patients with dyslipidemia.

Methods

The study design was a 6-month randomized parallel-group clinical trial. Methods and subjects were described in detail in our previous report13). Briefly, the recruited participants were Japanese patients with dyslipidemia aged between 30 and 65 years of age, with a body mass index (BMI) over 18.5 kg/m2, nonsmokers, receiving consistent drug regimens and permitted to participate in the program by doctors certified by the JAS. This study was conducted at Japan Women’s University according to the guidelines of the Declaration of Helsinki, and all procedures were approved by The Ethics Committee for Experimental Research Involving Human Subjects of Japan Women’s University (No.246). Written informed consent was obtained from all subjects prior to participation. The clinical trial registration number is UMIN000022955.

Participants were allocated to either the JD group or the PJD group. For both diet groups, reduced intakes of animal fat, fatty meat and poultry, confectionaries, and alcoholic drinks were recommended. In addition, consuming more fish (especially fatty fish), soybeans and soy products (especially natto, a fermented soy product), vegetables including green and yellow varieties, seaweed, mushrooms, konjak and unrefined cereals including barley were recommended for the JD group. Face-to-face nutritional education for each diet, at baseline and again at 3 months, was provided by registered dietitians. The participants were followed up for 6 months in total.

Weighted dietary records were kept for two weekdays and 1 weekend day at baseline and at the ends of the 3- and 6-month follow-up periods. Nutrient and fatty acid intakes were calculated employing Excel Eiyokun ver. 8.0 (Kenpaku-sha Co., Ltd., Tokyo, Japan) software.

At baseline, as well as at 3 and 6 months, height and weight were measured, and BMI was calculated as weight (kg)/height (m)2. Waist circumference was determined with a measuring tape during the late exhalation phase with the subject in the standing position. Blood was collected in the morning following a 12-hour fast and serum samples were obtained and stored at −80℃ until analyses. The data on total cholesterol (TC), LDL-C, high density lipoprotein-cholesterol (HDL-C), TG, malondialdehyde-modified low-density lipoprotein (MDA-LDL), oxygen radical absorbance capacity (ORAC), high-molecular-weight (HMW) adiponectin and leptin, and the measurement methods for each were described in our previous report in detail13, 14) and were also used in this study.

Fatty acid concentrations in serum phospholipids were measured. Serum lipids were extracted according to the method of Folch et al.15). The lipid fractions were separated with thin-layer chromatography on silica gel with petroleum ether, diethyl ether and acetic acid (84.15: 15.0: 0.85 v/v/v) as solvent systems. The phospholipids fraction was scraped into screw cap glass tubes and methylated under argon gas in a thermostat at 100℃ for 30 minutes with 3.5% sulfuric acid in methanol using C19:0 as internal standard. The fatty acid methyl esters were separated by gas–liquid chromatography (Shimadzu GC14B, Tokyo, Japan) using a 0.25 mm × 30 m capillary column containing DB-FFAP (J&W Scientific, CA, USA). The injector temperature was 230℃, and the column temperature rose at 2℃/min from 160℃ to 200℃. Nitrogen was employed as the carrier gas, and the split ratio was 22.0. The coefficient of variation in each fatty acid lower than 5% was acceptable.

Statistical Analysis

Statistical analyses were carried out using SPSS for Windows (ver. 26; IBM Japan, Inc.). The outcomes were analyzed according to the intention-to-treat principle. Imputations were used to replace missing data, employing the last observation carried forward method. Normality was assessed by applying the Shapiro-Wilk test. All values are presented as means with standard deviation for the normal distribution and as medians (IQR) for nonnormal distributions. The t-test or the Mann-Whitney U test was used to compare mean differences between the PJD and JD groups. The paired t-test or the Wilcoxon’s signed-rank test was used to analyze differences from the baseline concentrations. The statistical significances of correlations were assessed using the Pearson’s or nonparametric Spearman’s ranked test. P＜0.05 was considered to indicate statistical significance.

Results

Participant detail from enrollment, randomization and intervention were described in our previous report13). A total of 98 patients participated in the intervention at baseline. Among them, seven who had been treated with omega-3 fatty acid ethyl and three without fatty acid measurements due to lack of serum were excluded. The remaining 88 participants were included in the analysis. Baseline characteristics of the participants are shown in Supplementary Table 1. The age was 54±8 (mean±SD) years, and 41 (46.6%) and 10 (11.4%) participants were taking LDL-C-lowering and TG-lowering drugs, respectively, and these percentages did not differ between the JD and PJD groups. There were no changes in pharmacotherapy for lipid abnormalities during the study period.

Supplementary Table 1.Baseline characteristics of the participants

Partial Japan Diet Japan Diet
Men / Women (n) 21 / 23 21 / 23
Age (years) 54.3±7.7 a 53.5±8.2
Type 2 diabetes 9 (20.5) b 6 (13.6)
Medications 35 (79.5) 34 (77.3)
LDL-cholesterol lowering agents 22 (50.0) 19 (43.2)
Strong statin 15 (34.1) 12 (27.3)
Standard statin 2 ( 5.0) 5 (11.4)
Intestinal cholesterol transporter
inhibitor 5 (11.4) 3 (6.8)
Purobcol 0 ( 0.0) 1 (2.3)
TG-lowering agents
Fibrate 5 (11.4) 5 (11.4)
Oral hypoglycemic agents 7 (15.9) 2 (4.5)
Antihypertensive agents 11 (25.0) 13 (29.5)
Uric acid lowering agents 6 (13.6) 2 (4.5)

a : mean±SD, b : n (%)

Food Intakes (Table 1)

Table 1.Changes in daily food intakes in the Partial Japan Diet and Japan Diet groups

Partial Japan Diet (n = 44) Japan Diet (n = 44) Comparisons of differences from baseline between groups (Japan Diet - Partial Japan Diet)
Median (IQR) p† Median (IQR) p† Mean difference (95% CI) p‡
Refined cereals Baseline 302 (220 - 407) 345 (263 - 466)
3-month 296 (200 - 380) 283 (187 - 360) ＊＊＊ -54 (-103, -5) ＊
6-month 280 (216 - 362) 254 (159 - 380) ＊＊＊ -66 (-122, -9) ＊
Unrefined cereals Baseline 11 (0 - 58) 0 (0 - 0)
3-month 21 (0 - 64) 61 (33 - 33) ＊＊＊ 48 (20, 76) ＊＊
6-month 23 (0 - 61) 51 (17 - 17) ＊＊＊ 37 (6, 67) ＊
Potatoes & starches Baseline 20 (10 - 50) 23 (6 - 47)
3-month 27 (10 - 59) 14 (2 - 43) -14 (-31, 3)
6-month 22 (8 - 35) 17 (4 - 37) -5 (-19, 10)
Sugar & jam Baseline 6 (2 - 10) 5 (3 - 11)
3-month 5 (4 - 11) 6 (4 - 9) -1 (-4, 2)
6-month 4 (2 - 10) 5 (4 - 9) 0 (-3, 3)
Soy & soy products Baseline 40 (16 - 91) 51 (23 - 85)
3-month 46 (24 - 85) 53 (33 - 103) 6 (-19, 31)
6-month 63 (26 - 125) ＊ 56 (30 - 103) -15 (-53, 23)
Nuts Baseline 0 (0 - 1) 0 (0 - 1)
3-month 0 (0 - 3) 0 (0 - 3) 0 (-3, 4)
6-month 1 (0 - 3) 1 (0 - 2) 0 (-4, 3)
Green & yeallow vegetables Baseline 90 (51 - 132) 67 (41 - 105)
3-month 82 (50 - 131) 101 (67 - 180) ＊＊＊ 53 (22, 84) ＊＊
6-month 84 (48 - 142) 120 (63 - 206) ＊＊＊ 59 (26, 93) ＊＊
Other vegetables Baseline 157 (118 - 200) 157 (117 - 210)
3-month 157 (120 - 218) 193 (154 - 248) ＊＊ 30 (-4, 65)
6-month 142 (85 - 225) 190 (137 - 258) 22 (-21, 65)
Seaweed & mushrooms & konjak Baseline 24 (11 - 47) 23 (10 - 45)
3-month 22 (10 - 40) 34 (19 - 55) ＊ 13 (0, 26)
6-month 23 (12 - 55) 34 (14 - 60) 14 (-2, 29)
Fruits Baseline 64 (16 - 111) 19 (0 - 63)
3-month 49 (6 - 111) 15 (0 - 57) -1 (-33, 30)
6-month 55 (4 - 96) 24 (0 - 46) -12 (-42, 17)
Fish Baseline 32 (13 - 62) 42 (24 - 61)
3-month 49 (20 - 73) 84 (34 - 123) ＊＊ 23 (-2, 48)
6-month 24 (12 - 49) 61 (28 - 90) ＊＊ 26 (8, 45) ＊＊
Fatty fish Baseline 15 (0 - 33) 24 (0 - 40)
3-month 23 (3 - 47) 54 (22 - 88) ＊＊＊ 25 (5, 46) ＊
6-month 10 (0 - 24) 37 (1 - 73) ＊＊ 22 (9, 36) ＊＊
Other seafoods Baseline 16 (5 - 32) 11 (3 - 42)
3-month 23 (4 - 31) 17 (2 - 33) -4 (-17, 8)
6-month 9 (3 - 31) 16 (1 - 28) -4 (-16, 8)
SFA rich Fats Baseline 0 (0 - 2) 0 (0 - 2)
3-month 1 (0 - 4) 0 (0 - 2) 0 (-2, 1)
6-month 1 (0 - 2) 1 (0 - 2) 0 (-2, 1)
Meat & poultry Baseline 64 (45 - 97) 75 (41 - 110)
3-month 59 (38 - 109) 50 (30 - 78) ＊＊＊ -22 (-42, -3) ＊
6-month 56 (30 - 79) ＊ 49 (24 - 82) ＊＊ -9 (-28, 10)
Fatty meat & poultry Baseline 37 (20 - 57) 38 (9 - 70)
3-month 25 (5 - 46) ＊＊ 20 (2 - 33) ＊＊＊ -10 (-26, 6)
6-month 25 (9 - 48) ＊ 25 (4 - 47) ＊＊ -3 (-17, 10)
Meat products Baseline 11 (5 - 22) 12 (3 - 21)
3-month 9 (1 - 24) 5 (0 - 13) ＊＊ -4 (-4, 3)
6-month 7 (0 - 20) 7 (0 - 13) 1 (-8, 9)
Eggs Baseline 27 (17 - 51) 35 (23 - 51)
3-month 34 (19 - 51) 25 (15 - 45) -9 (-21, 4)
6-month 26 (15 - 46) 30 (18 - 47) -2 (-16, 11)
Milk & dairy products Baseline 71 (28 - 147) 67 (12 - 139)
3-month 67 (32 - 140) 53 (2 - 141) -7 (-43, 28)
6-month 69 (15 - 106) 42 (6 - 162) 10 (-25, 46)
Vegetable oils Baseline 9 (6 - 15) 12 (7 - 14)
3-month 8 (5 - 17) 9 (5 - 13) ＊ -2 (-6, 1)
6-month 7 (4 - 13) 9 (5 - 14) -1 (-4, 3)
Oil containing dressiong & Baseline 5 (2 - 13) 5 (3 - 9)
mayonnaise 3-month 7 (2 - 11) 6 (1 - 12) 0 (-4, 3)
6-month 6 (1 - 13) 6 (3 - 10) 0 (-4, 3)
Confections Baseline 33 (16 - 65) 33 (0 - 66)
3-month 19 (0 - 49) ＊ 13 (0 - 30) ＊＊＊ -11 (-27, 5)
6-month 33 (0 - 63) 6 (0 - 21) ＊＊＊ -19 (-39, 0)
Alcoholic beverages Baseline 0 (0 - 5) 0 (0 - 14)
3-month 0 (0 - 6) 0 (0 - 11) -2 (-6, 3)
6-month 0 (0 - 7) 0 (0 - 11) -4 (-9, 2)

Values are expressed in grams. Alchol beverages were shown as total ethanol intake.

†P values for within-group comparisons from baseline were determined using the Wilcoxon signed-rank test.

‡P values for between-group comparisons were determined using the Mann-Whitney U test.

＊, p＜0.05; ＊＊, p＜0.01; ＊＊＊, p＜0.001

Meat and poultry intakes decreased in both groups (PJD: P＜0.05 at 6 months, JD: P＜0.001 at 3 months and P＜0.01 at 6 months). Fatty meat intake especially was reduced (PJD: P＜0.01 at 3 months and P＜0.05 at 6 months, JD: P＜0.001 at 3 months and P＜0.01 at 6 months), but the intake of meat products was not changed and confections intake reduced only at 3 months (P＜0.05) in the PJD group. However, intake of refined cereals and confections both decreased (P＜0.001 at 3 and 6 months), consumption of unrefined cereals, green and yellow vegetables (P＜0.001 at 3 and 6 months), other vegetables (P＜0.01 at 3 months), and fish especially fatty fishes (P＜0.001 at 3 months, P＜0.01 at 6 months) all increased in the JD group. In the JD group, changes in reduced refined cereals and meat and poultry and increased unrefined cereals, green and yellow vegetables, fatty fish intake volumes from baseline were larger than those in the PJD group at 3 and 6 months. Notably in the JD group, the sum intake of meat, poultry and meat products were reduced from 91 (53, 121) (median (25th percentile, 75th percentile)) g at baseline to 58 (32, 86) g and 63 (36, 90) g at 3 months (P＜0.001) and 6 months (P＜0.01), respectively. While fish intake increased from 42 (24, 61) g to 84 (34, 123) g at 3 months, the total fish and fatty fish intake volume tended to be lower at 6 months than at 3 months (total fish: P=0.063, fatty fish: P=0.05).

Nutrient Intakes

Both groups showed decreases in total energy (PJD: P＜0.01 at 6 months, JD: P＜0.01 at 3 months and P＜0.001 at 6 months) with decreased lipid intakes (PJD: P＜0.01 at 6 months, JD: P＜0.05 at 3 months and P＜0.001 at 6 months).

The JD group also showed a decrease in carbohydrate intake (P＜0.001 both at 3 and 6 months), while intakes of dietary fiber (P＜0.001 at 3 months, P＜0.01 at 6 months), beta-carotene (P＜0.01 at both 3 and 6 months) and ascorbic acid (P＜0.01 at 3 months, P＜0.05 at 6 months) rose. Differences in change from the baseline were noted between the two groups in the intakes of dietary fiber (P＜0.001 at 3 months, P＜0.01 at 6 months), beta-carotene (P＜0.01 both at 3 and 6 months) and ascorbic acid (P＜0.01 at 3 months) (Supplementary Table 2).

Supplementary Table 2.Changes in daily nutrient intakes in the Partial Japan Diet and Japan Diet groups

Partial Japan Diet (n = 44) Japan Diet (n = 44) Comparisons of differences from baseline between groups (JapanDiet-PartialJapanDiet)
Mean (SD) Median (IQR) p† Mean (SD) Median (IQR) p† Mean difference (95% CI) p‡
Energy (kcal) Baseline 1829 (456) 1943 (428)
3-month 1740 (394) 1769 (304) ＊＊ -85 (-234, 64)
6-month 1670 (454) ＊＊ 1674 (406) ＊＊＊ -111 (-254, 33)
Protein (g) Baseline 67.5 (59.8 ‐ 81.2) 72.0 (60.7 ‐ 83.6)
3-month 70.5 (58.1 ‐ 82.0) 73.3 (61.9 ‐ 80.5) -1.2 (-8.8, 6.4)
6-month 64.0 (54.5 ‐ 75.4) ＊＊ 66.5 (60.4 ‐ 82.0) 3.0 (-3.0, 9.0)
Lipids (g) Baseline 61.6 (18.2) 66.4 (19.5)
3-month 58.6 (18.7) 59.5 (18.5) ＊ -3.9 (-12.3, 4.5)
6-month 53.4 (18.4) ＊＊ 56.1 (18.5) ＊＊＊ -2.2 (-8.9, 4.5)
(En%) Baseline 30.3 (6.0) 30.6 (4.8)
3-month 30.3 (6.4) 30.0 (6.8) -0.5 (-3.1, 2.0)
6-month 28.8 (6.8) 29.9 (5.3) 0.8 (-1.7, 3.3)
Saturated fatty acids (g) Baseline 18.3 (6.5) 18.8 (6.8)
3-month 16.5 (6.7) 15.0 (5.6) ＊＊ -2.1 (-4.8, 0.7)
6-month 15.2 (6.2) ＊＊ 14.7 (6.3) ＊＊＊ -1.1 (-3.4, 1.3)
(En%) Baseline 9.0 (2.4) 8.6 (2.0)
3-month 8.5 (2.6) 7.6 (2.3) ＊ -0.5 (-1.5, 0.4)
6-month 8.2 (2.3) ＊ 7.8 (2.2) ＊ -0.1 (-1.0, 0.9)
Cholesterol (mg) Baseline 285 (219 ‐ 383) 362 (229 ‐ 434)
3-month 299 (207 ‐ 419) 300 (216 ‐ 374) -51 (-115, 13)
6-month 244 (188 ‐ 341) ＊ 272 (193 ‐ 347) ＊ -32 (-90, 27)
Carbohydrate (g) Baseline 233.4 (69.5) 236.4 (57.5)
3-month 219.7 (58.3) 219.9 (46.1) ＊＊＊ -2.8 (-22.1, 16.6)
6-month 218.7 (67.2) ＊ 205.2 (55.9) ＊＊＊ -16.4 (-37.3, 4.5)
Total dietary fiber (g) Baseline 15.3 (5.1) 13.7 (4.0)
3-month 14.9 (3.8) 17.8 (6.2) ＊＊＊ 4.5 (2.4, 6.7) ＊＊＊
6-month 15.1 (4.3) 17.3 (5.8) ＊＊ 3.8 (1.7, 5.9) ＊＊
β-carotene equivalent (μg) Baseline 4025 (2415) 3033 (2153)
3-month 3651 (2479) 4718 (2817) ＊＊ 2059 (871, 3246) ＊＊
6-month 4078 (2797) 4940 (3549) ＊＊ 1854 (481, 3227) ＊＊
alpha-tocopherol (mg) Baseline 7.7 (2.2) 7.6 (2.5)
3-month 8.0 (3.6) 8.4 (3.4) 0.5 (-1.1, 2.1)
6-month 7.4 (2.4) 8.1 (3.3) 0.8 (-0.3, 2.0)
Ascorbic acid (mg) Baseline 108 (50) 96 (36)
3-month 98 (33) 115 (53) ＊＊ 28 (9, 46) ＊＊
6-month 113 (75) 111 (48) ＊ 10 (-16, 35)

†P values for within-group comparisons from baseline were determined using the paired t test or the Wilcoxon signed-rank test.

‡P values for between-group comparisons were determined using the unpaired t test or the Mann-Whitney U test.

＊, p＜0.05; ＊＊, p＜0.01; ＊＊＊, p＜0.001

Fatty Acid Intakes

Individual fatty acid intakes of which the mean or median volume was larger than 100 mg/day at least once during the intervention are shown in Table 2.

Table 2.Changes in fatty acid intakes in the Partial Japan Diet and Japan Diet groups

Partial Japan Diet (n = 44) Japan Diet (n = 44) Comparisons of differences from baseline between groups (Japan Diet - Partial Japan Diet)
Mean (SD) Median (IQR) p† Mean (SD) Median (IQR) p† Mean difference (95% CI) p‡
SFA subtotal Baseline 18230 (6417) 18814 (6787)
3-month 16479 (6623) 15013 (5555) ＊＊ -2051 (-4783, 681)
6-month 15161 (6123) ＊＊ 14656 (6302) ＊＊＊ -1090 (-3435, 1255)
C4:0 Baseline 167 (84 - 293) 158 (94 - 286)
3-month 124 (53 - 258) 111 (28 - 245) ＊ -26 (-93, 41)
6-month 133 (61 - 232) 98 (24 - 255) ＊ -21 (-89, 47)
C6:0 Baseline 124 (52 - 189) 101 (61 - 183)
3-month 85 (39 - 168) 75 (19 - 159) ＊ -19 (-61, 23)
6-month 90 (40 - 153) 64 (15 - 160) ＊ -14 (-57, 29)
C8:0 Baseline 97 (49 - 173) 74 (44 - 136)
3-month 65 (29 - 139) 54 (15 - 100) ＊＊ 25 (-51, 101)
6-month 66 (37 - 128) 55 (15 - 103) ＊＊ -65 (-206, 75)
C10:0 Baseline 185 (112 - 321) 153 (95 - 273)
3-month 133 (76 - 252) 109 (38 - 215) ＊＊ -7 (-82, 67)
6-month 138 (78 - 197) 97 (41 - 237) ＊＊ -52 (-160, 55)
C12:0 Baseline 365 (224 - 598) 296 (175 - 500)
3-month 334 (164 - 635) 208 (92 - 350) ＊＊ -119 (-316, 79)
6-month 257 (191 - 489) 230 (84 - 351) ＊ -26 (-231, 179)
C14:0 Baseline 1215 (546) 1320 (649)
3-month 1113 (630) 1178 (604) -39 (-302, 223)
6-month 997 (525) ＊ 1073 (677) ＊ -29 (-279, 221)
C15:0 Baseline 118 (76 - 152) 100 (80 - 148)
3-month 97 (61 - 156) 108 (76 - 149) 1 (-28, 30)
6-month 80 (59 - 141) 78 (58 - 152) 1 (-25, 27)
C16:0 Baseline 10393 (3445) 11292 (3911)
3-month 9550 (3489) 9130 (3170) ＊＊＊ -1319 (-2952, 313)
6-month 8678 (2937) ＊＊ 8912 (3542) ＊＊＊ -665 (-2006, 676)
C17:0 Baseline 143 (96 - 212) 142 (109 - 185)
3-month 126 (90 - 176) 147 (111 - 205) 12 (-29, 53)
6-month 111 (83 - 167) ＊＊ 125 (94 - 179) 11 (-23, 44)
C18:0 Baseline 4238 (1527) 4424 (1740)
3-month 3638 (1412) ＊ 3329 (1299) ＊＊＊ -496 (-1195, 204)
6-month 3319 (1283) ＊＊＊ 3333 (1422) ＊＊＊ -173 (-763, 417)
C20:0 Baseline 176 (131 - 208) 180 (131 - 214)
3-month 143 (124 - 201) 167 (114 - 198) ＊ -13 (-41, 14)
6-month 145 (109 - 199) ＊ 139 (115 - 188) ＊＊＊ -14 (-38, 10)
MUFA subtotal Baseline 23333 (7580) 25876 (8456)
3-month 21743 (7484) 21354 (7341) ＊＊ -2931 (-6497, 634)
6-month 19781 (7764) ＊＊ 20770 (8117) ＊＊＊ -1554 (-4678, 1570)
C16:1 Baseline 985 (508) 1173 (635)
3-month 916 (443) 1104 (547) 0 (-252, 251)
6-month 792 (395) ＊＊ 966 (499) ＊ -14 (-222, 195)
C17:1 Baseline 119 (73) 128 (95)
3-month 106 (70) 124 (80) 9 (-26, 44)
6-month 96 (71) ＊＊ 113 (82) 7 (-22, 36 )
C18:1 Baseline 21531 (15755 - 27113) 23220 (16785 - 28521)
3-month 18770 (15635 ‐ 23490) 18195 (14207 ‐ 22359) ＊＊＊ -3186 (-6407, 35)
6-month 17882 (12633 - 22534) ＊＊ 17998 (12222 - 22529) ＊＊＊ -1601 (-4518, 1317)
C18:1 cis vacssenic Baseline 142 (40 - 240) 163 (70 - 351)
3-month 88 (49 - 196) 98 (41 - 209) ＊ -59 (-155, 36)
6-month 153 (56 - 247) 113 (42 - 224) ＊ -83 (-170, 4)
C20:1 Baseline 370 (297 - 574) 529 (335 - 857)
3-month 484 (304 - 639) 597 (387 - 1279) 69 (-164, 303)
6-month 366 (243 - 550) 503 (359 - 731) 20 (-161, 201)
C22:1 Baseline 136 (33 - 248) 198 (76 - 743)
3-month 234 (85 - 340) 375 (153 - 1281) 157 (-175, 489)
6-month 115 (45 - 290) 299 (117 - 577) 26 (-248, 299)
n-6 PUFA subtotal Baseline 10989 (3915) 11246 (3251)
3-month 10758 (3780) 11012 (3822) -3 (-1638, 1632)
6-month 10325 (3970) 10704 (4034) 122 (-1366, 1609)
C18:2(n-6) (LA) Baseline 10689 (3877) 10921 (3202)
3-month 10433 (3746) 10694 (3788) 30 (-1584, 1643)
6-month 10078 (3959) 10417 (4013) 107 (-1372, 1586)
C20:4(n-6) (AA) Baseline 152 (57) 172 (70)
3-month 171 (81) 183 (73) 22 (0, 43) ＊
6-month 132 (56) ＊ 162 (62) 10 (-5, 26)
n-3 PUFA subtotal Baseline 2453 (1225) 2837 (1776)
3-month 2782 (1860) 3566 (1842) ＊ 400 (-498, 1299)
6-month 2289 (1291) 2933 (1221) 260 (-470, 990)
C18:3(n-3) Baseline 1550 (1077 - 1891) 1616 (1238 - 2085)
3-month 1407 (1049 - 1871) 1415 (1180 - 2033) -267 (-837, 304)
6-month 1327 (1004 - 1692) 1325 (986 - 2090) -124 (-650, 402)
C18:4(n-3) Baseline 30 (4 - 68) 39 (13 - 123)
3-month 40 (17 - 100) 114 (38 - 209) ＊ 66 (-8, 139)
6-month 29 (5 - 67) 78 (25 - 173) 25 (-32, 82)
C20:5(n-3) (EPA) Baseline 166 (38 - 251) 216 (111 - 398)
3-month 211 (80 - 432) 489 (194 - 878) ＊＊ 208 (29, 387) ＊
6-month 148 (40 - 304) 327 (121 - 739) ＊ 142 (14, 271) ＊
C22:5(n-3) Baseline 59 (24 - 89) 77 (41 - 118)
3-month 70 (36 - 110) 124 (58 - 226) ＊＊ 2 (-5, 8)
6-month 47 (17 - 91) 85 (47 - 178) 4 (-2, 10)
C22:6(n-3) (DHA) Baseline 324 (135 - 483) 407 (204 - 757)
3-month 385 (173 - 710) 899 (332 - 1431) ＊＊ 330 (40, 619) ＊
6-month 248 (94 - 519) 504 (231 - 1124) 200 (-9, 408)

LA, Linoleic acid; AA, Arachidonic acid; EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid

†P values for within-group comparisons from baseline were determined using the paired t test or the Wilcoxon signed-rank test.

‡P values for between-group comparisons were determined using the unpaired t test or the Mann-Whitney U test.

＊, p＜0.05; ＊＊, p＜0.01; ＊＊＊, p＜0.001

Saturated Fatty Acid (SFA)

Energy percent derived from SFA was 8.8 +/− 2.2% in all participants at baseline and was decreased to 8.2 +/− 2.3% at 6 months in the PJD group (P＜0.05), while being 7.6 +/− 2.3% and 7.8 +/− 2.2%, respectively, at 3 and 6 months in the JD group (P＜0.05). The sum of C12:0, C14:0 and C16:0 intakes decreased in both groups (PJD: P＜0.01 at 6 months, JD: P＜0.01 at 3 months and P＜0.001 at 6 months). Intakes of short and medium chain SFAs, such as C4:0–C12:0, were decreased in the JD group during the intervention.

Monounsaturated Fatty Acid (MUFA)

Both groups showed reduced intakes of C16:1 (PJD: P＜0.01, JD: P＜0.05) and C18:1 (PJD: P＜0.01 at 6 months, JD: P＜0.001 at 3 and 6 months). The PJD group also showed a reduction in C17:1 intake (P＜0.05) at 6 months.

Polyunsaturated Fatty Acid (PUFA)

The PJD group reduced C20:4 intake at 6 months (P＜0.05), but difference in changes from the baseline in C20:4 intake was larger in the JD group (P＜0.05 at 3 mouths). However, no change was observed in the total n-6 PUFA intake in either group. In the JD group, intakes of n-3 fatty acids such as C18:4(n-3) (P＜0.05), C20:5(P＜0.01), C22:5(n-3) (P＜0.01) and C22:6 (P＜0.01) were all increased at 3 months, and C20:5 intake was continued to increase at 6 months (P＜0.05). The sum of C20:5, C22:5(n-3) and C22:6 intakes was 707 (369, 1283) mg at baseline, then increased to 1593 (576, 2516) mg at 3 months (P＜0.01) and to 901 (410, 1991) mg at 6 months (P=0.072) in the JD group. Differences in changes from the baseline included greater increases in C20:5 and C22:6 at 3 months, as well as higher intakes of C20:5 at 6 months, in the JD group as compared with the PJD group (P＜0.05). The mean differences from baseline in the sum of C20:5, C22:5(n-3) and C22:6 were larger with 581 (72, 1090) (mean difference (95% confidence interval)) and 367 (1, 773) at 3 and 6 months, respectively, in the JD group (P＜0.05).

Metabolic Parameters

After the intervention, body weight reduction resulted in a BMI decrease in both groups (PJD: P＜0.001 at both 3 and 6 months, JD: P＜0.001 at 3 months and P＜0.01 at 6 months), and there was a greater decrease from baseline in waist circumference in the JD than in the PJD group (P＜0.01 at 3 months, P＜0.05 at 6 months). Leptin concentrations decreased in both groups (PJD: P＜0.01, JD: P＜0.01). HMW adiponectin rose early in the JD group (P＜0.01 at 3 month), but significant increases were then observed in both groups at 6 months (both PJD and JD: P＜0.05). TC, LDL-C and TG concentrations were decreased at 3 and 6 months in the JD group, and the differences of LDL-C and TG from baseline were significant as compared with those in the PJD group at 6 months (both LDL-C and TG: P＜0.05). MDA-LDL, 88 U/L at baseline was decreased to 80 U/L at 6 months in the JD group (P＜0.01). Serum ORAC was decreased in the JD group at 3 months (P＜0.001) and both groups showed a decrease at 6 months (P＜0.001) (Table 3). A positive correlation between MDA-LDL and serum ORAC was observed at baseline (r=0.224, P＜0.05), but there was no evidence of a correlation at either 3 or 6 months.

Table 3.Changes in anthropometric variables and circulating metabolic parameters and adipo-cytokine and inflammatory parameters in the Partial Japan Diet and Japan Diet groups

Partial Japan Diet (n = 44) Japan Diet (n = 44) Comparisons of differences from baseline between groups (Japan Diet - Partial Japan Diet)
Mean (SD) Median (IQR) p† Mean (SD) Median (IQR) p† Mean difference (95% CI) p‡
Height (cm) Baseline 162.9 (7.8) 162.2 (8.2)
Body weight (kg) Baseline 63.5 (11.6) 64.6 (13.2)
3-month 62.2 (11.3) ＊＊＊ 63.4 (13.4) ＊＊＊ 0.0 (-0.6, 0.7)
6-month 62.1 (11.7) ＊＊＊ 63.3 (13.4) ＊＊ 0.2 (-0.7, 1.1)
Body Mass Index Baseline 23.8 (3.2) 24.4 (3.6)
3-month 23.3 (3.1) ＊＊＊ 23.9 (3.7) ＊＊＊ 0.0 (-0.2, 0.3)
6-month 23.2 (3.2) ＊＊＊ 23.9 (3.7) ＊＊ 0.1 (-0.3, 0.4)
Waist circumference (cm) Baseline 85.7 (9.5) 88.4 (9.7)
3-month 85.4 (9.0) 86.7 (9.7) ＊＊＊ -1.4 (-2.5, -0.4) ＊＊
6-month 84.7 (9.1) ＊ 85.9 (9.7) ＊＊＊ -1.6 (-2.9, -0.2) ＊
Total Cholesterol (mg/dL) Baseline 224 (39) 212 (30)
3-month 219 (40) 204 (26) ＊ -2 (-13, 8)
6-month 224 (37) 203 (28) ＊＊ -9 (-19, 0) ＊
HDL-Cholesterol (mg/dL) Baseline 63 (17) 62 (18)
3-month 63 (19) 61 (16) -1 (-4, 2)
6-month 63 (19) 61 (16) -1 (-4, 2)
LDL-Cholesterol (mg/dL) Baseline 130 (33) 122 (25)
3-month 127 (29) 116 (21) ＊ -3 (-11, 6)
6-month 133 (31) 115 (23) ＊ -9 (-18, -1) ＊
Triglyceride (mg/dL) Baseline 89 (66 - 138) 102 (76 - 155)
3-month 89 (62 - 139) 90 (68 - 135) ＊ -7 (-31, 17)
6-month 87 (67 - 149) 86 (60 - 135) ＊＊ -10 (-35, 15) ＊
MDA-LDL(U/L) Baseline 94 (70 - 116) 88 (74 - 110)
3-month 86 (72 - 102) 88 (69 - 106) 5 (-7, 18)
6-month 91 (68 - 118) 80 (68 - 99) ＊＊ -4 (-17, 9)
ORAC (TE μmol/L) Baseline 274 (30) 275 (36)
3-month 268 (28) 264 (37) ＊＊＊ -5 (-13, 3)
6-month 253 (38) ＊＊＊ 251 (41) ＊＊＊ -4 (-15, 8)
Leptin (ng/mL) Baseline 7.51 (4.40 - 12.80) 7.27 (3.36 - 13.27)
3-month 6.28 (3.52 - 9.39) ＊＊ 5.75 (3.78 - 9.85) ＊＊ 0.48 (-1.34, 2.31)
6-month 6.18 (3.70 - 8.70) ＊＊ 6.69 (3.40 - 10.53) ＊＊ -0.05 (-1.60, 1.51)
HMW adiponectin (ng/mL) Baseline 3121 (1039 - 7399) 2863 (1538 - 5338)
3-month 2847 (1419 - 7014) 3080 (1721 - 6078) ＊＊ 185 (-135, 505)
6-month 3359 (1431 - 8300) ＊ 3589 (1709 - 5729) ＊ 88 (-404, 580)

MDA-LDL, Malondialdehyde modified-low density cholesterol; HMW adiponectin, High molecular weight adiponectin; ORAC, Oxygen radical absorbance capacity; TE, Trolox equivalent.

†P values for within-group comparisons from baseline were determined using the paired t test or the Wilcoxon signed-rank test.

‡P values for between-group comparisons were determined using the unpaired t test or the Mann-Whitney U test.

＊, p＜0.05; ＊＊, p＜0.01; ＊＊＊, p＜0.001

Serum Phospholipid Fatty Acid Compositions

Serum phospholipid fatty acid compositions are shown in Table 4. The abundant phospholipid FAs were C16:0, C18:2(n-6), C18:0, C20:4(n-6) and C18:1. In the PJD group, percentages of individual fatty acids were either unchanged or only slightly decreased in C18:4(n-3) during the intervention (P＜0.01 at 3 months, P＜0.05 at 6 months). On the other hand, in terms of SFA, the JD group showed a significant increase in C17:0 at 3 and 6 months (P＜0.01). The JD group, for MUFA, also showed decreases in C18:1 (P＜0.001 at both 3 and 6 months) and C16:1 (P＜0.01 at 3 months, P＜0.001 at 6 months) with increases in C24:1 and C22:1 (P＜0.01 at 3 months). The n-6 PUFA composition changes included a decrease in C20:4(n-6) (P＜0.001 at 3 months, P＜0.01 at 6 months), C20:3(n-6) (P＜0.001 at both 3 and 6 months) and C20:2 (P＜0.01 at both 3 and 6 months), while n-3 PUFA increases in C20:5 (P＜0.001 at 3 months, P＜0.05 at 6 months), C22:6 (P＜0.01 at 3 months, P＜0.05 at 6 months) and C20:4(n-3) (P＜0.05 at 3 months) were noted.

Table 4.Changes in serum phospholipid fatty acids composition in the Partial Japan Diet group and Japan Diet groups

Partial Japan Diet (n = 44) Japan Diet (n = 44) Comparisons of differences from baseline between groups (Japan Diet - Partial Japan Diet)
Mean (SD) Median (IQR) p† Mean (SD) Median (IQR) p† Mean difference (95% CI) p‡
SFA subtotal Baseline 43.89 (1.21) 44.09 (1.52)
3-month 43.59 (1.14) 43.71 (1.78) -0.09 (-0.83, 0.65)
6-month 43.70 (1.01) 44.03 (2.13) 0.12 (-0.68, 0.92)
C12:0 Baseline 0.01 (0.00) 0.01 (0.00)
3-month 0.01 (0.00) 0.01 (0.01) 0.00 (0.00, 0.00)
6-month 0.01 (0.00) 0.01 (0.00) 0.00 (0.00, 0.00)
C14:0 Baseline 0.22 (0.07) 0.21 (0.07)
3-month 0.21 (0.07) 0.22 (0.08) 0.02 (-0.01, 0.05)
6-month 0.20 (0.07) 0.20 (0.07) 0.01 (-0.02, 0.04)
C15:0 Baseline 0.10 (0.02) 0.10 (0.03)
3-month 0.10 (0.03) 0.11 (0.03) 0.01 (0.00, 0.01)
6-month 0.10 (0.02) 0.11 (0.03) 0.01 (0.00, 0.01)
C16:0 Baseline 25.21 (1.48) 25.22 (1.23)
3-month 25.09 (1.29) 25.08 (1.29) -0.01 (-0.62, 0.59)
6-month 25.10 (1.33) 25.17 (1.63) 0.07 (-0.57, 0.70)
C17:0 Baseline 0.35 (0.07) 0.35 (0.07)
3-month 0.36 (0.07) 0.38 (0.07) ＊＊ 0.02 (0.00, 0.04)
6-month 0.35 (0.07) 0.38 (0.08) ＊＊ 0.03 (0.01, 0.05) ＊＊
C18:0 Baseline 14.41 (1.16) 14.72 (1.03)
3-month 14.31 (0.95) 14.47 (1.06) -0.14 (-0.49, 0.20)
6-month 14.29 (0.98) 14.66 (1.28) 0.05 (-0.37, 0.48)
C20:0 Baseline 0.61 (0.14) 0.60 (0.11)
3-month 0.60 (0.14) 0.61 (0.11) 0.02 (-0.02, 0.05)
6-month 0.62 (0.13) 0.60 (0.11) 0.00 (-0.04, 0.04)
C22:0 Baseline 1.59 (0.37) 1.53 (0.26)
3-month 1.56 (0.31) 1.52 (0.32) 0.02 (-0.09, 0.14)
6-month 1.59 (0.35) 1.53 (0.29) 0.00 (-0.12, 0.11)
C24:0 Baseline 1.39 (0.29) 1.36 (0.24)
3-month 1.36 (0.26) 1.32 (0.26) -0.01 (-0.10, 0.08)
6-month 1.44 (0.32) 1.36 (0.24) -0.05 (-0.14, 0.05)
MUFA subtotal Baseline 12.22 (11.02 - 12.80) 11.96 (11.19 - 12.74)
3-month 11.99 (10.84 - 12.96) 11.81 (10.76 - 12.37) -0.25 (-0.64, 0.13)
6-month 12.11 (11.02 - 12.99) 11.52 (10.63 - 12.05) ＊＊＊ -0.34 (-0.74, 0.06)
C16:1 Baseline 0.38 (0.30 - 0.49) 0.37 (0.32 - 0.45)
3-month 0.36 (0.27 - 0.48) 0.35 (0.30 - 0.42) ＊＊ -0.02 (-0.06, 0.01)
6-month 0.35 (0.31 - 0.48) 0.34 (0.28 - 0.42) ＊＊＊ -0.04 (-0.06, -0.01) ＊
C17:1 Baseline 0.09 (0.07 - 0.11) 0.09 (0.08 - 0.11)
3-month 0.09 (0.07 - 0.10) 0.09 (0.07 - 0.11) -0.01 (-0.02, 0.01)
6-month 0.10 (0.07 - 0.11) 0.09 (0.07 - 0.11) -0.01 (-0.03, 0.01)
C18:1 Baseline 8.27 (7.89 - 9.34) 8.50 (7.72 - 8.93)
3-month 8.15 (7.59 - 9.40) 8.03 (6.67 - 8.55) ＊＊＊ -0.46 (-0.87, -0.05) ＊＊
6-month 8.38 (7.41 - 8.98) 7.93 (7.04 - 8.45) ＊＊＊ -0.34 (-0.79, 0.10)
C20:1 Baseline 0.23 (0.20- 0.25) 0.23 (0.20 - 0.26)
3-month 0.23 (0.19- 0.25) 0.22 (0.20 - 0.26) 0.00 (-0.03, 0.03)
6-month 0.22 (0.20- 0.26) 0.23 (0.19 - 0.26) 0.01 (-0.02, 0.04)
C22:1 Baseline 0.04 (0.04- 0.05) 0.04 (0.03 - 0.05)
3-month 0.04 (0.03- 0.05) 0.05 (0.04 - 0.06) ＊＊ 0.01 (0.01, 0.02) ＊＊
6-month 0.04 (0.04- 0.05) 0.05 (0.04 - 0.05) 0.01 (0.00, 0.01)
C24:1 Baseline 2.76 (2.31- 3.16) 2.78 (2.51 - 3.26)
3-month 2.78 (2.40- 3.24) 3.05 (2.62 - 3.67) ＊＊ 0.22 (-0.04, 0.48)
6-month 2.89 (2.50- 3.22) 2.98 (2.45 - 3.35) 0.04 (-0.21, 0.29)
n-6 PUFA subtotal Baseline 31.63 (2.18) 31.15 (2.85)
3-month 31.42 (2.98) 29.37 (3.20) ＊＊ -1.58 (-2.86, -0.30) ＊
6-month 31.69 (3.02) 30.27 (3.40) ＊ -0.95 (-2.13, 0.22)
C18:2 (LA) Baseline 18.60 (2.39) 17.98 (2.72)
3-month 18.46 (3.03) 17.31 (2.96) -0.53 (-1.75, 0.70)
6-month 18.53 (2.86) 18.00 (3.39) 0.08 (-1.00, 1.16)
C20:2 Baseline 0.31 (0.27 - 0.36) 0.31 (0.27 - 0.36)
3-month 0.30 (0.27 - 0.36) 0.29 (0.26 - 0.32) ＊＊ -0.02 (-0.04, 0.01)
6-month 0.32 (0.30 - 0.34) 0.29 (0.25 - 0.33) ＊＊ -0.03 (-0.05, -0.01) ＊
C20:3(n-6) (GLA) Baseline 2.25 (0.69) 2.31 (0.74)
3-month 2.18 (0.69) 1.93 (0.65) ＊＊＊ -0.30 (-0.52, -0.09) ＊＊
6-month 2.26 (0.71) 1.98 (0.73) ＊＊＊ -0.34 (-0.54, -0.14) ＊＊
C20:4(n-6) (AA) Baseline 9.82 (1.62) 9.93 (1.61)
3-month 9.84 (1.75) 9.20 (1.59) ＊＊＊ -0.75 (-1.19, -0.31) ＊＊
6-month 9.92 (1.60) 9.35 (1.68) ＊＊ -0.68 (-1.11, -0.24) ＊＊
C22:2 Baseline 0.64 (0.16) 0.62 (0.13)
3-month 0.63 (0.14) 0.63 (0.13) 0.02 (-0.02, 0.06)
6-month 0.65 (0.19) 0.64 (0.14) 0.01 (-0.04, 0.05)
n-3 PUFA subtotal Baseline 10.52 (2.33) 11.15 (3.11)
3-month 11.16 (2.65) 13.68 (4.09) ＊＊＊ 1.88 (0.46, 3.31) ＊
6-month 10.83 (2.93) 12.62 (3.74) ＊ 1.16 (-0.23, 2.55)
C18:3(n-3) Baseline 0.20 (0.16 - 0.27) 0.19 (0.17 - 0.23)
3-month 0.21 (0.17 - 0.27) 0.20 (0.17 - 0.24) 0.00 (-0.03, 0.03)
6-month 0.21 (0.16 - 0.27) 0.19 (0.16 - 0.23) 0.01 (-0.02, 0.04)
C18:4(n-3) Baseline 0.09 (0.08 - 0.11) 0.09 (0.08 - 0.11)
3-month 0.09 (0.07 - 0.10) ＊＊ 0.09 (0.08 - 0.10) 0.00 (0.00, 0.01)
6-month 0.08 (0.07 - 0.10) ＊ 0.09 (0.08 - 0.11) 0.01 (0.00, 0.02) ＊
C20:4(n-3) Baseline 0.12 (0.09 - 0.16) 0.13 (0.10 - 0.20)
3-month 0.11 (0.09 - 0.18) 0.15 (0.10 - 0.22) ＊ 0.03 (-0.01, 0.06)
6-month 0.13 (0.09 - 0.16) 0.14 (0.10 - 0.22) 0.01 (-0.02, 0.05)
C20:5 (EPA) Baseline 2.05 (1.25 - 2.81) 2.03 (1.24 - 3.55)
3-month 2.10 (1.67 - 3.02) 3.77 (2.01 - 5.33) ＊＊＊ 1.25 (0.40, 2.10) ＊＊
6-month 1.88 (1.31 - 2.74) 2.97 (1.89 - 4.92) ＊＊ 0.71 (-0.02, 1.45)
C22:5 Baseline 0.98 (0.16) 1.04 (0.21)
3-month 1.02 (0.20) 1.09 (0.26) 0.01 (-0.07, 0.10)
6-month 1.01 (0.22) 1.08 (0.27) 0.01 (-0.09, 0.10)
C22:6 (DHA) Baseline 6.86 (1.25) 7.14 (1.69)
3-month 7.22 (1.39) 8.09 (1.94) ＊＊ 0.59 (-0.04, 1.22)
6-month 7.09 (1.67) 7.78 (1.93) ＊ 0.40 (-0.28, 1.08)

GLA, γlinolenic acid; AA, Arachidonic acid; EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid

†P values for within-group comparisons from baseline were determined using the paired t test or the Wilcoxon signed-rank test.

‡P values for between-group comparisons were determined using the unpaired t test or the Mann-Whitney U test. ＊, p＜0.05; ＊＊, p＜0.01; ＊＊＊, p＜0.001

Differences in changes from baseline included tendency grater increase in C17:0 (P=0.078) and significant greater increases in C22:1 (P＜0.01) and C20:5 (P＜0.01), while decreases in C18:1 (P＜0.01), C20:3(n-6) (P＜0.01) and C20:4(n-6) (P＜0.01) were greater in the JD than in the PJD group at 3 months. Although, differences in changes from baseline between the two groups diminished at 6 months, but greater increases in C17:0 (P＜0.01) and C18:4(n-3) (P＜0.05), and greater decreases in C20:3(n-6) (P＜0.01), C20:2(n-6) and C20:4(n-6) (P＜0.01) persisted in the JD group.

Correlations between the Intake of Essential Fatty Acids and Serum Phospholipid Fatty Acid Concentrations (Table 5)

Table 5.Correlations among intake of essential fatty acids per body weight and those in serum phospholipid fatty acids concentration

n-6 n-3
C18:2 (LA) C20:4(AA) C18:3(ALA) C20:5 (EPA) C22:5 (DPA) C22:6 (DHA)
Baseline r 0.058 -0.099 0.155 0.600 0.234 0.387
p 0.591 0.360 0.149 0.000 0.028 0.000
Delta at 3-month r 0.040 -0.096 0.044 0.629 0.192 0.456
p 0.710 0.374 0.683 0.000 0.073 0.000
Delta at 6-month r 0.054 0.106 0.28 0.451 0.291 0.374
p 0.619 0.324 0.008 0.000 0.006 0.000

n = 88

LA, Linoleic acid; AA, Arachidonic acid; ALA, α-linolenic acid, EPA, Eicosapentaenoic acid; DPA, Docosapentaenoic acid; DHA, Docosahexaenoic acid Values are expressed as correlation coefficients; r or rs.

Values were determined using Pearson's or Spearman's rank correlation.

Correlation coefficients between the intake of essential fatty acids per body weight and their concentrations in phospholipids fraction were determined. The n-6 fatty acid concentrations of C18:2 and C20:4 did not correlate with those fatty acid intake volumes per body weight. In contrast, the essential n-3 fatty acids, C20:5 and C22:6 concentrations, showed positive correlations with those fatty acid intakes at baseline, delta at 3 months and delta at 6 months (P＜0.001). The concentration of C18:3 showed a positive correlation only in the delta with delta intake at 6 months (P＜0.01). The concentration of C22:5 showed positive correlations with intake at baseline (P＜0.05) and at delta at 6 months (P＜0.01).

Correlations among Serum Phospholipid Fatty Acids (Table 6)

Table 6.Correlation coefficients among percentages of phospholipid C20:5, C22:5, C22:6 and major fatty aicds of (MUFA and n-6 PUFA) at baseline, and changes at 3 month and at 6 month

C20:5 (EPA) C22:5 C22:6 (DHA)
Baseline Delta 3M Delta 6M Baseline Delta 3M Delta 6M Baseline Delta 3M Delta 6M
C18:1 rs -0.445 -0.474 -0.571 -0.120 -0.041 -0.324 -0.448 -0.407 -0.488
p ＜0.001 ＜0.001 ＜0.001 0.266 0.532 0.002 ＜0.001 ＜0.001 ＜0.001
C18:2 (LA) r -0.496 -0.703 -0.709 -0.499 -0.561 -0.553 -0.487 -0.678 -0.679
p ＜0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001 ＜0.001
C20:3(n-6) (GLA) r -0.511 -0.566 -0.361 -0.157 0.020 -0.059 -0.516 -0.419 -0.260
p ＜0.001 ＜0.001 ＜0.001 0.145 0.856 0.587 ＜0.001 ＜0.001 0.014
C20:4(n-6) (AA) r 0.047 -0.149 -0.159 0.015 -0.004 -0.094 -0.018 0.010 0.027
p 0.666 0.166 0.140 0.888 0.969 0.385 0.870 0.929 0.802

n = 88

LA, Linoleic acid; GLA, γ linolenic acid; AA, Arachidonic acid; EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid Values are expressed as correlation coefficients; r or rs.

Values were determined using Pearson's or Spearman's rank correlation.

Correlation coefficients between percentages of C20:5, C22:5 and C22:6 were determined. MUFA and PUFA in phospholipids fraction accounted for more than 1% at baseline and the changes at 3 and 6 months are shown in Table 6. C20:5 and C22:6 showed inverse correlations with C18:1, C18:2 and C20:3(n-6) at baseline, and these changes were still present at 3 and 6 months (P＜0.001), except for the small correlation between delta C22:6 and delta C20:3(n-6) at 6 months (P＜0.05). C22:5 was inversely related to C18:2 (P＜0.001), and the change correlated with delta C18:2 (P＜0.001 at both 3 and 6 months) and C18:1 at 6 months (P＜0.01). Delta C20:4(n-6) did not show any correlation with C20:5, C22:5 and C22:6.

Fatty Acid Ratios of Serum Phospholipids

The fatty acid concentration ratios of C16:1/C16:0 and C18:1/C18:0, as estimates of stearoyl-CoA desaturase (SCD) activity, decreased (P＜0.01 at both 3 and 6 months), while C20:4(n-6)/C20:3(n-6) ratio, as estimates of D5D activity, was increased (P＜0.05 at 3 months, P＜0.001 at 6 months) in the JD group (Table 7).

Table 7.Estimated desaturase activities at baseline and after intervention

Partial Japan Diet (n = 44) Japan Diet (n = 44) Comparisons of differences from baseline between groups (Japan Diet - Partial Japan Diet)
Median (IQR) p† Median (IQR) p† Mean difference (95% CI) p‡
C16:1/C16:0 (SCD) Baseline 0.015 (0.012 - 0.020) 0.015 (0.013 - 0.018)
3-month 0.015 (0.011 - 0.020) 0.014 (0.012 - 0.017) ＊＊ -0.001 (-0.002, 0.000)
6-month 0.014 (0.012 - 0.019) 0.013 (0.011 - 0.016) ＊＊＊ -0.001 (-0.003, 0.000) ＊
C18:1(n-9)/C18:0 (SCD) Baseline 0.596 (0.539 - 0.644) 0.582 (0.530 - 0.610)
3-month 0.572 (0.518 - 0.659) 0.554 (0.466 - 0.601) ＊＊ -0.023 (-0.053, 0.008)
6-month 0.573 (0.534 - 0.658) 0.518 (0.474 - 0.590) ＊＊ -0.022 (-0.054, 0.010)
C20:4(n-6)/C20:3(n-6) (D5D) Baseline 4.810 (3.417 - 5.892) 4.464 (3.651 - 5.551)
3-month 4.729 (3.715 - 6.034) 5.080 (3.903 - 6.697) ＊ 0.411 (-0.206, 1.027)
6-month 4.522 (3.579 - 5.552) 4.850 (3.900 - 6.317) ＊＊＊ 0.495 (-0.046, 1.037)

†P values for within-group comparisons from baseline were determined using the Wilcoxon signed-rank test.

‡P values for between-group comparisons were determined using the Mann-Whitney U test.

＊, p＜0.05; ＊＊, p＜0.01; ＊＊＊, p＜0.001

Discussion

This 6-month intervention for patients with dyslipidemia showed that JD produced essentially the same changes in phospholipid fatty acid profile as those observed in our previous pilot study12). As a control for this RCT, the PJD served as a model aimed at modifying excess consumption of animal fats and foods in the westernized Japanese diet, a strategy expected to decrease total energy and SFA intakes. The PJD group showed reduced meat and poultry and vegetable oil intakes but little change was observed in the percentages of phospholipids other than C18:4(n-3). On the other hand, the JD group replaced animal foods with marine and plant products and also reduced intakes of confectionaries. Compared with the PJD group, these changes resulted in increases in the percentages of serum phospholipids C17:0, C20:5 and C22:6, with concurrent decreases in C16:1, C18:1, C20:2(n-6), C20:3(n-6) and C20:4(n-6) in the JD group.

The participants in this study consumed 40 g/day of fish at baseline, which was comparable to the average amount in Japan16). Japanese nationwide cohort studies conducted baseline surveys around 1990 that showed 20% lower mortality rate from cardiovascular diseases in the highest quintile of median fish intake of 85 g/day compared with the lowest quintile of 20 g/day17), and nonfatal coronary disease was lower by 40% in the highest quintile intake of 180 g/day compared with the lowest quintile of 23 g/day18). In contrast, the Dietary Guidelines for Americans recommended only 1–2 seafood meals (3.5 oz=100 g per serving) per week, which provides 250 mg of EPA＋DHA to reduce the risk of coronary diseases19). The JD group increased their fish consumption to 80 g/day at 3 months, resulting in the increase in C20:5＋C22:5＋C22:6 (EPA＋DPA＋DHA) intake to 1600 mg (from 700 mg at baseline) and corresponding increase in serum phospholipid fatty acid concentrations.

Fish intake is well known to increase circulating C20:5 and C22:6 20-22), and these changes in fatty acids, with replacement of C20:3(n-6) and C20:4(n-6) by C20:5 and C22:6, are consistent with those in earlier fish oil supplementation23), cross-sectional24), and interventional fish meal25) studies. Dietary C20:5 and C22:6 have been reported to downregulate the activities of delta-6 desaturase, D5D, and elongases which convert C18:3(n-3) into long-chain n-3 fatty acids26-29). The C18:2(n-6) of essential n-6 PUFA is also converted to C20:3(n-6), C20:4(n-6), and other longer-chain n-6 PUFAs by the same enzymes. Therefore, rising C20:5 and C22:6 levels must be beneficial in terms of sparing D5D and prioritizing actions upon n-6 PUFA, possibly leading to decreases in C20:3(n-6) and C20:4(n-6).

Furthermore, both diet groups showed reductions in C16:1 and C18:1 consumption in this study, their percentages in phospholipids were decreased. However, the estimated SCD activity became lower only in the JD group, suggesting that the estimated SCD activity was downregulated in this group. The expression level of SCD, a rate-limiting enzyme that converts SFAs into MUFAs, is regulated at the transcriptional level by sterol regulatory element-binding protein, carbohydrate-responsive element-binding protein, liver X receptor and insulin signaling30), and is considered to be a central regulator of fuel metabolism as it pertains to obesity control and the progression of related metabolic diseases including type 2 diabetes mellitus and hepatic steatosis31). Furthermore, SCD inhibits SFA-induced lipotoxic cardiomyopathy in the unsaturated fatty acid-deficient state32). The expression of SCD was reportedly decreased in cells treated with serum from n3-PUFA-supplemented individuals in an ex vivo study33). Estimated SCD activity was positively associated with TG, while inverse association with TGs were observed for the estimated D5D activity in cross-sectional studies34-36) and a longitudinal Mediterranean population study37). These studies suggest that high SCD and low D5D levels are associated with metabolic syndrome. In an intervention study on patients who were slightly overweight and had moderate hyperlipidemia, the estimated SCD increased but the D5D decreased on a diet high in saturated fat (composed of butter, cheese, and cream) compared to a C18:2 and C18:3(n-3)-rich diet (with rapeseed oil)38). Furthermore, high estimated SCD and low estimated D5D were associated with increased cardiovascular mortality39). The results of this study on the JD are consistent with the findings in a previous study, suggesting that JD is beneficial for improving metabolic syndrome and may be a preventive diet for cardiovascular disease.

The major type of circulating phospholipids is glycerophospholipid, in which SFAs and MUFAs are usually esterified at the sn-1-position, whereas polyunsaturated acyl groups are esterified at the sn-2-position40). Therefore, unsaturated fatty acids compete with each other for the sn-2-position on glycerophospholipids41). The most abundant phospholipid unsaturated fatty acid was C18:2(n-6), followed by C20:4(n-6), C18:1, C22:6, C20:3, C20:5 and C24:1. The other unsaturated fatty acids were comprising small less than 2%. The negative correlations among n-3PUFA and other unsaturated fatty acids observed in this study suggest that C18:2 might be replaced by C20:5, C22:5, and C22:6, whereas C18:1 and C20:3(n-6) might be replaced by C20:5 and C22:6 at the sn-2-position. However, C20:4(n-6) did not show any correlation with these n-3 fatty acids, despite the significant decrease produced by the intervention in the JD group. We measured fatty acid concentrations of total serum phospholipids, which are composed of several classes. Phosphatidylcholine (PC) is a major component constituting 75%–85% of circulating phospholipids, and half of the sn-2-positions are occupied by C18:2, followed by C18:1 and C20:4(n-6), whereas C20:4(n-6) is a major component of phosphatidylserine and phosphatidylinositol42). Based on studies that examined the lipidomic approaches in patients after myocardial infarction or unstable ischemic attack, 100–150 g/day of fatty fish 4 times/week decreases bioactive ceramides and lysoPCs, which might be related to antiinflammatory effects of fish derived fatty acids43). Moreover, a healthy diet of whole grains, 100–150 g/day of fish 3 times/week, and 300 g of bilberries daily decreased lysoPC (20:3) but increased lysoPC (20:5) and lysoPC (22:6) in patients with impaired glucose metabolism44). Supplements of 1.6 g/day of C20:5＋C22:6 for 3 weeks were selectively incorporated into lysoPC45), while dietary C20:5 and C22:6 were not incorporated into lysophosphatidic acids46). Although differences in fatty acid distributions have not been clarified for all phospholipid classes, our data suggest increased C20:5 and C22:6 in response to the JD might not replace other unsaturated fatty acids in an equal ratio.

C20:3(n-6), C20:4(n-6), and C20:5 are well known to act as substrates for prostaglandins, thromboxanes, and leukotrienes of the “1”, “2”, and “3” series, respectively47), and are synthesized to serve as specialized pro-resolving lipid mediators such as lipoxins from C20:4(n-6) and resolvins, protectins, and maresins from C20:5 and C22:6 during the initiation and the resolution phases of inflammation48, 49). C20:5 competes with C20:4 for cyclooxygenase and lipoxygenase and blocks the productions of 2-series thromboxane, prostacyclin and leukotrienes50). The phospholipid fatty acid profiles of JD intake may produce differences in fatty acids incorporated into phospholipids and affect lipid metabolism, the production of bioactive substances that function in signaling pathways and the formation of lipid mediators involved in inflammation. To clarify the effect of the JD intake on lipid metabolism, further lipidomic approach is needed.

As for adverse effects of fish consumption, supplemented oils including large amounts of EPA＋DHA of 2.5–7.7 g/day51) and 3.4 g/day52) have been shown to increase the susceptibility of LDL to oxidative modification, and the consumption with antioxidants is thus recommended53, 54). However, conflicting data have been reported on the effects of n-3 PUFA, showing increased or no effect on the susceptibility of LDL to lipid peroxidation, or that C20:5 exerts direct antioxidant effects in a dose-dependent manner in an in vitro study55). In a Spanish cross-sectional study, the participants’ fish consumption ranged from 0 to 250 g/day, almost the same as our JD group at 3 months; n-6 PUFA showed a positive while C20:5 and C22:6 showed negative correlations with lipid oxidation parameters56). Despite the baseline MDA-LDL level of 88 U/L being lower than 142 U/L in younger participants in our previous pilot study13), the MDA-LDL in this study decreased slightly in the JD group, though there was no between-group difference. The JD group consumed more antioxidants such as beta-carotene and ascorbic acid and had stable alpha-tocopherol consumption as compared with the baseline, though there was no significant relationship between MDA-LDL and the intakes of these nutrients. Furthermore, serum ORAC, reflecting the combined effects of all antioxidants in serum, decreased in both groups. This is consistent with findings observed after weight loss in overweight and obese women57). Thus, the JD combination of fish and high green and yellow vegetable intakes might have prevented oxidative stress, even when the participants did not consume the recommended amounts of 350 g of vegetables. The Mediterranean diet, which includes lower fish intake than JD but high intake of vegetables (over 600 g/day) and polyphenols from fruit and extra virgin olive oil58, 59), has been advocated for its antioxidant properties59). Improved education and environmental support for increasing vegetable intake are required to confirm the beneficial effects of the JD on lipid oxidation.

As to odd-chain fatty acids, the percentages of C15:0 and C17:0 in serum phospholipids were low in participants with high TG levels and metabolic syndrome related fat storage34-36, 60, 61), plasminogen activator inhibitor-1, and tissue-type plasminogen activator62). In this study, there were no differences in terms of changes in C15:0 and C17:0 intakes from baseline, and the PJD group also showed reduced leptin and increased HMW adiponectin, while C15:0 and C17:0 percentages in serum phospholipids did not change in the PJD group. These circulating odd-chain fatty acids were believed to have been derived only from foods. It was, however, clarified that there are other endogenous sources including gut-derived propionates which are increased by a high dietary fiber intake63). Propionates are used for the hepatic synthesis of odd-chain fatty acids in humans and can be elongated to very-long-chain fatty acids in glycosphingolipids, particularly those found in brain tissues64). Recent cohort studies showed circulating odd-chain fatty acids to correlate positively with fiber-rich food intake, mainly whole grains with greater than 21 g/day of dietary fiber, and inversely with red meat consumption63, 65, 66). Although the intake of 18 g/day dietary fiber from unrefined cereals and vegetables in this study was lower than those in the former studies, increased dietary fiber may be one of the reasons for the higher odd-chain percentages in phospholipids in the JD group than in the PJD group. Large scale epidemiological studies have shown circulating C15:0 and C17:0 to be associated with reduced disease risks for type 2 diabetes65-68) and coronary heart disease39, 62, 69). Various beneficial effects of these odd-chain fatty acids, including those derived from dairy, have been noted in experimental research70), and further studies are needed to clarify the role of the JD in odd-chain fatty acid metabolism.

To summarize, fatty acid changes in phospholipids including increases in C17:0, C20:5 and C22:6, with concurrent decreases in C16:1, C18:1, C20:3(n-6) and C20:4(n-6) in the JD group, were reportedly related to low cardiovascular mortality rate40, 63, 70). Thus, beneficial polyunsaturated fatty acid changes in phospholipids might be achieved by the JD intake.

Limitations

This study has several limitations. First, the number of participants was relatively small due to the exclusion of several participants from the original study13), and the results have to be carefully interpreted. Furthermore, sub-analyses on medications and/or other complications could not be performed due to the same reason. Second, the circulating total phospholipid concentration could not be measured without performing mass spectrometry. Third, adherence to the recommended diet tended to be lower at 6 months as compared with that at 3 months in the JD group. The JD group participants were given a few boiled servings of rice with barley and canned mackerel to help them access these foods at baseline, and consuming these foods by themselves would presumably be difficult. Even though the volume of fish consumed was decreased at 6 months, the intake for the sum of C20:5 and C22:6 was 385 mg greater than at baseline. Skill training and environmental support for adopting behaviors aimed at consuming the JD are warranted for Japanese patients with dyslipidemia habituated to a westernized diet. Fourth, because consumptions of foods such as unrefined cereals and vegetables did not reach the recommended volumes11), the results obtained in this study were not the ideal for JD intake. In addition, we measured only MDA-LDL as a parameter of oxidized LDL, such that oxidation status was not fully evaluated. Additional parameters, such as 4-hydroxynonenal-LDL, should be measured. Fifth, medical diets for lifestyle-related diseases should be firmly established such that they can be continued throughout life, and this requires a longer intervention period with intensive education.

However, this is the first RCT examining the JD as a clinical dietary intervention for patients with dyslipidemia and investigated changes in phospholipid fatty acid profiles.

Conclusion

This 6-month intervention for dyslipidemia patients showed that the JD can change phospholipid fatty acid profiles to be protective against cardiovascular risk factors. Overall, intakes of recommended foods for the JD (unrefined cereals, fish, soybean and soybean products, vegetables, and seaweed/mushrooms/konjak) were increased, with reductions of animal foods and fats (meats, poultry, and confections). These changes, as a clinical diet for dyslipidemia, are suggested to be effective for improving the fatty acid profiles. Further studies involving a larger number of patients and patients with severe risk factors for atherosclerosis are warranted.

Acknowledgements

The authors thank all patients who participated in the present study, and all staff members who supported the survey. The authors also appreciate all nutrition study team members of the laboratory of Nutrition Education and Clinical Nutrition of Japan Women’s University, including Ms. Kanako Kamoshita, Ms. Seina Komine, Ms. Sayaka Hasegawa, Ms. Rina Ichiki, Ms. Kanako Chibai, Ms. Chieko Fukuda, Ms. Miyu Oshika, Ms. Sia SuHuai, Ms. Moe Matsumoto, Ms. Saaya Yamada, Ms. Sayuri Igawa, Ms. Hazuki Kitayama, Ms. Mayuka Kodama, Ms. Kirika Fujitani, Ms. Aoi Tokunaga, Ms. Akari Yasuda, Ms. Hinako Omata, Ms. Saori Toyota, Ms. Kana Kinugawa, Ms. Mai Murano, Ms. Chisato Ogawa, and Ms. Nana Mihara, all of whom made major contributions to calculating the nutrient intakes in this study.

Financial Support

This study was supported by a research grant from the SKYLARK Food Science Institute and the Rice Stable Supply Support Organization. This study was also supported by gifts of canned mackerel from Maruha Nichiro corporation, Tokyo, retort-packed rice with barley, Mochimugi Gohan from Hakubaku Co., Ltd. Tokyo and Omugi Gohan from Otsuka Pharmaceutical Co., Ltd. The SKYLARK Food Science Institute, the Rice Stable Supply Support Organization, Maruha Nichiro corporation and Hakubaku Co., Ltd. had no role in the design, analysis or writing of this article.

Conflicts of Interest

The authors report the following disclosures: Masako Waki has received clinical research funding from AstraZeneca KK and Eli Lilly Japan KK. Chizuko Maruyama has received scholarship grant from Rice Stable Supply Support Organization. Aisa Sato, Yui Nishikata, Mariko Nakazawa, Yuri Shijo, Noriko Kameyama, Ariko Umezawa, Ai Nishitani, Makoto Ayaori, Katsunori Ikewaki and Tamio Teramoto have no conflicts of interest.

References
 

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