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Comparison of plasma levels of different species of trans fatty acids in Japanese male patients with acute coronary syndrome versus healthy men

Open AccessPublished:March 12, 2019DOI:https://doi.org/10.1016/j.atherosclerosis.2019.02.025

      Highlights

      • There are several differences in trans-C18:1 positional isomers between acute coronary syndrome (ACS) and control.
      • Palmitelaidic acid, ruminant-derived TFA were lower in ACS patients, especially middle-aged patients.
      • Linoleic trans isomers, industrially-produced TFA (IP-TFAs) were higher in ACS.
      • IP-TFA to arachidonic acid (AA) ratio was significantly higher in ACS, especially elder ACS patients.
      • Palmitelaidic acid was significantly directly associated with HDL-C, EPA+DHA, and EPA+DHA/AA ratios.

      Abstract

      Background and aims

      It remains unclear how trans fatty acid (TFA) at low-level intake affect lipid levels and the development of acute coronary syndrome (ACS). The study aimed to investigate how plasma TFA composition differs between male patients with ACS and healthy men.

      Methods

      Plasma fatty acid (FA) composition (as determined by gas chromatography) was analyzed in ACS patients on hospital admission and compared to that of age-adjusted healthy men.

      Results

      Total FA and TFA levels were similar between ACS and control subjects. Palmitelaidic acid, ruminant-derived TFA (R-TFA), levels were lower in ACS patients (0.17 ± 0.06 vs. 0.20 ± 0.06 of total FA, in ACS and control, respectively, p<0.01), and were significantly directly associated with HDL cholesterol (HDL-C) (rho = 0.269) and n-3 polyunsaturated FA (n-3 PUFA) (rho = 0.442). Linoleic trans isomers (total C18:2 TFA), primary industrially-produced TFA (IP-TFAs), were significantly higher in ACS patients (0.68 ± 0.17 vs. 0.60 ± 0.20 of total FA, in ACS and control, respectively). Total trans-C18:1 isomers were comparable between ACS and control. Differences between ACS and controls in C18:1 trans varied by specific C18:1 trans species. Absolute concentrations of trans-C18:2 isomers were significantly directly associated with LDL-C and non-HDL-C in ACS men. The ACS patients showed significantly lower levels of both n-6 and n-3 PUFA (i.e., eicosapentaenoic, docosahexaenoic and arachidonic acids).

      Conclusions

      There were several case-control differences in specific TFA that could potential affect risk for ACS. Japanese ACS patients, especially middle-aged patients, may consume less R-TFA.

      Graphical abstract

      Keywords

      1. Introduction

      Fatty acids (FA) are biologically-active molecules with a wide array of effects [
      • Baum S.J.
      • Kris-Etherton P.M.
      • Willett W.C.
      • Lichtenstein A.H.
      • Rudel L.L.
      • et al.
      Fatty acids in cardiovascular health and disease: a comprehensive update.
      ]. FA are classified as saturated or unsaturated on the basis of the absence or presence of double bonds. Monounsaturated FA (MUFA) have one double bond; polyunsaturated FA (PUFA) have more than one double bond. UFA usually occur in the cis configuration, and trans FA (TFA) are UFA containing at least one double bond in trans configuration. Because humans cannot synthesize TFA, the plasma concentration of TFA is regulated by dietary TFA intake. TFAs are produced either by industrial partial hydrogenation of vegetable or fish oils or by biohydrogenation in ruminant animals [
      • Baum S.J.
      • Kris-Etherton P.M.
      • Willett W.C.
      • Lichtenstein A.H.
      • Rudel L.L.
      • et al.
      Fatty acids in cardiovascular health and disease: a comprehensive update.
      ]. Dietary recommendations for prevention of coronary heart diseases (CHD) include decreasing the intake of saturated FA (SFA) and TFA and replacing them with UFA [
      • Baum S.J.
      • Kris-Etherton P.M.
      • Willett W.C.
      • Lichtenstein A.H.
      • Rudel L.L.
      • et al.
      Fatty acids in cardiovascular health and disease: a comprehensive update.
      ].
      Brouwer et al. reported that all TFA raised the ratio of low-density lipoprotein cholesterol (LDL-C) to high-density lipoprotein cholesterol (HDL-C), and presumably the risk of CHD [
      • Brouwer I.A.
      • Wanders A.J.
      • Katan M.B.
      Effect of animal and industrial Trans fatty acids on HDL and LDL cholesterol levels in humans – a quantitative review.
      ]. There is some evidence that ruminant-derived TFA (R-TFA) do not increase risk for disease but industrially-produced TFA (IP-TFA) do [
      • Mozaffarian D.
      • Katan M.B.
      • Ascherio A.
      • Stampfer M.J.
      • Willett W.C.
      Trans fatty acids and cardiovascular disease.
      ,
      • Li H.
      • Zhang Q.
      • Song J.
      • Wang A.
      • Zou Y.
      • et al.
      Plasma trans-fatty acids levels and mortality: a cohort study based on 1999-2000 National Health and Nutrition Examination Survey (NHANES).
      ]. The prospective cohort study of Norwegian patients with suspected CHD failed to show the association between plasma concentrations of TFA (palmitelaidic acid, an R-TFA; and trans C18:1 isomers; primarily IP-TFA) and incidence of acute myocardial infarction (AMI) after multivariate adjustments [
      • Borgeraas H.
      • Hertel J.K.
      • Seifert R.
      • Berge R.K.
      • Bohov P.
      • et al.
      Serum trans fatty acids, asymmetric dimethylarginine and risk of acute myocardial infarction and mortality in patients with suspected coronary heart disease: a prospective cohort study.
      ]. The prospective cohort study of German patients with suspected CHD showed that higher levels of palmitelaidic acids in erythrocyte membranes were associated with lower risk for sudden cardiac death, and that three trans isomers of C18:2n6 were not related to fatal cardiovascular outcomes [
      • Kleber M.E.
      • Delgado G.E.
      • Lorkowski S.
      • März W.
      • von Schacky C.
      Trans-fatty acids and mortality in patients referred for coronary angiography: the Ludwigshafen Risk and Cardiovascular Health Study.
      ]. The cross-sectional study of Japanese patients undergoing coronary angiography (CAG) failed to show differences in levels of elaidic acid and linoelaidic acids (the two major IP-TFA, although the latter can be formed by frying in non-hydrogenated vegetable oils [
      • Sebedio J.
      • Chardigny J.
      Physiological effects of trans and cyclic fatty acids.
      ]) between patients with and without CHD [
      • Mori K.
      • Ishida T.
      • Yasuda T.
      • Hasokawa M.
      • Monguchi T.
      • et al.
      Serum trans-fatty acid concentration is elevated in young patients with coronary artery disease in Japan.
      ]. They showed significantly higher elaidic acid levels in younger patients with CHD (≤66 years) compared with elder CHD patients, and/or patients with metabolic syndrome compared with patients without metabolic syndrome [
      • Mori K.
      • Ishida T.
      • Yasuda T.
      • Hasokawa M.
      • Monguchi T.
      • et al.
      Serum trans-fatty acid concentration is elevated in young patients with coronary artery disease in Japan.
      ]. In that study, all subjects had undergone CAG and thus subjects without CHD were not generally healthy individuals. It remains controversial how R-TFA and IP-TFA are associated with acute coronary syndrome (ACS).
      The consumption of TFA is currently decreasing in many countries. According to the report of the total diet study (market basket method) from the Ministry of Agriculture, Forestry and Fisheries in Japan, estimated daily intake of TFA in the Japanese is 0.92–0.96 g, or 0.44–0.47% of the total energy intake [
      • Kinoshita M.
      • Yamashita S.
      • Yokote K.
      • Arai H.
      • Iida M.
      • et al.
      Committee for Epidemiology and Clinical Management of Atherosclerosis
      Japan atherosclerosis society (JAS) guidelines for prevention of atherosclerotic cardiovascular diseases 2017.
      ]. This is lower than the <1% of energy target recommended by the World Health Organization [
      • Kinoshita M.
      • Yamashita S.
      • Yokote K.
      • Arai H.
      • Iida M.
      • et al.
      Committee for Epidemiology and Clinical Management of Atherosclerosis
      Japan atherosclerosis society (JAS) guidelines for prevention of atherosclerotic cardiovascular diseases 2017.
      ] and is much lower than the average consumption in the Western countries [
      • Takeuchi H.
      • Sugano M.
      Industrial trans fatty acid and serum cholesterol: the allowable dietary level.
      ]. It remains unclear how low intakes of TFA affect lipid levels and the development of ACS. On the other hand, the Japanese dietary style has markedly changed from the 1960s, and fish to meat ratios in food consumption are decreasing in the younger generation, while the ratios in the Western countries stayed the same or slightly increased [
      • Yokoyama S.
      Beneficial effects of returning to “Japan Diet” for the Japanese.
      ,
      • Shijo Y.
      • Maruyama C.
      • Nakamura E.
      • Nakano R.
      • Shima M.
      • et al.
      Japan diet intake changes serum phospholipid fatty acid compositions in middle-aged men: a pilot study.
      ]. The age profile of the fish/meat >1.0 was ≥40 years in 2000, ≥50 years in 2005 and 2010, and ≥60 years in 2015 in Japan. The study aim was to compare plasma TFA composition such as R-TFA and twelve kinds of trans-C18 isomers (IP-TFA) in male patients with ACS and healthy men, and to investigate their difference by age.

      2. Patients and methods

      2.1 Subjects

      This study enrolled patients with ACS that followed successful percutaneous coronary intervention (PCI) at Showa University Hospital between July 2013 and December 2014. Controls were healthy, non-smoking males aged 40–80 years who were not receiving any pharmacological treatment. The fifty-five healthy men were recruited by advertisement among acquaintances of the research team, and six men were excluded because of their fasting blood glucose levels ≥126 mg/dL. Patients aged above 80, and those undergoing hemodialysis were excluded. The diagnoses of ACS were based on electrocardiographic changes and CAG findings. Serum and plasma samples were collected immediately before the emergency CAG on admission of ACS, and samples from the control subjects were collected after an overnight fast. The institutional review board of Showa University (1535) and Showa Women's University (13–02, 15–02, 17–12) approved this protocol. The investigation conformed to the principles of the Declaration of Helsinki, and the written informed consent was obtained from all subjects.

      2.2 Baseline evaluation

      Serum concentrations of total protein, albumin, total bilirubin, creatinine, total cholesterol, triglyceride, and HDL-C were measured using standard laboratory procedures. LDL-C levels were measured with a direct homogenous assay of the serum using detergents (Sekisui Medical, Tokyo, Japan). Non-HDL-C was estimated by subtracting the HDL-C concentration from the total cholesterol concentration.
      The diagnosis of hypertension was based on a history of hypertension or blood pressure >140 mmHg systolic or >90 mmHg diastolic [
      • Kinoshita M.
      • Yamashita S.
      • Yokote K.
      • Arai H.
      • Iida M.
      • et al.
      Committee for Epidemiology and Clinical Management of Atherosclerosis
      Japan atherosclerosis society (JAS) guidelines for prevention of atherosclerotic cardiovascular diseases 2017.
      ]. Diabetes mellitus was diagnosed as a fasting serum glucose value greater than 126 mg/dL, hemoglobin (Hb) A1c levels greater than 6.5%, or treatment with either oral hypoglycemic agents or insulin [
      • Kashiwagi A.
      • Kasuga M.
      • Araki E.
      • Oka Y.
      • Hanafusa T.
      • et al.
      Committee on the standardization of diabetes mellitus-related laboratory testing of Japan diabetes society (JDS): international clinical harmonization of glycated hemoglobin in Japan: from Japan diabetes society to national glycohemoglobin standardization program values.
      ]. Dyslipidemia was defined as the current use of lipid-lowering medications and/or meeting the criteria of the Japan Atherosclerosis Society for fasting serum lipid levels, i.e., LDL-C ≥140 mg/dL, HDL-C <40 mg/dL, or TG ≥ 150 mg/dL [
      • Kinoshita M.
      • Yamashita S.
      • Yokote K.
      • Arai H.
      • Iida M.
      • et al.
      Committee for Epidemiology and Clinical Management of Atherosclerosis
      Japan atherosclerosis society (JAS) guidelines for prevention of atherosclerotic cardiovascular diseases 2017.
      ]. Body mass index (BMI) was calculated as weight (kilograms) divided by height (meters) squared. Patients with a reported smoking habit of at least one cigarette per day on admission were classified as current smokers.

      2.3 Measurement of fatty acid composition

      Plasma FA levels were measured at Omegaquant, LLC (Sioux Falls, SD, USA). The Plasma samples were stored at −80 °C until the assay. After thawing, an internal standard (C23:0 in the triglyceride form) was added to an aliquot of plasma which was then combined (1:40 parts) with the methylating mixture (boron trifluoride in methanol [14%], toluene, and methanol [35/30/35 v/v]), shaken at 100 °C for 45 min. After cooling, 40 parts of both hexane and distilled water were added. After briefly vortexing, the samples were spun to separate layers, and an aliquot of the hexane layer that contained the FA methyl esters was analyzed by gas chromatography as previously described [
      • Harris W.S.
      • Pottala J.V.
      • Vasan R.S.
      • Larson M.G.
      • Robins S.J.
      Changes in erythrocyte membrane trans and marine fatty acids between 1999 and 2006 in older Americans.
      ,
      • Lemaitre R.N.
      • King I.B.
      • Mozaffarian D.
      • Kuller L.H.
      • Tracy R.P.
      • et al.
      Plasma phospholipid trans fatty acids, fatal ischemic heart disease, and sudden cardiac death in older adults: the Cardiovascular Health Study.
      ]. Identification, precision, and accuracy were evaluated with model mixtures of known FA methyl esters and an established in-house quality-control pool. The chromatographic conditions used in this study were sufficient to isolate the C16:1, C18:1, and C18:2 trans isomers. FA concentrations are expressed as percentages of total FAs by weight and/or as μg/dl. Total trans-C18:1 isomers are calculated as the sum of nine kinds of C18:1 trans isomers. Total trans-C18:2 isomers are calculated as the sum of three trans-linoleic isomers. Total trans C18 FA are the sum of total trans C18:1 isomers and trans C18:2 isomers. The sum of palmitelaidic acids and trans-C18:1 isomer (11-t, Vaccenic) are termed R-TFA, while the sum of the other C18:1 and C18:2 trans species are IP-TFA. Total TFA are calculated as the sum of total R-TFA and IP-TFA.

      2.4 Statistical analysis

      Statistical analysis was performed using the IBM SPSS Statistics for Macintosh, Version 23.0. (IBM Corp. Armonk, NY, USA). Baseline characteristics were compared between control and ACS patients using unpaired t-test for parametric variables, and Wilcoxon tests for non-parametric variables. Comparisons among the groups based on age and ACS or control were performed by one-way analysis of variance (ANOVA) with Tukey's honest significant difference test to identify differences among the four groups. Categorical variables were compared with chi-square tests. Correlation coefficients between lipid levels and fatty acid compositions among subjects who did not take any lipid-lowering drugs, were determined by Spearman's rank analyses. All the statistical analyses were two tailed. p < 0.05 was considered statistically significant.

      3. Results

      Table 1 compares the general characteristics and serum biomarkers between control and ACS subjects. Age, BMI, LDL-C, non-HDL-C, and triglyceride were comparable between the groups. Albumin and HDL-C were significantly lower in ACS patients compared with controls.
      Table 1Comparison of clinical characteristics between control and ACS patients.
      Control (n = 49)ACS (n = 66)p
      Age, years61.5 ± 10.162.2 ± 11.50.483
      BMI, kg/cm224.6 ± 3.424.4 ± 4.20.892
      AMI/UAP057/9<0.0001
      Prior MI020.327
      CVD030.185
      Prior coronary revascularization030.185
      Risk factors
      Smoker (current/former), n034/19<0.0001
      Hypertension, n (%)038 (58%)<0.0001
      Diabetes, n (%)018 (27%)<0.0001
      Dyslipidemia, n (%)22 (45%)50 (76%)0.001
      Prior medication, n (%)
      Prior any medication031 (47%)<0.0001
      Blood pressure lowering021 (32%)<0.0001
      Anti-diabetic therapy010 (15%)0.003
      Beta blocker05 (8%)0.058
      Anti-thrombotic drugs07 (11%)0.018
      Lipid-lowering drugs015 (23%)<0.0001
       Statin010 (15%)0.003
       Ezetimibe01 (2%)0574
       Fibrate01 (2%)0.574
       Omega-3 fatty acid04 (6%)0.104
      Laboratory findings
      Total protein, g/dL7.4 ± 0.2 (25)7.1 ± 0.70.006
      Albumin, g/dL4.7 ± 0.2 (25)4.1 ± 0.5<0.0001
      Total bilirubin,0.6 ± 0.3 (25)0.8 ± 0.40.028
      Creatinine, mg/dL0.88 ± 0.131.02 ± 0.540.943
      Glucose, mg/dL91.2 ± 16.6148.5 ± 60.9<0.0001
      Triglyceride, mg/dL125.8 ± 79.8111.3 ± 69.30.299
      Non-HDL-C, mg/dL148.1 ± 33.8143.8 ± 35.80.507
      LDL-C, mg/dL123.4 ± 29.5123.1 ± 34.70.954
      HDL-C, mg/dL59.8 ± 15.043.7 ± 11.2<0.0001
      Data are expressed as mean ± SD, or number (%). The number in parenthesis indicates the actual number of analyzed cases.
      AMI = acute myocardial infarction; BMI = body mass index; CVD = cerebral vascular disease; HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol; Non-HDL-C = non-high-density lipoprotein cholesterol; PCI = percutaneous coronary intervention; UAP = unstable angina pectoris.
      Table 2 compares TFA between control subjects and ACS patients. Total TFA, total trans-C18 isomers, total trans-C18:1 isomers, total R-TFA isomers, and total IP-TFA isomers were comparable between the groups. Palmitelaidic acid was significantly lower and total trans-C18:2 isomers were significantly higher in ACS patients. R-TFA/arachidonic acid (AA) were similar, while IP-TFA/AA were significantly higher in ACS patients. When the absolute concentrations of TFA were compared, palmitelaidic acid and R-TFA were significantly lower and 18:2 n6tt were significantly higher in ACS patients (Supplementary Table 1). These results were constant when patients treated with lipid-lowering, patients who took ezetimibe and/or n3 PUFA, and those with glucose ≥140 mg/dl were excluded (Supplementary Table 2).
      Table 2Comparison of plasma TFA composition between control and ACS men.
      Table thumbnail fx2
      Data are expressed as mean ± SD, and FA are presented as a percent of total plasma FA. Pink rows mean increase in ACS, and blue ones mean decrease in ACS.
      Spearman correlation coefficients were computed between TFA concentration and LDL-C, non-HDL-C, HDL-C, LDL-C to HDL-C ratios, or FA concentrations in 100 subjects not receiving lipid-lowering therapy. Palmitelaidic acid was significantly directly associated with HDL-C and significantly inversely associated with LDL-C/HDL-C ratio (Table 3). Total C18:1 trans isomers were significantly inversely associated with LDL-C and non-HDL-C, and total C18:2 trans isomers were significantly inversely associated with non-HDL-C. On the other hand, absolute concentrations of total C18:2 TFA were significantly directly associated with LDL-C and non-HDL-C in ACS men. Absolute concentrations of palmitelaidic acids and total C18:1 TFA were significantly positively associated with each FA concentration. Significantly positive association between palmitelaidic acids (either percentage of TFA or absolute levels) and total n-3 PUFA, eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA), or EPA plus DHA/AA were observed in all subjects, controls, and ACS men.
      Table 3Comparisons of correlation coefficients between TFA and various lipid levels based on TFA as percentage of total fatty acids or absolute amount.
      16:1 n7tTotal C18:1 TFATotal C18:2 TFA
      TFA as percentage of total fatty acids
      All subjects (n = 100), %0.20 ± 0.060.86 ± 0.210.64 ± 0.19
       LDL-C (mg/dl)−0.081−0.333***−0.151
       Non-HDL-C (mg/dl)−0.062−0.363***−0.330**
       HDL-C (mg/dl)0.269**0.039−0.172
       LDL-C/HDL-C−0.201*−0.1720.063
       Total SFA (%)−0.0330.126−0.091
       Total oleic cis isomers (%)−0.305**−0.184−0.127
       Total n-3 PUFA (%)0.442***0.170−0.181
       EPA plus DHA (%)0.458***0.185−0.163
       Total n-6 PUFA (%)0.136−0.1830.230*
       AA (%)−0.0390.0190.108
       EPA plus DHA/AA0.474***0.189−0.199
      Healthy men (n = 49), %0.20 ± 0.060.87 ± 0.190.60 ± 0.19
       LDL-C (mg/dl)0.167−0.261−0.260
       Non-HDL-C (mg/dl)0.163−0.324*−0.406**
       HDL-C (mg/dl)0.1420.175−0.101
       LDL-C/HDL-C0.037−0.194−0.003
       Total SFA (%)−0.179−0.0210.055
       Total oleic cis isomers (%)−0.180−0.337*−0.255
       Total n-3 PUFA (%)0.310*0.144−0.155
       EPA plus DHA (%)0.321*0.172−0.135
       Total n-6 PUFA (%)−0.0940.0120.241
       AA (%)−0.2190.1200.361*
       EPA plus DHA/AA0.385**0.140−0.266
      ACS men (n = 51), %0.16 ± 0.050.86 ± 0.230.67 ± 0.17
       LDL-C (mg/dl)−0.222−0.446**−0.120
       Non-HDL-C (mg/dl)−0.228−0.431**−0.310*
       HDL-C (mg/dl)0.122−0.179−0.027
       LDL-C/HDL-C−0.218−0.195−0.104
       Total SFA (%)0.1720.254−0.310*
       Total oleic cis isomers (%)−0.244−0.007−0.219
       Total n-3 PUFA (%)0.448**0.191−0.020
       EPA plus DHA (%)0.466**0.191−0.002
       Total n-6 PUFA (%)−0.230−0.332*0.266
       AA (%)−0.032−0.049−0.031
       EPA plus DHA/AA0.477***0.2290.008
      TFA as absolute amount
      All subjects (n = 100) μg/ml6.30 ± 2.6429.8 ± 10.321.4 ± 5.8
       LDL-C (mg/dl)0.1360.0070.192
       Non-HDL-C (mg/dl)0.296**0.1890.194
       HDL-C (mg/dl)0.216*0.086−0.196
       LDL-C/HDL-C−0.094−0.0710.210*
       Total SFA (μg/ml)0.611***0.601***0.221*
       Total oleic cis isomers (μg/ml)0.473***0.505***0.313**
       Total n-3 PUFA (μg/ml)0.654***0.545***0.117
       EPA plus DHA (μg/ml)0.620***0.505***0.069
       Total n-6 PUFA (μg/ml)0.487***0.431***0.259**
       AA (μg/ml)0.340**0.327**0.136
       EPA plus DHA/AA0.475***0.365***−0.016
      Healthy men (n = 49) μg/ml7.13 ± 2.8630.7 ± 7.920.5 ± 4.7
       LDL-C (mg/dl)0.332*0.1290.025
       Non-HDL-C (mg/dl)0.481***0.316*0.070
       HDL-C (mg/dl)0.0440.020−0.278
       LDL-C/HDL-C0.1300.0250.204
       Total SFA (μg/ml)0.624***0.637***0.256
       Total oleic cis isomers (μg/ml)0.610***0.635***0.319*
       Total n-3 PUFA (μg/ml)0.637***0.574***0.160
       EPA plus DHA (μg/ml)0.599***0.518***0.110
       Total n-6 PUFA (μg/ml)0.559***0.576***0.249
       AA (μg/ml)0.380**0.424**0.310*
       EPA plus DHA/AA0.447**0.371**−0.054
      ACS men (n = 51), μg/ml5.51 ± 2.1629.1 ± 12.122.3 ± 6.5
       LDL-C (mg/dl)0.048−0.0940.311*
       Non-HDL-C (mg/dl)0.1880.0750.305*
       HDL-C (mg/dl)0.1560.001−0.033
       LDL-C/HDL-C−0.073−0.0810.126
       Total SFA (μg/ml)0.588***0.551***0.206
       Total oleic cis isomers (μg/ml)0.384**0.412**0.305*
       Total n-3 PUFA (μg/ml)0.624***0.486***0.245
       EPA plus DHA (μg/ml)0.588***0.453**0.177
       Total n-6 PUFA (μg/ml)0.403**0.2530.305*
       AA (μg/ml)0.1570.1220.024
       EPA plus DHA/AA0.449**0.351*0.085
      Data are expressed as Spearman's Rho between TFA levels (% or μg/ml) and LDL-C, non-HDL-C, HDL-C, LDL-C/HDL-C ratio, total SFA, total oleic cis isomers, total n-3 PUFA, EPA plus DHA, total n-6 PUFA, AA, and EPA plus DHA/AA ratio in subjects who did not take any lipid-lowering drugs.
      *p<0.05, **p<0.01, ***p<0.001.
      Table 4 compares other FA compositions between the control subjects and ACS patients. ACS patients had significantly higher levels of saturated FA, mainly myristic and palmitic acids, and MUFA, mainly oleic acid, and lower levels of n-3 PUFA, mainly EPA and DHA, and AA, n-6 PUFA.
      Table 4Comparison of plasma fatty acid composition between the groups.
      Table thumbnail fx3
      Data are expressed as mean ± SD, and FAs are presented as a percent of total plasma FA. Pink rows mean increase in ACS, and blue ones mean decrease in ACS.
      AA = arachidonic acid; DGLA = dihomo-gamma linolenic acid; DHA = docosahexaenoic acid; DPA = docosapentaenoic acid; EPA = eicosapentaenoic acid.
      Table 5 compares FA composition and blood parameters between control subjects and ACS patients separated by age. In subjects <60 years old, palmitelaidic acid and certain trans-C18:1 isomers (4t and 5t) were significantly lower in ACS patients. In elder groups, IP-TFA/AA were significantly higher in ACS patients. Both total n-3 PUFA and EPA plus DHA were significantly lower in ACS patients and further lower in ACS patients <60 years old.
      Table 5Comparison of plasma TFA composition between control and ACS men by age.
      Middle-age, <60 yearsElder ≥60 years
      Control (n = 24)ACS men (n = 25)Control (n = 25)ACS men (n = 41)
      Age, years52.5 ± 3.250.0 ± 7.470.1 ± 6.0###69.6 ± 5.6###
      BMI, kg/m224.6 ± 3.026.3 ± 4.424.6 ± 3.023.2 ± 3.8*
      Diabetes, %016034
      Lipid-lowering, %012029
      Total FA, mg/L3772.3 ± 1201.33530.7 ± 1412.63514.7 ± 994.83206.3 ± 771.4
      Total TFAs, %1.71 ± 0.311.64 ± 0.291.63 ± 0.361.73 ± 0.33
      16:1 7t, %0.21 ± 0.060.16 ± 0.05**0.18 ± 0.050.18 ± 0.06
      Total 18:1 TFAs, %0.91 ± 0.180.81 ± 0.230.83 ± 0.200.87 ± 0.21
      Total 18:2 TFAs, %0.59 ± 0.200.67 ± 0.170.61 ± 0.190.68 ± 0.17
      Total R-TFA, %0.32 ± 0.080.27 ± 0.080.28 ± 0.080.29 ± 0.10
      Total IP-TFA, %1.40 ± 0.291.37 ± 0.241.35 ± 0.321.44 ± 0.30
      18:1 4t, %010 ± 0.050.06 ± 0.03*0.08 ± 0.050.08 ± 0.05
      18:1 5t. %0.09 ± 0.050.06 ± 0.02*0.07 ± 0.030.08 ± 0.04
      18:1 (6–8)t, %0.06 ± 0.020.05 ± 0.020.04 ± 0.020.05 ± 0.02
      18:1 9t, %0.11 ± 0.050.12 ± 0.060.09 ± 0.040.11 ± 0.04
      18:1 10t, %0.13 ± 0.060.12 ± 0.040.10 ± 0.050.12 ± 0.04
      18:1 11t, %0.11 ± 0.040.12 ± 0.060.09 ± 0.050.11 ± 0.05
      18:1 12t, %0.05 ± 0.020.07 ± 0.040.05 ± 0.030.07 ± 0.03
      18:1 13-14t, %0.15 ± 0.070.12 ± 0.050.17 ± 0.050.16 ± 0.06
      18:1 16t, %0.10 ± 0.050.10 ± 0.060.12 ± 0.060.10 ± 0.05
      18:2 n6tt, %0.17 ± 0.090.21 ± 0.090.19 ± 0.080.23 ± 0.11
      18:2 n6ct, %0.23 ± 0.100.27 ± 0.060.25 ± 0.110.26 ± 0.08
      18:2 n6tc, %0.19 ± 0.070.19 ± 0.080.17 ± 0.060.19 ± 0.08
      Total R-TFA/AA, %5.7 ± 2.05.0 ± 1.94.7 ± 1.85.7 ± 2.1
      Total IP-TFA/AA, %24.4 ± 6.024.9 ± 6.722.4 ± 6.028.4 ± 7.6**
      Total SFA, %30.0 ± 1.730.9 ± 2.130.5 ± 1.530.8 ± 1.9
      Total MUFA, %25.8 ± 3.627.6 ± 2.724.6 ± 2.027.5 ± 2.9**
      Total n3PUFA, %8.0 ± 2.95.4 ± 1.8**9.2 ± 2.67.5 ± 2.6*##
      Totaln6PUFA, %34.7 ± 4.634.5 ± 3.734.1 ± 3.432.5 ± 3.3
      AA, %5.85 ± 1.095.73 ± 1.186.16 ± 1.195.27 ± 1.15*
      EPA + DHA, %6.7 ± 2.84.2 ± 1.7**7.9 ± 2.46.0 ± 2.4*#
      EPA + DHA/AA1.19 ± 0.580.74 ± 0.27**1.32 ± 0.441.20 ± 0.55##
      Total protein, g/dL7.4 ± 0.27.2 ± 0.77.4 ± 0.37.1 ± 0.7
      Albumin, g/dL4.8 ± 0.24.3 ± 0.5*4.6 ± 0.24.0 ± 0.5**
      Total bilirubin,0.6 ± 0.30.9 ± 0.50.6 ± 0.20.8 ± 0.4
      Creatinine, mg/dL0.86 ± 0.070.79 ± 0.150.92 ± 0.181.16 ± 0.64##
      Glucose, mg/dL89.4 ± 18.2138.1 ± 58.9**92.9 ± 15.1154.9 ± 61.9***
      Triglyceride, mg/dL140.0 ± 101.1114.9 ± 74.6112.3 ± 50.5109.2 ± 66.7
      Non-HDL-C, mg/dL150.0 ± 37.1158.4 ± 40.3121.9 ± 29.4114.0 ± 29.7
      LDL-C, mg/dL125.1 ± 30.2138.0 ± 37.7121.9 ± 29.4114.0 ± 29.7#
      HDL-C, mg/dL61.2 ± 16.441.4 ± 9.5***58.5 ± 13.845.1 ± 12.1**
      Data are expressed as mean ± SD, and FA are presented as a percent of total plasma FA. *p < 0.05, **p < 0.01, ***p < 0.001 vs. non-ACS counterpart; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. younger counterpart by one-way ANOVA with Tukey's honest post hoc test.

      4. Discussion

      To the best of our knowledge, this is the first study to compare various TFA isomers between patients with ACS and age-matched healthy men in Japanese subjects. There are four novel findings in this report. First, palmitelaidic acid, a major R-TFA, was significantly lower in ACS patients, especially middle-aged ACS patients than in healthy controls, and was inversely associated with LDL-C to HDL-C ratios. Second, trans-C18:2 isomers (IP- or frying-derived TFA) and IP-TFA/AA ratios were significantly higher in ACS patients, especially elder ACS patients, than healthy men. Third, absolute concentrations of trans-C18:2 isomers were significantly directly associated with LDL-C and non-HDL-C in ACS men. Forth, both proportional and absolute concentrations of palmitelaidic acid were significantly directly associated with HDL-C, EPA + DHA, and EPA + DHA/AA ratios.
      Higher TFA levels have been reported to be associated with increased risk for CHD but this has mostly been observed in Western populations [
      • Mozaffarian D.
      • Katan M.B.
      • Ascherio A.
      • Stampfer M.J.
      • Willett W.C.
      Trans fatty acids and cardiovascular disease.
      ]. In the Nurses’ Health Study, increases in total TFA, trans-C18:1 isomers, and trans-C18:2 isomers in erythrocyte membrane were significantly associated with CHD [
      • Sun Q.
      • Ma J.
      • Campos H.
      • Hankinson S.E.
      • Manson J.E.
      • et al.
      A prospective study of trans fatty acids in erythrocytes and risk of coronary heart disease.
      ]. In the Cardiovascular Health study, a prospective cohort of older US adults, neither plasma trans-C18:1 isomers nor palmitelaidic acids, but linoelaidic acid (trans-C18:2 isomers), were significantly associated with fatal CHD [
      • Lemaitre R.N.
      • King I.B.
      • Mozaffarian D.
      • Kuller L.H.
      • Tracy R.P.
      • et al.
      Plasma phospholipid trans fatty acids, fatal ischemic heart disease, and sudden cardiac death in older adults: the Cardiovascular Health Study.
      ] and total mortality, mainly due to cardiovascular disease and increased risk of CHD [
      • Wang Q.
      • Imamura F.
      • Lemaitre R.N.
      • Rimm E.B.
      • Wang M.
      • et al.
      Plasma phospholipid trans-fatty acids levels, cardiovascular diseases, and total mortality: the Cardiovascular Health Study.
      ]. In recent studies comparing TFA levels between US and Japan, both we and others have observed much lower levels in Japan, consistent with lower CHD rates [18.19]. Our recent study with Japanese and American older men (>age 50) showed markedly lower levels of elaidic and linoelaidic acids (IP-TFA) and significantly higher levels of palmitelaidic acids (R-TFA), compared with American men [
      • Takada A.
      • Shimizu F.
      • Ishii Y.
      • Ogawa M.
      • Takao T.
      • et al.
      Plasma fatty acid composition in men over 50 in the USA and Japan.
      ]. The Hawaii-Los Angeles-Hiroshima Study reported that serum elaidic acid concentrations in the native Japanese living in Hiroshima were significantly lower than those in the Japanese-Americans living in Los Angeles [
      • Itcho K.
      • Yoshii Y.
      • Ohno H.
      • Oki K.
      • Shinohara M.
      Association between serum elaidic acid concentration and insulin resistance in two Japanese cohorts with different lifestyles.
      ]. In a German cohort with relatively low TFA levels, there was no association of IP-TFA with CHD outcomes [
      • Kleber M.E.
      • Delgado G.E.
      • Lorkowski S.
      • März W.
      • von Schacky C.
      Trans-fatty acids and mortality in patients referred for coronary angiography: the Ludwigshafen Risk and Cardiovascular Health Study.
      ]. Therefore, we undertook this study to see if in Japan, another country with relatively low TFA levels, there was a relationship of TFA with CHD risk. We found that not trans-C18:1 isomers but trans-C18:2 isomers were significantly higher in ACS, that was in good agreement with previous reports [
      • Baylin A.
      • Kabagambe E.K.
      • Ascherio A.
      • Spiegelman D.
      • Campos H.
      High 18:2 trans-fatty acids in adipose tissue are associated with increased risk of nonfatal acute myocardial infarction in costa rican adults.
      ,
      • Lemaitre R.N.
      • King I.B.
      • Raghunathan T.E.
      • Pearxe R.M.
      • Weinmann S.
      Cell membrane trans-fatty acids and the risk of primary cardiac arrest.
      ]. In the Costa Rican population, not trans-C18:1 isomers but high trans-C18:2 isomers in adipose tissue were significantly associated with increased risk of non-fatal AMI [
      • Baylin A.
      • Kabagambe E.K.
      • Ascherio A.
      • Spiegelman D.
      • Campos H.
      High 18:2 trans-fatty acids in adipose tissue are associated with increased risk of nonfatal acute myocardial infarction in costa rican adults.
      ]. Two population-based case-control studies in the US reported that neither total TFA nor trans-C18:1 isomers but high trans-C18:2 isomers in erythrocyte membrane were significantly associated with cardiac arrest [
      • Lemaitre R.N.
      • King I.B.
      • Mozaffarian D.
      • Kuller L.H.
      • Tracy R.P.
      • et al.
      Plasma phospholipid trans fatty acids, fatal ischemic heart disease, and sudden cardiac death in older adults: the Cardiovascular Health Study.
      ,
      • Lemaitre R.N.
      • King I.B.
      • Raghunathan T.E.
      • Pearxe R.M.
      • Weinmann S.
      Cell membrane trans-fatty acids and the risk of primary cardiac arrest.
      ]. Our present results showed trans-C18:2 isomers and IP-TFA were higher, and IP-TFA/AA were significantly higher in ACS patients. These results support that IP-TFA is associated with increased risk of CHD even in Japan.
      The previous cohort studies showed that higher plasma levels or higher intake of palmitelaidic acid was significantly associated with lower risk for sudden cardiac death [
      • Kleber M.E.
      • Delgado G.E.
      • Lorkowski S.
      • März W.
      • von Schacky C.
      Trans-fatty acids and mortality in patients referred for coronary angiography: the Ludwigshafen Risk and Cardiovascular Health Study.
      ] and diabetes [
      • Mozaffarian D.
      • Cao H.
      • King I.B.
      • Lemaitre R.N.
      • Song X.
      • et al.
      Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults: a cohort study.
      ,
      • Mozaffarian D.
      • de Oliveira Otto M.C.
      • Lemaitre R.N.
      • Fretts A.M.
      • Hotamisligil G.
      • et al.
      Trans-Palmitoleic acid, other dairy fat biomarkers, and incident diabetes: the Multi-Ethnic Study of Atherosclerosis (MESA).
      ]. Our finding of slightly but significantly lower palmitelaidic acid in ACS patients is generally consistent with these findings. The major sources of palmitelaidic acid are ruminant meat and milk, and it has been reported that the consumption of dairy products may help reduce risk for CHD and diabetes [
      • Prentice A.M.
      Dairy products in global public health.
      ,
      • Astrup A.
      Yogurt and dairy product consumption to prevent cardiometabolic diseases: epidemiologic and experimental studies.
      ,
      • de Souza R.J.
      • Mente A.
      • Maroleanu A.
      • Cozma A.I.
      • Ha V.
      • et al.
      Intake of saturated and trans unsaturated fatty acids and risk of all cause mortality, cardiovascular disease, and type 2 diabetes: systematic review and meta-analysis of observational studies.
      ].
      It has been well accepted that higher dietary intake of TFA raises LDL-C and decreases HDL-C, which results in an elevated risk of CHD [
      • Mozaffarian D.
      • Katan M.B.
      • Ascherio A.
      • Stampfer M.J.
      • Willett W.C.
      Trans fatty acids and cardiovascular disease.
      ,
      • Katan M.B.
      • Zock P.L.
      • Mensink R.P.
      Effects of fats and fatty acids on blood lipids in humans: an overview.
      ]. However, some studies pointed out that the dose-response relations between TFA and lipid levels were not observed when the dietary intake of TFAs was low [
      • Kinoshita M.
      • Yamashita S.
      • Yokote K.
      • Arai H.
      • Iida M.
      • et al.
      Committee for Epidemiology and Clinical Management of Atherosclerosis
      Japan atherosclerosis society (JAS) guidelines for prevention of atherosclerotic cardiovascular diseases 2017.
      ,
      • Hunter J.E.
      Dietary trans fatty acids: review of recent human studies and food industry responses.
      ,
      • Allen B.C.
      • Vincent M.J.
      • Liska D.
      • Haber L.T.
      Meta-regression analysis of the effect of trans fatty acids on low-density lipoprotein cholesterol.
      ]. This is consistent with a TFA intervention study in young Japanese women, which showed no relationship between TFA and LDL-C or HDL-C [
      • Takeuchi H.
      • Sugano M.
      Industrial trans fatty acid and serum cholesterol: the allowable dietary level.
      ]. The TRANSFAIR study, a cross-sectional study among middle-aged men and women in eight European countries, showed no associations between total TFA intake and LDL-C, HDL-C, or LDL-C/HDL-C ratio [
      • van de Vijver L.P.
      • Kardinaal A.F.
      • Couet C.
      • Aro A.
      • Kafatos A.
      • et al.
      Association between trans fatty acid intake and cardiovascular risk factors in Europe: the TRANSFAIR study.
      ]. In addition, the TRANSFAIR study reported two relationships between TFA intake and serum lipid levels: trans-C18:1 isomers or trans-C18:2 were significantly inversely associated with total cholesterol or LDL-C, and palmitelaidic acids were inversely associated with HDL-C and were positively associated with LDL-C/HDL-C ratio [
      • van de Vijver L.P.
      • Kardinaal A.F.
      • Couet C.
      • Aro A.
      • Kafatos A.
      • et al.
      Association between trans fatty acid intake and cardiovascular risk factors in Europe: the TRANSFAIR study.
      ]. On the other hand, the Cardiovascular Health study [
      • Mozaffarian D.
      • Cao H.
      • King I.B.
      • Lemaitre R.N.
      • Song X.
      • et al.
      Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults: a cohort study.
      ] showed that palmitelaidic acid was positively associated with HDL-C and inversely associated with triglyceride. The reason of this discrepancy remains unclear. Our present results support the inverse association between trans-C18:1 isomers or trans-C18:2 and non-HDL-C or LDL-C in the TRANSFAIR study [
      • van de Vijver L.P.
      • Kardinaal A.F.
      • Couet C.
      • Aro A.
      • Kafatos A.
      • et al.
      Association between trans fatty acid intake and cardiovascular risk factors in Europe: the TRANSFAIR study.
      ]; the direct association between palmitelaidic acids and HDL-C in the Cardiovascular Health study [
      • Mozaffarian D.
      • Cao H.
      • King I.B.
      • Lemaitre R.N.
      • Song X.
      • et al.
      Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults: a cohort study.
      ]; and the direct association between palmitelaidic acids and LDL-C in the previous intervention studies [
      • Kuhnt K.
      • Degen C.
      • Jahreis G.
      Evaluation of the impact of ruminant trans fatty acids on human health: important aspects to consider.
      ]. In addition, our present results showed inconsistent relationship between TFA and lipid parameters based on the proportional and absolute concentration of TFA. The absolute concentration of trans-C18:2 was significantly directly associated with LDL-C or non-HDL-C in ACS patients. On the other hand, both proportional and absolute concentration of palmitelaidic acids was significantly directly associated with HDL-C, n-3 PUFA, EPA + DHA or EPA + DHA/AA. Therefore, dietary patterns rich in EPA and DHA may be associated with higher intake of palmitelaidic acids, and these lifestyles were less observed in ACS patients, especially middle-aged patients. The present study supports that R-TFA is cardioprotective, although further studies are needed [
      • Kleber M.E.
      • Delgado G.E.
      • Lorkowski S.
      • März W.
      • von Schacky C.
      Trans-fatty acids and mortality in patients referred for coronary angiography: the Ludwigshafen Risk and Cardiovascular Health Study.
      ,
      • Mozaffarian D.
      • Cao H.
      • King I.B.
      • Lemaitre R.N.
      • Song X.
      • et al.
      Trans-palmitoleic acid, metabolic risk factors, and new-onset diabetes in U.S. adults: a cohort study.
      ,
      • Mozaffarian D.
      • de Oliveira Otto M.C.
      • Lemaitre R.N.
      • Fretts A.M.
      • Hotamisligil G.
      • et al.
      Trans-Palmitoleic acid, other dairy fat biomarkers, and incident diabetes: the Multi-Ethnic Study of Atherosclerosis (MESA).
      ].
      Although it is likely that most of TFA are of dietary origin [
      • Takeuchi H.
      • Sugano M.
      Industrial trans fatty acid and serum cholesterol: the allowable dietary level.
      ], we have very recently reported that various food intake except preference drinks such as tea or coffee, was not associated with plasma TFA levels in Japanese healthy old men [
      • Shimizu F.
      • Ishii Y.
      • Ogawa M.
      • Takao T.
      • Koba S.
      • et al.
      Effects of various foods intakes on plasma levels of trans fatty acids in Japanese old men.
      ]. A meta-analysis of seven cohorts with genome-wide association studies has not identified a significant genetic control for TFA, including palmitelaidc acids, trans-C18:1 isomers, trans/trans-C18:2, and trans/cis-C18:2 isomers [
      • Mozaffarian D.
      • Kabagambe E.K.
      • Johnson C.O.
      • Lemaitre R.N.
      • Manichaikul A.
      • et al.
      Genetic loci associated with circulating phospholipid trans fatty acids: a meta-analysis of genome-wide association studies from the CHARGE Consortium.
      ]. Gotoh et al. have very recently reported the distribution of trans-C18:1 positional isomers in various foods consumed in Japan [
      • Gotoh N.
      • Yoshinaga K.
      • Kagiono S.
      • Katoh Y.
      • Mizuno Y.
      • et al.
      Evaluating the content and distribution on trans fatty acid isomers in foods consumed in Japan.
      ]. They showed that some foods contained ≥1.0 g TFA/100 g food, and high content of trans-C18:2 in foods was attributed to the heating of oil. In addition, they described that difference between monoene-rich and polyene-rich TFA was attributed to diverse TFA formation mechanisms [
      • Gotoh N.
      • Yoshinaga K.
      • Kagiono S.
      • Katoh Y.
      • Mizuno Y.
      • et al.
      Evaluating the content and distribution on trans fatty acid isomers in foods consumed in Japan.
      ]. Further studies are required to investigate both dietary intake and plasma levels of TFA.
      We also found case-control differences in other plasma FA in this study, in particular, for palmitic, oleic, EPA, DHA and AA. According to a meta-analysis of prospective cohort studies investigating plasma FA and CHD outcome, relative risk and 95% of confidence interval (CI) for CHD for these five FAs was (respectively): 1.15 (CI 0.96–1.37), 1.09 (CI 0.97–1.23), 0.78 (CI 0.65–0.94), 0.79 (CI 0.67–0.93), and 0.83 (CI 0.74–0.92) [
      • Chowdhury R.
      • Warnakula S.
      • Kunutsor S.
      • Crowe F.
      • Ward H.A.
      • et al.
      Association of dietary, circulating, and supplement fatty acids with coronary risk: a systematic review and meta-analysis.
      ]. Our results are consistent with the statistically significant protective associations with EPA, DHA and AA, and with the trends toward adverse relationships with palmitic and oleic. The role of oleic acid in CHD is controversial [
      • Joris P.J.
      • Mensink R.P.
      Role of cis-monounsaturated fatty acids in the prevention of coronary heart disease.
      ]. Our finding of adverse associations for oleic acid between cases and controls is consistent with a recent report from the Multi-Ethnic Study of Atherosclerosis. In this prospective cohort study from the US with 6568 men and women aged 45–84 years without clinical evidence of cardiovascular disease, the top quartile of plasma oleic acid was linked to a significantly greater risk for all-cause mortality, cardiovascular disease, and heart failure after adjusting for typical cardiovascular risk factors, as well as plasma n-3 PUFA [
      • Steffen B.T.
      • Duprez D.
      • Szklo M.
      • Guan W.
      • Tsai M.Y.
      Circulating oleic acid levels are related to greater risks of cardiovascular events and all-cause mortality: the multi-ethnic study of atherosclerosis.
      ]. More studies are clearly needed to better understand the role of oleic acid in CHD prevention. The favorable association with n-6 PUFA, AA was perhaps unexpected given the popular view that this is a “proinflammatory” PUFA. Our findings, along with those of the Chowdhury meta-analysis cited above, indicate that this view needs to be re-considered. As noted above, the ACS patients, especially middle-aged patients, showed significantly lower levels of EPA and DHA, the two PUFAs coming from fish. It is well accepted that n3-PUFA has various beneficial effects in the prevention of CHD [
      • Lavie C.J.
      • Milani R.V.
      • Mehra M.R.
      • Ventura H.P.
      Omega-3 polyunsaturated fatty acids and cardiovascular diseases.
      ,
      • Saravanan P.
      • Davidson N.
      • Schmidt E.B.
      • Calder P.C.
      Cardiovascular effects of marine omega-3 fatty acids.
      ], but these effects have mostly been observed in Western populations. Our findings suggest that even in Japan, where average EPA and DHA levels are much higher than in the US [
      • Sekikawa A.
      • Curb J.D.
      • Ueshima H.
      • El-Saed A.
      • Kadowaki T.
      • et al.
      ERA JUMP (Electron-Beam tomography, risk factor Assessment among Japanese and U.S. Men in the post-world war II birth cohort) study group.: marine-derived n-3 fatty acids and atherosclerosis in Japanese, Japanese-American, and white men: a cross-sectional study.
      ], higher levels of the marine n-3 PUFA are associated with lower cardiovascular disease risk. However, the Japanese dietary style has changed markedly in the younger generation since 1990 [
      • Yokoyama S.
      Beneficial effects of returning to “Japan Diet” for the Japanese.
      ,
      • Shijo Y.
      • Maruyama C.
      • Nakamura E.
      • Nakano R.
      • Shima M.
      • et al.
      Japan diet intake changes serum phospholipid fatty acid compositions in middle-aged men: a pilot study.
      ]. Lack of fish intake and excessive oils and meat and poultry intakes have been recognized in subjects <60 years old at the subject recruitment of 2013–2014. Our present results show that palmielaidic acid and n-3 PUFA are markedly lower in middle-aged ACS patients.
      The major limitation of the present study is the single center cross sectional case-control analysis with very small sample size, and like all cross-sectional study, causal relationships cannot be established. Second, the intakes of dietary TFA and FA compositions were not measured. Although we had been able to do so, and the ACS patients were in fact eating more 18:2t (perhaps more fried foods), our conclusions would not have changed. Third, our control subjects were not randomly selected from either the population or patients admitted to the hospital for non-cardiac diagnoses. Fourth, the setting for collecting blood samples was different between controls and ACS patients, i.e., fasting vs. possibly non-fasting. Whether 18:2t levels in the cases were higher because of a recent meal cannot be excluded. Fifth, we could not measure TFA in erythrocyte membranes and/or lipoprotein fractions. These factors should be investigated in future studies.
      In conclusion, the present study demonstrated three findings. First, IP-TFA intake (estimated from plasma levels) is low in Japan, and accordingly, there is little difference in IP-TFA levels between Japanese ACS patients and healthy controls. Second, R-TFA intakes appear to be lower in ACS patients, possibly helping explain their increased risk for cardiac disease. Third, the association of TFA and CHD might differ depending on the TFA species, and the present study was too small to definitively answer this question. Future studies should be conducted to evaluate these issues in a larger number of patients.

      Conflicts of interest

      The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript.

      Financial support

      This study was supported by JSPS (Japan Society for the promotion of science), KAKENHI, Grants-in-Aid for Scientific Research Grant Number JP26460779 , Research Grants from the Ito Foundation (No. 105 ), and Ministry of Education, Culture, Sports, Science and Technology , Private University Research Branding Project, “Elucidation and Clinical Application of the Redox Regulation Systems Based on the Accomplishments of a Comprehensive Medical University Contributing to Health and Longevity”.

      Author contributions

      SK wrote the article, conducting the data analysis, contributed to study design, data interpretation, and critical revision of the article. TT, FS, MO, YI, YY, FF, FT, and MS contributed to acquisition of data and data analysis. WSH contributed to data analysis, drafting of the article, data interpretation, and critical revision of the article. AT contributed to study design, drafting of the article, data interpretation, and critical revision of the article.

      Acknowledgments

      We are grateful for the valuable help with this study of the nursing stuff of the catheterization laboratory and all of the cardiologists at the Department of Cardiology of Showa University Hospital.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:

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